| Glioma is the most common adult primary central nervous system tumors, includingvarious of histological types. The glioblastoma (GBM) is the highest degree of malignancy.The standard treatment of GBM includes operation excision, alkylating agents TMZ(Temozolomide, TMZ) and adjuvant radiotherapy combined with chemotherapy. However,these tumors is hardly worthy of belief resistance and frequent recurrence tumors,glioblastoma on surgical operation treatment, radiotherapy and chemotherapy are tolerated,the median survival of GBM is limited to about15months after diagnosis. In addition tothe tumor cell itself to radiotherapy, chemotherapy resistance, the interaction between thetumor and the internal environment through participation in the anti angiogenesis of tumor,hypoxia and immune suppression. These play an important role in the treatment ofchemotherapy in malignant glioma. A new generation of alkylating agents TMZ is presenton the standard chemotherapy of malignant glioma. It has been proved to have goodclinical result. Its main function is through the O6-G methylation mediated induction,mismatch repair mechanism of DNA activation induced injury. However, due to thepresence of chemotherapy drugs intrinsic and acquired drug resistance phenomenon andthe blood brain barrier, it has great influence on the effect of chemotherapy.AKT is a key molecule in the PI3K/AKT pathway, in promoting the growth,proliferation, invasion, metastasis, angiogenesis, apoptosis, and plays a key role in cellresistance to chemotherapy and radiotherapy. Many literatures reported that AKT plays animportant role in carcinogenesis and metastasis of tumor, molecular mechanisms of AKTaction further and reveal its relationship with malignant tumor, there may be gene therapyfor malignant tumor, ideas and provide new target and development studies on antitumordrugs. Our previous experiments showed that the29genes associated with TMZchemotherapy resistance and survival in patients with GBM microarray. AKT2gene is oneof these genes. In recent years, there have been a number of experimental study shows theclose relation between PI3K/AKT signal transduction pathway and tumor resistance tochemotherapy. AKT activation may be involved in tumor radiotherapy resistance, theresults show that AKT2appears to be an effective overcome resistance is an importanttarget for glioma treatment. According to the previous experimental results we speculatedthat silencing AKT2gene expression may be a feasible method to increase the sensitivityof glioma chemotherapy. This experiment will be divided into four parts to study the effectand its mechanism on chemotherapy sensitivity of GBM to TMZ. The first part, Effects of the expression of AKT2on sensitivity of glioma cell line U251to TMZ; the second part,The effect of sensitivity of human glioma cell line U251xenograft in nude mice toTMZ byRNA interference suppressing the expression of AKT2; the third part, establishment andcharacterization of temozolomide chemotherapy drug resistant cell lines U251; the fourthpart, the AKT2mechanism for temozolomide chemotherapy resistance in glioma cells.Through the above experiments elucidated AKT2gene in glioma for molecular biologymechanism of temozolomide chemotherapy resistance, so as to find the key genes at themolecular level in the future and develop targeted therapy, to provide theoretical basis toimprove the efficacy of chemotherapy of glioma.Part I Effects of the Expression of AKT2on Sensitivity of GliomaCell Line U251to TMZObjective: To investigate the effect of expression of AKT2in human glioblastoma U251cell to TMZ sensitivity.Methods: according to the gene AKT2interference lentiviral vector construction, in vitroAKT2specific shRNA expression vector AKT2-shRNA was transfected into U251cellline. Expression of AKT2gene mRNA and protein was detected after transfection ofshRNA U251cells in Real-time PCR, Western blot method, CCK8method was used todetect the RNA interference of AKT2U251cells to change TMZ sensitivity.Results: after transfection of AKT2-shRNA expression level of AKT2gene mRNA andprotein in U251cells compared with blank control and slow virus negative control groupdecreased significantly; the IC50of U251cell decreased from the blank control group(39.72±2.41)μg/ml, negative control group (39.43±2.24)μg/ml to (27.23±1.93) μg/ml,sensitivity of U251cell to TMZ decreased significantly.Conclusion: AKT2-shRNA can inhibit the expression of AKT2in glioblastoma cell lineU251, and can increase the sensitivity to TMZ. Part II The Effect of Sensitivity of Human Glioma Cell Line U251Xenograftin Nude Mice to TMZ by AKT2-shRNAObjective: To explore the effects of the