The Enhancing Effect Of The Chinese Medicine Prescription RTGD Combining With HSV1-tk/GCV System On Melanoma

Objective:Melanoma is a malignant cancer with poor prognosis and high mortality and incidence. With the development of molecular biology, gene therapy especially suicide gene therapy on the melanoma brings hope to patients and becomes a new anti-tumor treatment, following the traditional surgical resection, radiotherapy and chemotherapy. But how to enhance the destruction effect of suicide gene therapy is critical because of the shortcoming of low transfection and lack of targeting. Bystander effect, that gene trans fect ion-positive cells can kill gene transfection-negative cells, is a notable feature of suicide gene therapy. Enhancing the bystander effect has become an important strategy on improving the efficacy of tumor suicide gene therapy.Preliminary studies showed that:several active components of Chinese medicines such as curcumin、resveratrol、tanshinone IIA、diosgenin、Sarsasapogenin、lycium barbarum polysaccharides、luteolin and so on could enhance the bystander effect of the the suicide gene therapy by the mechanisms of GJIC (gap junctional intercellular communication) and apoptosis, at the same time, the study of Chinese medicine Liuweidihuang bolus showed that it also could enhance the bystander effect by GJIC and apoptosis besides of Cell-mediated immunity and humoral immunity. Based on preliminary studies and the synergy effect research of ginsenoside combining with HSV-tk/GCV gene suicide system on melanoma, according to the mechanisms of bystander effect and the pathogenesis of tumor in traditional Chinese medicine, we selected resveratrol、tanshinone IIA、ginsenoside to make up a Chinese medicine prescription RTGD, further validated its synergies and mechanisms on the suicide gene therapy of malignant melanoma in vivo and in vitro. Methods:1 The enhancing effect of ginsenoside combining with HSV-tk/GCV suicide gene system on melanoma.1.1 The enhancing effect of ginsenoside combining with HSV-tk/GCV suicide gene system on B16 cells in vivo:85 SPF-class male mice, except the normal group of 5 mice were injected with saline in the right forelimb armpit, 0. 1mL/only, the other mice were injected 2×105 cells (B16tk and B16 wild type cells were mixed in the proportion of 1:4) in the right forelimb armpit, 0. 1mL/only. In the 7 day after inoculation, when the masses were palpable in axillary, set the model group、the suicide gene treatment group (the GCVgroup)、the ginsenoside treatment group、the combined treatment group (the ginsenoside combining with suicide gene group) randomly. In 7～14 days after inoculated: the normal and model groups were injected intraperitoneally the mixed liquid of ethanol and saline 0.2mL/only, in which the proption of ethanol was 20%; the suicide gene treatment group were injected intraperitoneally the Ganciclovir injection 0.2mL/only (100mg/kg·d) in the afternoon; the ginsenoside treatment group were injected the ginsenoside 0.2mL/only (100mg/kg·d) in the morning; the combined treatment group were injected intraperitoneally the ginsenoside 0. 1mL/only (100mg/kg·d) in the morning, and injected intraperitoneally the Ganciclovir injection 0.1mL/only (100mg/kg·d) in the afternoon. When the masses were palpable in axillary, used the vernier caliper to measure the major axis and minor axis of each tumor and calculated the volumes of them; on the fourteenth day after inoculation, executed the mice and measured the tumors’weight and captured the tumors’ photos.1.2 The histopathological examination:fixing the masses with 10% neutral formaldehyde, slicing by paraffin, and then HE staining, last obsevering the necrosis and lymphocytic infiltration of the tumors and capturing the photos. The grade of lymphocytic infiltration:-:no infiltration of lymphocytes; +:seeing scattered lymphocytes;++:seeing some lymphocytes;+++: seeing intensive lymphocytes.1.3 The effect of ginsenoside on B16 cells:Treating B16 cells with ginsenoside at the final concentrations of Oug/mL、lug/mL、10ug/mL、100ug/mL 48h, and using MTT assay to detect the inhibition of each group. 