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Kim Y, Choi J, Kim EH, Park W, Jang H, Jang Y, Chi S, Kweon D, Lee K, Kim SH, Yang Y. Design of PD-L1-Targeted Lipid Nanoparticles to Turn on PTEN for Efficient Cancer Therapy. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2309917. [PMID: 38520717 PMCID: PMC11165541 DOI: 10.1002/advs.202309917] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/18/2023] [Revised: 03/15/2024] [Indexed: 03/25/2024]
Abstract
Lipid nanoparticles (LNPs) exhibit remarkable mRNA delivery efficiency, yet their majority accumulate in the liver or spleen after injection. Tissue-specific mRNA delivery can be achieved through modulating LNP properties, such as tuning PEGylation or varying lipid components systematically. In this paper, a streamlined method is used for incorporating tumor-targeting peptides into the LNPs; the programmed death ligand 1 (PD-L1) binding peptides are conjugated to PEGylated lipids via a copper-free click reaction, and directly incorporated into the LNP composition (Pep LNPs). Notably, Pep LNPs display robust interaction with PD-L1 proteins, which leads to the uptake of LNPs into PD-L1 overexpressing cancer cells both in vitro and in vivo. To evaluate anticancer immunotherapy mediated by restoring tumor suppressor, mRNA encoding phosphatase and tensin homolog (PTEN) is delivered via Pep LNPs to PTEN-deficient triple-negative breast cancers (TNBCs). Pep LNPs loaded with PTEN mRNA specifically promotes autophagy-mediated immunogenic cell death in 4T1 tumors, resulting in effective anticancer immune responses. This study highlights the potential of tumor-targeted LNPs for mRNA-based cancer therapy.
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Affiliation(s)
- Yelee Kim
- Biomedical Research DivisionKorea Institute of Science and Technology (KIST)Seoul02792Republic of Korea
- Department of Life SciencesKorea UniversitySeoul02841Republic of Korea
| | - Jiwoong Choi
- Biomedical Research DivisionKorea Institute of Science and Technology (KIST)Seoul02792Republic of Korea
| | - Eun Hye Kim
- Biomedical Research DivisionKorea Institute of Science and Technology (KIST)Seoul02792Republic of Korea
- Department of Life SciencesKorea UniversitySeoul02841Republic of Korea
| | - Wonbeom Park
- Department of Integrative BiotechnologySungkyunkwan UniversitySuwon16419Republic of Korea
| | - Hochung Jang
- Biomedical Research DivisionKorea Institute of Science and Technology (KIST)Seoul02792Republic of Korea
- Division of Bio‐Medical Science and TechnologyKIST SchoolKorea University of Science and TechnologySeoul02792Republic of Korea
| | - Yeongji Jang
- Biomedical Research DivisionKorea Institute of Science and Technology (KIST)Seoul02792Republic of Korea
- Department of Life SciencesKorea UniversitySeoul02841Republic of Korea
| | - Sung‐Gil Chi
- Department of Life SciencesKorea UniversitySeoul02841Republic of Korea
| | - Dae‐Hyuk Kweon
- Department of Integrative BiotechnologySungkyunkwan UniversitySuwon16419Republic of Korea
| | - Kyuri Lee
- College of Pharmacy and Research Institute of Pharmaceutical SciencesGyeongsang National UniversityJinju52828Republic of Korea
| | - Sun Hwa Kim
- Biomedical Research DivisionKorea Institute of Science and Technology (KIST)Seoul02792Republic of Korea
- KU‐KIST Graduate School of Converging Science and TechnologyKorea UniversitySeoul02841Republic of Korea
| | - Yoosoo Yang
- Biomedical Research DivisionKorea Institute of Science and Technology (KIST)Seoul02792Republic of Korea
- Division of Bio‐Medical Science and TechnologyKIST SchoolKorea University of Science and TechnologySeoul02792Republic of Korea
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Carlsen L, El-Deiry WS. Differential p53-Mediated Cellular Responses to DNA-Damaging Therapeutic Agents. Int J Mol Sci 2021; 22:ijms222111828. [PMID: 34769259 PMCID: PMC8584119 DOI: 10.3390/ijms222111828] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2021] [Revised: 10/29/2021] [Accepted: 10/29/2021] [Indexed: 01/01/2023] Open
Abstract
The gene TP53, which encodes the tumor suppressor protein p53, is mutated in about 50% of cancers. In response to cell stressors like DNA damage and after treatment with DNA-damaging therapeutic agents, p53 acts as a transcription factor to activate subsets of target genes which carry out cell fates such as apoptosis, cell cycle arrest, and DNA repair. Target gene selection by p53 is controlled by a complex regulatory network whose response varies across contexts including treatment type, cell type, and tissue type. The molecular basis of target selection across these contexts is not well understood. Knowledge gained from examining p53 regulatory network profiles across different DNA-damaging agents in different cell types and tissue types may inform logical ways to optimally manipulate the network to encourage p53-mediated tumor suppression and anti-tumor immunity in cancer patients. This may be achieved with combination therapies or with p53-reactivating targeted therapies. Here, we review the basics of the p53 regulatory network in the context of differential responses to DNA-damaging agents; discuss recent efforts to characterize differential p53 responses across treatment types, cell types, and tissue types; and examine the relevance of evaluating these responses in the tumor microenvironment. Finally, we address open questions including the potential relevance of alternative p53 transcriptional functions, p53 transcription-independent functions, and p53-independent functions in the response to DNA-damaging therapeutics.
