| Esophageal cancer is one kinds of malignant lesion which is caused byesophageal squamous epithelium or glandular epithelium abnormalproliferation. China is a high incidence area of esophageal cancer in the worldand squamous cell cancer is the most common pathological histology. Untilnow, there are several methods to treat esophageal cancer, the mainlytreatment is surgery with or without adjuvant radiotherapy or chemotherapy.But its early symptom is not obvious and imperceptible, patients are always ina middle-late period when they see the doctors, the prognosis is poor. A largenumber of molecular biology research data show that many factors includingmulti-stage, poly genes participated in the occurring of esophageal cancer, andthere are also many abnormal activation of singnaling pathways and relatedprotein over expression. Therefore, further study that antitumor drugs effect onthe esophageal cancer and related activation of signal transduction pathways,is still the goal of esophageal cancer treatment research.Many reports confirmed that abnormal activations of Akt/mTOR/P70S6Ksignaling pathways existied in the process of tumor. Akt/mTOR is consideredto be the main signal regulation pathway of protein synthesis, involved in cellproliferation, differentiation, migration, etc. As a kind of multi-functionalkinase, mTOR participated in the regulation of many important cellularfunctions. It actives phosphorylation P70S6K which can promote mRNAtranslation and produce a variety of proteins for the cell cycle across Gl period.Inhibiting mTOR can cause P70S6K dephosphorylation, thereby inhibiting thetranslation process, finally make the stagnation of the cell cycle and inducecell apoptosis.Bufalin is an effective anti-cancer substance, secreted from thepostauricular and glands of Bufo gargarizans Cantor or Bufo melanostictus Schneider. Researchs showed that Bufalin can induce many kinds of leukemiacells and some solid tumor cell apoptosis, and reduce the tumor related geneBcl-2, c-myc, WT-1expression. However, the exact mechanism of inducingthe apoptosis remain unclear.Currently, the reserches about Bufalin inducing cell apoptosis are very few.This experiment used the esophageal squamous cell cancer cell line ECA109toobserve the effects of Bufalin inducing cell apoptosis by inhibitingmTOR/P70S6K pathways, in order to expore the possible mechanisms ofBufalin on esophageal cancer.Objective：To investigate the effect of Bufalin inducing ECA109cellapoptosis by inhibiting the activation of mTOR/P70S6K pathways.Methods：1A plasmid containing wtmTOR gene was transformed into E.coli.DH5αand amplified. Then transfected the wtmTOR plasmid into esophageal cancercell ECA109by liposome method, examined the protein level of P70S6K andthe activation of P70S6K at0h,12h,24h,30h,36h,42h and48h by westernblot. The best transfection time was gained.2The expressions of cIAP-1, BAD in ECA109cells were examined byWestern blot after adding60nmol/l Bufalin2h,6h,12h,24h,36h,48h.3The expressions of P70S6K, p-P70S6K, cIAP-1, BAD in ECA109cellsin various groups (control group, empty vector group, wtmTOR plasmidtransfected group and adding Bufalin after transfected wtmTOR plasmidgroup) were examined by western blot. And the affects of wtmTOR plasmidand Bufalin on mTOR pathway and cell apoptosis were studied.4The apoptotic rate and the cell cycle were evaluated with Flowcytometry.5The cell apoptosis index was detected by TUNEL labeling method.6The changs of cell morphology were observed by inverted microscopeand Gimsa staining.Results:1The wtmTOR plasmid was amplificated successfully and transfected into esophageal cancer cell. The protein expressions of downstream wereexamined.(1)The expression of P70S6K was not different at respective times(0.599±0.011，0.594±0.013，0.606±0.012，0.608±0.010，0.592±0.017，0.599±0.021，0.600±0.036, P＞0.05). There was no significant difference.