| [Background]:Cervical cancer is the second more frequent type of cancer in women worldwide,preceded only by breast cancer, and a considerable cause of morbidity and mortalityamong them. Approximately500,000women worldwide are diagnosed with invasivecervical cancer each year,80%of which arise in less-developed countries, and morethan half of them die of this disease. Surgery is the recommended treatment forearly-stage cases of cervical cancer, while in advanced stage, recurrent and metastaticcervical cancer, surgery has very limited value. On the other hand, the tumors at thesestages are commonly resistant to chemotherapy, and furthermore, these patientsfrequently have a poor performance status that limits use of aggressive chemotherapy orradiation, thus the majority of patients die as a result of uncontrolled disease. Therefore,new therapeutic approaches to cervical cancer with less adverse effects are needed.In recent years, With the deep research of epigenetics, a number of studies confirmhistone deacetylases, HDACs in cancer occurrence plays an important role, and may bethe potential therapeutic agents for multiple human cancers. HDAC inhibitors killCancer cells by inducing gene expression which major regulate cell activity, inhibitionof cell proliferation and promote cell differentiation and apoptosis. In cervical cancer, ithas been reported that HDACs are overexpressed, so HDACs may be potentialtherapeutic agents of cervical cancer. Trichostatin A is Streptomyces metabolites, whichis a kind of antifungal, and also is a powerful HDAC inhibitor. All-trans retinoic acid could regulate cells growth and differentiation, and can control the differentiation ofnormal epithelial cells. The current clinical experiment has shown that ATRA havechemotherapy and induction chemotherapy in cervical cancer and pre-cancerous.This study choose HeLa and SiHa cell lines and people cervical cancer Nude MouseModel for the treatment. We want research the TSA combined with ATRA kills cericalcancer cells, and further discusses the related mechanism.[Objective]In the present study, we investigated the capacity of combinations of TSA and ATRAon cevical cancer cells, and discusses the related Molecular mechanism. It provide atheoretical basis and treatment of clues.[Methods]1. in vitro:(1) The CellTiter96AQueous cell proliferation use different concentration TSA andATRA (1μ g/mL) to treat cervical cancer HeLa and SiHa cell, and calculate growthinhibition rate. According to the results of proliferation experiment, TSA (500nmol/L)concentration was considered to effectively restrain cervical cancer cells proliferation.the next experiment will be divided into four groups: the control group, the TSA group,ATRA group, the TSA+ATRA group. The control group only treats with solvent.(2)7-AAD fluorescence staining method observation changes and apoptosis of cervicalcancer SiHa and HeLa cells.(3)Western blot were used to examine the expression of STAT3、p-STAT3、P53、P21、caspase-3、BCL-2in HeLa and SiHa cells after treatment.(4)FCM were used to examine cell apoptosis in HeLa and SiHa cells after treatment.2. in vivo Take fresh cervical cancer tissue mass, transplantation in left shoulder department skinof CD1nu/nu nude mouse, every example vaccination5mice. This is the firstgeneration cervical cancer mice.When CD1nu/nu nude mouse subcutaneous tumorgrow to greater than1cm3, open the skin, stripping subcutaneous tumor, part offreeze-stored and10%formalin fixed set aside, as part of transplantation oforganizational source. Take20only nu/nu CD1nude mouse, according to abovementioned method, establishes the second generation of cervical cancer mice(F2). Oncepalpable tumors were established, mice were Mice were administered with TSA (i.p.,1mg/kg/d), ATRA (p.o.,15mg/kg/d) daily. Alone or in combination, each group of5cases and the control group received the vehicle alone at the same schedule. Thetreatment is4weeks.(1) Tumor volumes were measured twice weekly and calculated using the followingformula:(length×width2)/2.(2) Animals were sacrificed after28days of treatment, and tumors were harvested forproteomic and pathology evaluation of STAT3、p-STAT3、P53、P21、BCL-2.[Results]1. in vitro:(1) The proliferation of HeLa and SiHa cells were significantly inhibited by TSA(500nmol/L) combined with ATRA,(P﹤0.05).(2) Compared with control group,7-AAD fluorescent dyes shows the cells have atypical apoptosis change treat with TSA, TSA combined with ATRA significantlyincreased the apoptotic cells, better than separate treatment group(P﹤0.05).(3) Western-blot showed that the treatment lead to increase of P53、 P21、active-caspase-3while reduction in STAT3, p-STAT3、BCL-2in cells.(4) FCM showed significant apotosis change in HeLa and SiHa cells after treatment2. in vivo (1) The inhibitory effect of TSA conbinated ATRA on growth of tumor xenografts wassignificantly higher than that treated with control and each drug alone (P<0.05).(2) Western-blot showed that the treatment lead to increase of P53、P21, whilereduction in STAT3, p-STAT3、BCL-2in tumor xenografts.[Conclusion]TSA alone or in combination with ATRA would inhibit the growth of HeLa and SiHacells in vitro and inhibit tumor xenografts effectively, which probably attributed toincrease P53,active-caspase-3, P21, while reduction in STAT3, p-STAT3,BCL-2incells. |
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