Simultaneous targeting of ATM and Mcl-1 increases cisplatin sensitivity of cisplatin-resistant non-small cell lung cancer

Abstract

Development of cisplatin-resistance is an obstacle in non-small cell lung cancer (NSCLC) therapeutics. To investigate which molecules are associated with cisplatin-resistance, we analyzed expression profiles of several DNA repair and anti-apoptosis associated molecules in parental (A549P and H157P) and cisplatin-resistant (A549CisR and H157CisR) NSCLC cells. We detected constitutively upregulated nuclear ATM and cytosolic Mcl-1 molcules in cisplatin-resistant cells compared with parental cells. Increased levels of phosphorylated ATM (p-ATM) and its downstream molecules, CHK2, p-CHK2, p-53, and p-p53 were also detected in cisplatin-resistant cells, suggesting an activation of ATM signaling in these cells. Upon inhibition of ATM and Mcl-1 expression/activity using specific inhibitors of ATM and/or Mcl-1, we found significantly enhanced cisplatin-cytotoxicity and increased apoptosis of A549CisR cells after cisplatin treatment. Several A549CisR-derived cell lines, including ATM knocked down (A549CisR-siATM), Mcl-1 knocked down (A549CisR-shMcl1), ATM/Mcl-1 double knocked down (A549CisR-siATM/shMcl1) as well as scramble control (A549CisR-sc), were then developed. Higher cisplatin-cytotoxicity and increased apoptosis were observed in A549CisR-siATM, A549CisR-shMcl1, and A549CisR-siATM/shMcl1 cells compared with A549CisR-sc cells, and the most significant effect was shown in A549CisR-siATM/shMcl1 cells. In in vivo mice studies using subcutaneous xenograft mouse models developed with A549CisR-sc and A549CisR-siATM/shMcl1 cells, significant tumor regression in A549CisR-siATM/shMcl1 cells-derived xenografts was observed after cisplatin injection, but not in A549CisR-sc cells-derived xenografts. Finally, inhibitor studies revealed activation of Erk signaling pathway was most important in upregulation of ATM and Mcl-1 molcules in cisplatin-resistant cells. These studies suggest that simultaneous blocking of ATM/Mcl-1 molcules or downstream Erk signaling may recover the cisplatin-resistance of lung cancer.

Keywords: ATM; Mcl-1; cisplatin-resistance; cisplatin-sensitivity; non-small cell lung cancer.

Figure 1.

ATM and Mcl-1 expression in parental and cisplatin-resistant lung cancer cells. A. Cisplatin-cytotoxicity tests of A549P/H157CisR and H157P/H157CisR cells. A549CisR and H157CisR cells were obtained by continuous treatment of cells with increasing dose of cisplatin. Cell cytotoxicities of A549P vs. A549CisR and H157P vs. H157CisR cells to varied concentrations of cisplatin were analyzed in MTT assay. B. Western blot analysis. Cytosolic and nucleic cell extracts were obtained from parental (A549P and H157P) and cisplatin-resistant cells (A549CisR and H157CisR) and western blot analyses were performed using antibodies of indicated molecules. C. Western blot analysis. Cytosolic and nucleic cell extracts of A549P and A549CisR were obtained after treatment with cisplatin (near IC₅₀ value of each cell line) for 48 hours and used in Western blot analyses.

Figure 2.

Investigations on ATM downstream signaling in A549P/A549CisR and H157P/H157CisR cells. A. Western blot analysis. Cytosolic and nucleic cell extracts were obtained from A549P/A549CisR and H157P/H157CisR cells (non-cisplatin treated) and used in Western blot analyses using antibodies of indicated molecules. B. IF staining. Cells (A549P/A549CisR and H157P/H157CisR) were plated in chamber slides (without treating with cisplatin) and IF staining was performed using antibodies of p-ATM and p-p53. Quantitation shown on right. Error bars and significance values were obtained by counting positively stained cells in one randomly chosen area of 3 different stainings. Magnification, 20X. *p < 0.05, **p < 0.01

Figure 3.

