| Esophageal cancer is one of the most common gastrointestinal malignanttumors in the world. About300,000people died because of esophageal cancereach year all over the world. The morbidity and mortality of esophagealcancer are differences in each country. China is the high-prone area ofesophageal cancer in the world, the male were more than female and the ageof most patients were above40. About150,000people died from esophagealcancer every year in China. Multiple genes and many factors participate in theoccurrence and development of esophageal cancer, but the exact pathogenesisis not yet clear. Numerous studies showed that the origin of esophageal canceroften from multiple adjacent cancerous stove, that is, multiple centre origin.Esophageal cancer was not easy to detect early, most patients were inadvanced stage and the prognosis was poor. Thus, target signal transductionpathways and the development of new anticancer drugs for esophageal cancerwere crucial. It was still the main goal of clinical work.Studies showed that mTOR/P70S6K signaling pathway was overactivation in the development of multiple tumors (including esophagealcancer). mTOR/P70S6K signalling pathway mediated tumor development bypromoting cell proliferation, inhibiting tumor cell apoptosis, promoting tumornew blood vessels, promoting invasion and metastasis. P70S6K was the keyprotein in the downstream, and it was the substrate of mTOR. mTOR canmake P70S6K phosphorylate into p-P70S6K. cIAP1was apoptosis inhibitorand caspase-3was an important member of the caspase family of apoptosisprotease, which played an important role in the development of esophagealcancer.The experiment inoculated esophageal squamous cell cancer cell line ECA109to nude mice and established orthotopic transplanted tumor ofesophageal squamous cell carcinoma. All animals were given low,medium and high dose of Bufalin (0.5mg/kg,1.0mg/kg，1.5mg/kg),rapamycin and the two drugs combined. Tumors were completed stripping.The expression of P70S6K, cIAP1and caspase-3mRNA were detected byRT-PCR. Western blot and immunohistochemical method were used to detectthe expression of P70S6K, cIAP1, active caspase-3and P70S6K activationlevel. And then learned more about Bufalin mediating the pathogenesis ofesophageal cancer by inhibiting P70S6K and the effects of Bufalin on tumorcells apoptosis, which may provide reliable theoretical basis for clinicaltreatment of esophageal cancer.Objective:To investigate the effects of Bufalin on P70S6K, cIAP1and caspase-3in orthotopic transplanted tumor of human esophageal squamous cellcarcinoma in nude mice.Methods:1Digested the ECA09cells with0.25%trypsin which in the logarithmicgrowth phase and adjusted to a final concentration of1.0×107/ml. InjectedECA109cells to right axilla subcutaneously in nude mice used1ml syringeand each of0.2ml,36nude mice were inoculated and feeding about15days(At this time the tumor size in nude mice were about1cm3).36nude micewhich orthotopic transplanted with esophageal cancer cell lines ECA109wererandomly divided into6groups, those were, model group (physiological salinegroup), low-dose Bufalin group (0.5mg/kg, BL), medium-dose Bufalin group(1.0mg/kg, BM), high-dose Bufalin group (1.5mg/kg, BH), rapamycin group(RAPA) and Bufalin with rapamycin combined group (BR). Each groupinoculated6mice and all mice were given intraperitoneal injection of drugsfor30days. Each mouse was injected0.2ml in model group and50μg/kg inRAPA group once a day. BR group injected once every other day. The diet andthe change of mental state of nude mice were recorded during the course ofmedication and the body weight and tumor size were measured. All the nude mice were put to death after medication. We stripped tumors completely,measured tumor size, calculated the tumor inhibition rate and observedmorphological changes by HE staining.2The apoptotic index in transplanted tumor cells were detected byTUNEL labeling method.3The expression of P70S6K, cIAP1and caspase-3mRNA in nude micetransplanted tumor were examined by RT-PCR method.4The expressions of protein P70S6K, cIAP1, active caspase-3andP70S6K activation in nude mice transplanted tumor were detected by westernblot and immunohistochemical method.Results：136nude mice all growed tumor, tumor formation rate were100%. Noobvious abnormality of the nude mice diet and mental state on each groups(model, BL, BM, BH, RAPA, BR) during the course of medication; Thedifferences of each groups nude mice body weight were no statisticallysignificant before treatment (20.9±2.4g,20.8±1.5g,20.8±2.1g,20.9±2.7g,20.9±1.9g,20.8±2.1g, P>0.05), and the differences were no statisticallysignificant in each groups nude mice body weight on the first day aftertreatment (27.9±3.0g,28.2±1.9g,27.9±2.9g,28.1±2.8g,27.9±2.0g,27.8±2.6g, P>0.05).2The tumor size were still increasing during the treatment, the tumor sizeof each groups (model, BL, BM, BH, RAPA, BR) were1.778±0.176cm3,1.714±0.188cm3,1.115±0.124cm3,0.713±0.122cm3,0.699±0.079cm3,0.506±0.076cm3respectively. Tumor inhibition rate were4.1%,37.6%,60.1%,60.9%,71.7%respectively in each treatment groups, significantdifferences were found in each groups (P<0.05), in addition to the model andBL group (P>0.05), BH and RAPA group (P>0.05).3Morphological observation results in nude mouse orthotopictransplanted tumor in each groups: HE staining sections were observedthrough microscope. The tumor tissues of nude mice orthotopic transplantedtumor were poorly differentiated squamous-cell carcinoma (PDSCC), the heteromorphism was obvious in cancer cells, karyomegaly and anachromasis,cell morphological were various, arranged in solid nests of unequal size, thenuclear chromatins showed coarse granular, nucleolus were obvious,infrequent ofintercellular bridge and keratosis. No necrosis were found inmodel group tumor tissues, BL group can visible spotty necrosis, BM groupmainly in focal necrosis, while, BH, RAPA and BR groups can visible largeflake necrosis.4TUNEL staining showed: Apoptotic nucleus demonstrated brownishyellow. The apoptotic indexes of each treatment groups (BL, BM, BH, RAPA,BR) were13.67%,16.17%,10.83%,10.33%,8.5%respectively, significantlyhigher than model group (2.17%), and BM group was the highest; Thedifferences were statistically significant in BR group compared with RAPAand BH group (P<0.05).5RT-PCR results showed:(1) The expression of cIAP1mRNA in eachgroups (model, BL, BM, BH, RAPA, BR) were0.929±0.044,0.889±0.048,0.664±0.044,0.503±0.046,0.496±0.045,0.357±0.038, mRNA expressiongradually decreased, significant differences were found in each groups(P<0.05), in addition to model and BL group (P>0.05), BH and RAPA group(P>0.05);(2) The expression of caspase-3mRNA in each groups (model, BL,BM, BH, RAPA, BR) were0.342±0.079,0.370±0.040,0.679±0.027,0.768±0.046,0.778±0.045,1.010±0.079, mRNA expression gradually increased,significant differences were found in each groups (P<0.05), in addition tomodel and BL group (P>0.05), BH and RAPA group (P>0.05);(3) Theexpression of P70S6K mRNA in each groups (model, BL, BM, BH, RAPA,BR) were no statistically significant (0.831±0.070,0.858±0.043,0.859±0.050,0.841±0.043,0.835±0.048,0.871±0.026, P>0.05).6Western blot results showed:(1) The expression of P70S6Kphosphorylation p-P70S6K in each groups (model, BL, BM, BH, RAPA, BR)were1.203±0.057,1.153±0.061,0.852±0.072,0.611±0.037,0.559±0.056,0.400±0.017; The expression of cIAP1in each groups (model, BL,BM, BH, RAPA, BR) were1.222±0.025,1.172±0.065,0.769±0.054,0.552 ±0.050,0.545±0.046,0.342±0.030, protein expression gradually decreased,significant differences were found in each groups (P<0.05), in addition tomodel and BL group (P>0.05), BH and RAPA group (P>0.05);(2) The modelgroup was not detect active caspase-3, the expression of active caspase-3ineach treatment groups (BL, BM, BH, RAPA, BR) were0.561±0.045,0.760±0.043,0.873±0.044,0.880±0.039,1.115±0.065, significant differenceswere found in each groups (P<0.05), in addition to BH and RAPA group(P>0.05);(3) The expression of P70S6K in each groups (model, BL, BM, BH,RAPA, BR) were no statistically significant (0.788±0.020,0.790±0.014,0.787±0.015,0.790±0.020,0.791±0.016,0.785±0.020, P>0.05).7Immunohistochemical results showed:(1) The immunoreactive score ofp-P70S6K in each groups (model, BL, BM, BH, RAPA, BR) were11.00±1.55,9.67±1.86,6.67±1.03,3.5±0.55,3.33±0.52,2.17±0.41; Theimmunoreactive score of cIAP1in each groups (model, BL, BM, BH, RAPA,BR) were11.50±1.22,10.00±2.19,7.00±1.10,3.83±0.41,3.67±0.52,2.67±0.52, protein expression gradually decreased, significant differenceswere found in each groups (P<0.05), in addition to model and BL group(P>0.05), BH and RAPA group (P>0.05);(2) The immunoreactive score ofactive caspase-3in model group was0, while the immunoreactive score ofactive caspase-3in each treatment groups (BL, BM, BH, RAPA, BR) were3.50±0.55,7.00±1.10,8.50±0.55,8.67±0.52,10.83±1.83, the proteinexpression gradually increased, significant differences were found in eachgroups (P<0.05), in addition to BH and RAPA group (P>0.05);(3) Theimmunoreactive score of P70S6K in each groups(model, BL, BM, BH, RAPA,BR) were no statistically significant (8.33±0.52,7.83±1.47,8.5±0.55,8.17±0.41,8.67±1.97,8.83±1.6, P>0.05).Conclusions:1Bufalin had significant antitumor effect on the orthotopic transplantedtumor of human esophageal squamous cell carcinoma in nude mice.2Bufalin could decrease the level of P70S6K phosphorylation, whileP70S6K was not affected, which prompted that Bufalin may inhibit tumor growth through inhibiting the activation of P70S6K.3Bufalin could reduce the expression of apoptosis inhibiting factorcIAP1, meanwhile up-regulated active caspase-3. It prompted that inducingtumor cell apoptosis may be one of the important anti-tumor mechanisms ofBufalin. |
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