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Alhammadi SHA, Baby B, Antony P, Jobe A, Humaid RSM, Alhammadi FJA, Vijayan R. Modeling the Binding of Anticancer Peptides and Mcl-1. Int J Mol Sci 2024; 25:6529. [PMID: 38928234 PMCID: PMC11203456 DOI: 10.3390/ijms25126529] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2024] [Revised: 05/22/2024] [Accepted: 05/27/2024] [Indexed: 06/28/2024] Open
Abstract
Mcl-1 (myeloid cell leukemia 1), a member of the Bcl-2 family, is upregulated in various types of cancer. Peptides representing the BH3 (Bcl-2 homology 3) region of pro-apoptotic proteins have been demonstrated to bind the hydrophobic groove of anti-apoptotic Mcl-1, and this interaction is responsible for regulating apoptosis. Structural studies have shown that, while there is high overall structural conservation among the anti-apoptotic Bcl-2 (B-cell lymphoma 2) proteins, differences in the surface groove of these proteins facilitates binding specificity. This binding specificity is crucial for the mechanism of action of the Bcl-2 family in regulating apoptosis. Bim-based peptides bind specifically to the hydrophobic groove of Mcl-1, emphasizing the importance of these interactions in the regulation of cell death. Molecular docking was performed with BH3-like peptides derived from Bim to identify high affinity peptides that bind to Mcl-1 and to understand the molecular mechanism of their interactions. The interactions of three identified peptides, E2gY, E2gI, and XXA1_F3dI, were further evaluated using 250 ns molecular dynamics simulations. Conserved hydrophobic residues of the peptides play an important role in their binding and the structural stability of the complexes. Understanding the molecular basis of interaction of these peptides will assist in the development of more effective Mcl-1 specific inhibitors.
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Affiliation(s)
- Shamsa Husain Ahmed Alhammadi
- Department of Biology, College of Science, United Arab Emirates University, Al Ain P.O. Box 15551, United Arab Emirates
| | - Bincy Baby
- Department of Biology, College of Science, United Arab Emirates University, Al Ain P.O. Box 15551, United Arab Emirates
| | - Priya Antony
- Department of Biology, College of Science, United Arab Emirates University, Al Ain P.O. Box 15551, United Arab Emirates
| | - Amie Jobe
- Department of Biology, College of Science, United Arab Emirates University, Al Ain P.O. Box 15551, United Arab Emirates
| | - Raghad Salman Mohammed Humaid
- Department of Biology, College of Science, United Arab Emirates University, Al Ain P.O. Box 15551, United Arab Emirates
| | - Fatema Jumaa Ahmed Alhammadi
- Department of Biology, College of Science, United Arab Emirates University, Al Ain P.O. Box 15551, United Arab Emirates
| | - Ranjit Vijayan
- Department of Biology, College of Science, United Arab Emirates University, Al Ain P.O. Box 15551, United Arab Emirates
- The Big Data Analytics Center, United Arab Emirates University, Al Ain P.O. Box 15551, United Arab Emirates
- Zayed Center for Health Sciences, United Arab Emirates University, Al Ain P.O. Box 15551, United Arab Emirates
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He W, Lin J, Yu F, Leng Y, Pan Z, Liang Q, Liu S, Huang X. Identification and function analysis of BCL2 in immune response of Pteria penguin. FISH & SHELLFISH IMMUNOLOGY 2024; 149:109574. [PMID: 38692379 DOI: 10.1016/j.fsi.2024.109574] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/21/2024] [Revised: 03/28/2024] [Accepted: 04/17/2024] [Indexed: 05/03/2024]
Abstract
B-cell lymphoma/leukemia-2 (BCL2), an anti-apoptotic factor in the mitochondrial regulatory pathway of apoptosis, is critically important in immune defenses. In this study, a novel BCL2 gene was characterized from Pteria penguin (P. penguin). The PpBCL2 was 1482 bp long, containing an open reading frame (ORF) of 588 bp encoding 195 amino acids. Four highly conserved BCL-2 homology (BH) domains were found in PpBCL2. Amino acid alignment and phylogenetic tree showed that PpBCL2 had the highest similarity with BCL2 of Crassostrea gigas at 65.24 %. Tissue expression analysis showed that PpBCL2 had high constitutive expression in gill, digestive diverticulum and mantle, and was significantly increased 72 h of Vibrio parahaemolyticus (V. parahaemolyticus) challenge in these immune tissues. Furthermore, PpBCL2 silencing significantly inhibited antimicrobial activity of hemolymph supernatant by 1.4-fold, and significantly reduced the survival rate by 51.7 % at 72 h post infection in P. penguin. These data indicated that PpBCL2 played an important role in immune response of P. penguin against V. parahaemolyticus infection.
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Affiliation(s)
- Wenhao He
- College of Fisheries, Guangdong Ocean University, Zhanjiang, Guangdong, 524088, China; Academician Joint Laboratory of Germplasm Resource Exploitation, Utilization and Health Assessment for Aquatic Animal, Guangdong Ocean University, Zhanjiang, Guangdong, 524088, China
| | - Jinji Lin
- College of Fisheries, Guangdong Ocean University, Zhanjiang, Guangdong, 524088, China; Academician Joint Laboratory of Germplasm Resource Exploitation, Utilization and Health Assessment for Aquatic Animal, Guangdong Ocean University, Zhanjiang, Guangdong, 524088, China
| | - Feifei Yu
- College of Fisheries, Guangdong Ocean University, Zhanjiang, Guangdong, 524088, China; Academician Joint Laboratory of Germplasm Resource Exploitation, Utilization and Health Assessment for Aquatic Animal, Guangdong Ocean University, Zhanjiang, Guangdong, 524088, China; Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture & Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes, College of Fisheries, Guangdong Ocean University, Zhanjiang City, 524088, Guangdong, China.
| | - Yang Leng
- Experiment Animal Center, Guangdong Medical University, Zhanjiang, Guangdong, 524023, China.
| | - Zhenni Pan
- Fangchenggang Marine Environmental Monitoring and Forecasting Center, Fangchenggang, Guangxi, 538000, China
| | - Qiwen Liang
- College of Fisheries, Guangdong Ocean University, Zhanjiang, Guangdong, 524088, China; Academician Joint Laboratory of Germplasm Resource Exploitation, Utilization and Health Assessment for Aquatic Animal, Guangdong Ocean University, Zhanjiang, Guangdong, 524088, China
| | - Siying Liu
- College of Fisheries, Guangdong Ocean University, Zhanjiang, Guangdong, 524088, China; Academician Joint Laboratory of Germplasm Resource Exploitation, Utilization and Health Assessment for Aquatic Animal, Guangdong Ocean University, Zhanjiang, Guangdong, 524088, China
| | - Xinyue Huang
- College of Fisheries, Guangdong Ocean University, Zhanjiang, Guangdong, 524088, China
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Tantawy SI, Timofeeva N, Sarkar A, Gandhi V. Targeting MCL-1 protein to treat cancer: opportunities and challenges. Front Oncol 2023; 13:1226289. [PMID: 37601693 PMCID: PMC10436212 DOI: 10.3389/fonc.2023.1226289] [Citation(s) in RCA: 25] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2023] [Accepted: 07/03/2023] [Indexed: 08/22/2023] Open
Abstract
Evading apoptosis has been linked to tumor development and chemoresistance. One mechanism for this evasion is the overexpression of prosurvival B-cell lymphoma-2 (BCL-2) family proteins, which gives cancer cells a survival advantage. Mcl-1, a member of the BCL-2 family, is among the most frequently amplified genes in cancer. Targeting myeloid cell leukemia-1 (MCL-1) protein is a successful strategy to induce apoptosis and overcome tumor resistance to chemotherapy and targeted therapy. Various strategies to inhibit the antiapoptotic activity of MCL-1 protein, including transcription, translation, and the degradation of MCL-1 protein, have been tested. Neutralizing MCL-1's function by targeting its interactions with other proteins via BCL-2 interacting mediator (BIM)S2A has been shown to be an equally effective approach. Encouraged by the design of venetoclax and its efficacy in chronic lymphocytic leukemia, scientists have developed other BCL-2 homology (BH3) mimetics-particularly MCL-1 inhibitors (MCL-1i)-that are currently in clinical trials for various cancers. While extensive reviews of MCL-1i are available, critical analyses focusing on the challenges of MCL-1i and their optimization are lacking. In this review, we discuss the current knowledge regarding clinically relevant MCL-1i and focus on predictive biomarkers of response, mechanisms of resistance, major issues associated with use of MCL-1i, and the future use of and maximization of the benefits from these agents.
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Affiliation(s)
- Shady I. Tantawy
- Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, United States
| | - Natalia Timofeeva
- Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, United States
| | - Aloke Sarkar
- Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, United States
| | - Varsha Gandhi
- Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, United States
- Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, United States
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Abstract
PURPOSE OF REVIEW While the treatment of acute lymphoblastic leukemia (ALL) has improved significantly over the last 30 years, the majority of adult patients will have their disease relapse. The BCL-2 gene was initially discovered from follicular lymphoma research; however, the BH3 family of proteins has is emerging to be crucial in patients with ALL due to their reliance on the balance of these pro-apoptotic and anti-apoptotic proteins in the BH3 family. We discuss apoptosis in ALL, the reliance mechanisms, drug development in this space, and areas for future research. RECENT FINDINGS The first drugs that were developed to inhibit the BCL-2 pathway include both venetoclax (BCL-2 specific inhibitor) and navitoclax (BCL-2, BCL-XL, and BCL-W). These drugs show promise and have obtained complete remissions, minimal residual disease negative status, and have been used as a bridge to allogeneic hematopoietic stem cell transplantation in acute myeloid leukemia and chronic lymphocytic leukemia. There are multiple ongoing clinical trials looking to assess the use of BCL-2 inhibition with chemotherapy, targeted therapies, and bi-specific T-cell engager therapies not only in both frontline and relapsed refractory ALL but also in consolidation and maintenance phases. There is still a large need for improvement of ALL outcomes in adult patients. Research has shown that ALL depends on the BCL-2 family of proteins for cell survival and proliferation. Targeting this pathway with BCL-2 inhibition has led to encouraging results, and future research is aimed at incorporating this targeted therapy into current treatment paradigms.
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Affiliation(s)
- Wesley M Smith
- Comprehensive Cancer Center of Wake Forest University, Winston Salem, NC, USA
| | - Daniel R Reed
- Comprehensive Cancer Center of Wake Forest University, Winston Salem, NC, USA.
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Zhu PJ, Yu ZZ, Lv YF, Zhao JL, Tong YY, You QD, Jiang ZY. Discovery of 3,5-Dimethyl-4-Sulfonyl-1 H-Pyrrole-Based Myeloid Cell Leukemia 1 Inhibitors with High Affinity, Selectivity, and Oral Bioavailability. J Med Chem 2021; 64:11330-11353. [PMID: 34342996 DOI: 10.1021/acs.jmedchem.1c00682] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Myeloid cell leukemia 1 (Mcl-1) protein is a key negative regulator of apoptosis, and developing Mcl-1 inhibitors has been an attractive strategy for cancer therapy. Herein, we describe the rational design, synthesis, and structure-activity relationship study of 3,5-dimethyl-4-sulfonyl-1H-pyrrole-based compounds as Mcl-1 inhibitors. Stepwise optimizations of hit compound 11 with primary Mcl-1 inhibition (52%@30 μM) led to the discovery of the most potent compound 40 with high affinity (Kd = 0.23 nM) and superior selectivity over other Bcl-2 family proteins (>40,000 folds). Mechanistic studies revealed that 40 could activate the apoptosis signal pathway in an Mcl-1-dependent manner. 40 exhibited favorable physicochemical properties and pharmacokinetic profiles (F% = 41.3%). Furthermore, oral administration of 40 was well tolerated to effectively inhibit tumor growth (T/C = 37.3%) in MV4-11 xenograft models. Collectively, these findings implicate that compound 40 is a promising antitumor agent that deserves further preclinical evaluations.
