From recent findings, conditionally replicating adenoviruses (CRAds) are expected to enhance the tumor specificity without any damage to other tissues and establish efficient tumor-killing action. In this study, we have evaluated the use of CRAds, showing oncolytic action in cancer cells with abnormal Rb activity, in combination with Ad/CMVp63γ and/or Ad/CMV-TRAIL against human cervical and ovarian cancer cells.
The cytotoxicity effect was evaluated with MTT and crystal violet assay. Cell cycle analysis of combination treated cells tested by flow cytometry. Western blotting determined BAX, p63γ, TRAIL, PARP, and cleaved caspase-3.
The combination of these viruses showed synergistic cell killing in vitro compared to single treatments. An increase in protein of p63γ and TRAIL was observed in Ad/CMVp63γ + CRAds or Ad/CMV-TRAIL + CRAds -infected human ovarian cancer cell line (SKOV3) and human cervical cancer cell line (C33A), compared to Ad/CMVp63γ or Ad/CMV-TRAIL alone. We found that two therapies combined (Ad/CMVp63γ and Ad/CMV-TRAIL) had greater protein level of BAX, cleaved caspase-3 and PARP than either one alone in C33A and SKOV3. Also, flow cytometry showed that the cell cycle analysis of combination treatments was arrested at the G2 phase and followed by the appearance of a sub-G1 peak in C33A and SKOV3 cells. Moreover, three Ad/CMV-LacZ + Ad/CMV-Luciferase + Ad/CMV-GFP combination with CRAds infected C33A and SKOV3 cells showed more increased level of LacZ, Luciferase, GFP gene than Ad/CMV-LacZ, Ad/CMV-Luciferase or Ad/CMV-GFP infected C33A and SKOV3 cells, but 3 combination therapy results showed similar to Ad/CMV-LacZ + CRAds, Ad/CMV-Luciferase + CRAds or Ad/CMV-GFP + CRAds. When compared to each of combination with CRAds, Ad/CMVp63γ + Ad/CMV-TRAIL + CRAds infected C33A and SKOV3 were enhanced cytotoxicity, and were found similar protein expression level, apoptotic protein, p63γ and TRAIL. The anti-cancer effect by combination therapy of CRAds and non-replicating adenovirus with therapeutic genes was more effective than that by single non-replicating adenovirus in SKOV3 and C33A. These results suggest that ‘triple’ gene therapy with CRAds and non-replicating adenovirus containing therapeutic genes (p63γ and TRAIL) can improve gene transfer and anti-cancer effect in some cancer cells with low transfection efficiency of adenoviral vectors.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA