The Anticancer Activity And Reversal Of Multidrug Resistance Of Xinghuayu Injection In Vitro And Its Mechanisms

Xinghuayu injection was a new Ginkgo biloba extract injection. The content of flavonoid glycoside was 49.79%, and terpene was 4.4% in the Ginkgo biloba extract. It was reported in many literature that flavanoids had goodish anticancer activity. Xinghuayu injection maybe has latent anticancer activity for it containing affluent flavonoids. The present study mainly explored the inhibitory effect of Xinghuayu injection and drug-containing serum on the MCF-7/S cells, and reverse effect of Xinghuayu injection on drug-resistant cells and the mechanisms of action to provide foundation for the clinical application of Xinghuayu injection.Objective: To investigate the inhibitory effect of Xinghuayu injection and drug-containing serum on the MCF-7/S cells, and reverse effect of Xinghuayu injection on multidrug resistance and its mechanisms.Methods:⑴Preparation of drug-containing serum: Twenty SD rats were divided randomly into 2 groups. Xinghuayu injection 1ml/100g and normal saline 1ml/100g were given once daily for three days by intrapentoneal injection respectively. Half an hour after the final injection, blood was collected and serum was extracted after anesthesia.⑵Effect of Xinghuayu injection on the proliferation of MCF-7/S cells in vitro: Different concentrations of Xinghuayu injection were added 24 hours after MCF-7/S cells were cultured in 96-well plates. The same volume of culture mediunm was added in solvent control group. OD value was determined at 570 nm by Fluo-star 3 days after drug administration. The inhibitory rate of Xinghuayu injection on proliferation of cells was calculated.⑶Measurement of the inhibitory effect of drug-containing serum on MCF-7/S cells by MTT method: MCF-7/S cells were divided into 6 groups: positive control group, solvent control group, 5% drug-containing serum group, 10% drug-containing serum group, 15% drug-containing serum group, 10% normal serum group. After 3 days, the OD value was detected at 570 nm by Fluo-star.⑷Reverse effect of Xinghuayu injection on drug resistance:Drug sensitivity and multiple of drug-resistance of cells were determined by the means of MTT. The concentration in which growth rate of cells was more than 95% was the non-cytotoxic concentration. The concentration in which growth rate of cells was in the range of 85% to 95% was the low-cytotoxic concentration. The IC50 value was calculated. The multiple of drug-resistance is the IC50 of drug-resistant cells to the IC50 of sensitive cells. This part of the experiment included four groups:①different concentrations of ADM acting on sensitive cells;②different concentrations of ADM acting on drug-resistant cells;③different concentrations ADM combined with low-cytotoxic concentration of Xinghuayu injection acting on drug-resistant cells ;④different concentrations of ADM combined with non-cytotoxic concentration of Xinghuayu injection acting on drug-resistant cells. OD value of each well was measured by the method mentioned above, and IC50 and reverse multiple of drug-resistance were calculated. Measurement of confocal microscope by laser scanning confocal microscope: Drug-resistant cells were cultured in 24-well plates. The next day, RPMI1640 medium+ADM was added in control group, ADM combined with non-cytotoxic concentration of Xinghuayu injection and ADM combined with low-cytotoxic concentration of Xinghuayu injection were added in the other two groups. Supematant was discarded and cells were washed with PBS 24 h after drug administration. Fluorescence intensity of cells was observed by laser scanning confocal microscope.⑸The mechanism of reversal of drug resistance by Xinghuayu injection: The expression of p-gp was determined by Western-blot.Results:⑴In MTT assay, after MCF-7/S cells were treated with Xinghuayu injection 22, 44, 88, 175 and 350mg/L for 3 d, the inhibition ratio was 2.50%, 6.25%, 8.72%, 18.72% and 26.63%,respectively. Compared with solvent control, Xinghuayu injection 350mg/L could inhibit the proliferation of MCF-7/S cells obviously (P<0.05).⑵In MTT assay, after MCF-7/S cells were treated with ADM, 5% drug-containing serum, 10% drug-containing serum, 15% drug-containing serum for 3d, the inhibition ratio was 91.21%, 7.46%, 19.36%,27.54%. Ten percent and fifteen percent drug-containing serum inhibited the proliferation of MCF-7/S cells obviously (P<0.01), but the effect was lower than that of ADM(P<0.01).⑶The IC50 values of Xinghuayu injection on MCF-7/A and MCF-7/S cells were (189.95±115.89)mg/L and (177.52±81.24) mg/L, and the multiple of drug resistance was 1.07. The IC50 values of ADM on MCF-7/A and MCF-7/S cells were (0.78±0.21) mg/L and (0.042±0.011) mg/L, the multiple of drug resistance was 18.57. The IC50 values of non-cytotoxic and low-cytotoxic concentration of Xinghuayu injectionon on MCF-7/A cells were (0.42±0.18) mg/L and (0.37±0.097) mg/L, the multiple of reversion was 1.87 and 2.12 respectively. It showed that Xinghuayu injection could decrease the IC50 of MCF-7/A cells (P <0.05).⑷The fluorescence intensity in drug-resistant cells treated with ADM only was (34±7), The fluorescence intensity in the cells was (73±11) in the group of ADM combined with non-cytotoxic concentration of Xinghuayu injection. The fluorescence intensity in the cells was (118±25) in the group of ADM combined with low-cytotoxic concentration of Xinghuayu injection. The result showed that Xinghuayu injection could increase intracellular concentration of ADM in drug-resistant cells. ⑸The optical density/β-actin was 0.364±0.061, 0.316±0.078, 0.159±0.036 and 0.214±0.021 in blank control group, verapamil group, low-cytotoxic concentration of Xinghuayu injection group and non-cytotoxic concentration of Xinghuayu injection group, respectively. Compared with blank control, Xinghuayu injection could down-regulate the expression of p-gp significantly(P <0.01).Conclusion:⑴Xinghuayu injection and drug-containing serum could singnigicantly inhibit the growth of MCF-7/S.⑵Xinghuayu injection could reverse the drug resistance of MCF-7/A cells.