| BackgroundCervical carcinoma is one of the most frequent female malignancy which is the first leading cause of death for gynecological cancer in China and many other countries. Although we have made big progress in its prevention and cure, the incidence of cervical carcinoma still rises steadily because of high HPV in-fection and changes in society life model. The proportion of young patients is on the rise, and the cervical adenocarcinoma is more and more familiar in recent 20 years. Because the adenocarcinoma is easy to occur lymphous nodes transfer early, the radiosensitivity of adenocarcinoma is low, and the chemotherapy is easy to be endured, it is difficult to treat cervical adenocarcinoma and the prog-nosis is not ideal. So, it has become a hotspot in the field of cervical cancer therapy to seek a scientific and applicable way to enhance the curative effect and improve the prognosis of cervical carcinoma. Glycogen synthase kinase-3(GSK-3) is a serine/threonine kinase,highly conserved and has been identified in every eukaryote investigated to date. It is encoded by two isoforms in mammals,termed GSK-3αand GSK-3β. GSK-3βis now recognized as not only a key rate-limiting enzyme of glycogen metabolic process, which phosphorylats hepatic glycogen synthase and makes it devitalize by insuline regulation, but also plays an important role in at least three signal transductory systems, namely, the Wnt/wingless, PI3'-kinase and Hedgehog pathways,which influence cellular processes, such as protein synthesis, cell proliferation, cell differentiation, cell motility and apoptosis. Dysregulation of the expression and activity of GSK-3βhas been linked to several prevalent pathological conditions, including tumorigenesis.It has been investigated that GSK-3βhas a close relationship with tumors-such as breast cancer, colon carcinoma and ovarian cancer, et al. And we find that GSK-3βis the crossing-point of the signaling pathways about cervical car-cinoma, which is showed that GSK-3βshould play an improtant role in the cer-vical tumorigeness. However, the mechanism of action of GSK-3βunder car-cinogenesis is not clear .Thus, GSK-3βgene is a promising target for cancer therapy. A better understanding of its physiologic and pathophysiologic roles in cervical carcinoma may open new opportunities for effective therapeutic inter-vention and better outcomes.ObjectiveTo explore the possible roles and mechanisms of GSK-3βplays in the pathogenesis, apoptosis, radiosensitivity and chemosensitivity of cervical carci-noma; and by seeking the possible mechanism to investigate the feasibility of exogenous changes of targeting GSK-3βin increasing radiosensitivity and chemosensitivity of cervical carcinoma. Thus, to lay a solid foundation for GSK-3βtherapy for cervical carcinoma.Methods1) Immunohistochemical method was employed to detect the expression of GSK-3βin normal cervix, precancerosis and carcinoma cervix tissues, then analyzed with regarding to clinical stage, pathological, histology type and metastatic;2) Differential expression of GSK-3βand pGSK-3βSer9 were examined by immunohistochemistry, RT-PCR and Western blot analysis in defferent cer-vical carcinoma cell lines;3) MTT assay was used to detect the the change of cisplatin chemoresistance of Hela and Siha cells pretreated with LiCl;4) Hoechst33342/PI staining was used to detect the changes of apoptosis to cisplatin of Hela and Siha cells pretreated with LiCl;5) Cervical adenocarcinoma Hela cells was transfected with GSK-3β-WT, GSK-3β-S9A and GSK-3β-K85R vectors by Lipofectin, the expression level and localization of GSK-3βprotein in each transfected cells were ex-amined by Western blot;6) Indirect immunofluorescence was used to observe subcellular localization of GSK-3βin transfected Hela cells and their phenotypic effects;7) Apoptosis of transfected Hela-S9A cell lines was observed by cell cycle distribution with FCM and transmission electron microscopy study;8) By G418 screening, stable transfectants of Hela-WT, Hela-S9A, Hela-K85R, Hela-Vec were obtained. MTT assay was performed to evaluate the effects of GSK-3βoverexpression on cell proliferation;9) Clonogenic assay and survival curve were carried out to evaluate the changes of cell radiosensitivity; MTT assay was used to measure influence on cell proliferation ability at 24, 36, 48, 72 and 96h after irradiated with 6Gy X-ray ;10) Western blot was performed to evaluate the expression level of GSK-3βandβ-catenin proteins at 48h after irradiated with different doses of X-ray ;11) MTT assay was performed in vitro to determine drug sensitivity of the Hela-S9A,Hela-WT,Hela-K85R,Hela-Vec and Hela cells, the IC50 values were calculated; measured influence on cell growth at 12, 24, 36, 48 and 72h after treated with 5μg/ml cisplatin by