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.

The anti-glioma effect of suicide gene therapy using BMSC expressing HSV/TK combined with overexpression of Cx43 in glioma cells

“…A bystander effect, meaning HSV-TK non-expressing cells near the HSV-TK expressing cells can also be killed, is mainly responsible for a dramatic tumor ablation or regression by HSV-TK/GCV treatment in animal studies. 7,8,[16][17][18] However, clinical trials for glioma using this treatment have failed. [19][20][21][22] One of the major reasons may be due to the delivery manner of HSV-TK into glioma cells located only in the direct vicinity of the injection site, but fail to transduce infiltrating tumor cells or distant tumor areas that eventually serve as the reservoirs for tumor recurrence.…”

“…[14][15][16][17][18] Owing to the loss of gap junction among C6 glioma cells, it is speculated that the second mechanism may have a major role in which, to a great extent, death of the neighboring C6 cells is induced by their uptake of cytotoxic GCV triphosphate released from BMSC-TK cells after GCV treatment. As transfection of Cx43 gene into C6 cells can restore gap junction, 27,28 cytotoxic phosphorylated GCV can be transported between C6 cells through gap junction, in addition to the uptake of phosphorylated GCV through apoptotic vesicles.…”

Direct evidence for the absence of intercellular trafficking of VP22 fused to GFP or to the herpes simplex virus thymidine kinase

“…[27][28][29] Therefore, pharmacological strategies aiming at upregulating the level of connexins have been proposed in order to increase the BE. [30][31][32][33][34][35] The delivery of connexin genes together with TK has also been evaluated, and it has been shown that the BE could be induced in gap junction deficient cancer cells. 26,36 A very different type of BE that is independent of the level of connexins has been recently reported.…”

Electroporation-mediated and EBV LMP1-regulated gene therapy in a syngenic mouse tumor model

“…44 However, this may be cell typespecific. [45][46][47][48] It is not clear whether this is effective in the cells examined in this study. This suggests that continuous treatment or a combination with other agents is required for the full regression of tumor growth, consistent with previous reports.…”