Natural product; matrix metallopeptidase 9 (MMP-9)

Sal A/salvianolic acid A inhibited the growth of A2058 and A375 cells dose-responsively and induced cell cycle arrest at the G2/M phase. Notably, Sal A selectively induces Chk-2 phosphorylation without affecting Chk-1, thereby degrading Chk-2-regulated genes Cdc25A and Cdc2. However, Sal A does not affect the Chk1-Cdc25C pathway. Conclusions: Salvianolic acids, especially Sal A/salvianolic acid A, effectively hinder melanoma cell growth by inducing Chk-2 phosphorylation and disrupting G2/M checkpoint regulation.[1]

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Sal A/salvianolic acid A Reduces Cell Viability and Inhibits Cell Proliferation of Melanoma Cancer Cells [1]
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.

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Sal A/Salvianolic acid A Induces Cell Cycle Arrest at the G2/M Phase in A2058 Cells [1]
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.

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Analysis of Target Proteins of Sal A/Salvianolic acid A in A2058 Cells Using Phospho-Kinase Array [1]
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.

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Sal A/Salvianolic acid A Induces Phosphorylation of Chk-2 Protein in Melanoma Cancer Cells [1]
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.

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Sal A/Salvianolic acid A Suppresses the Expression of Cdc25A and Cdc2 Proteins in Melanoma Cancer Cells [1]
Chk-1 and Chk-2 are recognized as inhibitors of Cdc25C and Cdc25A proteins, contributing to downstream signal transduction events. 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. 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.

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Interfering with the Expression of Cdc25A and Cdc2 Proteins Results in Cell Cycle Arrest in Melanoma Cancer Cells [1]
The effect of Sal A/Salvianolic acid 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).

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Expression of Cdc25A and Cdc2 in Melanoma Cancer Tissues [1]
The aforementioned cell line studies indicated that Salvianolic acid A/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).

In the salvianolic acid A treatment group, salvianolic acid A (SAA) had a substantial positive effect. Rats' stay on the plate can be considerably extended with salvianolic acid A (20 mg/kg). Salvianolic acid A (10 and 20 mg/kg) considerably reduced the brain water content, while the brain water content increased significantly in the model group when compared to the sham surgery group. Salvianolic acid A (5, 10, 20 mg/kg) can both increase the number of neurons and preserve their normal structure in comparison to the model group. It was also shown that salvianolic acid A (20 mg/kg) greatly decreased the overexpression of MMP-9 caused by I/R. MMP-2 expression was not significantly impacted by salvianolic acid A. By attaching themselves to the catalytic domain of MMPs with a strong affinity, tissue inhibitors of metalloproteinases, or TIMPs, can reduce the activity of MMPs [1].

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salvianolic acid A (SAA) is a water-soluble component from the root of Salvia Miltiorrhiza Bge, a traditional Chinese medicine, which has been used for the treatment of cerebrovascular diseases for centuries. The present study aimed to determine the brain protective effects of SAA against cerebral ischemia reperfusion injury in rats, and to figure out whether SAA could protect the blood brain barrier (BBB) through matrix metallopeptidase 9 (MMP-9) inhibition. A focal cerebral ischemia reperfusion model was induced by middle cerebral artery occlusion (MCAO) for 1.5-h followed by 24-h reperfusion. SAA was administered intravenously at doses of 5, 10, and 20 mg·kg−1. SAA significantly reduced the infarct volumes and neurological deficit scores. Immunohistochemical analyses showed that SAA treatments could also improve the morphology of neurons in hippocampus CA1 and CA3 regions and increase the number of neurons. Western blotting analyses showed that SAA downregulated the levels of MMP-9 and upregulated the levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) to attenuate BBB injury. SAA treatment significantly prevented MMP-9-induced degradation of ZO-1, claudin-5 and occludin proteins. SAA also prevented cerebral NF-κB p65 activation and reduced inflammation response. Our results suggested that SAA could be a promising agent to attenuate cerebral ischemia reperfusion injury through MMP-9 inhibition and anti-inflammation activities. [1]

Phospho-Kinase Array Analysis [1]
A2058 cells were placed in 6-cm culture dishes at a density of 1 × 106 and cultured overnight in complete serum. The culture medium was then replaced with 1% FBS-containing DMEM, and 50 µM of Salvianolic acid A/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. 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.

