Human A375 melanoma cells and murine B16F10 melanoma cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The two cell lines were routinely cultured in RPMI-1640 (Gibco, USA) containing 10% fetal bovine serum (FBS, Bioind, Israel) and maintained in an incubator with a humidified atmosphere of 5% CO₂ at 37°C. Wortmannin, cisplatin, cycloheximide (CHX) and RU486 were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and dissolved in dimethyl sulfoxide (DMSO) or ethanol. Recombinant human FN was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). For Dex treatment, cells were cultured in medium containing 10% Dextran-coated charcoal (DCC)-treated FBS to avoid possible interference from serum steroids and incubated with 100 nM Dex (Sigma-Aldrich; Merck KGaA) for different periods of time. Control cells were incubated with ethanol vehicle (<1‰ v/v).

Total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and 2 µg total RNA was reverse transcribed using Reverse Transcription Reagents (Takara Bio, Inc., Otsu, Japan) following the manufacturer's protocol. RT-qPCR was performed in triplicate using SYBR Green PCR Master Mix (Toyobo Life Science, Osaka, Japan) on a Mastercycler ep realplex (Eppendorf, Hamburg, Germany). The primer sequences used were as follows. FN (human): 5′-CGGTGGCTGTCAGTCAAAG-3′ (forward) and 5′-AAACCTCGGCTTCCTCCATAA-3′ (reverse). FN (mouse): 5′-GTCAGTGTCTCCAGTGTCTAC-3′ (forward) and 5′-TGGCTTGCTGGCCAATCAGT-3′ (reverse). GAPDH (human): 5′-CATGAGAAGTATGACAACAGCCT-3′ (forward) and 5′-AGTCCTTCCACGATACCAAAGT-3′ (reverse). β-actin (mouse): 5′-CTGTATGCCTCTGGTCGTAC-3′ (forward) and 5′-TGATGTCACGCACGATTTCC-3′ (reverse). Thermal cycling conditions consisted of an initial denaturing step (95°C, 2 min) followed by 40 cycles of denaturing (95°C, 15 sec), annealing (60°C, 15 sec) and extending (72°C, 45 sec). The level of FN mRNA was normalized to GAPDH or β-actin (internal control), and relative quantification was done using the 2^ΔΔCq formula. Changes in gene expression were expressed as the relative fold-increase in mRNA compared with a control.

Total cell lysates were prepared with 1× SDS lysis buffer with 100 mM Dithiothreitol and 2 µg/ml protease inhibitor solution containing 0.1 mM each leupeptin, aprotinin, and pepstatin. After electrophoresis, proteins were transferred to nitrocellulose membrane, blocked with 5% (v/v) nonfat milk in tris-buffered saline Tween-20 (TBST), and probed overnight at 4°C with primary antibodies against FN (sc-6953, 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), β-actin (1:10,000, Sigma-Aldrich; Merck KGaA), or p-Akt1/2/3 (Ser 473) (sc-7985, 1:1,000; Santa Cruz Biotechnology, Inc.), Akt1/2/3 (sc-8312, 1:1,000; Santa Cruz Biotechnology, Inc.). Then the membranes were washed three times and incubated with HRP-conjugated secondary antibodies (1:5,000; Rockland Immunochemicals, Inc., Gilbertsville, PA, USA) for 2 h. Finally HRP was detected using enhanced chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.). Protein bands were quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA) using β-actin as an internal control.

A375 melanoma cells were treated with or without 100 nM Dex for the indicated times, and then the conditioned media were collected and analyzed using human FN ELISA kits (R&D Systems, Inc.) according to the manufacturer's instructions. Absorbance of samples was read at 450 nm in a UV-visible spectrophotometer. The protein concentration was calibrated from a dose response curve based on reference standards.

SiRNAs were manufactured by GenePharma Co., Ltd. (Shanghai, China). The sequences were as follows. FN siRNA: 5′-GCAGUGGCUGAAGACACAAGGAAAU-3′; Control siRNA: 5′-CGCTTACCGATTCAGAATGG-3′. A375 cells were transfected with a final concentration of 10 nmol/l FN siRNA or control siRNA using INTERFERin™ (Polyplus Transfection, Strasbourg, France) following the manufacturer's instructions.