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Affiliation(s)
- Peng-Ju Zhu
- State Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China.,Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Ze-Zhou Yu
- State Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China.,Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Yi-Fei Lv
- State Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China.,Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Jing-Long Zhao
- State Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China.,Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Yuan-Yuan Tong
- State Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China.,Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Qi-Dong You
- State Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China.,Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Zheng-Yu Jiang
- State Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China.,Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
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Liou JW, Mani H, Yen JH, Hsu HJ, Chang CC. Hepatitis C virus core protein: Not just a nucleocapsid building block, but an immunity and inflammation modulator. Tzu Chi Med J 2021; 34:139-147. [PMID: 35465281 PMCID: PMC9020238 DOI: 10.4103/tcmj.tcmj_97_21] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2021] [Revised: 03/12/2021] [Accepted: 06/02/2021] [Indexed: 11/13/2022] Open
Abstract
Coevolution occurs between viruses and their hosts. The hosts need to evolve means to eliminate pathogenic virus infections, and the viruses, for their own survival and multiplication, have to develop mechanisms to escape clearance by hosts. Hepatitis C virus (HCV) of Flaviviridae is a pathogen which infects human liver and causes hepatitis, a condition of liver inflammation. Unlike most of the other flaviviruses, HCV has an excellent ability to evade host immunity to establish chronic infection. The persistent liver infection leads to chronic hepatitis, liver cirrhosis, hepatocellular carcinoma (HCC), as well as extrahepatic HCV-related diseases. HCV genomic RNA only expresses 10 proteins, many of which bear functions, in addition to those involved in HCV life cycle, for assisting the virus to develop its persistency. HCV core protein is a structural protein which encapsulates HCV genomic RNA and assembles into nucleocapsids. The core protein is also found to exert functions to affect host inflammation and immune responses by altering a variety of host pathways. This paper reviews the studies regarding the HCV core protein-induced alterations of host immunity and inflammatory responses, as well as the involvements of the HCV core protein in pro- and anti-inflammatory cytokine stimulations, host cellular transcription, lipid metabolism, cell apoptosis, cell proliferations, immune cell differentiations, oxidative stress, and hepatocyte steatosis, which leads to liver fibrosis, cirrhosis, and HCC. Implications of roles played by the HCV core protein in therapeutic resistance are also discussed.
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7
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Chen H, Sheng H, Zhao Y, Zhu G. Piperine Inhibits Cell Proliferation and Induces Apoptosis of Human Gastric Cancer Cells by Downregulating Phosphatidylinositol 3-Kinase (PI3K)/Akt Pathway. Med Sci Monit 2020; 26:e928403. [PMID: 33382670 PMCID: PMC7784594 DOI: 10.12659/msm.928403] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Background Piperine has been reported to inhibit proliferation and induce apoptosis in various cancer cells. This study aimed to explore the efficacy and underlying mechanism of piperine in human gastric cancer. Material/Methods MTT assay was performed to examine the effect of piperine (concentrations of 0–300 μM) on the proliferation of human gastric cancer SNU-16 cells and normal human gastric epithelial GES-1 cells. Flow cytometry and Western blot were used to determine cell apoptosis and the expression level of protein (Cyto C, cleaved PARP, cleaved caspase-3, Bax, Bcl-2, Bad, Bcl-xl, PI3K, pPI3K, Akt, and pAkt), respectively. To further investigate the anti-tumor mechanism of piperine in SNU-16 cells, we used a small-molecule Akt activator SC79 in this study. The in vivo mechanism of piperine against gastric cancer was evaluated using a xenograft tumor model. Results The results showed that piperine inhibited proliferation and induced apoptosis of SNU-16 cells. Piperine upregulated the protein expression of Bax, Bad, Cyto C, cleaved PARP, and cleaved caspase-3, but downregulated the protein expression of Bcl-2, Bcl-xl, pPI3k, and pAkt. However, SC79 reversed the function of piperine on the apoptosis-related proteins. An in vivo study revealed that, compared with the control group, the tumor volume of mice treated with piperine was significantly reduced. Piperine enhanced cleaved caspase-3 expression but decreased Ki-67 expression in a dose-dependent manner. Moreover, the nontoxicity effect of piperine was confirmed by H&E staining analysis in kidney and heart tissues of mice. Conclusions Our findings suggest that piperine inhibits proliferation and induces apoptosis of human gastric cancer cells through inhibition of the PI3K/Akt signaling pathway.
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Affiliation(s)
- Hanyu Chen
- Department of Pharmacy, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China (mainland)
| | - Hongqing Sheng
- Department of Pharmacy, Wenzhou People's Hospital, Wenzhou, Zhejiang, China (mainland)
| | - Yushuo Zhao
- Department of Pharmacy, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China (mainland)
| | - Guanghui Zhu
- Department of Pharmacy, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China (mainland)
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Sunisha C, Sowmya HD, Usharani TR, Umesha M, Gopalkrishna HR, Sriram S. Induction of Ced9 mediated anti-apoptosis in commercial banana cultivar Rasthali for stable resistance against Fusarium wilt. 3 Biotech 2020; 10:371. [PMID: 32832331 DOI: 10.1007/s13205-020-02357-4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2020] [Accepted: 07/24/2020] [Indexed: 11/30/2022] Open
Abstract
Anti-apoptotic gene Ced-9 enhanced resistance against Fusarium oxysporum f. sp. cubense (Foc) in the susceptible banana cultivar Rasthali by arresting tissue necrosis. The embryogenic cell suspension of banana cultivar Rasthali was stably transformed with Ced-9 gene and transformed lines were regenerated independently. The putative transgenic lines were analyzed with PCR using gene primers and further subjected to Southern blot to estimate copy number. The root-challenge bioassay with Foc showed 17-51% Vascular Discoloration Index in independent transformants compared to untransformed banana cv Rasthali (98% VDI). Four transgenic events showed a higher level of resistance over a period of 6 months. Overcoming tissue necrosis is the most ideal method to avoid Fusarium multiplication and spread in banana. Oxidative stress-induced cell necrosis is prevented by the activation of antiapoptotic pathways by Ced-9 and is proving to be an effective method to control this dreaded disease. This is the first report from India on the generation of transgenic banana cultivar Rasthali expressing antiapoptotic Ced-9 gene for resistance to Fusarium wilt.
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Affiliation(s)
- C Sunisha
- Division of Biotechnology, ICAR-Indian Institute of Horticultural Research, Hessaraghatta, Bangalore, 560 089 India
- Department of Biotechnology and Biochemistry, Centre for Postgraduate Studies, Jain University, Bangalore, India
| | - H D Sowmya
- Division of Biotechnology, ICAR-Indian Institute of Horticultural Research, Hessaraghatta, Bangalore, 560 089 India
| | - T R Usharani
- Division of Biotechnology, ICAR-Indian Institute of Horticultural Research, Hessaraghatta, Bangalore, 560 089 India
| | - M Umesha
- Division of Biotechnology, ICAR-Indian Institute of Horticultural Research, Hessaraghatta, Bangalore, 560 089 India
| | - H R Gopalkrishna
- Division of Biotechnology, ICAR-Indian Institute of Horticultural Research, Hessaraghatta, Bangalore, 560 089 India
| | - S Sriram
- Division of Biotechnology, ICAR-Indian Institute of Horticultural Research, Hessaraghatta, Bangalore, 560 089 India
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On the Stability and Degradation Pathways of Venetoclax under Stress Conditions. Pharmaceutics 2020; 12:pharmaceutics12070639. [PMID: 32645956 PMCID: PMC7407384 DOI: 10.3390/pharmaceutics12070639] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2020] [Revised: 07/02/2020] [Accepted: 07/03/2020] [Indexed: 12/18/2022] Open
Abstract
Venetoclax is an orally bioavailable, B-cell lymphoma-2 (BCL-2) selective inhibitor, used for the treatment of various types of blood cancers, such as chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). In this study we investigated the degradation of venetoclax under various stress conditions including acidic, basic, oxidative, photolytic and thermolytic conditions. We isolated and identified six of its main degradation products produced in forced degradation studies. The structures of the isolated degradation products were determined by using nuclear magnetic resonance (NMR) spectroscopy, high resolution mass spectrometry (HRMS) and infrared (IR) spectroscopy. Additionally, one oxidation degradation product was identified with comparison to a commercially obtained venetoclax impurity. We proposed the key degradation pathways of venetoclax in solution. To the best of our knowledge, no structures of degradation products of venetoclax have been previously published. The study provides novel and primary knowledge of the stability characteristics of venetoclax under stress conditions. Venetoclax is currently the only BCL-2 protein inhibitor on the market. In addition to single agent treatment, it is effective in combinational therapy, so future drug development involving venetoclax can be expected. A better insight into the stability properties of the therapeutic can facilitate future studies involving venetoclax and aid in the search of new similar therapeutics.
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10
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Rahimian A, Mahdavi M, Rahbarghazi R, Charoudeh HN. 4t-CHQ a Spiro-Quinazolinone Benzenesulfonamide Derivative Induces G 0/G 1 Cell Cycle arrest and Triggers Apoptosis Through Down-Regulation of Survivin and Bcl2 in the Leukemia Stem-Like KG1-a Cells. Anticancer Agents Med Chem 2020; 19:1340-1349. [PMID: 30868965 DOI: 10.2174/1871520619666190313165130] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2018] [Revised: 11/16/2018] [Accepted: 03/04/2019] [Indexed: 12/12/2022]
Abstract
OBJECTIVE Many experiments have revealed the anti-tumor activity of spiro-quinazolinone derivatives on different cell types. Exposing KG1-a cells to N-(4- tert- butyl- 4'- oxo- 1'H- spiro [cyclohexane- 1, 2'- quinazoline]- 3'(4'H)- yl)- 4- methyl benzenesulfonamide (4t-CHQ), as an active sub-component of spiroquinazolinone benzenesulfonamides, the experiment investigated the possible mechanisms that manifest the role of 4t-CHQ in leukemic KG1-a progenitor cells. Mechanistically, the inhibitory effects of 4t-CHQ on KG1-a cells emerge from its modulating function on the expression of Bax/Bcl2 and survinin proteins. METHODS Cell viability was assessed using MTT assay. The IC50 value of cells was calculated to be 131.3μM, after 72h-incubation with 4t-CHQ, ranging from 10 to 150μM. Apoptotic changes were studied using Acridine Orange/Ethidium Bromide (AO/EB) staining. DNA fragmentation was analyzed by agarose gel electrophoresis method. To evaluate the percentage of apoptotic cells and cell growth dynamic apoptotic features, we performed Annexin V/PI double staining assay and cell cycle analysis by flow cytometry. RESULTS According to the results, apoptosis induction was initiated by 4t-CHQ in the KG1-a cells (at IC50 value). Cell dynamic analysis revealed that the cell cycle at the G1 phase was arrested after treatment with 4t- CHQ. Western blotting analysis showed enhancement in the expression ratio of Bax/Bcl-2, while the expression of survinin protein decreased in a time-dependent manner in the KG1-a cells. According to the docking simulation data, the effectiveness of 4t-CHQ on KG1-a cells commenced by its reactions with the functional domain of BH3 and Bcl2 and BIR domains of survivin protein. CONCLUSION These results demonstrate a remarkable role of 4t- CHQ in arresting leukemia KG1-a stem cells both by induction of apoptosis as well as by down-regulating survivin and Bcl2 proteins.
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Affiliation(s)
- Arezoo Rahimian
- Department of Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran
| | - Majid Mahdavi
- Department of Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran
| | - Reza Rahbarghazi
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.,Department of Applied Cell Sciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Hojjatollah N Charoudeh
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.,Department of Applied Cell Sciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
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11
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Wei L, He L, Fu J, Liu Y, Ruan J, Liu L, Zhong Q. Molecular characterization of caspase-8-like and its expression induced by microcystin-LR in grass carp (Ctenopharygodon idella). FISH & SHELLFISH IMMUNOLOGY 2019; 89:727-735. [PMID: 30981886 DOI: 10.1016/j.fsi.2019.04.026] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/06/2019] [Revised: 03/31/2019] [Accepted: 04/10/2019] [Indexed: 06/09/2023]
Abstract
Caspase-8, an initiator caspase, plays a vital role in apoptosis. In this study, caspase-8-like (named as Cicaspase-8-like), a homologue of caspase-8, was identified in grass carp (Ctenopharygodon idella). The full-length cDNA sequence of CiCaspase-8-like was 1409 bp and contained a 162 bp 5'-UTR, a 239 bp 3'-UTR and a 1008 bp coding sequence. The putative amino acids sequence was 335 residues long, including a large subunit (P20) and a small subunit (P10), but lacking conserved death effector domains. A histidine active site DHSQMDAFVCCVLSHG and a cysteine active-site motif KPKLFFIQACQG were found in P20. Phylogenetic analysis showed that Cicaspase-8-like clustered with the caspase-8 and caspase-8-like of other fish and grouped closely with Carassius auratus caspase-8-like. Quantitative real-time PCR revealed that the Cicaspase-8-like mRNA were expressed constitutively in all tested tissues from healthy grass carp, with high expression level in the blood, spleen, liver and gill, indicating its role in immune reaction. The expression of Cicaspase-8-like mRNA was decreased significantly in the liver because of the stress caused by microcystin-LR (MC-LR) (75 and 100 μg MC-LR/kg BW) at 24 h and 96 h post injection (P < 0.05), but it was increased significantly in grass carp treated with 25 μg MC-LR/kg BW at 24 h (P < 0.05) post injection. Cleaved fragments of Cicaspase-8-like were observed using western blot analysis, and the expression of Cicaspase-8-like protein was increased after MC-LR treatments. Moreover, the expression of both caspase-9 and caspase-3 mRNA increased significantly after treatment with the three doses of MC-LR. TUNEL assay results showed remarkable changes in apoptosis after the MC-LR treatment. These results suggest that Cicaspase-8-like is an important caspase and plays an essential role in MC-LR-induced apoptosis.
