Synergistic Interaction Between Active Components And Monomer From Salvia Chinensisbeth. And Taxanes In Vitro And Its Probably Mechanism

Part1.Synergistic Interaction between ethanol extraction part、trichloromethane extraction part、ethylacetate extraction part、the monomer ursolic acid from Salvia chinensis beth. and taxanes in vitroObjective:Traditional Chinese medicine’s anti-tumor effect has a long history and wide range of use. To find one of the effective anti-tumor ingredient from natural products is the current direction of the development of anticancer drugs.In our lab the different extraction parts from Salvia chinensis beth. has been isolated and identificated.The different extraction parts and monomer has a valid anti-tumor activity.And further study demonstrate that they have valid anti-tumor activity in vitro, its anti-tumor mechanism involves the promotion of tumor cell apoptosis and antiangiogenesis. The purpose of this study is to investigate the different effective parts from Salvia chinensis beth. the monomer ursolic acid with docetaxel’s interactions and possible mechanisms.Further study will forcus on the effect of reversal drug resistance.and related mechanisms.Methods:1. The IC5o doses of each single drug docetaxel、ethanol extraction part、trichloromethane extraction part ethyl acetate extraction part from Salvia chinensis beth and the monomer ursolic acid were determined by the MTT assay. Based on their respective individual IC50values to cancer cells for48hr drugs with six different concentrations of both composition from Salvia chinensis beth. and docetaxel were added into96-well plates The inhibition of cell proliferation was calculated by comparison to the control group. Dose-response curves were got from each drugs, and for multiple dilutions of a fixed-ratio combination of each two drugs. Synergistic interactions between composition from Salvia chinensis beth. and chemotherapeutic agents on human gastric cancer cells BGC-823, were evaluated using the combination index (CI) method. Cls>1indicate antagonism, Cls<1indicate synergy, and Cls=1indicate additivity.2. Flow cytometry was used to study the cell apoptosis variability in each group3. Expression of Taxol treatment associated genes was measured by real-time quantitative RT-PCR.Results:1. MTT experimental results show that ethanol extraction part、trichloromethane extraction part、ethyl acetate extraction part from Salvia chinensis beth and the monomer ursolic acid with the Doc on the the of BGC-823, A549, A549/Taxol cell lines all have the concentration-dependented ability of inhibit proliferation.2. EAP40μg/ml、TCP25μg/ml、UA30μg/ml、Doc1μg/ml single or combined acted on BGC-823cell. Effects of apoptosis was detected:the control group, EAP, TCP, UA and Doc single-agent apoptosis rate were (4.633±1.81)%,(12.6±1.53)%,(10.7±2.41)%,(11.73±1.76)%,(35.4±1.42)%respectively. With the the Doc combination apoptosis rate(55.87±2.95)%,(41.97±1.44)%,(58.13±1.56)%. The apoptosis ratio Combination therapy compared with single-agent was significantly different (p<0.005).3. EAP, TCP, UA interact with BGC-823cell in a IC30concentration for48h, the EAP and TCP enable adjust BGC-823cell’s BRCA1, RAP80gene expression levels, which BRCA1were raised to1.08±0.03(P=0.049) and2.82±0.21(P=0.000); RAP80were raised to1.64±0.07(P=0.004) and3.66±0.51(P=0.012), but not significant downward effect on beta-tublin III gene level. Monomer UA has a significant role in increase of BRCAland RAP80’s gene expression levels (BRCA1, RAP80, respectively, on raised to1.33±0.15and1.76±0.20), significantly lowered level of gene beta-tublin III (down to0.63±0.16).Conclusion:1. Ethanol extraction part、trichloromethane extraction part from Salvia chinensis beth.in combination with Doc has synergistic sensitizing effects in low concentrations.2. The mechanism due to the change of chemotherapeutic agent resistance related genes and the synergistic apoptotic effect Part2.The effect of ethanol extraction