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Bing-Sang Wong,1 Ling-Yen Chiu,2 Dom-Gene Tu,2,3 Gwo-Tarng Sheu,4– 6,* Ting-Tat Chan7,* 1Division of Neurosurgery, Antai Medical Care Corporation Antai Tian-Sheng Memorial Hospital, Pingtung County, Taiwan; 2Department of Nuclear Medicine, Ditmanson Medical Foundation, Chia-Yi Christian Hospital, Chiayi City, Taiwan; 3Department of Biomedical Sciences, National Chung Cheng University, Chiayi 62102, Taiwan; 4Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan; 5Immunology Research Center, Chung Shan Medical University, Taichung, Taiwan; 6Department of Medical Oncology and Chest Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan; 7Palliative Care Unit, Department of Family Medicine, Ditmanson Medical Foundation, Chia-Yi Christian Hospital, Chiayi City, Taiwan*These authors contributed equally to this workCorrespondence: Gwo-Tarng SheuInstitute of Medicine, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Road, Taichung City 402, TaiwanTel +886-4-24730022 Ext 11692Fax +886-4-24751101Email raymondsheu@gmail.comPurpose: Hypertension and cancer are frequently found comorbidity occurring in same individual. This study was intended to evaluate the anticancer effects of commonly used antihypertensive medications and chemotherapy on chemoresistant lung cancer cells.Methods: Calcium channel blockers (CCBs), including Verapamil, Diltiazem, and Nifedipine, either alone or combined with docetaxel (DOC) or vincristine (VCR) were used to treat A549 lung adenocarcinoma chemoresistant sublines. Cell viability was determined by MTT assay, and colony formation assay was used to demonstrate the long-term effect of CCBs on proliferation of the sublines. Apoptosis was evaluated by Annexin V assay and autophagy intensity was quantitated from acidic vesicular organelle formation. Pan-caspase inhibitor, shATG5 interference and chloroquine were applied to study the roles of Verapamil on apoptosis and autophagy, with related proteins verified by Western blot analysis.Results: Results show that 10 μM of Verapamil and Diltiazem, but not Nifedipine, differentially induce autophagy in DOC-resistant or VCR-resistant A549 cells, respectively. When CCBs are combined with DOC or VCR to treat the sublines, 10 μM of Verapamil induces autophagy more significantly than Diltiazem and Nifedipine, respectively, in DOC-resistant (54.91± 0.76, 18.03± 0.69, 7.05± 0.30) or VCR-resistant A549 (32.41± 1.04, 21.51± 0.63, 7.14± 0.24) cells. Inhibition of apoptosis by pan-caspase inhibitor partly reduced cell death indicates association of caspase-dependent cell death but with persistence of autophagy. Inhibition of autophagy by interfering ATG5 expression reduced c-PARP level and apoptotic cells suggest a pro-death role of autophagy. Chloroquine treatment enhanced autophagosome accumulation and cell death but with reduced c-PARP level suggests that mechanism of caspase-independent cell death also contributes to Verapamil/chemotherapy-induced anticancer effects.Conclusion: Verapamil combined with DOC or VCR induces chemoresistant lung cancer cells to death through autophagy burst and apoptosis more strongly than Diltiazem and Nifedipine. Administering Verapamil or Diltiazem individually with chemotherapy, but not Nifedipine, can be considered in lung cancer patients with hypertension.Keywords: hypertension, calcium channel blockers, lung cancer, Verapamil, Diltiazem, Nifedipine, chemoresistance

Pemetrexed (MTA) is a multitargeted antifolate drug approved for lung cancer therapy. Clinically, supplementation with high doses of folic acid (FA) and vitamin B12 (VB12) lowers MTA cytotoxicities. An antagonistic effect of FA/VB12 on MTA efficacy has been proposed. However, patients who receive FA/VB12 show better tolerance to MTA with improved survival. The aims of this study are to investigate the modulation of FA and VB12 on MTA drug efficacy in human nonsmall cell lung cancer (NSCLC) cell lines. The sensitivities of cells, apoptosis, and MTA-regulated proteins were characterized to determine the possible effects of high doses of FA and VB12 on MTA efficacy. MTA has the lowest efficacy under 10% serum conditions. However, supplementation with FA and VB12 individually and additively reversed the insensitivity of NSCLC cells to MTA treatment with 10% serum. The enhanced sensitivities of cells following FA/VB12 treatment were correlated with increasing apoptosis and were specific to MTA but not to 5-fluorouracil (5-FU). Enhanced sensitivity was also associated with p21(WAF1/Cip1) expression level. Our results revealed no antagonistic effect of high doses of FA/VB12 on MTA efficacy in cancer cells grown in nutrient medium. Furthermore, these data may partially explain why supplementation of FA and VB12 resulted in better survival in MTA-treated patients.