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Affiliation(s)
- Lindsey Carlsen
- Laboratory of Translational Oncology and Experimental Cancer Therapeutics, The Warren Alpert Medical School, Brown University, Providence, RI 02903, USA;
- The Joint Program in Cancer Biology, Brown University and the Lifespan Health System, Providence, RI 02903, USA
- Department of Pathology and Laboratory Medicine, The Warren Alpert Medical School, Brown University, Providence, RI 02903, USA
- Pathobiology Graduate Program, The Warren Alpert Medical School, Brown University, Providence, RI 02903, USA
- Cancer Center, The Warren Alpert Medical School, Brown University, Providence, RI 02903, USA
| | - Wafik S. El-Deiry
- Laboratory of Translational Oncology and Experimental Cancer Therapeutics, The Warren Alpert Medical School, Brown University, Providence, RI 02903, USA;
- The Joint Program in Cancer Biology, Brown University and the Lifespan Health System, Providence, RI 02903, USA
- Department of Pathology and Laboratory Medicine, The Warren Alpert Medical School, Brown University, Providence, RI 02903, USA
- Pathobiology Graduate Program, The Warren Alpert Medical School, Brown University, Providence, RI 02903, USA
- Cancer Center, The Warren Alpert Medical School, Brown University, Providence, RI 02903, USA
- Department of Medicine, Hematology-Oncology Division, Rhode Island Hospital, Brown University, Providence, RI 02903, USA
- Correspondence:
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Mo J, Lin M, He B, Tan K, Jin C, Jiang H, Pan X, Lin W. Recombinant human adenovirus-p53 improves the outcome of mid-late stage pancreatic cancer via arterial infusion. Oncol Lett 2017; 14:6829-6832. [PMID: 29181104 DOI: 10.3892/ol.2017.7058] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2016] [Accepted: 06/21/2017] [Indexed: 11/05/2022] Open
Abstract
The present study aimed to investigate the therapeutic efficacy and clinical value of recombinant human adenovirus-p53 (rAd-p53) perfusion via the pancreatic artery for the treatment of mid-late stage pancreatic cancer. rAd-p53 (2×1012 virus particles) in 6 ml normal saline was pushed (intravenous bolus) into the gastroduodenal and superior pancreaticoduodenal arteries via interventional superselection, with the catheter retained for subsequent drug administration at a 3-day interval for 4 cycles. Tumor changes in all patients were observed to evaluate tumor response by computed tomography (CT) at 2, 8 and 16 weeks post-treatment. The following improvements were noted in the 23-patient cohort: A total of 73.9% (17/23) of patients demonstrated significant tumor shrinkage (>20%); the symptoms of abdominal and back pain were relieved in 15 patients; the survival time was >12 months in 1 patient and >6 months in 14 patients; the patient's general condition, including appetite, was improved in 13 patients; body weight was increased in 9 patients; jaundice was attenuated in 12 patients; and ascites subsided in 10 patients. However, the therapeutic outcome was poor in 2 patients whose tumors size did not show significant change after treatment as detected by CT. These 2 patients succumbed within 6 months. In conclusion, rAd-p53 perfusion via the pancreatic artery is a safe and minimally invasive option for the treatment of mid-late stage pancreatic cancer.
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Affiliation(s)
- Jinggang Mo
- Department of Hepatobiliary Surgery, First Clinical College, Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China.,Department of Hepatobiliary Surgery, Taizhou Central Hospital, Taizhou, Zhejiang 318000, P.R. China
| | - Meihua Lin
- Research Center of Clinical Pharmacy, State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China
| | - Bin He
- Department of Gastrointestinal Surgery, Taizhou Central Hospital, Taizhou, Zhejiang 325000, P.R. China
| | - Kai Tan
- Department of Radiology, Taizhou Central Hospital, Taizhou, Zhejiang 325000, P.R. China
| | - Chong Jin
- Department of Hepatobiliary Surgery, Taizhou Central Hospital, Taizhou, Zhejiang 318000, P.R. China
| | - Hao Jiang
- Department of Hepatobiliary Surgery, Taizhou Central Hospital, Taizhou, Zhejiang 318000, P.R. China
| | - Xuefeng Pan
- Department of Hepatobiliary Surgery, Taizhou Central Hospital, Taizhou, Zhejiang 318000, P.R. China
| | - Weidong Lin
- Department of Hepatobiliary Surgery, Taizhou Central Hospital, Taizhou, Zhejiang 318000, P.R. China
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Chen X. Prediction of optimal gene functions for osteosarcoma using network-based- guilt by association method based on gene oncology and microarray profile. J Bone Oncol 2017; 7:18-22. [PMID: 28443230 PMCID: PMC5396855 DOI: 10.1016/j.jbo.2017.04.003] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2017] [Revised: 04/05/2017] [Accepted: 04/06/2017] [Indexed: 01/21/2023] Open
Abstract
In the current study, we planned to predict the optimal gene functions for osteosarcoma (OS) by integrating network-based method with guilt by association (GBA) principle (called as network-based gene function inference approach) based on gene oncology (GO) data and gene expression profile. To begin with, differentially expressed genes (DEGs) were extracted using linear models for microarray data (LIMMA) package. Then, construction of differential co-expression network (DCN) relying on DEGs was implemented, and sub-DCN was identified using Spearman correlation coefficient (SCC). Subsequently, GO annotations for OS were collected according to known confirmed database and DEGs. Ultimately, gene functions were predicted by means of GBA principle based on the area under the curve (AUC) for GO terms, and we determined GO terms with AUC >0.7 as the optimal gene functions for OS. Totally, 123 DEGs and 137 GO terms were obtained for further analysis. A DCN was constructed, which included 123 DEGs and 7503 interactions. A total of 105 GO terms were identified when the threshold was set as AUC >0.5, which had a good classification performance. Among these 105 GO terms, 2 functions had the AUC >0.7 and were determined as the optimal gene functions including angiogenesis (AUC =0.767) and regulation of immune system process (AUC =0.710). These gene functions appear to have potential for early detection and clinical treatment of OS in the future.
