Enhancement of the Herpes Simplex Virus Thymidine Kinase/Ganciclovir Bystander Effect and Its Antitumor Efficacy In Vivo by Pharmacologic Manipulation of Gap Junctions

Abstract

Apigenin, a flavinoid, and lovastatin, an HMG-CoA reductase inhibitor, upregulated gap junction (GJ) function and dye transfer in tumors expressing GJ and were inactive in the GJ-negative tumor line N2a. N2a cells transfected with the connexin 43 gene showed restored cell-to-cell dye transfer, which could then be improved nearly fourfold by addition of apigenin. To test the drugs in HSV thymidine kinase/ganciclovir (HSV-tk/GCV) tumor killing, mixtures of 90% wild-type (WT) with 10% HSV-tk gene-modified MCA38 adenocarcinoma cells were exposed in vitro to GCV ± apigenin or lovastatin. A significant bystander effect (BSE) was seen following GCV treatment alone, while neither apigenin or lovastatin alone had any effect on the recovery of viable tumor colonies. However, GCV-treated cultures also exposed to apigenin or lovastatin showed an increased BSE and reduced tumor cell recovery. Thirty percent of mice bearing tumors from the same mixture of 90% WT and 10% HSV-tk MCA38 cells treated with GCV alone became tumor free. Tumor-bearing mice given only two or three injections of lovastatin or apigenin during GCV treatment had a doubling of the antitumor response rate, with 60–70% of the mice achieving complete remission. These results support the hypothesis that the transfer of phosphorylated GCV from HSV-tk gene-expressing cells to neighboring WT tumor cells is a major component of the BSE and that pharmacological manipulation of GJ function with lovastatin or apigenin can result in striking improvement in the antitumor response in mice with tumors modified to contain as few as 10% HSV-tk cells. Because the efficacy of gene delivery to tumor cells in vivo is inefficient, any treatment strategy employing direct gene modification of tumor in situ must have an amplifying mechanism to have a realistic chance for success as therapy for cancer. In the HSV-tk/GCV suicide gene system, one mechanism for such therapeutic amplification is the “bystander effect” (BSE) resulting from the cell-to-cell transfer of phosphorylated ganciclovir via cellular gap junctions. This article reports in vitro and in vivo evidence that gap junction function can be upregulated with the pharmacologic agents apigenin and lovastatin. Treatment with these compounds resulted in enhanced tumor killing in vitro and improved response rates and tumor-free survival in animals bearing tumors in which only 10% of the population was HSV-tk gene modified. These data strongly suggest that pharmacologic upregulation of gap junctions should be considered for inclusion in clinical trials testing suicide gene therapy for cancer in humans.