Human malignant melanoma cell lines A2058 and A375 were purchased from American Type Culture Collection (ATCC) and cultured in a medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/mL penicillin, and 100 µg/mL streptomycin. The cells were maintained in a humidified incubator at 37 °C with 5% CO${}_2$ and passaged every 3–4 days to sustain exponential growth. The cells were validated through Genomics by cross-referencing the short tandem repeat (STR) results with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) STR Profile database. Cell staining analysis using DAPI was performed monthly to exclude mycoplasma contamination.

For cell viability assessment, 2 $\times$ 10${}^4$ cells were seeded in triplicate in 24-well culture plates and cultured overnight in complete serum. After replacing the cell culture medium with 1% FBS-containing medium, the cells were treated with 0–100 µM of Sal A (Sigma-Aldrich, St. Louis, MO, USA) or Sal B (Sigma-Aldrich) for 24 h and 48 h. Water-soluble tetrazolium 1 (WST-1) reagent (Merck, Darmstadt, Germany) was added to the culture medium and incubated for an additional 4 h. Subsequently, the supernatant from each group of cells was transferred to a 96-well plate, and absorbance at 450 nm and 650 nm (as a reference) was measured using an EIA reader (Infinite F200, Tecan, Durham, NC, USA).

Cell proliferation was analyzed by incorporating bromodeoxyuridine (BrdU) into DNA. Briefly, cells were seeded in triplicate in 24-well culture plates at a density of 1 $\times$ 10${}^4$ and cultured overnight in complete serum. Subsequently, the cells were treated with 0–100 µM of Sal A or Sal B in a medium containing 1% FBS for 24 h. The proliferation status of cells was measured according to the manufacturer’s instructions (Roche Diagnostics, Mannheim, Germany). Cell death was assessed by measuring lactate dehydrogenase (LDH) released from cells. Cells were seeded at a density of 2 $\times$ 10${}^4$ in triplicate in 24-well culture plates and cultured overnight in complete serum. After treating the cells with 0–100 µM of Sal A or Sal B for 24 h, cell culture supernatant was collected, and cell death was measured following the manufacturer’s instructions (Cytotoxicity Detection Kit, Roche Diagnostics, Basel, Switzerland).

Cells were placed in 6-cm culture dishes at a density of 1 $\times$ 10${}^6$ and cultured overnight in complete serum. The culture medium was then replaced with 1% FBS-containing Dulbecco’s modified eagle medium (DMEM), and 50 µM of Sal A was added for an additional 24 h and 48 h. After cell collection, cells were fixed with 80% ethanol and stored at –20 °C for at least 24 h. Subsequently, cells were stained with 20 µg/mL propidium iodide (Thermo Fisher Scientific), 0.1% Triton-X 100 (Thermo Fisher Scientific), and 0.2 mg/mL RNase A for 15 min at 37 °C. Flow cytometry analysis was performed using a CytoFLEX S flow cytometer (Beckman Coulter, Brea, IN, USA), and cell cycle status was analyzed.

A2058 cells were placed in 6-cm culture dishes at a density of 1 $\times$ 10${}^6$ and cultured overnight in complete serum. The culture medium was then replaced with 1% FBS-containing DMEM, and 50 µM of Sal A was added for 12–24 h. Cell lysates were collected using the lysis buffer provided in the Proteome Profiler™ Human Phospho-Kinase Array Kit (Catalog No: ARY003C, R&D Systems, Minneapolis, MN, USA). Each array was loaded with 500 µg of protein, and subsequent analysis was performed according to the manufacturer’s instructions. Imaging and film scanning were carried out on the Bio-Rad ChemiDoc XRS${}^{+}$ system (Hercules, CA, USA).

Cells were placed in 6-cm culture dishes at a density of 1 $\times$ 10${}^6$ and cultured overnight in complete serum. The culture medium was then replaced with 1% FBS-containing DMEM, and 50 µM of Sal A was added for an additional 24 h. Cell lysis was performed using radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and a phosphatase inhibitor. After collecting cell lysates, 40–100 µg of protein from each cell extract was separated by electrophoresis on a 12% polyacrylamide gel. Subsequently, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, blocked, and incubated with various primary antibodies at 4 °C for 12 h. Afterward, horseradish peroxidase-conjugated secondary antibodies (Calbiochem, Cambridge, MA, USA) were applied for 2 h at room temperature. Finally, enzyme detection was performed using the Amersham enhanced chemiluminescence (ECL) reagent kit (Amersham, Bucks, UK), followed by imaging and film scanning.

Antibodies used in this study included anti-phospho-Chk-1 (Ser 345), anti-phospho-Chk-2 (Thr68), anti-Chk-1, anti-Chk-2, anti-phospho-p53 (Ser 15), anti-Cdc25A, anti-Cdc25C, anti-Cyclin A, anti-Cyclin B, anti-Cdc2, all purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-$\beta$-actin antibody and horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies were purchased from Sigma-Aldrich.

