Cisplatin-induced GADD34 upregulation potentiates oncolytic viral therapy in the treatment of malignant pleural mesothelioma

Abstract

Background: NV1066, a replication-competent oncolytic herpes simplex virus type 1 (HSV-1) attenuated by a deletion in the gene gamma(1)34.5, preferentially replicates in and kills malignant cells. gamma(1)34.5 encodes ICP34.5, a viral protein essential for productive replication, which has homology with mammalian stress response induced GADD34 (growth arrest and DNA damage-inducible protein). We hypothesized that cisplatin upregulates GADD34 expression, which enhances NV1066 replication and oncolysis.

Methods: Ten human malignant pleural mesothelioma (MPM) cell lines were infected with NV1066 at multiplicities of infection (MOI; ratio of viral particles per tumor cell) 0.005 to 0.8 in vitro, with and without cisplatin (1 to 4 microM). In the MPM cell line VAMT, viral replication was determined by plaque assay, cell kill by lactate dehydrogenase assay, and GADD34 induction by quantitative RT-PCR and Western blot. Synergistic efficacy was confirmed by the isobologram and combination index methods of Chou-Talalay. GADD34 upregulation by cisplatin was inhibited with GADD34 siRNA to further confirm the synergistic efficacy dependence with GADD34.

Results: Combination therapy with NV1066 and cisplatin showed strong synergism in epithelioid (H-2452, H-Meso), sarcomatoid (H-2373, H-28), and biphasic (JMN, Meso-9, MSTO-211H) MPM cell lines, and an additive effect in others. In VAMT cells combination therapy enhanced viral replication 4 to 11-fold (p < 0.01) and cell kill 2 to 3-fold (p < 0.01). Significant dose reductions for both agents (2 to 600-fold) were achieved over a wide range of therapeutic-effect levels (LD50-LD99) without compromising cell kill. Synergistic cytotoxicity correlated with GADD34 upregulation (2 to 4-fold, p < 0.01) and was eliminated following transfection with GADD34 siRNA.

Conclusion: Cisplatin-induced GADD34 expression selectively enhanced the cytotoxicity of the gamma(1)34.5-deficient oncolytic virus, NV1066. This provides a cellular basis for combination therapy with cisplatin and NV1066 to treat MPM and achieve synergistic efficacy, while minimizing dosage and toxicity.

Figure 1.

Cytotoxic effect of cisplatin, NV1066, or both on VAMT cells in vitro. VAMT cells were treated with cisplatin (1 μM), NV1066 (MOI 0.03), or the combination. Results for the treated groups are expressed as cell survival compared to untreated control cells grown under identical conditions. MOI: multiplicity of infection, ratio of viral particles to tumor cells.

Figure 2.

Isobolograms demonstrate dose-reductions achieved due to synergism of cisplatin with NV1066 in VAMT cells. The doses of cisplatin and NV1066 necessary to achieve 50% cell kill (open triangles), 75% cell kill (open squares), and 95% cell kill (open circles) are plotted on the axes, and the connecting solid lines represent the expected additive effects for combination therapy. Experimental combination therapy doses necessary to generate actual LD values of 50% (filled triangle), 75% (filled square), and 95% (filled circle) all lie to the lower left of the corresponding lines, indicating synergism.

Figure 3.

(A) GADD34 upregulation following cisplatin therapy and inhibition by GADD34 siRNA. LacZ transfected (control) and GADD34 siRNA-transfected VAMT cells were treated with cisplatin. Untreated cells served as control. GADD34 expression was measured by real-time RT-PCR at 0 to 72 h, standardized by an 18S control, and was upregulated in cisplatin-treated cells. This upregulation was inhibited in GADD34 siRNA-transfected cells. GADD34 upregulation was expressed as fold upregulation compared to untreated control cells. (B) GADD34 upregulation and its inhibition by siRNA transfection were confirmed at the protein level with Western blot analysis. (C) GADD34 upregulation inhibition by siRNA eliminated synergistic cytotoxicity. VAMT cells were treated with cisplatin (1 μM), NV1066 (MOI 0.06), or the combination, with or without GADD34 siRNA transfection. MOI = multiplicity of infection, ratio of viral particles to tumor cells. GADD34, Growth Arrest and DNA Damage-Inducible Protein 34, siRNA: small inhibitory RNA.