The Enhancing Utility And Mechanism Research Of Curcumin On Bystander Effect Of HSV-tk/GCV Suicide Gene System

Malignant tumor is a kind of common deadly diseases.Along with the development of modern medicine,gene therapy,as a new treatment,is of great concern.Abroad, gene therapy has been a standard therapeutic trial of malignant tumor after the failure of conventional treatment.However,due to the low transfection efficiency, weak in targeting,the toxic side-effect,and not high enough efficiency to kill tumor cell,it is very urgent to be solved that how to reduce the dosage and toxicity of carrier and precursors effectively,enhance lethality of gene therapy system to carcinoma.Chinese medicine has the advantage of little toxic side-effect,lessening the side-effects of radiotherapy and chemotherapy.Searching suicide gene synergies drugs in Chinese medicines,treating tumor with integrated traditional Chinese and western medicine has been increasingly reecognized and accepted by the world medical and acceptance.Objective:To investigated synergy effect of curcumin on the suicide gene system and its mechanism,explore the pharmacological target of enhancing suicide gene bystander effect by GJIC or apoptosis mechanism,clarify the mechanism of its synergy effect,and provide experimental basis for establishing cancer gene therapy integrated with Chinese medicine therapy,promoting the clinical application of cancer gene therapy and the development of new anti-tumor drugs.Methods:1.Used MTT method to screen the concentration for the synergy with tumor suicide gene bystander effect from curcumin(with Kim Jong-Q values as evaluation criteria). First of all,to examine the effect of curcumin on CBRH7919 and B16 cells: The cells CBRH7919 were treated seperately by curcumin with final concentration at 0μM,3.125μM,6.25μM,12.5μM,25μM 50μM for 48h and the cells B16 were treated seperately by curcumin with final concentration at 0μM,6.25μM,12.5μM,25μM,50μM for 72h.The quantity and the form of cells in each group were observed under inverted phase contrast microscope.The viability of cell in each group was detected by MTT assay.And secondly,to choose the best work concentration of GCV on B16 cells,they were treated by GCV with concentration at 0μM,3.925μM,7.85μM,15.7μM,31.4μM,62.8μM,for 72h;To select perfect ratio of tk⁺:tk^- in the therapy of HSV-tk/GCV system,the tk⁺:tk^- cell proportion of 0%,10%,20%, 30%,50%,100%on B16 cells affects 48h.The quantity and the form of cells in each group were observed under inverted phase contrast microscope.The viability of cell in each group was detected by MTT assay.The last,to examine the influence of curcumin on the bystander effect on HSV-tk/GCV mechanism in cells CBRH7919 and B16:the mixed cells with 10%tk⁺ on the cells CBRH7919 and the mixed cells with 20%tk⁺ on the cells B16 were treated seperately by curcumin with final concentration at 6.25μM,12.5μM and 25μM and GCV with final concentration at 15.7μM,or treated by curcumin plus GCV with the same concentration,for 48h,then the quantity and form of the cells in each group were observed under inverted phase contrast microscope before and after the treatment.The inhibition rate of cells in each group was detected by MTT assay.The Q-value was used to test the synergistic effects on curcumin combined with the HSV-tk/GCV system.2.Used flow cytometry to detect the effect of curcumin on cancer cell cycle and to analyze its impact on the apoptotic index.Single-PI staining dectect the influence of curcumin on 10%tk⁺ cell cycle:the mixed cells with 10%tk⁺on the cells CBRH7919 were treated seperately by curcumin with final concentration at 6.25μM,12.5μM,25μM,and GCV with final concentration at 15.7μM,or treated by curcumin plus GCV with the same concentration,for 48h.FACS analyse cell cycle phase in each group.Annexin V/PI made impact on 10%tk⁺ cell apoptosis index:the mixed cells with 10%tk⁺ on the cells CBRH7919 were treated seperately by curcumin with final concentration at 6.25μM,12.5μM,25μM and GCV with final concentration at 15.7μM,or treated by curcumin plus GCV with the same concentration,for 48h.FACS analyse cell apoptosis index in each group.The Q-value was used to test the synergistic effects on curcumin combined with the HSV-tk/GCV system.3.Used dual-dye-transfer technology to observe the effect of curcumin on the GJIC function of CBRH7919 and B16.The cells CBRH7919 and B16 were treated by curcumin with final concentration at 6.25μM,12.5μM and 25μM for 48h,the function of GJIC in each group was detected by dual-dye-transfer technology and flow cytometry.4.Used RT-PCR and westen-blot to detect the effect of curcumin on the gap junction cx26 and Cx43.The cells CBRH7919 and B16 were