Despite its low transfer efficiency, suicide gene therapy with HSV-TK is known for its bystander killing effect. its long-term inhibitor alpha-glycyrrhetinic acid had a negative influence around the bystander effect of gene therapy with HSV-TK/GCV. In addition, combined resveratrol and GCV treatment in tumor-bearing mice with CBRH7919TK and CBRH7919WT cells at a ratio of 2:3 resulted in a significant decrease in the volume and weight of the tumor in comparison to GCV or only resveratrol. The present results demonstrate that resveratrol can enhance the bystander effect exerted by the HSV-TK/GCV system by enhancing connexin-mediated gap junctional communication. low transfection rate. As the half maximal inhibitory concentration (IC50) of resveratrol required for killing cancer cells is usually relatively high,19 to efficiently kill malignancy cells, in general, a high dose of resveratrol is required. The cell viability assay using CCK8 showed that resveratrol inhibits CRBH7919 cells at an IC50 of less than 40 M. However, since HSV-TK and GCV treatment are known to result in hepatic dysfunction,20 we used a low dose of resveratrol in combination with GCV treatment. On the one hand, a low dose Rabbit Polyclonal to OR10D4 of resveratrol reduces the burden on normal hepatocytes; on the other hand, it can effectively increase upregulation of Cxs and thereby enhance GJIC. In our study, combined treatment with resveratrol and GCV had TGX-221 reversible enzyme inhibition a synergistic killing effect on CRBH7919 cells under both and conditions. Therefore, using a low dose of resveratrol combined with HSV-TK/GCV seems to be safe and also feasible for the treatment of HCC. Cxs are capable of localizing in the cytoplasm of tumor cells; this results in the dysfunction of GJIC. TGX-221 reversible enzyme inhibition 21 Our immunofluorescence results showed that Cx43 is mainly localized in the cell membrane of CBRH7919 cells, and that its expression remains at basal levels. Following resveratrol treatment, however, Cx43 is usually upregulated and predominantly localized in the cell membrane.22 Cxs not only mediate GJIC, but also function as tumor suppressor proteins. In the case of TGX-221 reversible enzyme inhibition malignancy cells with abnormal communication, their tumorigenicity is usually lost or growth is downregulated when they are transfected with the appropriate Cx genes.23,24 Thus, Cx expression in tumor cells might have the following benefits: (a) mediation of the bystander effect and (b) tumor suppression. The bystander effect can also be achieved in a GJ-independent manner.25 In order to investigate whether a non-GJ mechanism was involved, we treated cancer cells with a long-term inhibitor of GJ (AGA) and found that the bystander effect of HSV-TK/GCV dramatically decreased. The results indicate that GJIC represents the main mechanism for the killing of neighboring cells by GCV-P. We used a GJ inhibitor and not siRNA to repress GJIC function because knock down of only one Cx in the GJ channels does not predominantly block GJIC. The data showed that inhibition of GJIC significantly decreased the bystander effect of HSV-TK/GCV therapy whether or not resveratrol treatment was also used. To conclude, the present study shows that when administered at low doses, resveratrol worked with HSV-TK/GCV therapy in a synergistic manner to induce killing of HCC cells, and that the underlying mechanism predominantly involved GJIC. Materials and methods Material GCV (?99% purity, for the in vitro experiments), resveratrol ( 98% purity), alpha-glycyrrhetinic acid (AGA) and Lucifer yellow (#L0259) were obtained from Sigma (St. Louis, MO, USA). Calcein AM and DiI were obtained from Invitrogen (Carlsbad, CA, USA). CCK8 was obtained from Dojindo (Tokyo, Japan). For the mouse experiments, GCV was obtained from Hubei Qianlong Pharmaceutical Co. (Qianjiang, China). Cell lines and culture Mouse malignant hepatoma cell line CBRH7919 (wild type, WT) was bought from the Laboratory Animal Center of Sun-yat Sun University. CBRH7919tk cells, which are CBRH7919 cells that stably express HSV-TK by lentiviral contamination, were established in our lab with the lentiviral system. RPMI 1640 medium with 10% fetal bovine serum and penicillin and streptomycin (100?U/mL each) was used for cell culture. Western blot analysis CBRH7919 cells were cultured in 6-well plates, treated with resveratrol, and lysed in RIPA buffer (0.25?M Tris-HCl [pH 6.8], 8% SDS, 1?mM phenylmethylsulfonyl fluoride, 10?mg/ml aprotinin and 1.0?mg/ml leupeptin). The cellular extract obtained after lysing was separated by 10% TGX-221 reversible enzyme inhibition sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) and immunoblotted using polyclonal antibodies against Cx26 (ABclonal, Wuhan, China) and polyclonal antibodies against Cx43 (Cell Signaling Technology, Danvers, MA, USA). The internal reference used was the anti-actin antibody (Cell Signaling Technology, Danvers, MA, USA). Immunofluorescence analysis CBRH7919 cells that were cultured on cover slips were fixed by adding PBS with 4% paraformaldehyde for 20?min, permeabilized in 0.1% Triton X-100 for 5?min, and incubated in blocking buffer (3% horse serum in TBST) for 1?h. The cells were incubated in antibody dilution buffer (3% bovine serum albumin in TBS) made up of antibodies against Cx43 for 2?h at room temperature. They were washed well in.