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Affiliation(s)
- LiLi Wei
- College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, Jiangxi Province, 330045, PR China.
| | - Li He
- College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, Jiangxi Province, 330045, PR China
| | - Jianping Fu
- College of Life Sciences, Jiangxi Normal University, Nanchang, Jiangxi Province, 330022, PR China
| | - Yi Liu
- College of Life Sciences, Jiangxi Normal University, Nanchang, Jiangxi Province, 330022, PR China
| | - Jiming Ruan
- College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, Jiangxi Province, 330045, PR China
| | - Lin Liu
- College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, Jiangxi Province, 330045, PR China
| | - Qiwang Zhong
- College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang, Jiangxi Province, 330045, PR China.
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Kashyap D, Tuli HS, Garg VK, Goel N, Bishayee A. Oncogenic and Tumor-Suppressive Roles of MicroRNAs with Special Reference to Apoptosis: Molecular Mechanisms and Therapeutic Potential. Mol Diagn Ther 2018; 22:179-201. [PMID: 29388067 DOI: 10.1007/s40291-018-0316-1] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
MicroRNAs (miRNAs) are the non-coding class of minute RNA molecules that negatively control post-transcriptional regulation of various functional genes. These miRNAs are transcribed from the loci present in the introns of functional or protein-coding genes, exons of non-coding genes, or even in the 3'-untranslated region (3'-UTR). They have potential to modulate the stability or translational efficiency of a variety of target RNA [messenger RNA (mRNA)]. The regulatory function of miRNAs has been elucidated in several pathological conditions, including neurological (Alzheimer's disease and Parkinson's disease) and cardiovascular conditions, along with cancer. Importantly, miRNA identification in cancer progression and invasion has evolved as an incipient era in cancer treatment. Several studies have shown the influence of miRNAs on various cancer processes, including apoptosis, invasion, metastasis and angiogenesis. In particular, apoptosis induction in tumor cells through miRNA has been extensively studied. The biphasic mode (up- and down-regulation) of miRNA expression in apoptosis and other cancer processes has already been determined. The findings of these studies could be utilized to develop potential therapeutic strategies for the management of various cancers. The present review critically describes the oncogenic and tumor suppressor role of miRNAs in apoptosis and other cancer processes, therapy resistance, and use of their presence in the body fluids as biomarkers.
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Affiliation(s)
- Dharambir Kashyap
- Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, 160012, Punjab, India
| | - Hardeep Singh Tuli
- Department of Biotechnology, Maharishi Markandeshwar University, Mullana-Ambala, 133207, Haryana, India.
| | - Vivek Kumar Garg
- Department of Biochemistry, Government Medical College and Hospital, Chandigarh, 160030, Punjab, India
| | - Neelam Goel
- Department of Information Technology, University Institute of Engineering and Technology, Panjab University, Chandigarh, 160014, Punjab, India
| | - Anupam Bishayee
- Department of Pharmaceutical Sciences, College of Pharmacy, Larkin University, Miami, FL, 33169, USA.
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Xie Q, Xu Y, Gao W, Zhang Y, Su J, Liu Y, Guo Y, Dou M, Hu K, Sun L. TAT‑fused IP3R‑derived peptide enhances cisplatin sensitivity of ovarian cancer cells by increasing ER Ca2+ release. Int J Mol Med 2017; 41:809-817. [PMID: 29207009 PMCID: PMC5752180 DOI: 10.3892/ijmm.2017.3260] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2016] [Accepted: 10/25/2017] [Indexed: 01/04/2023] Open
Abstract
Ovarian cancer is the most common gynecological malignancy. At present, cisplatin is used to treat ovarian cancer; however, the development of cisplatin resistance during therapy is a common obstacle to achieving favorable outcomes. Recently, the B‑cell lymphoma 2 (Bcl‑2) BH4 domain has been reported to mediate the prosurvival activity of Bcl‑2 in cancer; however, the involvement of the BH4 domain of Bcl‑2 in the cisplatin resistance of ovarian carcinoma cells is not entirely clear. In this study, we observed the cytoplasmic and mitochondrial levels of Ca2+ by confocal laser microscopy. We also detected cell apoptosis using western blot analysis and flow cytometry. The present study demonstrated that TAT‑fused inositol 1,4,5‑trisphosphate receptor‑derived peptide (TAT‑IDPS), which targets the BH4 domain of Bcl‑2, increased cisplatin‑induced Ca2+ flux from the endoplasmic reticulum (ER) into the cytosol and mitochondria. In addition, TAT‑IDPS increased cisplatin‑induced expression of mitochondrial apoptosis‑associated proteins and ER stress‑associated proteins. These results indicated that TAT‑IDPS may enhance the cytotoxicity of cisplatin toward ovarian carcinoma cells by increasing ER Ca2+ release.
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Affiliation(s)
- Qi Xie
- Department of Pathophysiology, Basic College of Medicine, Jilin University, Changchun, Jilin 130021, P.R. China
| | - Ye Xu
- Department of Histology and Embryology, Basic College of Medicine, Jilin Medical University, Jilin, Jilin 132013, P.R. China
| | - Weinan Gao
- Department of Clinical Medicine, College of Clinical Medicine, Jilin University, Changchun, Jilin 130021, P.R. China
| | - Yong Zhang
- Department of Pathophysiology, Basic College of Medicine, Jilin University, Changchun, Jilin 130021, P.R. China
| | - Jing Su
- Department of Pathophysiology, Basic College of Medicine, Jilin University, Changchun, Jilin 130021, P.R. China
| | - Yanan Liu
- Department of Pathophysiology, Basic College of Medicine, Jilin University, Changchun, Jilin 130021, P.R. China
| | - Yuting Guo
- Department of Histology and Embryology, Basic College of Medicine, Jilin Medical University, Jilin, Jilin 132013, P.R. China
| | - Minghan Dou
- Department of Histology and Embryology, Basic College of Medicine, Jilin Medical University, Jilin, Jilin 132013, P.R. China
| | - Kebang Hu
- Department of Urology, First Hospital of Jilin University, Changchun, Jilin 130031, P.R. China
| | - Liankun Sun
- Department of Pathophysiology, Basic College of Medicine, Jilin University, Changchun, Jilin 130021, P.R. China
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A co-evolutionary arms race: trypanosomes shaping the human genome, humans shaping the trypanosome genome. Parasitology 2017; 142 Suppl 1:S108-19. [PMID: 25656360 PMCID: PMC4413828 DOI: 10.1017/s0031182014000602] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Trypanosoma brucei is the causative agent of African sleeping sickness in humans and one of several pathogens that cause the related veterinary disease Nagana. A complex co-evolution has occurred between these parasites and primates that led to the emergence of trypanosome-specific defences and counter-measures. The first line of defence in humans and several other catarrhine primates is the trypanolytic protein apolipoprotein-L1 (APOL1) found within two serum protein complexes, trypanosome lytic factor 1 and 2 (TLF-1 and TLF-2). Two sub-species of T. brucei have evolved specific mechanisms to overcome this innate resistance, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. In T. b. rhodesiense, the presence of the serum resistance associated (SRA) gene, a truncated variable surface glycoprotein (VSG), is sufficient to confer resistance to lysis. The resistance mechanism of T. b. gambiense is more complex, involving multiple components: reduction in binding affinity of a receptor for TLF, increased cysteine protease activity and the presence of the truncated VSG, T. b. gambiense-specific glycoprotein (TgsGP). In a striking example of co-evolution, evidence is emerging that primates are responding to challenge by T. b. gambiense and T. b. rhodesiense, with several populations of humans and primates displaying resistance to infection by these two sub-species.
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15
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Structural Insight into the Mechanism of Dibenzo[a,l]pyrene and Benzo[a]pyrene-Mediated Cell Proliferation Using Molecular Docking Simulations. Interdiscip Sci 2017; 10:653-673. [DOI: 10.1007/s12539-017-0226-7] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2016] [Revised: 03/04/2017] [Accepted: 03/14/2017] [Indexed: 01/08/2023]
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16
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Jayash SN, Hashim NM, Misran M, Baharuddin NA. Formulation and in vitro and in vivo evaluation of a new osteoprotegerin-chitosan gel for bone tissue regeneration. J Biomed Mater Res A 2017; 105:398-407. [PMID: 27684563 DOI: 10.1002/jbm.a.35919] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2016] [Revised: 09/23/2016] [Accepted: 09/27/2016] [Indexed: 12/17/2022]
Abstract
The osteoprotegerin (OPG) system plays a critical role in bone remodelling by regulating osteoclast formation and activity. The study aimed to determine the physicochemical properties and biocompatibility of a newly formulated OPG-chitosan gel. The OPG-chitosan gel was formulated using human OPG protein and water-soluble chitosan. The physicochemical properties were determined using Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Gel morphology was determined using scanning electron microscopy (SEM) and then it was subjected to a protein release assay and biodegradability test. An in vitro cytotoxicity test on normal human periodontal ligament (NHPL) fibroblasts and normal human (NH) osteoblasts was carried out using the AlamarBlue assay. In vivo evaluation in a rabbit model involved creating critical-sized defects in calvarial bone, filling with the OPG-chitosan gel and sacrificing at 12 weeks. In vitro results demonstrated that the 25 kDa OPG-chitosan gel had the highest rate of protein release and achieved 90% degradation in 28 days. At 12 weeks, the defects filled with 25 kDa OPG-chitosan gel showed significant (p < 0.05) new bone formation and the highest expression of osteocalcin and osteopontin compared to controls. Thus, the 25 kDa OPG-chitosan gel could be a promising new biomaterial for tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 398-407, 2017.
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Affiliation(s)
- Soher Nagi Jayash
- Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, Kuala Lumpur, 50603, Malaysia
| | - Najihah Mohd Hashim
- Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, 50603, Malaysia
- Centre for Natural Products And Drug Discovery (CENAR), Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur, 50603, Malaysia
| | - Misni Misran
- Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur, 50603, Malaysia
| | - N A Baharuddin
- Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, Kuala Lumpur, 50603, Malaysia
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17
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Bassiouni R, Nemec KN, Iketani A, Flores O, Showalter A, Khaled AS, Vishnubhotla P, Sprung RW, Kaittanis C, Perez JM, Khaled AR. Chaperonin Containing TCP-1 Protein Level in Breast Cancer Cells Predicts Therapeutic Application of a Cytotoxic Peptide. Clin Cancer Res 2016; 22:4366-79. [PMID: 27012814 DOI: 10.1158/1078-0432.ccr-15-2502] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2015] [Accepted: 02/21/2016] [Indexed: 01/01/2023]
Abstract
PURPOSE Metastatic disease is a leading cause of death for patients with breast cancer, driving the need for new therapies. CT20p is a peptide previously discovered by our group that displays cancer-specific cytotoxicity. To design the optimal therapeutic use of the peptide, we identified the intracellular target of CT20p in breast cancer cells, correlating expression patterns of the target with susceptibility to CT20p. EXPERIMENTAL DESIGN Using polymeric nanoparticles to deliver CT20p, we assessed cytoskeletal changes, cell migration, adhesion, and viability in cells treated with the peptide. Protein pull-down experiments, coupled to mass spectrometry, enabled identification of the peptide's intracellular target. Biochemical and histologic techniques validated target identity in human cell lines and breast cancer tissue microarrays and revealed susceptibility patterns to CT20p. RESULTS Chaperonin containing TCP-1 (CCT) was identified as the intracellular target of CT20p. Cancer cells susceptible to CT20p had increased CCT, and overexpression of CCTβ, a subunit of the CCT complex, enhanced susceptibility to CT20p. Susceptible cells displayed reduced tubulin, a substrate of CCT, and inhibition of migration upon CT20p treatment. CCTβ levels were higher in invasive ductal carcinomas than in cancer adjacent tissues and increased with breast cancer stage. Decreased breast cancer patient survival correlated with genomic alternations in CCTβ and higher levels of the chaperone. CONCLUSIONS Increased CCT protein in breast cancer cells underlies the cytotoxicity of CT20p. CCT is thus a potential target for therapeutic intervention and serves as a companion diagnostic to personalize the therapeutic use of CT20p for breast cancer treatment. Clin Cancer Res; 22(17); 4366-79. ©2016 AACR.
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Affiliation(s)
- Rania Bassiouni
- Burnett School of Biomedical Science, College of Medicine, University of Central Florida, Orlando, Florida
| | - Kathleen N Nemec
- Burnett School of Biomedical Science, College of Medicine, University of Central Florida, Orlando, Florida
| | - Ashley Iketani
- Burnett School of Biomedical Science, College of Medicine, University of Central Florida, Orlando, Florida
| | - Orielyz Flores
- Nanoscience Technology Center, University of Central Florida, Orlando, Florida
| | - Anne Showalter
- Burnett School of Biomedical Science, College of Medicine, University of Central Florida, Orlando, Florida
| | | | | | | | - Charalambos Kaittanis
- Gordon Center for Medical Imaging, Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts
| | - Jesus M Perez
- Cedars-Sinai Medical Center, Los Angeles, California
| | - Annette R Khaled
- Burnett School of Biomedical Science, College of Medicine, University of Central Florida, Orlando, Florida.
