CTD and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA), fetal calf serum (FCS) and L-glutamine were purchased from Gibco (Thermo Fisher Scientific, Inc.). Primary antibodies against PTEN, protein kinase B (AKT), protein kinase B (p) AKT, Bax, B-cell lymphoma-2 (bcl-2), caspase-3, anti-GAPDH and the appropriate secondary antibodies were purchased from ProteinTech Group, Inc. (Chicago, IL, USA). The enhanced chemiluminescence (ECL) detection system was obtained from Millipore (EMD Millipore, Billerica, MA, USA).

The A375 human melanoma cancer cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured at 37°C in a 5% CO₂ humidified incubator in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin.

Micron^™ hsa-miR-21-5p agomir (5′-UAGCUUAUCAGACUGAUGUUGA-3′) or agomir control (sequence unavailable), micrOFF^™hsa-miR-21-5p antagomir (5′-UCAACAUCAGUCUGAUAAGCUA-3′) or antagomir control (sequence unavailable) were obtained from Guangzhou RiboBio Co., Ltd., (Guangzhou, China; 10 nM). SignalSilence^® PTEN siRNA (5′-GCATATGGAAAGCTTCATT-3′) and its negative control oligonucleotides (5′-ACACGTCCGAACATACTAC-3′) were purchased from Cell Signaling Technology (50 nM). Transfections were performed using Lipofectamine^™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. For overexpression, the full-length PTEN sequence was cloned into the lentiviral vector [Obio Technology (Shanghai) Corp., Ltd., Shanghai, China]. Following amplification, lentiviral-PTEN (LV-PTEN) or empty vector (Vector) as a control was transduced into A375 cells at a concentration of 5×10⁴ transducing units/ml using polybrene [Obio Technology (Shanghai) Corp.] according to the manufacturer's protocol. Next, 5×10⁵ cells were seeded into 6-well plates. A total of 48 h following transfection, the cells were harvested for subsequent experiments.

Total RNA was isolated from the A375 cell line using Trizol (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration and purity of these RNA samples were detected. The total RNA samples were reverse transcribed into complementary DNA (cDNA) using PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). qPCR was performed using the MyiQ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the SYBR-Green I premix (Takara Biotechnology Co., Ltd.). U6 or GAPDH mRNA were used as an endogenous control. The thermocycling conditions were as follows: 95°C for 10 min, 40 cycles of 95°C for 15 sec and 60°C for 1 min. The experiment was repeated three times and the levels of miR were normalized for each well to the levels of U6 using the 2^−ΔΔCqmethod (14). The forward (F) and reverse (R) primers used in this study were as follows: miR-21, F: 5′-GGACTAGCTTATCAGACTG-3′ and R: 5′-CATCAGATGCGTTGCGTA-3′; U6 F: 5′-ATTGGAACGATACAGAGAAGAT-3′ and R, 5′-GGAACGCTTCACGAATTT-3′; PTEN F: 5′-CGGCAGCATCAAATGTTTCAG-3′ and R: 5′-AACTGGCAGGTAGAAGGCAACTC-3′.

Cells were collected and homogenized in ice-cold RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China). The amounts of protein from each treatment were detected and normalized using a bicinchoninic acid assay. Proteins (20 µg/lane) were separated by 10% SDS-PAGE and were subsequently transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk at room temperature for 2 h and then separately probed overnight at 4°C with the following primary antibodies: Anti-GAPDH (1:5,000; cat. no. 10494-1-AP), Bcl-2 (1:1,000; cat. no. 12789-1-AP), Bax (1:1,000; cat. no. 50599-2-Ig), caspase-3 (1:1,000, cat. no. 19677-1-AP), PTEN (1:1,000, cat. no. 22034-1-AP), AKT (1:1,000, cat. no. 10176-2-AP), p AKT (1:1,000; cat. no. 66444-1-Ig). After washing with TBST buffer (0.1% Tween-20), the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Ig)G or goat anti-rabbit IgG (1:5,000; cat. no. SA00001-1 or SA00001-2) for 1 h at room temperature. Images were then captured of the membranes using the ECL detection system (EMD Millipore). Densitometry analysis of the bands was performed using Quantity One (version 4.5.0; Bio-Rad Laboratories, Inc.). The proteins were quantified and expressed as their ratio to GAPDH.

