| Objective:Malignant melanoma is extremely high incidence, prone to metastasis, poor prognosis, high mortality,and clinical treatment is very difficult. With the development of molecular biology, gene therapy especially suicide gene therapy on malignant melanoma brings hope to patients. following the traditional surgical resection, radiotherapy and chemotherapy,suicide gene therapy becomes to the method of a new anti-tumor treatment. But as a result of issues are still not resoled such as suicide gene therapy vector transfection efficiency is currently low and targeting not enough, there are still some residual tumor cells leading to tumor recurrence, which is the treatment of major defects exist. Bystander effect,that gene transfection-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 efficacy of tumor suicide gene therapy. Preliminary studies showed that:curcumin and resveratrol have obvious synergies on the suicide gene therapy of malignant tumors in vitro,and it related to gap junctions. Be based on preliminary studies,we further validated their synergies on the suicide gene therapy of malignant tumors in vivo and in vitro.In this study, killing effect and its mechanism were discussed through curcumin and resveratrol combined with HSV1-tk/GCV suicide gene system in vitro to B16 melanoma cells. Killing effect were discussed through curcumin and resveratrol combined with HSV1-tk/GCV suicide gene system in vivo to mouse malignant melanoma tumor.Through research we want to provide experimental data and scientific evidence for establishment the programs of suicide gene therapy for clinical compound combined with traditional Chinese medicine.Methods: 1. The effect of curcumin and resveratrol on B16 cells:Setting suitable final concentration of curcumin and resveratrol, MTT assay to detect the inhibition of each group. The final concentration of curcumin were 0μM,5μM,10μM,20μM,40μM,80μM; The final concentration of resveratrol were 0μM,25μM,50μM,100μM,200μM.2. The work concentration of the proportion of tk+:Mixing B16/tk and B16 with the proportion of 0,10%,20%,40%,80%,100%,adding the final concentration of GCV(15.7μM), MTT assay was detected; 60 C57BL/6J mice which are SPF-class healthy,male, setting control group,model group,0%tk/B16 group,20%tk/B16 group,40%tk/B16 group,80% tk/B16 group,0.1ml normal saline were inoculated subcutaneously into C57 mouse under their arms each of control group,while other groups 0.1ml B16 cell suspension(2×106 cells/ml) with each mouse. From the eighth day to the fourteenth day, treated each mouse with 0.2ml·d-1 normal saline in control group with intraperitoneal injection,while 0.2ml·d-1 GCV(10g/L) in other groups. Measured the tumor major axis and minor axis of each tumor, executed mice and measured tumor weight at the fifteenth day,took photos.3. The effect of curcumin and resveratrol combined with tk/GCV system on B16:B16tk mixed with B16 cells at the proportion of 20%, the mixed cells were treated seperately by curcumin with final concentration at 5μM, 10μM,20μM and GCV with final concentration at 15.7μM, or treated by curcumin plus GCV with the same concentration; the mixed cells were treated seperately by resveratrol with final concentration at 12.5μM, 25μM,50μM and GCV with final concentration at 15.7μM, or treated by resveratrol plus GCV with the same concentration,MTT assay to detect the inhibition of B16;using Q-analysis of Kim Jong-kwan to analysis the effect of curcumin or resveratrol combined with HSV-tk/GCV suicide gene system.4. The effect of curcumin and resveratrol on GJ of B16 cells:Setting control group,5uM curcumin group,10μM curcumin group,20μM curcumin group,parachute assay detected the impact of GJ with curcumin combined with tk/GCV system on B16;Setting control group,12.5μ.M resveratrol group,25μM resveratrol group,50μM resveratrol group,parachute assay detected the impact of GJ with curcumin combined with tk/GCV system on B16.5.The synergy effect of curcumin and resveratrol combined with HSV-tk/GCV system on mouse malignant melanoma tumor:170 C57BL/6J rats which are SPF-class healthy,male and female were even.Setting control group,model group,GCVgroup, curcumin group (100mg/kg), curcumin (100mg/kg) joint GCV group, resveratrol (100mg/kg) group, resveratrol(100mg/kg) joint GCV group, resveratrol(200mg/kg) group, resveratrol (200mg/kg) joint GCV group.0.1ml normal saline were inoculated subcutaneously into C57 mice under their