| Background:Glioblastoma multiforme is regarded as the most malignant primary brain tumor in the CNS(central nervous system).Due to the characteristics of the invasive growth mode and often adjacent to important brain functional areas or nerve fiber bundles,it is difficult to resect tumors completely and postoperative tumor recurrence is inevitable.The current standard therapy for primary glioblastoma is a comprehensive treatment plan of maximal surgical resection + postoperative radiotherapy and chemotherapy.Even so,the prognosis of patients with glioblastoma multiforme is still unsatisfactory,and the median survival time after diagnosis of the disease is limited to only 14.6-16.7months.The value of chemotherapy in the treatment of glioblastoma multiforme has been confirmed by multiple clinical studies.However,due to the existence of the blood-brain barrier,many chemotherapeutic agents commonly used in clinical practice cannot achieve effective concentrations in brain.At present,the first-line chemotherapeutic agent for the treatment of glioblastoma multiforme is temozolomide(TMZ).Temozolomide can effectively prolong the median survival time of patients by about 2months.However,it has been found in clinical treatment that the sensitivity of glioblastoma multiforme to temozolomide decreases with time,which may be related to a variety of inherent or acquired chemotherapy resistance mechanisms in tumor cells.Recent years,with the deepening understanding of the structure and function of mitochondria,we come to realize that mitochondria may be involved in the process of chemo-resistance of tumor cells in various forms.This also provides us with the opportunity to overcome the chemotherapeutic resistance of glioblastoma multiforme in a new field.At the same time,the phenomenon of multiple chemotherapy resistance in tumor chemo-treatment suggests that there may exist some common and core I mechanisms in the process of chemo-resistance.Considering the core role of energy metabolism in cells,we are trying to figure out the changes of energy metabolism mode induced by chemotherapy and the mechanism behind in order to find new ways to sensitize the glioblastoma multiforme to chemotherapy.When exposed to internal or external environmental stresses,tumor cells will deploy cell energy metabolism in a variety of ways to adapt to different stimulies.Mitochondrial dynamics is an important player in the process of energy metabolism reprogramming.Mitochondrial dynamics includes processes such as mitochondrial fission and fusion,mitochondrial transport and mitophagy.Among them,mitochondrial fission and fusion and mitophagy are important cellular events that participate in the quality and quantity control of the mitochondrial system.Mitochondrial fission is mediated by GTPase dynein Drp1 and its receptor Mff,Mid49/51,etc;mitochondrial fusion is mediated by Mfn1/2,an important molecule involved in mitochondrial outer membrane fusion and Opa1 related to inner membrane fusion.Mitochondrial fission and fusion can achieve structural and functional complementarity between damaged mitochondria,and can discard severely damaged mitochondria from the mitochondrial system to optimize mitochondrial function and control the production of harmful metabolites.When demonstrated at the organelle level,it shows changes in mitochondrial morphology.Mitophagy can be carried out by multiple pathways,and the PINK1-Parkin pathway is the dominant one.In the process of mitophagy,the damaged mitochondria fuse with lysosomes,and be degraded.Mitophagy regulates the mass of mitochondria directly and accurately,helping to maintain the healthy functioning of the mitochondrial system.There have been many research reports on the changes of mitochondrial dynamics induced by chemotherapeutic agents,but the mechanism is still inconclusive.As to the changes in the quality and quantity of mitochondria will have an important impact on metabolism,it may provide us new options to overcome cancers.What role these metabolic changes play in tumor cells in response to chemotherapy agents,and whether they can provide new strategies for tumor chemotherapeutic sensitization? These inconclusive questions constitute the main theme of this study.That is,to explore the changes in the mitochondrial dynamics of glioblastoma cells under the treatment of temozolomide,the underlying mechanisms and its impact on tumor cell metabolism.Besides,we tried to intervene the process of mitochondrial dynamics to sensitize glioblastoma multiforme to temozolomide.Methods:1.Lactic acid,glucose,ATP and ROS detection kits and oxygen consumption rate(OCR)measurement kit were used to evaluate the energy metabolism state of SHG44,U87 cells and allogeneic tumor tissues under different treatment conditions.2.Western blotting(WB)and Immunofluorescence was used as a semi-quantitative method to study of the expression levels of multiple targeted proteins in tumor cells under basic and different treatments3.The qPCR was used to evaluate the expression of targeted genes on transcription level under different treatments;total cell DNA was extracted and qPCR was also used to evaluate the mitochondrial DNA copies and the level of mitochondrial DNA damage under the treatment of temozolomide.4.Marking mitochondria with specific mitochondrial fluorescent probes to evaluate the number of mitochondria and the level of co-localization between mitochondria and various targeted proteins.In addition,the fluorescently-labeled mitochondria were photographed by a microscope and then images were processed with Image J to achieve quantitative evaluation of mitochondrial morphology.5.In order to study the regulatory effect of P53 protein,over expression of TP53 was carried out by transfecting tumor cells with plasmid.6.Immunofluorescence co-localization assay was conducted to analyze the subcellular localization of the targeted proteins.7.Mitochondria and nucleus extraction and separation kits were used to extract cell substructures under different treatments,and semi-quantitatively study of the targeted proteins was conducted by WB.8.The MTT assay was used to evaluate the viability of tumor cells under different treatments.9.Flow cytometry was used to detect the cellular apoptosis levels under different treatments.10.Clone formation assay was conducted to evaluate the inhibitory effect of temozolomide and(or)WY14643 on SHG44 and U87 cells in vitro.11.Establishing an allogeneic subcutaneous