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Pochini L, Tedesco GE, Mazza T, Scalise M, Indiveri C. OCTN1 mediates acetylcholine transport in the A549 lung cancer cells: possible pathophysiological implications. Front Mol Biosci 2024; 11:1512530. [PMID: 39719963 PMCID: PMC11666908 DOI: 10.3389/fmolb.2024.1512530] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2024] [Accepted: 11/25/2024] [Indexed: 12/26/2024] Open
Abstract
A role for acetylcholine in cell proliferation, epithelial mesenchymal transition and invasion has been well assessed and related to the presence of the non-neuronal cholinergic system in lung cancer. For the operation of this non-neuronal system, acetylcholine should be released by a transporter mediated non-quantal process. OCTN1 is one of the transporters able to catalyse acetylcholine efflux in vitro and ex vivo. Using the A549 cell line as a lung cancer model, it has been found that these cells express OCTN1 at a higher level with respect to other cancer cells. The transport capacity of OCTN1 extracted from A549 and reconstituted into proteoliposomes reflects the protein expression profile. The properties of the acetylcholine transport mediated by OCTN1 of A549 in terms of specificity to ligands and ability to catalyse efflux of acetylcholine correspond to those previously described for the same transporter in other cells or to those of the human recombinant protein. OCTN1 is the major player in acetylcholine release in A549 and, therefore, may represent a target for inhibitors able to block the acetylcholine action in this type of aggressive tumors.
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Affiliation(s)
- Lorena Pochini
- Laboratory of Biochemistry, Molecular Biotechnology and Molecular Biology, Department DiBEST (Biologia, Ecologia, Scienze Della Terra), University of Calabria, Arcavacata di Rende, Italy
- Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), National Research Council (CNR), Bari, Italy
| | - Giusi Elisabetta Tedesco
- Laboratory of Biochemistry, Molecular Biotechnology and Molecular Biology, Department DiBEST (Biologia, Ecologia, Scienze Della Terra), University of Calabria, Arcavacata di Rende, Italy
| | - Tiziano Mazza
- Laboratory of Biochemistry, Molecular Biotechnology and Molecular Biology, Department DiBEST (Biologia, Ecologia, Scienze Della Terra), University of Calabria, Arcavacata di Rende, Italy
| | - Mariafrancesca Scalise
- Laboratory of Biochemistry, Molecular Biotechnology and Molecular Biology, Department DiBEST (Biologia, Ecologia, Scienze Della Terra), University of Calabria, Arcavacata di Rende, Italy
| | - Cesare Indiveri
- Laboratory of Biochemistry, Molecular Biotechnology and Molecular Biology, Department DiBEST (Biologia, Ecologia, Scienze Della Terra), University of Calabria, Arcavacata di Rende, Italy
- Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), National Research Council (CNR), Bari, Italy
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Muñoz JP, Calaf GM. Acetylcholine, Another Factor in Breast Cancer. BIOLOGY 2023; 12:1418. [PMID: 37998017 PMCID: PMC10669196 DOI: 10.3390/biology12111418] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/03/2023] [Revised: 11/08/2023] [Accepted: 11/09/2023] [Indexed: 11/25/2023]
Abstract
Acetylcholine (ACh) is a neurotransmitter that regulates multiple functions in the nervous system, and emerging evidence indicates that it could play a role in cancer progression. However, this function is controversial. Previously, we showed that organophosphorus pesticides decreased the levels of the enzyme acetylcholinesterase in vivo, increasing ACh serum levels and the formation of tumors in the mammary glands of rats. Furthermore, we showed that ACh exposure in breast cancer cell lines induced overexpression of estrogen receptor alpha (ERα), a key protein described as the master regulator in breast cancer. Therefore, here, we hypothesize that ACh alters the ERα activity through a ligand-independent mechanism. The results here reveal that the physiological concentration of ACh leads to the release of Ca+2 and the activity of MAPK/ERK and PI3K/Akt pathways. These changes are associated with an induction of p-ERα and its recruitment to the nucleus. However, ACh fails to induce overexpression of estrogen-responsive genes, suggesting a different activation mechanism than that of 17ß-estradiol. Finally, ACh promotes the viability of breast cancer cell lines in an ERα-dependent manner and induces the overexpression of some EMT markers. In summary, our results show that ACh promotes breast cancer cell proliferation and ERα activity, possibly in a ligand-independent manner, suggesting its putative role in breast cancer progression.