MTT assay;12) Western blot was performed to evaluate the expression level of GSK-3βandβ-catenin proteins at 48h after treated with 5μg/ml cisplatin;13) To detect the expression of Bcl-2 and Bax proteins by Western blot.Results1) GSK-3βwas a ubiquitous protein, whose level was lower in adenocarci-noma tissues than that in normal cervix, the difference was significant (p<0.05). But the expression of pGSK-3βSer9 was much higher in precancer and carcinoma than that in nomal cervix;2) By immunocytochemistry, GSK-3βwas predominantly cytoplasmic in Hela cells, while primarily localized in the nuclear in Siha cells;3) Cisplatin-resistant was increased in Hela and Siha cell lines pretreated with lithium, and the resistance to cisplatin in Hela was much higher than that in Siha cell;4) Hela and Siha cell lines pretreated with lithium led to apoptotic morpho-logical alteration observed by Hoechst nuclear staining;5) Western blot analysis showed that both Hela and Siha expressed GSK-3β, and the levels of total GSK-3βin two cell lines were almost aequalis, how-ever, the level of pGSK-3βSer9 protein in Hela was lower than that in Siha , indicating that Hela might have more active form of GSK-3β;6) Successfully transfected by pcDNA3.0/GSK-3βplasmids, Western blot study suggested the transfection plasmids of each GSK-3βinto Hela sig-nificantly increased total GSK-3βexpression ,but decreased pGSK-3βSer9 expression;7) MTT assay showed overexpression of GSK-3β-S9A considerably inhibited Hela cell growth, which led to apoptotic morphological alteration observed by electron microscopy study, as well as the G2-M arrest with FCM;8) Immunofluorescence study suggested Hela-S9A almost localized in the nu- clear, whereas Hela-WT primalily localized in the cytoplasmic;9) With the rise of radiation dose, cell clone formation rate declined in all transfected cells and at the same dose of radiation, clone formation rate of Hela-S9A declined notably, whose cell survival curve also showed a sig-nificant decrease with a dose-dependent way. With time went by, cell pro-liferation inhibitive rate rose up significantly after irradiated with 6Gy X-ray, which was in a good time-dependant way, and the cell apoptotic rates and proliferation inhibitive rates of Hela-S9A at every checkpoint were obviously lower than those of untransfected Hela cells.10) Western blot showed that: with the rise of radiation dose, expression level of GSK-3βup-regulated in Hela-S9A cells and at the same dose of radiation, levels of up-regulation notably with a dose-dependent way; whereas the ex-pression level ofβ-catenin significantly decreased;11) With the rise of cisplatin concentration, cell viability declined in all trans-fected cells and at the same concentration of cisplatin, cell viability of Hela-S9A declined sharply, the IC50 at 48h after delt with cisplatin was (0.38±0.02)μg/ml. With time went by, cell proliferation inhibitive rate rose up significantly after delt with 5μg/ml cisplatin with a good time-dependent way, and cell proliferation inhibitive rates of Hela-S9A at every checkpoint were obviously higher than those of untransfected Hela cells.12) Western blot showed that: with the rise of cisplatin concentration, expres-sion level of GSK-3βup-regulated in Hela-S9A cells and at the same con-centration of cisplatin, levels of up-regulation notably with a dose-depen dent way; whereas the expression level ofβ-catenin significantly decrease;13) As showed by western blot assay, compared with control group, from pro-tein level, the expression of Bax significant increased, but the expression of Bcl-2 significant decreased, which was benefit for inhibition ofβ-catenin. These showed that GSK-3βcould induce cell apoptosis, which benefit from the regulation of Bcl-2/Bax.Conclusions: Under this experimental condition :1) In malignant tumors, the expression of GSK-3βprotein was high by using im-munohistochemical methods, and the expression of GSK-3βwas correlated with TNM stages;2) The Hela cell distinguished from Siha cell with different expression level of pGSK-3βSer9 localized in nucleus;3) Different function yield different GSK-3βprotein in Hela cells with special subcellular localization. Lithium was able to promote the chemoresistance to cisplatin through induction of inactive phosphoralation of GSK-3βat Serine9;4) Constitutely active of GSK-3βcould increase cell apoptosis induced by X-ray irradiation and cisplatin treatment .There were some relationships on the dose-effect and time-effect of the inhibitive efficiency;5) In the Wnt/β-catenin signaling pathway, constitutely active of GSK-3βcould inhibit growth and invasion of cervical carcinoma cells by inducing cell apoptosis signaling pathway regulated by Bax and Bcl-2. .
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