Trifluoperazine,A Novel Autophagy Inhibitor, Increases Radiosensitivity In Glioblastoma By Impairing Homologous Recombination

BackgroundGlioblastoma(GBM)is the most aggressive of all intracranial tumors.Despite multimodal treatment including surgical resection,chemotherapy and radiotherapy,the median survival is only 14.6 months.Radiotherapy targets cancer cells by causing DNA damage and is a highly cost-effective treatment.However,DNA damage induced by radiation triggers a series of signaling cascades promoting cell survival,including DNA repair,cell cycle arrest,and autophagy,all of which mediate radioresistance and prevent further clinical efficacy.Previous studies have shown that fractionated radiotherapy and intensity modulated radiation therapy dose do not improve the survival of patients with glioblastoma.While the presence of blood-brain barrier(BBB)prevents traditional radiotherapeutic sensitizers,such as carboplatin,from penetrating the brain.Autophagy is a highly conserved process produced by eukaryotic cells to adapt to the environment.In this process,damaged proteins and disordered organelles are first wrapped in a double-layer membrane structure,which is called autophagosome.Then autophagosome is fused with lysosome and is degraded by lysosomal proteases,to pro-vide raw materials and energy for the synthesis of macromolecules.Autophagy is involved in various cellular physiological and pathological processes,such as adaptation to hunger,resistance to aging,presentation of antigens,maintenance of the stability of genetic information.And the dysfunction of autophagy will lead to neurodegenerative diseases,cardiovascular diseases,tumors and other diseases.Recently,an increasing number of studies found that autophagy greatly contributes to the radioresistance of tumor cells.Basic studies showed that radiotherapy significantly increased autophagy,while combining autophagy inhibitors increase radiosensitivity.Radiation-induced autophagy is recognized as a protective effect of tumor cells.And autophagy inhibitors are believed to be promising reagents to enhance the anti-tumor effect of radiotherapy.However,due to the blood-brain-barrier(BBB),most autophagy inhibitors will not effectively benefit GBM patients.Therefore,identifying new autophagy inhibitors with improve pharmacokinetics for diseases in the central nervous system(CNS)is urgently needed.Trifluoperazine(TFP)is a typical antipsychotic compound of the phenothiazine chemical class.It has been used in the treatment of schizophrenia for more than 50 years and relieves agitation in patients with behavioral problems,severe nausea and vomiting as well as severe anxiety.Recently,an increasing number of studies have found that TFP has potent anti-tumor effects in lung cancer,malignant peripheral nerve sheath tumors and leukemia.In our study,we found that TFP inhibited the autophagy in GBM cells.Here,modern techniques in molecular and cellular biology were used to explore the role of TFP on the autophagy and radiosensitivity of GBM cells.An intracranial xenograft model was established to test the radiosensitive effect of TFP in vivo.And underlying mechanisms will be explored.Part ? TFP decreased GBM cell viability and inhibited cell proliferationObjectiveTo investigate the effect of TFP on the cell viability and cell proliferation of GBM cells.Methods1.Cell-Counting-Kit8(CCK8)was used to test the cytotoxic effects of different doses of TFP on GBM cell lines,U87,U251,primary tumor cells,P3 and human normal astrocytes(NHA).2.EdU assay was used to test the effect of TFP on cell proliferation.Results1.TFP decreased cell viability of GBM cellsThe results of CCK-8 assay showed that the IC50 values of TFP for U251,U87 and P3 cells were 16 ?M,15 ?M,15.5 ?M,respectively.And GBM cells were significantly more sensitive to TFP compared to normal human astrocytes(IC50 22.5?M,P<0.05).2.TFP significantly decreased cell proliferation of GBM cellsThe results of EdU assay showed that TFP decreased the cell proliferation of GBM cells in a dose-dependent manner.Conclusion1.TFP decreased cell viability of GBM cells;2.TFP decreased cell proliferation of GBM cells.Part ? TFP interferes with autophagy flux in GBM cellsObjectiveTo explore the role of TFP on autophagy in GBM cellsMethods1.Western blot analysis was performed to examine the protein level of LC3B of GBM cell lines U251 and U87 after treatment of different doses of TFP.2.GBM cells were transiently transfected with a GFP-LC3 expression construct and treated with or without TFP.24 h later,Confocal microscopy was used to count the green dots from two different groups.3.Transmission electron microscopy(TEM),the gold standard for detecting autophagic vacuoles(AVs)was used to count the number of AVs in TFP treated GBM cells compared to the control group.4.Western blot analysis was performed to examine the protein level of P62 of GBM cell lines U251 and U87 treated with different doses of TFP,autophagy inhibitor Bafilomycin A1(BAF),and autophagy inducer,Rapamycin.5.Fluorescence microscopy was used to detect red fluorescence intensity of LysoTracker Red in GBM cells treated with TFP and BAF.Results1.TFP increased the protein level of LC3B-?,the number of GFP-LC3B dots and AVsWestern blot analysis showed that after treatment of TFP,the protein level of LC3B-? increased in GBM cell lines U251 and U87,which is in a dose-dependent manner.GBM cell lines U251 and U87 were transiently transfected with a GFP-LC3 expression construct and confocal microscopy showed an increase in fluorescence puncta in TFP treated cells after 24 h.Transmission electron microscopy(TEM)represents the gold standard for detecting autophagic