Flavokawain B Regulates Autophagy,Senescence And Apoptosis In Glioblastoma Via ATF4/DDIT3/TRIB3/AKT/MTOR/RPS6KB1 Signaling Pathway

BackgroudGlioblastoma multiforme(GBM)is the most malignant primary human brain tumor.Tumors are characterized by a high proliferation rate and chemoresistance.Despite advances in combination treatments consisting of radiation and chemotherapy folloxwing surgical resection,the 5-year survival rate of WHO grade IV glioblastoma remains at less than 5%.Although significant advances have been made in our understanding of the molecular status of this tumor type,novel efficacious therapeutic avenues are critically neededAlthough cancers,such as GBM,may be intrinsically resistant to therapy,the ability to engage survival mechanisms in response to treatment potentially further reduces efficacy of any promising agent.Autophagy is one such conserved cellular pathway.The process removes dysfunctional or damaged organelles through lysosomal degradation and recycles the products for cellular metabolic needs.Autophagy is thus essential for maintaining homeostasis and has been shown to mediate resistance to anticancer therapies such as radiation,chemotherapy and some targeted therapies Increasing evidence supports that treatment with autophagy inhibitors such as Bafilomycin Al(BAF)and chloroquine(CQ)potentiates the effects of different cancer treatments.These studies have led to the initiation of multiple clinical trials combining chemotherapeutic agents and autophagy inhibitors for various cancer types.However,the role of autophagy in cancer is still controversial as it may suppress tumors during cancer development but promote cell survival during cancer progression.Thus,the specific role of autophagy seems to be highly cell type and context dependent.Natural products have received recent interest in the discovery of novel anti-cancer therapeutic agents as they have long been used as alternative remedies for a variety of diseases,including cancer,with relatively few side effects.Flavokawain B(FKB),a natural kava chalcone,has displayed anticancer activity in various types of cancer,such as osteosarcoma,lung cancer,leiomyosarcoma,and prostate cancer.The cancer specific cytotoxic activity of FKB has been mainly attributed to induction of cell cycle arrest and apoptosis characterized by the generation of intracellular reactive oxygen species(ROS)and the upregulation of BIM(BCL2L11),a proapoptotic molecule.The role that FKB plays in cell death in GBM cells and whether it induces autophagy remain largely unclear.Here,we investigated the chemotherapeutic potential of FKB in human GBM cell populations in vitro and in vivo.While we report that FKB does inhibit GBM cell growth largely through the processes of senescence and autophagy,we were able to promote apoptosis by combining treatment with autophagy inhibitors.These results support the strategy of combination therapy of FKB and autophagy inhibitors in the treatment of human GBM.Part I Flavokawain B inhibits proliferation of glioblastoma cells in vitro by inducing senescenceObjectiveTo explore the effect and mechanism of flavokawain B on the proliferation of GBM cells.Methods1.The effect of flavokawain B on the activity of GBM cell lines U251,U87 and T98 and primary GBM P3 cells was investigated by Cell Counting Kit-8(CCK-8).2.The effect of flavokawain B on the proliferation of GBM cells was investigated by using the EdU kit.3.The effect of flavokawain B on apoptosis of GBM cells was investigated by Annexin V/PI staining.4.Using PI staining to investigate the effect of flavokawain B on the cell cycle of GBM.5.Immunofluorescence staining of yH2AFX was used to investigate the effect of flavokawain B on DNA damage in GBM cells.6.To investigate the effect of flavokawain B on the senescence of GBM cells using SA-GLB1 staining.Results1.Flavokawain B significantly inhibits proliferation of GBM cells in vitro.1.1 To begin to determine whether flavokawain B might be effective against GBM,flavokawain B treatment was first evaluated in U25 1,U87,T98,and P3 cells in vitro,using the cell viability assay CCK-8.Cells were treated with differing concentrations of flavokawain B in vitro,and viability was assessed at 12,24,and 48 h.Decreases in cell viability(?50%)relative to untreated cells were statistically significant at 48 h in 3 ?g/mL flavokawain B for all cell lines.1.2 Quantification of EdU incorporation also revealed a statistically significant decrease in proliferation for U251,U87,and T98 cell lines after exposure to flavokawain B at 3 ?g/mL for 48 h(?45%vs?15%,untreated vs