Cells were transfected with siRNA using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) and according to the manufacturer’s recommendations. Briefly, 30 µM of siRNA was first dissolved in opti-minimum essential medium (MEM) culture medium and left at room temperature for 15 minutes, then placed into cells that had been cultured in Opti-MEM and cultured at 37 °C for 6 h. After removing the siRNA and Opti-MEM, the culture medium was then replaced with 1% FBS-containing DMEM, and the cells were cultured for an additional 48 h. Cdc25A and Cdc2 siRNAs were purchased from Sigma-Aldrich and the sequences of the siRNAs targeted to Cdc25A and Cdc2 are 5${}^{\prime }$-GAUACAAGGAGUUCUUUAU[dT][dT]-3${}^{\prime }$ inter associated with 5${}^{\prime }$-AUAAAGAACUCCUUGUAUC[dT][dT]-3${}^{\prime }$ and 5${}^{\prime }$-AGCCUAGCAUCCCAUGUCA[dT][dT]-3${}^{\prime }$ inter associated with 5${}^{\prime }$-UGACAUGGGAUGCUAGGCU[dT][dT]-3${}^{\prime }$, respectively.

The melanoma tissue cDNA array (MERT301) was purchased from Origene Technologies (Rockville, MD, USA) and supplied the melanoma tissue cDNA array (MERT301). Specific primer mixtures and SYBR Green Master Mix (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) in a real-time polymerase chain reaction (PCR) machine (StepOne PlusTM, Waltham, MA, USA) were used to detect gene expression. The primer sequence for Cdc25A is as follows: forward primer: 5${}^{\prime }$-CGTCGTGAAGGCGCTATTTG-3${}^{\prime }$ and reverse primer: 5${}^{\prime }$-ACAGCTTCTGAGGTAGGGAA-3${}^{\prime }$. The primer sequence for Cdc2 is as follows: forward primer: 5${}^{\prime }$-CTTGGCTTCAAAGCTGGCTC-3${}^{\prime }$ and reverse primer: 5${}^{\prime }$-CATGGCTACCACTTGACCTGT-3${}^{\prime }$. Finally, Cdc25A and Cdc2 expressions were normalized to individual actin expression.

Cell viability, proliferation, and death metrics were reported as mean $\pm$ standard deviations based on a minimum of three replicates per condition. Statistical significance was assessed via Student’s t-test, with a p-value threshold of less than 0.05.

This study assessed the influence of salvianolic acids, specifically Sal A and Sal B, on melanoma cell viability using A2058 cells as a model. Fig. 1A showed that a 48-hour exposure to Sal A at concentrations ranging from 10 to 100 µM notably reduced A2058 cell viability by 17.7 to 38.4%. In contrast, Sal B did not significantly affect viability. Subsequent analysis revealed that Sal A diminished A2058 cell proliferation to 62.3%–77.6% relative to the control, as depicted in Fig. 1B. Notably, Sal A did not trigger apoptosis in these cells (Fig. 1C). Conversely, Sal B exhibited no appreciable impact on either cell proliferation or death. These results suggest that the decrease in A2058 cell viability caused by Sal A is mainly due to the inhibition of cell proliferation.

Fig. 1.

The effects of Sal A and Sal B on the viability, proliferation, and death of A2058 cells. A2058 cells were treated with different concentrations of Sal A and Sal B for 24 h and 48 h, and the impact on cell viability (A), cell proliferation (B), and cell death (C) was measured using water soluble tetrazolium salt-1 (WST-1), BrdU incorporation assay, and LDH release, respectively. *p 0.05 compared with the control group. Sal A, salvianolic acid A; Sal B, salvianolic acid B; BrdU, bromodeoxyuridine; LDH, lactate dehydrogenase.

Parallel observations were recorded with A375 cells, where Sal A elicited a decline in both cell viability and proliferation without inducing apoptosis, as delineated in Supplementary Fig. 1. Similarly, Sal B had no significant effect on A375 cell viability, cell proliferation, or cell death (Supplementary Fig. 1). Since Sal B did not have any impact on melanoma cancer cells. We focused on Sal A for the subsequent experiments; a 50 µM concentration of Sal A was selected to optimize potential therapeutic dosages.

Given the inhibitory effect of Sal A on melanoma cell proliferation, further investigation into its impact on cell cycle progression was conducted. Fig. 2 reveals that a 48-hour treatment with Sal A precipitated a reduction in the G1 phase population and an augmentation in the G2/M phase population of A2058 cells relative to controls, suggesting an induced cell cycle arrest at the G2/M transition. Concordant findings were documented in A375 cells, as presented in Supplementary Fig. 2.

Fig. 2.

The effect of Sal A on the cell cycle of A2058 cells. A2058 cells were treated with 50 µM Sal A for 24 h and 48 h, fixed and stained with propidium iodide (PI), and the cell cycle was analyzed using flow cytometry. The presented data are representative graphs and summarized as mean $\pm$ standard deviations in percentage. *p 0.05 compared with the control group.