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5
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Wang Q. CpG methylation patterns are associated with gene expression variation in osteosarcoma. Mol Med Rep 2017; 16:901-907. [DOI: 10.3892/mmr.2017.6635] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2015] [Accepted: 01/27/2017] [Indexed: 11/05/2022] Open
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Huang Z, Fan G, Wang D. Downregulation of calbindin 1, a calcium-binding protein, reduces the proliferation of osteosarcoma cells. Oncol Lett 2017; 13:3727-3733. [PMID: 28529588 PMCID: PMC5431599 DOI: 10.3892/ol.2017.5931] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2015] [Accepted: 01/13/2017] [Indexed: 12/25/2022] Open
Abstract
Osteosarcoma is the most common type of primary malignant bone tumor and has a high propensity to metastasize to the lungs and bones. Calbindin 1 (CALB1) is a constituent Ca2+ binding protein, which can prevent apoptotic death in several cell types induced through various pro-apoptotic signaling pathways. To investigate whether CALB1 is implicated in the tumor growth of human osteosarcoma, two different short hairpin RNAs (shRNAs) against CALB1 were used for CALB1-knockdown in osteosarcoma U2OS cells. The U2OS cells were divided into three groups: Two groups with CALB1 knockdown (CALB1-shRNA 1 and CALB1-shRNA 2) and one control group (Con-shRNA). Reverse transcription-quantitative polymerase chain reaction and western blot analysis confirmed that the CALB1-shRNA 1- and 2-infected cells exhibited significantly lower levels of CALB1 gene and protein expression compared with the Con-shRNA group. The proliferation and colony formation abilities were significantly inhibited in CALB1-deficient U2OS cells compared with the control, as measured using an MTT assay and crystal violet staining. Flow cytometry revealed that the number of CALB1-shRNA 2-injected cells was increased in the G0/G1 and G2/M phases, but decreased in the S phase, compared with the control group. The assessment of apoptosis and necrosis using Annexin V/7-aminoactinomycin D demonstrated that there was a significantly higher percentage of necrotic, early apoptotic, and late apoptotic cells, but a significantly lower percentage of viable cells in U2OS cells with CALB1-knockdown compared with the control group. In conclusion, CALB1 contributes to protecting osteosarcoma cells from apoptosis and provides a potential novel target for gene therapy to treat patients with osteosarcoma.
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Affiliation(s)
- Zhengxiang Huang
- Department of Orthopedic Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, P.R. China
| | - Guojun Fan
- Department of Orthopedic Surgery, The First People's Hospital of Urumqi, Urumqi, Xinjiang 830000, P.R. China
| | - Dongliang Wang
- Department of Orthopedic Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, P.R. China
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McErlean EM, McCrudden CM, McCarthy HO. Delivery of nucleic acids for cancer gene therapy: overcoming extra- and intra-cellular barriers. Ther Deliv 2016; 7:619-37. [PMID: 27582234 DOI: 10.4155/tde-2016-0049] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
The therapeutic potential of cancer gene therapy has been limited by the difficulty of delivering genetic material to target sites. Various biological and molecular barriers exist which need to be overcome before effective nonviral delivery systems can be applied successfully in oncology. Herein, various barriers are described and strategies to circumvent such obstacles are discussed, considering both the extracellular and intracellular setting. Development of multifunctional delivery systems holds much promise for the progression of gene delivery, and a growing body of evidence supports this approach involving rational design of vectors, with a unique molecular architecture. In addition, the potential application of composite gene delivery platforms is highlighted which may provide an alternative delivery strategy to traditional systemic administration.
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8
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Liu Y, Yang P, Chen N, Lin S, Liu M. Effects of recombinant human adenovirus-p53 on the regression of hepatic fibrosis. Int J Mol Med 2016; 38:1093-100. [PMID: 27572658 PMCID: PMC5029955 DOI: 10.3892/ijmm.2016.2716] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2015] [Accepted: 08/10/2016] [Indexed: 01/28/2023] Open
Abstract
Hepatic fibrosis is a scarring process that may progress to hepatic cirrhosis and even hepatic carcinoma if left untreated. Hepatic stellate cells (HSCs) play essential roles in the development of hepatic fibrosis. The tumor suppressor protein p53 is a transcription factor that is involved in cell proliferation, cell cycle regulation, apoptosis and DNA repair. Recombinant human adenovirus-p53 (Ad-p53) has been demonstrated to act as a promising antitumor gene therapy in various types of cancer. However, there is limited infomration regarding the therapeutic effect of Ad-p53 on the regression of hepatic fibrosis. In order to examine the underlying molecular mechanism responsible for the effects of Ad-p53 on HSCs, a rat model of hepatic fibrosis was established and HSC-T6 cells were cultured under different conditions. The expression of p53, transforming growth factor (TGF-β1) and α-smooth muscle actin (α-SMA), which is a marker of activated HSCs, was detected by immunohistochemical assays and RT-qPCR. In vitro, five different concentrations (1×106, 5×106, 1×107, 2×107 and 5×107 PFU/ml) of Ad-p53 were selected for use in the MTT assay to analyze the proliferation of HSCs at 0, 24, 48 and 72 h. Flow cytometric analysis was applied to determine the effect of three different concentrations of Ad-p53 (5×106, 1×107 and 2×107 PFU/ml) on the cell cycle and the apoptosis of HSC-T6 cells at 24 and 48 h. The results of immunohistochemical studies and RT-qPCR showed that Ad-p53 upregulated the expression of p53, and downregulated the expression of TGF-β1 and α-SMA. The MTT assay revealed that when treated with various doses of Ad-p53, the proliferation of HSCs was inhibited within a certain range of concentrations and time periods. Analysis of flow cytometric data showed that Ad-p53 arrested the cell cycle in G1 phase and significantly induced apoptosis. Taken together, these findings suggest that Ad-p53 promotes apoptosis and inhibits the proliferation of HSCs in a time- and dose-dependent manner by modulating the expression of p53, TGF-β1 and α-SMA.
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Affiliation(s)
- Yehong Liu
- Department of Infectious Diseases, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China
| | - Puye Yang
- Department of Infectious Diseases, Xi'an North Hospital of Xi'an Medical University, Xi'an, Shaanxi 710043, P.R. China
| | - Na Chen
- Department of Infectious Diseases, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China
| | - Shumei Lin
- Department of Infectious Diseases, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China
| | - Min Liu
- Department of Infectious Diseases, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China
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Tazawa H, Kagawa S, Fujiwara T. Advances in adenovirus-mediated p53 cancer gene therapy. Expert Opin Biol Ther 2014; 13:1569-83. [PMID: 24107178 DOI: 10.1517/14712598.2013.845662] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
INTRODUCTION The tumor suppressor p53 gene regulates diverse cellular processes, such as cell-cycle arrest, senescence, apoptosis and autophagy, and it is frequently inactivated by genetic alterations in ∼ 50% of all types of human cancers. To restore wild-type p53 function in p53-inactivated tumors, adenovirus-mediated p53 gene therapy has been developed as a promising antitumor strategy in preclinical experiments and clinical studies. AREAS COVERED This review focuses on the clinical relevance of replication-deficient adenovirus vectors that carry the wild-type p53 gene (Ad-p53; Advexin, Gendicine and SCH-58500) in clinical studies of patients with various cancers and the future perspectives regarding conditionally replicating adenovirus vectors expressing the wild-type p53 gene (CRAd-p53; AdDelta24-p53, SG600-p53, OBP-702) in preclinical experiments. Moreover, the recent advances in our understanding of the molecular basis for the p53-mediated tumor suppression network induced by Ad-p53 and CRAd-p53 vectors and the combination therapies for promoting the therapeutic potential of adenovirus-mediated p53 gene therapy are discussed. EXPERT OPINION Exploration of the molecular mechanism underlying the p53-mediated tumor suppression network and the effective strategy for enhancing the p53-mediated cell death signaling pathway would provide novel insights into the improvement of clinical outcome in p53-based cancer gene therapy.
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Affiliation(s)
- Hiroshi Tazawa
- Okayama University Hospital, Center for Innovative Clinical Medicine , Okayama 700-8558 , Japan
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10
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Hasei J, Sasaki T, Tazawa H, Osaki S, Yamakawa Y, Kunisada T, Yoshida A, Hashimoto Y, Onishi T, Uno F, Kagawa S, Urata Y, Ozaki T, Fujiwara T. Dual programmed cell death pathways induced by p53 transactivation overcome resistance to oncolytic adenovirus in human osteosarcoma cells. Mol Cancer Ther 2013; 12:314-25. [PMID: 23315976 DOI: 10.1158/1535-7163.mct-12-0869] [Citation(s) in RCA: 51] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Tumor suppressor p53 is a multifunctional transcription factor that regulates diverse cell fates, including apoptosis and autophagy in tumor biology. p53 overexpression enhances the antitumor activity of oncolytic adenoviruses; however, the molecular mechanism of this occurrence remains unclear. We previously developed a tumor-specific replication-competent oncolytic adenovirus, OBP-301, that kills human osteosarcoma cells, but some human osteosarcoma cells were OBP-301-resistant. In this study, we investigated the antitumor activity of a p53-expressing oncolytic adenovirus, OBP-702, and the molecular mechanism of the p53-mediated cell death pathway in OBP-301-resistant human osteosarcoma cells. The cytopathic activity of OBP-702 was examined in OBP-301-sensitive (U2OS and HOS) and OBP-301-resistant (SaOS-2 and MNNG/HOS) human osteosarcoma cells. The molecular mechanism in the OBP-702-mediated induction of two cell death pathways, apoptosis and autophagy, was investigated in OBP-301-resistant osteosarcoma cells. The antitumor effect of OBP-702 was further assessed using an orthotopic OBP-301-resistant MNNG/HOS osteosarcoma xenograft tumor model. OBP-702 suppressed the viability of OBP-301-sensitive and -resistant osteosarcoma cells more efficiently than OBP-301 or a replication-deficient p53-expressing adenovirus (Ad-p53). OBP-702 induced more profound apoptosis and autophagy when compared with OBP-301 or Ad-p53. E1A-mediated miR-93/106b upregulation induced p21 suppression, leading to p53-mediated apoptosis and autophagy in OBP-702-infected cells. p53 overexpression enhanced adenovirus-mediated autophagy through activation of damage-regulated autophagy modulator (DRAM). Moreover, OBP-702 suppressed tumor growth in an orthotopic OBP-301-resistant MNNG/HOS xenograft tumor model. These results suggest that OBP-702-mediated p53 transactivation is a promising antitumor strategy to induce dual apoptotic and autophagic cell death pathways via regulation of miRNA and DRAM in human osteosarcoma cells.
