The Effect Of Bufalin In ERK/p-ERK And Cell Migration In Human Esophageal Cancer Cells

Esophageal cancer is one of the common malignant tumours in human. Itis commonly accepted that many factors participated in the occurring ofesophageal cancer including multi-factor, multi-stage and polygenes. But theexact mechanism is still undefined. China is one of the countries with thehighest mortality of esophageal carcinoma worldwide. There were so manypersons died of esophageal cancer each year. The esophageal cancer, whoseearly symptom is not obvious and diagnose at the later period of diseaseprocess is always associated with poor prognosis in China. At recent years,although the levels of early find, diagnosis and treatment were improvedthrough general survey in esophageal cancer, the prognosis of esophagealcancer was poor at the rate of10%-15%. Therefore, it is necessary andimportant for discovering new anticancer drugs, and researching the target ofthe drug at signaling pathway in esophageal cancer.This experiment mainly studied the ERK signaling pathway namedRas-Raf-MEK-ERK enzymatic cascade reactions, and investigated theactivated state of ERK and cell migration after transfected MEK-ca plasmidinto esophageal cancer cells. Further more, the biological activity of ERKsignaling pathway was studied in esophageal cancer cells also. Bufalin, as thesecretion from the postauricular and glands of Bufo gargarizans Cantor orBufo melanostictus Schneider, was used to block abnormal intercellularconduction and then reduced the activation level of various factors, so as toachieve the function of anti-cancer. Although Bufalin was reported by clinicalresearch frequently, it had not been used in the treatment of esophageal cancer.Before, a vast amount of in vitro experiments about Bufalin had been done inour laboratory. They all confirmed that Bufalin can adjust down the levels ofp-Raf, p-MEK, p-ERK in cells through ERK signal transduction, but Raf, MEK, ERK had no obvious changes. So, Bufalin played an essential role inanti-tumor through inhibiting the activation of various factors in ERK pathway,and then restrained the growth of esophageal cancer cells. This experimentstudied clearly about Bufalin aiming to provide reliable experimental basis forbetter treatment in esophageal cancer.What was the specific mechanism in the genesis process of esophagealcancer? This experiment used the esophageal squamous cell carcinoma lineTE13to study deeply in vitro in order to realize the effect of Bufalin in theaction of ERK pathway in the process of esophageal cancer. Then it provided asolid foundation for clinical treatment.Objective: To explore the effect of Bufalin in ERK/p-ERK and cellmigration in human esophageal cancer cellsMethods:1A plasmid containing MEK-ca gene was transformed into E.coli.DH5αand amplified. Then transfected the MEK-ca plasmid into esophageal cancercell TE13by liposome and examined the state of the activation of ERK after0h,24h,30h,36h,42h and48h by western blot. And then the best transfectiontime was gained.2The activation of ERK was examined in various groups (control group,empty plasmid transfected group, MEK-ca plasmid transfected group and addBufalin after transfected MEK-ca plasmid) by western blot. Further, the affectof ERK/p-ERK was investigated after transfected MEK-ca plasmid and addedBufalin in human esophageal cancer cells.3The migratory range was measured in different groups through woundhealing assay. Then the effect of cell migration was investigated in each groupin human esophageal cancer cells.Results:1The MEK-ca plasmid was amplificated successfully and transfectedinto esophageal cancer cell. The expression in each groups was not statisticallysignificant difference about ERK(0.542±0.015,0.687±0.020,0.576±0.019,0.579±0.016,0.475±0.011,0.611±0.006, P＞0.05), but the active state of p-ERK was statistically significant difference in MEK-ca plasmid transfectedgroup comparing with other three groups, and the active state of p-ERKincreased along with the time0h,24h,30h,36h and then decreased at42h,48h after transfected. The active state of p-ERK was the highest at36h(0.756±0.035,0.663±0.040,0.714±0.053,1.314±0.053,0.663±0.034,0.704±0.009, P＜0.05).2Western Blot results showed that: the active state of p-ERK wassignificantly higher in MEK-ca plasmid transfected group comparing withother three groups, and then reduced after adding Bufalin(0.381±0.026,0.400±0.017,0.698±0.010,0.432±0.007, P＜0.05). The change of ERK was notsignificant difference in different groups(0.800±0.003,0.714±0.034,0.759±0.022,0.750±0.026, P＞0.05).3Wound healing assay: After scratched36h, the migratory range indifferent groups was compared with control group. The range of MEK-caplasmid transfected group was statistically significant larger than others. Theeffect of inhibition was markedly clearer in the group of using Bufalin andUo126in the meantime than single using any one(4.100±0.200,4.200±0.265,10.333±0.252,6.683±0.058,7.033±0.158,3.933±0.153).Conclusions:1ERK, which located downstream of MEK, was activated aftertransfected plasmid MEK-ca. Meanwhile, the level of p-ERK inside the cellwas the highest after transfected36h. But the change of ERK was notstatistically significant.2The active state of phospho-ERK was statistically significant elevatedin MEK-ca plasmid transfected group, and then the activity was restrained byBufalin. The change of ERK was not significant difference in different groups.3Bufalin had vital effect on inhibiting the migration of TE13cell.