ATRA enhances bystander effect of suicide gene therapy in the treatment of prostate cancer

Objectives: All-trans retinoic acid (ATRA) has been shown to inhibit the growth of many malignancies by altering gap junctional intercellular communication (GJIC) and the expression of connexin (Cx) 43. Here, we report that the alteration of GJIC by ATRA may directly enhance the bystander effect (BE) of suicide gene therapy against prostate cancer in vitro and in vivo. Methods: PC-3 cells were exposed to different concentrations of ATRA for varying lengths of time in culture. Flow cytometry was performed to measure Cx43-positive cells and the GJIC function of the cells was examined with the scrape-loading dye transfer assay. Cells were treated with ATRA in combination with an adenovirus/ganciclovir (Ad-TK/GCV) system encoding herpes simplex virus-thymidine kinase, and the BE was assessed in the treatment of androgen-independent prostate cancer both in vitro and in vivo. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were performed to assess the expression of Cx43 mRNA and protein in tumor tissues. Results: ATRA significantly increased the amount of Cx43-positive cells in a time- and dose-dependent manner (P < 0.05). GJIC functions were enhanced 3- to 5-fold in the presence of ATRA, although ATRA did not augment GCV toxicity of PC-3 cells. In the mixing assay, ATRA significantly increased cell killing when the ratio of TK-positive cells in the coculture ranged from 30% to 60% compared with ATRA-untreated cell (P < 0.05), and attained 50% cell killing cells when the ratio of TK-positive cell was 30%, but the same result did not appear until the ratio of TK-positive cell was up to 60% in the ATRA-untreated cell. Mice treated with a combination of ATRA and GCV had significantly smaller Ad-TK infected tumors than those treated with GCV or ATRA alone after 3-weeks of therapy (P < 0.05). However, from the fourth-week of therapy, there was no difference in tumor growth inhibition between GCV treatment and GCV + ATRA treatment (P > 0.05), as two tumors in the latter group started to grow more quickly than tumors in the control group. This phenomenon was not found in other groups. Conclusions: ATRA could enhance the efficiency of cell killing in suicide gene therapy against prostate cancer by strengthening the BE in vitro and in vivo. Induction of Cxs and GJIC by ATRA might provide an element of selectivity to suicide gene therapy. Future studies should focus on safety and tailoring this cooperative therapy to the patient. (C) 2008 Elsevier Inc. All rights reserved.