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Affiliation(s)
- Joe Hasei
- Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
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Spina A, Sorvillo L, Di Maiolo F, Esposito A, D'Auria R, Di Gesto D, Chiosi E, Naviglio S. Inorganic phosphate enhances sensitivity of human osteosarcoma U2OS cells to doxorubicin via a p53-dependent pathway. J Cell Physiol 2012; 228:198-206. [PMID: 22674530 DOI: 10.1002/jcp.24124] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Osteosarcoma is the most common malignant primary bone tumor in children and adolescents. The clinical outcome for osteosarcoma remains discouraging despite aggressive surgery and intensive radiotherapy and chemotherapy regimens. Thus, novel therapeutic approaches are needed. Previously, we have shown that inorganic phosphate (Pi) inhibits proliferation and aggressiveness of human osteosarcoma U2OS cells identifying adenylate cyclase, beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected in response to Pi. In this study, we investigated whether Pi could affect chemosensitivity of osteosarcoma cells and the underlying molecular mechanisms. Here, we report that Pi inhibits proliferation of p53-wild type U2OS cells (and not of p53-null Saos and p53-mutant MG63 cells) by slowing-down cell cycle progression, without apoptosis occurrence. Interestingly, we found that Pi strongly enhances doxorubicin-induced cytotoxicity in U2OS, and not in Saos and MG63 cells, by apoptosis induction, as revealed by a marked increase of sub-G1 population, Bcl-2 downregulation, caspase-3 activation, and PARP cleavage. Remarkably, Pi/doxorubicin combination-induced cytotoxicity was accompanied by an increase of p53 protein levels and of p53 target genes mdm2, p21 and Bax, and was significantly reduced by the p53 inhibitor pifithrine-alpha. Moreover, the doxorubicin-induced cytotoxicity was associated with ERK1/2 pathway inhibition in response to Pi. Altogether, our data enforce the evidence of Pi as a novel signaling molecule capable of inhibiting ERK pathway and inducing sensitization to doxorubicin of osteosarcoma cells by p53-dependent apoptosis, implying that targeting Pi levels might represent a rational strategy for improving osteosarcoma therapy.
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Affiliation(s)
- Annamaria Spina
- Department of Biochemistry and Biophysics, Medical School, Second University of Naples, Naples, Italy
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Hao H, Chen C, Rao XM, Gomez-Gutierrez JG, Zhou HS, McMasters KM. E2F-1- and E2Ftr-mediated apoptosis: the role of DREAM and HRK. J Cell Mol Med 2012; 16:605-15. [PMID: 21564512 PMCID: PMC3822935 DOI: 10.1111/j.1582-4934.2011.01338.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
E2F-1-deleted mutant, ‘truncated E2F’ (E2Ftr, E2F-1[1–375]), lacking the carboxy-terminal transactivation domain, was shown to be more potent at inducing cancer cell apoptosis than wild-type E2F-1 (wtE2F-1; full-length E2F-1). Mechanisms by which wtE2F-1 and E2Ftr induce apoptosis, however, are not fully elucidated. Our study demonstrates molecular effects of pro-apoptotic BH3-only Bcl-2 family member Harakiri (Hrk) in wtE2F-1- and E2Ftr-induced melanoma cell apoptosis. We found that Hrk mRNA and Harakiri (HRK) protein expression was highly up-regulated in melanoma cells in response to wtE2F-1 and E2Ftr overexpression. HRK up-regulation did not require the E2F-1 transactivation domain. In addition, Hrk gene up-regulation and HRK protein expression did not require p53 in cancer cells. Hrk knockdown by Hrk siRNA was associated with significantly reduced wtE2F-1- and E2Ftr-induced apoptosis. We also found that an upstream factor, ‘downstream regulatory element antagonist modulator’ (DREAM), may be involved in HRK-mediated apoptosis in response to wtE2F-1 and E2Ftr overexpression. DREAM expression levels increased following wtE2F-1 and E2Ftr overexpression. Western blotting detected increased DREAM primarily in dimeric form. The homodimerization of DREAM resulting from wtE2F-1 and E2Ftr overexpression may contribute to the decreased binding activity of DREAM to the 3′-untranslated region of the Hrk gene as shown by electromobility shift assay. Results showed wtE2F-1- and E2Ftr-induced apoptosis is partially mediated by HRK. HRK function is regulated in response to DREAM. Our findings contribute to understanding the mechanisms that regulate wtE2F-1- and E2Ftr-induced apoptosis and provide insights into the further evaluation of how E2Ftr-induced apoptosis may be used for therapeutic gain.
