Influences Of Chinese Herbal Medicine Active Components On Suicide Gene Bystander Effect And GJIC

The suicide-gene therapy is a promising project of treating tumor, but the problem in this remedy is insufficient killing ability, low efficiency of transfection and uncertain safety. It's reported that increasing the bystander effect is a good approach to resolve this problem. Considering the mechanism of bystander effect conducted by the Gap junction intercellular communication (GJIC) of cell, apoptosis of cell and immune factors, researchers today enhance the bystander effect by methods of twin genes transfection and medical inducement to elevate GJIC, induce apoptosis and raise the function of immune system. The method of two genes transfection means that we should transfer the suicide gene with another effect-increasing gene at the same time, but in actual practice there is a low efficiency of transfection in vivo, so the difficulty of transfection practice of two genes simultaneously is far high, and the potential side-effect is much high either. In comparison, much merit such as simple practice, small expense, aiming much more target cells, etc. existed in the later method. So we adapted it.There were not many medicines especially natural medicine with this effect had been found until today, but we knew some advantages and potentials have been illustrated by in apoptosis inducing, cell communication and adjustment of immune system, even some ingredients of TCM had been proved by the clinic or basic investigations on the effects of inducing apoptosis of tumor cells and increasing immunity of the patients. But few articles were released about the investigation on increasing bystander effect and cooperating the tumor gene therapy with TCM or their ingredients.Therefore, according to the development and trend of the studies in the mechanism of bystander effect and bystander effect-increasing induced by medicine, we presented the following hypothesis, there were some active ingredients in the Traditional Chinese Medicine could improve the GJIC, induce the apoptosis of tumor cells, improve the local inflammatory immune environment of tumor or enhance the function of immune system, and exert the effect of anti-tumor cooperated with other components by the manner of increasing bystander effect of suicide-gene which maybe increase GJIC, induce apoptosisof tumor cells and immunity so on. ObjectiveIn our research, all above served as the purpose to investigate the possibility improving the effect of suicide gene therapy cooperated with Traditional Chinese Medicine. After getting the several effective components of TCM on the cell level, we would validate them on the whole level repeatedly to find the real effective ingredients of TCM. And at the same time, we wanted to compose those ingredients acted on different mechanism a complex dose with known components, clarify pharmacology, stable quantity and maybe have a higher effect.During the research, we hoped to explore the possible mechanism of adjusting cell GJIC and inducing of cell apoptosis with TCM, then we could get the further comprehension about the mechanism of increasing bystander effect with TCM and constructed the foundation of investigating the pharmacology effect in signal transduction of tumor-cell, regulating the cell cycle, communication of cell and immunity of tumor cell etc. with the ingredients of TCM. Methods1. Screening active compounds of TCM in vitro. The sensibilities of rat hepatocarcinoma cell CBRH7919 to apigenin, resveratrol and curcumin were detected by MTT assay. Then the cell CBRH7919, and the mixed cell with 5%or 10% tk+ and tk' are treated separately by apigenin, resveratrol, curcumin in were treated with diverse concentrations of apigenin, resveratrol, curmin and GCV separately, or apigenin plus GCV , resveratrol plus GCV , curmin plus GCV (n = 3,6). Viability of cells was determined by MTT assay. The Q-value analysis was used to estimate the synergistic effects of the active components on the suicide gene system. The Q-value was equal to the ratio of the actual effect of combined treatment to its theoretical effect. The effect was classified into three categories: antagonistic effect( Q <0.85), additive effect(0.85 1.15).2. Exploring the mechanism of the synergistic effects of the active components: The cell CBRH7919, and the mixed cell with 5%or 10% tk+ and tk" are treated separately by apigenin, resveratrol, curcumin in were treated with diverse concentrations of apigenin, resveratrol, curmin and GCV separately, or apigenin plus GCV , resveratrol plus GCV , curmin plus GCV ( n = 3,).Then the cell cycle analysis and cell apoptotic index were performed using a FACScan cell analyzer. AGA was added in the combination therapeutic system in vitro and cell viability in every group was detected by MTT assay. The effects of resveratrol or curcumin