part、trichloromethane extraction part、 ethylacetate extraction part、the monomer ursolic acid from Salvia chinensis beth. on paclitaxel-resistant lung cancer cells A549/TaxolObjective:Our previous research under the guidance of different extraction parts and monomer from Salvia chinensis beth. Interaction with docetaxel in vitro experimental results also show that the mechanism due to the change of chemotherapeutic agent related genes and the synergistic apoptotic effect. This part of the experiment will further explore the effect of different extraction parts and monomer from Salvia chinensis beth.to paclitaxel resistant cell A549/Taxol and whether they can change the taxane efficacy related gene expression, to play the reversal of drug resistance effect.Methods:1. Morphological differences was observed by light microscopy2. MTT assay was used to compare the differences in sensitivity of the two cells A549and A549/Taxol to traditional Chinese medicineand chemotherapy drug paclitaxel.3. Flow cytometry was applied to study the cell apoptosis rate and cell cycle4. Flow cytometry was applied to study the cell cycle5. Transwell chamber was employed to investigate the invasion ability of cells6. Expression of Taxol treatment associated genes and MDR1was measured by real-time quantitative RT-PCR.Results:1. A549/Taxol cell growth was significantly faster than the A549cell, and has an enhanced invasion.The cell cycle analysis showed that proportion of A549/Taxol in the G1phase compared with the A549were increase d and S phase were decreased. The A549/Taxol cell’s BRCA1and RAP80mRNA expression were significantly reduced compared with the A549cells, but multidrug resistance gene MDR1and beta-tublin Ⅲ mRNA expression levels were significantly increased. BRCA1mRNA decreased to (0.0046±0.019)(p= 0.001), RAP80mRNA decreased to (0.0037±0.017)(p=0.000), MDR1mRNA elevated to(44.54±3.27) times (p=0.034), beta-tublin III mRNA increased (4.71±0.98) times (p=0.003).2. The IC50of paclitaxel to A549/Taxol was48.358times that of its parental cell A549and the drug resistant index (RI) to docetaxel was27.208..3. EAP, EYP, TCP and UA were able to significantly reduce the force of A549/Taxol cell’s invasion (p=0.000), and the strongest drug was UA.4. With EAP12.5μg/ml TCP6.25μg/ml UA6.25μg/ml and Taxol400ng/ml, single or combined acted on A549/Taxol cells after48h, the control group’s natural apoptosis rate was:(7.8±2.21)%the monotherapy group apoptosis rate were (10.3±3.66)%,(8.9±3.73)%,(13.1±2.29)%,(16.4±3.15)%. The apoptosis rate of the combined treatment group (42.2±7.73)%,(32.8±6.69)%,(66.8±10.68)%. The apoptosis ratio Combination therapy compared with single-agent was significantly different (p<0.005).5. The impact on cycle of A549/Taxol was mainly for the blok of S phase. EAP, EYP, TCP didn’t show cell cycle blockade effect. In the role of combination therapy, only EAP increased Taxol’s G2/M phase blok effect,(p<0.05).6. With EAP12.5μg/ml TCP6.25μg/ml UA6.25μg/ml acted on A549/Taxol cells for48h the RAP80mRNA expression levels were raised to8.210±1.750=0.000),5.654±1.75(p=0.015)3.331±1.23(p=0.000) respectively; the beta-tublin Ⅲ’s expression level were brought to0.496±0.14(p=0.004),0.551±0.51(p=0.014),0.507±0.23(p=0.032) and only TCP can upregulate BRCA1’s expression level{raised to2.226±0.789(p=0.039)}.The role of EAP, TCP, UA’s regulation effect on gene of MDR1were not significant (p>0.05).Conclusion:1. Compared with the parental cells, paclitaxel-resistant cells A549/Taxol has a unique morphology and biological behavior and have good resistance to paclitaxel and docetaxel.2. Ethanol extraction part、trichloromethane extraction partfrom Salvia chinensis beth and the monomer ursolic acid can effectively reduce A549/Taxol cell’s invasion ability increase the sensitivity to paclitaxel.The mechanisms involved in their efficacy of changing the expression of chemotherapeutic agent genes and has nothing to do with the changes in the level of MDR1gene.