Effects Of Bufalin On ERK Signal Transduction In Human Esophageal Cancer Cell

Esophageal cancer is a common primary gastrointestinal maligant tumor,and it is the eighth most common cancer cause of cancer death in the word.China is one of the country with the highest mortality of Esophageal cancerworldwide, and about150000people dues to esophageal cancer each year.Since most esophageal cancer patients remain clinically silent until late in theprocess of disease, they are often diagnosed at an advanced stage and oftenassociated with poorer prognoses in China. Although prognoses of earlyesophageal cancer has greatly improved after general survey, surgery,chemotheyapy, radiation, immune, gene therapy, but the prognosis ofadvanced esophageal cancer is poor. Therefore, searching new anticancer drug,study of the role of the anticancer drug target and esophageal cancer-relatedsignal transduction pathways, are still the emphasis in the treatment ofesophageal cancer.There are multiple signal transduction pathways involved in theoccurrence and development of esophageal cancer, but the mitogen-activatedprotein kinase(MAPK) is the main pathways. Extracellular signal-regulatedkinase (ERK) is a import member of mitogen-activated protein kinase(MAPK)family. It can be activated into the state of phosphorylation (p-ERK) byvariety of stressor and mitogen-activated signaling. Activated ERK regulatescell proliferation, differentiation, apoptosis and malignant transformation ofcell. P-ERK transducts signal into the nucleus to regulate the expression ofrelated genes through phosphorylasing transcription factors. A lot of previousstudies reported that the level of p-ERK involved in the occurrence anddevelopment of maligant tumor including esophageal cancer. ERK is the thefocus of the study currently,it can provide new target for clinical treatment.Bufalin is secreted from the postauricular and glands of Bufo gargarizans Cantor or Bufo melanostictus Schneider, wich belonged to cardiotonic steroidand has been used as a traditional Chinese medicine for the treatment ofinfection, anesthesia and tumor. Bufalin, a bufadienolide compound, was oneof the main active constituent of toad venom. It has been conifimed thatBufalin was a Na+–K+pump inhibitor that depressed the delayed K+rectifiercurrent in myocytes. A lot of previous studies reported that Bufalin suppresseda variety of the growth of tumor cells in vitro studies. It has been used to treatmany tumors clinically. However, the effects of Bufalin on esophageal cancerhad not yet been reported. In the study, we researched the effects of Bufalinon hunman esophageal cancer cell and the possible molecular mechanisms,and provide basis for clinical treatment.In order to research the effects and mechanisms of Bufalin on ERKsignal pathways in human esophageal cancer cell.we investigate theexpression of ERK and phosphoralation ERK (p-ERK) protein in hunmanesophageal cancer cell, to expore the mechanisms of Bufalin on esophagealcancer cell, and to observe the effects of Bufalin and Uo126interact to inhibithuman esophageal cancer cell.Objective: To investigate the effects of Bufalin on ERK andphosphoralation ERK (p-ERK) protein expression in hunman esophagealcancer cell, and to observe the effects of Bufalin and Uo126interact to inhibithuman esophageal cancer cell line (TE13), and discussed the possiblemechanism.Methods:1. The expression of phosphoralation ERK (p-ERK), incentived by fatalbovine serum in esophageal cancer cell line TE13,were detected by WesternBlot method.2. The Immunocytochemistry and Western Blot methods were used todetect the amount and expression of ERK and p-ERK of the esophagealcancer cell line TE13by different Bufalin concentration.3. The expression of phosphoralation ERK (p-ERK), treated with acardiotonic steroid Bufalin and the ERK inhibitor Uo126interact to esophageal cancer cell line TE13,were examined by Western Blot method.Results:1. Western Blot results showed that: the expression of phosphoralationERK (p-ERK) increased and then decreased (0.582±0.005；0.641±0.005；0.680±0.003；0.567±0.003；0.579±0.004；P＜0.05)along with the time (0min,15min,30min,60min and120min) of fatal bovine serum stimulation. Theexpression of phosphoralation ERK (p-ERK) was highest at30min.2. The Immunocytochemistry method showed that ERK priteinexpression mainly in the cytoplasm of the esophageal TE13,for tan and at highmagnification in a granular,observation, the activation ERK(p-ERK) mainlyexpress in the nuclear or nuclear-plasma,for tan particles. The effects ofBufalin on ERK protein in esophageal cancer cell line TE13was notsignificantly increased or decreased(76.40%；76.80%；75.60%；77.60%；76.20%),but the hydroxycamptotheich had reduced the amount ofphosphoralation ERK (p-ERK) of the esophageal cancer cell line TE13. Withthe concentration of Bufalin increased, the number of positive cells reducedgradually, brown yellow particles became smaller, the density reducedgradually(79.60%；68.00%；57.00%；44.20%；11.00%；P＜0.05).3. Western Blot results showed that：The expression of ERK protein wasnot significantly increased or decreased (0.703±0.020；0.706±0.020；0.711±0.018；0.716±0.007；0.708±0.005), while it could be decreased theexpression of phosphoralation ERK (p-ERK) gradually with the increase ofconcentration of Bufalin (0.686±0.046；0.608±0.029；0.523±0.032；0.348±0.017；0.273±0.022；P＜0.05).4. Western Blot results showed that：Compared with the Bufalin andUo126as single group alone, the expression of phosphoralation ERK (p-ERK)was significantly decreased in the combination group(0.713±0.010；0.662±0.002；0.610±0.007；0.403±0.010；P＜0.05).Conclusion:1. The phosphoralation ERK (p-ERK) play an important role inesophageal cancer cell line TE13growth. 2. Bufalin decreased the expression of phosphoralation ERK (p-ERK)through ERK signaling pathway,while the ERK protein was unchanged. Thestudy showed that Bufalin inhibited cell growth though the process ofERK-activated.3. Bufalin and Uo126synergistically inhibited cell growth by decreasingthe phosphoralation ERK (p-ERK) at low dose. So it can expand theapplication of the cardiac glycoside, at the same time,it also can strengthen theeffect of Uo126,with potential value of clinical application.