Overexpression of tumor suppressor TSLC1 by a survivin-regulated oncolytic adenovirus significantly inhibits hepatocellular carcinoma growth

Abstract

Purpose: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Oncolytic viruses represent a promising therapeutic agent or vehicle to human cancers due to their ability of selectively lysing cancer cells but not in normal cells. TSLC1, a novel tumor suppressor gene, was loss in many human cancers including HCC, not in normal cells. The current study is focused on the antitumor effect of TSLC1-armed survivin-regulated oncolytic adenovirus for HCC and to explore their molecular mechanism.

Methods: The expression of tumor suppressor TSLC1 and survivin was detected by quantitative PCR. The recombinant virus Ad.SP-E1A-E1B((Δ55))-TSLC1 (brief name as SD55-TSLC1) was constructed by inserting TSLC1 gene into the dual-regulated oncolytic adenovirus vector Ad.SP-E1A-E1B((Δ55)). Then, we performed the antitumor experiments of SD55-TSLC1 in vitro and in nude mice xenografted with Huh7 liver cancer.

Results: The expression of TSLC1 was lower in HCC cells than in normal cells, which implied TSLC1 is a tumor suppressor of liver cancer. Survivin expression is higher in detected HCC cells than in normal cells. The SD55-TSLC1 exhibited an excellent antitumor effect on HCC cell growth in vitro but does no or little damage to normal liver cells. Animal experiment further confirmed that SD55-TSLC1 achieved significant inhibition of Huh7 liver cancer xenografted growth. Furthermore, the mechanism of antitumor efficacy by SD55-TSLC1 was elucidated to be due to the activation of caspase apoptotic pathway including the inducement of caspase-3, caspase-8, and poly (ADP-ribose) polymerase cleavage. This is the first report of TSLC1 by oncolytic adenovirus with an excellent antitumor effect to liver cancer growth.

Conclusion: These data suggest that an oncolytic adenovirus expressing TSLC1 is effective and support that SD55-TSLC1 may be a potent antitumoral agent for future clinical trials of liver cancer.

Fig. 1

Schematic diagram of recombinant oncolytic adenovirus structure. All viruses were constructed using the backbone of wild-type Ad5. ITR, inverted terminal repeat

Fig. 2

Characterization of oncolytic adenovirus SD55-TSLC1 and its specific expression in tumor cells. a TSLC1 expression level in HCC cells. TSLC1 mRNAs or proteins extracted from HCC cells (Huh7, PLC, and Bel-7404) and human normal liver cells L02 and QSG-7701 were subjected to real-time quantitative PCR [the upper left, data are presented as means ± SD (bars) (n = 3, *P < 0.05, **P < 0.01)] and Western blot analysis (the lower left). Characterization of SD55-TSLC1 was assayed by Western blot. HCC cell lines and normal liver cells were infected with SD55-TSLC1 at a multiplicity of infection (MOI) of 10 pfu/cell, and the E1A protein (the upper right) and TSLC1 expression (the lower right) were detected after 48 h. b Relative expression of survivin and assay of virus infection with reporter gene. The relative level of survivin mRNA in three tumor cells and two normal cells was examined by real-time quantitative PCR. GAPDH was used as an internal control. Data are presented as means ± SD (bars) (n = 3, *P < 0.05, **P < 0.01). Cells were plated in 24-well plates and then infected with SD55-EGFP at a MOI of 10. After virus infection for the indicated time, EGFP expression was detected using a fluorescence microscope

Fig. 3

Viral progeny assay. Tumor cells and normal cells were seeded in 6-well plates and were infected with SD55-TSLC1 and SD55-EGFP or with ONYX015 at a multiplicity of infection (MOI) of 10. After 5 h, medium was removed, cells were washed with phosphate-buffered saline (PBS) 3 times, and then 2 ml of fresh medium was added. Two days later, cells were collected and virus was released by three freeze-thawing cycles and centrifuged to collect the supernatant. Virus production was determined by TCID50 assay in HEK293 cells