A375 cells were seeded in 96-well plates at a density of 1,000 cells/well in 100 µl culture medium with different doses (0, 0.2, 1 or 5 µM) of CTD (dissolved with 0.5% dimethyl sulfoxide; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) treatments. Next, at 24, 48, 72 and 96 h following treatment, CCK-8 solution was added to each well. The optical density of the cells was measured using a microplate reader (Bio-Rad Laboratories, Inc.) at 450 nm.

A375 cells were seeded in a 6-well plate (600 cells/well) in complete medium and were allowed to grow for 24 h. The cells were then exposed to 5 µM CTD or vehicle for 48 h. After the drug was removed, the cells were washed with PBS and incubated for another 14 days in complete medium. The cells were stained with 1% crystal violet solution at room temperature. Following 10 min incubation, the excess crystal violet was washed out and the stained colonies were counted under an inverted microscope (Nikon Corporation, Tokyo, Japan).

Male BALB/C Nu mice were obtained from Vital River Laboratory Animal Technology Co., Ltd., (Beijing, China) at 3- to 4-weeks old and weighing 18–20 g. Five mice per cage were housed together under a 12/12 h light/dark cycle in standard laboratory specific pathogen free cages (22±2°C, 50–60% humidity), with food and water ad libitum. A375 cells were digested using 0.25% pancreatin and were counted. The cell concentration was adjusted to 1×10⁷ cells/ml and a 0.1 ml cell suspension was subcutaneously injected into the right armpit of the mice. The volume of tumors was measured with a caliper every 3 days using the following equation: Tumor volume=width (W)²xlength (L)/2. When the tumor volumes reached approximately 100 mm³, the mice were randomly and equally divided into 2 groups and were treated with control or CTD (intraperitoneal injected with 200 µl vehicle or CTD, 0.5 mg/kg/day) for 3 weeks. In addition, certain mice in the CTD-treated group were intratumorally injected with miR21 agomir (10 nM diluted with 50 µl of PBS, three times per week; Guangzhou Ribobio Co., Ltd.) or agomir control accompanied with CTD treatment (n=6 per group). A total of 30 mice were included in the present study. All the experiments were performed in accordance with the relevant guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of Taishan Medical University.

Following treatment with CTD for 48 h, the cells were harvested and reconstituted to 1–5×10⁶/ml. Next, 100 µl cell suspension was transferred into a 5 ml flow tube, followed by staining with 5 µl of Annexin V/fluorescein isothiocyanate (FITC; Beijing 4A Biotech Co., Beijing, China) for 5 min at room temperature in the dark. Next, the cells were stained with 10 µl propidium iodide (PI) and 400 µl PBS, and then collected and detected by flow cytometry. The results were analyzed using Flowjo software (version 9.3.2; FlowJo LLC, Ashland, OR, USA).

Data analysis was performed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). All the data were presented as the mean ± standard deviation. The data of only two groups were analyzed using a Student's t-test. One-way analysis of variance followed by the Student Newman Keuls test was used for the analysis of three or more groups. P<0.05 was considered to indicate a statistically significance difference.

