Reversal Of Multidrug Resistance In SGC7901 / ADR Cells By Total Flavonoids Of Sanghuang Fermentation Broth

Phellinus linteus is a well-known traditional Chinese medicinal fumgi. Over the past several decades, Phellinus linteus possesses many effects, such as antioxidant, antitumer, liver protection and immunity enhancement activities. Chemotherapy and radiotherapy have many side-effects, such as lack of appetite, vomiting, high fever and lipsotrichia, which can be reduced by Phellinus linteus. At present, wild Phellinus linteus resource is deficient, meanwhile the market demand for Phellinus linteus is growing which lead to promote its artificial culture technology progress. The researches show that there are no difference between artificial and wild Phellinus linteus in many kinds of effective components.In this research, we chose PLTF (Total flavonoids from the zymotic fluid of Phellinus linteus mycelium, PLTF) as experimental drug, researched its reversal effect on human gastric cancer cell line SGC7901/ADR, and then explored the possible mechanism. The results of this study could provide the theoretical basis for the Phellinus linteus clinical application.The main research methods and results were as follows:(1) MTT assay was used to detect the resistance of human gastric cancer cell line SGC7901/ADR to adriamycin, the results showed that the resistance index(RI) to adriamycin was 126.77. And then we observed the morphological differences between the two cell lines using optical microscope. We found that SGC7901 cells were close to round, while SGC7901/ADR cells were nearly long spindle, in addition, the wire drawing structure in SGC7901/ADR cells was more than in SGC7901 cells.(2) The cytotoxicity experiment was determined by MTT assay, the results showed that 20% inhibitive concentration(IC20) and 50% inhibitive concentration(IC50) of PLTF were 86.46,302.57 μg/mL, respectively. Then we chose concentration below IC20 as the non-cytotoxicity concentration for the following experiments.(3) MTT assay was applied to detect the reversal effect of PLTF. SGC7901/AD cells were exposed to 20,40,80μg/mL PLTF for 48 hours, and then incubated with ADR for 36 hours. The results indicated that PLTF could reverse MDR of SGC7901/ADR cells in a concentration-depent manner, and the drug resistance reversal fold were 5.09,6.41, 2.89, respectively.(4) Flow cytometry was used to detect the accumulation of Rh123 and ADR in SGC7901/ADR cells treated with PLTF. After SGC7901/ADR cells were exposed to 20, 40,80μg/mL PLTF for 48 hours, Rh123 accumulation was significantly enhanced by 8.19-,9.44-and 11.03-fold, respectively. However, SGC7901/ADR cells treated by PLTF for 1 hour, PLTF did not significantly alter the intracellular accumulation of Rh123. SGC7901/ADR cells were treated by PLTF for 36 hours, and then incubated with ADR(6μg/mL), the fluorescence intensity of ADR showed a remarkable increase compared with the control group.(5) Utilizing Semi-quantitative reverse transcription PCR technology to determine the regulation of PLTF on P-glycoprotein MDR1 gene transcription. The results indicated that, PLTF could decrease MDR1 gene transcription level in SGC7901/ADR cells. The ratio of MDR1 grey value/(3-actin grey value was 1.35% in control cells, while the radios were 1.68%,1.48%,1.12%,0.581% and 0.37% when SGC7901/ADR cells were treated with 5,10,20,40 and 80μg/mL PLTF.(6) Flow cytometry in combination with immunofluorescence method were used to detect the expression of P-glycoprotein, the results showed that PLTF could significantly decrease the expression of P-glycoprotein (P<0.01) in a concentration-dependent manner.(7) Western blot method was applied to detect the regulation of PLTF of on NF-κB signaling pathway in SGC7901/ADR cells. The results demonstrated that treatment with PLTF for 12 hours, the expression of p-IκBα in cell plasma and NF-κB p65 in nucleus gradually reduced, meanwhile the expression of IκBα in cell plasma increased.Conclusion:PLTF could concentration-dependently enhance the intracellular accumulation of Rh123 and ADR, and reverse MDR of tumor. PLTF could inhibit IκBα be phosphorylated, which decreased the quantity of NF-κB p65 in nuclear, down-regulated the transcription level of the MDR1 gene and the expression level of P-glycoprotein. The results indicated that PLTF may be the potential agent which can reverse the P-glycoprotien mediated multidrug resistance effectively.