Drug Resistance Reversing Effects Of Chinese Medicine Hedyotis Diffusa On Hypoxic HepG-2Cells

ObjectiveTo establish hypoxic HepG-2cell modle,proved its drug resistance forADM.Investigated whether Hedyotis diffusa reversing the drug resisitance of hypoxicHepG-2cells for ADM, involved in the formation of multidrug resistance ofHepG-2/tt,And to further explore the specific mechanisms involved.MethodHepG-2cell survival rate was observed after CoCl2affected on by the trypan bluedye exclusion assay;MTT assay determined the inhibition rate for hypoxic HepG-2cells affected by Hedyotis diffusa;The IC50value of ADM for common HepG-2andhypoxic HepG-2cells were analyzed by MTT assay,And measured its multiple ofdrug resistance; MTT assay derermined the IC50value of ADM for hypoxic HepG-2cells which affected by Hedyotis diffusa,and measured multiple of drug resistancereversal.flow cytometry detected the cell apoptosis rate,and the expression level ofHIF-1a, P-gp and MRP detected by Western Blot.Results1.The medium with CoCl2(100umol/L) cultured the HepG-2cells can estabilshhypoxic HepG-2cells model.To culture the HepG-2cell with diffirent concentation of CoCl2from0umol/L to200umol/L for4days.After3days,the cell cultured by CoCl2150umol/L or more hasdead more than40%, the cell viability was more than90%which cultured by CoCl2 concentration100umol/L or less. Therefore, we choose the maximum tolerated dose100umol/L as the follow-up experiment dosage.2.The HIF-1a expression increased in Hypoxic HepG-2cells which induced byCoCl2.Western Blot results showed that,The expression of HIF-1a in hypoxic HepG-2cells which established by100umol/L CoCl2is more than that in HepG-2cells (P<0.01), and its expression is time-dependent.3.No significant morphological differences between hypoxic HepG-2cells andcommon HepG-2.Wright-Giemsa’s stained the HepG-2cells and hypoxic HepG-2cells induced by100umol/L CoCl2, then observed the morphologic changes under invertedmicroscope,no significant morphological differences were found.4.The maximal non-toxic concentration of Hedyotis diffusa on hypoxic HepG-2cells.Set25,50,75----250mg/L those ten concentrations of Hedyotis Diffusa acted onhypoxic HepG-2cells, and measured its inhibition rate by MTT assay,and draw outthe concentration effect curve. Selected maximum non-toxic dose (the inhibition rateless than5%)25mg/L of Hedyotis diffusa as the follow-up experimentconcentration.5.Hedyotis diffusa partially reverse the drug resisitance of hypoxic HepG-2cellsto ADM.5.1The IC50value of ADM on hypoxic HepG-2cells reduced by Hedyotisdiffusa.The MTT assay showed that: ADM acted on hypoxic HepG-2cells for48h, theIC50value was19.21±0.32mg/L, Obviously higher than the IC50value of2.31±0.23mg/L (P<0.001) on common HepG-2cells. While added Hedyotis diffusa,theIC50value reduced to7.27±0.41mg/L, had significant difference compared tohypoxic HepG-2cells group (P<0.01), respectively the drug resistance times andreversal times were8.32and2.64. The effects showed that the hypoxic HepG-2cellsare resistant to ADM, and Hedyotis diffusa partly reverse the resistance. 5.2Hedyotis diffusa aggravated the toxic effect of ADM on hypoxic HepG-2cells.2.5mg/L ADM acted on those three groups of cells: HepG-2cells,hypoxicHepG-2cells and Hedyotis diffusa+hypoxic HepG-2cells, flow cytometry(FCM)was used to measure the apoptosis rate were58.20±3.76%,10.05±1.25%and25.42±2.34%. The effects showed that Hedyotis diffusa partly reverse the resistant toADM on hypoxic HepG-2cells, and aggravate the toxic effect of ADM.6.Hedyotis diffusa reverse the resistance to ADM on hypoxic HepG-2cells,Thedown-regulation of HIF-1a,P-gp,MRP protein maybe the possible mechanisms.Western-blot results showed that hypoxic HepG-2cells compared with HepG-2cells, the expression of HIF-1a, P-gp and MRP protein were increased significantly(P<0.01, P<0.01, P<0.001), but the hypoxic HepG-2cells acted by Hedyotis diffusa,the expression of those proteins significantly reduced(P<0.05, P<0.05, P<0.05). Theresults explained the HIF-1a, P-gp and MRP protein may be involved in the hypoxicHepG-2cells resistant to ADM, and Hedyotis diffusa down regulate the expression ofthese three proteins to reverse the drug resistance.Conclusion1.The hypoxic HepG-2cells modle induced by CoCl2with resistance to ADM, andHedyotis diffusa partly reverse the resistance.2.The mechanisms of Hedyotis diffusa reverses resistance of hypoxic HepG-2cells toADM maybe the down-regulation of HIF-1a, P-gp, MRP protein expression.