Cell adhesive ability was determined by cell adhesion assay. After cells were treated with Dex or transiently transfected with siRNA for the indicated times, cells were digested into single cell suspension and 8×10⁴ cells were seeded onto non-coated 96-well plates in triplicate and incubated at 37°C for 60 min. The plates were gently washed thrice with 1X PBS to remove the non-adherent cells. The number of remaining cells in the 96-well plates was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the remaining cells were incubated in medium supplemented with 50 µg/ml methylthiazole tetrazolium for 3 h. Cells were then solubilized by adding 200 µl DMSO. The absorbance was measured at 570 nm in a UV-visible spectrophotometer.

Cell adhesion ability was also determined in 96-well plates coated with or without human FN purchased from CORNING (USA). Before the cell adhesion assay, cells were pre-treated with a CD44 primary antibody (103014; BioLegend, Inc., San Diego, CA, USA) and Con IgG antibody (400622; BioLegend, Inc.) for 1 h.

Cells were seeded in 96-well culture plates at a density of 3×10³ cells per well in triplicate, allowed to attach overnight, and then treated with the indicated chemicals or reagents. Cisplatin, a chemotherapeutic drug applied in melanoma therapy, was used to induce cell death. At the indicated time, cell viability was evaluated using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) following the standard procedures provided by the manufacturer. The optical density (OD) was measured at a wavelength of 450 nm using a Labsystem multiskan microplate reader (Merck Eurolab, Dietikon, Switzerland).

Statistical significances between multiple experimental groups were analyzed by one-way analysis of variance and Tukey's post hoc tests. The Student's t-test was used to compare the difference between two different groups. P<0.05 was considered to indicate a statistically significant difference. Quantitative data were expressed as the mean ± standard deviation of at least three determinations.

We first examined the expression of FN in A375 and B16F10 melanoma cells treated with 100 nM Dex for different periods of times by RT-qPCR and western blotting. We did not observe a significant change in the level of FN mRNA (Fig. 1A and B), but we found that Dex significantly upregulated FN protein in A375 and B16F10 cells in a time-dependent fashion, with the maximal induction of FN protein (3.9-fold of control and 3.5-fold of control, respectively, P<0.01) observed at 48 h after Dex treatment (Fig. 1C and D). A significant increase in the level of secreted FN was also observed at 48 h and 72 h after Dex treatment in A375 cells (Fig. 1E).

Since upregulation of FN by Dex occurred at the post-transcriptional level, we investigated whether Dex-induced upregulation of FN was mediated through GR and caused by an increase in the protein stability in A375 melanoma cells. As shown in Fig. 2A, RU486, an antagonist of GR, dramatically reversed the upregulation of FN protein, indicating that the effect of Dex was mediated through GR.

We next examined the protein stability of FN in the presence and absence of Dex by western blotting. A375 cells were pre-treated with Dex or vehicle for 48 h and further treated with 100 µg/ml cycloheximide (CHX, a protein synthesis inhibitor) for different amounts of times. As shown in Fig. 2B, Dex significantly extended the half-life of FN protein (from 21.11 to 39.87 h, a 1.89-fold increase, P<0.01). These results indicate that Dex-induced upregulation of FN was achieved by preventing protein degradation.

As a multifunctional ECM glycoprotein and a core component of many extracellular matrices, FN plays an important role in regulating intracellular signal transduction and tumor behaviors, including cell adhesion and cell survival (28–30). Therefore, we investigated whether the effect of Dex on melanoma biology was due to upregulation of FN. We silenced the expression of FN using specific small RNA interference (si-FN) and determined the effects of knock-down of FN on melanoma cell adhesion. We found that 100 nM Dex significantly enhanced the adhesive ability of B16F10 and A375 cells (Fig. 3A). Western blotting confirmed that knock-down of FN expression with si-FN almost abolished the Dex-induced expression of FN in A375 cells (Fig. 3B). As shown in Fig. 3C, Dex significantly enhanced the adhesive ability of A375 cells transfected with control small RNA interference (si-con). However, the pro-adhesive effect of Dex in FN knock-down cells was almost completely inhibited, indicating that upregulation of FN contributed to the pro-adhesive effect of Dex in melanoma cells.