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Affiliation(s)
- Juan P. Muñoz
- Laboratorio de Bioquímica, Departamento de Química, Facultad de Ciencias, Universidad de Tarapacá, Arica 1000007, Chile;
| | - Gloria M. Calaf
- Instituto de Alta Investigación, Universidad de Tarapacá, Arica 1000000, Chile
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Calaf GM. Breast carcinogenesis induced by organophosphorous pesticides. ADVANCES IN PHARMACOLOGY (SAN DIEGO, CALIF.) 2023; 96:71-117. [PMID: 36858780 DOI: 10.1016/bs.apha.2022.10.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Breast cancer is a major health threat to women worldwide and the leading cause of cancer-related death. The use of organophosphorous pesticides has increased in agricultural environments and urban settings, and there is evidence that estrogen may increase breast cancer risk in women. The mammary gland is an excellent model for examining its susceptibility to different carcinogenic agents due to its high cell proliferation capabilities associated with the topography of the mammary parenchyma and specific stages of gland development. Several experimental cellular models are presented here, in which the animals were exposed to chemical compounds such as pesticides, and endogenous substances such as estrogens that exert a significant effect on normal breast cell processes at different levels. Such models were developed by the effect of malathion, parathion, and eserine, influenced by estrogen demonstrating features of cancer initiation in vivo as tumor formation in rodents; and in vitro in the immortalized normal breast cell line MCF-10F, that when transformed showed signs of carcinogenesis such as increased cell proliferation, anchorage independence, invasive capabilities, modulation of receptors and genomic instability. The role of acetylcholine was also demonstrated in the MCF-10F, suggesting a role not only as a neurotransmitter but also with other functions, such as induction of cell proliferation, playing an important role in cancer. Of note, this is a unique experimental approach that identifies mechanistic signs that link organophosphorous pesticides with breast carcinogenesis.
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Affiliation(s)
- Gloria M Calaf
- Instituto de Alta Investigación, Universidad de Tarapacá, Arica, Chile.
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Español A, Salem A, Sanchez Y, Sales ME. Breast cancer: Muscarinic receptors as new targets for tumor therapy. World J Clin Oncol 2021; 12:404-428. [PMID: 34189066 PMCID: PMC8223712 DOI: 10.5306/wjco.v12.i6.404] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/17/2021] [Revised: 03/26/2021] [Accepted: 06/02/2021] [Indexed: 02/06/2023] Open
Abstract
The development of breast cancer is a complex process that involves the participation of different factors. Several authors have demonstrated the overexpression of muscarinic acetylcholine receptors (mAChRs) in different tumor tissues and their role in the modulation of tumor biology, positioning them as therapeutic targets in cancer. The conventional treatment for breast cancer involves surgery, radiotherapy, and/or chemotherapy. The latter presents disadvantages such as limited specificity, the appearance of resistance to treatment and other side effects. To prevent these side effects, several schedules of drug administration, like metronomic therapy, have been developed. Metronomic therapy is a type of chemotherapy in which one or more drugs are administered at low concentrations repetitively. Recently, two chemotherapeutic agents usually used to treat breast cancer have been considered able to activate mAChRs. The combination of low concentrations of these chemotherapeutic agents with muscarinic agonists could be a useful option to be applied in breast cancer treatment, since this combination not only reduces tumor cell survival without affecting normal cells, but also decreases pathological neo-angiogenesis, the expression of drug extrusion proteins and the cancer stem cell fraction. In this review, we focus on the previous evidences that have positioned mAChRs as relevant therapeutic targets in breast cancer and analyze the effects of administering muscarinic agonists in combination with conventional chemotherapeutic agents in a metronomic schedule.
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Affiliation(s)
- Alejandro Español
- Laboratory of Immunopharmacology and Tumor Biology, CEFYBO CONICET University of Buenos Aires, Buenos Aires C1121ABG, Argentina
| | - Agustina Salem
- Laboratory of Immunopharmacology and Tumor Biology, CEFYBO CONICET University of Buenos Aires, Buenos Aires C1121ABG, Argentina
| | - Yamila Sanchez
- Laboratory of Immunopharmacology and Tumor Biology, CEFYBO CONICET University of Buenos Aires, Buenos Aires C1121ABG, Argentina
| | - María Elena Sales
- Laboratory of Immunopharmacology and Tumor Biology, CEFYBO CONICET University of Buenos Aires, Buenos Aires C1121ABG, Argentina
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Calaf GM. Role of organophosphorous pesticides and acetylcholine in breast carcinogenesis. Semin Cancer Biol 2021; 76:206-217. [PMID: 33766648 DOI: 10.1016/j.semcancer.2021.03.016] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2021] [Accepted: 03/18/2021] [Indexed: 12/16/2022]
Abstract
Breast cancer is the leading cause of cancer-related death in women worldwide. Several studies have addressed the association between cancer in humans and agricultural pesticide exposure. Evidence indicates that exposure to organophosphorous pesticides such as parathion and malathion occurs as a result of occupational factors since they are extensively used to control insects. On the other hand, estrogens have been considered beneficial to the organism; however, epidemiological studies have pointed out an increased breast cancer risk in both humans and animals. Experimental female rat mammary gland cancer models were developed after exposure to parathion, malathion, eserine, an acetylcholinesterase inhibitor, and estrogen allowing the analysis of the signs of carcinogenicity as alteration of cell proliferation, receptor expression, genomic instability, and cell metabolism in vivo and in vitro. Thus, pesticides increased proliferative ducts followed by ductal carcinoma; and 17β-estradiol increased proliferative lobules followed by lobular carcinomas. The combination of both pesticides and either eserine or estrogen induced tumors with both types of structures followed by mammary gland tumors and metastasis to the lung and kidneys after 240 days of a 5-day treatment. Studies also showed that these pesticides and eserine decreased three to five times the acetylcholinesterase activity in the serum compared to controls whereas terminal end buds increased in number, being inhibited by atropine. Genomic instability was analyzed in such tissues (mp53, CYP1A2, c-myc, c-fos, ERα, M2R) and pesticides increased protein expression that was stimulated by estrogens but inhibited by atropine. Eserine also transformed the epithelium of the rat mammary gland in the presence of estrogen and increased the number of terminal end buds after treatment inducing mammary carcinomas. Then, enzymatic digestion of such structures gave rise to cells with increased DNA synthesis and induced anchorage independence. Thus, there were changes in the epithelium of the mammary gland influencing breast carcinogenesis. Furthermore, these substances and acetylcholine also showed the signs of carcinogenicity in vitro as cell proliferation, receptor expression (ERα, ErbB2, M2R), genomic instability (c-myc, mp53, ERα, M2R), and cell metabolism. A unique cellular model is also presented here based on the use of MCF-10 F, a non-tumorigenic cell line that represents a valuable clinically translatable experimental approach that identifies mechanistic links for pesticides and estrogen as suspect human carcinogenic agents.