expression of AKT2on sensitivity of glioma cellline U251to TMZ.Methods: U251cell inplaned tumor model in nude mice was established. By the intratumoral injection and intraperitoneal administration, the nude mice tumor were treatedwith TMZ chemotherapy drugs and AKT2-shRNA expression vector. Observe the nudemice tumor accumulation conditions. The expression level of AKT2of tumor cells weredetected in nude mice in each group by method of Real-time PCR andImmunohistochemistry, DNA in situ end labeling method (TUNEL) was used to analyzethe apoptosis of tumor cells in each group.Results: the blank control group, TMZ chemotherapy group, the TMZ+negative controlgroup, TMZ+AKT2interference group nude mice tumor volume were:(669.34±98.73)mm3,(399.86±55.26)mm3,(383.81±34.01)mm3,(297.72±41.49)mm3; quality were:(1.25±0.26)g,(0.72±0.11)g,(0.69±0.07)g,(0.52±0.07)g. TMZ+AKT2interference group, thetumor volume and tumor weight were significantly less than the blank control group andnegative control group, there was significant difference (P<0.05). Immunohistochemistryand Real-time PCR results indicated that TMZ+AKT2interference group was comparedwith other control group decreased AKT2expression level. TUNEL experimental resultsshow: the blank control group, TMZ chemotherapy group, the TMZ+negative controlgroup, TMZ+AKT2interference body apoptosis index group of tumor were:(7.15±1.04)%,(25.26±2.71)%,(26.63±3.46)%,(42.81±5.97)%. TMZ+AKT2interference group theapoptosis was significantly higher than that in the blank control group and negative controlgroup, there was significant difference (P <0.05).Conclusion: RNA interference of AKT2can significantly improve the sensitivity ofglioma chemotherapy to TMZ. Part III Development and characterization of TMZ-resistant humanglioma cell line U251Objective：To establish the resistance to TMZ in human glioma cell line U251anddescribe the characteristics of its resistant variant.Methods: By gradually increasing chemotherapy drugs TMZ concentrations, gliomaresistant cell line U251/TMZ will be established. The four methyl thiazolyl tetrazolium(MTT) method calculated the IC50of U251and U251/TMZ cell line and resistancecoefficient (RI). Flow cytometry analysis investigate the cell cycle of cell lines.1358common protein of U251and U251/TMZ two cell lines were determined and analyzed byprotein chip technology. Results: the IC50of U251cell line was (10.78±0.72)μg/ml, and the IC50of U251/TMZwas (45.42±3.17)μg/ml, resistance index RI by MTT measured value is4.21(P<0.05).Flow cytometry analysis showed that the cell cycle of U251/TMZ occurred mainly in G2phase arrest.Determination results of protein chip showed DNA damage repair proteins ofthe Bcl-2family, caspase family, P53and other apoptosis protein and were obviouslyup-regulated or down regulated.Conclusion: GBM chemotherapy resistance cell line to TMZ (U251/TMZ) wasconstructed successfully, it’s resistance is very obvious and have stable biologicalcharacteristics. The cell line U251/TMZ is suitable for research for temozolomidechemotherapy resistance in glioma. Part IV The role of AKT2in resistance to TMZ in glioma cell line U251Objective: To study AKT2, the multi drug resistance gene (MDR-1), multidrug resistanceassociated protein (MRP-1), apoptosis and autophagy related protein DNA damage repairprotein as well as Bcl-2, Caspase-3, beclin-1etc., in glioblastoma cell line U251fortemozolomide (TMZ) chemotherapy resistance in the mutual relation and regulation.Methods: by gradually increasing chemotherapy drugs TMZ concentrations, gliomaresistant cell line U251/TMZ was established successfully. related protein on U251cellline and U251/TMZ resistant cell lines were detected by protein chip technology andanalyed by computer software singal-net. Apoptosis of cells of each group was detected byflow cytometry cell technology. further analysis and evaluation of the AKT2and theMDR-1, MRP-1, MGMT, and apoptosis related protein expression regulatory interactionused by Real-time PCR and Western Blot.Results: singal-net analysis showed that U251resistant strains of AKT2targeting Bcl-2,Caspase-3, P53regulation, survivin, beclin-1, apoptosis and autophagy related protein,apoptosis increased significantly after AKT2-shRNA interference of U251, Real-time PCRand Western blot results showed Bcl-2, survivin, MGMT was down regulated, andCaspase-3, PTEN, P53, beclin-1increased.Conclusion: AKT2may be involved in the regulation of expression of apoptosis andautophagy related protein Bcl-2, Caspase-3, P53, survivin, Beclin1, and DNA damagerepair protein MGMT, which mediates U251chemotherapy resistance to TMZ. |
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