Arsenic trioxide amplifies cisplatin toxicity in human tubular cells transformed by HPV-16 E6/E7 for further therapeutic directions in renal cell carcinoma

Abstract

Human papillomavirus (HPV) DNA integrations may affect therapeutic responses in cancers through ATM network-related DNA damage response (DDR). We studied whether cisplatin-induced DDR was altered in human HK-2 renal tubular cells immortalized by HPV16 E6/E7 genes. Cytotoxicity assays utilized thiazolyl blue dye and DDR was identified by gene expression differences, double-strand DNA breaks, ATM promoter activity, and analysis of cell cycling and side population cells. After cisplatin, HK-2 cells showed greater ATM promoter activity indicating activation of this network, but DDR was muted, since little γH2AX was expressed, DNA strand breaks were absent and cells continued cycling. When HK-2 cells were treated with the MDM2 antagonist inducing p53, nutlin-3, or p53 transcriptional activator, tenovin-1, cell growth decreased but cisplatin toxicity was unaffected. By contrast, arsenic trioxide, which by inhibiting wild-type p53-induced phosphatase-1 that serves responses downstream of p53, and by depolymerizing tubulin, synergistically enhanced cisplatin cytotoxicity including loss of SP cells. Our findings demonstrated that HPV16 E6/E7 altered DDR through p53-mediated cell growth controls, which may be overcome by targeting of WIP1 and other processes, and thus should be relevant for treating renal cell carcinoma.

Keywords: Ataxia telangiectasia mutated; Chemotherapy; DNA damage response; Nephrotoxicity; Renal cell carcinoma.

Figure 1. Cis-P cytotoxicity in HuH-7 and HK-2 cells

A. Phase contrast microscopy for morphological changes in Cis-P-treated HuH-7 and HK-2 cells. Shown are cells with or without IC50 doses of Cis-P. These were calculated with dose response analysis of Cis-P cytotoxicity by MTT cell viability assays and were 15 μM and 25 μM for HuH-7 and HK-2 cells, respectively (B).

Figure 2. Differences in expression of DNA damage-associated genes in Cis-P-treated HuH-7 and HK-2 cells

A. Overview showed significant divergence in ≥2 fold up- or down-regulated genes in HuH-7 cells versus HK-2 cells, p<0.05. B. Representation of DNA damage genes in various ontological classes indicated fewer DDR genes were upregulated in Cis-P-treated HK-2 cells compared to Cis-P-treated HuH-7 cells.

Figure 3. Evidences for ATM-related DDR in Cis-P-treated HuH-7 and HK-2 cells

A. HuH-7 and HK-2 cells with or without Cis-P showed extensive γH2AX expression by immunostaining in the former, whereas γH2AX was expressed less frequently in the latter, as shown by quantitative analysis (B), 70–80% versus 20–40%, p<0.05 (asterisks). C. Analysis of ATM promoter activity with transgene reporter assay using tdT/Hoechst fluorescence with significant increase in Cis-P-treated HuH-7 cells but not in HK-2 cells, p<0.05 (asterisk). D. Photomicrographs showing formation of Comets in Cis-P-treated HuH-7 cells and not in HK-2 cells. E. Quantitation of Comets and Comet tail lengths in Cis-P-treated HuH-7 and HK-2 cells indicated this parameter of double-stranded DNA breaks was also significantly different in these cell lines, p<0.05 (asterisks).

Figure 4. Cell cycle and side population analyses

A. Shows cell cycle profiles in HuH-7 and HK-2 cells with or without Cis-P. The fractions in G0/G1, S or G2/M are given and indicated interference with cycling in both cell lines although the fraction in S was relatively more depleted in HK-2 cells. B. Both HuH-7 and HK-2 cells contained SP cell population as indicated by loss of these cells after treatment with verapamil, which characterizes Hoechst dye efflux in this cell population. After Cis-P, SP cells declined in HuH-7 as well as HK-2 cells, which indicated cytotoxicity in this relatively slower or noncycling cell population.

Figure 5. Effects on Cis-P cytotoxicity with regulation of p53 activity by nutlin-3 or tenovin-1

A. MTT cell viability assays in HuH-7 or HK-2 cells with limited dose-dependent cytostatic effects of nutlin-3, which was consistent with MDM2 antagonism-based activation of p53, as well as of tenovin-1, which indicated possible further transcriptional regulation of p53 activity, although these differences did not reach statistical significance by ANOVA. However, neither nutlin-3 nor tenovin-1 had synergistic effects upon Cis-P cytotoxicity. Asterisks indicate p<0.05.

Figure 6. Regulation in HuH-7 and HK-2 cells of WIP1 by ATO and effects on Cis-P cytotoxicity

A. MTT cell viability assays in HuH-7 and HK-2 cells showed ATO was cytotoxic in both cell lines in 10 μM or 5 μM and 10 μM concentrations, respectively, p<0.05, asterisks, Chi-square tests. Moreover, ATO increased Cis-P cytotoxicity in HK-2 cells, asterisks, p<0.05, whereas that was not the case in HuH-7 cells. B. Western blot showing inhibition of WIP1 compared with control cells (top panel, lanes 1, 2) in ATO-treated HK-2 cells either with or without Cis-P (top panel, lanes 3, 4). Also, reprobing of the blot showed ATO decreased β-tubulin in these cells (middle panel, lanes 3, 4). Ponceau red staining after completion of westerns confirmed proteins were present in all samples (bottom panel). C. Analysis of SP cells after ATO and Cis-P in HuH-7 and HK-2 cells indicated these were depleted in both cases, asterisk, p<0.05. In HK-2 cells, SP fractions were no longer detectable.