For cell viability assessment, 2 × 104 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 Salvianolic acid A/Sal A or Sal B for 24 h and 48 h. Water-soluble tetrazolium 1 (WST-1) reagent 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.

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Cell Proliferation and Cell Death Assay [1]
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 × 104 and cultured overnight in complete serum. Subsequently, the cells were treated with 0–100 µM of Salvianolic acid A/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. Cell death was assessed by measuring lactate dehydrogenase (LDH) released from cells. Cells were seeded at a density of 2 × 104 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.

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Cell Cycle Analysis [1]
Cells were placed in 6-cm culture dishes at a density of 1 × 106 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 Salvianolic acid A/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, 0.1% Triton-X 100, and 0.2 mg/mL RNase A for 15 min at 37 °C. Flow cytometry analysis was performed using a CytoFLEX S flow cytometer, and cell cycle status was analyzed.

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Western Blotting [1]
Cells were placed in 6-cm culture dishes at a density of 1 × 106 and cultured overnight in complete serum. The culture medium was then replaced with 1% FBS-containing DMEM, and 50 µM of Salvianolic acid A/Sal A was added for an additional 24 h. Cell lysis was performed using radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail 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 were applied for 2 h at room temperature. Finally, enzyme detection was performed using the Amersham enhanced chemiluminescence (ECL) reagent kit, followed by imaging and film scanning.

Preparation of rat middle cerebral artery occlusion (MCAO) model and salvianolic acid A/SAA administration [1]
The rats were randomly divided into sham operation group (n = 35), I/R group (n = 40), I/R + SAA (5, 10, and 20 mgkg–1) groups (n = 40) and Edaravone (5 mgkg–1) group (n = 20). The salvianolic acid A/SAA doses were selected based on our previous pharmacokinetic study. Because SAA was rapidly eliminated from animal body, relatively high doses (5, 10, and 20 mgkg–1) were selected to maintain a high plasma and tissue concentration. SAA was diluted with normal saline. Focal cerebral I/R model was established by MCAO for 1.5-h followed by 24-h reperfusion as described before. First, the rats were fasted overnight with free access to tap water. After anesthetized with 10% chloral hydrate (380 mgkg–1, i.p.), the right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) of the rats were isolated. An 18-mm length of nylon suture ( : 0.2 mm) was i ϕ ntroduced into the ECA lumen and advanced into the ICA to block the origin of the middle cerebral artery. Occlusion was performed for 1.5-h, then the nylon suture was withdrawn for reperfusion. The sham operation rats received all surgical procedures but without the suture inserted. Right after the reperfusion, I/R + SAA groups were intravenously given different concentrations of salvianolic acid A/SAA (5, 10, and 20 mgkg–1). The positive group received 5 mgkg–1 of Edaravone. The sham operation group and I/R group received equal volume of normal saline. After 12-h of the reperfusion, the above administration method was performed again to maintain relatively high plasma concentrations of SAA and Edaravone. During the operation procedure, the rectal temperature of the rats was kept between 36.5 and 37.5 °C. Rat body temperature was carefully monitored during the post-operation period. After the experiment, the animals were housed individually with free access to food and tap water.

[1]. Salvianolic acid A attenuates ischemia reperfusion induced rat brain damage by protecting the blood brain barrier through MMP-9 inhibition and anti-inflammation. Chin J Nat Med. 2018 Mar;16(3):184-193.

[2]. Inhibition of Melanoma Cell Growth by Salvianolic Acid A through CHK2-CDC25A Pathway Modulation. Front Biosci (Landmark Ed). 2024 Jun 12;29(6):213.

Salvianolic acid A is a stilbenoid.
Salvianolic acid A is under investigation in clinical trial NCT03908242 (Phase I Study of Continuous Administration of Salvianolic Acid A Tablet).
Salvianolic acid A has been reported in Salvia miltiorrhiza, Origanum vulgare, and other organisms with data available.