In order to further evaluate the role of FN in the adhesion of A375 cells, we examined cell adhesive capacity on uncoated and FN-coated wells (to imitate the over-expression of extracellular FN). The number of adhering cells in the FN-coated group was significantly higher than the uncoated control group (Fig. 3D), indicating that augmentation of extracellular FN enhanced A375 cell adhesive capacity.

Studies have shown that cell adhesion to the ECM is pivotal for survival and growth of most of solid malignant cells, so we further investigated the role of FN upregulation in the effect of Dex on melanoma cell survival in the presence of the chemotherapeutic agent cisplatin. We found that treatment with cisplatin for 24 h decreased A375 cell viability in a dose-dependent manner (Fig. 4A). The survival rate of A375 cells treated with 40 µM cisplatin was decreased to 39% (P<0.01) (Fig. 4B). However, we found that 100 nM Dex significantly weakened the cytotoxic effect of cisplatin and increased the cell survival rate to 68% (P<0.01) (Fig. 4B). Similar results were observed for B16F10 cells (Fig. 4C), indicating that Dex exerted a pro-survival effect on melanoma cells under unfavorable conditions (Fig. 4B and C). Next, we observed the effect of Dex and cisplatin treatment on survival in cells where FN expression was silenced by si-FN. We found that Dex also enhanced cell resistance to cisplatin in A375 cells transfected with si-con (Fig. 4D), but inhibiting FN expression with si-FN significantly reduced the pro-survival effect of Dex in the presence of cisplatin (Fig. 4D). These results indicate that upregulation of FN contributes to Dex-induced melanoma cell survival and chemo-resistance.

The interaction of extracellular FN with one of its receptors, such as CD44, mediates cell adhesion by triggering several signaling pathways, and adhesion consequently regulates cell behaviors, including cell survival (31,32). Therefore, we further investigated the correlation between FN-enhanced cell adhesion and FN-enhanced melanoma cell survival and resistance to chemotherapeutics. We used FN-coated wells to mimic extracellular FN. As shown in Fig. 4E, blocking CD44 signaling with an anti-CD44 antibody significantly abrogated the effect of FN-enhanced cell adhesion in A375 cells, suggesting the partial involvement of extracellular FN/CD44 signaling in enhanced cell adhesion. Furthermore, extracellular FN protected A375 cells against chemotherapeutics, further validating the effect of FN on enhanced cell survival under unfavorable conditions (Fig. 4F). Inhibiting extracellular FN signaling with an anti-CD44 antibody not only reduced cell adhesion, but also significantly reduced the FN-mediated pro-survival effect in the presence of cisplatin (Fig. 4F). These data indicate that cell adhesion is positively linked to melanoma cell survival and that Dex-induced survival and chemo-resistance is mediated through FN upregulation and enhancement of cell adhesion.

The PI3K/AKT pathway plays a critical role in modulating tumor cell proliferation, adhesion and survival (33–35). Therefore, we examined whether the PI3K/AKT pathway was involved in the pro-survival effect of FN on melanoma cells. We found that treatment with recombinant human FN (rh-FN; 10 µg/ml) significantly increased the level of phosphorylated AKT at S473 in A375 cells in a time-dependent manner (Fig. 5A). Treatment with rh-FN also significantly increased cell viability from 36 to 61% (P<0.01) (Fig. 5B). However, inhibiting the PI3K/AKT pathway with wortmannin almost abolished the pro-survival effect of rh-FN in the presence of cisplatin in A375 cells (Fig. 5B). These results indicate that activation of PI3K/AKT by extracellular FN is involved in the survival and enhancement of cisplatin resistance in melanoma cells.