treated by curcumin with final concentration at 6.25μM,12.5μM and 25μM for 48h,then RT-PCR and Western-blot examine the expression level of the Connexin26 and Connexin43 in each group.Results:1.Impact of curcumin on HSV-tk/GCV system bystander effectObserved under inverted phase contrast microscope,the cells in the control group were found in good state with enhanced adherence,high density,displaying spindle, diamond,triangle or polygon in shape.Compared with the cells in the control group, the quantity of cells in the treated groups decreased and less cells adhered. Furthermore,cell debris were seen and cells were found dead(the cells turned round and dropped).The viability of cell in the curcumin of CBRH7919 group was 96.0±2.45%,96.01±3.05%,92.69±2.32%,52.54±3.35%,33.62±5.44%respectively, for 48h.The viability of cell in the curcumin of B16 group was 71.35±1.35%,62.40±0.30%,36.0±7.0%,10.0±0.20%,respectively,for 72h.Experiments show:the suitable concentration was 15.7μM of GCV for avoiding the inhibiting effect of on B16 cells.The effect of drugs under 20%tk⁺of B16 can be relatively large,conducive to observation of drug-induced bystander.We used in vitro MTT method to investigate the effect of curcumin on the bystander effect of suicide gene,when combining the curcumin at 6.25μM,12.5μM and 25μM with 10%tk⁺/GCV, the inhibition rate(%) was 23.12±8.49,32.34±4.07,35.48±2.53,which was higher remarkably than that when only treated by 10%tk⁺/GCV(10.80±5.03) and the curcumin at 6.25μM,12.5μM and 25μM respectively(20.00±5.27,5.90±8.54,22.47±2.37).When comparing the actual inhibition rate with the theoretical one, the Q-value analysis indicated that when combining the curcumin with concentration at 6.25μM,12.5μM and 25μM with 10%tk⁺/GCV,the actual inhibition rate was 23.12%,32.34%,35.48%respectively,which were remarkable higher than the theoretical ones(10.80%,16.07%,30.84%respectively),and the Q-value was 2.14,2.01,1.15 respectively.All were higher than or equal to 1.15,which indicated synergistic effect.When combining the curcumin at 6.25μM,12.5μM and 25μM with 20%tk⁺/GCV,the inhibition rate was 32.15±0.81%,39.92±0.48%,67.98±0.67% respectively,which was higher remarkably than that when only treated by 20%tk⁺/GCV(13.96±1.93%) and the curcumin at 6.25μM,12.5μM and 25μM respectively (6.27±1.15%,22.20±0.96%,51.36±0.93%).When comparing the actual inhibition rate with the theoretical one,the Q-value analysis indicated that when combining the curcumin with concentration at 6.25μM,12.5μM and 25μM with 20%tk⁺/GCV,the actual inhibition rate was 32.15%,39.92%,67.98%respectively, which were remarkable higher than the theoretical ones(19.36%,33.07%,58.15% respectively),and the Q-value wasl.66,1.21,1.17.All were higher than 1.15, which indicated synergistic effect.2.Effect of curcumin on cancer cell cycle and cancer cell apoptosis index flow cytometry analysis showed that:after using rcumin at 6.25μM,12.5μM and 25μM to effect on 10%tk⁺ cells of CBRH7919 for 48 hours,the ratio decreased at G₀/G₁ phase,and increased at S phase and G₂/M phases in a dose-dependent manner, suggesting that curcumin might promote the development from G₀/G₁ to S phase; combination of curcumin with HSV-tk/GCV system could make significantly more cells remain in S phase,and induce apoptosis of some cells.When comparing the actual inhibition rate with the theoretical one,the Q-value analysis indicated that when combining the curcumin with concentration at 6.25μM,12.5μM and 25μM with 10%tk⁺/GCV,the actual inhibition rate was 14.20%,15.65%,18.05%,which were remarkable higher than the theoretical ones(10.85%,12.10%,13.60%),and the Q-value was 1.31,1.29,1.33.All higher than 1.15,which indicated synergistic effect.3.Effect of Curcumin on GJIC function of CBRH7919 and B16 Dual-Fuel technology transfer flow cytometry results showed that:curcumin has the trend to enhance the gap junctional intercellular communication function of CBRH7919 and B16.4.Effect of Curcumin on the gap junction Cx26 and Cx43RT-PCR and Western-blot results showed that:Curcumin could enhance the expression of CBRH7919 and B16 cell gap junction protein Cx26 and Cx43.Showing a tissue-specific expression and the different level from transcription to translation.Conclusions:This investigation explored the synergy effect of cuicumin,the active ingredient of traditional Chinese medicine,on HSV-tk/GCV suicide gene therapy in cellular level.We found that luteolin could enhance the lethality of suicide gene on cells effectively,improve the bystander effect,and have the synergy effect.Analysis of its mechanism showed it was likely to play a role by enhancing GJIC or inducing apoptosis,regulating cell cycle,or some of the above mechanisms together.