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Chloroquine potentiates the anti-cancer effect of lidamycin on non-small cell lung cancer cells in vitro.

Liu F, Shang Y, Chen SZ - Acta Pharmacol. Sin. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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Effect of LDM and CQ on the proliferative activity of A549 and H460 cells. (A) The proliferative activity of A549 and H460 cells treated with LDM (0.0001, 0.001, 0.01, 0.1, 1, or 10 nmol/L) for 48 h assessed by the MTT assay. The y-axis represents the survival rate, calculated as the ratio to the control (untreated cells). (B) The proliferative activity of A549 and H460 cells treated with CQ (20–120 μmol/L) for 24, 48 and 72 h assessed by the MTT assay. The y-axis represents survival rate, calculated as the ratio to the control (untreated cells). Values were given as mean±SD. n=3. Each independent experiment was performed in triplicate.
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fig1: Effect of LDM and CQ on the proliferative activity of A549 and H460 cells. (A) The proliferative activity of A549 and H460 cells treated with LDM (0.0001, 0.001, 0.01, 0.1, 1, or 10 nmol/L) for 48 h assessed by the MTT assay. The y-axis represents the survival rate, calculated as the ratio to the control (untreated cells). (B) The proliferative activity of A549 and H460 cells treated with CQ (20–120 μmol/L) for 24, 48 and 72 h assessed by the MTT assay. The y-axis represents survival rate, calculated as the ratio to the control (untreated cells). Values were given as mean±SD. n=3. Each independent experiment was performed in triplicate.

Mentions: Cell viability decreased after treatment with LDM for 48 h in a dose-dependent manner (Figure 1A). The IC50 values (95% confidence limits) were (1.70±0.75) nmol/L for A549 cells and (0.043±0.026) nmol/L for H460 cells. On the other hand, CQ inhibited the proliferation of A549 and H460 cells in a time- and dose-dependent manner. The IC50 values of CQ at 48 h were 71.3±6.1 μmol/L for the A549 cells and 55.6±12.5 μmol/L for H460 cells (Figure 1B). The H460 cells were more sensitive to LDM or CQ than the A549 cells. Based on these results, we selected the dose of 25 or 50 μmol/L of CQ in a 48-h-treatment for the further experiments.


Chloroquine potentiates the anti-cancer effect of lidamycin on non-small cell lung cancer cells in vitro.

Liu F, Shang Y, Chen SZ - Acta Pharmacol. Sin. (2014)

Effect of LDM and CQ on the proliferative activity of A549 and H460 cells. (A) The proliferative activity of A549 and H460 cells treated with LDM (0.0001, 0.001, 0.01, 0.1, 1, or 10 nmol/L) for 48 h assessed by the MTT assay. The y-axis represents the survival rate, calculated as the ratio to the control (untreated cells). (B) The proliferative activity of A549 and H460 cells treated with CQ (20–120 μmol/L) for 24, 48 and 72 h assessed by the MTT assay. The y-axis represents survival rate, calculated as the ratio to the control (untreated cells). Values were given as mean±SD. n=3. Each independent experiment was performed in triplicate.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4814038&req=5

fig1: Effect of LDM and CQ on the proliferative activity of A549 and H460 cells. (A) The proliferative activity of A549 and H460 cells treated with LDM (0.0001, 0.001, 0.01, 0.1, 1, or 10 nmol/L) for 48 h assessed by the MTT assay. The y-axis represents the survival rate, calculated as the ratio to the control (untreated cells). (B) The proliferative activity of A549 and H460 cells treated with CQ (20–120 μmol/L) for 24, 48 and 72 h assessed by the MTT assay. The y-axis represents survival rate, calculated as the ratio to the control (untreated cells). Values were given as mean±SD. n=3. Each independent experiment was performed in triplicate.
Mentions: Cell viability decreased after treatment with LDM for 48 h in a dose-dependent manner (Figure 1A). The IC50 values (95% confidence limits) were (1.70±0.75) nmol/L for A549 cells and (0.043±0.026) nmol/L for H460 cells. On the other hand, CQ inhibited the proliferation of A549 and H460 cells in a time- and dose-dependent manner. The IC50 values of CQ at 48 h were 71.3±6.1 μmol/L for the A549 cells and 55.6±12.5 μmol/L for H460 cells (Figure 1B). The H460 cells were more sensitive to LDM or CQ than the A549 cells. Based on these results, we selected the dose of 25 or 50 μmol/L of CQ in a 48-h-treatment for the further experiments.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

Show MeSH
Related in: MedlinePlus