tumor-bearing model in nude mice with U87 cell to verify the inhibitory effect of temozolomide and(or)WY14643 on tumor growth in vivo.12.Transmission electron microscopy was used to detect the changes in mitochondrial morphology of allogeneic tumor tissues in different treatment groups.13.Detecting the expression levels of TP53 and PINK1 in vivo under the treatment of temozolomide by immunohistochemistry.Results:1.Under the treatment of temozolomide,the glioblastoma cell lines SHG44 and U87 showed obvious changes in the energy metabolic pattern,manifested by increased oxygen consumption rate,elevated glucose uptake and consumption,increased intracellular ATP level,which indicated the increased oxidative phosphorylation level;what's more,the ROS level in SHG44 and U87 cells was not elevated significantly.2.In the evaluation of the mass of mitochondria under temozolomide treatment,it was found that the mass of mitochondria of cell lines SHG44 and U87 increased significantely when compared to control group.And in the process of figuring out the mechanism behind,it was found that the expression level of PINK1 in SHG44 and U87 cells was down-regulated,and the expression level of phosphorylated ubiquitin,the phosphorylated substrate of PINK1,was also down-regulated,and the recruitment of Parkin to mitochondria was impired.What's more,the fusion between mitochondria and lysosomes was impired.The treatment of temozolomide down-regulated the flux of mitophagy in SHG44 and U87 cells and it may be the mechanism of the accumulation of mitochondria.3.Temozolomide induced nuclear DNA damage in glioblastoma cell lines SHG44 and U87 and at the same time mitochondrial DNA damage was found in SHG44 cells,which was accompanied by significant high expression of TP53;further the bioinformatics study found that the expression of TP53 is negatively correlated with the expression of PINK1,and the same result was confirmed in the IHC evaluation of GBM tissues.4.Further study on the expression of PINK1 regulated by TP53 found that the elevated expression of wild-type TP53 caused by DNA damage can down-regulate the expression level of PINK1,thereby down-regulating the flux of mitophagy;while in cells overexpressing TP53,the down-regulation of PINK1 expression was also observed;in the TP53 mutant cell line U251,temozolomide induced the upregulation of mutant TP53 and PINK1 expression;under low-dose temozolomide treatment conditions that can induce DNA damage without elevated expression of TP53,the expression of PINK1 was not down-regulated.These results confirmed that the wild-type P53 protein regulated the expression of PINK1 negatively.5.Temozolomide treatment resulted in the elevated level of mitochondrial fusion in SHG44 and U87 cells and further study revealed that the translocation of Drp1 to mitochondria was impired in SHG44 and U87 cells.6.In the process of revealing the mechanism behind the Drp1 translocation,it found that temozolomide treatment decreased the ATP levels in tumor cells and the activated AMPK in a short time.In addition,temozolomide treatment elevated the threshold of AMPK energy sensing system to sense ATP fluctuation.And the activated AMPK promoted the up-regulation of P53 protein levels in the nucleus on the one hand,and on the other hand also promoted the occurrence of mitochondrial fusion by impiring the translocation of Drp1 to mitochondria.7.There was difference in the subcellular distribution of Drp1 molecules in SHG44 and U87 cells.Drp1 is diffusely distributed in SHG44 cells while concentrated in the nucleus of U87 cells.8.In in vitro experiments,the combined use of mitochondrial fission inducer WY14643 and temozolomide shifted the temozolomide-tumor viability curve downward and increased the sensitivity of tumor cells to temozolomide.The use of mitochondrial fission inhibitor Mdivi1 in combination with temozolomide yielded the opposite results.What's more,the use of WY14643 offseted the mitochondrial fusion and up-regulation of ATP synthesis in SHG44 and U87 cells induced by temozolomide to a certain extent;and the ROS level was elevated compared to temozolomide treatment group.The use of WY14643 on the basis of temozolomide treatment enhanced the effectiveness of temozolomide in inducing tumor cell apoptosis,and inhibited the ability of tumor cell clone formation.9.In the study of GBM tumor xenograft nude mice model,it was found that the combination of WY14643 and temozolomide significantly inhibited tumor growth.Detection of the expression of apoptosis-related proteins in tumor tissues revealed that the expression level of apoptosis-related proteins was higher in the two-agents combined treatment group.The detection of mitochondrial morphology by transmission electron microscopy found that WY14643 offseted the mitochondrial fusion effect induced by temozolomide treatment,and ATP assay of tumor tissues revealed that the tumor tissue of WY14643+ temozolomide group was with the decreased ATP level when compared with temozolomide group.Conclusion:1.The DNA damage induced by temozolomide treatment in SHG44 and U87 cells and the reactive high expression of TP53 impaired the recruitment of Parkin molecules to mitochondria by down-regulating the expression level of PINK1,and down-regulated the flux of mitophagy,leading to the accumulation of mitochondrial mass in tumor cells.2.The imbalance of intracellular energy supply and demand caused by temozolomide treatment in SHG44 and U87 cells leaded to the activation of AMPK,and the activated AMPK promoted the elevated expression of TP53 in the nucleus which further promoted the down-regulation of mitophagy flux.What's more,the activated AMPK also promoted the occurrence of mitochondrial fusion by reducing the translocation of Drp1 to mitochondria,so as to optimize the quality of the mitochondrial system.3.Interfering with the mitochondrial fusion process increased the level of ROS in tumor cells under temozolomide treatment,reduced the level of intra-cellular ATP,and increase the sensitivity of glioblastoma to temozolomide.4.The accumulation and quality optimization of mitochondria in SHG44 and U87 cells under temozolomide chemotherapy stress is an important mechanism behind the evaleted level of oxidative phosphorylation,which is involved in the chemotherapy resistance process of tumor cells and blocking this compensatory response can enhance chemotherapy efficacy. |
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