The Study On The Relationship Between Sorafenib Effect On Cell Proliferation And Basal Level Of Phosphorylated Extracellular Signal-regulated Kinase In Different HCC Cell Lines

Objective:To investigate the relationship between effects of sorafenib on cell proliferation and basal phosphorylated extracellular signal-regulated kinase(pERK) levels in different HCC cell lines.Methods:The effects of sorafenib and 5-Fluorouracil on cell proliferation were evaluated by cell viability assay in four HCC cell lines(SMMC-7721,MHCC97-L,MHCC97-H and HCCLM6),with different metastatic potential and basal pERK expression. Expression levels of pERK were determined by immunocytochemical quantification along with Western Blot analysis and pERK density values were also calculated. Correlation analyses were then carried out between the IC₅₀ values of drugs and pERK density values.After basal ERK phosphorylation was down-regulated with U0126 in MHCC97-H cells,cellular responsiveness to sorafenib was then assessed by cell viability assay.Results:(1) The results of immunocytochemistry and image quantification,confirmed by Western Blot analysis,revealed that baseline pERK was differentially expressed in these HCC cell lines(P＜0.0001,n=6) and seemed to be correlated with their metastatic potential.The pERK density in SMMC-7721,MHCC97-L,MHCC97-H and HCCLM6 cells was 0.042±0.006,0.081±0.007,0.329±0.037 and 0.463±0.084, respectively.In metastatic MHCC97-H and HCCLM6 cells,pERK levels were significantly higher than that in non-metastatic SMMC-7721 cells(P＜0.0001,n=6). Even among the three metastatic cell lines,pERK levels were differentially expressed and increased stepwise with their metastatic potential(P＜0.0001,n=6),which indicated that the RAF/MEK/ERK pathway may be involved in tumor invasion and metastasis in HCC. (2) In this study,sorafenib could inhibit ERK phosphorylation in all four HCC cell lines dose-dependently at a concentration between 5 and 20μM.Take SMMC-7721 and HCCLM6 cells for instance,after exposure to 5,10 or 20μM sorafenib for 24 hours,the expression rate of pERK in SMMC-7721 fell gradually to 81.88±7.65%,71.63±10.80%and 17.47±1.34%,respectively,and in HCCLM6 to 78.06±4.66%,28.12±1.36%and 3.99±0.19%,respectively.The expression rates in both cell lines were significantly reduced when compared to each DMSO control group(P=0.0043,n=6 and P＜0.0001,n=6,respectively).However,further statistical analyses revealed the significant difference in the degree of the sorafenib effects in these HCC cell lines.Interestingly,the sorafenib pERK inhibition effect in SMMC-7721 cells with lower level of pERK was significantly weaker when compared to the other three HCC cell lines with relatively higher basal pERK levels (P＜0.0001,n=6).These results suggested that the effects of sorafenib on ERK phosphorylation inhibition were significantly associated with basal pERK levels in HCC cell lines.(3) On the contrary,no significant change was observed after 5-FU treatment in MHCC97-H cells,pERK expression rate was 102.3±7.88%,110.8±6.60%, 101.1±5.12%,respectively,after exposure to 10,20 or 50mg/l 5-FU for 48 hours,with no statistical difference to the control group(P＞0.05,n=6).(4) Effects of sorafenib on cell proliferation were measured by the CCK-8 cell viability assay.According to our results,sorafenib inhibited all four HCC cell lines proliferation in a dose-dependent characteristic,with an IC₅₀ of 20.85±2.81μM, 10.38±1.52μM,10.70±2.35μM and 9.11±2.44μM in SMMC-7721,MHCC97-L, MHCC97-H and HCCLM6 cells,respectively.As expected,SMMC-7721 cells containing lower level of pERK were significantly less sensitive to sorafenib-mediated growth inhibition than the other three HCC cell lines with higher basal pERK levels(P=0.0018,n=4).Meanwhile,a significant negative correlation (Spearman r=-0.8671,P=0.0003,n=12) was observed between the IC₅₀ values of sorafenib in these HCC cell lines and their pERK density values,indicating that the effects of sorafenib on cell proliferation were significantly correlated with basal pERK levels in these HCC cell lines.(5) While opposite results were observed in treatment with traditional chemotherapy drug 5-FU.5-FU inhibited HCC cell proliferation with an IC₅₀ of 4.24±0.87mg/l,79.71±24.49mg/l,41.21±21.55mg/l and 187.45±78.05mg/l in SMMC-7721,MHCC97-L,MHCC97-H and HCCLM6 cells,respectively,with significant statistical differences(P＜0.0001,n=4).The SMMC-7721 cells with lower pERK expression demonstrated a higher-sensitivity to 5-FU.However,MHCC97-L, MHCC97-H and HCCLM6 cells with higher pERK expression exhibited more resistance to this drug.The ultimate inhibition rate before reaching platform in these three cell lines was about 35%,40%and 45%,respectively,each compared with the control group.Furthermore,a significant correlation(Spearman r=0.7846,P=0.0009, n=12) was observed between the IC₅₀ values of 5-FU and their pERK density values, indicating that the resistance to 5-FU was significantly associated with basal pERK expression in these HCC cell lines.(6) To more directly determine the relationship between pERK expression and sensitivity to sorafenib,we inhibited the MEK/ERK pathway and reduced basal pERK expression in MHCC97-H cells via U0126,a selective inhibitor of MEK1 and MEK2, and then compared cellular responsiveness to sorafenib with that of the untreated cells. Quantification of cellular pERK level by immunocytochemical analysis indicated that constitutive ERK phosphorylation was strongly reduced in MHCC97-H cells after treatment with 20μM U0126 for 6 hours relative to the level observed in the untreated cells(60%inhibition),which induced almost no detectable systemic toxicity on cell proliferation.In the following experiments,we compared sorafenib responsiveness of MHCC97-H cells pretreated with 20μM U0126 for 6 hours to an untreated control. Cell viability assay revealed that the pretreated cells were significantly less sensitive to sorafenib-mediated growth inhibition,with an IC₅₀ of 17.31±1.62μM versus 10.81±1.24μM(P=0.0281,n=6).These results confirmed that the RAF/MEK/ERK signaling pathway was essential for sorafenib-mediated growth inhibition,and that the sensitivity to sorafenib was directly related to the activation of this pathway and basal pERK expression in MHCC97-H cells.Conclusion:In this vitro study,pERK was confirmed to be a useful biomarker predictive of sensitivity in treating HCC with sorafenib.The RAF/MEK/ERK pathway may be involved in invasion,metastasis and drug resistance to traditional chemotherapy in HCC.