| Esophageal cancer is a kind of common malignant tumor. In China,About250,000people develop esophageal cancer one year in China. It isabout more than50%of the average number of this disease around the world.It is widely recognized that the occurrence of esophageal cancer is amulti-factored, polygenic, multi-phase complex development process. Whilethe exact pathogenesis is not fully elucidated. Therefore, the main researchdirection of esophageal cancer treatment is still the abnormal activation ofvarious signaling pathway in esophageal cancer and the target of newantitumor drugs.Multiple researches confirmed that, mTOR/4EBP1signaling transductionpathway exists abnormal activation(in the development of tumor).mTOR/4EBP1is considered to be the main signal regulation pathway ofprotein synthesis, involved in cell proliferation, differentiation, migration andso on. mTOR is a kind of multi-functional kinase, involved in the regulation ofmany important cellular functions. Activation of mTOR makes thephosphorylation of its downstream4EBP1, the affinity of phosphorylated4EBP1falls, and then eIF4E separates from it. It promotes the formation oftranslation initiation complex, thus accelerates protein synthesis. In contrast,when mTOR is inhibited,4EBP1dephosphorylate, and combine with eIF4E,that inhibits the starting of translation process and induces cell apoptosis.This experiment mainly studied the change of4EBP1/p-4EBP1inesophageal cancer cells after the transfection of wtmTOR plasmid, and theeffect of the wtmTOR plasmid in mTOR signaling pathways in humanesophageal cancer cells. At the same time, It investigated the influence ofBufalin on mTOR/4EBP1pathway and the esophageal cancer cell apoptosis.The research further reveals the mechanism of the occurrence and development of esophageal cancer and provides new ideas for the treatment.Objective: to study the effect of Bufalin on the actvivation of4EBP1and cell apoptosis in human esophageal cancer cells.Methods:1A plasmid containing wtmTOR gene was transformed into E.coli.DH5αand amplified. Then the wtmTOR plasmid was transfected into esophagealcancer cell Eca109by liposome and the state of the activation of4EBP1at0h,12h,24h,30h,36h,42h and48h was examined by western blot. The besttransfection time was gained.2The expression of mTOR and activation of4EBP1were examined invarious groups (control group, empty plasmid transfected group, wtmTORplasmid transfected group, adding Bufalin after transfected group, addingRapamycin after transfected group) by Western blot. Further, the effect ofBufalin on mTOR pathway was investigated after being transfected wtmTORplasmid in human esophageal cancer cells.3The expression of the molecular level of mTOR was examined invarious groups (control group, empty plasmid transfected group, wtmTORplasmid transfected group, add Bufalin after transfected wtmTOR plasmid,add Rapamycin after transfected wtmTOR plasmid) by RT-PCR. Theexpression of the molecular level of4EBP1was examined in various groupsby RT-PCR.4The expression of Bad and Bcl-2were examined in various groups(control group, empty plasmid transfected group, wtmTOR plasmidtransfected group, the group of adding Bufalin after being transfectedwtmTOR plasmid, the group of add adding Rapamycin after being transfectedwtmTOR plasmid) by immunocytochemistry method.Results:1The wtmTOR plasmid was amplificated successfully and transfectedinto esophageal cancer cell. RT-PCR results showed that, after beingtransfected wtmTOR plasmid, Eca109cell lines molecular expression ofmTOR in wtmTOR plasmid transfection group (1.274±0.006) was significantly higher than the control group (0.422±0.012) and empty vectortransfection group (0.450±0.025), but the expression of mTOR wassignificantly lower in adding Bufalin after being transfected wtmTOR plasmidgroup (0.304±0.026) and adding Rapamycin after being transfected wtmTORplasmid group (0.244±0.015), statistically significant difference (P <0.05).And4EBP1molecular expression in the control group (1.015±0.021), theempty vector transfection group (1.085±0.017), wtmTOR plasmid transfectiongroup (1.004±0.016), the group of adding Bufalin after being transfectedwtmTOR plasmid (1.077±0.024), the group of adding Rapamycin after beingtransfected wtmTOR plasmid (1.046±0.020) had no obvious difference, nostatistically significant difference (P>0.05).2The wtmTOR plasmid was amplificated successfully and transfectedinto esophageal cancer cell. The expression of4EBP1was not statisticallysignificant difference at0h,12h,24h,30h,36h,42h,48h after transfected（0.872±0.021，0.854±0.016，0.796±0.011，0.787±0.015，0.831±0.034，0.835±0.032，0.788±0.036，P＞0.05）, but the active state of p-4EBP1wasstatistically significant higher in wtmTOR plasmid transfected groupcomparing with other groups, and the active state of4EBP1increased alongwith the time0h,12h,24h,30h (0.357±0.025，0.572±0.022，0.655±0.017，0.895±0.021) and then decreased at36h,42h,48h (0.641±0.014，0.425±0.031，0.352±0.034) after transfected. The active state of4EBP1wasthe highest at30h（0.895±0.021）, statistically significant difference (P <0.05).3Western Blot results showed that: the expression of mTOR wassignificantly higher in wtmTOR plasmid transfected group (0.967±0.015)comparing with the control group (0.472±0.033) and empty vector transfectiongroup (0.495±0.028), and then reduced in the group of adding Bufalin afterbeing transfected wtmTOR plasmid (0.398±0.031), and the group of addingRapamycin after being transfected wtmTOR plasmid (0.381±0.024),statistically significant difference (P <0.05). the active state of4EBP1wassignificantly higher in wtmTOR plasmid transfected group (0.825±0.016)comparing with the control group (0.502±0.031) and empty vector transfection group (0.521±0.027), and then reduced in the group of adding Bufalin afterbeing transfected wtmTOR plasmid (0.376±0.015), and the group of addingRapamycin after being transfected wtmTOR plasmid (0.352±0.022),statistically significant difference (P <0.05). The change of4EBP1was notsignificant difference in the control group (0.655±0.013), the empty vectortransfection group (0.642±0.026), wtmTOR plasmid transfection group(0.670±0.015), the group of adding Bufalin after being transfected wtmTORplasmid (0.674±0.023), the group of adding Rapamycin after being transfectedwtmTOR plasmid (0.645±0.033) had no obvious difference, no statisticallysignificant difference (P>0.05).4Immunocytochemistry method results showed that Bad and Bcl-2protein were mainly expressed in cytoplasm or cytomembrane of esophagealcancer cells Eca109, as tan, granular in observed at high magnification, theresults showed that the positive expression rate of Bad was16.53%inwtmTOR plasmid transfected group, which was significantly lower comparingwith the control group (35.67%) and empty vector transfection group(37.81%),and then higher in the group of adding Bufalin after beingtransfected wtmTOR plasmid (64.22%) and the group of adding Rapamycinafter being transfected wtmTOR plasmid (65.30%), statistically significantdifference (P <0.05). and the positive expression rate of Bcl-2was86.67%in wtmTOR plasmid transfected group, which was significantly highercomparing with the control group (64.43%) and empty vector transfectiongroup (62.75%),and then reduced in the group of adding Bufalin after beingtransfected wtmTOR plasmid (37.26%) and the group of adding Rapamycinafter being transfected wtmTOR plasmid (36.07%), statistically significantdifference (P <0.05).Conclusion:1Bufalin could inhibit the growth of Eca109esophagus carcinoma celllines through inhibiting the activation of mTOR/4EBP1.2Bufalin could promote cell apoptosis of esophageal Eca109cells byinhibiting Bcl-2and promoting Bad protein expression. |
PDF Full Text Request