(2) The expression of p-P70S6K was increased along with the time (0h,12h,24h), and then decreased at (30h,36h,42h,48h) after transfected. Theexpression of p-P70S6K was the highest at24h (0.389±0.013，0.411±0.019，0.609±0.016，0.573±0.015，0.394±0.013，0.383±0.006，0.262±0.018，P＜0.05). The difference was statistically significant.2The protein expressions of cIAP-1and BAD after adding60nmol/lBufalin for2h,6h,12h,24h,36h and48h were examined by Western Blot.(1) The expression of cIAP-1was gradually decreased along with thetime after adding Bufalin (0.542±0.003,0.517±0.007,0.455±0.002,0.414±0.004,0.369±0.026,0.218±0.015, P＜0.05). The difference wasstatistically significant.(2) The expression of BAD was gradually increased along with the timeafter adding Bufalin (0.456±0.009,0.659±0.042,0.750±0.023,0.813±0.019,0.937±0.013,1.047±0.013, P＜0.05). The difference was statisticallysignificant.3Western Blot results showed the protein expressions of P70S6K,p-P70S6K, cIAP-1and BAD in different groups.(1) The change of P70S6K was not significant different in differentgroups (0.901±0.045,0.914±0.023,0.900±0.020,0.898±0.022, P＞0.05).There was no significant difference.(2) The expression of p-P70S6K was significantly higher in wtmTORplasmid transfected group comparing with control and empty vector group,and then reduced after adding Bufalin2h (0.761±0.085,0.766±0.068,0.952±0.059,0.762±0.019, P＜0.05). The difference was statisticallysignificant.(3) The expression of cIAP-1was significantly higher in wtmTOR plasmid transfected group comparing with control and empty vector group,and then reduced after adding Bufalin24h (0.721±0.019,0.731±0.248,0.840±0.010,0.742±0.021, P＜0.05). The difference was statisticallysignificant.(4) The expression of BAD was significantly lower in wtmTOR plasmidtransfected group comparing with control and empty vector group, and thenincreased after adding Bufalin24h（0.929±0.046,0.944±0.060,0.779±0.182,1.029±0.049, P＜0.05）. The difference was statistically significant.4Flow cytometry results showed that:(1) The apoptotic rate was gradually increased after adding Bufalin0,20,40,60,80and100nmol/l for24h (3.01±0.317%,3.67±0.306%,6.74±0.198%,7.59±0.340%,18.22±0.651%,28.60±1.737%, P＜0.05）. The cell cycle ofECA109appear obvious G2/M phase retardation, and the cell percentage ofG2/M is increased from9.24±1.919%to70.5±2.934%. The difference wasstatistically significant.(2) The apoptotic rate was significantly lower in wtmTOR plasmidtransfected group comparing with control and empty vector group, and thenincreased after adding Bufalin24h (5.60±0.411%,5.46±0.341%,4.19±0.210%,10.12±0.325%, P＜0.05). The difference was statistically significant.5TUNEL staining showed that: the cell nucleus with brownish yellowwas positive cell, however, the normal cell nucleus was not dyed. Theapoptotic index was gradually increased after adding Bufalin0,20,40,60,80and100nmol/l for24h (2.13±0.431%,5.56±1.345%,7.87±1.443%,15.17±2.657%,36.69±2.986%,50.98±2.978%, P＜0.05). The difference wasstatistically significant.6The morphological changes of ECA109cells after adding Bufalin.(1) Histological appearance was observed by inverted microscope. Withthe action time of the drug extending, many cells shrinkaged, cell shapes werechanged from polygon into circle. The cell surface gradually raised smallbubbles, cells constantly shedding suspended in cell nutrient solution. Thenumber of living cells decreased significantly, and dying cells increased. (2) The typical nucleus morphological changes of apoptosis wereobserved by Gimsa staining. After adding Bufalin for24h, the cell membranewas integrity, vacuoles appeared in the cytoplasm, nuclear chromatincondensed and broken into pieces dispersion in the cytoplasm, and theapoptotic body was visible. Meantime, we can also see a small amount of deadcells, cell outline was not clear, the nucleus dissolved away.Conclusions:1Bufalin could promote esophageal cancer cell ECA109apoptosis intime dependent manner by inhibiting cIAP-1and promoting BAD proteinexpression.2Bufalin induced ECA109apoptosis by inhibiting the activation ofmTOR/P70S6K pathways. |
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