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18
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Gulbake A, Jain A, Jain A, Jain A, Jain SK. Insight to drug delivery aspects for colorectal cancer. World J Gastroenterol 2016; 22:582-599. [PMID: 26811609 PMCID: PMC4716061 DOI: 10.3748/wjg.v22.i2.582] [Citation(s) in RCA: 82] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/29/2015] [Revised: 08/29/2015] [Accepted: 11/30/2015] [Indexed: 02/06/2023] Open
Abstract
Colorectal cancer (CRC) is the third most common cancer diagnosed worldwide in human beings. Surgery, chemotherapy, radiotherapy and targeted therapies are the conventional four approaches which are currently used for the treatment of CRC. The site specific delivery of chemotherapeutics to their site of action would increase effectiveness with reducing side effects. Targeted oral drug delivery systems based on polysaccharides are being investigated to target and deliver chemotherapeutic and chemopreventive agents directly to colon and rectum. Site-specific drug delivery to colon increases its concentration at the target site, and thus requires a lower dose and hence abridged side effects. Some novel therapies are also briefly discussed in article such as receptor (epidermal growth factor receptor, folate receptor, wheat germ agglutinin, VEGF receptor, hyaluronic acid receptor) based targeting therapy; colon targeted proapoptotic anticancer drug delivery system, gene therapy. Even though good treatment options are available for CRC, the ultimate therapeutic approach is to avert the incidence of CRC. It was also found that CRCs could be prevented by diet and nutrition such as calcium, vitamin D, curcumin, quercetin and fish oil supplements. Immunotherapy and vaccination are used nowadays which are showing better results against CRC.
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19
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Ying HQ, Chen J, He BS, Pan YQ, Wang F, Deng QW, Sun HL, Liu X, Wang SK. The effect of BIM deletion polymorphism on intrinsic resistance and clinical outcome of cancer patient with kinase inhibitor therapy. Sci Rep 2015; 5:11348. [PMID: 26076815 PMCID: PMC4466895 DOI: 10.1038/srep11348] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2015] [Accepted: 05/08/2015] [Indexed: 11/25/2022] Open
Abstract
A common deletion polymorphism within B-cell chronic lymphocytic leukemia-lymphoma like 11 gene (BIM) was deemed to be a genetic cause leading to compromised kinase inhibitor therapeutic efficacy in cancer individuals. However, the results reported were not consistent. Thus, a comprehensive meta-analysis containing 12 eligible studies including 1,532 Asian patients was conducted to investigate a steady and reliable conclusion. The results showed that BIM deletion polymorphism was significantly associated with tyrosine kinase inhibitor (TKI) clinical efficacy in term of response rate (Ph = 0.349, HR = 0.438, 95%CI = 0.274-0.699) and disease control rate (Ph = 0.941, HR = 0.370, 95%CI = 0.202-0.678) in EGFR-mutated NSCLC population, not in CML and HCC subgroups. Additionally, EGFR-mutated NSCLC patient harbored BIM deletion polymorphism was associated with a shorter progression-free survival (PFS) than those with BIM wild polymorphism (Ph = 0.580, adjusted HR = 2.194, 95%CI = 1.710-2.814). However, no significant association was examined between BIM deletion polymorphism and overall survival (OS) and toxic adverse events in EGFR-mutated NSCLC population and it was not associated with PFS and OS in HCC subgroup. These findings revealed that BIM deletion polymorphism might be a genetic cause of intrinsic resistance to TKI therapy and it could be emerged as an independent predictor to identify patients who would benefit from TKI targeted therapy in EGFR-mutated NSCLC.
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MESH Headings
- Antineoplastic Agents/therapeutic use
- Apoptosis Regulatory Proteins/deficiency
- Apoptosis Regulatory Proteins/genetics
- Bcl-2-Like Protein 11
- Carcinoma, Hepatocellular/drug therapy
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/mortality
- Carcinoma, Hepatocellular/pathology
- Carcinoma, Non-Small-Cell Lung/drug therapy
- Carcinoma, Non-Small-Cell Lung/genetics
- Carcinoma, Non-Small-Cell Lung/mortality
- Carcinoma, Non-Small-Cell Lung/pathology
- Drug Resistance, Neoplasm/genetics
- ErbB Receptors/genetics
- ErbB Receptors/metabolism
- Gene Expression Regulation, Neoplastic
- Humans
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/mortality
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology
- Membrane Proteins/deficiency
- Membrane Proteins/genetics
- Neoplasms/drug therapy
- Neoplasms/genetics
- Neoplasms/mortality
- Neoplasms/pathology
- Polymorphism, Genetic
- Protein Kinase Inhibitors/therapeutic use
- Proto-Oncogene Proteins/deficiency
- Proto-Oncogene Proteins/genetics
- Sequence Deletion
- Signal Transduction
- Survival Analysis
- Treatment Outcome
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Affiliation(s)
- Hou-Qun Ying
- Medical College, Southeast University, Nanjing 210009, Jiangsu, China
- Central Laboratory, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, Jiangsu, China
| | - Jie Chen
- Life Scientific College, Nanjing Normal University, Nanjing 210023, Jiangsu, China
- Central Laboratory, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, Jiangsu, China
| | - Bang-Shun He
- Central Laboratory, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, Jiangsu, China
| | - Yu-Qin Pan
- Central Laboratory, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, Jiangsu, China
| | - Feng Wang
- Central Laboratory, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, Jiangsu, China
| | - Qi-Wen Deng
- Central Laboratory, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, Jiangsu, China
| | - Hui-Ling Sun
- Life Scientific College, Nanjing Normal University, Nanjing 210023, Jiangsu, China
- Central Laboratory, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, Jiangsu, China
| | - Xian Liu
- Central Laboratory, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, Jiangsu, China
| | - Shu-Kui Wang
- Central Laboratory, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, Jiangsu, China
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Saleh AM, El-Abadelah MM, Aziz MA, Taha MO, Nasr A, Rizvi SAA. Antiproliferative activity of the isoindigo 5'-Br in HL-60 cells is mediated by apoptosis, dysregulation of mitochondrial functions and arresting cell cycle at G0/G1 phase. Cancer Lett 2015; 361:251-261. [PMID: 25790909 DOI: 10.1016/j.canlet.2015.03.013] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2015] [Revised: 03/09/2015] [Accepted: 03/10/2015] [Indexed: 12/25/2022]
Abstract
Our new compound, 5'-Br [(E)-1-(5'-bromo-2'-oxoindolin-3'-ylidene)-6-ethyl-2,3,6,9-tetrahydro-2,9-dioxo-1H-pyrrolo[3,2-f]quinoline-8-carboxylic acid], had shown strong, selective antiproliferative activity against different cancer cell lines. Here, we aim to comprehensively characterize the mechanisms associated with its cytotoxicity in the human promyelocytic leukemia HL-60 cells. We focused at studying the involvement of apoptotic pathway and cell cycle effects. 5'-Br significantly inhibited proliferation by inducing caspase-dependent apoptosis. Involvement of caspase independent mechanism is also possible due to observed inability of z-VAD-FMK to rescue apoptotic cells. 5'-Br was found to trigger intrinsic apoptotic pathway as indicated by depolarization of the mitochondrial inner membrane, decreased level of cellular ATP, modulated expression and phosphorylation of Bcl-2 leading to loss of its association with Bax, and increased release of cytochrome c. 5'-Br treated cells were found arrested at G0/G1 phase with modulation in protein levels of cyclins, dependent kinases and their inhibitors. Expression and enzymatic activity of CDK2 and CDK4 was found inhibited. Retinoblastoma protein (Rb) phosphorylation was also inhibited whereas p21 protein levels were increased. These results suggest that the antiproliferative mechanisms of action of 5'-Br could involve apoptotic pathways, dysregulation of mitochondrial functions and disruption of cell cycle checkpoint.
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Affiliation(s)
- Ayman M Saleh
- Department of Basic Medical Sciences, College of Medicine, King Saud Bin Abdulaziz University for Health Sciences (KSAU-HS), National Guard Health Affairs, P.O. Box: 3660, Mail Code: 3127, Riyadh 11481, Saudi Arabia; King Abdullah International Medical Research Center (KAIMRC), National Guard Health Affairs, P.O. Box 22490, Riyadh 11426, Saudi Arabia.
| | - Mustafa M El-Abadelah
- Department of Chemistry, Faculty of Science, The University of Jordan, Amman 11942, Jordan
| | - Mohammad Azhar Aziz
- King Abdullah International Medical Research Center (KAIMRC), National Guard Health Affairs, P.O. Box 22490, Riyadh 11426, Saudi Arabia
| | - Mutasem O Taha
- Department of Pharmaceutical Sciences, Faculty of Pharmacy, The University of Jordan, Amman 11942, Jordan
| | - Amre Nasr
- Department of Basic Medical Sciences, College of Medicine, King Saud Bin Abdulaziz University for Health Sciences (KSAU-HS), National Guard Health Affairs, P.O. Box: 3660, Mail Code: 3127, Riyadh 11481, Saudi Arabia; Department of Microbiology, Faculty of Sciences and Technology, Al-Neelain University, Khartoum, Sudan
| | - Syed A A Rizvi
- Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University (NSU), Fort Lauderdale, Florida 33328, USA
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Wang M, Lu X, Dong X, Hao F, Liu Z, Ni G, Chen D. pERK1/2 silencing sensitizes pancreatic cancer BXPC-3 cell to gemcitabine-induced apoptosis via regulating Bax and Bcl-2 expression. World J Surg Oncol 2015; 13:66. [PMID: 25880226 PMCID: PMC4337256 DOI: 10.1186/s12957-015-0451-7] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2014] [Accepted: 01/08/2015] [Indexed: 11/10/2022] Open
Abstract
Background Our previous study has demonstrated that knockdown of activated ERK1/2(pERK1/2) sensitizes pancreatic cancer cells to chemotherapeutic drug gemcitabine (Gem) treatment. However, the details of this survival mechanism remain undefined. It has also shown that Bcl-2 confers resistance and Bax sensitizes to gemcitabine-induced apoptosis in pancreatic cancer cells. Furthermore, the extracellular signaling-regulated kinase (ERK) signaling pathway regulates Bcl-2/Bax expression ratio. We therefore tested the hypothesis that pancreatic cancer cells are resistant to gemcitabine and this resistance is due to activation of ERK1/2 and subsequent upregulation of Bcl-2 and downregulation of Bax. Methods Pancreatic cancer cell BXPC-3 was used in the study. The effect of pharmacological inhibition of ERK1/2 on resistance of pancreatic cancer cells to apoptosis induced by treatment with gemcitabine was analyzed. The following methods were utilized: TUNEL and ELISA were used to detect apoptosis. Western blot was used to detect the protein expression. Results Gemcitabine treatment enhanced the activity of ERK1/2 in the BXPC-3 cells. Inhibition of the ERK1/2 by PD98059 could downregulate Bcl-2 and upregulate Bax and was associated with restoration of sensitivity to gemcitabine in BXPC-3 cells. Depletion of endogenous Bcl-2 expression by specific small interfering RNA transfection significantly increased gemcitabine-induced cell apoptosis. Combined treatment with PD98059 and Bax siRNA transfection could decrease gemcitabine-induced ERK1/2 and Bax activation, which subsequently resulted in decreased apoptosis. Conclusions The upregulation of ERK1/2-dependent Bcl-2 and downregulation of ERK1/2-dependent Bax can protect human pancreatic cancer cells from gemcitabine-induced apoptosis. Targeting the ERK1/2-Bax/Bcl-2 pathway may in part lead to sensitization of pancreatic cancer to gemcitabine.
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Affiliation(s)
- Min Wang
- Department of Clinical Laboratory, People's Hospital of Laiwu, Laiwu, Shangdong, China.
| | - Xingjiao Lu
- Department of Internal Neurology, People's Hospital of Zhangqiu, ZhangQiu, Shangdong, China.
| | - Xueguang Dong
- Department of Clinical Laboratory, People's Hospital of Laiwu, Laiwu, Shangdong, China.
| | - Fengyun Hao
- Department of General Surgery, The Affiliated Hospital of Qingdao University, Qingdao, China. .,Department of Clinical Laboratory, People's Hospital of Weifang, Weifang, Shangdong, China.
| | - Zimin Liu
- Department of Clinical Laboratory, People's Hospital of Laiwu, Laiwu, Shangdong, China. .,Department of General Surgery, The Affiliated Hospital of Qingdao University, Qingdao, China.
| | - Guangzhen Ni
- Department of Clinical Laboratory, People's Hospital of Weifang, Weifang, Shangdong, China.
| | - Dong Chen
- Department of General Surgery, The Affiliated Hospital of Qingdao University, Qingdao, China.
