| Mammalian target of rapamycin(mTOR) is an atypical serine-threonine phosphatase, belonging to the family of phosphoinositide kinase—related kinase(PIKK). mTOR has an regulative effect on cell growth, cell differentiation, cell proliferation, cell migration and cell survival. mTOR exists in the form of two kinds of compound. They are mTORC1(mTOR-raptor compound) and mTORC2(mTOR-rictor compound).And mTORC1consists of mTOR Raptor(regulatory-associated protein of mTOR), unit-like protein), PRAS40and Deptor, which is mLS8/GβL (G protein sensitive to rapamycin and stimulated by hormone, growth factor, nutrition, energy and stress to be active. The activation of mTORC1leads to the phosphorylation of S6K1(4OS ribosomal protein S6kinase1) and4E-BP1(eukaryotic initiation factor-4E binding protein1). Consequently, mTORC1can be involved in the biological process of cell growth, cell proliferation, cell survival, protein interpretation, ribosome generation, autophagy and so on. In addition, mTORC2consists of mTOR, Rictor (rapamycin-insensitive companion of mTOR), mLS8/G(3L, mSin1(SAPK interacting protein1). PRR5/Protor and Deptor, which is have a high tolerance of rapamycin. mTORC2has an effect on Akt, but it’s only in view of the site of Ser473, not other sites of Akt. After the activation of Akt, mTORC2can regulate the growth, metabolism and proliferation of cells. Otherwise, The research says that mTORC2also take a significant role in various biological process as formation of cytoskeleton system.It was found out that the mTOR signaling appears the state of overactivity in angiogenesis, insulin resistance, adipose synthesis, T lymphocyte activation and so on. besides. Research finds the mTOR signaling can be highly actived in carcinomatosis in recent years, which controls variouscrucial biological process on tumorigenesis and progression of tumour. Therefore, the mTOR signaling has become a hot spot in life science. With the the progress of study, the mTOR signaling has witnessed huge improvements in incidence and development of multiple common tumors as mammary cancer, colorectal cancer, prostatic carcinoma(PC), ovarian cancer(OC), liver cancer, lung cancer, leukemia and lymphoma. Although control of rapamycin and its analogs by mTOR has entered the clinical test stage, the control mechanism of the mTOR signaling is still at the exploration stage.Mammary cancer is one of the most common female malignant tumors, and the incidence is trending to rapidly ascend. In China, the incidence of mammary cancer has been the top one of malignant tumors among women in big cities. Her-2is one kind of transmembrane glycoprotein edited by the gene of c-erbB2which is located in the chromosome of17q21, it possesses the activity of protein tyrosine kinase (PTK), in short of P185. The clinical trial has verified that Her-2affects gene amplification and hyperproteosis in15%to25%of mammary cancer, it also raises the risk of tumour cells’recurrencing and transferring, shorts the time of disease free survival (DFS), the prognosis is not good too. Trastuzumab is a kind of recombinant humanized monoclonal antibody which takes Her-2as a target, combining with c-erbB2’s recipient cell externally. Trastuzumab has high affinity, specificity and antitumous effect caused by interruptting c-erbB2acceptor and it is widely used in curing patients who have mammary cancer caused by over-presentation of Her-2.Nowadays, there is no specific research on the correlation among Trastuzumab, the mTORC1signaling and the mTORC2signaling. The purpose of this paper is to probe into the influence of Trastuzumab to the mTORC1signaling and the mTORC2signaling and observe whether Trastuzumab is better than rapamycin to the extent of the mTOR signaling’s significant impact by using cell line of mammary cancer which is Her-2-overpresented.PurposeTo probe into the influence of Trastuzumab to the mTORC1signaling and the mTORC2signaling and observe whether Trastuzumab is better than rapamycin to the extent of the mTOR signaling’s significant impact by using cell line of mammary cancer which is Her-2-overpresented.MethodsIn laboratory, MDA-MB-453, the cell line of mammary cancer caused by over-presentation of Her-2that is dashed with10%fetal calf serum(FSC) will be kept in5%CO2incubator which is set to37℃with1640nutrient medium. The nutrient medium will be replaced timely according to the speed of cell growth, and the0.25%parenzyme and the0.02%fluid will be used to digest and disperse cells. Thus, serial subcultivation and inoculated culture can be guaranteed.Western Blot is used to detect the effect of Trastuzumab to the mTORC1signaling and the mTORC2signaling. Inoculate the MDA-MB-453cell into12pores plate,and add Trastuzumab with different concentration after cells enter into logarithmic growth phase. Then, set blank control group and rapamycin control group, and add equivalent normal saline in blank control group. Next, extract proteinum, captain, electrophoretic separation and trarsmembrane after2hours. What next is incubating at4℃for one night after1hour’s sealing, then hatching it in normal room temperature for12hours. The last step is ECL coloration, exposure and film processing.Cell Counting Kit-8(CCK-8) is used to detect the effect of