The Enhancing Utility And Mechanism Research Of Tanshinone IIA On Bystander Effect Of Suicide Gene

Malignant tumor seriously threatens human health and life. With the development of molecular biology, gene therapy has brought the light of hope to malignant tumor patients. In foreign countries, gene therapy has been adopted as a standard therapeutic trial of malignant tumor after the failure of conventional treatment. Suicide gene therapy at present is the most promising therapeutically method for tumor gene treatment. It has specificness for tumor cells and produces bystander effect. Theoretically, it can completely eliminate the tumor cells and cure patients. The expected effects haven't been achieved due to problems like low transfection efficiency of carrier and weak targeting, ect. The key problems in urgent need to be solved are how to enhance curative effect and decrease the toxic side-effects. Enhancing the bystander effect can lower the requirement for high transfection efficiency, lower the dosage and toxicity of carrier and precursors and enhance lethality of gene therapy system on tumor. Therefore enhancing the bystander effect has become an important strategy to raise curative effect of the suicide gene therapy. The current methods adopted to raise bystander effect of the suicide gene are applying gene transfection methods or medicine revulsion methods to promote the GJIC, inducing apoptosis or improving the immune function of tumor mechanism. As a treasure house of natural medicine, Chinese medicine has a long history of phymatosis. Up to now, many reports have confirmed Chinese Herbal Medicine's unique advantages or potentials in promoting GJIC and inducing apoptosis, etc. It's economical, convenient and has little toxic side-effect. It has great potential of becoming the utility synergy element of suicide gene therapy together with which it can exert the cooperativity in tumor treatment.Objective:This research selects the Chinese Herbal Medicine elements with anti-tumor effect which are Tanshinone IIA, Salvianic acid A sodium and total saponins of Panax Notognseng to see whether they have the synergy effect on the suicide gene system. On the basis of acquiring the Chinese Herbal Medicine elements of significant effect, further exploration is made about their influence on tumor cell growth, the inducer cell apoptosis and the strengthening of GJIC, so as to understand the functional mechanism of enhancing the bystander effect by the effective element of Chinese Herbal Medicine and attempt to establish an Integrated Chinese and Western Medicine cancer gene therapy combining the suicide gene therapy with the Chinese Herbal Medicine which will offer a new thoughtway and model for tumor treatment.Methods:1. The vitro selection of suicide gene bystander effect by Chinese Medicine active components:Tanshinone IIA, Salvianic acid A sodium and total saponins of Panax Notognseng work on the 10% tk+/tk- mixed CBRH7919 rats'hepatoma carcinoma cells (HCC) at different density separately or together with GCV. Use MTT method to examine inhibition ratio of each group. Compare the difference of inhibition ratio and bystander effect of each group. Adopt JIN Zhengjun's Q Value formula to determine whether the Chinese Herbal Medicine and suicide gene system have cooperativity.2. Observation of mice's transplanting melanoma cell treatment by Tanshinone IIA together with suicide gene system. The trial has 4 groups: Controlled Model Group, Tanshinone IIA Group, GCV Group and Tanshinone IIA+GCV Group. Mix the tk+ and tk- cells at the rate of 1:4 and inoculate at the armpit hypoderma of C57BL/6J mice. Each mouse has been inoculated 2×105 tumor cells and the next day the male and female mice are grouped at random. Traet with Chinese Herbal Medicine 1-7 days after inoculation. Treat with suicide gene system for 7 days and observe the curative effect.3. Effect of Tanshinone IIA on rats'hepatoma carcinoma cells and mice's melanoma cells:Take the two kinds of cell lines which are the rats'hepatoma carcinoma cells CBRH7919 and mice's melanoma cells B16 as subjects. Adopt the MTT method to observe the Chinese Medicine active components'effect on the two kinds of cells'growth from density dependence and time dependence. Observe the cells'morphologic change after medication. Examine the effect of medicine on cell DNA by comet assay method.4. Adopt the flow cytometry technique to observe the effect of Tanshinone on HSV-tk/GCV system. Observe the influence of cell cycle and apoptosis of rats'HCC and mice's carcinoma nigrum by Tanshinone IIA together with 10% tk+/GCV. Examine TanshinoneⅡA's effect on 10% CBRH7919/tk+ and B16/tk+ by PI simple staining method. Apply Tanshinone IIA with final concentration of 12.5μM,25μM and 50μM and GCV with final concentration of 15.7μM separately or together on the 10% CBRH7919/tk+ and B16/tk+ cell 72h. Wash twice the cells with PBS, fix with precooling 70% alcohol, dye with PI and use flow cytometry to make apoptosis rate and cell cycle analysis.5. Observe the effect of Tanshinone IIA on the gap junction of rats' HCC CBRH 7919 cells and mice's carcinoma nigrum B 16 cells. Adopt parachute method to observe the effect of Tanshinone IIA on the cell GJIC. Incubate the cells within culture board by Calecin-AM. This green fluorescent dye metabolizes in cells and the metabolic products can be transmitted by GJ. Make cell