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Affiliation(s)
- Hongying Hao
- Department of Surgery, University of Louisville School of Medicine, and J. Graham Brown Cancer Center, Louisville, KY, USA
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Broadhead ML, Clark JCM, Choong PFM, Dass CR. Making gene therapy for osteosarcoma a reality. Expert Rev Anticancer Ther 2010; 10:477-80. [PMID: 20397911 DOI: 10.1586/era.10.18] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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Hu X, Yu AX, Qi BW, Fu T, Wu G, Zhou M, Luo J, Xu JH. The expression and significance of IDH1 and p53 in osteosarcoma. JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2010; 29:43. [PMID: 20459648 PMCID: PMC2873426 DOI: 10.1186/1756-9966-29-43] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/29/2010] [Accepted: 05/07/2010] [Indexed: 02/01/2023]
Abstract
Background To detect the expression of isocitrate dehydrogenase 1 (IDH1) and transformation-related protein 53 (p53) in osteosarcoma and analyze the correlation between them and the clinico-pathological features. Methods The expressions of IDH1 and p53 were detected in human osteosarcoma cell lines (MG-63 and U2OS) by immunocytochemistry, Real-time PCR and Western Blotting. The expressions of IDH1 and p53 in formalin-fixed paraffin-embedded tissue sections from 44 osteosarcoma patients were determined by immunohistochemistry, and the correlation between them and clinicopagthological features were analyzed. None of these patients received chemotherapy prior to surgery. Results IDH1 is detected in osteosarcoma cell lines and biopsies. IDH1 expresses higher in U2OS cells with wild type p53 than in MG-63 cells with mutation p53. IDH1 correlates with histological Rosen grade and metastasis negatively. P53 correlates with histological Rosen grade, metastasis and overall survival in clinical osteosarcoma biopsies. Osteosarcoma patients with High IDH1 expression have a very high p53 expression. Conclusion IDH1 may correlate with p53 and be a candidate biomarker for osteosarcoma correlate with histological Rosen grade and metastasis.
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Affiliation(s)
- Xiang Hu
- Department of Orthopedics, Zhongnan Hospital of Wuhan University, No 169 Donghu Road, Wuchang District, 430071, Wuhan, China
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Ottaviano L, Schaefer KL, Gajewski M, Huckenbeck W, Baldus S, Rogel U, Mackintosh C, de Alava E, Myklebost O, Kresse SH, Meza-Zepeda LA, Serra M, Cleton-Jansen AM, Hogendoorn PCW, Buerger H, Aigner T, Gabbert HE, Poremba C. Molecular characterization of commonly used cell lines for bone tumor research: a trans-European EuroBoNet effort. Genes Chromosomes Cancer 2010; 49:40-51. [PMID: 19787792 DOI: 10.1002/gcc.20717] [Citation(s) in RCA: 130] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
Usage of cancer cell lines has repeatedly generated conflicting results provoked by differences among subclones or contamination with mycoplasm or other immortal mammalian cells. To overcome these limitations, we decided within the EuroBoNeT consortium to characterize a common set of cell lines including osteosarcomas (OS), Ewing sarcomas (ES), and chondrosarcomas (CS). DNA fingerprinting was used to guarantee the identity of all of the cell lines and to distinguish subclones of osteosarcoma cell line HOS. Screening for homozygous loss of 38 tumor suppressor genes by MLPA revealed deletion of CDKN2A as the most common event (15/36), strictly associated with absence of the CDKN2A (p16) protein. Ten cell lines showed missense mutations of the TP53 gene while another set of nine cell lines showed mutations resulting in truncation of the TP53 protein. Cells harboring missense mutations expressed high levels of nuclear TP53, while cell lines with nonsense mutations showed weak/absent staining for TP53. TP53(wt) cell lines usually expressed the protein in 2-10% of the cells. However, seven TP53(wt) osteosarcomas were negative for both mRNA and protein expression. Our analyses shed light on the correlation between immunohistochemical and genetic data for CDKN2A and TP53, and confirm the importance of these signaling pathways. The characterization of a substantial number of cell lines represents an important step to supply research groups with proven models for further advanced studies on tumor biology and may help to make results from different laboratories more comparable.
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Affiliation(s)
- Laura Ottaviano
- Institute of Pathology, University Medical Center Duesseldorf, Duesseldorf, Germany
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16
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Schotanus BA, van den Ingh TSGAM, Penning LC, Rothuizen J, Roskams TA, Spee B. Cross-species immunohistochemical investigation of the activation of the liver progenitor cell niche in different types of liver disease. Liver Int 2009; 29:1241-52. [PMID: 19490419 DOI: 10.1111/j.1478-3231.2009.02024.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
BACKGROUND When hepatocyte replication during liver disease is insufficient for regeneration, liver progenitor cells (LPCs) are activated. The cells and stroma in the immediate environment of LPCs, together termed the LPC niche, are thought to play an important role in this activation. Among these cells are the hepatic stellate cells (HSCs)/myofibroblasts (MFs). AIMS/METHODS We assessed the activation of HSC/MFs and LPCs in relation to the histological location and extent of liver disease in immunohistochemically (double) stained serial sections. Markers of HSC/MFs [alpha-smooth muscle actin, glial fibrillary acidic protein (GFAP), neurotrophin 3 and neural-cell adhesion molecule], markers of LPCs (keratin 7 and keratin 19) and a proliferation marker (Ki67) were used. A very relevant spontaneous model to evaluate LPC niche activation in a translational approach seems to be the dog. Therefore, both human and canine liver diseases with different degree of fibrosis and disease activity were included. RESULTS In human and canine liver disease, type and extent of LPC niche activation depended on type and severity of disease (P<0.05) and corresponded to the main location of disease. Activated HSCs surrounded the activated LPCs. In chronic hepatitis and non-alcoholic steatohepatitis lobular-type HSCs were activated, while during biliary disease portal/septal MFs were mainly activated. In canine liver, GFAP further presented as an early marker of HSC activation. Activation of the LPCs correlated with disease location and severity (P<0.01), and was inversely related to hepatocyte proliferation, as was previously shown in man. CONCLUSION A shared involvement of HSC/MFs, LPCs and disease severity during hepatic disease processes is shown, which is highly similar in man and dog.
