Synergistic suppression effect on tumor growth of hepatocellular carcinoma by combining oncolytic adenovirus carrying XAF1 with cisplatin

Abstract

Purpose: The potent anticancer efficacy of oncolytic viruses has been verified in Clinic in recent years. Cisplatin (DDP) is one of most common chemotherapeutic drugs, but is accompanied by side effects and drug resistance. Our previous studies have shown the strategy of cancer -targeting gene-viro-therapy (CTGVT) mediated by the oncolytic virus ZD55 containing the XAF1 cDNA (ZD55-XAF1), which exhibited potent antitumor effects in various tumor cells and no apparent toxicities on normal cells. In the study, the CTGVT strategy is broadened by combining DDP with ZD55-XAF1 for growth inhibition of hepatocellular carcinoma (HCC) cells.

Methods: The transgenic expression was evaluated by both in vitro and in vivo experiments, and the enhanced inhibitory effect of ZD55-XAF1 combined with cisplatin was assessed in HCC cells. The cytotoxicity on normal liver cells was evaluated by MTT assay and apoptotic cell staining. Activation of caspase-9 and PARP for apoptosis was further detected by Western blot analysis. The in vivo antitumor efficacy of combination treatment with cisplatin and ZD55-XAF1 was estimated in an HCC xenograft mouse model.

Results: We found that the combination of ZD55-XAF1 and cisplatin showed enhanced inhibitory effects on the proliferation of HCC cells in vitro and tumor growth in mice. Furthermore, the combined treatment of ZD55-XAF1 and DDP decreases the chemotherapy dose needed to achieve the same inhibitory effect without overlapping toxicities on normal liver cells and induces tumor cell apoptosis via the activation of caspase-9/PARP pathway.

Conclusion: Thus, these data suggest that the chemo-gene-viro-therapeutic strategy by combining ZD55-XAF1 and DDP reveals a novel therapeutic strategy for hepatocellular carcinoma.

Fig. 1

Characterization of ZD55-XAF1. a The expression of XAF1 was detected by RT-PCR. Lane M: DNA marker; lane 1: negative control; lane 2: DDP; lane 3: ZD55-XAF1; lane 4: ZD55-XAF1 + DDP. b Detection of XIAP in L-02, BEL-7404, SMMC-7721 and Hhu7. Lane 1: L-02; lane 2: BEL-7404; lane 3: SMMC-7721; lane 4: Huh7. c Expression of E1A in BEL-7404. Lane 1: negative control; lane 2: DDP; lane 3: ZD55-XAF1 + DDP; lane 4: ZD55-XAF1. d Expression of HA-XAF1 in BEL-7404. Lane 1: negative control; lane 2: DDP; lane 3: ZD55-XAF1 + DDP; lane 4: ZD55-XAF1

Fig. 2

Enhanced suppression of HCC cell proliferation by the combination of ZD55-XAF1 and DDP. Three liver cancer cell lines BEL-7404, SMMC-7721, and Huh7 were treated with ZD55-XAF1 or ZD55-XAF1 + DDP at various MOIs. Cell viability was determined by MTT assay, and data are presented as mean ± SD of three independent experiments

Fig. 3

The cytotoxicity of ZD55-XAF1 and/or DDP or DOX. BEL-7404 and L-02 cells were treated with ZD55-XAF1 (1 MOI), DDP (3 μg/mL), DOX (3 μg/mL), ZD55-XAF1 (1 MOI) + DDP (3 μg/mL), or ZD55-XAF1 (1 MOI) + DOX (3 μg/mL). Cell viability was determined by MTT assay at consecutive 4 days, and data are presented as mean ± SD of three independent experiments

Fig. 4

The decrease of DDP dose by combining ZD55-XAF1 with DDP. a The combination decreases the dose of DDP. BEL-7404 and L-02 cells were treated with ZD55-XAF1 + DDP or DDP with indicated concentration, respectively, and 48 and 72 h later. Cell viability was determined by MTT assay. b Suppression of BEL-7404 cell growth by various combinations. BEL-7404 cells were infected with ZD55-XAF1 at different MOI (MOI = 0, 1, 5, 10), DDP at different doses (0, 1, 3, 5 ug/mL) or their combination. Cell viability was determined by MTT assay, and the above data are presented as mean ± SD of three independent experiments

Fig. 5

Apoptosis detection of tumor cells induced by ZD55-XAF1 or combined with DDP/DOX. A Apoptosis detection by Hoechst 33342 staining. BEL-7404 and L-02 cells were treated with a PBS. b ZD55-XAF1 (1 MOI). c DDP (3 μg/mL). d ZD55-XAF1 (1 MOI) + DDP (3 μg/mL). e DOX (2 μg/mL). f ZD55-XAF1 (1 MOI) + DOX (2 μg/mL). Condensation and fragmentation of nuclei were observed under a fluorescence microscope as arrowed (original magnification: ×200). B Flow cytometric analysis of apoptosis. BEL-7404 and L-02 were treated with PBS, ZD55-XAF1 (1 MOI), DDP (3 ug/mL), or both. The cells were harvested 3 days later and stained with PI before flow cytometry. The group of ZD55-XAF1 plus DDP was used to compare with the other groups. *p < 0.05; **p < 0.01, NS nonsignificance (p > 0.05)

Fig. 6

Activation of caspase-mediated apoptosis signaling pathway. BEL-7404 cells were treated with PBS, DDP (3 μg/mL), ZD55-XAF1 (4 MOI), or ZD55-XAF1 (4 MOI) + DDP (3 μg/mL). 48 h later, cells were harvested and examined by Western blot analysis. Activation of caspase-9 and the downstream apoptotic protein PARP was also detected. GAPDH was used as the internal control

Fig. 7

Antitumor effect of ZD55-XAF1 and cisplatin in nude mice. Female BALB/c nude mice were subcutaneously inoculated with HCC Bel-7404 cells (1 × 10⁷). After 10 days, when tumors reached a size of 70–120 mm³, the animals were treated with an intratumoral injection of PBS, DDP (4 mg/kg of body weight for four consecutive day), ZD55-XAF1 (1 × 10⁹ PFU dose per mice for four times), ZD55-XAF1 (1 × 10⁹ PFU dose per mice for four times every other day) followed by DDP (4 mg/kg of body weight). Tumor volume (V) was calculated according to the formula: V (mm³) = 1/2 × length × width²

Fig. 8

Evidence for ZD55-XAF1 in combination with cisplatin on tumor growth inhibition and toxicity in vivo. Subcutaneous Bel-7404 tumors and liver tissue receiving various treatments were harvested 7 days after injection, and sections were treated by different methods. The upper row is hematoxylin and eosin (HE) staining analysis. Tumor tissues treated with ZD55-XAF1 in combination with DDP showed more cell death than other groups (original magnification: ×400). In the middle, the IHC analysis showed XAF1 was effectively expressed in tumors regardless of the presence of DDP. TUNEL was performed and showed enhanced apoptotic treated with ZD55-XAF1 plus DDP. The lowest row is HE staining for mice liver section, and no obvious toxicity was found in liver treated by ZD55-XAF1 alone or with DDP, compared with control animals

Fig. 9

Morphological observation of Bel-7404 xenograft tumor by TEM analysis. The more obvious apoptotic phenomena, such as nuclear collapse, appearance of nucleus deformation, and the chromatin condensed in lumps, were detected in tumor samples treated with ZD55-XAF1 plus DDP, over the indications seen in other treated groups