Norcantharidin inhibits tumor growth and vasculogenic mimicry of human gallbladder carcinomas by suppression of the PI3-K /MMPs /Ln-5γ2 signaling pathway

Background:Vasculogenic mimicry(VM)is a novel tumor blood supply in some highly aggressive malignant tumors.Recently,we reported VM existed in gallbladder carcinomas(GBCs)and the formation of the special passage through the activation of the PI3K/MMPs/Ln-5γ2 signaling pathway.GBC is a highly aggressive malignant tumor with disappointing treatments and a poor prognosis.Norcantharidin(NCTD)has shown to have multiple antitumor activities against GBCs,etc;however the exact mechanism is not thoroughly elucidated.In this study,we firstly investigated the anti-VM activity of NCTD as a VM inhibitor for GBCs and its underlying mechanisms.Methods:In vitro and in vivo experiments to determine the effects of NCTD on proliferation,invasion,migration,VM formation,hemodynamic and tumor growth of GBC-SD cells and xenografts were respectively done by proliferation,invasion,migration assays,HE staining and CD31-PAS double staining,optic/electron microscopy,tumor assay,and dynamic microMRA.Further,immunohistochemistry,immunofluorescence,Western blotting and RT-PCR were respectively used to examine expression of VM signaling-related markers PI3-K,MMP-2,MT1-MMP and Ln-5γ2 in GBC-SD cells and xenografts in vitro and in vivo.Results:After treatment with NCTD,proliferation,invasion,migration of GBC-SD cells were inhibited;GBC-SD cells and xenografts were unable to form VM-like structures;tumor center-VM region of the xenografts exhibited a decreased signal in intensity;then cell or xenograft growth was inhibited.Whereas all of untreated GBC-SD cells and xenografts formed VM-like structures with the same conditions;the xenograft center-VM region exhibited a gradually increased signal;and facilitated cell or xenograft growth(Figure 1-6).Furthermore,expression of MMP-2 and MT1-MMP products from sections/supernates of 3-D matrices and the xenografts,and expression of PI3-K,MMP-2,MM1-MMP and Ln-5γ2 proteins/mRNAs of the xenografts were all decreased in NCTD or TIMP-2 group(Figure 7-10;all P<0.01,vs.control group);NCTD down-regulated expression of these VM signaling-related markers in vitro and in vivo.Conclusions:NCTD inhibited tumor growth and VM formation of human gallbladder carcinoma GBC-SD cells and xenografts by suppression of the PI3-K/MMPs/Ln-5γ2 signaling pathway.It is firstly concluded that NCTD may be a potential anti-VM agent for human GBCs.

Background：Vasculogenic mimicry （VM） is a novel tumor blood supply in some highly aggressive malignant tumors. Recently,we reported VM existed in gallbladder carcinomas （GBCs） and the formation of the special passage through the activation of the PI3K/ MMPs/Ln-5γ2 signaling pathway. GBC is a highly aggressive malignant tumor with disappointing treatments and a poor prognosis. Norcantharidin （NCTD） has shown to have multiple antitumor activities against GBCs, etc; however the exact mechanism is not thoroughly elucidated. In this study, we firstly investigated the anti-VM activity of NCTD as a VM inhibitor for GBCs and its underlying mechanisms. Methods ： In vitro and in vivo experiments to determine the effects of NCTD on proliferation, invasion, migration, VM formation, hemodynamic and tumor growth of GBC-SD cells and xenografts were respectively done by proliferation, invasion,migration assays,HE staining and CD31-PAS double staining, optic/electron microscopy, tumor assay, and dynamic micro- MRA. Further, immunohistochemistry, immunofluorescence, Western blotting and RT-PCR were respectively used to examine expression of VM signaling-related markers PI3-K,MMP-2 ,MT1-MMP and Ln-5γ2 in GBC-SD cells and xenografts in vitro and in vivo. Results： After treatment with NCTD, proliferation, invasion, migration of GBC-SD cells were inhibited; GBC-SD ceils and xenografts were unable to form VM-like structures; tumor center-VM region of the xenografts exhibited a decreased signal in intensity; then cell or xenograft growth was inhibited. Whereas all of untreated GBC-SD cells and xenografts formed VM-like structures with the same conditions; the xenograft center-VM region exhibited a gradually increased signal; and facilitated cell or xenograft growth （Figure 1-6 ）. Furthermore, expression of MMP-2 and MT1-MMP products from sections/supemates of 3-D matrices and the xenografts, and expression of PI3-K, MMP-2, MM1-MMP and Ln-5γ2 proteins/mRNAs of the xenografts were all decreased in NCTD or TIMP-2 group （Figure 7-10; all P 〈 0.01, vs. control group） ; NCTD down-regulated expression of these VM signaling-related markers in vitro and in vivo. Conclusions：NCTD inhibited tumor growth and VM formation of human gallbladder carcinoma GBC-SD cells and xenografts by suppression of the PI3-K/MMPs/Ln-5γ2 signaling pathway. It is firstly concluded that NCTD may be a potential anti-VM agent for human GBCs.