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22
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Dariushnejad H, Zarghami N, Rahmati M, Ghasemali S, Sadeghi Z, Davoodi Z, Jafari Tekab H, Gandomkar Ghalhar M. ABT-737, Synergistically Enhances Daunorubicin-Mediated Apoptosis in Acute Myeloid Leukemia Cell Lines. Adv Pharm Bull 2013; 4:185-9. [PMID: 24511483 DOI: 10.5681/apb.2014.027] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2013] [Revised: 10/07/2013] [Accepted: 10/19/2013] [Indexed: 11/17/2022] Open
Abstract
PURPOSE Intensive chemotherapy with daunorubicin (DNR) is associated with serious side effects in acute myeloid leukemia (AML) patients. In this study the effect of small-molecule BH3-mimetic, ABT-737, on the sensitivity of HL60 and U937 AML cell lines was investigated. METHODS The cytotoxic effects of DNR and ABT-737, alone or in combination were assessed using MTT assay and combination index analysis. The effects of treatments on the cell proliferation was determined by trypan blue assay. ELISA cell death assay was used for measurement of apoptosis. RESULTS IC50 values of DNR and ABT-737 were 2.52 and 0.59 µM for HL-60 cells line and 1.31 and 0.80 µM for U937 cell line at 24 h, respectively. Surprisingly, combination treatment significantly lowered the IC50 values in a synergic manner in both cell lines. Moreover, treatment with a mixture of two agents had more growth inhibition effect relative to the monotherapy. RESULTS of apoptosis assay showed that the cytotoxic effects are related to the enhancement of apoptosis. CONCLUSION Our study suggests that ABT-737 synergistically enhances the cytotoxic effect of DNR in AML cell lines and therefore may be useful to overcome chemoresistance of leukemia patients.
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Affiliation(s)
- Hassan Dariushnejad
- Department of Medical Biotechnology, Faculty of Advance Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Nosratallah Zarghami
- Department of Medical Biotechnology, Faculty of Advance Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Clinical Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mohammad Rahmati
- Department of Medical Biotechnology, Faculty of Advance Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Clinical Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Samaneh Ghasemali
- Department of Medical Biotechnology, Faculty of Advance Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Zohreh Sadeghi
- Department of Medical Biotechnology, Faculty of Advance Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Zahra Davoodi
- Department of Medical Biotechnology, Faculty of Advance Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Hossein Jafari Tekab
- Department of Medical Genetics, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Masoud Gandomkar Ghalhar
- Department of Medical Biotechnology, Faculty of Advance Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
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Brazel CY, Alaythan AA, Felling RJ, Calderon F, Levison SW. Molecular features of neural stem cells enable their enrichment using pharmacological inhibitors of survival-promoting kinases. J Neurochem 2013; 128:376-90. [PMID: 24032666 DOI: 10.1111/jnc.12447] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2012] [Revised: 09/05/2013] [Accepted: 09/10/2013] [Indexed: 11/28/2022]
Abstract
Isolating a pure population of neural stem cells (NSCs) has been difficult since no exclusive surface markers have been identified for panning or FACS purification. Moreover, additional refinements for maintaining NSCs in culture are required, since NSCs generate a variety of neural precursors (NPs) as they proliferate. Here, we demonstrate that post-natal rat NPs express low levels of pro-apoptotic molecules and resist phosphatidylinositol 3'OH kinase and extracellular regulated kinase 1/2 inhibition as compared to late oligodendrocyte progenitors. Furthermore, maintaining subventricular zone precursors in LY294002 and PD98059, inhibitors of PI3K and ERK1/2 signaling, eliminated lineage-restricted precursors as revealed by enrichment for Nestin(+)/SOX-2(+) cells. The cells that survived formed neurospheres and 89% of these neurospheres were tripotential, generating neurons, astrocytes, and oligodendrocytes. Without this enrichment step, less than 50% of the NPs were Nestin(+)/SOX-2(+) and 42% of the neurospheres were tripotential. In addition, neurospheres enriched using this procedure produced 3-times more secondary neurospheres, supporting the conclusion that this procedure enriches for NSCs. A number of genes that enhance survival were more highly expressed in neurospheres compared to late oligodendrocyte progenitors. Altogether, these studies demonstrate that primitive neural precursors can be enriched using a relatively simple and inexpensive means that will facilitate cell replacement strategies using stem cells as well as other studies whose goal is to reveal the fundamental properties of primitive neural precursors.
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Affiliation(s)
- Christine Y Brazel
- Department of Neurology and Neurosciences, Rutgers University-New Jersey Medical School, Newark, New Jersey, USA
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24
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Ring BA, Ferreira Lacerda A, Drummond DJ, Wangen C, Eaton HE, Brunetti CR. Frog virus 3 open reading frame 97R localizes to the endoplasmic reticulum and induces nuclear invaginations. J Virol 2013; 87:9199-207. [PMID: 23760249 PMCID: PMC3754063 DOI: 10.1128/jvi.00637-13] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2013] [Accepted: 06/08/2013] [Indexed: 12/18/2022] Open
Abstract
Frog virus 3 (FV3) is the type species of the genus Ranavirus, family Iridoviridae. The genome of FV3 is 105,903 bases in length and encodes 97 open reading frames (ORFs). The FV3 ORF 97R contains a B-cell lymphoma 2 (Bcl-2) homology 1 (BH1) domain and has sequence similarity to the myeloid cell leukemia-1 (Mcl-1) protein, suggesting a potential role in apoptosis. To begin to understand the role of 97R, we characterized 97R through immunofluorescence and mutagenesis. Here we demonstrated that 97R localized to the endoplasmic reticulum (ER) at 24 h posttransfection. However, at 35 h posttransfection, 97R localized to the ER but also began to form concentrated pockets continuous with the nuclear membrane. After 48 h posttransfection, 97R was still localized to the ER, but we began to observe the ER and the outer nuclear membrane invaginating into the nucleus. To further explore 97R targeting to the ER, we created a series of C-terminal transmembrane domain deletion mutants. We found that deletion of 29 amino acids from the C terminus of 97R abolished localization to the ER. In contrast, deletion of 12 amino acids from the C terminus of 97R did not affect 97R localization to the ER. In addition, a hybrid protein containing the 97R C-terminal 33 amino acids was similarly targeted to the ER. These data indicate that the C-terminal 33 amino acids of 97R are necessary and sufficient for ER targeting.
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Affiliation(s)
- Brooke A Ring
- Department of Biology, Trent University, Peterborough, Ontario, Canada
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25
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Zhang J, Yan H, Wu YP, Li C, Zhang GY. Activation of GluR6-containing kainate receptors induces ubiquitin-dependent Bcl-2 degradation via denitrosylation in the rat hippocampus after kainate treatment. J Biol Chem 2011; 286:7669-80. [PMID: 21148565 PMCID: PMC3045021 DOI: 10.1074/jbc.m110.156299] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2010] [Revised: 12/09/2010] [Indexed: 11/06/2022] Open
Abstract
We previously showed that Bcl-2 (B-cell lymphoma 2) is down-regulated in a kainate (KA)-induced rat epileptic seizure model. The underlying mechanism had remained largely unknown, but we here report for the first time that denitrosylation and ubiquitination are involved. Our results show that the S-nitrosylation levels of Bcl-2 are down-regulated after KA injection and that the GluR6 (glutamate receptor 6) antagonist NS102 can inhibit the denitrosylation of Bcl-2. Moreover, the ubiquitin-dependent degradation of Bcl-2 was found to be promoted after KA treatment, which could be suppressed by the proteasome inhibitor MG132 and the NO donors, sodium nitroprusside and S-nitrosoglutathione. In addition, experiments based on siRNA transfections were performed in the human SH-SY5Y neuroblastoma cell line to verify that the stability of Bcl-2 is causal to neuronal survival. At the same time, it was found that the exogenous NO donor GSNO could protect neurons when Bcl-2 is targeted. Subsequently, these mechanisms were morphologically validated by immunohistochemistry, cresyl violet staining, and in situ TUNEL staining to analyze the expression of Bcl-2 as well as the survival of CA1 and CA3/DG pyramidal neurons. NS102, GSNO, sodium nitroprusside, and MG132 contribute to the survival of CA1 and CA3/DG pyramidal neurons by attenuating Bcl-2 denitrosylation. Taken together, our data reveal that Bcl-2 ubiquitin-dependent degradation is induced by Bcl-2 denitrosylation during neuronal apoptosis after KA treatment.
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MESH Headings
- Animals
- Brain Ischemia/chemically induced
- Brain Ischemia/metabolism
- Brain Ischemia/pathology
- CA1 Region, Hippocampal/drug effects
- CA1 Region, Hippocampal/metabolism
- CA1 Region, Hippocampal/pathology
- CA3 Region, Hippocampal/drug effects
- CA3 Region, Hippocampal/metabolism
- CA3 Region, Hippocampal/pathology
- Caspase 3/metabolism
- Cell Line, Tumor
- Dentate Gyrus/drug effects
- Dentate Gyrus/metabolism
- Dentate Gyrus/pathology
- Disease Models, Animal
- Epilepsy/chemically induced
- Epilepsy/metabolism
- Epilepsy/pathology
- Excitatory Amino Acid Agonists/toxicity
- Hippocampus/drug effects
- Hippocampus/metabolism
- Hippocampus/pathology
- Humans
- Kainic Acid/toxicity
- Male
- Neuroblastoma
- Nitric Oxide/metabolism
- Nitric Oxide Donors/pharmacology
- Nitrogen/metabolism
- Proteasome Endopeptidase Complex/metabolism
- Proteasome Inhibitors
- Protein Processing, Post-Translational/physiology
- Proto-Oncogene Proteins c-bcl-2/metabolism
- Rats
- Rats, Sprague-Dawley
- Receptors, Kainic Acid/genetics
- Receptors, Kainic Acid/metabolism
- Ubiquitin/metabolism
- GluK2 Kainate Receptor
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Affiliation(s)
- Jia Zhang
- From the Research Center of Biochemistry and Molecular Biology, Jiangsu Province Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou, Jiangsu 221002, China and
| | - Hui Yan
- From the Research Center of Biochemistry and Molecular Biology, Jiangsu Province Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou, Jiangsu 221002, China and
| | - Yong-Ping Wu
- the Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou, Jiangsu 221002, China
| | - Chong Li
- From the Research Center of Biochemistry and Molecular Biology, Jiangsu Province Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou, Jiangsu 221002, China and
| | - Guang-Yi Zhang
- From the Research Center of Biochemistry and Molecular Biology, Jiangsu Province Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou, Jiangsu 221002, China and
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26
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Boohaker RJ, Zhang G, Carlson AL, Nemec KN, Khaled AR. BAX supports the mitochondrial network, promoting bioenergetics in nonapoptotic cells. Am J Physiol Cell Physiol 2011; 300:C1466-78. [PMID: 21289292 DOI: 10.1152/ajpcell.00325.2010] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The dual functionality of the tumor suppressor BAX is implied by the nonapoptotic functions of other members of the BCL-2 family. To explore this, mitochondrial metabolism was examined in BAX-deficient HCT-116 cells as well as primary hepatocytes from BAX-deficient mice. Although mitochondrial density and mitochondrial DNA content were the same in BAX-containing and BAX-deficient cells, MitoTracker staining patterns differed, suggesting the existence of BAX-dependent functional differences in mitochondrial physiology. Oxygen consumption and cellular ATP levels were reduced in BAX-deficient cells, while glycolysis was increased. These results suggested that cells lacking BAX have a deficiency in the ability to generate ATP through cellular respiration. This conclusion was supported by detection of reduced citrate synthase activity in BAX-deficient cells. In nonapoptotic cells, a portion of BAX associated with mitochondria and a sequestered, protease-resistant form was detected. Inhibition of BAX with small interfering RNAs reduced intracellular ATP content in BAX-containing cells. Expression of either full-length or COOH-terminal-truncated BAX in BAX-deficient cells rescued ATP synthesis and oxygen consumption and reduced glycolytic activity, suggesting that this metabolic function of BAX was not dependent upon its COOH-terminal helix. Expression of BCL-2 in BAX-containing cells resulted in a subsequent loss of ATP measured, implying that, even under nonapoptotic conditions, an antagonistic interaction exists between the two proteins. These findings infer that a basal amount of BAX is necessary to maintain energy production via aerobic respiration.
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Affiliation(s)
- Rebecca J Boohaker
- Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, 6900 Lake Nona Blvd., Orlando, FL 32827, USA
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27
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Thomas LW, Lam C, Edwards SW. Mcl-1; the molecular regulation of protein function. FEBS Lett 2010; 584:2981-9. [PMID: 20540941 DOI: 10.1016/j.febslet.2010.05.061] [Citation(s) in RCA: 423] [Impact Index Per Article: 28.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2010] [Revised: 05/25/2010] [Accepted: 05/28/2010] [Indexed: 10/19/2022]
Abstract
Apoptosis, an essential and basic biological phenomenon, is regulated in a complex manner by a multitude of factors. Myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the B-cell lymphoma 2 (Bcl-2) family of apoptosis-regulating proteins, exemplifies a number of the mechanisms by which a protein's contribution to cell fate may be modified. The N-terminus of Mcl-1 is unique amongst the Bcl-2 family, in that it is rich in experimentally confirmed and putative regulatory residues and motifs. These include sites for ubiquitination, cleavage and phosphorylation, which influence the protein's stability, localisation, dimerization and function. Here we review what is known about the regulation of Mcl-1 expression and function, with particular focus on post-translational modifications and how phosphorylation interconnects the complex molecular control of Mcl-1 with cellular state.
