The fucoidan from Fucus vesiculosus was purchased from Sigma (St. Louis, MO, USA). Live and dead viability/cytotoxicity kits and MTT assay kits were purchased from Thermo Fisher Scientific (Rockford, IL, USA). MS Collagen (type 1 atelo-collagen from porcine skin) was obtained from MS Bio, Inc. (Gyeonggi, Korea). Antibodies recognizing p-Bcl-2 (Ser70) (sc-293128), Bcl-2 (sc-7382), H2B (sc-515808), caspase-3 (SC-56052) and actin (SC-58673) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies recognizing p-AKT (Ser473) (#9271), p-PTEN (Ser380) (#9551), p-ERK (#9101), p-p38 (#9211), p-JNK (#9251), and p-Bcl-2 (Thr56) (#2875) were obtained from Cell Signaling Technology (MA, USA). Apoptosis western blot cocktail (pro/p17-caspase-3, cleaved PARP1, muscle actin) (ab136812) was obtained from Abcam (Cambridge, UK). The A735 human malignant melanoma cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). The human fibroblast cell line CCD-986 SK cells were obtained from the Korea Cell Line Bank (KCLB, Seoul, Korea). The A375 and CCD-986 SK cells were cultured in complete medium [DMEM-high glucose supplemented with 10% FBS and 1% PS (penicillin-streptomycin)]. To assess the dose-dependent effects of LMW-F on A375 cell viability, cells were treated with LMW-F at concentrations ranging from 0, 1, 5, 10, 20, 50 μg/mL and incubated for 24 h. Cell viability was then assessed using a live/dead assay and MTT assay, as described previously [13–15]. 1–2 mm PDME fragments were obtained from patient-derived melanoma xenograft models, which were sourced from Seoul St. Mary’s Hospital (Seoul, Korea), as described in our prior studies [13–15].

We prepared stock concentrations of LMW-F (10 and 50 mg/mL) using a modified ultrasonication method and PBS buffer, as previously described [8,9]. LMW-F was dissolved in PBS buffer and treated in an ultrasonic bath (DATHAN Scientific, Korea) under conditions of 60 kHz frequency, 25°C temperature, 30 min time, 117 W power, 100% amplitude, and sweep mode. The LMW-F stock concentrations were further diluted for subsequent experiments.

3D-PCS-PDMEs were prepared as previously described [13,14]. Uniform and stable 3D-PCSs were manufactured using an extrusion-based 3D printing method with an X Printer (T&R Biofab Co., Ltd., Gyeonggi-do, Korea). 3D-PCS-PDMEs were cultured in a-Minimum Essential Medium with 10% fetal calf serum, 2 mM L-glutamine, and 1% PS in the presence or absence of LMW-F in a dose-dependent manner. They were then incubated for 14 days at 37°C in a 5% CO₂ incubator.

Cell viability and proliferation assays were conducted using a live and dead assay and MTT assay, as previously described [13–15]. Absorbance of MTT assay was measured using a spectrometer (Emax; Molecular Devices, Sunnyvale, CA, USA). To further assess the viability of A375 cells, a double staining kit of calcein-acetoxymethyl (AM) and propidium iodide (PI) was used to fluorescently label live and dead cells, respsectively [13,14]. Cells were incubated at 37°C in a 5% CO₂ incubator for 30 min with protection from light. Images were acquired using an Olympus FV1200 confocal microscope with laser lines at 405, 473, 559, and 635 nm.

The H&E staining protocol was performed as Formalin-fixed paraffin-embedded tissue sections were deparaffinized and rehydrated through xylene and graded alcohol washes [13]. The sections were then immersed in Harris hematoxylin solution for a duration of approximately 5–10 min to facilitate nuclear staining. Following hematoxylin staining, the slides were differentiated using acid alcohol or Scott’s tap water for a few seconds. The slides were rinsed with distilled water thereafter. For cytoplasmic and extracellular staining, the slides were treated with eosin Y solution for a brief period of 1–5 min, and subsequently rinsed with distilled water. Dehydration of the slides was achieved through sequential immersion in increasing concentrations of ethanol (70%, 95%, and 100%). Finally, clearing of the tissue sections was accomplished using xylene or a xylene substitute. Following dehydration and clearing, coverslips were mounted onto the tissue sections using DPX mountant. The mountant was allowed to dry, and the edges of the coverslips were sealed using clear nail polish or a similar sealant. The slides were then ready for observation and analysis under a light microscope.