1.4 The damage effect of the ginsenoside combining with HSV-tk/GCV suicide gene system on B16 and B16tk mixed cells:Mixing the B16tk cells with B16 cells at the proportion of 20% and planking, setting the ginsenoside groups of 0ug/mL、5ug/mL、25ug/mL、125ug/mL, the suicide gene group, the 5ug/mL、25ug/mL 125ug/mL of ginsenoside combining with GCV 15.7μM groups (the concentration had been confirmed by preliminary study),48h, using MTT assay to detect the inhibition of each group and using Q-analysis of Kim Jong-kwan to analysis the effect of ginsenoside combining with suicide gene system.2 The enhancing effect of the presciption of resveratrol、tanshinoneⅡA and ginsenoside combining with HSV-tk/GCV suicide gene system on melanoma.2.1 The effect of the presciption of resveratrol、tanshinoneⅡA and ginsenoside on B16 cells:Treating B16 cells with the three-drug presciption at the final concentrations of 12.5μg/mL (light concentration)、25μg/mL (medium concentration)、50μg/mL (high concentration) 24h、48h, and using MTT assay to detect the inhibition of each group.2.2 The damage effect of the presciption of resveratrol、tanshinoneⅡA and ginsenoside combining with HSV-tk/GCV suicide gene system on B16 and B16tk mixed cells:Mixing the B16tk cells with B16 cells at the proportion of 20% and planking, setting the cell control group(0μg/mL), the three-drug presciption groups with the final concentrations of 12.5μg/mL、25μg/mL, 50μg/mL, the suicide gene group, the 12.5μg/mL、25μg/mL、50μg/mL groups of the presciption combining with GCV 15.7μM(the concentration of GCV had been confirmed by preliminary study),48h, using MTT assay to detect the inhibition of each group and using Q-analysis of Kim Jong-kwan to analysis the effect of the three-drug presciption combining with HSV-tk/GCV suicide gene system.2.3 The apoptosis inducing effect of the presciption of resveratrol、tanshinoneⅡA and ginsenoside combining with HSV-tk/GCV suicide gene system on B16/bl6tk cells:Mixing the B16tk cells with B16 cells at the proportion of 20% and planking, setting the cell control group(0μg/mL), the three-drug presciption group with the final concentration of 50μg/mL, the suicide gene group, the combined treatment group (the concentration of GCV was 15.7μM), 48h. Flow cytometry was used to detect the cell cycle and apoptosis.2.4 The enhancing effect of the presciption of resveratrol、tanshinoneⅡA and ginsenoside combining with HSV-tk/GCV suicide gene system to B16 cells in vivo:85 SPF-class male mices, except the normal group of 5 mice were injected with saline in the right forelimb armpit 0. mL/only, the other mice were injected 2X105 cells (B16tk and B16 wild type cells were mixed in the proportion of 1:4) in the right forelimb armpit 0.1mL/only. In the 7 day after inoculation, when the masses were palpable in axillary, set the model group、the suicide gene treatment group、the traditional Chinese medicine treatment group (the presciption of resveratrol、tanshinone IIA and ginsenoside group)、the combined treatment group (the traditional Chinese medicine combining with suicide gene treatment group) randomly. In the 7～14 days after inoculation: the normal and model groups were injected intraperitoneally the mixed liquid of ethanol and saline 0.2mL/only, in which the proption of ethanol was 20%; the suicide gene group were injected intraperitoneally the Ganciclovir injection 0.2mL/only (100mg/kg·d) in the afternoon; the traditional Chinese medicine treatment group were injected the resveratrol 0.05mL/only (100mg/kg·d)、tanshinone IIA 0.05mL/only (100mg/kg·d)、ginsenoside 0. 1mL/only (100mg/kg·d) respectively in the morning; the combined treatment group were injected intraperitoneally the resveratrol 0.05mL/only (100mg/kg·d)、tanshinone IIA 0.05mL/only (100mg/kg·d)、ginsenoside 0.05mL/only (100mg/kg·d) respectively in the morning, and injected intraperitoneally the Ganciclovir injection 0. 1mL/only (100mg/kg·d) in the afternoon. When the masses were palpable in axillary, using the vernier caliper to measure the major axis and minor axis of each tumor and calculating the volumes of them; on the fourteenth day after inoculation, executed mice and measured tumors’weight, and then captured the tumors’photos.Results:1 The enhancing effect of ginsenoside combining with HSV-tk/GCV suicide gene system on melanoma.1.1 The enhancing effect of ginsenoside combining with HSV-tk/GCV suicide gene system on B16 cells in vivo:the animal experimental result showed that, the tumor weight of each treatmental groups were smaller than the model group; compared to the model group, there was statistically significant in the combined treatmental group (P<0.05), there was no statistically significant in the ginsenoside treatmental group and suicide gene treatmental group (p>0.05). The tumoral inhibitional rates of the suicide gene treatment group、the ginsenoside treatmental group、the combined treatment group were 16.22%、 17.26%、32.29% respectively, the tumoral inhibitional rate of the combined treatment group was highest, indicating that the ginsenoside had synergy effect ion on HSV-tk/GCV suicide gene system in vivo.1.2 The histopathological examination:the result of pathological section showed that, the phenomenon of lymphocyte infiltration was obvious in the ginsenoside group and the combined treatment group, especially the latter group. Sorting in degree according to the infiltration of lymphocytes and