Combination of bifunctional alkylating agent and arsenic trioxide synergistically suppresses the growth of drug-resistant tumor cells

Abstract

Drug resistance is a crucial factor in the failure of cancer chemotherapy. In this study, we explored the effect of combining alkylating agents and arsenic trioxide (ATO) on the suppression of tumor cells with inherited or acquired resistance to therapeutic agents. Our results showed that combining ATO and a synthetic derivative of 3a-aza-cyclopenta[a]indenes (BO-1012), a bifunctional alkylating agent causing DNA interstrand cross-links, was more effective in killing human cancer cell lines (H460, H1299, and PC3) than combining ATO and melphalan or thiotepa. We further demonstrated that the combination treatment of H460 cells with BO-1012 and ATO resulted in severe G(2)/M arrest and apoptosis. In a xenograft mouse model, the combination treatment with BO-1012 and ATO synergistically reduced tumor volumes in nude mice inoculated with H460 cells. Similarly, the combination of BO-1012 and ATO effectively reduced the growth of cisplatin-resistant NTUB1/P human bladder carcinoma cells. Furthermore, the repair of BO-1012-induced DNA interstrand cross-links was significantly inhibited by ATO, and consequently, gammaH2AX was remarkably increased and formed nuclear foci in H460 cells treated with this drug combination. In addition, Rad51 was activated by translocating and forming foci in nuclei on treatment with BO-1012, whereas its activation was significantly suppressed by ATO. We further revealed that ATO might mediate through the suppression of AKT activity to inactivate Rad51. Taken together, the present study reveals that a combination of bifunctional alkylating agents and ATO may be a rational strategy for treating cancers with inherited or acquired drug resistance.

Figure 1

Enhanced cytotoxicity of BO-1012 by ATO in human cancer cell lines. (A) Chemical structure of BO-1012. (B) Cytotoxicity of human cancer cell lines to BO-1012 and ATO. Six human cancer cell lines, H460, H1299, PC3, U87, MCF7, and OEC-M1, were treated with various concentrations of BO-1012 or ATO for 72 hours. Cell viability was determined by the WST-1 assay. (C) Enhanced cytotoxicity of BO-1012, melphalan, and thiotepa by ATO cotreatment in three relatively resistant cell lines. Three relatively resistant cells, H460, H1299, and PC3, were exposed to ATO with BO-1012, melphalan, or thiotepa for 72 hours. The cell viabilities and IC₅₀ values of BO-1012 and other two bifunctional alkylating agents in combination with or without ATO were calculated. *P < .05, **P < .001, compared with the IC₅₀ values of each bifunctional alkylating agent alone. (D) Synergistic cytotoxicity of BO-1012 and ATO in H460 cells. H460 cells were treated with BO-1012 for 1 hour, washed, and then treated with ATO for 72 hours. Left: Cell viability analysis was performed. *P < .05, **P < .001 compared with the viability of cells treated with ATO alone. Right: The isobologram analysis of CI against affected fraction (Fa) was obtained by constant ratio combination method as described in Materials and Methods.

Figure 2

Cell cycle perturbation and apoptotic cell death induced by BO-1012, ATO and in combination. H460 cells were treated with 10 or 20 µM BO-1012 for 1 hour, washed, and then treated with 8 µM ATO for 24, 48 or 72 hours. (A) Cell cycle analysis. At the end of treatment, cell cycle analysis was performed by flow cytometry as described in Materials and Methods. Representative DNA histograms of three independent experiments with similar results are shown. Cell cycle distribution was determined as described in Materials and Methods and shown on top of each histogram. (B) Apoptotic cell analysis. At the end of treatment, the cells were subjected to analysis of apoptosis using annexin V staining. Percentages of annexin V⁺ cells were calculated. *P < .05, compared with control at each time point.

