| [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 mortality among them. Approximately 500,000 women worldwide are diagnosed with invasive cervical cancer each year, 80% of which arise in less-developed countries, and more than half of them die of this disease. Surgery is the recommended treatment for early-stage cases of cervical cancer, while in advanced stage, recurrent and metastatic cervical cancer, surgery has very limited value. On the other hand, the tumors at these stages are commonly resistant to chemotherapy, and furthermore, these patients frequently have a poor performance status that limits use of aggressive chemotherapy or radiation, 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, Histone Deacetylases (HDAC) inhibitors (HDACi) have emerged as the potential therapeutic agents for multiple human cancers due to their capability to induce cell differentiation, growth arrest and apoptosis. valproic acid (VPA), a potent anti-convulsant widely used to treat epilepsy and mood disorders that acts as an HDACi at therapeutic concentrations, produces mild adverse effects in man even when serum levels exceed the normal therapeutic range In cervical cancer, it has been reported that HDACs are overexpressed, and phosphorylated and acetylated forms of histone H3 in cytologic smears shows a marked association of histone H3 modifications with progression of the disease from cervical intraepithelial neoplasia (CIN) I to CIN II and CIN III. Thus HDACs are also seen as a potential target for cervical cancer treatment. All-trans retinoic acid (ATRA) is a potent regulator of cellular growth and differentiation, and regulates the normal pathway of epithelial-cell differentiation, and can act as chemopreventive agents and effective inhibitors of chemical and viral carcinogenesis. Emerging clinical trials have shown the chemotherapeutic and chemopreventive potential of ATRA in cancerous and precancerous conditions of the uterine cervix. The signaling actions of ATRA are mediated by RA receptors, Of all the different isoforms of RA receptors, RARβ(particularly theβ2 isoform) seems to have the dominant role as a tumor suppressor and its loss is associated with tumor progression. Downregulation of RARβ2 has been shown in several tumors, including lung, breast, head and neck cancers, and cervical carcinomas. The loss of expression of this receptor in tumor cells has been attributed to the silencing of the promoter region of its gene through histone hypermethylation and deacetylation. Therefore, the use of HDAC inhibitors or combined with ATRA has been shown to restore the expression of silenced RARβ2, growth inhibitory and proapoptotic actions of ATRA in many types of cancer cells.[Objective]In the present study, we investigated the capacity of combinations of VPA and ATRA on cercical cn HeLa and SiHa (human cervical cancer cell lines)growth inhibition, and to observe their Molecular mechanism for cervical cancer treatment and provide a theoretical basis and treatment of clues.[Methods]1. in vitro:HeLa cells were treated with combined use of 3mM VPA, 1μM ATRA, 1μg/ml DDP, or respectively. The control group was treated with vehicle only.(1) MTT assay was used to assess proliferation inhibition. (2) IHC was used to examine the expression of Ki67 in cells after treatment(3)Western blot were used to examine the expression of AC-H3, RARβ2, p53,p21,STAT3,p-STAT3 and cyclinD1 in HeLa and SiHa cells after treatment.(4)FCM were used to examine cell cycle analysis in HeLa and SiHa cells after treatment.2. in vivoCervical cancer cells were harvested with trypsin/EDTA and resuspended in PBS,and injected (5×10~6cells per injection) s.c. into mice in the dorsal side between the scapulae. Once palpable tumors were established, mice were Mice were administered with VPA (i.p., 300mg/kg/d), ATRA (p.o., 15mg/kg/d) daily, or cisplatin (i.v., 2mg/kg) twice weekly The control group received the vehicle alone at the same schedule.(1) Tumor volumes were measured twice weekly and calculated using the following formula: (length×width~2)/2 (23).(2) Animals were sacrificed after 28 days of treatment, and tumors were harvested for proteomic and pathology evaluation of AC-H3, RARβ2, p53,p-STAT3 and Ki67.[Results]1. in vitro:(1) The proliferation of HeLa and SiHa cells were significantly inhibited by VPA combined with ATRA, P﹤0.05.(2) The expression of Ki67 in HeLa and SiHa cells after treatment is lower than the control group, P﹤0.05.(3) Western-blot showed that the treatment lead to increase of AC-H3, RARβ2, p53, p21while reduction in STAT3, p-STAT3 and cyclinD1 in cells.(4) FCM showed significant G1 phase arrest in HeLa and SiHa cells after treatment2. in vivo(1) The inhibitory effect of VPA conbinated ATRA on growth of tumor xenografts was significantly higher than that treated with control and each drug alone (P<0.05).while no significant difference existed with cisplatin administered alone (P > 0.05).(2) Western-blot showed that the treatment lead to increase of AC-H3, RARβ2, p53, p21while reduction in STAT3, p-STAT3 and cyclinD1 in tumor xenografts.(3) The expression of Ki67 in tumor xenografts after treatment is lower than the control group, (P﹤0.05)[Conclusion]VPA alone or in combination with ATRA would inhibitor the growth of HeLa and cells in vitro and inhibitor tumor xenografts effectively, which probably attributed to the histone H3 hyperacetylation and up-regulation of RARβ2, p53 and down-regulation of p-STAT3 after treatment.
|
PDF Full Text Request