arms each of control group,while other groups 0.1ml B16 cell suspension(2×106 cells/ml) with each mice.Then treated each mice with 0.2ml·d-1 normal saline in control group from the second day to the fourteenth day with intraperitoneal injection.From the second day,0.2ml·d-1 normal saline in model group and GCV group with intraperitoneal injection; 0.2ml·d-1 curcumin(20g/L) in curcumin group (100mg/kg) and curcumin (100mg/kg) joint GCV group;0.2ml·d-1 resveratrol (20g/L)/ resveratrol (40g/L) in resveratrol group (100mg/kg) and resveratrol (100mg/kg) joint GCV group/in resveratrol group(200mg/kg)and resveratrol(200mg/kg) joint GCV group. From the eighth day,0.2ml·d-1 GCV(10g/L)in GCV group;0.1ml·d-1 curcumin(40g/L) and 0.1ml·d-1 GCV(20g/L) in curcumin (100mg/kg) joint GCV group; 0.1ml·d-1 resveratrol (40g/L) and 0.1ml·d-1 GCV(20g/L) in resveratrol(100mg/kg) joint GCV group;0.1ml·d-1 resveratrol (80g/L) and 0.1ml·d-1 GCV(20g/L) in resveratrol(200mg/kg) joint GCV group. Measured the tumor major axis and minor axis, executed mice and measured tumor weight at the fifteenth day,took photos.Results:1. The effect of curcumin and resveratrol on B16 cells:MTT results showed that, 5μM. 10μM.20μM.40μM.80μM curcumin group,cell inhibition rates were respectively: 10.82%.18.92%.41.24%.65.74%.89.08%,and compared to control group, there were significant reduction in inhibition rate of each curcumin group (P<0.01); 25μM.50μM. 100μM.200μM resveratrol group, cell inhibition rates were respectively:27.22%.46.97%. 48.10%.62.63%, and compared to control group, there were significant reduction in inhibition rate of each curcumin group (P<0.01).2. The work concentration of the proportion of tk +:MTT results showed that, 0%tk/B16 group,20%tk/B16 group,40%tk/B16 group,80% tk/B16 group,cell inhibition mice were respectively:11.27%.27.40%.35.73%,57.51%,71.21%. The effect of cell inhibition with the proportion of B16/tk cells increased, the tumor inhibitory effect was observed by measuring tumor size and weight significantly when the proportion of B16/tk cells was 80%(p<0.01). In order to allow space for Chinese medicine ingredients to increase bystander effect and simulate low vector transfection efficiency,the mixture of 20% B16/tk cells were used.3. The effect of curcumin and resveratrol combined with tk/GCV system on B16: 5μM, 10μM,20μM curcumin Joint 20% tk+ /GCV group,the actual inhibition rate (38.84%,43.13%,54.31%)was significantly higher than the theoretical inhibition rate (21.04%,30.20%,46.70%), Kim Jung-kwan, Q values were 1.85,1.43,1.16,are greater than 1.15, with synergy.12.5μM,25μM,50μM resveratrol Jiont 20% tk+/GCV group, the actual inhibition rate (30.11%,45.89%,55.72%) was significantly higher than the theoretical inhibition rate (12.99%,38.71%,39.79%), Kim Jung-kwan, Q values were 2.31,1.19,1.40, are greater than 1.15, with synergy.4. The effect of curcumin and resveratrol on GJ of B16 cells:curcumin and resveratrol can advance GJ of B16 cells,with dose-dependency.5. The synergy effect of curcumin and resveratrol combined with HSV-tk/GCV system on mouse malignant melanoma tumor:There were significantly killing effects on B16 cells compared with model group(p<0.05):GCVgroup, curcumin group(100mg/kg), curcumin(100mg/kg)jointGCV group, resveratrol (100mg/kg) group, resveratrol(100mg/kg) joint GCV group, resveratrol(200mg/kg) group, resveratrol (200mg/kg) joint GCV group, cell inhibition rates were respectively:38.88%,35.14%,60.99%,45.81%,60.14%, 54.94%,70.92%;Tumor weight were reduced compared with model group(p<0.05): Thereinto curcumin(100mg/kg) joint GCV group,resveratrol(100mg/kg) joint GCV group,resveratrol(200mg/kg) group,resveratrol (200mg/kg) joint GCV group were significantly reduced compared with model group(p<0.01). Compared with GCV group, tumor weight were significantly reduced at resveratrol(100mg/kg) joint GCV group(p<0.05), curcumin(100mg/kg) joint GCV group,resveratrol (200mg/kg) joint GCV group(p<0.01). There were no statistical significance between other groups.Conclusion:This study found that curcumin and resveratrol on growth inhibition of B16 cells in vitro, synergy existed in suicide gene therapy combined with curcumin and resveratrol,the synergy maight be relevant with the gap junction mechanism; And we found that curcumin and resveratrol on suicide gene therapy to mouse malignant melanoma tumor also has a synergistic effect.
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