on the GJIC of CBRH7919 were detected by SL/DT. The expression of Cx43 in cells were determined by FITC indirect immuno-fluorescent assay.3. Cloning of the tkgfp Fusion Gene and its expression: The cDNA encoding the thymidine kinase gene of HSV-1 was obtained by digestion of pLXSN-tk withEcoR I /BamHI. Thel.7-kb tk fragment was isolated and digested with Xma I. Xmal cuts 22 bp in front of the STOP codon. The resulting 1.3-kb tk fragment was isolated and ligated to the 4.7-kb DNA fragment obtained after plasmid pEGFP-Nl was digested sequentially with BamH I and Xmal. After ligation, the open reading frames of both tk and gfp are in-frame within the pTKGFP expression plasmid 6.0 kb, placing the tkgfp cDNA under control of the cytomegalo-virus CMV immediate early IE 1 promoter. Cells of B16, CBRH7919 and NIH3T3 were transfected with the plasmid pTKGFP or pEGFP-Nlor pDsRed2-Nl, with polyfect transfection reagent, according to the manufacturer's protocol. For fluorescent detection of g#>-expressing cells or DsRed2-expressing cells, culture plates were examined with a standard fluorescence microscope 24 hours after transfection. For determination of the ganciclovir sensitivity, GCV-treatment was carried out at different final concentrations of medium for half of the dishes starting 24 hours after transfection. Three days later, surviving cells were determined as a percentage of non - GCV-treated transfected cells. 2. Result 2.1 The results of screening active compounds of TCM in vitro.Apigenin showed an inhibition effect on CBRH7919 when the final concentration was higher than 50uM but showed low inhibiting effect at 50uM.When combining with HSV-tk/GCV system, apigenin showed a synergistic effect at 50uM(Q value >1.15) but showed an additive effect on HSV-tk/GCV therapeutic system at 25uM(0.851.15).Curcumin showed a distinct inhibition effect on CBRH7919 in a range of concentration from lOuM to 50uM. IC50 was 24uM .And the effect showed in a dose-dependent manner. When combining with HSV-tk/GCV system, curcumin showed a synergistic effect at 5uM and 10pM(Q values >1.15).2.2 The results of the mechanisms researchs of the synergistic effects of the active components:Results of FACS showed that when combining with HSV-tk/GCV system resveratrol showed an apoptotic rate-elevating effect on CBRH7919 but showed no effect on cells cell cycle. Curcumin showed an apoptotic rate-elevating effect on CBRH7919 when combiningwith HSV-tk/GCV system.Furthermore, it showed a S-phase lagging effect on CBRH7919 cell cycle. GJ inhibitor AGA showed a down-regulation effect on the killing effect in the combination group of resveratrol or curcum with tk/GCV system. These results indicated that mechanisms of the augmentation effect of resveratrol or curcum on tk/GCV system were related to GJIC. The result of SL/DT showed that ability of GJIC function of CBRH7919 was up-regulated by resveratrol or curcumin. The result of Cx43 expression detection by FTTC indirect-immuofluorescent assay showed a low expression of Cx43 in CBRH7919 control groups and a higher expression of Cx43 in resveratrol or curcumin treated groups.2.3 The levels of ^-expression were assessed days after transfection with a standard fluorescence mi croscope and were visually of similar intensity in tkgfp and native gfp transfected cells . The data indicate that tkgfp transduced cells can be readily identified by fluorescent light at least to the same extent as gfp transduced cells. The intracellular expression pattern of the TKGFP seems different than the native GFP. TKGFP is mostly concentrated in the nucleus, whereas the native GFP and DsRed displays a predominantly cytoplasmic distribution. GCV sensibility examination showed that the pTKGFP transfected cells were much more sensitive to GCV than the pEGFP-Nl transfected cells in a dose-dependent manner. Cone I us i onWe explore the effects of resveratrol, apigenin and curcumin on tumor HSV-TK /GCV suicide gene therapy system. Results show that all of them exibit synergistic killing effects on suicide gene system. Both of the effective concentration of resveratrol and curcumin are lower than apigenin. Mechanisms of the synergistic effect of resveratrol and curcum are related to the restoration of GJIC, or/and the apoptotic inducement or the regulation of cellular cycle.We construct a TKGFP fusion protein expression vector which is transfected into CBRH7919, B16, and NIH3T3 and expressed in these cells successively. The expressed fusion protein exibit the activity of thymidine kinase(TK) and green fluorescence of EGFP. The tkgfp fusion gene itself may be useful in developing novel cancer gene therapy approaches. Valuable information about the efficiency of gene transfer and expression could be obtained by non-invasive imaging of tkgfp expression with direct visualization of gfp expression in situ by fluorescence microscopy or endoscopy.