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Affiliation(s)
- Luke W Thomas
- School of Biological Sciences, University of Liverpool, Liverpool, UK
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28
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Karnak D, Xu L. Chemosensitization of prostate cancer by modulating Bcl-2 family proteins. Curr Drug Targets 2010; 11:699-707. [PMID: 20298153 DOI: 10.2174/138945010791170888] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2009] [Accepted: 12/27/2009] [Indexed: 01/16/2023]
Abstract
A major challenge in oncology is the development of chemoresistance. This often occurs as cancer progresses and malignant cells acquire mechanisms to resist insults that would normally induce apoptosis. The onset of androgen independence in advanced prostate cancer is a prime example of this phenomenon. Overexpression of the pro-survival/anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 are hallmarks of this transition. Here we outline the evolution of therapeutics designed to either limit the source or disrupt the interactions of these pro-survival proteins. By either lessening the stoichiometric abundance of Bcl-2/xL/Mcl-1 in reference to their pro-apoptotic foils or freeing these pro-apoptotic proteins from their grip, these treatments aim to sensitize cells to chemotherapy by priming cells for death. DNA anti-sense and RNA interference have been effectively employed to decrease Bcl-2 family mRNA and protein levels in cell culture models of advanced prostate cancer. However, clinical studies are lagging due to in vivo delivery challenges. The burgeoning field of nanoparticle delivery holds great promise in helping to overcome the challenge of administering highly labile nucleic acid based therapeutics. On another front, small molecule inhibitors that block the hetero-dimerization of pro-survival with pro-apoptotic proteins have significant clinical advantages and have advanced farther in clinical trials with promising early results. Most recently, a peptide has been discovered that can convert Bcl-2 from a pro-survival to a pro-apoptotic protein. The future may lie in targeting multiple steps of the apoptotic pathway, including Bcl-2/xL/Mcl-1, to debilitate the survival capacity of cancer cells and make chemotherapy induced death their only option.
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Affiliation(s)
- David Karnak
- Department of Radiation Oncology, Division of Radiation and Cancer Biology, Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI 48109-5637, USA
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29
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The Interplay between BCL-2 Family Proteins and Mitochondrial Morphology in the Regulation of Apoptosis. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2010; 687:97-114. [DOI: 10.1007/978-1-4419-6706-0_6] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/07/2023]
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30
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Raucher D, Moktan S, Massodi I, Bidwell GL. Therapeutic peptides for cancer therapy. Part II - cell cycle inhibitory peptides and apoptosis-inducing peptides. Expert Opin Drug Deliv 2009; 6:1049-64. [PMID: 19743895 DOI: 10.1517/17425240903158909] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
BACKGROUND Therapeutic peptides have great potential as anticancer agents owing to their ease of rational design and target specificity. However, their utility in vivo is limited by low stability and poor tumor penetration. OBJECTIVE The authors review the development of peptide inhibitors with potential for cancer therapy. Peptides that arrest the cell cycle by mimicking CDK inhibitors or induce apoptosis directly are discussed. METHODS The authors searched Medline for articles concerning the development of therapeutic peptides and their delivery. RESULTS/CONCLUSION Inhibition of cancer cell proliferation directly using peptides that arrest the cell cycle or induce apoptosis is a promising strategy. Peptides can be designed that interact very specifically with cyclins and/or cyclin-dependent kinases and with members of apoptotic cascades. Use of these peptides is not limited by their design, as a rational approach to peptide design is much less challenging than the design of small molecule inhibitors of specific protein-protein interactions. However, the limitations of peptide therapy lie in the poor pharmacokinetic properties of these large, often charged molecules. Therefore, overcoming the drug delivery hurdles could open the door for effective peptide therapy, thus making an entirely new class of molecules useful as anticancer drugs.
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Affiliation(s)
- Drazen Raucher
- The University of Mississippi Medical Center, Department of Biochemistry, Jackson, 39216, USA.
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31
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Saad M, Garbuzenko OB, Minko T. Co-delivery of siRNA and an anticancer drug for treatment of multidrug-resistant cancer. Nanomedicine (Lond) 2009; 3:761-76. [PMID: 19025451 DOI: 10.2217/17435889.3.6.761] [Citation(s) in RCA: 278] [Impact Index Per Article: 17.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
AIMS To develop a novel nanomedicine approach for the treatment of multidrug-resistant (MDR) cancer by combining an anticancer drug and suppressors of cellular resistance within one multifunctional nanocarrier-based delivery system (NDS). MATERIALS & METHODS The NDS consisted of cationic liposomes (carrier, 100-140 nm), doxorubicin (DOX, anticancer drug), siRNA targeted to MRP1 and BCL2 mRNA (suppressors of pump and nonpump cellular-resistance, respectively). The resulting approximately 500 nm complex has a zeta potential of +4 mV. RESULTS & DISCUSSION The NDS provides an effective co-delivery of DOX and siRNA as well as cell-death induction and suppression of cellular resistance in MDR lung cancer cells. CONCLUSION We demonstrate NDS-enhanced efficiency of chemotherapy to a level that cannot be achieved by applying its components separately.
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Affiliation(s)
- Maha Saad
- Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-08020, USA
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32
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Mikami Y, Somei M, Takagi M. A tryptamine derivative, SST-VEDI-1, inhibits apoptosis and stimulates mineralization in osteoblasts. Endocr J 2009; 56:665-78. [PMID: 19461163 DOI: 10.1507/endocrj.k08e-340] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
SST-VEDI-1(VEDI-1) is a new synthetic compound that is synthesized from tryptamine, and has structural similarity to the SSH-BM family of compounds. However, the biological effects of VEDI-1 have yet to be well characterized. A recent report has demonstrated that SSH-BM-type compounds can stimulate osteoblast activity in cultured scales of goldfish. In this study, we examined the effects of VEDI-1 on osteoblastic differentiation as well as its effects on apoptosis, which is known to be closely related to osteoblastic differentiation. We found that VEDI-1 enhanced the formation of mineralized nodules in rat osteoblast cell lines, including ROS17/2.8 cells, and in mouse pre-osteoblast cell lines, including MC3T3-E1 cells, in a dose dependent manner, which was accompanied by increased expression of late osteoblast markers, bone sialoprotein (BSP) and osteocalcin (OC). Furthermore, VEDI-I inhibited apoptotic cell death and regulated the expression of proteins in the Bcl-2 family. These results suggest that VEDI-1 may facilitate late differentiation of osteoblasts and may have an inhibitory effect on apoptosis.
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Affiliation(s)
- Yoshikazu Mikami
- Department of Anatomy, Nihon University School of Dentistry, Tokyo, Japan.
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33
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Pike CJ, Nguyen TVV, Ramsden M, Yao M, Murphy MP, Rosario ER. Androgen cell signaling pathways involved in neuroprotective actions. Horm Behav 2008; 53:693-705. [PMID: 18222446 PMCID: PMC2424283 DOI: 10.1016/j.yhbeh.2007.11.006] [Citation(s) in RCA: 103] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/11/2007] [Revised: 10/31/2007] [Accepted: 11/05/2007] [Indexed: 11/15/2022]
Abstract
As a normal consequence of aging in men, testosterone levels significantly decline in both serum and brain. Age-related testosterone depletion results in increased risk of dysfunction and disease in androgen-responsive tissues, including brain. Recent evidence indicates that one deleterious effect of age-related testosterone loss in men is increased risk for Alzheimer's disease (AD). We discuss recent findings from our laboratory and others that identify androgen actions implicated in protecting the brain against neurodegenerative diseases and begin to define androgen cell signaling pathways that underlie these protective effects. Specifically, we focus on the roles of androgens as (1) endogenous negative regulators of beta-amyloid accumulation, a key event in AD pathogenesis, and (2) neuroprotective factors that utilize rapid non-genomic signaling to inhibit neuronal apoptosis. Continued elucidation of cell signaling pathways that contribute to protective actions of androgens should facilitate the development of targeted therapeutic strategies to combat AD and other age-related neurodegenerative diseases.
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Affiliation(s)
- Christian J Pike
- Davis School of Gerontology, University of Southern California, Los Angeles, CA 90089, USA.
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34
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Minko T, Khandare JJ, Vetcher AA, Soldatenkov VA, Garbuzenko OB, Saad M, Pozharov VP. Multifunctional Nanotherapeutics for Cancer. MULTIFUNCTIONAL PHARMACEUTICAL NANOCARRIERS 2008. [DOI: 10.1007/978-0-387-76554-9_10] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/08/2022]
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35
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Muzio G, Maggiora M, Oraldi M, Trombetta A, Canuto RA. PPARalpha and PP2A are involved in the proapoptotic effect of conjugated linoleic acid on human hepatoma cell line SK-HEP-1. Int J Cancer 2007; 121:2395-401. [PMID: 17691108 DOI: 10.1002/ijc.23004] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Conjugated linoleic acid (CLA), found in dairy products, in beef and lamb has been demonstrated to possess anticancer properties protecting several tissues from developing cancer. Moreover, it has been shown to modulate apoptosis in several cancer cell lines. The aim of this study was to investigate which signaling transduction pathways were modulated in CLA-induced apoptosis in human hepatoma SK-HEP-1 cells. The cells exposed to CLA were evaluated for PPARalpha, PP2A, pro-apoptotic proteins Bak, Bad and caspases, and anti-apoptotic proteins Bcl-2 and Bcl-X(L). Cells were also treated with okadaic acid, a PP2A inhibitor, or with Wy-14643, a specific PPARalpha agonist. The CLA-induced apoptosis was concomitant to the increase of percentage of cells in the S phase, PPARalpha, PP2A and pro-apoptotic proteins; simultaneously, antiapoptotic proteins decreased. Inhibition of PP2A prevented apoptosis, and PPARalpha agonist showed similar effect as CLA. The increased PP2A could be responsible for the dephosphorylation of Bcl-2 and Bad, permitting apoptotic activity of Bax and Bad. The increase of caspase 8 and 9 suggested that both the intrinsic and extrinsic apoptotic pathways were induced. PP2A was probably increased by PPARalpha, since putative PPRE sequences were found in genes encoding its subunits. In conclusion, CLA induces apoptosis in human hepatoma SK-HEP-1 cells, by increasing PPARalpha, PP2A and pro-apoptotic proteins.
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Affiliation(s)
- Giuliana Muzio
- Dipartimento di Medicina ed Oncologia Sperimentale, Università di Torino, Corso Raffaello Torino, Italy
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36
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Torrecillas A, Martínez-Senac MM, Ausili A, Corbalán-García S, Gómez-Fernández JC. Interaction of the C-terminal domain of Bcl-2 family proteins with model membranes. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2007; 1768:2931-9. [PMID: 17905195 DOI: 10.1016/j.bbamem.2007.08.014] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/18/2007] [Revised: 07/23/2007] [Accepted: 08/13/2007] [Indexed: 11/19/2022]
Abstract
Bcl-2 family proteins are involved in the cell homeostasis by regulating programmed cell death. Some of these proteins promote apoptosis, while others inhibit the same process. The C-terminal hydrophobic domain of some of these proteins is predicted to be involved in anchoring them to a variety of cell membranes, such as mitochondrial, endoplasmic reticulum and nuclear membranes. We have used five synthetic peptides imitating the C-terminal domain from both anti-apoptotic (Bcl-2) and pro-apoptotic members (Bak, Bax, and two mutants of this last protein) of this family to study their interaction with model membranes. Some differences were detected in the interaction with these peptides. The addition of all the peptides to large unilamellar vesicles destabilized them and released encapsulated carboxyfluorescein to different degrees, so that fluidity and the increase in negative curvature favoured the extent in the release of carboxyfluorescein. Bcl-2-C and Bax-C peptides produced the highest release levels in most cases, while BaxS184K-C was the least efficient in this respect. These results indicate that these C-terminal domains are able to insert themselves in the membranes, each in a different way that is probably related with their different way which can be related to their differing locations within the cell and their different roles in regulating apoptosis.