To identify the protein targets of Sal A in melanoma cells, a human phospho-kinase array was employed to profile the expression of key signaling proteins in A2058 cells post Sal A exposure. Fig. 3 indicated that a 12-hour treatment with Sal A upregulated the levels of phosphorylated p53 and Chk-2 compared to untreated controls. Notably, after 24 hours of Sal A treatment, the phosphorylation of Chk-2 remained pronounced, whereas phosphorylated p53 levels did not differ substantially from controls. Sal A appeared to selectively modulate the phosphorylation status of p53 and Chk-2 without broadly impacting other phosphorylated proteins within the cellular signaling network.

Fig. 3.

The effect of Sal A on protein phosphorylation in A2058 cells. A2058 cells were treated with 50 µM Sal A for 12–24 h, and cell extracts were collected for human phospho-kinase array assays.

To corroborate the phospho-kinase array findings, Western blot analyses were conducted on cell lysates from Sal A-treated melanoma cells. The results from Fig. 4A indicate that treatment with the chemotherapeutic drug Cisplatin (CDDP) significantly improved the phosphorylation of Chk-1 and Chk-2 levels in A2058 cells after 24 h, whereas treatment with Sal A only slightly increased the phosphorylated Chk-1 levels but markedly increased the phosphorylated Chk-2 levels in A2058 cells. A similar upregulation of phosphorylated Chk-2 was detected in A375 cells (Fig. 4B). Furthermore, we observed that 6-h Sal A treatment of A2058 cells with Sal A for 6 h induced phosphorylation of Chk-2 in the cells and maintained it for at least 24 h (Supplementary Fig. 3). Additionally, the phosphorylated p53 expression was evaluated after 24 h of Sal A treatment. Fig. 4A,B indicate no notable alteration in phosphorylated p53 levels, suggesting that p53 does not play a major role in Sal A modulation of the melanoma cell cycle.

Fig. 4.

The effect of Sal A on the expression of phospho-Chk-1, phospho-Chk2, and phospho-p53 in melanoma cells. A2058 cells (A) or A375 cells (B) were treated with 50 µM Sal A along with 10 µM Cisplatin (CDDP) for 24 h, and cell extracts were collected. Levels of Chk-1, Chk-2, and p53 were analyzed using western blotting.

Chk-1 and Chk-2 are recognized as inhibitors of Cdc25C and Cdc25A proteins, contributing to downstream signal transduction events [19, 20]. Considering the upregulation of phosphorylated Chk-2 by Sal A, the investigation was expanded to examine its effects on Cdc25A and Cdc25C. Fig. 5A,B showed that Sal A suppressed Cdc25A expression in both A2058 and A375 cell lines, while Cdc25C levels were unaltered. Given the pivotal function of Cyclin A, Cyclin B, and Cdc2 in the G2/M phase transition, with Cdc2 being modulated by Cdc25A and Cdc25C [21, 22, 23]. Their expressions post Sal A exposure were also evaluated. Although Cyclin A and Cyclin B responses to Sal A varied across melanoma cell types, a consistent downregulation of Cdc2 was observed in both A2058 and A375 cells. Hence, the findings suggest that Sal A attenuates the production of both Cdc25A and Cdc2 proteins, implicating a targeted disruption of cell cycle regulation in melanoma cells.

Fig. 5.

The effect of Sal A on the expression of G2 checkpoint-related proteins in melanoma cells. A2058 cells (A) or A375 cells (B) were treated with 50 µM Sal A for 24 h, and cell extracts were collected. Levels of Cdc25A, Cdc25C, Cyclin A, Cyclin B, and Cdc2 were analyzed using western blotting.

The effect of Sal A, an inhibitory compound, on cell viability and proliferation in melanoma cancer cells was explored by investigating its impact on the degradation of Cdc25A and Cdc2 via the Chk2 pathway. Additionally, the direct influence of Cdc25A and Cdc2 degradation on cell growth was examined. To achieve this, A2058 cells were transfected with Cdc25A or Cdc2 siRNA, resulting in a decrease in cell viability by 17.2% and 26.1%, respectively (Fig. 6A). Moreover, cell proliferation was reduced by 13.9% and 21.7%, respectively (Fig. 6B). Similar trends were observed in A375 cells (Supplementary Fig. 4). Furthermore, the impact of inhibiting Cdc25A and Cdc2 on cell cycle progression was investigated. The results suggest that although the effect on cell cycle arrest is not as prominent as that seen with Sal A treatment, inhibiting the expression of either Cdc25A or Cdc2 significantly induces cell arrest in the G2/M phase (Fig. 6C).

Fig. 6.

The effects of Cdc25A and Cdc2 siRNA on the cell cycle of A2058 cells. A2058 cells were transfected with Cdc25A or Cdc2 siRNA for 48 h, and cell viability (A), cell proliferation (B), and the influence on the cell cycle (C) were measured using WST-1, BrdU incorporation assay, and PI staining, respectively. *p 0.05 compared with the control group.

The aforementioned cell line studies indicated that Sal A inhibits the growth of melanoma cancer cells through the Chk-2 pathway. Therefore, we further investigated the activation status of Chk-2 during melanoma cancerization by analyzing Cdc25A and Cdc2 expression. Analysis results from purchased melanoma cancer tissue arrays indicate no significant difference compared with normal cells despite an increasing trend in intracellular Cdc25A and Cdc2 expression in cancer tissues (Supplementary Fig. 5).