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Affiliation(s)
- Gloria M Calaf
- Instituto de Alta Investigación, Universidad de Tarapacá, Arica, 1000000, Chile; Center for Radiological Research, Columbia University Medical Center, New York, NY, 10032, USA.
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Kato M, Kolotuev I, Cunha A, Gharib S, Sternberg PW. Extrasynaptic acetylcholine signaling through a muscarinic receptor regulates cell migration. Proc Natl Acad Sci U S A 2021; 118:e1904338118. [PMID: 33361149 PMCID: PMC7817160 DOI: 10.1073/pnas.1904338118] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Acetylcholine (ACh) promotes various cell migrations in vitro, but there are few investigations into this nonsynaptic role of ACh signaling in vivo. Here we investigate the function of a muscarinic receptor on an epithelial cell migration in Caenorhabditis elegans We show that the migratory gonad leader cell, the linker cell (LC), uses an M1/M3/M5-like muscarinic ACh receptor GAR-3 to receive extrasynaptic ACh signaling from cholinergic neurons for its migration. Either the loss of the GAR-3 receptor in the LC or the inhibition of ACh release from cholinergic neurons resulted in migratory path defects. The overactivation of the GAR-3 muscarinic receptor caused the LC to reverse its orientation through its downstream effectors Gαq/egl-30, PLCβ/egl-8, and TRIO/unc-73 This reversal response only occurred in the fourth larval stage, which corresponds to the developmental time when the GAR-3::yellow fluorescent protein receptor in the membrane relocalizes from a uniform to an asymmetric distribution. These findings suggest a role for the GAR-3 muscarinic receptor in determining the direction of LC migration.
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Affiliation(s)
- Mihoko Kato
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125
| | - Irina Kolotuev
- Electron Microscopy Facility, University of Lausanne, Quartier Sorge-Biophore, CH-1015 Lausanne, Switzerland
| | - Alexandre Cunha
- Center for Advanced Methods in Biological Image Analysis, California Institute of Technology, Pasadena, CA 91125
| | - Shahla Gharib
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125
| | - Paul W Sternberg
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125;
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Role of the parasympathetic nervous system in cancer initiation and progression. Clin Transl Oncol 2020; 23:669-681. [PMID: 32770391 DOI: 10.1007/s12094-020-02465-w] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2020] [Accepted: 07/21/2020] [Indexed: 12/29/2022]
Abstract
The nervous system plays an important role in cancer initiation and progression. Accumulated evidences clearly show that the sympathetic nervous system exerts stimulatory effects on carcinogenesis and cancer growth. However, the role of the parasympathetic nervous system in cancer has been much less elucidated. Whereas retrospective studies in vagotomized patients and experiments employing vagotomized animals indicate the parasympathetic nervous system has an inhibitory effect on cancer, clinical studies in patients with prostate cancer indicate it has stimulatory effects. Therefore, the aim of this paper is a critical evaluation of the available data related to the role of the parasympathetic nervous system in cancer.
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Sales ME, Español AJ, Salem AR, Pulido PM, Sanchez Y, Sanchez F. Role of Muscarinic Acetylcholine Receptors in Breast Cancer: Design of Metronomic Chemotherapy. CURRENT CLINICAL PHARMACOLOGY 2019; 14:91-100. [PMID: 30501602 PMCID: PMC7011678 DOI: 10.2174/1574884714666181203095437] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/21/2018] [Revised: 11/15/2018] [Accepted: 11/15/2018] [Indexed: 12/14/2022]
Abstract
BACKGROUND muscarinic acetylcholine receptors (mAChRs) have attracted interest as targets for therapeutic interventions in different illnesses like Alzheimer´s disease, viral infections and different tumors. Regarding the latter, many authors have studied each subtype of mAChRs, which seem to be involved in the progression of distinct types of malignancies. METHODS We carefully revised research literature focused on mAChRs expression and signaling as well as in their involvement in cancer progression and treatment. The characteristics of screened papers were described using the mentioned conceptual framework. RESULTS Muscarinic antagonists and agonists have been assayed for the treatment of tumors established in lung, brain and breast with beneficial effects. We described an up-regulation of mAChRs in mammary tumors and the lack of expression in non-tumorigenic breast cells and normal mammary tissues. We and others demonstrated that muscarinic agonists can trigger anti-tumor actions in a dose-dependent manner on tumors originated in different organs like brain or breast. At pharmacological concentrations, they exert similar effects to traditional chemotherapeutic agents. Metronomic chemotherapy refers to the administration of anti-cancer drugs at low doses with short intervals among them, and it is a different regimen applied in cancer treatment reducing malignant growth and angiogenesis, and very low incidence of adverse effects. CONCLUSION The usage of subthreshold concentrations of muscarinic agonists combined with conventional chemotherapeutic agents could be a promising tool for breast cancer therapy.
