Abstract
Vasculogenic mimicry (VM) is a newly-defined tumor microcirculation pattern in highly aggressive malignant tumors. We recently reported tumor growth and VM formation of gallbladder cancers through the contribution of the ephrin type a receptor 2 (EphA2)/focal adhesion kinase (FAK)/Paxillin signaling pathways. In this study, we further investigated the anti-VM activity of norcantharidin (NCTD) as a VM inhibitor for gallbladder cancers and the underlying mechanisms. In vivo and in vitro experiments to determine the effects of NCTD on tumor growth, host survival, VM formation of GBC-SD nude mouse xenografts, and vasculogenic-like networks, malignant phenotypes i.e., proliferation, apoptosis, invasion and migration of GBC-SD cells. Expression of VM signaling-related markers EphA2, FAK and Paxillin in vivo and in vitro were examined by immunofluorescence, western blotting and real-time polymerase chain reaction (RT-PCR), respectively. The results showed that after treatment with NCTD, GBC-SD cells were unable to form VM structures when injecting into nude mouse, growth of the xenograft was inhibited and these observations were confirmed by facts that VM formation by three-dimensional (3-D) matrix, proliferation, apoptosis, invasion, migration of GBC-SD cells were affected; and survival time of the xenograft mice was prolonged. Furthermore, expression of EphA2, FAK and Paxillin proteins/mRNAs of the xenografts was downregulated. Thus, we concluded that NCTD has potential anti-VM activity against human gallbladder cancers; one of the underlying mechanisms may be via blocking the EphA2/FAK/Paxillin signaling pathway.
Highlights
Gallbladder cancer (GBC), a lethal aggressive malignant neoplasm, is the most common malignancy of the biliary tract, the 5th or 6th common malignant neoplasm of the digestive tract and the leading cause of cancer-related deaths in Western countries and China [1,2,3]
We recently reported that Vasculogenic mimicry (VM) existed in human gallbladder cancers and gallbladder cancers by both 3-D matrix of highly aggressive GBCSD cells in vitro and GBC-SD nude mouse xenografts in vivo and correlated with the patient’s poor prognosis and that poorly aggressive SGC-996 cells did not form the vasculogenic-like networks when cultured under the same conditions [12,13,17,18]
VM is a newly-defined tumor microcirculation pattern in some highly aggressive malignant tumors, which differs from endothelium-dependent angiogenesis, and is associated with a poor prognosis for the patients with some aggressive malignancies such as melanoma and gallbladder cancers [11,14,15,16,17,18]
Summary
Gallbladder cancer (GBC), a lethal aggressive malignant neoplasm, is the most common malignancy of the biliary tract, the 5th or 6th common malignant neoplasm of the digestive tract and the leading cause of cancer-related deaths in Western countries and China [1,2,3]. We identified that VM formation in gallbladder cancers through the activation of a key VM-related signaling pathway—the EphA2/FAK/Paxillin signaling pathway in the 3-D matrix of highly aggressive GBC-SD cells in vitro and GBC-SD nude mouse xenografts in vivo [42]. Using TIMP-2 or NCTD for 2,4 days after network formation, the already formed vasculogenic-like network structures from the 3-D matrix of GBC-SD cells were inhibited or destroyed, with visible cell aggregation, float, deformed collagen framework, decreased microvilli, vacuolar degeneration, nuclear fragmentation and typical apoptotic bodies (Fig.4A,D). NCTD downregulated expression of these VM signaling-related markers in vivo, to TIMP-2, inhibited VM formation of GBC-SD cells and growth of GBC-SD xenografts These findings demonstrated that NCTD inhibits tumor growth and VM of human gallbladder cancers, probably via blocking the EphA2/ FAK/Paxillin signaling pathway
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