The effects of different doses of CTD (0.2, 1 and 5 µM) were first evaluated on the growth of A375 cells using the CCK-8 assay. The results demonstrated that the OD value of 5 µM CTD-treated A375 cells was significantly decreased compared with the vehicle control at 48, 72 and 96 h (P<0.05). Next, 1 µM CTD significantly decreased the OD value of A375 cells only at 96 h (P<0.01) and significantly increased compared with 5 µM CTD (P<0.01). Additionally, 0.2 µM CTD demonstrated no effect on the OD value (Fig. 1A). These results suggest that CTD had an inhibitory effect on the proliferation of A375 cells in a dose-dependent manner. Therefore, 5 µM CTD was used in subsequent in vitro experiments. The colony formation assay was also used to detect the roles of CTD in the growth of A375 cells. The plates with 5 µM CTD treatment exhibited significantly fewer cell clones compared with the control group (P<0.01; Fig. 1B and C). All these results indicated that CTD inhibits the proliferation of A375 cells effectively in vitro. Whether CTD had antitumor effects on A375 cell xenografts in vivo was next investigated. The results demonstrated that CTD retarded the growth of the xenograft tumor significantly and the final tumor volume was significantly smaller compared with the vehicle-treated group (P<0.01; Fig. 1D and E), suggesting that CTD inhibited the tumorigenesis of melanoma in vivo.

To observe the effect of CTD on cell apoptosis in A375 cells, an Annexin V-FITC/PI staining assay was conducted. As shown in Fig. 2A and B, CTD increased the number of apoptotic cells compared with those in the vehicle control group (P<0.01). These data suggest that CTD may promote human melanoma cell apoptosis. To study further the effects of CTD on cell apoptosis western blot analysis was performed. The expression level of Bcl-2, an anti-apoptosis protein, was significantly decreased following CTD treatment (P<0.01), while the expression levels of pro-apoptosis proteins Bax and active caspase-3 were significantly increased (P<0.01; Fig. 2C and D). These results indicate that CTD could effectively induce the apoptosis of A375 cells.

Next, alterations in the relative expression levels of miR-21 and its target gene PTEN following CTD administration were measured. A higher miR-21 expression in A375 cells was decreased significantly following CTD treatment (P<0.01; Fig. 3A). Furthermore, PTEN, the potential downstream gene of miR-21, was upregulated in both the mRNA and protein expression levels following CTD treatment (P<0.01; Fig. 3B and C). These results suggested that miR-21 and PTEN may be involved in the effects of CTD on A375 cells. The AKT kinase is the one of the important downstream effectors of PTEN. Accompanied with the increased expression of PTEN following CTD treatment, the expression of the active form of AKT, pAKT, was decreased (Fig. 3C). These results proved that CTD may suppress the AKT pathway by increasing PTEN expression.

To further verify the association between miR-21 downregulation and CTD-induced antitumor effects, miR-21 agomir and antagomir were used to observe the CTD effects on A375 proliferation and tumorigenesis. The results demonstrated that the miR-21 agomir or antagomir could significantly increase or decrease, respectively, the expression of miR-21 in A375 cells (both P<0.01; Fig. 4A). Compared with the CTD+antagomir/agomir control, the miR-21 agomir (P<0.01) or antagomir (P<0.05) impeded or promoted the effect of CTD on the OD values, respectively (Fig. 4B). Furthermore, it was demonstrated that miR-21 agomir administration could significantly reverse the decrease in the xenograft tumor volumes caused by CTD treatment on the 21st day (P<0.01; Fig. 4C). These results suggest that miR-21 is involved in the antitumor effect of CTD both in vitro and in vivo. Next, the roles of PTEN in the antitumor effect of CTD were evaluated. First, the effectiveness of the tools used to regulate PTEN expression was confirmed (Fig. 4D and E). Similarly, it was observed that PTEN overexpression or siRNA PTEN treatment could significantly enhance or impede the roles of CTD in A375 cell viability, respectively (P<0.01; Fig. 4F). Finally, it was demonstrated that miR-21 agomir or antagomir administration blocked or enhanced the decreased effect of CTD on OD values, respectively, whereas a combination with PTEN overexpression or knockdown could, respectively, reverse these results (all P<0.05; Fig. 4G). These results indicate that the antitumor effects of CTD occurred via attenuating miR-21-mediated PTEN suppression.