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Affiliation(s)
- María E. Sales
- Centro de Estudios Farmacológicos y Botánicos (CEFYBO)-CONICET. 2da Cátedra de Farmacología, Facultad de Medicina, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina
| | - Alejandro J. Español
- Centro de Estudios Farmacológicos y Botánicos (CEFYBO)-CONICET. 2da Cátedra de Farmacología, Facultad de Medicina, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina
| | - Agustina R. Salem
- Centro de Estudios Farmacológicos y Botánicos (CEFYBO)-CONICET. 2da Cátedra de Farmacología, Facultad de Medicina, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina
| | - Paola M. Pulido
- Centro de Estudios Farmacológicos y Botánicos (CEFYBO)-CONICET. 2da Cátedra de Farmacología, Facultad de Medicina, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina
| | - Y. Sanchez
- Centro de Estudios Farmacológicos y Botánicos (CEFYBO)-CONICET. 2da Cátedra de Farmacología, Facultad de Medicina, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina
| | - Francisco Sanchez
- Centro de Estudios Farmacológicos y Botánicos (CEFYBO)-CONICET. 2da Cátedra de Farmacología, Facultad de Medicina, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina
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Wei W, Wang M, Li Y, Meng Q, Tang Y, Lu H, Yu W, Cheng Q, Li Y, Xu L, Jian S, Wu Y, Yi X, Xie K. Muscarinic cholinergic signaling and overactive bladder-like symptoms associated with invasive bladder cancer. Oncol Lett 2018; 16:775-784. [PMID: 29963145 PMCID: PMC6019950 DOI: 10.3892/ol.2018.8715] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2017] [Accepted: 03/16/2018] [Indexed: 11/06/2022] Open
Abstract
The objective of the present study was to explore the association between muscarinic cholinergic signaling and urothelial bladder tumors. Possible associations among overactive bladder (OAB) symptoms and bladder tumors were retrospectively investigated using a multicenter Chinese database with prospectively collected data since 2010. Firstly, it was demonstrated that OAB symptoms, such as urgency, were more severe in patients with invasive bladder cancer and were associated with a reduced prognosis. Following this, muscarinic cholinergic receptor 3 (M3R) expression in urothelium was determined to be lower in invasive cancer tissue than in adjacent non-cancerous tissue, yet M3R upregulation was associated with a reduced progression free survival (PFS) time. Additionally, it was also demonstrated that muscarinic cholinergic receptor 2 (M2R) was upregulated in the sub-urothelium, and this was also associated with a reduced PFS time. Furthermore, it was determined that cholinesterase and acetylcholinesterase were lower in invasive cancer than in non-invasive cancer. In conclusion, the results indicated that M3R expression was downregulated in invasive bladder cancer, which may have a role as a protective anti-oncogene, in contrast to its oncogenic role in numerous other cancer types. Therefore, muscarinic cholinergic signaling may be a novel therapeutic target for treating bladder cancer.