Trastuzumab to the MDA-MB-453cell’s proliferation. Adjust the MDA-MB-453cell to2×104/ml, then add it to96pores plate,about200ml per pore, and each parallel bore was5bores. After24-hour culture, adherent cells were treated with culture medium containing physiological saline (the blank control group), culture medium containing rapamycin(the rapamycin control group) and culture medium containing Trastuzumab with different concentrations. After3-day culturing, detect the absorptance value (λ=450nm) with microplate-reader for ELISA according to the instruction of CCK8kit.Use microscope to observe death of Breast Cancer Cell. Adjust the concentration of the MDA-MB-453cell to2×104个/ml, then add it to24pores plate, change new culture medium when cell fusion raise to30%after24hours, which are culture medium containing physiological saline(the blank control group), culture medium containing rapamycin(the rapamycin control group), culture medium containing Trastuzumab with different concentrations(the experimental group) and culture medium containing rapamycin as well as Trastuzumab with different concentrations(the combined modality group). Furthermore, photograph each pore at the same time ecery day randomly, about three prints of photo a pore, and the observation needs to last6days.The experimental data are processed by SPSS13.0, and all data of the cell proliferation experiment and the clone formation experiment are presented by x±s. If there is variance equality existed, analysis of variance should be adopted, and then the two-two comparisons with LSD method should be choosed. If there is no variance equality existed. Welch method should be adopted in multiple groups, then he two-two comparisons with Dunnet’s T3method should be used. Ps:a value less than0.05was considered as significance.ResultsThe results of Western Blot indicate that the phosphorylation of mTOR, S6and 4EBP1in the rapamycin control group are inhibited distinctly. In the wake of the increasing Trastuzumab concentration, the level of the phosphorylation is lowered, and the the controlling effect of mTOR, S6,4EBP1and Akt to the mTOR signaling is more distinct than the rapamycin control group.The results of CCK8kit indicate that OD value of the blank control group is1.79±0.07, the same one of the rapamycin control group is1.47±0.08, and the OD values of the Trastuzumab experimental group are1.37±0.11,1.28±0.03,1.03±0.09,0.68±0.09and0.37±0.03separately in concordance with the variation of concentration. Homogeneity of variance test (Levene’s test) is conducted among the five experimental groups and the blank control group, which confirms the assumption of equal variance (P=0.083).One-way ANOVA Analysis is performed then, indicating significant difference exists(F=221.863, P=0.000). Thus post hoc Analysis is processed with the LSD method:Compared with the blank control group, all Trastuzumab groups(P=0.000) can inhibit proliferation of the MDA-MB-453cell. The differences have statistical significance. According to the formula of cell proliferation, we can learn that the rates of cell proliferation in the Trastuzumab group are (77±6.3)%,(71±1.5)%,(57±4.9)%,(38±5.1)%and (21±1.5)%separately, and the rate of cell proliferation in the rapamycin control group is (82±4.6)%. Compared with the blank control group, the rapamycin control group significantly inhibits the MDA-MB-453cell proliferation (P=0.000).The data of6groups of clone formation indicate that the mean of the control group and the experimental group are (519±32),(283±23),(140±18),(110±12),(83±10) and (19±6) per pore separately, and the cloning efficiency are (100±6)%,(55±4)%,(27±3)%,(21±2)%,(16±2)%and (4±1)%respectively. Homogeneity of variance test (Levene’s test) is conducted among the five experimental groups and the blank control group, which confirms the assumption of equal variance (P=0.052).One-way ANOVA Analysis is performed then, indicating significant difference exists (F=372.643, P=0.000). Thus post hoc Analysis is processed with the LSD method:all all Trastuzumab groups are P=0.000Compared with those of the control group.The observation on death of Breast Cancer Cell under microscope last6days indicate that cells of mammary caner MDA-MB-453is semipearl round cells growing as grape-like aggregation. After6-day drug treatment, no matter the single Trastuzumab group or the combined modality group, the numbers of cells are decreased with the increase of the Trastuzumab concentration. However, the effect of using rapamycin singly is not better than which of Trastuzumab, the differences between the effect of combined modality and the impact of single Trastuzumab treatment are not so significant. Moreover, number of the2.5mg/ml Trastuzumab+0.1mg/ml rapamycin group is more than the Trastuzumab group.ConclusionsAs the effective medicine in view of the Her-2target, Trastuzumab can inhibit the concentration dependence of the MDA-MB-453cell line of mammary cancer by the mTORC1and mTORC2signaling. Consequently, the growth and proliferation of tumor cell can be controlled. Otherwise, the effect of Trastuzumab is better than rapamycin to breast cancer cell that is Her-2-overpresented to the extent of the mTOR signaling. |
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