Enhancement Of Kaempferol Combined With HSV1-tkGFP/GCV System On Murine Melanoma Cell Line B16

ObjectiveTo confirm that kaempferol enhances bystander effect of recombinant Herpes simplex virus type1thymidine kinase/ganciclovir (HSV1-tkGFP/GCV) tumor suicide gene system with fluorescent protein monitoring function on murine melanoma B16cells.Methods1. The positive clone was propagated and the plasmid pLXSN-tkGFP was extracted by the Ultra Pure QIAGEN Plasmid Midi Kit, and then the constructed recombinant plasmid (pLXSN-tkGFP or pLXSN-DsRed2) was transfected into the murine melanoma B16cells via Lipofectamine2000reagent, and anti-G418monoclone named B16-tkGFP and B16-RFP respectively were obtained after G418selection, fluorescence-activated cell sorting and limited dilution assay. The presence and expression of tk gene in B16-tkGFP cells were confirmed by the killing effects of GCV. The sensitivity of B16-tkGFP cells to GCV, the "bystander effect (BE)" of HSV1-tkGFP/GCV system and enhancement of kaempferol on the bystander effect were measured by MTT assay and mixed co-culture experiment with B16-RFP cells. Finally,We observe the cells, especially the B16-RFP cells without tk gene, by fluorescence image, in order to determine whether the apoptosis involved in tk gene-expressing cells (B16-tkGFP cells) induces the apoptosis of their neighboring cells only with a red fluorescent protein gene(B16-RFP cells) directly.2. Effects of kaempferol on the proliferation of B16cells were determined by MTT assays. The gap junction intercellular communication was analyzed by fluorescent tracer with flow cytometry. Enhancement of kaempferol on the bystander effect of HSV1-tkGFP/GCV system were measured by MTT assay and mixed co-culture experiment(100%tk+and50%tk+) with B16-RFP cells,and then the combined effect of kaempferol and HSV1-tkGFP/GCV system was evaluated by "Q Value" method. The effect of kaempferol on the bystander effect of HSV1-tkGFP/GCV system was observed by fluorescence image directly.3. In the in vivo study, a mixture of activated B16-tkGFP and B16-RFP cells at the ratio of1:1were inoculated subcutaneously into C57BL/6J mice. The tumor-bearing mice were randomly divided into tumor model group, GCV treated group, kaempferol treated group and kaempferol/GCV combined treated group (n=10).Then kaempferol(40mg/kg) and GCV(50mg/kg) were administrated intraperitoneally every day, and the tumor inhibitory effects were observed by measuring tumor sizes every three days. After the animal experiment, tumor tissues, thymus and spleen of all mice were dissected and fixed in the10%methanol. Some tumor tissues were embedded in paraffin and cut into sections, and then the sections were used for hematoxylin-eosin (HE) staining. The staining effect was observed under the light microscope.Results1. Anti-G418monoclone named B16-tkGFP (tk gene-expressing cells with stable green fluorescence) and B16-RFP (only with stable green fluorescence) respectively were obtained. The killing effect of GCV confirmed transfection of tk gene in B16-tkGFP mono-clone. The MTT array’s results and fluorescent microscope images indicated some bystander effect of the HSV1-tkGFP/GCV system.2.0μmol/L～12μmol/L kaempferol has no effect on the propagation of B16cells after48h. Flow cytometry analysis showed that the radio of Calcein labeled cell group (A4) to double negative group (A3) was0.056±0.006、0.072±0.013、0.079±0.009and0.094±0.013respectively in B16cells group treated with0μmol/L、3μmol/L、6μmol/L and12μmol/L kaempferol after48h.3μmol/L kaempferol showed synergetic effects, combined with12μmol/L or below GCV on100%B16-tkGFP cells after72h;6.25μmol/L and12.5μmol/L (<25μmol/L) kaempferol also showed synergetic effects, combined with6.25μmol/L GCV on100%B16-tkGFP cells after96h;6.25μmol/L kaempferol showed synergetic effects, combined with6.25μmol/L GCV on a mixture of B16-tkGFP and B16-RFP cells with50%B16-tkGFP cells after96h. Fluorescent microscope images indicated that3μmol/L,6μmol/L and12μmol/L kaempferol enhanced the bystander effect of HSV1-tkGFP/GCV system with50%B16-tkGFP cells.3. In the in vivo study, the tumor growth rate in the GCV/kaempferol combined treated group was smaller than that in the GCV treated group(P< 0.05). Differential HE staining patterns revealed less density of tumor cells in the GCV treated group and the GCV/kaempferol combined treated group, more lymphocyte infiltration in the kaempferol group and the GCV/kaempferol combined treated group. The phenomenon above was more significant in the GCV/kaempferol combined treated group.ConclusionKaempferol enhances the effect of recombinant Herpes simplex virus type1thymidine kinase/ganciclovir (HSV1-tkGFP/GCV) tumor suicide gene system on murine melanoma B16cells,maybe via GJIC or kaempferol’s immune regulation to the mice, which deserves further studies for possible applications in suicide gene therapy of melanoma.