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Affiliation(s)
- Wei Wei
- Department of Radiology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China
| | - Minggang Wang
- Department of Urology, Affiliated Hospital of Qinghai University, Xining, Qinghai 810016, P.R. China
| | - Yunglong Li
- Department of Urology, First People's Hospital of Kunshan, Jiangsu 215300, P.R. China
| | - Qinggui Meng
- Department of Radiology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China
| | - Yong Tang
- Department of Urology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China
| | - Haoyuan Lu
- Department of Urology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China
| | - Wenchao Yu
- Department of Radiology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China
| | - Qiwei Cheng
- Department of Urology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China
| | - You Li
- Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan, Hubei 430075, P.R. China
| | - Long Xu
- Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan, Hubei 430075, P.R. China
| | - Shaojun Jian
- Department of Urology, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180, P.R. China
| | - Yuexian Wu
- Department of Respiratory, Nanfang Hospital of Southern Medical University, Guangzhou, Guangdong 510515, P.R. China
| | - Xianlin Yi
- Department of Urology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China.,Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan, Hubei 430075, P.R. China
| | - Keji Xie
- Department of Urology, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180, P.R. China
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Wang Y, Cui P, Liu J, Wu H, Ma J. Aclidinium bromide inhibits the growth and metastasis of gastric cancer MKN‑28 cells via the PI3K signaling pathway. Mol Med Rep 2018; 18:2263-2268. [PMID: 29956761 DOI: 10.3892/mmr.2018.9220] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2017] [Accepted: 05/05/2018] [Indexed: 11/06/2022] Open
Abstract
The present study investigated the effect and underling mechanisms of aclidinium bromide, a novel, inhaled long‑acting muscarinic antagonist, on the development of gastric cancer. Human gastric cancer MKN‑28 cells, as a model in vitro, were treated with aclidinium bromide and dimethyl sulfoxide. Cell Counting Kit‑8 assay, transwell assay and flow cytometry were used to assess cell proliferation, invasion/migration and apoptosis, respectively. In addition, western blotting was performed to determine the relative expression of proteins associated with apoptosis and the phosphatidylinositol‑3‑kinase (PI3K) signaling pathway. Optical density values of MKN‑28 cells were decreased in a time‑ and dose‑dependent manner in the aclidinium bromide treated group. Matrigel invasion analysis demonstrated the number of invasive cells were significantly decreased in the aclidinium bromide‑treated group when compared with the control group. Furthermore, aclidinium bromide led to the marked reduction of the number of MKN‑28 cells passing though the microwells of the transwell chamber. The expression levels of the anti‑apoptotic protein B‑cell lymphoma 2 (Bcl‑2) decreased, and the expression of pro‑apoptotic proteins active Caspase3 and Bcl‑2‑associated X protein increased concurrently following aclidinium bromide stimulation using western blotting. The phosphorylation of protein kinase B and mechanistic target of rapamycin were significantly inhibited in MKN‑28 cells treated with aclidinium bromide; and the activity of the downstream proteins such as p70S6K and Cyclin D1 were also significantly decreased. In conclusion, aclidinium bromide could inhibit gastric cancer cell proliferation and metastasis, which may be associated with the enhancement of apoptosis induced by the PI3K signaling pathway.
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Affiliation(s)
- Yuanzhi Wang
- Department of Operating, Binzhou Central Hospital, Binzhou, Shandong 251700, P.R. China
| | - Ping Cui
- Department of Oncology, Binzhou City TB Prevention and Control of Hospital, Binzhou, Shandong 251700, P.R. China
| | - Jingjing Liu
- Department of Neurosurgery, Binzhou Central Hospital, Binzhou, Shandong 251700, P.R. China
| | - Hongxia Wu
- Department of Nursing, Binzhou Central Hospital, Binzhou, Shandong 251700, P.R. China
| | - Jun Ma
- Department of Infectious Diseases, Binzhou City TB Prevention and Control of Hospital, Binzhou, Shandong 251700, P.R. China
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Li G, Cao X, Li Y, Qiu YY, Li Y, Liu X, Sun X. MicroRNA‐374b inhibits cervical cancer cell proliferation and induces apoptosis through the p38/ERK signaling pathway by binding to JAM‐2. J Cell Physiol 2018; 233:7379-7390. [PMID: 29575013 DOI: 10.1002/jcp.26574] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2017] [Accepted: 02/27/2018] [Indexed: 02/03/2023]
Affiliation(s)
- Guang‐Cai Li
- Medical Insurance Management OfficeLinyi People's HospitalLinyiP.R. China
| | - Xiao‐Yun Cao
- Medical Insurance Management OfficeEconomic and Technological Development Zone People's Hospital of LinyiLinyiP.R. China
| | - Ying‐Ni Li
- Department of Obstetrics and GynecologyEconomic and Technological Development Zone People's Hospital of LinyiLinyiP.R. China
| | - Yu‐ Yan Qiu
- Department of Obstetrics and GynecologyEconomic and Technological Development Zone People's Hospital of LinyiLinyiP.R. China
| | - Ying‐Na Li
- Department of Obstetrics and GynecologyEconomic and Technological Development Zone People's Hospital of LinyiLinyiP.R. China
| | - Xing‐Jie Liu
- Residency Office of Educational DepartmentLinyi People's HospitalLinyiP.R. China
| | - Xiang‐Xiu Sun
- Department of Obstetrics and GynecologyLinyi People's HospitalLinyiP.R. China
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Janovick JA, Spicer TP, Bannister TD, Scampavia L, Conn PM. Pharmacoperone rescue of vasopressin 2 receptor mutants reveals unexpected constitutive activity and coupling bias. PLoS One 2017; 12:e0181830. [PMID: 28767678 PMCID: PMC5540481 DOI: 10.1371/journal.pone.0181830] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2017] [Accepted: 07/08/2017] [Indexed: 12/30/2022] Open
Abstract
Pharmacoperones are small molecules that diffuse into cells and rescue misfolded, mistrafficked protein mutants, restoring their function. These substances act with high target specificity, serving as templates to fold (or refold) receptors, enzymes, ion channels or other proteins and enable them to pass the scrutiny of the cellular quality control system ("rescue"). In the present study we demonstrate that a rescued mutant (L83Q) of the vasopressin type 2 receptor (V2R), shows a strong bias for Gs coupling unlike the WT V2 receptor, which couples to both Gs and Gq/11. Failure of the mutant to couple to Gq/11 was not due to a limiting quantity of G-proteins since other Gq/11-coupled receptors (WT V2R, histamine receptor and muscarinic receptor) responded appropriately to their ligands. Transfection with DNA encoding Gq enabled the V2 receptor mutant to couple to this G protein, but only modestly compared with the WT receptor. Fourteen V2R mutant pharmacoperones, of multiple chemical classes, obtained from a high throughput screen of a 660,000 structure library, and one V2R peptidomimetic antagonist rescues L83Q. The rescued mutant shows similar bias with all pharmacoperones identified, suggesting that the bias is intrinsic to the mutant protein's structure, rather than due to the chemical class of the pharmacoperone. In the case of V2R mutant Y128S, rescue with a pharmacoperone revealed constitutive activity, also with bias for Gs, although both IP and cAMP were produced in response to agonist. These results suggest that particular rescued receptor mutants show functional characteristics that differ from the WT receptor; a finding that may be important to consider as pharmacoperones are developed as therapeutic agents.