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Affiliation(s)
- Baukje A Schotanus
- Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.
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Zuffa E, Mancini M, Brusa G, Pagnotta E, Hattinger CM, Serra M, Remondini D, Castellani G, Corrado P, Barbieri E, Santucci MA. P53 oncosuppressor influences selection of genomic imbalances in response to ionizing radiations in human osteosarcoma cell line SAOS-2. Int J Radiat Biol 2009; 84:591-601. [DOI: 10.1080/09553000802195349] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
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Valerie K, Graves PR. Strategies of Gene Transfer and Silencing, and Technical Considerations. THE IMPACT OF TUMOR BIOLOGY ON CANCER TREATMENT AND MULTIDISCIPLINARY STRATEGIES 2009. [PMCID: PMC7120147 DOI: 10.1007/978-3-540-74386-6_18] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
Cancer gene therapy is a relatively new modality that might ultimately revolutionize oncology. The basic principle is to alter the tumor genetically to enhance more traditional chemo- and radiation therapy schema. The last decade has seen tremendous progress and development of new technologies in the areas of vector delivery, tumor targeting, and numerous clever ways to increase tumor killing, including early attempts to modulate tumor gene expression by RNA interference. In recent years, attempts to image affected cells have also been part of these efforts. Many studies have proceeded to the preclinical stage and a fair number to early clinical testing with some showing encouraging results. However, real impact on patient survival remains to be seen. One major problem still to be overcome is the quantitative delivery of the vector into the tumor mass. The next decade is expected to resolve many of these technical issues and improve the treatment of patients. This chapter will discuss new technologies and provide a brief overview of the field.
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Shen C, Zhou Y, Zhan J, Reske SN, Buck AK. Chromosome instability and tumor lethality suppression in carcinogenesis. J Cell Biochem 2008; 105:1327-41. [DOI: 10.1002/jcb.21937] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
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Hori M, Suzuki K, Udono MU, Yamauchi M, Mine M, Watanabe M, Kondo S, Hozumi Y. Establishment of ponasterone A-inducible the wild-type p53 protein-expressing clones from HSC-1 cells, cell growth suppression by p53 expression and the suppression mechanism. Arch Dermatol Res 2008; 301:631-46. [PMID: 19009304 DOI: 10.1007/s00403-008-0915-5] [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: 04/24/2008] [Revised: 10/09/2008] [Accepted: 10/20/2008] [Indexed: 01/21/2023]
Abstract
Gene therapy for a variety of human cancers containing the mutant p53 (mt-p53) gene has been performed by direct injection of a retroviral or adenoviral vector containing the wild-type p53 (wt-p53) gene. Because many individuals with skin squamous cell carcinoma (SCC) have been shown to carry the p53 gene mutation, these patients are candidates for p53 gene therapy. For this reason, we established ponasterone A-inducible the wild-type p53 (wt-p53) protein-expressing clones by transfecting a ponasterone-inducible vector containing the wt-p53 gene into HSC-1 cells, which harbor the mutated p53 (m/w) at codon 173 (GTG --> TTG in one allele). Upon the induction of the wt-p53 protein, severe growth suppression was observed. Based on the results of the expression patterns of the p21, p16, RB, BAX and Bcl-2 proteins, as well as on the results of senescence-associated beta-galactosidase staining, the suppression was caused by senescence-like growth arrest of the cells. Although it is generally accepted that the suppression of tumor cell growth is caused by p53-induced apoptosis, permanent G1 arrest induced by p53 is also an important part of the growth-suppression mechanism in p53 gene therapy. The present results should expand the possibilities for p53 gene therapy for human skin SCCs containing the mutant p53 gene.
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Affiliation(s)
- Makoto Hori
- Hori Dermatology Clinic, Nagasaki 852-8134, Japan.