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Affiliation(s)
- Alejandro Torrecillas
- Departamento de Bioquímica y Biología Molecular (A), Facultad de Veterinaria, Universidad de Murcia, Apartado de Correos 4021, E-30080, Murcia, Spain
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37
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Chandna P, Saad M, Wang Y, Ber E, Khandare J, Vetcher AA, Soldatenkov VA, Minko T. Targeted Proapoptotic Anticancer Drug Delivery System. Mol Pharm 2007; 4:668-78. [PMID: 17685579 DOI: 10.1021/mp070053o] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
A novel targeted proapoptotic anticancer drug delivery system (DDS) was developed and evaluated both in vitro and in vivo. The system contains poly(ethylene glycol) polymer (PEG) as a carrier, camptothecin (CPT) as an anticancer drug/cell death inducer, a synthetic analogue of luteinizing hormone-releasing hormone (LHRH) peptide as a targeting moiety/penetration enhancer, and a synthetic analogue of BCL2 homology 3 domain (BH3) peptide as a suppressor of cellular antiapoptotic defense. The design of the multicomponent DDS allowed for a conjugation of one or two copies of each active ingredient (CPT, LHRH, and BH3) to one molecule of PEG carrier. The complex structure of the PEG conjugates was visualized at nanometer resolution using atomic force microscopy. We found that the ligand-targeted DDS for cancer cells preferentially accumulated in the tumor and allowed the delivery of active ingredients into the cellular cytoplasm and nuclei of cancer cells. Simultaneous apoptosis induction through the caspase-dependent signaling pathway and inhibition of cellular antiapoptotic defense by the suppression of BCL2 protein enhanced cytotoxicity and antitumor activity of the entire DDS to a level which could not be achieved by individual components applied separately. The DDS containing two copies of each active component (CPT, LHRH, and BH3) per molecule of PEG polymer had the highest anticancer efficiency in vitro and in vivo.
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Affiliation(s)
- Pooja Chandna
- Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA
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38
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Wang Y, Cao R, Liu D, Chervin A, Yuan J, An J, Huang Z. Oligomerization of BH4-truncated Bcl-x(L) in solution. Biochem Biophys Res Commun 2007; 361:1006-11. [PMID: 17692289 DOI: 10.1016/j.bbrc.2007.07.122] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2007] [Accepted: 07/21/2007] [Indexed: 11/16/2022]
Abstract
BH4 domain is critical for the anti-apoptotic functions of Bcl-2 and Bcl-x(L) and their binding abilities with other members of the Bcl-2 family. The cleavage of the BH4 domain in Bcl-x(L) and Bcl-2 by caspase 1 or 3 converts the anti-apoptotic Bcl-x(L) and Bcl-2 into pro-apoptotic proteins that potently induce apoptosis. Herein, we report that recombinant Bcl-x(L) proteins without N-terminal 61 residues, His(6)-NDelta61-Bcl-x(L)-CDelta21 and NDelta61-Bcl-x(L)-CDelta21, form oligomers in solution, whereas Bcl-x(L)-CDelta21 exists as a monomer. The oligomerization of the truncated proteins is independent of protein-lipid interaction, protein concentration or the ion strength of the solution. Circular dichroism spectrum shows a significant decrease in the content of alpha-helices upon deletion of N-terminal residues. NDelta61-Bcl-x(L)-CDelta21 also loses its heterodimerization capability with the BH3 peptide derived from Bak. This newly acquired property might be linked to its ability to induce apoptosis in cells.
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Affiliation(s)
- Youli Wang
- Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
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Zhu L, Rorke EA, Eckert RL. DeltaNp63alpha promotes apoptosis of human epidermal keratinocytes. J Invest Dermatol 2007; 127:1980-91. [PMID: 17392828 DOI: 10.1038/sj.jid.5700797] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
In this study we show that deltaNp63alpha overexpression in primary human epidermal keratinocytes causes decreased cell proliferation and increased apoptosis. These changes are associated with increased levels of p21 and p27, decreased cyclin D1 and cyclin E levels, reduced mitochondrial membrane potential, and enhanced procaspase and poly(ADP-ribose) polymerase cleavage. Bcl-xS and Bax levels are increased and Bcl-xL level is reduced. p53 levels are increased in the deltaNp63alpha-expressing cells and p53 overexpression reproduces features of the deltaNp63alpha phenotype. Increased p53 expression results in reduced deltaNp63alpha, suggesting that p53 may negatively regulate deltaNp63alpha level. DeltaNp63alpha also induces apoptosis in HaCaT and SCC-13 cells, which encode inactive p53 genes, suggesting that the response is p53 independent in these cell lines. Both deltaNp63alpha and TAp63alpha reduce SCC-13 cell survival. These studies indicate that both deltaNp63alpha and TAp63alpha can negatively regulate keratinocyte survival.
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Affiliation(s)
- Ling Zhu
- Department of Physiology and Biophysics, Case School of Medicine, Cleveland, Ohio, USA
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Mitsunaga M, Tsubota A, Nariai K, Namiki Y, Sumi M, Yoshikawa T, Fujise K. Early apoptosis and cell death induced by ATX-S10Na (II)-mediated photodynamic therapy are Bax- and p53-dependent in human colon cancer cells. World J Gastroenterol 2007; 13:692-8. [PMID: 17278191 PMCID: PMC4066001 DOI: 10.3748/wjg.v13.i5.692] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the roles of Bax and p53 proteins in photosensitivity of human colon cancer cells by using lysosome-localizing photosensitizer, ATX-S10Na (II).
METHODS: HCT116 human colon cancer cells and Bax-null or p53-null isogenic derivatives were irradiated with a diode laser. Early apoptosis and cell death in response to photodynamic therapy were determined by MTT assays, annexin V assays, transmission electron microscopy assays, caspase assays and western blotting.
RESULTS: Induction of early apoptosis and cell death was Bax- and p53-dependent. Bax and p53 were required for caspase-dependent apoptosis. The levels of anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-xL, were decreased in Bax- and p53-independent manner.
CONCLUSION: Our results indicate that early apoptosis and cell death of human colon cancer cells induced by photodynamic therapy with lysosome-localizing photosensitizer ATX-S10Na (II) are mediated by p53-Bax network and low levels of Bcl-2 and Bcl-xL proteins. Our results might help in formulating new therapeutic approaches in photodynamic therapy.
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Affiliation(s)
- Makoto Mitsunaga
- Institute of Clinical Medicine and Research, Jikei University School of Medicine, 163-1 Kashiwa-shita, Kashiwa, Chiba 277-8567, Japan
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Chao OSP, Clément MV. Epidermal growth factor and serum activate distinct pathways to inhibit the BH3 only protein BAD in prostate carcinoma LNCaP cells. Oncogene 2006; 25:4458-69. [PMID: 16767165 DOI: 10.1038/sj.onc.1209421] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
A better understanding of pathways involved in survival of prostate cancer cells is the key to develop effective and target-selective therapies. Presence of serum or epidermal growth factor in the culture medium of LNCaP cells decreases apoptosis induced by the inhibition of phosphatidylinositol 3-kinase with LY294002. However, intracellular pathway(s) involved in this survival signaling are not well defined. Here, we investigated the mechanism(s) involved in serum or epidermal growth factor-mediated inhibition of LY294002-induced death in LNCaP cells. Cell death was assessed by the percentage of cells in sub-G1 phase and caspase 3 activity. Phosphorylation status of BAD, ERK1/2 and RSKs were assessed by Western blot. Specific gene expression knock down of BAD, BAX, RSK1 and RSK2 were performed using siRNA transfections. Our results demonstrate that cell death induced by LY294002 is mediated by translocation of BAD and BAX proteins from the cytosol to the mitochondria. Whereas, epidermal growth factor activates a MAPK/ERK/RSK1 module leading to inactivation of BAD via Ser(75) phosphorylation, the presence of serum, on the other hand, induces a nonconducive intracellular environment for mitochondrial translocation of dephosphorylated BAD. Taken together, these results indicate that phosphorylation of BAD or inhibition of its translocation to the mitochondria are critical phosphatidylinositol 3-kinase-independent survival pathways in LNCaP cells.
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Affiliation(s)
- O S P Chao
- Department of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore
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Dharap SS, Chandna P, Wang Y, Khandare JJ, Qiu B, Stein S, Minko T. Molecular targeting of BCL2 and BCLXL proteins by synthetic BCL2 homology 3 domain peptide enhances the efficacy of chemotherapy. J Pharmacol Exp Ther 2006; 316:992-8. [PMID: 16291730 DOI: 10.1124/jpet.105.094243] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Chemotherapeutic agents are known to induce programmed cell death or apoptosis. The activation of cellular antiapoptotic defense that prevents the translation of drug-induced damage into cell death is the key factor in cellular antiapoptotic resistance that decreases the chemotherapeutic effectiveness of a broad spectrum of anticancer drugs. A novel proapoptotic anticancer drug delivery system (DDS) was designed to simultaneously induce apoptosis and suppress antiapoptotic cellular defense. The system includes three main components: 1) anticancer drug camptothecin (CPT) as an apoptosis inducer, 2) synthetic BCL2 homology 3 domain (BH3) peptide as a suppressor of cellular antiapoptotic defense, and 3) poly(ethylene glycol) (PEG) polymer as a carrier. The above DDS was studied in vitro using A2780 human ovarian carcinoma cells and in vivo on nude mice bearing xenografts of human ovarian tumor. The results obtained in both series of experiments corroborate each other. They show that the designed DDS provided intracellular delivery of active components and suppressed cellular antiapoptotic defense, leading to the more pronounced induction of caspase-dependent signaling pathway of apoptosis compared with CPT alone and simple CPT-PEG conjugate. Including BH3 peptide in complex DDS decreased apoptotic cellular defense, substantially increased toxicity of the whole complex, and provided high antitumor activity. Therefore, the proposed novel multicomponent proapoptotic anticancer drug delivery system has high potential to enhance the efficacy of chemotherapy.
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Affiliation(s)
- Sonia S Dharap
- Department of Pharmaceutics, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA
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Styles NA, Zhu W, Li X. Phosphorylation and down-regulation of Bim by muscarinic cholinergic receptor activation via protein kinase C. Neurochem Int 2005; 47:519-27. [PMID: 16183168 DOI: 10.1016/j.neuint.2005.08.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2005] [Revised: 07/26/2005] [Accepted: 08/01/2005] [Indexed: 11/27/2022]
Abstract
Bim is one of the proapoptotic BH3-only homologs of the Bcl-2 family proteins, which interacts with other Bcl-2 family proteins to activate the intrinsic apoptotic pathway. The expression and protein level of Bim are highly regulated in cells at both transcriptional and post-translational levels, and inadequate control of Bim level may largely determine its pro-apoptic activity. In the present study, we reported that carbachol, a muscarinic cholinergic receptor agonist, regulated Bim in human SH-SY5Y neuroblastoma cells. Carbachol rapidly induced an upward gel mobility shift of Bim, which was abolished by protein phosphatase treatment, indicating an increased Bim phosphorylation by carbachol. The effect of carbachol was mimicked by the protein kinase C activator 12-myristate 13-acetate (PMA) and was blocked by the protein kinase C inhibitor rottlerin, suggesting that activation of protein kinase C was required for carbachol-induced phosphorylation of Bim. Prolonged treatment with carbachol and PMA significantly decreased Bim protein levels in total cell lysates and mitrochondria. Carbachol and PMA had no effect in the transcriptional regulation of Bim, whereas the reduction of Bim by both carbachol and PMA was reversed by the proteosome inhibitors, suggesting that carbachol and PMA facilitated the proteosome-dependent Bim degradation. Thus, this study identified the muscarinic receptor-protein kinase C signaling pathway as a regulator of Bim in neuroblastoma cells, and activation of muscarinic receptor and protein kinase C functions to induce Bim phosphorylation, followed by down-regulation of the proapoptotic protein.
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Affiliation(s)
- Nathan A Styles
- Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA
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Madureira PA, Matos P, Soeiro I, Dixon LK, Simas JP, Lam EWF. Murine gamma-herpesvirus 68 latency protein M2 binds to Vav signaling proteins and inhibits B-cell receptor-induced cell cycle arrest and apoptosis in WEHI-231 B cells. J Biol Chem 2005; 280:37310-8. [PMID: 16150693 DOI: 10.1074/jbc.m507478200] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
The MHV-68 latent protein, M2, does not have homology to any known viral or cellular proteins, and its function is unclear. To define the role played by M2 during MHV-68 latency as well as the molecular mechanism involved, we used M2 as bait to screen a yeast two-hybrid mouse B-cell cDNA library. Vav1 was identified as an M2-interacting protein in two independent screenings. Subsequent yeast two-hybrid interaction studies showed that M2 also binds to Vav2, but not Vav3, and that three "PXXP" motifs located at the C terminus of M2 are important for this interaction. The interactions between M2 and Vav proteins were also confirmed in vivo in 293T and WEHI-231 B-cells by co-immunoprecipitation assays. Rac1/GST-PAK "pull-down" experiments and Western blot analysis using a phospho-Vav antibody demonstrated that expression of M2 in WEHI-231 cells enhances Vav activity. We further showed in WEHI-231 cells that M2 expression promotes proliferation and survival and is associated with enhanced cyclin D2 and repressed p27(Kip1), p130, and Bim expression. Taken together, these experiments suggest that M2 might have an important role in disseminating the latent virus during the establishment and maintenance of latency by modulating B-cell receptor-mediated signaling events through Vav to promote B-cell activation, proliferation, and survival.
