The efficacy of combination therapy using adeno-associated virus-TRAIL targeting to telomerase activity and cisplatin in a mice model of hepatocellular carcinoma

Abstract

Purpose: TNF-related apoptosis-inducing ligand (TRAIL) functions as a soluble cytokine and has been demonstrated significant antitumor activity against a variety of cancer cell lines without toxicity to most normal cells. Cisplatin is a potent anticancer agent and is widely used in the clinical for treatment of human cancers. Adeno-associated virus (AAV2) is a promising gene delivery vehicle for its advantage of low pathogenicity and long-term gene expression. However, lack of tissue specificity caused low efficiency of AAV transfer to target cells. The promoter of human telomerase reverse transcriptase (hTERT) is a good candidate to enhance targeting efficiency of AAV in cancer cells. Although AAV-mediated TRAIL controlled by hTERT promoter (AAV-hTERT-TRAIL) has obvious antitumor activity, the tumor cannot be completely eradicated. In this study, we first examined the effectiveness of combination therapy of cisplatin and AAV-hTERT-TRAIL on human hepatocellular carcinoma (HCC) in vitro and in vivo.

Methods: For in vitro experiments, tumor cell lines were treated with cisplatin, virus, or both. The transgene TRAIL expression controlled by hTERT promoter was evaluated in BEL7404 HCC cell line. Cytotoxicity was performed by MTT analysis. Cell apoptosis was detected by flow cytometry analysis. The in vivo antitumor efficacy of combination treatment with cisplatin and AAV-hTERT-TRAIL was assessed in human hepatocellular carcinoma xenografts mouse model.

Results: The enhanced TRAIL expression was observed in BEL7404 cells treated with AAV-hTERT-TRAIL plus cisplatin. Treatment with both AAV-hTERT-TRAIL and cisplatin exhibited stronger cytotoxicity and induced more significant apoptosis in cancer cells compared with AAV-hTERT-TRAIL or cisplatin alone, respectively. Moreover, in animal experiments, the combined treatment greatly suppressed tumor growth and resulted in tumor cell death.

Conclusions: AAV-mediated therapeutic gene expression in combination with chemotherapy provides a promising therapeutic strategy for human cancers. These data suggest that combined use of AAV-hTERT-TRAIL and cisplatin may have potential clinical application.

Fig. 1

Identification of AAV-hTERT-TRAIL. a RT–PCR analysis for detection of TRAIL expression. RNA was extracted from BEL7404 cells infected with AAV-hTERT-TRAIL (5 × 10⁴v.g/cell), cisplatin 3 μg/ml, PBS or AAV-hTERT-TRAIL plus cisplatin and TRAIL expression was detected by RT–PCR two days later. β-actin served as an internal control. The DNA fragments of 846 bp (TRAIL) and 378 bp (β-actin) were amplified. M, DNA marker (DL 2000, Takara); (1) treated with AAV-hTERT-TRAIL plus cisplatin; (2) treated with AAV-hTERT-TRAIL; (3) cisplatin; (4) PBS. b Identification of TRAIL protein in BEL7404 cells by Western blot. BEL7404 cells were infected with AAV-hTERT-TRAIL (5 × 10⁴ v.g/cell) alone, cisplatin (3 μg/ml) alone, or the combination for 3 days, and cell lysates were harvested for analysis. The β-actin expression was used as protein internal control

Fig. 2

Inhibitory effects of combined AAV-hTERT-TRAIL and cisplatin on BEL7404 cell line by MTT viability assay. L-02 and BEL7404 cell line were infected with AAV-hTERT-TRAIL at different MOI (MOI = 0, 5 × 10³, 5 × 10⁴, 5 × 10⁵v.g/cell), cisplatin at different concentrations (0, 1, 3, 5 μg/ml) or both. After 48 h of post-infection, cell viability was determined by MTT assay. Experiments were repeated three times in quadruplicates. The results are presented as mean ± SD (bars)

Fig. 3

Enhanced suppression effects of tumor cell proliferation by combination of AAV-hTERT-TRAIL with cisplatin. The normal cell line L-02 and three tumor cell lines BEL7404, Hep3B, H1299 were treated with AAV-hTERT-TRAIL (5 × 10⁴v.g/cell), cisplatin (3 μg/ml), or both. Then, cell viability was measured by MTT assay from day 1 to day 4. Data were represented as mean ± SD (n = 4). The experiment was repeated twice with essentially the same results

Fig. 4

Apoptosis detection of tumor cells mediated by combined AAV-hTERT-TRAIL and cisplatin. BEL7404 cells were treated with PBS, AAV-hTERT-TRAIL (5 × 10⁴v.g/cell), cisplatin (3 μg/ml), or both, and three days later, the cells were harvested and stained with annexin V-FITC and immediately followed by flow cytometry for apoptosis assay. The percentage of apoptotic cells was calculated with CellQuest software. Each value represents the mean of 3 wells

Fig. 5

Morphological evaluation. a L-02 cells and BEL7404 cells were treated with AAV-hTERT-TRAIL (5 × 10⁴v.g/cell), cisplatin (3 μg/ml), or both and morphological changes of cell death or apoptosis were analyzed by microscopy after 72 h. (original magnification: 100×). b Cells were treated as mentioned earlier and incubated with Hoechst 33342 for 30 min, and condensation and fragmentation of nuclei were observed under a fluorescence microscope (as the arrow indicated). (Hoechst 33342 stain; original magnification: 400×)

Fig. 6

Antitumor effect of AAV-hTERT-TRAIL and cisplatin in nude mice. Tumor xenografts were established by subcutaneous injection of BEL7404 cells (5 × 10⁶) into the right flank of nude mice. When the mean tumor size reached 100–150 mm³, the mice were divided into four groups (n = 6) and intratumorally injected with PBS and AAV-hTERT-TRAIL (5 × 10¹⁰ v.g dose per mice) for three consecutive daily, intraperitoneally administered cisplatin (4 mg/kg of body weight) for five consecutive daily, AAV-hTERT-TRAIL (5 × 10¹⁰ v.g dose per mice, days 1–3) followed by cisplatin (4 mg/kg of body weight, days 4–8) (treatment indicated by arrow). a Tumor growth after various treatments. Tumor volumes were estimated as: tumor volume V (mm³) = 1/2 × Length (mm) × Width (mm)². Points, mean; bars, SEM. NS, P > 0.05; *, P < 0.05; **, P < 0.01. b Kaplan–Meier survival of animals after the different treatments. Statistical significance: a, P < 0.05 compared with PBS; b, P < 0.05, compared with cisplatin; c, P < 0.02, compared with AAV-hTERT-TRAIL

Fig. 7

Evidence for AAV-hTERT-TRAIL in combination with cisplatin on tumor cell death in vivo. a Hematoxylin and eosin (HE) staining analysis. BEL7404 xenograft tumors were treated as mentioned earlier. Tumor sections were excised, fixed, dewaxed, followed by standard HE staining. Tumor tissues treated with AAV-hTERT-TRAIL plus cisplatin showed more cell death than others groups. The arrow denotes the death of tumor cells. (original magnification 200×). b Detection of apoptotic cells in tumor tissue by TUNEL assay. Tumor sections were processed as described in Materials and Methods. The combined treatment with AAV-hTERT-TRAIL and cisplatin induced more tumor cell apoptosis in tumor mass than AAV-hTERT-TRAIL alone (as indicated by the arrows; original magnification 400×)