vacuoles(AVs).We found that More AVs were detected in TFP treated cells compared to the control group.2.TFP increased the protein level of P62,and decreased the fluorescence intensity of LysoTracker Red in GBM cellsAn accumulation of AVs in cells can occur either as a consequence of an increased autophagosome formation or decreased degradation.The levels of p62,one of the most important long-lived proteins critical for autophagy,should be degraded in autophagy.And its accumulation indicated inhibiting of autophagic flux.Western blot analysis showed that the level of p62 accumulated in a dose-dependent manner in response to TFP in GBM cell lines U251 and U87.With the help of rapamycin,which induces autophagy,and Bafilomycin A1(BAF),which blocks autophagy,we found that p62 levels decreased in rapamycin treated cells whereas it was clearly increased in cells treated with TFP or BAF.Previous studies showed that BAF blocks autophagic flux by reducing the acidification of lysosomes.Altered lysosome function prevents fusion of autophagosomes with lysosomes.Then we used LysoTracker Red,which labels highly acidic lysosomal vacuoles,to determine the status of lysosomes after TFP or BAF treatment.Under fluorescence microscopy,lysosomes could not be detected with LysoTracker Red in TFP and BAF treated cellsConclusionTFP blocks autophagy by inhibiting the acidification of lysosomes.Part ? The Role and Mechanism of TFP in the radiosensitivity of GBM cell lines.ObjectiveTo explore the role and mechanism of TFP in the radiosensitivity of GBM cell lines.Methods1.Comet assay,western blot analysis of protein level of ?-H2AX were uses to detect the level of DNA damage in four treatment groups:control group,TFP treatment group,radiation treatment group,TFP combined with radiation treatment group.2.We next quantified ?-H2AX foci at 2,6,12,24 h after radiation treatment or TFP combined with radiation using immunofluorescence.3.Western blot analysis was used to detect the protein levels of Rad51?BRCA1?BRCA2 in GBM cells treated with different doses of TFP.4.Using a PCR-based HR assay kit,Homologous recombination(HR)efficiency was calculated in GBM cell lines U251 and U87 treated with or without TFP.5.GBM cell lines U251 and U87 were divided into four groups:control group,TFP treatment group,radiation treatment group,TFP combined with radiation treatment group.Then colony formation assay and Caspase3/7 activity were tested.6.The enzymatic activities of two main cathepsins(B and L),in response to TFP was tested with a fluorometric-based kit.7.Western blot analysis was performed to test the protein level of Cathepsin B and L in GBM cell lines U251 and U87 treated with different doses of TFP.8.Western blot analysis was performed to test the protein level of Cathepsin L?P62?Rad51 and ?-H2AX in GBM cell lines treated with siRNA of Cathepsin L.9.HR efficiency was calculated in GBM cell lines U251 and U87 treated with or without siRNA of Cathepsin L.10.We established orthotopic xenograft models with U251 and P3 cells and animals were devided into different treatment groups:control group(treated with PBS),TFP treatment group,radiation treatment group,TFP combined with radiation treatment group.Then survival was calculated from each group.And immunohistochemistry was done with tumors tissues from each group.Results1.TFP increased radiation-induced DNA damagePrevious studies have demonstrated that inhibitors of autophagy enhance the sensitivity of cells to radiation therapy.To investigate whether TFP affected radiosensitivity,we used the comet assay to measure DNA integrity in U251 and U87 cells.TFP or radiation treatment alone slightly increased the levels of DNA tails above controls,whereas combined treatment caused a significant increase in the appearance of DNA tails.Protein levels of ?-H2AX,a gold standard to detect the presence of DSBs,were correspondingly increased following combination treatment2.TFP decreased DNA damage repairAfter treatment of TFP,we found that the expression of Rad51 and the associated DNA repair proteins BRCA1 and BRCA2,decreased,while ?-H2AX increased in a dose-dependent manner.In addition,HR efficiency was calculated using a PCR-based HR assay kit.We found that after TFP treatment,the HR efficiency decreased significantly(P<0.05)compared to the control group.3.TFP decreased the proliferation of GBM cells and increased apoptosis after radiationWe also found that TFP combining with radiation reduced clonogenic ability compared with other treatments.And caspase activity increased the most in combined treatment group.4.TFP decreased activity of lysosomal proteases in GBM cellsWith a fluorometric-based kit,we found that the enzymatic activities of two main cathepsins(B and L)decreased significantly after TFP treatment.Western blot analysis also showed that the protein levels of cathepsin B and L decreased after TFP treatment.5.Knocking down Cathepsin L decreased DNA damage repair and inhibited autophagyWestern blot analysis showed that the protein levels of Rad51 decreased after knocking down Cathepsin L with siRNA,while DNA damage marker,?-H2AX and autophagy-related protein P62,increased.And we also found that HR efficiency decreased after knocking down Cathepsin L.6.TFP increased radiosensitivity of GBM cells in vivoAnimal experiments showed that combining treatment increased survival compared with other treatment group.And the results of immunohistochemistry demonstrated that combined treatment decreased cell proliferation,DNA damage repair and increased DNA damage.Conclusion1.TFP could increase the radiosensitivity in vitro and in vivo.2.TFP decreased the protein level of Cathepsin L,so as to impair HR efficiency.