Inhibitory effect of norcantharidin on melanoma tumor growth and vasculogenic mimicry by suppressing MMP-2 expression

Abstract

A form of microcirculation called vasculogenic mimicry (VM), which constitutes a novel approach for tumor blood supply in certain highly aggressive malignant tumors, was recently reported to contribute to tumor metastasis and poor prognosis in melanoma patients. Development of strategies to target tumor VM may be significant to reduce the recurrence and metastasis of melanoma. Norcantharidin (NCTD) has been shown to inhibit tumor growth and VM of human gallbladder carcinomas. Besides, NCTD could induce melanoma cell apoptosis. However, whether NCTD can inhibit the growth and VM formation of melanoma has not been evaluated. The present study aims to investigate the anti-VM activity of NCTD as a VM inhibitor for melanoma and its potential mechanisms. The anti-VM activity of NCTD was determined in human melanoma A375 cells and xenografts in vitro and in vivo. The findings indicate that NCTD inhibits tumor growth and VM formation of melanoma both in vitro and in vivo by suppressing matrix metalloproteinase-2 expression. The results suggest that NCTD is a potential therapeutic agent targeting VM in melanoma.

Keywords: matrix metalloproteinases; melanoma; norcantharidin; vasculogenic mimicry.

Figure 1.

NCTD inhibits the invasion and capillary-like tube formation abilities of A375 melanoma cells in vitro. (A) Inhibitory effect of NCTD on the invasive ability of A375 melanoma cells in vitro. Upper panel, representative images of cells from the transwell invasion assay. Magnification, ×200. Lower panel, quantitative analysis indicating that fewer melanoma cells invaded through the Matrigel matrix following NCTD treatment compared with those in the control group. (B) NCTD decreases the capillary-like tube formation ability of melanoma cells in vitro. Upper panel, NCTD impaired the vessel-like networks formed by A375 melanoma cells cultured on Matrigel at 12 and 24 h. Magnification, ×400. Lower panel, quantitative analysis indicating that less capillary-like tubes were formed in cells subjected to NCTD treatment compared with those in the control group. *P<0.05 compared with the control group (n=3). NCTD, norcantharidin.

Figure 2.

Norcantharidin inhibits A375 human melanoma tumor growth. Plot of tumor volume (upper panel) or tumor weight (lower panel) vs. the number of days post-tumor inoculation of melanoma-bearing mice in different groups. *P<0.05 compared with the control group. Data are shown as the mean ± standard error of the mean (n=10 mice/group).

Figure 3.

NCTD decreases the capillary-like tube formation ability of A375 melanoma xenografts in vivo. Upper panel, representative images of the presence of VM in tissue samples of A375 melanoma xenografts with or without treatment with NCTD. In the control group, CD31-periodic acid-Schiff staining was used for the morphological observations of VM formed by tumor cells without CD31-positively stained endothelial cells (black arrow). Red blood cells were present in the center of the channels. The yellow arrow points to CD31-positively stained endothelium-dependent vessels in the same field. In the NCTD-treated group, VM was not observed, while large areas of necrosis were easily noticed (red arrow). Yellow arrow, endothelium-dependent vessels. Magnification, ×400. Lower panel, quantitative analysis indicating that less capillary-like tubes were formed upon NCTD treatment in the treated group compared with those in the control group. *P<0.05 compared with the control group. NCTD, norcantharidin; CD, cluster of differentiation; VM, vasculogenic mimicry.

Figure 4.

Expression and activity of MMP-2 protein from (A) sections of A375 melanoma xenografts and (B) serum-free conditioned medium from melanoma cells cultured on Matrigel. (A) Upper panel, representative images of MMP-2 expression in A375 melanoma xenografts. Positive staining of MMP-2 could be observed in the cytoplasm of tumor cells. The lower panel indicates the quantitative analysis of MMP-2 expression, and lower expression was observed in the NCTD group compared with that in the control group (*P<0.05). (B) The concentration and activity of MMP-2 protein from supernatants of three-dimensional culture samples were measured either by MMP-2 Biotrak Activity Assay (left) or by zymography assay (right). Both the expression and activity of MMP-2 protein in the NCTD group were significantly lower than those in the control group (*P<0.05). NCTD, norcantharidin; MMP, matrix metalloproteinase.