| Object Radiotherapy is one of the most important treatments of malignancies, and it is involved in about65%-75%of total malignancies in our country. Cerebral gliomas are neurogliocytoma, which are accounted for30%-50%of brain tumors. Cerebral gliomas especially malignant gliomas have high ability to proliferate and infiltrate. Radiation is the most effective auxiliary treatment to cerebral gliomas, but cerebral gliomas cells have larger resistion to radiation, in addition, brain is a radiation dose restrictive organ, which is difficult to increase local control through increase radiation dose. Therefore, it is extremely urgent to explore effective brain radiotherapy sensitization agent. Histone deacetylase inhibitors (HDACIs) are new antitumor agents, the research of the agent that was just beginning. Valproic acid (VPA), which belongs to HDACIs, is clinical commonly used antiepileptic drug, and recenyl reports find that it can inhibit histone deacetylase (HDAC) activity to make it be the theme of many experiental studies, therefore, it is demonstrated to be relaxative and harmfulless. At present, researches on VPA and other HDACIs are limited to peripheral tumors such as cervical cancer lung cancer colon cancer pancreas cancer, however, researches on radiation sensitizers of central nervous system tumors are less. Even so, studies have found that VPA can both increase radiosensitivity of human brain tumors in vitro and vivo. So, we choose VPA as object of our study, to observe radiosensitivity to C6cells line in vivo, which may provide experimental evidences for VPA to the therapy of cerebral gliomas.Methods Rat C6glioma cells were cultured commonly in vitro. MTT assays was used to measure the proliferation inhibition of VPA in different concentrations (Ommol/L、0.25mmol/L、0.5mmol/L、1mmol/L、2mmol/L、4mmol/L) and different durations (24h、48h、72h) to choose appropriate response concentration and response time. The cell morphology was observed by inverted microscope after it incubated with different concentration VPA for2days. C6cells incubated with0.5mmol/L VPA were exposed to different doses X-ray irradiation (IR)(0、2、4、6、8Gy). Cloning experiments were used to determine the radiosensitizing effect of VPA. Flow cytometry was used to determine the effects of various concentrations VPA on apoptosis double staining with Annexin V-FITC and propidium iodide (PI). Trials are divided into several groups, control group, medication group(V group)、X-ray group (X group) and joint group(V+X group), real time PCR and Western blot were used to detect the expression of bcl-2and bax to find the pathway of VPA to induce C6to apoptosis.Results The HDAC inhibitor VPA inhibited the proliferation of rat C6glioma cells in vitro significantly with time and dose dependent(p<0.05). After a2days incubated with various concentration VPA, the cells exhibited deflate, the cell membrane shrinkage and bad adherence, appearing to be apoptosis. VPA at the concentration of0.5mmol/L enhanced the killing effect on C6cells and the cloning efficiency decreased obviously (p<0.05). The sensitization enhancement ratio (SER) was1.30according Do. VPA induced apoptosis on C6cells, the apoptosis rate is increasing along with the concentration of VPA (p<0.05). Regardless of level of transcription or level of translation, after treatment with VPA, expression of bcl-2mRNA and protein decreased, whereas up regulation was detected with bax.Conclusions Histone deacetylase inhibitor VPA inhibited the growth of rat C6glioma cell line at a dose-and time-dependent manner. VPA at the concentration of0.5mmol/L is low-toxic and can enhance the radiosensitivity of C6cells, and the mechanisms for this action might include inhibition of direct cellular proliferation、 inducing apoptosis and regulating apoptosis related genes. |
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