| Esophageal carcinoma is one of the most common malignant tumors inour country, and ranked the fourth currently. It occupyed22.4%of malignanttumors in China, and about250,000new patients were observed every year.Despite clinical treatment of esophageal cancer have progressed,5-yearsurvival rate is still low. Therefore, the research of development interrelatedsignal transduction pathway and drug targets for new antitumor drugs becomea focus in the study of esophageal cancer treatment.Mammalian target of rapamycin protein (mTOR) and4E-BP1isdownstream of phosphatidyl inositol3-kinase/protein kinase B (P13K/PKB)signalling pathway, which plays an important role in cell differentiation andgrowth. When mTOR is activated, it makes4E-BP1phosphorylation, theneIF-4E has been released and formed complex with other translation initiationfactor, so as to release its inhibition of translation, translation initiation,accelerate the growth of the related protein expression. When mTOR isinhibited, dephosphorylation of4E-BP1combined with eIF4E, and eIF4E-4E-BP1complex formation.It inhibits translation initiation. Studies showed thatphosphorylation of4E-BP1played a vital role in the development of tumor.Therefore the pathway is the focus of the present study, which provide newtargets for clinical treatment of tumor.Bufalin is a kind of toxic aglycone, which extracted from the traditionalChinese medicine-bufotoxin. It belongs to cardiac glycoside content. It has thefunction of anti-cancer, strong heart, anti-inflammatory, and enhances theimmune function. Bufalin has been already widely used in clinical treatmentincluding variety of solid tumors, blood disease and chronic hepatitis B. Ourearly in vitro studies confirmed that Bufalin effectively inhibited the growth and proliferation of esophageal cancer cells. This experiment was to studythe effect of Bufalin on esophageal cancer animal models, and provideexperimental basis for clinical treatment of esophageal cancer.Objective:To investigate the mechanisms of Bufalin inhibiting tumors and inducingcell apoptosis in the orthotopic transplantation model of human esophagealcaner in nude mice.Methods:136nude mice which established as orthotopic transplantation tumormodels divided into model group, the low-dose Bufalin group(0.5kg/ml, BL),the middle-dose Bufalin group(1.0kg/ml, BM), the high-dose Bufalingroup(1.5kg/ml, BH), rapamycin group(RAPA), high-dose Bufalin andrapamycin combined group. Each group had6mice. All mice were takenabdominal cavity injection for30days, and put into death after medication.All tumors were completed removed, and measured nude mice body weight,tumor size and tumor inhibition rate.2Necrosis degree of tumor tissue observed by HE staining.3The mRNA expression of mTOR and4E-BP1in tumor tissues wasdetected by RT-PCR.4The protein expression of mTOR,4E-BP1, p4E-BP1, Bad and Bcl-2was detected by Western Blot and immunohistochemistry method.5Apoptosis index of each transplanted tumor groups analysed by flowcytometry technology.Results:1The animal body weight of model, BL, BM, BH, RAPA and BR groupsbefore treatment were20.9±2.4g,20.8±1.5g,20.8±2.1g,20.9±2.7g,20.9±1.9g,20.8±2.1g, and27.9±3.0g,28.2±1.9g,27.9±2.9g,28.1±2.8g,27.9±2.0g,27.8±2.6g after treatment, There was no statistically significant differencebetween each other. Tumor volume in all groups after treatment were1.791±0.087cm3,1.720±0.088cm3,1.137±0.060cm3,0.702±0.054cm3,0.699±0.079cm3,0.506±0.076cm3, and tumor inhibition rate was4.0%,36.5%, 60.8%,61.0%,71.7%in experimental groups. The tumor volume in BM, BH,RAPA and BR groups demonstrated significantly reduced after treatmentcompared with model group (P<0.05). The tumor volume in BR wassignificant reduced compared with other groups (P <0.05).2Tumor tissues HE staining showed that: tumour necrosis appeared in