Test effects of selective inhibitors or knockdown strategy of ATM and/or Mcl-1 molcules in altering cisplatin-sensitivity and apoptotic death of cisplatin-resistant cells upon cisplatin treatment. A. Western blot analysis showing inhibition of ATM signaling and Mcl-1 expression in A549CisR cells upon incubation with ATM/Mcl-1 inhibitor(s) (veh; vehicle, CP; CP466722, UMI; UMI77, C + U, combined use of CP466722 plus UM177). B. Cisplatin-cytotoxicity tests of A549P and A549CisR in the absence and presence of ATM/Mcl-1 inhibitor(s). Cell survival upon treatment with varied concentrations of cisplatin was analyzed in MTT assay. C. Western blot analysis of apoptotic markers in A549P and A549CisR cells upon cisplatin treatment, in the absence and presence of ATM/Mcl-1 inhibitors. Cell (A549P and A549CisR) were treated with cisplatin (10 μM and 30 μM cisplatin were used for treating A549P and A549CisR cells, respectively) for 48 hours, in the absence and presence of inhibitors, and cell extracts were obtained. Expression of apoptotic markers were examined in Western blot analyses. D. Western blot analysis testing ATM and Mcl-1 levels in A549CisR-sc, A549CisR-siATM, A549CisR-shMcl1, and A549CisR-siATM/shMcl1 cells. Cell extracts were obtained from A549CisR-sc, A549CisR-ATMsi, A549CisR-Mcl1si, and A549CisR-ATMsi/Mcl1si cells and ATM-1 and Mcl-1 levels were examined by Western blot analysis. E. Cisplatin-cytotoxicity tests of A549CisR-sc, A549CisR-siATM, A549CisR-shMcl1, and A549CisR-siATM/shMcl1 cells. Cells were treated with indicated concentration of cisplatin and cell survival was analyzed by MTT assay. F. Western blot analyses investigating expression of the apoptotic markers A549CisR-sc, A549CisR-siATM, A549CisR-shMcl1, and A549CisR-siATM/shMcl1 cells upon cisplatin treatment. Cell extracts were obtained from and expression of apoptotic markers were examined by Western blot analyses. G. Flow cytometric analysis of apoptotic cells. A549CisR-sc and A549CisR-siATM/shMcl1 cells were treated with cisplatin (30 μM) for 24 hours and apoptotic cells (%) were analyzed in AnnexinV based flow cytometric analyses. The graph shown right indicates average values obtained from 3 independent experiments. *p < 0.05

Figure 4.

In vivo mice studies. A. IHC staining of tumor tissues (ATM/Mcl-1). Tumor tissues of each mouse group were obtained at sacrifice of mice and 5 μm tissues sections were used in staining using antibodies of ATM and Mcl-1. Quantitation of positively stained cells was shown on right. B. Graphs showing tumor regression upon cisplatin injection into tumor bearing mice. Mice bearing A549CisR-sc and A549CisR-siATM/shMcl1 cells derived tumors were treated with cisplatin (5 mg/kg, vehicle as control) and tumor volume at indicated time points were measured. C. IHC staining of tumor tissues. Tumor tissues of vehicle or cisplatin treated mice in A549CisR-sc and A549CisR-siATM/shMcl1 xenografts were obtained and 5 μm tissues sections were used in staining using Ki67 antibody. Quantitation of positively stained cells was shown on right. Error bars and significance values in IHC staining (A and C) were obtained by counting positively stained cells in one randomly chosen area of slides of 3 different stains. Magnification, 40X. *p < 0.05, **p < 0.01.

Figure 5.

Investigation of signaling pathways responsible for the upregulated ATM/Mcl-1 in cisplatin-resistant cells. A. Western blot analysis investigating signaling pathways responsible for upregulated ATM/Mcl-1 in cisplatin-resistant cells. Cytosolic and nucleic cell extracts were obtained from parental (A549P and H157P) and cisplatin-resistant cells (A549CisR and H157CisR) and western blot analyses were performed using antibodies of indicated signaling molecules. B. Western blot analysis of ATM/Mcl-1 in A549CisR and H157CisR cells upon treatment with inhibitors of each signaling pathway. Cytosolic and nucleic cell extracts were obtained from parental (A549P and H157P) and cisplatin-resistant cells (A549CisR and H157CisR), in the presence of inhibitors of each signaling pathway and levels of ATM and Mcl-1 were analyzed in Western blot analyses. (V: vehicle, SB; SB203850, LY; LY24002, U; U0126) C. Cisplatin-cytotoxicity test of A549P and A549CisR in the presence of inhibitors of each signaling pathway. Cells were treated with indicated concentration of cisplatin, in the presence of inhibitors of each signaling pathway and cell survival was analyzed in MTT assay. Different cisplatin concentration was used in parental cisplatin-resistant cells according to the IC₅₀ value of each cell line.