capsazepine and Necrosis

Radiosensitizers are used in cancer therapy to increase the γ-irradiation susceptibility of cancer cells, including radioresistant hypoxic cancer cells within solid tumors, so that radiotherapy can be applied at doses sufficiently low to minimize damage to adjacent normal tissues. Radiation-induced DNA damage is repaired by multiple repair systems, and therefore these systems are potential targets for radiosensitizers. We recently reported that the transient receptor potential vanilloid type 1 (TRPV1) channel is involved in early responses to DNA damage after γ-irradiation of human lung adenocarcinoma A549 cells. Therefore, we hypothesized that TRPV1 channel inhibitors would have a radiosensitizing effect by blocking repair of radiation-induced cell damage. Here, we show that pretreatment of A549 cells with the TRPV1 channel inhibitors capsazepine, AMG9810, SB366791 and BCTC suppressed the γ-ray-induced activation of early DNA damage responses, i.e., activation of the protein kinase ataxia-telangiectasia mutated (ATM) and accumulation of p53-binding protein 1 (53BP1). Further, the decrease of survival fraction at one week after γ-irradiation (2.0 Gy) was enhanced by pretreatment of cells with these inhibitors. On the other hand, inhibitor pretreatment did not affect cell viability, the number of apoptotic or necrotic cells, or DNA synthesis at 24 h after irradiation. These results suggest that inhibition of DNA repair by TRPV1 channel inhibitors in irradiated A549 cells caused gradual loss of proliferative ability, rather than acute facilitation of apoptosis or necrosis. TRPV1 channel inhibitors could be novel candidates for radiosensitizers to improve the efficacy of radiation therapy, either alone or in combination with other types of radiosensitizers.

Topics: Acrylamides; Anilides; Animals; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Capsaicin; Cell Line, Tumor; Cell Survival; Cinnamates; DNA Damage; Gamma Rays; Humans; Mice; Necrosis; Neoplasms; Pyrazines; Pyridines; Radiation-Sensitizing Agents; TRPV Cation Channels

Herein, we provide the first evidence on the capsaicin (CPS) receptor vanilloid receptor type-1 (VR1) by rat thymocytes, and its involvement in CPS-induced apoptosis. VR1 mRNA was identified by quantitative RT-PCR in CD5(+) thymocytes. By immunofluorescence and flow cytometry, we found that a substantial portion of CD5+ thymocytes, namely CD4+ and double negative (DN) cell subsets, express VR1 that was present on plasma membrane on discrete spots. By Western blot, VR1 protein was identified as a single band of 95 kDa. We also described that CPS could trigger two distinct pathways of thymocyte death, namely apoptosis and necrosis depending on the dose of CPS exposure. CPS-induced apoptosis involved intracellular free calcium (Ca2+) influx, phosphatidylserine exposure, mitochondrial permeability transmembrane pore (PTP) opening and mitochondrial transmembrane potential (Delta Psi m) dissipation leading to cytochrome c release, activation of caspase-9 and -3 and oligonucleosomal DNA fragmentation. VR1 was functionally implicated in these events as they were completely abrogated by the VR1 antagonist, capsazepine (CPZ). Finally, we demonstrated that VR1 expression on distinct thymocytes was associated with the selective ability of CPS to trigger DNA fragmentation in VR1+ CD4+ and DN thymocytes. Overall, our results suggest that the expression of VR1 on thymocytes may function as a sensor of harmful stimuli present in the thymic environment.

Topics: Animals; Apoptosis; Calcium Signaling; Capsaicin; Caspases; CD4 Antigens; CD4-Positive T-Lymphocytes; CD5 Antigens; Cell Membrane; Cells, Cultured; DNA Fragmentation; Dose-Response Relationship, Drug; Intracellular Membranes; Lymphocyte Subsets; Male; Mitochondria; Necrosis; Rats; Rats, Wistar; Receptors, Cell Surface; Receptors, Drug; RNA, Messenger; T-Lymphocytes