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Affiliation(s)
- Jo Ann Janovick
- Departments of Internal Medicine and Cell Biology/Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas, United States of America
| | - Timothy P. Spicer
- Lead Identification Division, Translational Research Institute and Department of Molecular Therapeutics, Scripps Research Institute, Jupiter, Florida, United States of America
| | - Thomas D. Bannister
- Department of Chemistry, Scripps Research Institute, Jupiter, Florida, United States of America
| | - Louis Scampavia
- Lead Identification Division, Translational Research Institute and Department of Molecular Therapeutics, Scripps Research Institute, Jupiter, Florida, United States of America
| | - P. Michael Conn
- Departments of Internal Medicine and Cell Biology/Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas, United States of America
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The insect repellent N,N-diethyl-m-toluamide (DEET) induces angiogenesis via allosteric modulation of the M3 muscarinic receptor in endothelial cells. Sci Rep 2016; 6:28546. [PMID: 27345502 PMCID: PMC4921870 DOI: 10.1038/srep28546] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2016] [Accepted: 06/06/2016] [Indexed: 12/12/2022] Open
Abstract
The insect repellent N,N-diethyl-m-toluamide (DEET) has been reported to inhibit AChE (acetylcholinesterase) and to possess potential carcinogenic properties with excessive vascularization. In the present paper, we demonstrate that DEET specifically stimulates endothelial cells that promote angiogenesis which increases tumor growth. DEET activates cellular processes that lead to angiogenesis including proliferation, migration and adhesion. This is associated with an enhancement of NO production and VEGF expression in endothelial cells. M3 silencing or the use of a pharmacological M3 inhibitor abrogates all of these effects which reveals that DEET-induced angiogenesis is M3 sensitive. The experiments involving calcium signals in both endothelial and HEK cells overexpressing M3 receptors, as well as binding and docking studies demonstrate that DEET acts as an allosteric modulator of the M3 receptor. In addition, DEET inhibited AChE which increased acetylcholine bioavailability and binding to M3 receptors and also strengthened proangiogenic effects by an allosteric modulation.
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Chotirat S, Suriyo T, Hokland M, Hokland P, Satayavivad J, Auewarakul CU. Cholinergic activation enhances retinoic acid-induced differentiation in the human NB-4 acute promyelocytic leukemia cell line. Blood Cells Mol Dis 2016; 59:77-84. [PMID: 27282572 DOI: 10.1016/j.bcmd.2016.04.009] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2015] [Revised: 04/16/2016] [Accepted: 04/17/2016] [Indexed: 01/28/2023]
Abstract
The non-neuronal cholinergic system (NNCS) has been shown to play a role in regulating hematopoietic differentiation. We determined the expression of cholinergic components in leukemic cell lines by Western blotting and in normal leukocyte subsets by flow cytometry and found a heterogeneous expression of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), choline transporter (CHT), M3 muscarinic acetylcholine receptor (M3-mAChR) and α7 nicotinic acetylcholine receptor (α7-nAChR). We then evaluated NNCS role in differentiation of human NB-4 acute promyelocytic leukemia cell line and discovered a dramatic induction of M3-mAChR after all-trans retinoic acid (ATRA) treatment (p<0.0001). Adding carbachol which is a cholinergic agonist to the ATRA treatment resulted in an increase of a granulocytic differentiation marker (CD11b) as compared with ATRA treatment alone (p<0.05), indicating that cholinergic activation enhanced ATRA in inducing NB-4 maturation. The combination of carbachol and ATRA treatment for 72h also resulted in decreased viability and increased cleaved caspase-3 expression when compared with ATRA treatment alone (p<0.05). However, this combination did not cause poly (ADP-ribose) polymerase (PARP) cleavage. Overall, we have shown that NB-4 cells expressed M3-mAChR in a differentiation-dependent manner and cholinergic stimulation induced maturation and death of ATRA-induced differentiated NB-4 cells.
