Study On The Effects To U251Cells Jointly By Temozolomide (TMZ) And Ad-rBmK CTa In Vitro

Glioma is a common malignant tumor of the central nervous system, originated in theneural epithelium, accounting for about44%of brain tumor incidence in China. Like otherhuman tumors, it’s a serious threat to the health and lives of the people. Gliomas resultedfrom a variety of factors, among which, the most fundamental ones are anti oncogenesinactivation, oncogenes being activated, and the changing of some related molecularsignaling pathways. Surgical excision, radiosurgery, chemotherapy and other traditionaltreatment methods are not so satisfactory. Except for the traditional methods, there aremany biological treatments, such as gene-targeted therapy, immunotherapy, andcombination therapy, which have become the research focuses of glioma treatment.In recent years, some scholars have found that the scorpion toxin can reduce tumorcell growth, and through in-depth study, they found that, some elements of the scorpiontoxins have targeted effect of glioma cells and can act on different types of ion channels inthe glioma cell membrane, which can work for tumor inhibition. Domestic scholars focusmore on the study of BmK CT, a kind of chloride channel scorpion toxin extracted from thevenom of Buthus martensii (Buhtus maretnsii Karhsch, BmK), which specifically targetingglioma cells, and with the effects of inhibiting cell growth, invasion and metastasis. FuYuejun and other scholars, from Institute of Molecular Biology of China Shanxi University,made an optimal reorganization on the basis of BmKCT cDNA sequence and got thechloride channel neurotoxin gene of Buthus martensii (recombinant of BmK the CT artiafet,rBmK CTa), and applied for patent.Under the guidance and help of the Institute of Molecular Biology, Shanxi University,the study used the extracted chloride channels neurotoxin gene of Buthus martensii, orrBmK CTa, and packaged it with adenovirus. By in vitro experiments, the scholarspreliminarily studied the inhibition of the gene to U251glioma cells; and through the jointuse of cytotoxic drug temozolomide in the glioma cells, to explore the associated tumorinhibition.Firstly, the subject constructed the adenovirus-mediated chloride channel neurotoxinsof Buthus martensii, namely Ad-rBmK CTa. Through the Application of PAdEasy-1 System adenovirus vector system, rBmK CTa gene was cloned into the shuttle plasmidpAdTrack-CMV, to obtain recombinant shuttle plasmid pAdTrack-CMV-rBmK CTa. Then,through restriction enzyme and other functions, the pre-transformed BJ5183bacteria ofpAdEasy-1adenoviral backbone plasmid was transformed for homologous recombination,to build the recombinant adenovirus plasmid pAd-rBmK CTa. The PacI digested linearizedrecombinant plasmid was transfected into293A cells, and successfully constructed theAd-rBmK CTa. By the PCR preliminary identification, the repeated infected293A cellsmassively proliferated. The virus was purified and concentrated by cesium chloridegradient centrifugation. Through determination, the virus titer was3.5x1012pfu／ml.Secondly, the subject made Ad-rBmK CTa, TMZ, as well as the both work in U251cells in vitro, to observe their inhibition of the cell. The results showed that with theincrease of the virus titer and TMZ concentration, the inhibition to U251cells enhanced.The separate effect of Ad-rBmK CTa and TMZ to apoptosis was not so significant, whileboth combined, the effect enhanced. Western blot experiments showed that proteinexpression of apoptosis related proteins Bax and caspase-3increased, but Bcl-2decreased,which further confirmed that the combined one has stronger effect of inducing apoptosisthan the single one. The same time, because Bcl-2protein is the link to two kinds ofprogrammed cell death-apoptosis and autophagy, both the inhibition and autophagypromoting effect to U251cells should be considered. The present study has confirmedthat TMZ promoting glioma cell autophagy is one of the main mechanisms of tumorsuppressor. We suppose that the mechanism of the combined effects for cell inhibition maybe: The synergistic effect greatly increasing the pro-apoptotic effect; through inhibition ofMMP-2activity, Ad-rBmK CTa thereby inhibited the invasion and metastasis of tumor cell,to prompt the cells being blocked at the early phase of the cell cycle, increase the sensitivityof glioma cells to TMZ, so the autophagy enhanced.In summary, adenovirus-mediated gene rBmK CTa can inhibit the proliferation ofU251glioma cells; combined targeting function of Ad-rBmK CTa and TMZ can createsynergistic effect and further enhance the inhibition. The mechanism for synergistic effectis not clear, so we will continue to do in-depth studies to further clarify the mechanism ofjoint effect of the Ad-rBmK CTa and TMZ. Meanwhile, the next step will be the mice vivo study, to provide a strong theoretical basis for the targeted therapy of theadenovirus-mediated rBmK CTa gene therapy and combined chemotherapy. With thecontinuous development of the related research at home and abraod, rBmK CTa will mostlikely develop into a new cytotoxic drug, and provide new hope for the treatment of glioma.