Nutlin-3 overcomes arsenic trioxide resistance and tumor metastasis mediated by mutant p53 in Hepatocellular Carcinoma

Abstract

Background: Arsenic trioxide has been demonstrated as an effective anti-cancer drug against leukemia and solid tumors both in vitro and in vivo. However, recent phase II trials demonstrated that single agent arsenic trioxide was poorly effective against hepatocellular carcinoma (HCC), which might be due to drug resistance.

Methods: Mutation detection of p53 gene in arsenic trioxide resistant HCC cell lines was performed. The therapeutic effects of arsenic trioxide and Nutlin-3 on HCC were evaluated both in vitro and in vivo. A series of experiments including MTT, apoptosis assays, co-Immunoprecipitation, siRNA transfection, lentiviral infection, cell migration, invasion, and epithelial-mesenchy-mal transition (EMT) assays were performed to investigate the underlying mechanisms.

Results: The acquisition of p53 mutation contributed to arsenic trioxide resistance and enhanced metastatic potential of HCC cells. Mutant p53 (Mutp53) silence could re-sensitize HCC resistant cells to arsenic trioxide and inhibit the metastatic activities, while mutp53 overexpression showed the opposite effects. Neither arsenic trioxide nor Nutlin-3 could exhibit obvious effects against arsenic trioxide resistant HCC cells, while combination of them showed significant effects. Nutlin-3 can not only increase the intracellular arsenicals through inhibition of p-gp but also promote the p73 activation and mutp53 degradation mediated by arsenic trioxide. In vivo experiments indicated that Nutlin-3 can potentiate the antitumor activities of arsenic trioxide in an orthotopic hepatic tumor model and inhibit the metastasis to lung.

Conclusions: Acquisitions of p53 mutations contributed to the resistance of HCC to arsenic trioxide. Nutlin-3 could overcome arsenic trioxide resistance and inhibit tumor metastasis through p73 activation and promoting mutant p53 degradation mediated by arsenic trioxide.

Figure 1

Influence of arsenic trioxide or Nutlin-3 on HCC cell viability and apoptosis. All HCC cell lines were treated with arsenic trioxide or Nutlin-3 for 48 h and 72 h respectively. (A and B) The cell viability was determined using MTT assay. Data are presented as mean ± SD, ***: P < 0.001. (C) The apoptosis was examined using Annexin V-FITC Apoptosis Detection Kit. ***: P < 0.001 compared with the control. (D) The expression of MDM2, p53 and p-gp was investigated by Western blot in indicated cell lines.

Figure 2

Mutp53 plays an important role in HCC arsenic trioxide resistance. (A) Knockdown of mutp53 in HCC resistant cells. (B) MTT assays were performed after cells were treated with arsenic trioxide for 48 h. ***: P < 0.001 versus negative siRNA-transfected cells. (C) Cells were treated with arsenic trioxide (2 μM) for 48 h before performing apoptosis assay. **, P < 0.01 versus negative siRNA-transfected cells. Histograms represent averages of three independent experiments. (D) Cells were treated with arsenic trioxide (2 μM) for 24 h after siRNA transfection (48 h). The target proteins were detected by Western blot analyses. (E) One representative Western blot from three independent experiments demonstrates the accumulation of the V5-tagged exogenous mutant p53 in each cell lines. (F) MTT assays were performed after cells were treated with arsenic trioxide for 48 h. ***: P < 0.001 versus vector control cell lines.

Figure 3

Mutp53 contributed to the increased metastatic potential of HCC resistant cells. (A) The migration and invasive abilities of HCC parental and arsenic trioxide resistant cells were determined by trans-well assays in chambers coated with matrigel (for invasion assays) or without matrigel (for migration assays). Left panels: representative images; right panels: quantifications of average number of cells/field. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Bonferroni post-test. (B) Results of the migration and invasion assays for the HepG2/As and SMMC7721/As cells transfected with p53 siRNA or control siRNA. (**P < 0.01, ***P < 0.001, two-way ANOVA with Bonferroni post-test). (C) Single and merged images were taken to show immunofluorescence staining of N-cadherin (green) and vimentin (red) accompanied by the cell nucleus (blue) stained by DAPI.

Figure 4

The synergetic effects of arsenic trioxide and Nutlin-3 on HCC resistant cells. (A and B) The concentration of arsenic trioxide applied here was 2 μM, and Nutlin-3 was 20 μM. Cell viability and apoptosis were determined after treatment for 48 h. **P < 0.01, ***P < 0.001, versus cells treated with arsenic trioxide or Nutlin-3 alone. (C) Protein expression was compared among untreated, arsenic trioxide, Nutlin-3 and Nutlin-3/arsenic trioxide groups in HepG2/As and SMMC7721/As cells. Cells had been cultured in the medium without any arsenic trioxide for at least one week. The concentration of rhodamine 123 and JC-1 was 0.1 μM. (D) The fluorescence of rhodamine 123 and JC-1 in HepG2/As and SMMC7721/As cells. Cells were incubated with rhodamine 123 and JC-1 for 60 min, and for another 60 min to efflux. **P < 0.01,***P < 0.001. (E) The inhibition of efflux was depended on the concentration of Nutlin-3. Nutlin-3 at different concentration was added with rhodamin 123 or JC-1 into medium and incubated for 60 min. Rho123, rhodamine 123. *P < 0.05, **P < 0.01, ***P < 0.001, versus control cells. (F) Nutlin-3 and R(+) verapamil assisted to increase the intracellular arsenic. All the cells were cultured with 2 μM arsenic trioxide, while treated groups were added 20 μM Nutlin-3 or 40 μM verapamil respectively and incubated for 2 h. *P < 0.05, **P < 0.01, ***P < 0.001. (G) Nutlin-3 inhibits binding of p73 to MDM2 when in combination with arsenic trioxide. Untreated cells, cells treated with 2μM arsenic trioxide, cells treated with 20 μM Nutlin-3, and cells treated with arsenic trioxide/Nutlin-3 combination for 24 h were immune-precipitated with an anti-MDM2 antibody. Immunocomplexes were subjected to immunoblotting with anti-MDM2 and anti-p73 antibodies.

Figure 5

Nutlin-3 potentiates the anti-tumor effects of arsenic trioxide in vivo. (A) Representative bioluminescence images corresponding to the SMMC7721/As orthotopic hepatic tumors formed in the liver of the nude mice. (B) Volume of SMMC7721/As orthotopic tumors was determined at different time points. Data points represent the mean ± SD. (***P < 0.001, two-way ANOVA with Bonferroni post-test). (C) Representative images of sections stained with anti-Ki-67 antibody, or with anti-cleaved caspase-3 antibody. (D) Cells expressing Ki-67 were counted to calculate the proliferation index; cleaved caspase-3-positive cells were counted to give the apoptosis index. (*P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Bonferroni post-test). (E) The representative H&E staining of pulmonary metastasis (arrows) on 5wk (left panel) and the average number of foci per mouse were calculated (right panel); (***P < 0.001, two-way ANOVA with Bonferroni post-test).

Figure 6

A schematic presentation for the possible mechanisms of arsenic trioxide resistance in HCC and the synergistical anticancer mechanisms of arsenic trioxide/Nutlin-3 combination in the current study.