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Affiliation(s)
- Sadudee Chotirat
- Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
| | - Tawit Suriyo
- Laboratory of Pharmacology, Chulabhorn Research Institute, Bangkok 10210, Thailand.
| | - Marianne Hokland
- Department of Biomedicine, Faculty of Health, Aarhus University, Aarhus 8000, Denmark.
| | - Peter Hokland
- Department of Haematology, Aarhus University Hospital, Aarhus 8000, Denmark.
| | - Jutamaad Satayavivad
- Laboratory of Pharmacology, Chulabhorn Research Institute, Bangkok 10210, Thailand; Center of Excellence on Environmental Health and Toxicology, Office of Higher Education Commission, Ministry of Education, Bangkok 10400, Thailand.
| | - Chirayu U Auewarakul
- Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
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15
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Pochini L, Scalise M, Indiveri C. Immuno-detection of OCTN1 (SLC22A4) in HeLa cells and characterization of transport function. Int Immunopharmacol 2015; 29:21-6. [DOI: 10.1016/j.intimp.2015.04.040] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2015] [Revised: 04/01/2015] [Accepted: 04/17/2015] [Indexed: 11/27/2022]
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16
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Suriyo T, Tachachartvanich P, Visitnonthachai D, Watcharasit P, Satayavivad J. Chlorpyrifos promotes colorectal adenocarcinoma H508 cell growth through the activation of EGFR/ERK1/2 signaling pathway but not cholinergic pathway. Toxicology 2015; 338:117-29. [PMID: 26514924 DOI: 10.1016/j.tox.2015.10.009] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2015] [Revised: 10/19/2015] [Accepted: 10/19/2015] [Indexed: 01/08/2023]
Abstract
Aside from the effects on neuronal cholinergic system, epidemiological studies suggest an association between chlorpyrifos (CPF) exposure and cancer risk. This in vitro study examined the effects of CPF and its toxic metabolite, chlorpyrifos oxon (CPF-O), on the growth of human colorectal adenocarcinoma H508, colorectal adenocarcinoma HT-29, normal colon epithelial CCD841, liver hepatocellular carcinoma HepG2, and normal liver hepatocyte THLE-3 cells. The results showed that CPF (5-100 μM) concentration-dependently increased viability of H508 and CCD841 cells in serum-free conditions. This increasing trend was not found in HT-29, HepG2 and THLE-3 cells. In contrast, CPF-O (50-100 μM) reduced the viability of all cell lines. Cell cycle analysis showed the induction of cells in the S phase, and EdU incorporation assay revealed the induction of DNA synthesis in CPF-treated H508 cells indicating that CPF promotes cell cycle progression. Despite the observation of acetylcholinesterase activity inhibition and reactive oxygen species (ROS) generation, atropine (a non-selective muscarinic acetylcholine receptor antagonist) and N-acetylcysteine (a potent antioxidant) failed to inhibit the growth-promoting effect of CPF. CPF increased the phosphorylation of epidermal growth factor receptor (EGFR) and its downstream effector, extracellular signal regulated kinase (ERK1/2), in H508 cells. AG-1478 (a specific EGFR tyrosine kinase inhibitor) and U0126 (a specific MEK inhibitor) completely mitigated the growth promoting effect of CPF. Altogether, these results suggest that EGFR/ERK1/2 signaling pathway but not cholinergic pathway involves in CPF-induced colorectal adenocarcinoma H508 cell growth.
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Affiliation(s)
- Tawit Suriyo
- Laboratory of Pharmacology, Chulabhorn Research Institute, Bangkok 10210, Thailand
| | - Phum Tachachartvanich
- Environmental Toxicology Program, Chulabhorn Graduate Institute, Bangkok 10210, Thailand
| | | | - Piyajit Watcharasit
- Laboratory of Pharmacology, Chulabhorn Research Institute, Bangkok 10210, Thailand; Environmental Toxicology Program, Chulabhorn Graduate Institute, Bangkok 10210, Thailand
| | - Jutamaad Satayavivad
- Laboratory of Pharmacology, Chulabhorn Research Institute, Bangkok 10210, Thailand; Environmental Toxicology Program, Chulabhorn Graduate Institute, Bangkok 10210, Thailand; Center of Excellence on Environmental Health and Toxicology, Office of Higher Education Commission, Ministry of Education, Bangkok 10400, Thailand.
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17
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Amonyingcharoen S, Suriyo T, Thiantanawat A, Watcharasit P, Satayavivad J. Taurolithocholic acid promotes intrahepatic cholangiocarcinoma cell growth via muscarinic acetylcholine receptor and EGFR/ERK1/2 signaling pathway. Int J Oncol 2015; 46:2317-26. [PMID: 25815516 PMCID: PMC4441291 DOI: 10.3892/ijo.2015.2939] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2014] [Accepted: 02/10/2015] [Indexed: 01/25/2023] Open
Abstract
Cholangiocarcinoma (CCA) is a malignant cancer of the biliary tract and its occurrence is associated with chronic cholestasis which causes an elevation of bile acids in the liver and bile duct. The present study aimed to investigate the role and mechanistic effect of bile acids on the CCA cell growth. Intrahepatic CCA cell lines, RMCCA-1 and HuCCA-1, were treated with bile acids and their metabolites to determine the growth promoting effect. Cell viability, cell cycle analysis, EdU incorporation assays were conducted. Intracellular signaling proteins were detected by western immunoblotting. Among eleven forms of bile acids and their metabolites, only taurolithocholic acid (TLCA) concentration dependently (1–40 μM) increased the cell viability of RMCCA-1, but not HuCCA-1 cells. The cell cycle analysis showed induction of cells in the S phase and the EdU incorporation assay revealed induction of DNA synthesis in the TLCA-treated RMCCA-1 cells. Moreover, TLCA increased the phosphorylation of EGFR, ERK 1/2 and also increased the expression of cyclin D1 in RMCCA-1 cells. Furthermore, TLCA-induced RMCCA-1 cell growth could be inhibited by atropine, a non-selective muscarinic acetylcholine receptor (mAChR) antagonist, AG 1478, a specific EGFR inhibitor, or U 0126, a specific MEK 1/2 inhibitor. These results suggest that TLCA induces CCA cell growth via mAChR and EGFR/EKR1/2 signaling pathway. Moreover, the functional presence of cholinergic system plays a certain role in TLCA-induced CCA cell growth.