suspension with the cells marked by the green fluorescent dye, cause the green fluorescent cells fall into the unmarked cell groups like parachute and determine the effect of medicine on cell GJIC by observing the intracellular transmission of the green fluorescent dye. Add the enoxolone, the prolonged-action inhibitor of GJ, to the integrated system of Tanshinone IIA and HSV-tk/GCV and observe the effect of enoxolone on the integrated system with MTT method:add Tanshinone IIA with the final concentration of 0μM,12.5μM,25μM, and 50μM to four groups of different density. The final concentrations of GCV are 0μM and 15.7μM, and the densities of enoxolone are 0 and 15μM. Integrate medicine with GCV at two densities separately. Integrate Medicine and GCV and enoxolone at two densities. After medication for 72 hours, use MTT method to determine the sensitivity of tumor cells to different groups. Apply Tanshinone IIA with the final concentration of 12.5μM,25μM, and 50μM to CBRH 7919 and B 16 for 72 hours. Adopt RT-PCR and Western-blot techniques to determine the expression value of Cx43 in each group. Results:1. The vitro selection of the effect of the active elements of Chinese Herbal Medicine on the bystander effect of the suicide gene:The cell inhibiting ratio (%) of Tanshinone IIA of different densities plus 10% tk+/GCV group are 30.38±5.28,33.22±5.21,40.58±4.49 and 73.04±2.85 which is obviously higher than that of the pure 10%tk+/GCV group (21.04±3.96) and the Tanshinone IIA of different densities group (3.65±2.84,4.74±4.48,17.34±5.08 and 52.81±5.95). Form 2 shows that the actual inhibiting ratio of the group of Tanshinone IIA at density of 6.25μM,12.5μM,25μM and 50μM plus 10%tk+/GCV is significantly higher than the theoretical inhibiting ratio. JIN Zhengjun's Q Values are 1.270, 1.341,1.168 and 1.164 and all are above 1.15. Repeat the experiment with mice's carcinoma nigrum cells by Tanshinone IIA of the same density together with suicide gene. The result shows that the actual inhibiting ratio of the group of Tanshinone IIA at density of 6.25μM,12.5μM,25μM and 50μM plus 10%tk+/GCV is significantly higher than the theoretical inhibiting ratio. JIN Zhengjun's Q Values are 2.276,1.892,1.150 and 1.211 and all are above 1.15. It proves that Tanshinone IIA has the synergy effect to the suicide gene system and the synergy effect is the cooperativity. Salvianic acid A sodium at density of 25μM plus 10%tk+/GCV is significantly higher than the theoretical inhibiting ratio. JIN Zhengjun's Q Values are above 1.15. It proves that Salvianic acid A sodium has the synergy effect to the suicide gene system. However, Salvianic acid A sodium at density of 50μM, 100μM and 200PM plus 10%tk+/GCV is lower than the theoretical inhibiting ratio. JIN Zhengjun's Q Values are lower than 1.15. It proves that Salvianic acid A sodium at these densities has not the synergy effect to the suicide gene system. Total saponins of Panax notognseng at density of 100 mg/L,200 mg/L,400 mg/L and 800 mg/L plus 10%tk+/GCV is lower, than the theoretical inhibiting ratio. JIN Zhengjun's Q Values are lower than 1.15. It proves that the total saponins of Panax notognseng at these densities have not the synergy effect to the suicide gene system.2. The observation of mice's transplanting melanoma cell treatment by Tanshinone IIA plus suicide gene system:On the ninth or the tenth day, tumors at right armpit can be touched in mice of each group. After being treated by suicide gene system plus Tanshinone IIA, the tumor growth slows down and is inhibited. The mean tumor volume is 200.2 mm3, and decreases 43.24%(P<0.05) compared with 352.7 mm3 of the Controlled Model Group. The mean tumor quality is 0.278g and decreases 50.6%(P＜0.05) compared with 0.5628 of the Controlled Model Group. There's statistical significance. In the GCV group, the mean tumor volume decreases 34.76% (P<0.05) compared with the Controlled Model Group. The mean tumor quality decreases 38.98%(P>0.05) compared with the Controlled Model Group and the result has no statistical significance. In the Tanshinone IIA group, the mean tumor volume decreases 29.94% compared with the Controlled Model Group. The mean tumor quality decreases 21.22% compared with the Controlled Model Group. There's no statistical significance (P >0.05)by comparing the tumor volume and quality with the Controlled Group. The above results show that Tanshinone IIA can raise the lethality of suicide gene system to mice's transplanted tumor and has synergy effect; therefore it can enhance the effect of suicide gene.3. Tanshinone IIA's effect on rats'HCC CBRH7919 and mice's carcinoma nigrum B16:Growth inhibition at different degree has appeared in CBRH 7919 and B16 cells after being applied with TanshinoneⅡA at gradient densities (12.5μM,25μM and 50μM). The related analysis shows that this inhibition has certain density and time dependence. The increased density or the prolonged medication will increase the inhibition ratio. It can be found with inverted phase contrast microscope that the cells of the Controlled Group of CBRH 7919 and B16 grow well, has good adherence and high density. The cells have the shape of shuttle, lozenge, triangle or polygon. After medication, the growth is inhibited obviously and grows slowly or the growth even stops. Cells shed at different level with medication density. After Hoechst dying, CBRH 7919 cells turn circular