Study On The Method And Mechanism Of Reversal Of Drug Resistance To Bevacizumab And Temozolomide Of Glioma

Part Ⅰ. Study on the mechanism and reversal of bevacizumab resistance induced by autophagy of glioma stem cellsGlioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor. Surgical resection, postoperative temozolomide (TMZ)chemotherapy combined with concurrent radiotherapy plus adjuvan chemotherapy has become a clinically common standard treatment for newly diagnosed GBM patients.However, even after positive treatment, the median survival of GBM patients was only 14.6 months, and the overall survival rate of 1-year and 5-yaer for adults high-grade gliomas was only about 30% and 13% respectively.Bevacizumab is a recombinant human VEGFA targeting monoclonal antibody,approved in 2009 for recurrent glioblastoma. Clinical studies have shown that the use of bevacizumab despite prolonged progression-free survival,but has no significant improvement in overall survival.The characteristics and mechanism of vascular mimicry in glioma have been studied in our study group. We found that glioma stem cells (GSCs) have the ability to form vascular mimicry and VEGFR-2 plays a key role in the formation of vascular mimicry by GSCs. At present, vasculogenic mimicry is lack of response to antiangiogenic therapies such as bevacizumab. Our previous experiments also show that in the absence of VEGF, GSCs can form vasculogenic mimicry.Autophagy is an evolutionary conserved biological process for cells. The cell cytoplasm and organelles are encapsulated into autophagosomes, then autophagosomes fuse with lysosomes to form autolysosome which can degrade the substance inside it. Previous studies have shown that hypoxia induced autophagy promotes the survival of tumor cells in antiangiogenic therapy. Moreover, anti angiogenic drugs can promote the enrichment of cancer stem cells.There are many factors that may contribute the poor efficacy of anti-angiogenic therapy (eg bevacizumab), and vascular bypass is one of the most important reasons.Vasculogenic mimicry is mainly composed of tumor cells, and its structural characteristics determine its insensitivity to vascular therapy. In addition, application of anti angiogenic drugs decreased the number of tumor blood vessels and caused insufficient blood supply,resulting in hypoxia, increased lactic acid and other stress conditions, triggering tumor cell autophagy activity. Previous studies on autophagy and vasculogenic mimicry have shown that there are many similarities between them in terms of formation conditions and mechanisms. It is suggested that autophagy may be involved in the formation of tumor vasculogenic mimicry, which leads to the poor effect of bevacizumab.Reactive oxygen species (ROS) are a group of heterogeneous and highly reactive ions or molecules derived from oxygen molecules. A growing body of evidence suggests that the role of ROS is complex and contradictory, in particular, that ROS is involved in the maintenance of cellular homeostasis. Studies have shown that ROS regulates VEGF-VEGFR pathway,and autophagy can be associated with ROS by PI3K / AKT.The main content of this section and the issues to be discussed include: (1) To investigate the role of autophagy in the anti-angiogenesis therapy of bevacizumab in glioma.(2) To investigate the mechanism of bevacizumab resistance induced by autophagy of GSCs.Results: (1) Combined use of chloroquine improves the efficacy of bevacizumab in mice transplanted with GSCs. (2) Autophagy activates VEGFR-2 in GSCs via ROS-PI3K/AKT pathway.Conclusion: In this study, we clarify the relationship between autophagy, ROS,PI3K/AKT and phosphorylation of VEGFR-2 in GSCs and elucidate the molecular mechanism of activation of VEGFR-2 by autophagy of GSCs. Our findings highlight that vasculogenic mimicry,a kind of vascular bypass is an important reason for the poor effect of bevacizumab in patients with glioma. Our results provide a theoretical and experimental basis for the development of individualized anti-angiogenic therapy for glioma patients.Part Ⅱ. Study on the effect and mechanism of inflammasome - IL-1β pathway in the temozolomide resistance of glioblastomaChemoresistance has become the main reason for the poor efficacy or even failure of tumor chemotherapy drugs, and its mechanism is complicated, involving a variety of factors.Therefore, the study of mechanism of tumor chemoresistance should be considered from multi-channel, multi-link, multi-level and multi-target, so as to improve the therapeutic efficiency of tumor chemotherapy drugs.Temozolomide, an imidazole derivative, was approved by the FDA as a second-generation alkylating agent for recurrent GBM in 1999 and was used as first-line chemotherapeutic drugs for newly diagnosed GBM in 2005. TMZ chemotherapy combined with concurrent radiotherapy plus adjuvan chemotherapy can promote the median survival time of GBM patients from 12.1 months to 14.6 months, and the two-year survival rate was increased from 10.4% to 26.5%. However, 55% of GBM patients did not benefit from TMZ chemotherapy because of the high expression of 06-methylguanine DNA methyl-transferase (MGMT). Due to the diversity, heterogeneity and complexity of the molecular characteristics of GBM, the mechanism of resistance to TMZ treatment and the corresponding therapeutic strategy still need to be further explored.Inflammasome is a platform of multiprotein complex which can activate caspase-1.The activation of inflammasome make these cells to secrete and release mature IL-1β and IL-18 to the extracellular microenvironment, and then exert the corresponding biological function. The role of inflammasome in occurrence and development of tumor has been reported. Studies have shown that IL-1β can directly drive the occurrence or immune surveillance mechanisms of gastric cancer and breast cancer to promote tumor progression.Some studies have confirmed the facilitating roles of inflammasome in tumor angiogenesis of melanoma and lung cancer ,and its role in the invasion and metastasis of breast cancer,prostate cancer and non-small cell lung cancer have also been reported.As far as GBM , the current first-line chemotherapeutic agent, TMZ, through its alkylation, makes the DNA mismatch repair of tumor cells, leading to apoptosis of glioma cells. This condition is a kind of great stress for glioma cells, so it is possible to activate the inflammasome pathway in glioma cells. And then activation of downstream signaling pathway by the secreted IL-1β of these cells can exert a certain biological function.Combined with the results of our previous microarray, the expression of IL-1β in glioma stem cells is higher than correlative non stem cells, we speculate that the NLRP3 inflammasome-IL-1β pathway may be one of the mechanisms of the chemoresistance of TMZ treatment for glioma.The main contents of this part and the issues to be discussed include: (1) To investigate the effect of TMZ on the NLRP3 inflammasome-IL-1β pathway in glioma cell,and the role of this pathway on the survival of glioma cells when treated with TMZ. (2) To study the downstream pathway by which IL-1β give play to its biological function.Results: (1) TMZ was able to activate the NLRP3 inflammasome in glioma cells, and promote the secretion of IL-1β in these cells. (2) Blocking IL-1β improved the sensitivity of glioma cells to TMZ. (3) Blockade of IL-1β pathway decreased the expression of MGMT ,stemness and the expression of IL-6 in glioma cells treated with TMZ.Conclusion: This study illustrates the effect and mechanism of NLRP3 inflammasome-IL-1β pathway in TMZ resistance of glioma . Our findings highlight the significance of NLRP3 inflammasome - IL-1β pathway in glioma chemoresistance and provide the theoretical and experimental basis for glioma treatment strategies targeting this pathway.