Tan IIA enhances the bystander effect.

<p>Stable HSV-tk-EGFP and RFP B16 cell lines were mixed at a ratio of 1∶1. The mixture of cells was left untreated or treated with Tan IIA (10 or 20 µM) for 24 h, and then was cultured with or without GCV (15.7 µM) for 48 h. (A) Representative images as shown by fluorescence microscopy. The red fluorescence in living RFP cells mainly localizes in cytoplasm; the aggregation of red fluorescence indicates cell shrinkage which is the hallmark of apoptosis (white arrows); the clustered light spots indicate the formation of apoptosis bodies (yellow arrows). (B) The apoptosis of RFP cells was analyzed by flow cytometry with annexin V stain. (C) Quantification of three independent experiments. *p<0.05, **p<0.01 compared with control; ^#p<0.05, ^#p<0.01 compared with Tan IIA or GCV treatment alone.</p

Tanshinone IIA Increases the Bystander Effect of Herpes Simplex Virus Thymidine Kinase/Ganciclovir Gene Therapy via Enhanced Gap Junctional Intercellular Communication

<div><p>The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV). This effect is reported to be mediated by gap junctional intercellular communication (GJIC), and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA), a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy.</p></div

Tan IIA in combination with GCV causes greater growth inhibition on a mixture of HSV-tk and WT B16 cells.

<p>HSV-tk B16 cells were mixed with WT B16 cell line at a ratio of 1∶9. The mixture of cells was left untreated or treated with Tan IIA (10 or 20 µM) for 24 h, and then was cultured with or without GCV (15.7 µM) for 48 h followed by MTT assay. *p<0.05, **p<0.01 compared with control; ^##p<0.01 compared with Tan IIA or GCV treatment alone.</p

The combination effects of Tan IIA and GCV on tumor formation for a mixed population of HSV-tk and WT B16 cells in vivo.

<p>Subcutaneous B16 tumors were established in C57BL/6J mice using a 1∶9 mixture of HSV-tk and WT B16 cells. Mice were then divided into four treatment groups: control (saline only), Tan IIA only, GCV only, and Tan IIA plus GCV. The tumor weight was measured on 14 days following treatment. *p<0.05 compared with control; ^#p<0.05 compared with Tan IIA treatment alone.</p

Tan IIA treatment of B16 cells results in the upregulation of Cx26 and Cx43 proteins.

<p>B16 cells were treated with Tan IIA (0, 5, 10 or 20 µM) for 72 h. (A) Immunoblotting was performed using antibody against Cx26, Cx30 and Cx43. Actin was also tested as a loading control. (B) Relative quantification of the immunoblotting results as calculated by gray scanning (*p<0.05). The results shown are representative of three independent experiments.</p

Tan IIA promotes GJIC of B16 cells, as measured by fluorescent dye transfer.

<p>B16 cells were left untreated (control) or treated with Tan IIA (5, 10 or 20 µM) for 18 h. Donor cells were pre-loaded with two fluorescent dyes: DiI and Calcein AM. A 1∶100 mixture of labeled donor cells and unlabeled recipient cells was co-cultured for 6 h in the presence of Tan IIA followed by flow cytometry analysis. (A) Flow cytometric analysis for fluorescent dye transfer.G1: Cell populations positive for DiI; G2: Cell populations positive for both DiI and Calcein; G3: Double negative cells; G4: Cell populations positive for Calcein only. (B) Quantification of three independent experiments. The G4/(G3+G4) of experimental groups was higher than that of the control, indicating more efficient cell-to-cell spread of Calcein after Tan IIA treatment. **p<0.01 compared with control.</p