Sample sections were deparaffinized and subsequently rehydrated via successive washes in xylene and graded alcohols. Following these preparations, the sections were immersed in Weigert’s iron hematoxylin solution, imparting a deep blue-black hue to the nuclei. To achieve optimal staining contrast, an acid-alcohol rinse was then performed, effectively facilitating the differentiation of nuclei. Subsequently, the sections were stained in a solution comprising Biebrich scarlet-acid fuchsin, which imparts a vivid red coloration to the cytoplasm and muscle fibers. To ensure specificity in highlighting the connective tissue, a phosphomolybdic acid solution was applied, selectively binding to collagen fibers. This acid solution enhances the staining precision by accentuating the collagen fibers within the tissue. Lastly, counterstaining was performed using aniline blue solution, effectively coloring the background and muscle fibers. Aniline blue demonstrates preferential affinity towards collagen fibers and the extracellular matrix, resulting in their conspicuous blue appearance. The stained sections were subsequently subjected to dehydration, clearing, and finally mounted with coverslips, enabling microscopic examination.

The samples were disrupted using a homogenizer, after which an ice-cold RIPA protein extraction solution with a protease inhibitor cocktail (iNtRON biotechnology, Inc., Seoul, Korea) was added, and the samples were homogenized with stainless steel beads (Qiagen, Cam, USA). Protein concentrations were assessed using a BCA-kit (Thermo Scientific, Rockford, IL, USA). An equal amount of protein (50 μg) from each sample was loaded onto 10% to 12% SDS gel, and transferred to a PVDF membrane (Merk Millipore, MA, USA). The membranes were blocked for 2 h at room temperature with 5% nonfat dry milk in PBS containing 0.1% Tween-20 and incubated with anti-bodies (1:1000) overnight at 4°C (Suppl. Table 1). After washing three times, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000) at RT for 2 h and visualized with a chemiluminescence substrate. Quantitative analysis of the western blot images was performed using ImageJ Software.

To investigate the effects of LMW-F on melanoma cell viability and proliferation, we treated A375 cells with LMW-F at concentrations ranging from 0 to 50 μg/mL for 24 and 72 h. We evaluated cell viability and proliferation using MTT assays and live/dead staining. Treatment with 50 μg/mL LMW-F did not affect A375 cell viability (Fig. 1A). However, treatment with 5 μg/mL LMW-F resulted in a 40% inhibition of cell proliferation, while treatment with 50 μg/mL LMW-F led to an approximately 80% inhibition of cell proliferation (Fig. 1B). These effects of LMW-F on A375 cell viability and proliferation were confirmed by live/dead staining (Fig. 1C). To examine potential dose-dependent effects of LMW-F on normal cells, we treated CCD-986 SK fibroblast with various concentrations of LMW-F for 24 and 72 h. Our results showed that LMW-F treatment did not affect CCD-986 SK fibroblast viability or proliferation in a dose-dependent manner (Suppl. Fig. S1), indicating that LMW-F has an anti-proliferation effect on A375 melanoma cells. These findings suggested that LMW-F inhibits the proliferation of A375 melanoma cells in a dose-dependent manner, while having no significant effect on the viability or proliferation of CCD-986 SK fibroblasts.

To investigate the effects of LMW-F on proliferation-related signaling pathways in melanoma cells, we performed western blot analyses to detect key cellular pathways, including AKT, ERK, p38, and JNK, which are important signaling transduction pathways, and types of protein kinases in cells (Figs. 2A–2F). Untreated A375 cells exhibited AKT phosphorylation at Ser473 for at least 8 h. The phosphorylation of PTEN at Ser473 increased for 24 h, whereas LMW-F related phosphorylation of PTEN at Ser380 decreased after 16 h (Figs. 2A–2D). Treatment with LMW-F in A375 cells induced phosphorylation of AKT at Ser475 after 10 min, followed by increased phosphorylation at 1 h. After 4 h, LMW-F related phosphorylation of AKT at Ser473 was downregulated, and phosphorylation of PTEN at Ser380 increased (Figs. 2B–2D). These findings might be because phosphorylation of PTEN at Ser380 regulates PTEN stability and AKT dephosphorylation at (Ser473), consequently suppressing tumorigenesis [16,17]. In the presence of LMW-F, the PTEN pathway at Ser380 negatively regulates the AKT pathway (Figs. 2A, 2C, and 2D). Treatment with LMW-F slightly affects the EKR, p38, or JNK pathways compared to untreated A375 cells (Suppl. Figs. S2 and S3). Furthermore, LMW-F treatment downregulated the phosphorylation of Bcl-2 at Thr56 and the expression of Bcl-2 (Figs. 2B, 2E, and 2F). Phosphorylation of Bcl-2 at Ser70 has been associated with cell survival and proliferation by enhancing the anti-apoptotic activity of Bcl-2, whereas phosphorylation of Bcl-2 at Thr56 has been reported to play a role in promoting apoptosis [18,19]. These results suggest that LMW-F may inhibit cell proliferation and promote apoptosis in melanoma cells by regulating PTEN/AKT signaling and Bcl-2 phosphorylation.

