Synergistic antitumor effect of AAV-mediated TRAIL expression combined with cisplatin on head and neck squamous cell carcinoma

Abstract

Background: Adeno-associated virus-2 (AAV-2)-mediated gene therapy is quite suitable for local or regional application in head and neck cancer squamous cell carcinoma (HNSCC). However, its low transduction efficiency has limited its further development as a therapeutic agent. DNA damaging agents have been shown to enhance AAV-mediated transgene expression. Cisplatin, one of the most effective chemotherapeutic agents, has been recognized to cause cancer cell death by apoptosis with a severe toxicity. This study aims to evaluate the role of cisplatin in AAV-mediated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and the effect on HNSCC both in vitro and in vivo.

Methods: Five human HNSCC cell lines were treated with recombinant soluble TRAIL (rsTRAIL) and infected with AAV/TRAIL to estimate the sensitivity of the cancer cells to TRAIL-induced cytotoxicity. KB cells were infected with AAV/EGFP with or without cisplatin pretreatment to evaluate the effect of cisplatin on AAV-mediated gene expression. TRAIL expression was detected by ELISA and Western blot. Cytotoxicity was measured by MTT assay and Western blot analysis for caspase-3 and -8 activations. Following the in vitro experiments, TRAIL expression and its tumoricidal activity were analyzed in nude mice with subcutaneous xenografts of HNSCC.

Results: HNSCC cell lines showed different sensitivities to rsTRAIL, and KB cells possessed both highest transduction efficacy of AAV and sensitivity to TRAIL among five cell lines. Preincubation of KB cells with subtherapeutic dosage of cisplatin significantly augmented AAV-mediated transgene expression in a heparin sulfate proteoglycan (HSPG)-dependent manner. Furthermore, cisplatin enhanced the killing efficacy of AAV/TRAIL by 3-fold on KB cell line. The AAV mediated TRAIL expression was observed in the xenografted tumors and significantly enhanced by cisplatin. AAV/TRAIL suppressed the tumors growth and cisplatin augmented the tumoricidal activity by two-fold. Furthermore, Combination treatment reduced cisplatin-caused body weight loss in nude mice.

Conclusion: The combination of AAV-mediated TRAIL gene expression and cisplatin had synergistic therapeutic effects on head and neck cancers and reduced the potential toxicity of cisplatin. These findings suggest that the combination of AAV/TRAIL and cisplatin may be a promising strategy for HNSCC therapy.

Figure 1

Effects of rsTRAIL and AAV/TRAIL on cell viability in HNSCC cell lines. (A) Treatment with rsTRAIL (50-1000 ng/mL) for 24 h; (B) transfected with AAV/TRAIL (MOI = 1 × 10⁵ v.g.) for 72 h. Values are mean of three independent experiments with error bar representing standard deviation of the mean.

Figure 2

Cisplatin exhibits dose-dependent cytotoxicity and augments AAV/EGFP transduction in KB cell line. (A) Treatment with cisplatin (50-5000 ng/mL) for 72 h. (B) Fluorescence microscopy of KB cells without (a) or with treatment of cisplatin for 2 h before (b) and after (c) AAV/EGFP infection. For the competition experiments, KB cells were infected with the virus and incubated for 2 h at 37°C in the presence of 500 IU heparin sulfate (d and e). Values are mean of three independent experiments with error bar representing standard deviation of the mean.

Figure 3

Cisplatin enhances AAV-mediated TRAIL expression and apoptosis in KB cells. KB cells pre-treated or post-treated with cisplatin were transducted with AAV/TRAIL for 72 h, the cells were harvested and TRAIL expression was analyzed by (A) ELISA assay and (B) Western blot assay. Purified rsTRAIL was used as a standard. The cells pre-treated with cisplatin (200 ng/mL for 2 h) and apoptosis was detected by MTT assay (C) and (D) Western blot analysis for the activation of caspase-8 and -3 in KB cells. Densitometric analysis showing change in optical density of TRAIL, cleaved caspase-3 and -8 bands. GAPDH expression was used as adjustment. Values are mean of three independent experiments with error bar representing standard deviation of the mean. **P < 0.01 compared to the values obtained with the equivalent dose of AAV/TRAIL alone. ##P < 0.01 compared to the sum effect of AAV/TRAIL and cisplatin.

Figure 4

Infection of AAV/TRAIL suppresses tumor formation and growth in BALB/c nude mice. KB cells were infected with AAV/TRAIL or AAV/null for 4 h in vitro, and then injected into nude mice subcutaneously. The tumor formation was observed for 33 days. Values are mean with error bar representing standard deviation of the mean (n = 6).

Figure 5

Synergistic antitumor effect of the combination of AAV/TRAIL and cisplatin in vivo. (A) KB cells were inoculated into the right thighs of BALB/c nude mice. When the tumor size was reached to 60 mm³, the mice were received an intratumoral injection of AAV/TRAIL, intraperitoneal injection of cisplatin and the combination treatment. The tumor volume was measured and calculated. The animals were euthanized on the day 33 post-treatment. The tumor weights were determined (B) and processed for immunohistochemistry analysis (× 200) (C). Values are mean with error bar representing standard deviation of the mean (n = 6). *P < 0.05, **P < 0.01 compared to AAV/null.

Figure 6

The changes of body weight (A) and net body weight (the tumor weight subtracted from the total body weight) (B) of the mice in the respective treatment groups. Values are mean with error bar representing standard deviation of the mean (n = 6). **P < 0.01 compared to AAV/null, #P < 0.05 compared to the combination group.