The A375 human melanoma cell line was obtained from the Cell Resource Center (Beijing, China) and was maintained in RPMI 1640 medium (Beijing Neuronbc Laboratories, Co. Ltd., Beijing, China) supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) in an incubator with high-efficiency particulate arrestance class 100 filter (Forma™ Series II; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C with 5% CO₂.

Transwell membranes (8-µm pore size; Costar; Corning, Life Sciences, Cambridge, UK) were coated with Matrigel (1 mg/ml; BD Biosciences, Franklin Lakes, NJ, USA) prior to cell passage. A375 cells (1×10⁵) were seeded into the upper wells in RPMI 1640 medium supplemented with 0.2% FBS. Cells were untreated (control group) or treated with 28 µg/ml (1/2 half maximal inhibitory concentration) NCTD (NCTD group). NCTD was purchased from Jiangsu Kangxi Pharmaceutical Works (Jiangsu, China) in fresh culture medium. Lower wells contained RPMI 1640 medium supplemented with 20% FBS. After 24 h of incubation at 37°C, non-invading cells were removed from the upper surface of the membrane, and the cells that invaded each membrane were stained with a crystal violet solution and counted as described previously (7).

Matrigel was thawed at 4°C, and 20 µl was quickly added to each well of a 96-well plate and allowed to solidify for 40 min at 37°C in a humidified 5% CO₂ incubator. Tumor cells were seeded in complete RPMI 1640 medium onto the plate and incubated with or without NCTD (28 µg/ml) at 37°C for 12 and 24 h, respectively. Vasculogenic-like structure formation was filmed under an inverted phase-contrast light microscope.

Mice (4–6 week-old BALB/c nu/nu male mice; Shanghai Laboratory Animal Center, Shanghai, China) weighing 18–24 g were randomly divided into the control group and the NCTD group (n=10/group). The mice were housed according to the official recommendations of the Chinese Community Guidelines (14). Tumors were established by inoculation of 1×10⁶ A375 cells suspended in 100 µl normal saline into the right back of the mice by subcutaneous injection. After the tumors had grown to a size of ~100 mm³, NCTD was administered by intraperitoneal injection at 28 mg⁄kg (1/5 half maximal lethal dose) in 0.1 ml normal saline for 13 consecutive days. Control animals were administered 0.1 ml normal saline as a vehicle control. The maximum diameter (a) and the minimum diameter (b) were measured with calipers every 2 days. The tumor volume (V) was calculated by the following formula: V = ab² x 0.5. The tumor weight was also evaluated once the mice were sacrificed. All animal procedures were performed according to an approved protocol by the Animal Ethical Committee of the Tianjin International Joint Academy of Biotechnology and Medicine (Tianjin, China).

Xenografted tumors from the mice were excised, fixed in 4% paraformaldehyde for 24 h, and then embedded in paraffin for histological studies. Paraffin-embedded tissues were sectioned into slices of 4-µm thickness for histological studies. Dewaxed and rehydrated tissue sections were subjected to antigen retrieval processes. Upon blocking, the sections were incubated overnight at 4°C with anti-cluster of differentiation (CD) 31 antibody (dilution 1:50; catalogue number JC70; Neomarkers, Fremont, CA, USA) or anti-MMP-2 antibody (dilution 1:200; catalogue number ab37150; Abcam, Cambridge, UK). Negative controls were prepared using PBS instead of the primary antibodies. Upon washing with PBS, the sections were incubated with a goat anti-mouse EnVision kit (Genentech, South San Francisco, CA, USA) for 40 min at 37°C, and then incubated with 3,3′-diaminobenzidine chromogen for 10 min. The slides for CD31-periodic acid-Schiff (PAS; Beijing Zhongshan Jinqiao Biotechnology Co. Ltd., Beijing, China) double staining were then exposed to a 0.5% periodic acid solution for 15 min and subsequently incubated in Schiff solution for 20 min in a dark chamber. Subsequently, the slides were washed with distilled water for 3 min and counterstained with hematoxylin. Multiplication of intensity and percentage scores was utilized to determine the staining index result.

Gelatin zymography was used to examine the levels of MMP-2 activity in A375 cells that were either untreated (control group) or treated with 28 µg/ml NTCD for 48 h. The culture media were collected and subjected to 10% SDS-PAGE using 0.01% w/v gelatin. The gel was washed twice in 2.5% (w/v) Triton X-100 solution and incubated overnight at 37°C in developing buffer [50 mmol/l Tris/HCl (pH 7.4), 10 mmol/l CaCl₂, 5 mmol/l ZnCl₂ and 0.05% Brij™ 35 Surfact-Amps™ Detergent Solution (Thermo Fisher Scientific, Inc.)]. The gels were subsequently stained with Coomassie Brilliant Blue R250 and destained until the wash remained clear and cleared zones associated with MMP activity were apparent. The concentrations of exogenous pro-MMP-2 and active MMP-2 in cell culture supernatants were assessed using the MMP-2 Biotrak Activity Assay (GE Healthcare Life Sciences, Chalfont, UK) according to the manufacturer's guidelines.

All data were reported as the mean ± standard error of the mean and evaluated using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Student-Newman-Keuls t tests were used to evaluate differences between two groups. P<0.05 was considered to indicate a statistically significant difference.

Matrigel-coated transwell plates were used to assess the effects of NCTD on the ability of A375 melanoma cells to invade a basement membrane matrix. The results revealed that NCTD significantly reduced the number of cells that invaded through the Matrigel matrix after 24 h of treatment (P= 0.005) (Fig. 1A).

The vasculogenic-like network formation ability of melanoma cells was assessed in vitro by seeding the cells onto Matrigel-coated plates and then observing the cells under an inverted phase-contrast light microscope. As shown in Fig. 1B, the formation of VM networks by A375 melanoma cells was disrupted by the addition of NCTD for 12 or 24 h.

To investigate the efficacy of NCTD in inhibiting melanoma growth, the tumor sizes of melanoma-bearing mice were measured once every 2 days throughout the experiment. At the end of the experiment, the volume and weight of the xenografts in the NCTD group decreased significantly with increased tumor inhibition in comparison with those of the control group (Fig. 2). CD31-PAS double staining was used to identify VM in the xenografts on day 21 of tumor inoculation. Microscopically, the xenografts in the control group exhibited tumor cell-lined channels containing red blood cells (Fig. 3) without any evidence of tumor necrosis. The channels consisted of tumor cells negative for CD31 and positive for PAS. By contrast, VM could hardly be observed in the tumor tissues treated with NCTD, while large areas of necrosis were easily detected, suggesting that NCTD inhibits the VM formation of melanoma xenografts in vivo.

MMP-2 is a key player in VM formation (15). Thus, to explore the possible mechanisms of NCTD effects on tumor growth and VM of human melanoma in vitro and in vivo, in the present study, the expression and activity of MMP-2 protein from sections of melanoma xenografts and supernatants of 3-D culture samples were determined. The results indicated that MMP-2 expression in the in vivo xenografts of the NCTD group was significantly lower than that of the control group (Fig. 4A). Furthermore, the addition of NCTD decreased both the expression and activity of MMP-2 in the 3-D culture samples (Fig. 4B and Table I).