treated cells).These results indicated that flavokawain B potently arrested proliferation in GBM cells and in a dose-dependent manner.2.Flavokawain B mediates G2/M phase cell cycle arrest in GBM cells.PI staining showed that G2/M phase arrest was observed in the cell cycle of GBM cells after treatment with 3 ?g/ml flavokawain B for 48 h.3.Flavokawain B mediates DNA damage in GBM cells.,yH2AFX fluorescence staining showed that 7H2AFX nuclear positive cells in GBM cells increased significantly after treatment with 3 ?g/ml flavokawain B for 48 h.4.Flavokawain B induces senescence in GBM cells.The results of SA-GLB1 staining showed that flavokawain B increased the SA-GLB1 GBM positive cells in a time-and dose-dependent manner.ConclusionFlavokawain B inhibits proliferation of GBM cells by inducing senescence.Part II Flavokawain B induces protective autophagy via ATF4/DDIT3/TRIB3/AKT/MTOR/RPS6KB1 signaling pathway in glioblastoma cellsObjectiveTo investigate the effect and molecular mechanism of flavokawain B on autophagic flux in GBM cells.Methods1.The number of autophagosomes in the typical bilayer membrane structure of GBM cells treated with flavokawain B was observed by transmission electron microscopy(TEM).2.Western blot was used to investigate the expression of autophagy-related genes SQSTM1,MAP1LC3B,ATG5 and ATG7 in GBM cells by different concentrations of flavokawain B and different treatment time.3.After GFP-MAP1LC3B plasmid was transfected into GBM cells,the fluorescence treatment was used to observe the changes of GFP fluorescence spots in GBM cells treated with different concentrations of flavokawain B and different treatment time.4.The effect of flavokawain B on autophagic flux of GBM cells was investigated by using autophagy inhibitor 3-MA/chloroquine(CQ)and knockdown autophagy-related gene ATG5/ATG7.5.Western blot was used to investigate the effects of different concentrations of flavokawain B and different treatment time on ER-stress related genes HSPA5,EIF2A,P-EIF2A,p-EIF2AK3?ATF4 and DDIT3 in GBM cells.6.After knocking down the ER-stress related genes ATF4 and DDIT3,the effect of flavokawain B on autophagic flow of GBM cells was detected by Western blot.7.Western blot was used to investigate the effects of different concentrations of flavokawain B and different treatment time on the expression of classical MTOR pathway-related genes AKT,p-AKT,MTOR,p-MTOR,RPS6KB1 and p-RPS6KB1 in autophagy of GBM cells.8.Western blot was used to investigate the effects of different concentrations of flavokawain B and different treatment time on TRIB3 in GBM cell lines.The effect of flavokawain B on autophagy and autophagy classical pathway was detected by small interference knockdown of TRIB3.9.Using the autophagy inhibitor 3-MA/chloroquine(CQ)and knockdown autophagy-related gene ATG5/ATG7 to investigate the effect of flavokawain B on senescence and apoptosis of GBM cells to determine flavokawain B-mediated whether it is autophagic death or protective autophagyResults1.Flavokawain B mediates autophagy in GBM cells.1.1 TEM results showed that after treatment of GBM cells with flavokawain B,the number of autophagosomes increased significantly.1.2 Western blot results showed that the expression of MAP1LC3BII increased and the expression of SQSTM1 decreased with the flavokawain B treatment in a dose-and time-dependent manner in GBM cells1.3 Under fluorescence microscopy,we found that GFP-MAPILC3B fluorescence dots increased after flavokawain B treatment of GBM cells1.4 Western blot and fluorescence microscopy showed that autophagy inhibitors 3-MA/CQ and knockdown autophagy-related genes ATG5/ATG7 inhibited flavokawain B-mediated autophagy in GBM cells.2.Flavokawain B induces autophagy via ATF4/DDIT3/TRIB3/AKT/MTOR/RPS6KB1 signaling pathway in GBM cells.2.1 Western blot results showed that the expression levels of ER-stress related genes HSPA5,EIF2A,p-EIF2A,p-EIF2AK3,ATF4 and DDIT3 increased with the flavokawain B treatment in a dose-and time-dependent manner in GBM cells2.2 Knockdown of the ER-stree-related genes ATF4 and DDIT3 or the application of the ER-stress inhibitor 4-PBA inhibited flavokawain B-mediated autophagy in GBM cells2.3 Western blot results further showed that the autophagy classical AKT/MTOR/RPS6KBB1 signaling pathway was inhibited with the flavokawain B treatment in a dose-and time-dependent manner in GBM cells.As a downstream molecule of ATF4/DDIT3 and upstream of AKT/MTOR/RPS6KB1,TRIB3 expression increased after treatment of GBM cells with flavokawain B in a dose-dependent manner,and decreased TRIB3 expression