Anti-Glioma Effect And Molecular Mechanism Of Resibufogenin

BackgroundGlioblastoma multiforme(GBM,WHO IV)accounts for nearly 51.4%of all kinds of brain cancer and remains the leading cause of cancer-related death due to intracranial malignant disease.Currently,the standard of care for newly diagnosed patients is maximum safe resection followed by adjuvant ionizing radiotherapy and temozolomide chemotherapy.However,median survival has remained stagnant over the last decade,remaining at 14.6 months.Developing novel anticancer strategies in addition to chemotherapy,radiation,and immunotherapy will be critical for patients with glioma.Therefore,low-toxicity,effective drugs to treat glioma and prolong the survival of patients are urgently needed.Natural ingredient extracts have historically been an important resource for potential cancer therapies.Chansu,a dried secretion of Bufo gargarizans Cantor or B.melanostictus Schneider,is a widely used traditional Chinese medicine that exhibits a variety of pharmacological activities,including anti-inflammatory,detoxifying,analgesic,stimulatory,and anticancer properties.Chansu was reported to significantly inhibit the malignant progression of glioma,lung cancer and breast cancer.Resibufogenin,alternatively known as bufogenin and recibufogenin,is a major active ingredient in cinobufacini.RB has been commonly used for the treatment of a variety of tumors in recent years.For instance,RB was demonstrated to inhibit TAK1-mediated NF-κB activity through protein kinase C-dependent repression of GSK-3 in pancreatic cancer.Moreover,RB inhibited the malignant progression of ovarian clear cell carcinoma by suppressing the PI3K/AKT and actin cytoskeleton signaling pathways.Nevertheless,the effect and the molecular mechanisms of RB on glioma are currently unclear.The dysregulated cell cycle of glioma cells is the main cause of their malignant proliferation and is a classic therapeutic target for oncology drug development.The cell G2/M phase transition is mediated by the activation of the CDK1-Cyclin B complex.Activation of CDK1(cyclin-dependent kinase 1)is mediated by dephosphorylation of CDK1 at Tyr15 by CDC25C(cell division cycle 25).Additionally,the elevated expression of p21 can downregulate the expression of CDK1-Cyclin B complex to induce G2/M phase arrest.It has been demonstrated that signaling mediated by mitogen-activated protein kinases(MAPKs)plays an important role in regulating the cell cycle and responding to DNA damage.MAPKs are classified into three subfamilies based on their sequence similarity and the nature of their upstream activators:JNK/SAPK(c-Jun N-terminal kinase/stress-activated protein kinase),ERK1/2 and p38.Extracellular signal-regulated kinase(ERK1/2)is a major transmitter of extracellular signals that connect cellular membrane receptor stimulation to alterations in cellular function.The transient activation of ERK is critical in cell proliferation,whereas continuous ERK activation triggers cell G2/M phase arrest by promoting CDC25C ubiquitination and proteasomal degradation and by upregulating p21 expression.Additionally,the newly developed clinical drug Ibrance,the first CDK4/6 inhibitor approved by the FDA in 2015,targets the cell cycle and may be effective and safe against certain types of breast cancer.In addition to the uncontrollable proliferation of glioma,invasion is another distinctive feature of its malignant phenotype.The focal adhesion complex composed of integrins serves as the primary site of cell adhesion to the extracellular matrix and interacts with the actin cytoskeleton to regulate cell motility.The dynamic regulation of focal adhesion and the associated actin cytoskeleton reorganization are critical factors for cell invasion.Focal adhesion kinase(FAK)was discovered to associate with SRC via a SRC homology domain-mediated interaction after it was initially identified as a tyrosine phosphorylated protein in cells transformed by the SRC oncogene.The FAK-SRC complex interacts with and phosphorylates several adaptor proteins,including P130cas and Paxillin,to promote tumor invasion.Furthermore,the ever-present second messenger Ca2+has been identified as a vital regulator of cancer cell invasion by regulating the dynamic cycle of focal adhesion complex assembly and disassembly.Exploring RB’s target proteins in glioma cells will help us better understand RB’s anticancer mechanisms and generate new treatment options for glioma.Chansu has been reported to inhibit Na+-K+-ATPase.Na+-K+-ATPase is a classic ion transporter enzyme that exchanges Na+for K+in cells.ATP1A1(Na+-K+-ATPase α1)is one of 4 isoforms of the Na+K+-ATPase alpha subunit identified in humans.When Na+-K+-ATPase is only partially inhibited,there is no significant disruption of the homeostasis of