Previous studies in our laboratory utilized 3D-PCS and PDME fragments to generate 3D-PCS-PDMEs [13,14]. To assess the effects of LMW-F (0, 10, 50 μg/mL) for 14 days (Fig. 3), we applied 3D-PCS-PDMEs. We observed distinct, dose-dependent morphological outgrowths of PDME (Fig. 3A). At a concentration of 50 μg/mL, LMW-F inhibited the morphological outgrowth of 3D-PCS-PDMEs compared to the untreated group (Fig. 3A). The WHO-RECIST measurement demonstrated a greater outgrowth of PDME in the absence of LMW-F (50 μg/mL) (Fig. 3B). We also measured the diameter and thickness of the 3D-PCS in each group. It is well-known that melanoma cells can secrete enzymes, such as matrix metalloproteinase (MMPs), that can degrade components of the extracellular matrix, including collagen [20]. As shown in Fig. 4, the diameter and thickness of the 3D-PCS-PDMEs remained unchanged in the presence of LMW-F, while the untreated group exhibited a decrease in the thickness of 3D-PCS. To further confirm these observations, we evaluated the cell viability and histological staining of the 3D-PCS-PDMEs in the presence or absence of LMW-F using MTT assays and H&E/MT staining, respectively. The results of the MTT assays and H&E/MT staining are consistent with the WHO-RECIST measurement (Fig. 5). Therefore, these observations indicate that LMW-F has potential as a candidate for inhibiting melanoma outgrowth.

If the anti-melanoma effects of LMW-F contribute to cell proliferation in melanoma, one of the key regulators of apoptosis, such as caspase-3, could be associated with the anti-melanoma performance of LMW-F. To investigate this hypothesis, we performed western blot analysis on A375 cells and 3D-PCS-PDMEs treated with varying concentrations of LMW-F (0, 10, 50 μg/mL) at the indicated time points (Figs. 6A–6F). In both the A375 cells and 3D-PCS-PDMEs, LMW-F (50 μg/mL) resulted in changes in the activity of caspase-3 (Figs. 6A, 6B, 6D, and 6E). Cleaved PARP was markedly observed in LMW-F-treated 3D-PCS-PDMEs compared to the untreated group (Figs. 6B and 6E). However, there was no detection of cleaved PARP and cleaved caspase-3 in LMW-F-treated A375 cells. H2B is a histone protein that plays an essential role in the process of programmed cell death, including apoptosis. Interestingly, the expression of H2B dramatically decreased in both A375 cells and 3D-PCS-PDMEs in the absence of LMW-F (Figs. 6A, 6C, 6D, 6F, and Suppl. Fig. S4). Overall, our results indicate that LMW-F may have anti-melanoma effects in A375 cells and 3D-PCS-PDMEs by changing the activity of caspase-3 and inhibiting H2B expression.

Student’s t-tests (for comparisons of two groups) or a one-way analysis of variance (ANOVA, for comparisons of three or more groups) followed by Tukey post hoc tests were used for the statistical analyses. SPSS software ver. 17.0 (SPSS, Chicago, IL, USA) was used. A value of p < 0.05 was considered significant. Data are compressed as means ± standard error of the mean (SEM). Data analysis was carried out using GraphPad Prism software (GraphPad Software Inc. Sandie go, CA, USA). *p < 0.05–0.01, **p < 0.01–0.001, and ***p < 0.001 vs. corresponding controls. All errsssor bars represent the standard deviation of three or more biological replicates.