after knockdown of ATF4,and AKT/MTOR/RPS6KB1 signaling pathway is activated after TRIB3 knockdown.3.Flavokawain B induces protective autophagy in GBM cells.3.1 The results of CCK-8 and EdU showed that the activity and proliferation of GBM cells were further decreased after flavokawain B was combined with autophagy inhibitor 3-MA/CQ or knockdown autophagy-related gene ATG5/ATG7.3.2 SA-GLB1 staining showed that flavokawain B no longer mediates GBM cell senescence after combined use of autophagy inhibitor 3-MA/CQ or knockdown of autophagy-related gene ATG5/ATG7.3.3 Annexin V/P1 staining showed that after the combination of autophagy inhibitor 3-MA/CQ or knockdown autophagy-related gene ATG5/ATG7,the apoptotic cells of GBM were significantly increased after flavokawain B treatment.3.4 Western blot analysis showed that the expression of apoptotic markers Cleaved CASP3 and Cleaved PARP1 was significantly increased after flavokawain B combined with autophagy inhibitor 3-MA/CQ or knockdown autophagy related gene ATG5/ATG7 in GBM cells.ConclusionFlavokawain B induces protective autophagy in GBM cells via the ATF4/DDIT3/TRIB3/AKT/MTOR/RPS6KB1 signaling pathway.In combination with the autophagy inhibitor 3-MA/CQ or knockdown of the autophagy-related gene ATG5/ATG7,flavokawain B no longer mediates GBM cell senescence,but directly mediates GBM cell apoptosis.Part ? Flavokawain B inhibits glioblastoma growth in vivoObjectiveTo explore the effect of flavokawain B on the growth of GBM cells in vivoMethods1.The luciferase-stable U251 cells were implanted into the brain of 4-week old nude mice by stereotaxic apparatus,and PBS,flavokawain B(50 mg/kg/day),CQ(25 mg/kg/Day)or flavokawain B(50 mg/kg/day)+ CQ(25 mg/kg/day)were injected intraperitoneally every other day from day three.The intracranial tumor growth of nude mice was monitored weekly using a small animal imager.2.We firstly established the ATG5 stable knockdown U251 cell line and implanted sh-ATG5 or sh-NC luciferase-stable U251 cells into the brain of 4-week old nude mice with stereotactic instrument,and injected PBS or flavokawain B(50 mg/kg/day)into the abdominal cavity every other day from the third day.The intracranial tumor growth of nude mice was monitored weekly using a small animal imager3.We monitored the weight of nude mice daily,anesthetized the nude mice when the weight loss is ? 20%,and took the brain out after infusion of 4%paraformaldehyde The brain was made into frozen sections and paraffin sections.4.SA-GLB1 staining was used to investigate the effect of flavokawain B on senescence in GBM cells.5.TUNEL staining was used to investigate the effect of flavokawain B on apoptosis in GBM cells.6.Immunohistochemical Ki67 and MAP1LC3B staining were used to investigate the effects of flavokawain B on proliferation and autophagy in GBM cellsResults1.Flavokawain B inhibits the growth of GBM cells in vivo and prolongs the survival of tumor-bearing nude mice.1.1 Small animal imaging results showed that the volume of intracranial tumors in the flavokawain B group was lower than that in the control group.1.2 The survival curve of nude mice showed that the survival time of the flavokawain B group was longer than that of the control group1 3 The results of immunohistochemistry showed that the Ki67-positive cells of the flavokawain B group were decreased compared with the control group.2.Flavokawain B combined with autophagy inhibitor CQ or ATG5 knockdown can more effectively inhibit the growth of GBM cells in vivo and prolong the survival of tumor-bearing nude mice.2.1 Small animal imaging results showed that the intracranial tumor volume of the nude mice in the combined autophagy inhibitor CQ or ATG5 knockdown group was significantly reduced compared with the flavokawain B group.2.2 The survival curve of nude mice showed that the survival time of the combined autophagy inhibitor CQ or ATG5 knockdown group was longer than that of the flavokawain B group.2.3 Immunohistochemistry results showed that the Ki67-positive cells in the combined autophagy inhibitor CQ or ATG5 knockdown group were significantly reduced compared with the flavokawain B group.2.4 TUNEL results showed that the TUNEL-positive cells in the combined autophagy inhibitor CQ or ATG5 knockdown group were significantly increased compared with the flavokawain B group.ConclusionFlavokawain B inhibits growth of GBM cells in vivo.Combined with autophagy inhibitor CQ or ATG5 knockdown,flavokawain B can no longer mediate senescence but directly mediate apoptosis,thereby significantly enhancing the anti-tumor effect of flavokawain B.