Fig. 4

Cytotoxicity of SD55-TSLC1 in tumor cells in vitro. The HCC cell lines (Huh7, PLC, and Bel-7404) and normal liver cell lines (L02 and QSG-7701) were infected with SD55-TSLC1, SD55-EGFP, or ONYX-015 at a MOI of 1, 2, 5, and 10. 96 h later, and cell viability rate was measured by MTT assay. The results are presented as the mean ± SD (n = 4) and are expressed as the percentage relative to mock-treated control cells

Fig. 5

Tumor-specific cytopathic effect induced by SD55-TSLC1. Three HCC cell lines (Huh7, PLC, and Bel-7404) and two normal cell lines (L02 and QSG-7701) were seeded in 24-well plates as a density of 5 × 10⁴ cells per well and infected with SD55-TSLC1, SD55-EGFP, and ONYX-015 at the indicated MOIs. Four days later, cells were stained with crystal violet

Fig. 6

Antitumor efficacy of SD55-TSLC1 in nude mice. Female BALB/c nude mice were subcutaneously inoculated with Huh7 cells (5 × 10⁶). When tumors reached 100–130 mm³, the animals were treated with SD55-TSLC1, SD55-EGFP, ONYX-015, or PBS via intratumoral injection. a Tumor size was measured, and tumor volume was calculated. Data are presented as means ± SD (n = 8, **P < 0.01, compared with PBS, ONYX-015, SD55-EGFP). b Survival rate of animals was monitored before the end of the animal experiment. A pair-wise log-rank test was used to analyze survival rates in the different groups. Statistical significance: a, P < 0.05, compared with PBS; b P < 0.05, compared with ONYX015; c P < 0.05, compared with SD55-EGFP; d P > 0.05, SD55-EGFP compared ONYX015

Fig. 7

SD55-TSLC1 induces apoptosis of HCC cells and activates caspase-mediated apoptosis signaling pathway. a Apoptosis detection by Hoechst 33342 staining. Cells were plated in 6-well plates and infected with SD55-TSLC1, SD55-EGFP, and ONYX-015 at a MOI of 10, and uninfected cells served as control. 72 h later, cells were treated with Hoechst 33342 staining at 1 mg/ml for 30 min and then observed under the inverted fluorescence microscope. Arrows indicate positive apoptotic cells. Original magnification, ×200. b Activation of caspase pathway member by SD55-TSLC1. The Huh7 cells were treated with the SD55-TSLC1 at a MOI of 10. 48 h later, cells were harvested and examined by Western blot analysis. Activation of caspase-3, caspase-8, and the downstream apoptotic protein poly (ADP-ribose) polymerase (PARP) was also detected. Actin was used as the internal control

Fig. 8

SD55-TSLC1 induced cell death in vivo. Subcutaneous Huh7 tumors receiving various treatments were harvested 4 days after infection, and tumor sections were treated as described in “Materials and methods”. The upper row is hematoxylin and eosin (HE) staining analysis. Tumor tissues treated with SD55-TSLC1 showed more cell death than other groups. Vessel growth in tumors treated with SD55-TSLC1 was significantly suppressed, while some blood vessels are clearly seen in control-treated groups. The middle row showed adenovirus hexon and TSLC1 expression by IHC analysis in tumor tissues. The lower row is TUNEL assay for detecting apoptotic cells in tumor tissues treated by indicated treatment. SD55-TSLC1 induced more apoptosis of tumor cells. The brown color represents the apoptotic cells. Original magnification: ×200

Fig. 9

Morphological observation of cell apoptosis and detection of maculae adherens by TEM analysis. a Morphological observation of cell apoptosis. The significant apoptosis in tumors treated with SD55-TSLC1 was detected than other treatments, including nuclear collapse, an increased nuclear-to-cytoplasmic ratio, appearance of nucleus deformation, and chromatin condensed in lumps. b Detection of maculae adherens in tumor tissues. The enhancement of maculae adherens was observed in tumor tissues treated with SD55-TSLC1