allgroups. Necrosis in model group was rare; BL group was existed priority tofocal necrosis; BM group had a large patch of necrosis; BH and RAPA andBR group were visible large area of necrosis in the tumor tissue. Cancer cellclump were scattered in the necrotic tissue.3RT-PCR results showed that: the mRNA expression of4E-BP1inModel, BL, BM, BH, RAPA and BR groups after treatment were1.032±0.061,1.039±0.082,1.036±0.093,1.036±0.081,1.044±0.092and1.037±0.087. Therewas no statistically significant difference between each other.The mTORexpression in all groups after treatment were1.136±0.083,1.124±0.044,0.994±0.142,0.823±0.113,0.787±0.074and0.661±0.070. The expression inBM, BH, RAPA and BR groups demonstrated significantly reduced aftertreatment compared with Model group (P<0.05). The expression in BR wassignificant reduced compared with other groups (P <0.05).4Western Blot showed that: the protein expression of mTOR in Model,BL, BM, BH, RAPA and BR groups after treatment were1.58±0.121,1.52±0.110,1.03±0.141,0.81±0.150,0.79±0.110and0.61±0.171. Theexpression in BM, BH, RAPA and BR groups demonstrated significantlyreduced after treatment compared with Model group (P<0.05).The expressionin BR was significant reduced compared with other groups (P<0.05). Theprotein expression of4E-BP1in Model, BL, BM, BH, RAPA and BR groupsafter treatment were1.205±0.143,1.320±0.131,1.310±0.142,1.217±0.133,1.315±0.134and1.214±0.121. There was no statistically significant differencebetween each other.The expression in BR was significant reduced comparedwith other groups (P <0.05). The protein expression of p4E-BP1in Model,BL, BM, BH, RAPA and BR groups after treatment were1.662±0.110,1.513±0.095,1.082±0.150,0.767±0.070,0.732±0.071and0.627±0.063. The expression in BM, BH, RAPA and BR groups demonstrated significantlyreduced after treatment compared with Model group(P<0.05). The expressionin BR was significant reduced compared with other groups (P<0.05). Theprotein expression of Bad in Model, BL, BM, BH, RAPA and BR groups aftertreatment were0.610±0.054,0.695±0.055,0.818±0.151,1.037±0.123,1.056±0.105and1.180±0.160. The expression in BM, BH, RAPA and BRgroups demonstrated significantly raised after treatment compared with Modelgroup (P<0.05). The expression in BR was significant raised compared withother groups (P<0.05). The protein expression of Bcl-2in Model, BL, BM,BH, RAPA and BR groups after treatment were1.630±0.070,1.520±0.092,1.052±0.07g,0.785±0.080,0.743±0.081and0.556±0.058. The expression inBM, BH, RAPA and BR groups demonstrated significantly reduced aftertreatment compared with Model group(P<0.05). The expression in BR wassignificant reduced compared with other groups (P<0.05).Tumor tissue immunohistochemical results showed the same result asWestern Blot.5Flow cytometry showed that: apoptotic index of Model, BL, BM, BH,RAPA and BR groups were2.58±0.16%,5.48±0.27%,11.05±1.12%,23.47±1.05%,22.29±1.20%and20.32±1.06%respectively. It wassignificantly higher in each experimental groups compared with model group(P＜0.05); BH group’s apoptosis index is the highest; and it was highercompared with other groups. The difference was statistically significant(P＜0.05).Conclusions:1Bufalin can effectively inhibit the growth of nude mice tumor.2Bufalin can inhibit tumor growth through inhibiting mTOR/4E-BP1signaling pathway. The result provided an important molecular targets for thetreatment of esophageal squamous cell carcinoma.3Bufalin can promote the expression of Bad gene, and inhibit Bcl-2gene.It prompted that Bufalin promoting tumor cell apoptosis through increasingBad gene and reducing the expression of Bcl-2. 4The antitumor function of the combined group was stronger than theothers, which provided the basis for clinical combination treatment. |
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