Figure 3

Synergistic anticancer activity of ATO and BO-1012 combination on the H460 xenograft. The nude mice with H460 xenograft were injected daily i.v. with 5 mg/kg ATO, 2.5 mg/kg BO-1012, or a combination of both agents for 5 days. (A) Tumor volumes. The numbers of animal in each group are six to seven. The tumor volumes were measured with calipers every 2 or 3 days, and these are expressed as mean ± SE. (B) Representative images of the mice bearing the tumors one the 25th day. The average tumor weights are shown at the bottom. *P < .05 compared with control. (C) Body weight change of mice. The body weights were measured every 2 or 3 days. (D) PCNA immunohistochemistry and TUNEL assay. Xenograft tumor sections from each group at 1 day after the last treatment (i.e., the sixth day after the first treatment) were taken out, sectioned, and subjected for PCNA immunohistochemistry and TUNEL assay as described in Materials and Methods.

Figure 4

Synergistic anticancer activity of ATO and BO-1012 combination on human bladder cancer cells, NTUB1, and derived cisplatinresistant NTUB1/P cells. (A and B) Cell viability analysis of BO-1012, ATO, and the combination of NTUB1 (A) and NTUB1/P (B) cells. Cell viability was assayed as described in Figure 1B. *P < .05 compared with ATO alone at each concentration. Bottom: The isobologram analysis of CI against affected fraction (Fa) was obtained by the constant-ratio combination method as described in Materials and Methods. (C and D) The anticancer activity of BO-1012, ATO, and in combination against NTUB1 (C) and NTUB1/P (D) tumors. The mice bearing NTUB1 or NTUB1/P xenografts were treated with individual drugs or in combination as described in Figure 3A. The numbers of animal in each group are four to six. The tumor volumes were measured with calipers at the indicated time points, and these are expressed as mean ± SE.

Figure 5

Inhibition of the repair of BO-1012 induced DNA damage and exaggeration of DSB formation by ATO. (A) Induction of ICLs by BO-1012, melphalan, and thiotepa. H460 cells were treated with various concentrations of BO-1012, melphalan, or thiotepa for 1 hour and then subjected for a modified comet assay as described in Materials and Methods. Percentage of DNA ICLs was calculated by the percentage of decrease in tailmoment. (B) Inhibition of the repair of BO-1012-induced DNA ICLs by ATO. H460 cells were treated with 40 µM BO-1012 for 1 hour, washed, and then treated with 8 µM ATO for 16, 24, and 48 hours. *P < .05 compared with BO-1012 alone at the indicated time point. (C) Enhanced formation of BO-1012-induced γH2AX nuclei foci by ATO. H460 cells were treated with BO-1012, ATO, or in combination as described previously. After treatment, the detection of γH2AX (green) immunofluorescence staining was performed as described in Materials and Methods. Nuclei were counterstained with DAPI (blue). (D) The percentages of the cells containing four or more γH2AX nuclei foci were determined under a fluorescent microscope. *P < .05 compared with control at each time point.

Figure 6

Inhibition of BO-1012-triggered Rad51 activation by ATO through decreased AKT activity. H460 cells were treated with 40 µM BO-1012 for 1 hour, washed, and then treated with 8 µM ATO for 24 hours. (A) Western blot analysis of Rad51 and DNA-PKcs in nuclear extracts (NE) and whole-cell extracts (WCE). At the end of treatment, an aliquot of NE and WCE were separated on a 10% SDS-polyacrylamide gel. Rad51 and DNA-PKcs were visualized by immunoblot analysis technique as described in Materials and Methods. Oct-1 and β-actin were included on the blot as loading controls for NE and WCE, respectively. (B) Immunofluorescence staining of Rad51. At the end of treatment, the cells were fixed and stained with primary antibody against Rad51 and then Alexa Fluor 555-conjugated secondary antibody (red) as described in Materials and Methods. Nuclei were counterstained with DAPI (blue). (C) Western blot analysis of AKT and pAKT. AKT and pAKT in WCE were analyzed as described in panel A using antibodies against AKT and p-AKT, respectively. (D) Inhibition of BO-1012 induced Rad51 translocation by wortmannin and AKTi. H460 cells were treated with 40 µM BO-1012 for 1 hour and followed with wortmannin (1 µM) or AKTi (20 µM) for 24 hours. After treatments, the nuclear extracts were subjected to Western blot analysis for Rad51 as described in Materials and Methods.