Nutlin-3 cooperates with doxorubicin to induce apoptosis of human hepatocellular carcinoma cells through p53 or p73 signaling pathways

Abstract

Purpose: Despite recent advances in chemotherapeutic agents for Hepatocellular carcinoma (HCC) treatment, the results of chemotherapy remain unsatisfactory. Doxorubicin (DOX) still represents the cornerstone in HCC chemotherapy, but resistance and toxicity to normal cells are major obstacles to successful chemotherapy. Therefore, new active agents in HCC chemotherapy and agents that increase the chemosensitivity of HCC cells to DOX are still urgently required. Nutlin-3 is a small-molecule inhibitor that acts to inhibit murine double minute-2 (MDM2) binding to p53 or p73, and subsequently activates p53- or p73-dependent apoptosis signaling pathway. This study was designed to investigate whether Nutlin-3 alters cell toxicity to HCC cells following DNA damage and to assess the suitability of DOX/Nutlin-3 as a chemotherapeutic combination in HCC chemotherapy.

Methods: Four human HCC cells were analyzed using cell proliferation assay, apoptosis assay, western blotting, co-immunoprecipitation and siRNA experiments. Anti-tumoral effects of Nutlin-3/DOX targeting the p53/MDM2 and p73/MDM2 pathways were evaluated in HCC cell lines.

Results: Nutlin-3 enhances the growth inhibition by DOX and potentates the apoptotic effect in all HCC cell lines with different p53 types. Nutlin-3 acts through the disruption of p53-MDM2 binding in HepG2, and the disruption of p73-MDM2 in Huh-7 and Hep3B cell lines with subsequent activation of the apoptotic pathway, which leads to the increase in chemosensitivity to DOX in HCC cells.

Conclusions: Taken together, our findings suggest that Nutlin-3 will be active in the treatment of HCC and offers new prospects for overcoming DOX resistance.

Fig. 1

Inhibitory effects of DOX/Nutlin-3 on the growth of HCC cells. SK-Hep-1, HepG2, Huh-7 and Hep3B cells were exposed to increasing concentrations (0–5 μg/ml) of DOX in the absence or presence of Nutlin-3 (10 μM) for 48 h followed by the CCK-8 assay. All assays were done in triplicates. Error bars represent the standard deviation of at least three independent experiments done in triplicates

Fig. 2

Induction of apoptosis in HCC cells and p73/p53 siRNA blocks Nutlin-enhanced apoptosis induced by DOX. Quantification of apoptotic cells (cell death) determined by flow cytometry. Error bars represent the standard deviation of at least three independent experiments done in triplicates. *P < 0.05 versus untreated control, **P < 0.05 versus cells treated with DOX alone; ***P < 0.05 versus negative siRNA-transfected cells treated with DOX/Nutlin-3

Fig. 3

Effects of Nutlin-3 and DOX on apoptosis-related proteins. HepG2, Huh-7 and Hep3B cells were exposed to various concentrations of drugs for 24 h, and the target proteins were detected by western blot analyses. Western blots were prepared as described in “Materials and methods”

Fig. 4

a Nutlin-3 inhibits binding of p53 and p73 to MDM2 when combined with DOX and increases p53 and p73 activity in human HCC cell lines. Untreated HCC cells, cells treated with 1.25 μg/ml DOX and cells treated with DOX/Nutlin-3 (10 μM) for 24 h were immunoprecipitated with an anti-MDM2 antibody. Immunocomplexes were subjected to immunoblotting with anti-MDM2, p53 and p73 antibodies. Equal amounts of whole-cell lysates were blotted with the same antibodies to demonstrate equal loading. b A schematic presentation for the synergistical anticancer mechanisms of DOX/Nutlin-3 combination in the current study