| Glioma is the most common primary malignant tumor in the central nervous system. Owing to the feature of its invasive growth, fast cell proliferation and high recurrence rate, glioma is hard to be removed completely by the surgical resection. The postoperative radiotherapy, chemotherapy and immunotherapy can improve the life quality and overall survival of the patients. The molecular development of glioma is a complex process which involved the activation of proto-oncogenes and inactivation of tumor suppressor genes and the abnormal expression of mutiple cytokines in the cellular message transmission pathway. Therefore, seeking more genes associated with the gliomagenesis, a comprehensive understanding of the molecular pathology of gliomas, and optimizing treatment strategies and developing novel therapeutic approaches for glioma, have become an important research project for glioma. It can be said that the gene therapy for correcting the aberration of genetic events in glioma and small molecular inhibitor targeting to the abnormal cellular message transmission pathway have been the hot spots in the study of glioma.Studies in recent years have revealed that P13K/Akt signal transduction Pathway is the most important survival pathway for the survival of tumor cells. Akt occupies hinge location in the cell signal transductions, which plays an important role in tumorogenesis and development via multiple functions and can be phosphorylated by PI3K activated by many growth factors and cytokines on the upstream. The increase of p-Akt(PhosPho-Akt) level contributes to activate the PI3K/Akt pathway. Activation of this pathway plays a pivotal role in enhancing tumor cell growth, proliferation, invasion and migration. Cell and animal experiments have shown that PI3K inhibitor (LY294002) has suppressing effect on tumor cells. However, the Phase III clinical trials in recent years show that the single or multi-target small molecule inhibitors targetting to the cell signal pathway, have not been made so ideal effect as that in early vitro and animal experiments.PTEN is the first tumor-repressor with double specific phosphatase which has been found. PTEN can exerts its tumor suppression effect through suppress PI3K/Akt pathway. It has been confirmed that the loss and mutation of PTEN gene in many human malignant tumors can leads to activation of PI3K/Akt cell signaling pathway. Especially the expression and function of phosphor-Akt are usually increased greatly, which result in tumor cell growth, proliferation, invasion and migration. Therefore, PTEN has become an important target for gene therapy for glioma.In this study, PI3K inhibitor (LY294002) combined with wild-type PTEN gene recombinant adenovirus vector (Ad-PTEN) were actted on the malignant glioma cell lines, then we observed whether there are jointly or collaborative enhancement to suppress the proliferation and invasion of tumor, and then explored the possible mechanisms with a view to provide a new direction for the comprehensive treatment of glioma.The present study was divided into two parts.In the first part of this study, MTT method was used to evaluate the IC50 of LY294002 alone and LY294002 combined AD-PTEN in human glioma U251, LN229 cell line. MTT method was used to evaluate cell proliferation rate. Flow cytometry and Annexin V-FITC were used for cell cycle apoptosis analysis respectively. The invasion and migration ability was detected by Transwell analysis and Scarification test. Western blot was used to evaluate the expression of proteins. The result showed that the IC50 of LY294002 alone and LY294002 combined AD-PTEN in human glioma U251,LN229 cell line were 12.7,16.9umol/L; 6.30,8.11umol/L, which were in the safety range of experimental use. The cell multiplication rate in combination group presented decreasing trend since the sixth day in culture (P<0.05), as compared with DMSO,Ad-vector and LY294002 group. The number of cells inhibited at G0/G1 phase in combination group was more than any other group (P<0.05). The apoptosis rate in combination group was higher than that in the other three groups (P<0.05). Transwell and Scarification test indicated that the invasion and migration ability of the cells in combination group were decreased obviously (P<0.05). The protein expression of PTEN in combination group were significantly higher, while PCNA,CyclinD1,bcl-2 and MMP9,p-FAK were lower than those in the other three groups (P<0.05).In the second part, in vivo study was carried out to further investigate the combination or synergy effect of LY294002 and Ad-PTEN and its concrete mechanism. Twenty-four athymic mice were randomly divided into 4 groups (DMSO,Ad-vector plus DMSO,LY294002 alone and Ad-PTEN plus LY294002 group), and were treated respectively. Athymic mice xenogeneic transplant model was established by inoculation (sc) with LN229 glioma cells. Body mass (BM) and diameter of tumor mass were measured. Furthermore, The protein expressions of PTEN,p-Akt,CyclinD1,Caspase-3,MMP-2,p-FAK in tumor tissues were analyzed with immunohistochemistry. The results show that the tumor-inhibiting rate of Ad-PTEN plus LY294002 (92.47%) was significantly higher than the LY294002 alone (65.59%) (P<0.05).The protein expression of PTEN and Caspase-3 in Ad-PTEN plus LY294002 group were significantly higher, while PCNA,CyclinD1,bcl-2 and MMP-2,p-FAK was significantly lower than that in the other three groups (P<0.05).Conclusion:The suppressive effect of LY294002 combined with Ad-PTEN is better than LY294002 using alone, which indicate the combination effect in vitro and vivo. Its molecular mechanism may be as followed:1. The combination therapy can reinforce the inhibitory to the PI3K/Akt pathway.2. The Ad-pten can decreased the invasion through the FAK pathway. |
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