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Affiliation(s)
- Patrícia A Madureira
- Cancer Research-UK Laboratories, Department of Cancer Medicine, MRC Cyclotron Building, Imperial College London, Hammersmith Hospital
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Abedin N, Ashraf Q, Mishra OP, Delivoria-Papadopoulos M. Effect of hypoxia on the expression of pro- and anti-apoptotic proteins in neuronal nuclei of the guinea pig fetus during gestation. BRAIN RESEARCH. DEVELOPMENTAL BRAIN RESEARCH 2005; 156:32-7. [PMID: 15862625 DOI: 10.1016/j.devbrainres.2005.01.006] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/30/2004] [Revised: 01/24/2005] [Accepted: 01/26/2005] [Indexed: 01/03/2023]
Abstract
The present study investigates the expression of apoptotic proteins Bax, Bad, Bcl-2, and Bcl-xl following hypoxia in the cerebral cortex of the guinea pig fetus as a function of gestational age. Normoxic (Nx, n = 6) and hypoxic (Hx, n = 6) guinea pig fetuses at 35 and 60 days gestation were studied. Bax expression (OD X mm(2)) was 96.9 +/- 9.5 (Nx 35 days), 116.5 +/- 8.3 (Hx 35 days), P < 0.05 and 116.2 +/- 3.4 (Nx 60 days, 144.6 +/- 11.7 (Hx 60 days), P < 0.05. Bad expression (OD X mm(2)) was 78.6 +/- 2.6 (Nx 35 days), 102.9 +/- 5.8 (Hx 35 days), P < 0.05 and 101.5 +/- 4.3 (Nx 60 days), 139.8 +/- 7.9 (Hx 60 days), P < 0.05 vs. Nx 60 days, also significantly higher from preterm hypoxia P < 0.007. Expression of Bcl-2 (OD X mm(2)) was 27.4 +/- 2.0 (Nx 35 days), 28.0 +/- 2.4 (Hx 35 days), and 27.4 +/- 2.7 (Nx 60 days), 29.7 +/- 2.3 (Hx 60 days). Expression of Bcl-xl (OD X mm(2)) was 51.0 +/- 4.4 (Nx 35 days), 46.1 +/- 8.0 (Hx 35 days) and 50.0 +/- 1.4 (Nx 60 days), 54.9 +/- 7.4 (Hx 60 days). Hypoxia resulted in increased expression of the proapoptotic proteins Bax and Bad by 20% and 30% in the preterm as compared to 24% and 38% at term, without altering the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl. We conclude that the hypoxia-induced increased expression of Bax and Bad is greater at term compared to preterm. Furthermore, the hypoxia-induced increase in proapoptotic as compared to antiapoptotic proteins at term will accelerate the ongoing active process of programmed cell death at term compared to preterm gestation.
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Affiliation(s)
- Naheed Abedin
- Drexel University College of Medicine, MCP Hospital, Neonatal Research, 3300 Henry Avenue Philadelphia, PA 19133, USA.
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46
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Huang YC, Guh JH, Shen YC, Teng CM. Investigation of anticancer mechanism of clavulone II, a coral cyclopentenone prostaglandin analog, in human acute promyelocytic leukemia. J Biomed Sci 2005; 12:335-45. [PMID: 15920677 DOI: 10.1007/s11373-005-3009-9] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2004] [Accepted: 02/05/2005] [Indexed: 10/25/2022] Open
Abstract
The marine prostanoid clavulones were shown to exert cytotoxicity against several cancer cells. In the present study, we illustrate the pathways utilized by clavulone II to trigger apoptotic signaling in human acute promyelocytic leukemia HL-60 cells. Exposure of cells to clavulone II resulted in early induction of phosphatidylserine externalization, mitochondrial dysfunction, and alteration of the cell cycle. Down-regulated expression of cyclin D1 explained the effect of clavulone II on G1 phase arrest of the cell cycle. Clavulone II induced the disruption of mitochondrial membrane potential and activation of caspase-8, -9 and -3 in a time- and concentration-dependent manner. Furthermore, the effect of 3 microM clavulone II was accompanied by the up-regulation of Bax, down-regulation of Mcl-1, and cleavage of Bid. Taken together, it is suggested that low concentrations of clavulone II induce the antiproliferative effect through the down-regulation of cyclin D1 expression and G1 arrest of the cell cycle, while that of high concentration induce the apoptotic cell death via the modulation of members of caspases and Bcl-2 family proteins in HL-60 cells.
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Affiliation(s)
- Yu-Chun Huang
- Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
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47
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Hayward RL, Schornagel QC, Tente R, Macpherson JS, Aird RE, Guichard S, Habtemariam A, Sadler P, Jodrell DI. Investigation of the role of Bax, p21/Waf1 and p53 as determinants of cellular responses in HCT116 colorectal cancer cells exposed to the novel cytotoxic ruthenium(II) organometallic agent, RM175. Cancer Chemother Pharmacol 2005; 55:577-83. [PMID: 15726367 DOI: 10.1007/s00280-004-0932-9] [Citation(s) in RCA: 64] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2004] [Accepted: 11/25/2004] [Indexed: 12/20/2022]
Abstract
Ruthenium(II) organometallic complexes form monofunctional adducts with guanine in DNA in vitro and have a cytotoxic anticancer activity spectrum in preclinical models suggesting lack of cross-resistance with cisplatin. The primary cytotoxic lesion remains to be identified but the downstream mechanism of action is nevertheless of interest. Using isogenic derivatives of the HCT116 colorectal cancer cell line, we investigated the role of p53, p21/WAF1 and Bax in the cellular response to the novel ruthenium(II) organometallic complex RM175, [(eta(6)-C(6)H(5)C(6)H(5))RuCl (H(2)NCH(2)CH(2)NH(2)-N,N')](+) PF(6)(-). Western blotting demonstrated dose-dependent accumulation of p53, Bax and p21/WAF1 within 48 h of the start of RM175 treatment in wild-type HCT116 cells. HCT116 wild-type and Bax-null cells arrested in the G(1) and G(2) phases of the cell cycle. This pattern of cell cycle arrest was not observed in p53-null or in p21/WAF1-null cells. Following RM175 treatment, HCT116 wild-type and p21/WAF1 null cells underwent a dose-dependent induction of apoptosis (Annexin-V and sub-G(1) apoptosis assays). This apoptotic response was not observed in p53-null or Bax-null cells. In short-term sulphorhodamine B assays, the IC(50) for RM175 was 16 microM for p53-null HCT116, and 8 microM for wild-type cells (P<0.05). However, the sensitivity to RM175 in clonogenic assays at 16 days was independent of p53 status. These results identify determinants of the short-term in vitro response to RM175 demonstrating a role for p53 and p21/WAF1 in the growth arrest and for p53 and Bax in the apoptotic response. The mechanism of p53-independent suppression of long-term clonogenicity remains to be determined.
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Affiliation(s)
- R L Hayward
- Cancer Research UK Edinburgh Oncology Unit, University of Edinburgh Cancer Research Centre, Edinburgh, EH4 2XR, UK
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Bhuvaneswari V, Rao KS, Nagini S. Altered expression of anti and proapoptotic proteins during chemoprevention of hamster buccal pouch carcinogenesis by tomato and garlic combination. Clin Chim Acta 2004; 350:65-72. [PMID: 15530461 DOI: 10.1016/j.cccn.2004.07.002] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2004] [Accepted: 07/05/2004] [Indexed: 10/26/2022]
Abstract
BACKGROUND Effective combinations of dietary agents are promising candidates for cancer chemoprevention because of their safety and the fact that they are not perceived as medicine. The present study was designed to investigate the apoptosis-inducing effect of combined administration of tomato and garlic during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. METHODS Hamsters were divided into four groups. The right buccal pouches of animals in group 1 were painted with 0.5% DMBA three times a week. Animals in group 2 painted with DMBA as in group 1, received in addition intragastric administration of a combined dose of tomato and garlic on days alternate to DMBA application. Group 3 animals were given chemopreventive agents alone. Animals in group 4 served as control. All the animals were sacrificed after an experimental period of 14 weeks. DNA fragmentation and the apoptosis-associated proteins-Bcl-2, Bax, Bim, P53 as well as caspases 8 and 3 were used as markers of apoptosis. RESULTS Topical application of DMBA for 14 weeks resulted in well-developed squamous cell carcinomas (SCCs) associated with increased expression of Bcl-2 and decreased expression of Bax, Bim, P53 and caspases 8 and 3. Combined administration of tomato and garlic significantly inhibited the development of HBP carcinomas and induced apoptosis. This was evidenced by downregulation of Bcl-2 and upregulation of Bax, Bim, P53 and caspases 8 and 3. CONCLUSION The induction of apoptosis may be one of the mechanisms through which functional foods such as tomato and garlic exert their anticancer properties.
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Affiliation(s)
- V Bhuvaneswari
- Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar-608 002, Tamil Nadu, India
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Xu P, Rogers SJ, Roossinck MJ. Expression of antiapoptotic genes bcl-xL and ced-9 in tomato enhances tolerance to viral-induced necrosis and abiotic stress. Proc Natl Acad Sci U S A 2004; 101:15805-10. [PMID: 15505199 PMCID: PMC524858 DOI: 10.1073/pnas.0407094101] [Citation(s) in RCA: 84] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2004] [Indexed: 12/28/2022] Open
Abstract
D satellite RNA (satRNA) is a strain of cucumber mosaic virus (CMV) satRNA that induces an epidemic lethal disease in tomato. No natural resistance or tolerance has ever been found. Previously, we demonstrated the involvement of programmed cell death in disease development. Here, transgenic tomato plants expressing animal antiapoptotic genes bcl-xL and ced-9 were generated through agrobacterium-mediated transformation. High expression of bcl-xL or ced-9 affected plant growth and seed development. Inoculation of seedlings with CMV/D satRNA at T(1) and T(2) generations resulted in delayed cell-death symptoms or absence of symptoms. The degree of symptom suppression was correlated with increasing expression levels of the transgenes. Survival rates were compared among inoculated transgenic lines expressing bcl-xL, ced-9, and bcl-xL (G138A), a loss-of-function mutant of bcl-xL. More than 80% of the bcl-xL and ced-9 T(1) transgenic lines showed higher survival rates than the average for bcl-xL (G138A) transgenic lines. Total RNA extracted from surviving plants contained D satRNA, indicating systemic accumulation of D satRNA. Thus, expression of bcl-xL and ced-9 improved tolerance to, rather than resistance to, CMV/D satRNA infection. In addition, expression of bcl-xL and ced-9 specifically abrogated the formation of necrotic lesions, but not other symptoms, in tomato leaves during chilling at 4 degrees C. At 7 degrees C, temperature-induced leaf senescence was dramatically delayed in bcl-xL and ced-9 transgenic plants, and high levels of anthocyanins accumulated, possibly limiting oxidative stress. Hence, expression of these animal antiapoptotic genes improved plant survival under abiotic or biotic stress.
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Affiliation(s)
- Ping Xu
- Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA
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Li D, Ueta E, Kimura T, Yamamoto T, Osaki T. Reactive oxygen species (ROS) control the expression of Bcl-2 family proteins by regulating their phosphorylation and ubiquitination. Cancer Sci 2004; 95:644-50. [PMID: 15298726 PMCID: PMC11158795 DOI: 10.1111/j.1349-7006.2004.tb03323.x] [Citation(s) in RCA: 184] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2004] [Revised: 05/25/2004] [Accepted: 06/01/2004] [Indexed: 12/31/2022] Open
Abstract
We examined the influence of ROS on the phosphorylation and complex formation of Bcl-2 family proteins in Mn-superoxide dismutase (SOD) antisense-transfected squamous cell carcinoma cells, OSC-4 cells. The increase of intracellular ROS level induced by cis-diamminedichloroplatinum (CDDP) and gamma-ray treatment was greater in antisense-transfected cells than in control vector-transfected cells, and apoptosis was more extensively induced in the former. Antisense-transfected cells expressed high levels of Bax and Bak, but low levels of Bcl-2 and Bcl-XL when treated with CDDP, peplomycin, 5-fluorouracil or gamma-rays. After treatment with these agents, the phosphorylation of protein kinase A, Bcl-2 (Thr56) and Bad (Ser155) was increased, especially in antioxidant (N-acetylcysteine and pyrrolidine dithiocarbamate)-pretreated control cells, but the phosphorylation levels were very low in the antisense-transfected cells. Bcl-2 ubiquitination was increased, but ubiquitination of Bad and Bax was decreased in the antisense-transfected cells, although their ubiquitination was increased by the antioxidants. These results reveal that ROS induce apoptosis by regulating the phosphorylation and ubiquitination of Bcl-2 family proteins, resulting in increased proapoptotic protein levels and decreased antiapoptotic protein expression.
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Affiliation(s)
- Dechao Li
- Department of Oral Oncology, Kochi Medical School, Kohasu, Nankoku City, 783-8505, Japan
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