Anti-glioblastoma Mechanism Of STELB

Objective: Glioblastoma is a very aggressive tumor with high mortality.The current mainstay of the treatment is very limited with restricted options for radio-and chemotherapy,which are especially prone to develop therapeutic resistances.Therefore,making efforts to develop new therapeutic agents that can overcome resistance to radio-and chemotherapy is the key point to resolve the current dilemma of glioblastoma treatment,which may determine the therapeutic prospect of glioblastoma worldwide.In this paper,we intend to investigate the specific molecular mechanism of STELB(Stellettin B),a triterpene isolated from marine sponge,on glioblastoma,and to explore its potential effects on sensitizing glioblastoma to radio-and chemotherapy.This paper aims to provide compelling preclinical evidence for STELB or its derivatives to be developed into novel drugs for glioblastoma treatment.Methods: 1.The effect of STELB and ionizing radiation(IR)on survival fraction of glioblastoma cells was detected by colony formation assay.Annexin V/PI assay and Western blot were used to analyze the effect of STELB and IR on apoptosis.Comet assay and immunofluorescence were used to examine DNA damage in glioblastoma cells.2.The effect of STELB and temozolomide(TMZ,a first-line drug for glioblastoma)on cell viability was detected by WST assay and colony formation assay.Annexin V/PI assay and Western blot were used to analyze the effect of STELB and TMZ on apoptosis.Comet assay and immunofluorescence were used to examine DNA damage in glioblastoma cells.The antitumour effect of STELB and TMZ in vivo was investigated by using xenografts in nude mice and orthotopic xenografts in zebrafish.3.The effect of STELB on the efficiency of homologous recombination(HR)was detected by HR reporter assay.The effect of STELB on HR related proteins were examined by Western blot and q RT-PCR.Western blot,immunofluorescence and immunohistochemistry were used to investigate the combination effect of STELB with IR or TMZ on expression of HR related proteins.4.The effect of STELB on PI3 K pathway was examined by western blot.The effects of knockdown or PI3 K inhibitors and overexpression of PIK3 CA on HR related proteins and HR efficiency were examined by western blot,q RT-PCR and HR reporter assay.The effects of STELB on m RNA expression and kinase activity of PI3 K were detected by q RT-PCR and Adapta kinase assay respectively.The effect of STELB on protein stability of PI3K-p110α was investigated by using the eukaryote protein synthesis inhibitor CHX and the proteasome inhibitor MG132.5.The combination effect of STELB and PARP inhibitors(PARPi)Olaparib or Rucaparib on cell viability was detected by WST assay and colony formation assay.Chou-Talalay assay was employed to calculate combination index.Annexin V/PI assay and Western blot were used to analyze the effect of STELB and PARPi on apoptosis.Comet assay and immunofluorescence were used to examine DNA damage in glioblastoma cells.The combination effects of STELB and PARPi on HR efficiency and HR related proteins were examined by HR reporter assay,Western blot and immunohistochemistry.The potential targets of STELB to sensitize glioblastoma cells to PARPi were investigated by using si-PIK3 CA and HA-PIK3 CA.The antitumour effect of STELB together with PARPi in vivo was investigated by using xenografts in nude mice and orthotopic xenografts in zebrafish.6.The transcriptome change of SF295 cells after STELB treatment was analyzed by RNA-Seq.q RT-PCR and Western blot were used to verify the effect of STELB on DNA replication pathway and Fanconi anemia pathway.Results: 1.Combination of STELB and IR significantly reduced the survival rate of SF295,U87,U251 and T98 G cells,synergistically induced apoptosis,and increased the degree of DNA damage in glioblastoma cells,especially DNA double-strand breaks.2.Combination of STELB and TMZ potently inhibited the proliferation of SF295,U87 and U251 cells,synergistically induced apoptosis,and increased the degree of DNA damage in glioblastoma cells,especially DNA double-strand breaks.Besides,combination of STELB and TMZ significantly inhibited the growth of xenografts in nude mice and orthotopic xenografts in zebrafish in vivo with high tolerance.3.STELB reduced HR efficiency in SF295 and U87 cells,downregulated the expression of BRCA1/2,Rad51,and reversed the activation of HR induced by IR and TMZ in vitro and in vivo.4.STELB inhibited PI3 K pathway dose-dependently.Knockdown of PIK3 CA and PI3 K inhibitor BKM-120 downregulated the expression of BRCA1/2,Rad51 and reduced HR efficiency in glioblastoma cells;while overexpression of PIK3 CA partially antagonized the inhibition effects of STELB on the expression of BRCA1/2,Rad51 and HR efficiency.According to the study of PI3K-p110α,STELB might compromise the protein stability of PI3K-p110α by promoting its degradation through ubiquitin-proteasome pathway.5.Combination of STELB and PARPi synergistically inhibited the proliferation of SF295,U87 and U251 cells,induced apoptosis and DNA damage,and significantly inhibited the growth of xenografts in nude mice and orthotopic xenografts in zebrafish in vivo.Further mechanism study showed that STELB inhibited HR by regulating PI3 K pathway,reversed the activation of HR induced by PARPi,and resulting in "synthetic lethality" in glioblastoma when combined with PARPi.6.STELB regulated various signaling pathways including HR pathway,and downregulated gene expression of DNA replication pathway(MCM6,RFC2,POLA2,PCNA,etc.)and Fanconi anemia pathway(UBE2T,FANCD2,FANCB,etc.).On one hand,these pathways have a wide range of crosstalk with HR pathway;and on the other hand,they may also contribute to the sensitizing effects of STELB for radio-and chemotherapy.Conclusion: STELB leads to a HR deficiency phenotype(reflected in impaired HR efficiency)in glioblastoma through blocking the PI3 K mediated BRCA1/2 and RAD51 expression,which making a great contribution on sensitizing glioblastoma to IR and TMZ therapy,and resulting in "synthetic lethality" in glioblastoma when combined with PARPi.As a potential anti-glioblastoma drug lead,STELB is worthy to be further developed and will be of great innovation and clinical value in glioblastoma treatment.