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In conclusion, our results from the present study suggested that SAA owned excellent neuroprotective effects against I/R injury, including BBB protection and anti-inflammation effects. These findings indicated a promising therapeutic potential for SAA as a leading compound towards the treatment of ischemic stroke. [1]

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Background: This study investigated the impact of salvianolic acids, derived from Danshen, on melanoma cell growth. Specifically, we assessed the ability of Salvianolic acid A (Sal A) to modulate melanoma cell proliferation. Methods: We used human melanoma A2058 and A375 cell lines to investigate the effects of Salvianolic acid A/Sal A on cell proliferation and death by measuring bromodeoxyuridine incorporation and lactate dehydrogenase release. We assessed cell viability and cycle progression using water soluble tetrazolium salt-1 (WST-1) mitochondrial staining and propidium iodide. Additionally, we used a phospho-kinase array to investigate intracellular kinase phosphorylation, specifically measuring the influence of Sal A on checkpoint kinase-2 (Chk-2) via western blot analysis.
Sal A/Salvianolic acid A and B constitute primary salvianolic acid derivatives from Danshen, with Sal A uniquely demonstrating melanoma growth inhibition properties. Furthermore, the Chk2-Cdc25A-Cdc2 signaling pathway plays a role in the G2 checkpoint regulation of the melanoma cell cycle induced by Sal A. The results of this study define the pathways involved in the anticancer efficacy of Danshen and suggest Sal A as a therapeutic agent for melanoma. [2]

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Sal A is structurally analogous to Danshensu and can be derived by adding ortho-vanillin to Danshensu. The anticancer properties of Danshensu were extensively documented despite a limited focus on melanoma. Danshensu does not reduce the proliferation of B16F10 melanoma cells; however, it inhibits VEGF, MMP-2, and MMP-9 secretion, thereby attenuating cellular invasiveness. These results reveal that Danshensu’s influence on melanoma may be independent of cell proliferation, indicating a divergent mechanism from Sal A’s regulation of the G2 checkpoint in the cell cycle. The potential parallelism between Sal A and Danshensu in impeding melanoma cell invasion warrants further investigation. [2]

C26H22O10

494.4469

494.121

96574-01-5

5281793

Light yellow to yellow solid powder

1.6±0.1 g/cm3

858.7±65.0 °C at 760 mmHg

164-167ºC

292.9±27.8 °C

0.0±0.3 mmHg at 25°C

1.790

4.23

7

10

9

36

798

1

C1=CC(=C(C=C1C[C@H](C(=O)O)OC(=O)/C=C/C2=C(C(=C(C=C2)O)O)/C=C/C3=CC(=C(C=C3)O)O)O)O

YMGFTDKNIWPMGF-UCPJVGPRSA-N

InChI=1S/C26H22O10/c27-18-7-2-14(11-21(18)30)1-6-17-16(4-9-20(29)25(17)33)5-10-24(32)36-23(26(34)35)13-15-3-8-19(28)22(31)12-15/h1-12,23,27-31,33H,13H2,(H,34,35)/b6-1+,10-5+/t23-/m1/s1

(2R)-3-(3,4-dihydroxyphenyl)-2-[(E)-3-[2-[(E)-2-(3,4-dihydroxyphenyl)ethenyl]-3,4-dihydroxyphenyl]prop-2-enoyl]oxypropanoic acid

Salvianolic acid A; 96574-01-5; SALVIANOLIC ACID; (R)-3-(3,4-Dihydroxyphenyl)-2-(((E)-3-(2-((E)-3,4-dihydroxystyryl)-3,4-dihydroxyphenyl)acryloyl)oxy)propanoic acid; Dan Phenolic Acid A; (+)-Salvianolic acid A; (2R)-3-(3,4-dihydroxyphenyl)-2-[(E)-3-[2-[(E)-2-(3,4-dihydroxyphenyl)ethenyl]-3,4-dihydroxyphenyl]prop-2-enoyl]oxypropanoic acid; Salvianolic-acid-A;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~83.33 mg/mL (~168.53 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.06 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 2.17 mg/mL (4.39 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.17 mg/mL (4.39 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)