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Affiliation(s)
| | - Tawit Suriyo
- Laboratory of Pharmacology, Chulabhorn Research Institute, Bangkok 10210, Thailand
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18
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Ockenga W, Kühne S, Bocksberger S, Banning A, Tikkanen R. Non-neuronal functions of the m2 muscarinic acetylcholine receptor. Genes (Basel) 2013; 4:171-97. [PMID: 24705159 PMCID: PMC3899973 DOI: 10.3390/genes4020171] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2013] [Revised: 03/10/2013] [Accepted: 03/25/2013] [Indexed: 12/26/2022] Open
Abstract
Acetylcholine is an important neurotransmitter whose effects are mediated by two classes of receptors. The nicotinic acetylcholine receptors are ion channels, whereas the muscarinic receptors belong to the large family of G protein coupled seven transmembrane helix receptors. Beyond its function in neuronal systems, it has become evident that acetylcholine also plays an important role in non-neuronal cells such as epithelial and immune cells. Furthermore, many cell types in the periphery are capable of synthesizing acetylcholine and express at least some of the receptors. In this review, we summarize the non-neuronal functions of the muscarinic acetylcholine receptors, especially those of the M2 muscarinic receptor in epithelial cells. We will review the mechanisms of signaling by the M2 receptor but also the cellular trafficking and ARF6 mediated endocytosis of this receptor, which play an important role in the regulation of signaling events. In addition, we provide an overview of the M2 receptor in human pathological conditions such as autoimmune diseases and cancer.
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Affiliation(s)
- Wymke Ockenga
- Institute of Biochemistry, Medical Faculty, University of Giessen, Friedrichstrasse 24, 35392 Giessen, Germany.
| | - Sina Kühne
- Institute of Biochemistry, Medical Faculty, University of Giessen, Friedrichstrasse 24, 35392 Giessen, Germany.
| | - Simone Bocksberger
- Institute of Biochemistry, Medical Faculty, University of Giessen, Friedrichstrasse 24, 35392 Giessen, Germany.
| | - Antje Banning
- Institute of Biochemistry, Medical Faculty, University of Giessen, Friedrichstrasse 24, 35392 Giessen, Germany.
| | - Ritva Tikkanen
- Institute of Biochemistry, Medical Faculty, University of Giessen, Friedrichstrasse 24, 35392 Giessen, Germany.
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Calleja-Macias I, Osann K, Remedios-Chan M, Barrera-Saldana HA, Illades-Aguiar B, Anton-Culver H, Chikova AK, Grando SA, Bernard HU. Association of single nucleotide polymorphisms of nicotinic acetylcholine receptor subunits with cervical neoplasia. Life Sci 2012; 91:1099-102. [PMID: 22406075 DOI: 10.1016/j.lfs.2012.02.015] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2011] [Revised: 02/13/2012] [Accepted: 02/17/2012] [Indexed: 11/16/2022]
Abstract
AIMS Cholinergic signaling, particularly in response to non-physiological ligands like nicotine, stimulates carcinogenesis of a variety of tissue types including epithelia of the cervix uteri. Cholinergic signaling is mediated by nicotinic acetylcholine receptors (nAChRs), which are pentamers formed by subsets of 16 nAChR subunits. Recent literature suggests that single nucleotide polymorphisms (SNPs) of some of these subunits, notably alpha5, are risk factors for developing lung cancer in smokers as well as in non-smokers. MAIN METHODS We have studied the prevalence of four SNPs in the alpha5, alpha9, and beta1 subunits, which are expressed in cervical cells, in 456 patients with cervical cancers, precursor lesions, and healthy controls from two cohorts in Mexico. KEY FINDINGS A SNP in the alpha9 subunit, the G allele of rs10009228 (alpha9, A>G) shows a significant trend in the combined cohort, indicating that this allele constitutes a risk factor for neoplastic progression. The A allele of the SNP rs16969968 (alpha5, G>A), which correlates with the development of lung cancer, shows a non-significant trend to be associated with cervical lesions. Two other SNPs, rs55633891 (alpha9, C>T) and rs17856697 (beta1, A>G), did not exhibit a significant trend. SIGNIFICANCE Our study points to a potential risk factor of cervical carcinogenesis with importance for DNA diagnosis and as a target for intervention.
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Affiliation(s)
- Itzel Calleja-Macias
- Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine, CA 92697, USA
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