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21
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Kim E, Giese A, Deppert W. Wild-type p53 in cancer cells: when a guardian turns into a blackguard. Biochem Pharmacol 2008; 77:11-20. [PMID: 18812169 DOI: 10.1016/j.bcp.2008.08.030] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2008] [Revised: 08/25/2008] [Accepted: 08/27/2008] [Indexed: 10/21/2022]
Abstract
The tumor suppressor p53 controls a broad range of cellular responses. Induction of a transient (cell cycle arrest) or a permanent (senescence) block of cell proliferation, or the activation of cell death pathways in response to genotoxic stress comprise the major arms of the survival-death axis governed by p53. Due to these biological properties, inactivation of p53 is a crucial step in tumor development and progression, reflected by the high incidence of TP53 mutations in different types of human cancers. The remarkable potency of p53 in suppressing tumorigenic outgrowth has promoted the expectation that tumor cells expressing wild-type p53 (wtp53) should be more prone to elimination by cytotoxic treatments than tumor cells expressing mutant p53 (mutp53) with defunct wtp53 activities. However, recent findings yielded somewhat unexpected insights concerning the preponderance of the survival-promoting effects of wtp53 in cancer cells, a rather undesired property from the therapeutic point of view. In this commentary we will discuss the possibility that the developmentally established distinct patterns of wtp53 mediated responses in different tissues are an important factor in determining the ultimate outcome of cellular responses mediated by wtp53 in different types of tumor cells, with a particular focus on the divergent impact of wtp53 in malignant tumors of the central nervous system. We infer that a selective gain of pro-survival functions of wtp53 in cancer cells will confer a survival advantage that counteracts tumor therapy.
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Affiliation(s)
- Ella Kim
- The Translational Neurooncology Research Group, Department of Neurosurgery, Georg-August-University of Göttingen, Robert-Koch-Strasse 40, 37074 Göttingen, Germany.
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22
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Dass CR, Choong PFM. Gene therapy for osteosarcoma: steps towards clinical studies. J Pharm Pharmacol 2008; 60:405-13. [PMID: 18380911 DOI: 10.1211/jpp.60.4.0001] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/31/2022]
Abstract
Gene therapy, an applied form of biotechnology, relies on the delivery of foreign DNA into cells. More than 50% of all reported clinical trials for gene therapy are for cancer, though only a scant number for osteosarcoma. Osteosarcoma is a neoplasm afflicting young adults, who in their prime years of life suffer debilitation if not death. The disease is not entirely curable, even with surgery combined with aggressive chemotherapy. Thus, other forms of therapies are being evaluated, including gene therapy. There exist two major forms of gene transfer: viral and non-viral. This review only covers proof-of-principle work carried out in cancer beyond the cell culture stage, in animals. Drawing from the experiences of gene therapy against other cancers, studies for which have already reached the clinical phase, the review discusses potential pitfalls and solutions to enhance gene therapy for osteosarcoma.
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Affiliation(s)
- Crispin R Dass
- Department of Orthopaedics, University of Melbourne, St. Vincent's Hospital Melbourne, Australia.
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Abstract
Human sarcoma cells can be killed by radio- and chemotherapy, but tumor cells acquiring resistance frequently kill the patient. A keen understanding of the intracellular course of oncogenic cascades leads to the discovery of small molecular inhibitors of the involved phosphorylated kinases. Targeted therapy complements chemotherapy. Oncogene silencing is feasible by small interfering RNA. The restoration of some of the mutated or deleted tumor-suppressor genes (p53, Rb, PTEN, hSNF, INK/ARF and WT) by demethylation or reacetylation of their histones has been accomplished. Genetically engineered or naturally oncolytic viruses selectively lyse tumors and leave healthy tissues intact. Adeno- or retroviral vectors deliver genes of immunological costimulators, tumor antigens, chemo- or cytokines and/or tumor-suppressor proteins into tumor (sarcoma) cells. Suicide gene delivery results in apoptosis induction. Genes of enzymes that target prodrugs as their substrates render tumor cells highly susceptible to chemotherapy, with the prodrug to be targeted intracellularly. It will be combinations of sophisticated surgical removal of the nonencapsulated and locally invasive primary sarcomas, advanced forms of radiotherapy to the involved sites and immunotherapy with sarcoma vaccines that will cure primary sarcomas. Adoptive immunotherapy with immune lymphocytes will be operational in metastatic disease only when populations of regulatory T cells are controlled. Targeted therapy with small molecular inhibitors of oncogene cascades, the driving forces of sarcoma cells, alteration of the tumor stroma from a supportive to a tumor-hostile environment, reactivation or replacement of wild-type tumor-suppressor genes, and radio-chemotherapy (with much reduced toxicity) will eventually accomplish the cure of metastatic sarcomas.
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Affiliation(s)
- Joseph G Sinkovics
- The University of South Florida, Cancer Institute of St Joseph's Hospital, HL Moffitt Cancer Center, The University of South Florida College of Medicine, FL, USA.
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Shu KX, Li B, Wu LX. The p53 network: p53 and its downstream genes. Colloids Surf B Biointerfaces 2007; 55:10-8. [PMID: 17188467 DOI: 10.1016/j.colsurfb.2006.11.003] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2006] [Revised: 10/17/2006] [Accepted: 11/03/2006] [Indexed: 12/13/2022]
Abstract
The tumor-suppressor gene p53 and its downstream genes consist of a complicated gene network. p53 is a key molecular node in the network, which is activated in response to several cellular signals resulting in the maintenance of genetic stability. Several cellular signals may activate the p53 network. When the expression of P53 is elevated, P53-MDM2 module and the ubiquitin system can accurately regulate the expression level of P53. P53 can bind to specific DNA sequence, activate its downstream genes expression, and control cell-cycle arrest, DNA repair, and apoptosis. Elucidating the function of p53 gene network will help understand the interaction mechanisms of p53 and its downstream genes.
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Affiliation(s)
- Kun-Xian Shu
- College of Bioinformation, Chongqing University of Posts and Telecommunications, Chongqing 400065, China.
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