Fang Liu1, Yue Shang1, Shu-zhen Chen1. 1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Abstract
AIM: To assess the synergistic actions of lidamycin (LDM) and chloroquine (CQ), a lysosomal enzyme inhibitor, in human non-small cell lung cancer (NSCLC) cells, and to elucidate the potential mechanisms. METHODS: Human NSCLC cell lines A549 and H460 were treated with CQ and/or LDM. Cell proliferation was analyzed using MTT assay, and apoptosis was quantified using flow cytometry. Western blotting was used to detect the protein levels of caspase 3, PARP, Bcl-2, Bax, p53, LC3-I and LC3-II. A H460 cell xenograft model in BALB/c nude mice was used to evaluate the anticancer efficacy of CQ and LDM in vivo. RESULTS: Both LDM and CQ concentration-dependently suppressed the proliferation of A549 and H460 cells in vitro (the IC50 values of LDM were 1.70 ± 0.75 and 0.043 ± 0.026 nmol/L, respectively, while the IC50 values of CQ were 71.3 ± 6.1 and 55.6 ± 12.5 μmol/L, respectively). CQ sensitized both NSCLC cell lines to LDM, and the majority of the coefficients of drug interaction (CDIs) for combination-doses were less than 1. The ratio of apoptosis of H460 cells induced by a combined treatment of CQ and LDM (77.0% ± 5.2%) was significantly higher than those caused by CQ (23.1% ± 4.2%) or by LDM (65.1% ± 4.1%) alone. Furthermore, the combined treatment markedly increased the cleaved PARP and cleaved caspase 3 in H460 cells, which were partly reversed by pretreatment with the caspase inhibitor zVAD.fmk. zVAD.fmk also partially reversed the inhibitory effect of the combination treatment on the proliferation of H460 cells. The combination therapy group had a notable increase in expression of Bax and a very slight decrease in expression of Bcl-2 and p53 protein. LDM alone scarcely affected the level of LC3-II in H460 cells, but slightly reduced CQ-induced LC3-II expression. 3-MA, an autophagy inhibitor also sensitized H460 cells to LDM. In nude mice bearing H460 cell xenograft, administration of LDM (25 μg/kg, iv) and CQ (60 mg/kg, ip) suppressed tumor growth by 57.14% and 73.02%, respectively. CONCLUSION: The synergistic anticancer effect of LDM and CQ in vitro results from activation of a caspase-dependent and p53-independent apoptosis pathway as well as inhibition of cytoprotective autophagy.
AIM: To assess the synergistic actions of lidamycin (LDM) and chloroquine (CQ), a lysosomal enzyme inhibitor, in humannon-small cell lung cancer (NSCLC) cells, and to elucidate the potential mechanisms. METHODS:HumanNSCLC cell lines A549 and H460 were treated with CQ and/or LDM. Cell proliferation was analyzed using MTT assay, and apoptosis was quantified using flow cytometry. Western blotting was used to detect the protein levels of caspase 3, PARP, Bcl-2, Bax, p53, LC3-I and LC3-II. A H460 cell xenograft model in BALB/c nude mice was used to evaluate the anticancer efficacy of CQ and LDM in vivo. RESULTS: Both LDM and CQ concentration-dependently suppressed the proliferation of A549 and H460 cells in vitro (the IC50 values of LDM were 1.70 ± 0.75 and 0.043 ± 0.026 nmol/L, respectively, while the IC50 values of CQ were 71.3 ± 6.1 and 55.6 ± 12.5 μmol/L, respectively). CQ sensitized both NSCLC cell lines to LDM, and the majority of the coefficients of drug interaction (CDIs) for combination-doses were less than 1. The ratio of apoptosis of H460 cells induced by a combined treatment of CQ and LDM (77.0% ± 5.2%) was significantly higher than those caused by CQ (23.1% ± 4.2%) or by LDM (65.1% ± 4.1%) alone. Furthermore, the combined treatment markedly increased the cleaved PARP and cleaved caspase 3 in H460 cells, which were partly reversed by pretreatment with the caspase inhibitor zVAD.fmk. zVAD.fmk also partially reversed the inhibitory effect of the combination treatment on the proliferation of H460 cells. The combination therapy group had a notable increase in expression of Bax and a very slight decrease in expression of Bcl-2 and p53 protein. LDM alone scarcely affected the level of LC3-II in H460 cells, but slightly reduced CQ-induced LC3-II expression. 3-MA, an autophagy inhibitor also sensitized H460 cells to LDM. In nude mice bearing H460 cell xenograft, administration of LDM (25 μg/kg, iv) and CQ (60 mg/kg, ip) suppressed tumor growth by 57.14% and 73.02%, respectively. CONCLUSION: The synergistic anticancer effect of LDM and CQ in vitro results from activation of a caspase-dependent and p53-independent apoptosis pathway as well as inhibition of cytoprotective autophagy.
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