Bruceine D Inhibits Hepatocellular Carcinoma By Targeting ?-Catenin/Jagged1 Pathways

Primary liver cancer is among mostly common malignant neoplasms worldwide and approximately 90% of which is Hepatocellular carcinoma(HCC).Despite improvements in detection and clinical treatment strategies,the 5-year survival rate for HCC is less than 17% for all stages combined.Sorafenib remains the first-line treatment for advanced HCC,with only 2.8 months increased survival compared with placebo.Recent studies attempted to identify new drugs that could be categorized as sorafenib-synergetic agents or compounds targeting HCC-involving pathways.Aberrant activation of the Wnt and Notch signaling pathways is critical to liver carcinogenesis and progression.In addition,?-catenin gains somatic activating mutations in 20 to 40% of patients with HCC,and no FDAapproved drug targeting ?-catenin is available for clinical use.Preliminary study screened a Notch inhibitor Bruceine D(BD)which showed great inhibitory effects on HCC in vitro and in vivo,through RBP-J?-dependent luciferase-reporter system.BD significantly decreased expression of Jagged1 which is required for anti-HCC effects.Here,we carry out luciferase-expressing orthotopic HCC model to identify BD anti-HCC effects and synergistic effects with sorafenib in vivo.Mechanistically,further study demonstrate the directly target pathways of BD.Methods: 1.Luciferase-expressing orthotopic HCC model was used to test BD therapeutic effects and synergistic effects with sorafenib of anti-HCC in vivo.H&E and immunohistochemistry were used to test inhibitory effects of tumor growth in vivo.2.Jagged1 is downregulated in BD-treated HCC cells and required for BD-mediated proliferation inhibition of HCC.The knockdown of Jagged1 and ?-catenin by siRNA respectively,inhibitors and agonists of Wnt pathway treatments were carried out to test the regulation of ?-catenin on expression of Jagged1 on HCC.3.Co-immunoprecipitation,qPCR and western blot were used to test the effects on transcription activity of Wnt/TCF4 pathways.4.Immunofluorescence analysis and western blot analysis of ?-catenin levels in cytoplasmic and nucleus fractions were carried out to identify nuclear accumulation levels of ?-catenin in BD-treated HCC cells.5.Western blot was used to test total levels of ?-catenin and active ?-catenin(ABC)in whole cells of BD-treated HCC.MG132 was used to test effects of BD on proteasomes dependent degradation pathways of ?-catenin.6.Wnt agonists Wnt3 a and GSK3? inhibitors were used to test effects on BD inhibitory of nuclear ?-catenin.7.The inhibitors of nucleo-cytoplasmic shuttling pathway CRM1 was used to identified BD effects on nucleo-cytoplasmic shuttling of ?-catenin.8.Immunohistochemistry and western blot were used to test BD effects on expressions of Jagged1,?-catenin and ABC in tumor tissues.9.Biacore,MST and Thermal shift assays were used to test the interaction between BD and ?-catenin.Results: 1.Previous study shown that BD is anti-HCC agent in vivo and in vitro.BD treatment significantly inhibited Huh7 tumor growth rate and size compared with vehicle treatment in orthotopic liver cancer model.In an HCC murine model,treatment with BD at 0.75 mg/kg alone or a low dose of sorafenib(10 mg/kg)alone showed weak and little therapeutic effect,respectively.Notably,a combination of BD(0.75 mg/kg)and low dose sorafenib(10 mg/kg)exhibited tumor inhibition that was comparable to inhibition observed after a high dose of sorafenib(30 mg/kg).H&E and immunohistochemistry showed BD alone and combined with sorafenib caused significant tumor cell necrosis comparing with high dose sorafenib treatment,reduced the number of ki67-positive cells, and increased the number of TUNEL-positive cells in tumor tissues.Together,these findings suggest that BD enhanced the anti-tumor effects of sorafenib in vivo.2.As previous work shown that Jagged1 is required for anti-HCC effects of BD and was reported to be a target gene of ?-catenin in colorectal and breast carcinoma.?-catenin knockdown assay showed inhibition of ?-catenin expression using shRNA(sh?-catenin) downregulated Jagged1 expression and promoted BD-mediated Jagged1 inhibition. However,the amount of ?-catenin was unchanged in Jagged1 knockdown cells.In addition,Wnt signaling inhibitors FH535 and ICG-001 showed similar effects to sh?-catenin on Jagged1 level,while Wnt activators Wnt3 a,BIO and CHIR99021 enhanced Jagged1 expression.These observations suggest that Jagged1 could be the link between Notch and Wnt/?-catenin in HCC cells.3.?-catenin co-immunoprecipitation assay found that BD disrupted ?-catenin/TCF4 complex formation in dose-dependent manner.In addition,BD significantly decreased the expression levels of Wnt/?-catenin target genes,including LEF1,survivin,AXIN2, and c-Myc in Huh7 cells in a dose-dependent manner.Protein levels of downregulated target genes were also reduced in a dose-and time-dependent manner after BD treatment. These results strongly support that BD suppresses Wnt transcriptional activities and reduces Wnt-target gene expression.4.The nuclear ?-catenin level decreased significantly after BD treatment,while the cytoplasmic level was not affected.Immunofluorescence staining of Huh7 cells confirmed the reduction in nuclear ?-catenin accumulation.5.BD treatment decreased the total ?-catenin and active-?-catenin(ABC)in whole cell lysates,indicating that BD downregulated the overall level of ?-catenin,leading to the loss of nuclear ?-catenin and ABC in Huh7 cells.And BD showed little effects to ?-catenin and ABC in total fractions and nuclear fractions of MG132-treated Huh7 cells. These results showed that BD induces ?-catenin proteasome-dependent degradation to decrease total levels of ?-catenin.6.BD decreased total ?-catenin in Wnt3a-,BIO-,and CHIR99021-treated Huh7 cells, indicating that BD inhibited ?-catenin independent of GSK3? and Wnt ligand.7.BD decreased nuclear translocation of BD in CRM1 inhibitors LMB and Selinexor-treated Huh7 cells.8.Immunohistochemistry staining and Western blot confirmed that BD reduced expressions of ?-catenin,ABC,and Jagged1 in BD-treated Huh7 orthotopic hepatic tumors.9.Cellular thermal shift assay showed that BD has little interaction with ?-catenin in Huh7 cells.BD also showed little binding abilities to ?-catenin identified by thermal shift, Biacore and MST assays in vitro.Conclusion:Prevously,we identified BD shows great anti-HCC effects in vitro and targets for reduction of Jagged1.Here,we found that BD significantly inhibited liver tumor growth and enhanced the therapeutic effects of sorafenib in a murine HCC model.Mechanistically,BD promotes proteasomal degradation of ?-catenin and the depletion of its nuclear accumulation,which in turn disrupts the Wnt/?-catenin-dependent transcription of the Notch ligand Jagged1 in HCC.Our findings provide important information about a novel Wnt/Notch crosstalk inhibitor that is synergistic with sorafenib for treatment of HCC,and therefore have high clinical impact.