intracellular Na+and K+.Depending on the cell after activation by the appropriate ligand,Na+-K+-ATPase stimulates the proliferation of healthy cells or,in contrast,inhibits the proliferation of tumor cells.Moreover,ATP1A1 can activate the MAPK/ERK pathway,and Na+-K+-ATPase can alter intracellular Ca2+concentrations.Therefore,exploring the mechanisms of RB in glioma will identify new clinical agents for the targeted treatment of glioma.Taken together,our study shows that upon RB binds to ATP1A1,Na+-K+-ATPase is activated as a receptor,accompanied by activation of the intracellular MAPK/ERK pathway and inhibition of the Ca2+-regulated SRC/FAK/Paxillin focal adhesion pathway,after which GBM cells undergo G2/M phase arrest and inhibition of invasion.ObjectivesTo explore the effect and mechanism of resibufogenin on proliferation and invasion of glioblastoma.Methods(1)IC50 assay was used to detect the toxicity of RB on primary GBM cells,P3#GBM,GBM cell lines,U251 and A172,and human astrocytes.The effects of RB on GBM cell proliferation were evaluated in vitro by CCK-8,EdU,cell cloning,cell cycle assay,etc.,and the effects of RB on GBM cell invasion were evaluated by transwell,3D-spheroid invasion and GBM-brain organoid coculture invasion assay.Western blot was used to detect the effects of RB on GBM cell proliferation,cell cycle distributions and invasion.(2)Whole transcriptome sequencing was performed on RB-treated P3#GBM cells(4μM RB)and U251 cells(4 μM RB)and their parent cell lines.KEGG enrichment analysis was performed on the intersecting genes.Western Blot was used to detect the effects of RB on the expression levels of proteins on KEGG enrichment signaling pathway.Cell cycle assay,transwell and western Blot assay confirmed the relationship between enrichment pathways and RB-induced cycle arrest and invasion inhibition.(3)BioSolveIT SeeSAR software was used to predict the target proteins of RB.Knockdown or overexpression of target proteins in GBM cells verified its relationship with RB in inhibiting proliferation and invasion of GBM cells.(4)Orthotopic tumor model of P3#GBM cell in nude mice was established to text the effect of RB on the survival of tumor-bearing mice in vivo.The expression levels of target proteins and Ki-67 were observed by IHC staining.Results(1)The results of CCK-8,cell clonal formation and EdU assays showed that 2 and 4 μM RB significantly inhibited the proliferation of P3#GBM,U251 and A172.The results of cell cycle distribution test indicated that RB induced G2/M phase arrest of P3#GBM,U251 and A172 cells.(2)Transwell,3D-spheroid invasion assay and GBM-brain organoid coculture invasion assay showed that RB inhibited GBM cell invasion.Western blot analysis showed that RB inhibited the invasion of GBM cells by down-regulating the expression of EMT-related proteins.(3)Transcriptome sequencing showed that MAPKs pathway was enriched after RB treatment.Western blot showed that RB activated MAPK/ERK pathway.ERK1/2 inhibitor(U0126)rescues RB-induced cycle arrest.(4)Transcriptome sequencing showed that the focal adhesion signal pathway was enriched after RB treatment.Western blot assay showed that RB inhibited the SRC/FAK/Paxillin signaling pathway.Intracellular Ca2+assay showed that RB inhibited SRC/FAK/Paxillin focal adhesion signaling pathway by inducin1 intracellular Ca2+accumulation.(5)BioSolvelT SeeSAR predicted that RB targets ATP1A1 protein in GBM cells.(6)Na+-K+-ATPase activity assay showed that RB increased the activity of Na+-K+ATPase in GBM cells.Reactive oxygen species(ROS)assay showed that RB closed the ROS/Na+-K+-ATPase amplification loop by activating the Na+-K+-ATPase activity.Western blot showed that RB promoted the protein expression level of ATP1A1 in GBM cells.Upon RB binding to ATP1A1,Na+-K+-ATPase was activated as a receptor and then triggered the intracellular MAPK/ERK pathway and Ca2+-mediated SRC/FAK/Paxillin focal adhesion pathway,which led to the arrest of the G2/M phase and inhibited the invasion of GBM cells.(7)Tumor growth was suppressed in an orthotopic tumor model by RB,which also resulted in a considerable increase in the survival of tumor bearing mice.Treatment with RB(10mg/kg/day)dramatically increased the median survival of tumor-bearing mice(n=10,26 days vs 29 days,P=0.0082).The fluorescence signal in the RB treatment group was nearly 42%lower than that in the control group(n=10,P=0.0012).At the same time,IHC assay showed that Ki-67 was significantly decreased in RB treatment group,ConclusionUpon RB binds to ATP1A1,Na+-K+-ATPase is activated as a receptor,accompanied by activation of the intracellular MAPK/ERK pathway and inhibition of the Ca2+-regulated SRC/FAK/Paxillin focal adhesion pathway,after which GBM cells undergo G2/M phase arrest and inhibition of invasion.