and have poor refraction. Their volume decrease, the cellular nucleus crenate, and the chromatin condense. Granules can be seen in the cells. In the group of high density, several circular apoptosis body surrounds the cell, but there's no death or apoptosis of B16 cells. The comet assay results show that the after applying TanshinoneⅡA (12.5μM,25μM and 50μM) to rats'HCC CBRH7919 and mice's carcinoma nigrum B16 cell lines, DNA is injured and long trailing is formed behind the cells and takes the typical form of comet apoptosis. The average optical decreases compared with the negative control and the difference has statistical significance. The comet back range rises compared with the negative control and the difference has statistical significance. And the changes are related to the functional density that is the higher the density the more the cells with injured DNA.4. Observe the Tanshinone IIA's effect on HSV-tk/GCV system by flow, cytometry. Apply Tanshinone IIA plus 10%tk+/GCV to HCC CBRH7919 cells of rats, S-phase cells increase significantly compared with the Controlled Group and the GCV group which indicates that Tanshinone IIA plus 10%tk+/GCV can effectively block rats'HCC CBRH7919 cells at the S-phase and inhibit the composition of DNA. The apoptosis rates of the integrated group of the density of 25μM and of 50μM are 23% and 35% which are 5 times of the apoptosis rate of the separate application of Tanshinone IIA or GCV of the same density. It indicates that Tanshinone IIA can greatly increase the lethality of suicide gene system, inhibit HCC CBRH7919's growth and promote apoptosis. Medicine has little effect on the B16 cell cycle, but from the flowing chart of the integrated group of density of 25μM and of 50μM, subdiploid apoptotic peak can be observed and the apoptosis rate of the integrated group of density of 50μM reaches 34%. It proves that Tanshinone IIA can promote the apoptosis of B16 (10%tk+/GCV) cell system and has synergy effect to the suicide gene system.5. Tanshinone IIA's effect on the gap junction protein of rats'HCC CBRH7919 and mice's carcinoma nigrum cells. The parachute experiment shows that after applying Tanshinone IIA, there's dye transmission between cells and with the increase of density, the unmarked cells of green fluorescent adherence increase which indicates that Tanshinone IIA has high possibility of enhancing GJIC between cells and the effect rises with density. After adding GJ prolonged-action inhibitor of enoxolone to the integrated Tanshinone IIA and HSV-tk/GCV system, the result shows that enoxolone can significantly decrease the joint action of Tanshinone IIA and HSV-tk/GCV and lower the inhibition ratio of medicine which indicates that Tanshinone IIA's synergy effect to the suicide gene system is highly possible to be related with improving the GJIC. The RT-PCR and Western-blot examinations show:high-density (≥50μM) Tanshinone IIA promotes the expression of Cx43mRNA and protein, while the medium and low density groups don't have obvious promotion effect; Tanshinone IIA at density of 12.5μM,25μM and 50μM promotes the expression of Cx43 protein of B16 cells, while only the high density group has the enhancement property of mRNA of Cx43.Conclusions:This investigation explored the bystander effect of Tanshinone IIA, Salvianic acid A sodium and total saponins of Panax Notognseng, the active ingredient of Chinese Herbal Medicine on the suicide gene therapy and finds that Tanshinone IIA can effectively enhance the lethality of suicide gene, promote the bystander effect and has the synergy enhancement property. The curative effect of TanshinoneⅡA on the mice's transplanting tumor model proves that by integrating with the suicide gene system, it can significantly prohibit the mice's transplanting melanoma. By vivo experiment, the author makes preliminary research of the mechanism of Tanshinone IIA's promotion of bystander effect of suicide gene and infer that the synergy mechanism can be:Tanshinone IIA recovers Cx-mediated GJIC function of tumor cells, recovers or enhances the communication between cells, blocks the tumor cells at S-phase, exert the effect of cytotoxin GCV-TP, hinders the composition of DNA of tumor cells, injures DNA and finally induces the apoptosis of cells. Cytotoxin enters more cells by apoptosis or cell junction and communication; therefore it greatly improves the lethality of suicide gene.This thesis lays foundation for researching on the feasibility and mechanism of bystander effect enhancement of suicide gene with Chinese Herbal Medicine. In the future, the author will select the effective ingredients for bystander effect mechanism research from the Chinese Herbal Medicine to be selected, make further study on the pharmacological mechanism of the active ingredients, and clarify the anti-tumor mechanism of Chinese Herbal Medicine from the new field of GJIC, cell apoptosis, immunological adjustment and the relationship with bystander effect, etc. Also, the author hopes to make a compound formula with multiple Chinese Herbal Medicine ingredients of synergy effect which are more effective than single ingredient and to form the cocktail-form synergy compound of clear pharmacology and pharmacodynamic action as well as stable quality. The research is beneficial for the clinical promotion of tumor gene therapy and provides trial basis for new integrated tumor gene therapy.