Study On The Role Of Lipid Rafts In Epidermal Extracellular Signal-regulated Kinase(Erkl/2) Pathway And The Multidrug Resistance Of Human Hepatoma Cells

ObjectiveThe present study assessed the effect of lipid raft to the activation of receptor tyrosine kinase signaling and wheather lipid raft produce an effect on the multidrug resistance(MDR)of human hepatoma cells.Methods1.The SMMC7721/ADM and BEL 7402/ADM cells were harvested after a long period of culture using pulse treatment with high concentration of Adriamycin(ADM),and the sensitivities were evaluated by CellTiter 96R AQueous One Solution in different concentrations through calculating the half maximal inhibitory concentration(IC50)of ADM.Values of IC50 were evaluated by the probit analysis using SPSS.Resistance index(RI)was calculated according to the formula:RI =(IC50 for MDR cells)/(IC50 for parental cells).2.M?CD binds specifically to cholesterol to disturb the association of proteins with lipid rafts.Alexa Fluor 488 is able to mark CtxB dyed lipid rafts green,the content of lipid rafts can be determined by the intensity of green fluorescence under the laser confocal microscopy.3.After M?CD or PD98059 treatment,western blot was used to detect the expression levels of protein.4.After M?CD or PD98059 treatment,an annexin V-FITC/PI staining Kit was used to measure apoptosis caused by 1 mg/1 ADM incubation 24h.5.After M?CD treatment,flow cytometry was used to detect rhodamine 123 accumulation caused by 5 ?g/ml rhodamine 123 incubation 45min.Results1.The SMMC7721/ADM and BEL7402/ADM cells showed a stable drug resistance.Compared with their parent strain cells,the cell proliferation inhibition rates in resistant cells incubated with 0.25,1,4,16 and 64 ?g/ml ADM for 24 h were reduced and the IC50 values of ADM were obviously increased.The RI of the SMMC7721/ADM and BEL7402/ADM cells was 17.07 and 14.28 respectively.The data showed the expressions of Erkl/2,MEK,c-Raf,p-Erkl/2,p-MEK and p-c-Raf was significantly higher than that of non-resistant parent cells.2.After cells were treated with 5mM M?CD,green fluorescence decreased significantly,demonstrating definitely the destructive effect of M?CD on lipid rafts.3.After M?CD treatment and EGF stimulation,the expression of p-Erk 1/2,p-MEK,p-c-Raf showed obvious increases,while the total Erkl/2,MEK and c-Raf hardly changed and increased phosphorylation of EGF receptor.The phosphorylation of Erkl/2 was reduced after PD98059 treatment.4.After M?CD treatment,the sensitivity to ADM was significantly raised.In the SMMC7721/ADM and BEL7402/ADM cells,the IC50 values to ADM were decreased to 1.71 ±0.142 ?g/ml and 1.467±0.173 ?g/ml,meanwhile,the reversal levels of resistance to ADM were 2.79-fold and 1.77-fold respectively.After the PD98059 treatment,the IC50 values in the human hepatoma resistant cells were reduced to 3.324±0.184?g/ml and 1.879±0.174?g/ml,meanwhile,the reversal levels of resistance to ADM were 1.43-fold and 1.38-fold respectively.After the M?CD and PD98059 treatment jointly,the IC50 values of ADM in the cells were reduced to 1.552 ± 0.037?g/ml?1.334 ± 0.079?g/ml,meanwhile,the reversal levels of resistance to ADM were 3.07-fold and 1.95-fold respectively.5.After M?CD or PD98059 treatment,the sensitivity to ADM was obviously increased and the percentage of apoptosis was enhanced.6.The accumulation values of intracellular Rh-123 in the cells treated with M? CD was elevated than that of the control cells respectively.It indicated that the efflux function mediated by P-gp was significantly downregulated after Mp CD treatment.Further,we found the expression of P-gp hardly changed.Conclusions1.The the expression of Erkl/2,MEK,c-Raf,p-Erkl/2,p-MEK,p-c-Raf was increased in the resistant cells.2.lipid rafts destruction by M?CD significantly increased phosphorylation of EGF receptor and its downstream Erkl/2 pathway.3.lipid rafts destruction by M?CD reduced the resistance of SMMC7721/ADM and BEL7402/ADM cells and the activity of P-glycoprotein(P-gp).4.lipid rafts destruction by M?CD and PD98059 reduced the resistance of SMMC7721/ADM and BEL7402/ADM cells,the results assessed Erkl/2 pathway produce an effect on the decrease of resistance after lipid rafts destruction.