The Enhancing Utility Reserch Of Luteolin Baicalin And Emodin On "Bystdander Effect" Of HSV1-tk/GCV System

Malignant tumors belong to a kind of serious disease which endangerhuman's health and even their lives. Gene therapy sheds lights on patientswho are suffering from this kind of disease. In foreign countries, gene therapyhas already become a standard trail after the failure of conventionalchemotherapy, but over a decade past, gene therapy is not as effective aspeople's expectation. Although the existing of bystander effect makes it thepotential of killing all tumor cells theoretically, the low efficiency oftransfection and lack of targeting lead to its insufficient killing ability.Moreover, the use of virus vector may bring some potential side-effects topatients. Therefore, it is important to improve the therapy effect,eliminating its side-effect. The bystander effect is indicated in almost everysuicide gene system and so increasing the bystander effect is a good approachto solve this problem. Considering the mechanism of bystander effect conductedby the Gap junction intercellular communication (GJIC), apoptosis of cell andimmune factors, researchers today enhance the bystander effect by methods oftwin genes transfection and medical inducement to elevate GJIC, induceapoptosis and raise the function of immune system. Chinese medicine is anatural medical treasury which has relatively light side-effect and someadvantages and potentials in apoptosis inducing, cell communication andadjustment of immune system. It is feasible to search some effective componentsin traditional Chinese medicine for improving the effect of suicide genetherapy and to establish a combination therapy of suicide gene and Chinesemedicine.Objective:To investigate the effects of luteolin, baichlin and emodin, which are components of some tumor-inhibiting herbs and have the potential of enhancingGJIC or inducing tumor cell apoptosis, on tumor suicide gene therapy systemto see if they have the synergistic effects on suicide gene therapy, and toexplore and clarify the possible pharmacological targets of the real effectivecomponents to enhance the suicide gene bystander effect through the GJIC orcellular apoptosis mechanisms. Further more, to construct the foundation ofdeveloping a combination therapy of tumor suicide gene and Chinese medicine,triggering the clinical applying of tumor gene therapy and exploring newanti-tumor Chinese medicine.Method:Rat hepatocarcinoma cell CBRH7919/CBRH7919tk~+ were used in the screeningof active compounds of TCM in vitro, and the tk~- control group, GCV group, TCMgroup, TCM plus GCV group, 10%tk~+ control group, 10%tk~+/GCV group, TCM and10%tk~+/GCV combination group were set in the screening experiment. MTT assaywere first used in the screening of active compounds with synergistic effecton tumor suicide gene from luteolin, bachlin and emodin. The Q-value analysiswas used to estimate the synergistic effects of the active components on thesuicide gene system. The Q-valuewas equal to the ratio of the actual effectof combined treatment to its theoretical effect. The effect was classifiedinto three categories: antagonistic effect (Q≤0.85), additive effect (0.85≤Q＜1.15), and synergistic effect (Q≥1.15). Then the cell cycle analysisand cell apoptotic index were performed using a FACScan cell analyzer. Methylgreen-pyronin staining and acri dine orange (AO) staining were used in thecellular apoptotic morphology observation. DNA of the cells was extracted andidentified by gel electrophoresis for evidence "DNA ladder" The effectsof the effective component on the GJIC of CBRH7919 were detected bySL/DT(scrape loading/dye transfer) technique. The expressions of Cx43 and Cx32in cells were determined by western bloting.Result:1. The results of screening active compounds of TCM in vitro.When the final concentration of luteolin was 5μM and 10μM, inhibition rateof luteolin on CBRH7919 was 13.8±2.95% and 28.93±5.49%, respectively. IC50was 26μM. When combining with HSV-tk/GCV system, luteolin showed asynergistic effect at 5μM and 10μM(Q values＞1.15) and the actualinhibition rate was 42.33±4.89% and 48.79±6.89% respectively, which was much higher than the theoretical inhibition rate and that in the Luteolin groupand the 10%tk~+/GCV group. But it showed an additive effect on HSV-tk/GCVtherapeutic system at 20μM and 40μM (0.85＜Q value＜1.15). Both bachlin andemodin showed an additive effect on HSV-tk/GCV therapeutic system at a rangeof concentration (0.85＜Q value＜1.15).2. The results of the apoptotic inducing mechanisms research of theinhibition effects of luteolin on CBRH7919.2.1 Luteolin showed an apoptotic rate-elevating effect on CBRH7919 whencombining with HSV-tk/GCV system. Furthermore, it showed a S-phase laggingeffect on CBRH7919 cell cycle. When working alone, luteolin did not showed asignificant effect on the apoptotic rate and the cellular cycle of CBRH7919.2.2 The results of methyl green-pyronin staining and acri dine orangestaining showed evident apoptotic morphology in luteolin treated CBRH7919.2.3 The DNA extracted from the cells showed apparent "DNA ladder".3. The results of the research on the influence of luteolin on GJIC andthe expression of associated connexin in CBRH7919.When CBRH7919 were scraped and incubated in presence of Lucifer yellow,we observed extensive Lucifer yellow diffusion through CBRH7919 treated byluteolin for 32 hrs, whereas lower dye transfer of Lucifer yellow were observedin the control group. This result showed that ability of GJIC function ofCBRH7919 was up-regulated by luteolin.The results of western-blotting showed that the sizes and color of theconnexin bands in luteolin group were bigger and deeper than that in controlgroup. The quantities of expressed connexins were greater in 48h luteolintreated group than that in 24h luteolin treated group. These results showedthat luteolin improved the expression of Cx43 and Cx32 in CBRH7919. The effectshowed in a time-dependent manner.Conclusion:We explore the effects of luteolin, bachlin and emodin on tumor HSV-TK/GCV suicide gene therapy system. Results show that luteolin exibitssynergistic killing effect on suicide gene system. Whereas bachlin and emodinexibit only additive effects on suicide gene system. Mechanisms of thesynergistic effect of luteolin are related to the restoration of GJIC, or/andthe apoptotic inducement or the regulation of cellular cycle.