using the rank test, compared to the model group there were statistically significant in the suicide gene treatment group、the ginsenoside treatment group、the combined treatment group (p<0.05), the infiltration of lymphocytes of each treatmental groups replied that the body’s immune system of the ginsenoside group and the combined treatment group (the ginsenoside combining with suicide gene group) was higher than the model group, which indicating that synergy effect of ginsenoside on HSV-tk/GCV suicide gene system realized by the immunologic mechanism of the bystand effect of suicide gene.1.3 The effect of ginsenoside on B16 cells:MTT results showed that, after the ginsenosid effected on B16 cells 24h, the 1ug/mL、10ug/mL、100ug/mL groups’ growth inhibitional rates were respectively:-9.8%、2.0%、8.8%; after 48h, the growth inhibition rates were respectively:2.3%、2.7%、10.3%, indicating that there was no significantly growth inhibition.1.4 The damage effect of the ginsenoside combining with tk/GCV suicide gene system on B16 and B16tk mixed cells:5ug/mL、25ug/mL、125ug/mL of the ginsenoside combining with GCV 15.7μM acted on the cells 48h, the inhibition rates were 19.5%、25.2%、39.6% respectively, there were no stastically significant in all groups compared to the suicide gene group(p>0.05); and the Kim Jung-kwan Q values were 0.67、0.69、0.85, less than 1.15, the results concluded that the ginsenoside had no synergy effect on HSV-tk/GCV suicide gene system in vitro.2 The enhancing effect of resveratrol、tanshinoneⅡA and ginsenoside combining with HSV-TK/GCV suicide gene system on melanoma.2.1 The effect of resveratrol、tanshinoneⅡA and ginsenoside on B16 cells: MTT results showed that, after the three-drug presciption effected on B16 cells 24h,12.5μg/mL (light concentration)、25μg/mL (medium concentration)、50μg/mL (high concentration) groups’inhibition rates were respectively: 16.2%、29.1%、44.3%,24h IC50is 61.7μg/mL; after 48h, the growth inhibition rates were respectively:32.0%、59.6%、67.0%、48h IC50 is 22.4μg/mL, there wore significant time and dose relationships2.2 The damage effect of the presciption of resveratrol、tanshinoneⅡA and ginsenoside combining with HSV-tk/GCV suicide gene system on B16 and B16tk mixed cells:12.5ug/mL、25ug/mL、50ug/mL of the three-drug presciption combining with GCV 15.7μM acted on the cells 48h, the inhibition rates were 30.8%、44.7%、65.3% respectively, there were statistically significant in all groups compared with the suicide gene group (p<0.05); the Kim Jung-kwan Q values were 0.91、0.96、1.16, the high concentration group was greater than 1.15, which concluded that the presciption of resveratrol、tanshinone IIA and ginsenoside had synergy effect on HSV-tk/GCV suicide gene system.2.3 The apoptosis inducing effect of the presciption of resveratrol、tanshinone IIA and ginsenoside combining with HSV-tk/GCV suicide gene system on B16/B16tk cells:The result of flow cytometry showed that the three-drug presciption combing with suicide gene system could promote the apoptosis of B16 cells, the result indicated that the synergy effect of the three-drug presciption combining with HSV-tk/GCV system maybe achieved by prmoting the apopotosis of B16 cells.2.4 The enhancing effect of the presciption of resveratrol、tanshinone IIA and ginsenoside combining with IISV-k/GCV suicide gene system on B16 cells in vivo:the animal experimental result showed that, the quatities of each treatment group were lighter than the model group; compared to the model group, there was statistically significant in the combined treatment group (the traditional Chinese medicine combining with suicide gene treatment group) p<0.05) and there was no statistically significant in the traditional Chinese medicine treatment group (p>0.05). The tumoral inhibitional rates of the suicide gene group、the traditional Chinese medicine treatment group、the combined treatment group were 16.22%、21.62%、44.22% respectively, the tumoral inhibition rate of the combined treatment group was highest and the Q value was greater than 1.15, indicating that the three-drug presciption of resveratrol、tanshinoneⅡA and ginsenoside had synergy effection on HSV-tk/GCV suicide gene system in vivo, the method of compound of several Chinese medicines combining with HSV-tk/GCV suicide gene system was viable in vivo.Conclusion: This study find that the ginsenoside can enhance the immunity of mice to achieve the synergy effect of suicide gene on murine transplanted malignant melanoma; the prescription of several Chinese medicines such as resveratrol、tanshinone IIA and ginsenoside has synergy effect on HSV-tk/GCV suicide gene system in vitro and vivo. The enhancing effect has business with the interaction of multiple mechanisims such as GJIC、apoptosis and cellular immunity.