The Combination Of Cantharidin And Anti-angiogenic Therapeutics Presents Synergistic Antitumor Effects Against Pancreatic Cancer

Objective:Cantharidin is an active constituent of mylabris,a traditional Chinese medicine.Previously,we demonstrated that cantharidin could represse the growth and metastasis of pancreatic cancer cells in vitro.However,the antitumor effect of cantharidin against pancreatic cancer has not been evaluated in vivo.Therefore,in the present study,we investigated the effects of cantharidin on pancreatic cancer xenografts in vivo and the mechanism involved,providing a more scientific basis for the application of cantharidin in the treatment of pancreatic cancer.Methods:1.Nude mouse subcutaneous/orthotopic xenograft models were made by injecting PANC-1 cells stably expressing luciferase into the left flanks or the body-tail of the pancreas of nude mice.2.Nude mouse subcutaneous/orthotopic xenograft models were used to evaluate the effects of cantharidin on pancreatic cancer in vivo,and mean fluorescence intensity was calculated by Luminex Technology.3.Immunohistochemistry was performed to test the expression of CD34 in pancreatic xenografts,and then microvessel density(MVD)was calculated.4.Milliplex assay was used to measure the levels of VEFF,IL-6,LI-8,and TNF-? in the culture of PANC-1 cells after treatment with cantharidin.5.Microarray,RNA-seq,and Ray Bio antibody array were used to analysis the expression of target genes in PANC-1 cells at both m RAN and protein levels.6.Real-time PCR was performed to determine the expression of target genes in PANC-1 cells which were pretreated with inhibitors of ERK,JNK,NF-?B,PKC pathways and cantharidin.7.Proliferation of PANC-1 cells treated with Ginsenoside Rg3 and PKC pathway inhibitor(Tamoxifen)separately was tested by MTT assay.8.Nude mouse subcutaneous xenograft model was used to evaluate the effect of cantharidin co-treatment with Tamoxifen or anti-angiogenic therapeutics(Ginsenoside Rg3,Bevacizumab,Apatinib,Endostar)on pancreatic cancer in vivo.9.The effect of cantharidin derivatives co-treatment with Bevacizumab on pancreatic cancer in vivo was also evaluated by using nude mouse subcutaneous xenograft model.Results:1.In cantharidin-treated group,the tumor volume,weight and bioluminescence of subcutaneous/orthotopic xenografts were significantly increased,and the bioluminescence peak value emerged earlier,decayed faster than that of the control group.MVD levels were also significantly elevated,indicating that cantharidin could promote the proliferation of pancreatic cancer in vivo,and induce tumor angiogenesis.2.Both cantharidin and OA could elevate the secretion of IL-6,IL-8,TNF-?,and VEGF in the culture of PANC-1,and increase the expressions of angiogenesis-related genes IL-8/CXC L8,CXCL1/GRO-?,VEGF/VEGFA,GM-CSF/CSF2,and PLAUR/u PAR at both the m RNA and protein levels.3.Cantharidin upregulated the expression of pro-angiogenic genes in PANC-1 cells which were pretreated with ERK,JNK,NF-?B,and PKC signaling pathway inhibitors,suggesting that all these pathways contributed to the pro-vascularizing process of cantharidin.4.Both Ginsenoside Rg3 and Tamoxifen exhibited a dose-and time-dependent repression on PANC-1 cell growth.5.The tumor volume,weight and bioluminescence of subcutaneous xenografts were significantly reduced in the groups treated with cantharidin in combination with tamoxifen or antiangiogenic therapeutics(ginsenoside Rg3,bevacizumab,apatinib,and Endo)separately,suggesting that PKC inhibitor and anti-angiogenic therapeutics could antagonize the growth-promoting effect of cantharidin and show a synergistic anti-tumor effect in vivo.6.The tumor volume,weight and bioluminescence of subcutaneous xenografts were significantly reduced in the groups treated with cantharidin derivatives in combination with bevacizumab,further indicating that cantharidin derivatives co-treated with anti-angiogenic therapeutics presented a remarkable synergistic antitumor effect in vivo as well.Conclusion:Our present results indicated that cantharidin promoted the proliferation of pancreatic cancer via targeting the tumor angiogenic microenvironment in vivo.ERK,JNK,NF-?B,and PKC kinase pathways may participate in the cantharidin-induced tumor vascular remodeling process.Anti-angiogenic therapeutics or inhibitors of pro-angiogenic kinase pathways could antagonize the growth-promoting effect of cantharidin and present synergistic anti-tumor effects.Therefore,clinical application of cantharidin should be performed on the premise of anti-vascularization therapy.