Natural product; STAT3

Danshen (Salvia miltiorrhiza Bunge) is widely used in traditional Chinese medicine. In our study, the in vivo protective effect of danshen in prostate cancer patients was validated through data from the National Health Insurance Research Database in Taiwan. In vitro, we discovered that dihydroisotanshinone I (DT), a bioactive compound present in danshen, can inhibit the migration of both androgen-dependent and androgen-independent prostate cancer cells. In addition, we noted that DT substantially inhibited the migratory ability of prostate cancer cells in both a macrophage-conditioned medium and macrophage/prostate cancer coculture medium. Mechanistically, DT both diminished the ability of prostate cancer cells to recruit macrophages and reduced the secretion of chemokine (C-C motif) ligand 2 (CCL2) from both macrophages and prostate cancer cells in a dose-dependent manner. Moreover, DT inhibited the protein expression of p-STAT3 and decreased the translocation of STAT3 into nuclear chromatin. DT also suppressed the expression of tumor epithelial-mesenchymal transition genes, including RhoA and SNAI1. In conclusion, danshen can prolong the survival rate of prostate cancer patients in Taiwan. Furthermore, DT can inhibit the migration of prostate cancer cells by interrupting the crosstalk between prostate cancer cells and macrophages via the inhibition of the CCL2/STAT3 axis. These results may provide the basis for a new therapeutic approach toward the treatment of prostate cancer progression[1].

Dihydroisotanshinone I is a phenanthrenequinone derivative isolated from the roots of Salvia trijuga Diels. The present study demonstrated the hepatoprotective effect of dihydroisotanshinone I against menadione-induced cytotoxicity in a primary culture of rat hepatocytes. Pretreating the cells with dihydroisotanshinone I at concentrations ranging from 2.5 microM to 20 microM for 24 hours caused dose-dependent protection against hepatotoxicity induced by menadione. Intracellular glutathione level and activity of DT-diaphorase have been suggested to play important roles in menadione-induced cytotoxicity. However, treating the hepatocytes with 20 microM dihydroisotanshinone I for 24 hours did not cause a significant change in glutathione level and DT-diaphorase activity. On the contrary, adding dihydroisotanshinone I to freshly isolated hepatocytes at concentrations between 50 nM to 200 nM inhibited NADH-induced superoxide production dose-dependently as indicated by the decrease of lucigenin-amplified chemiluminescence. In addition, dihydroisotanshinone I at concentrations ranging from 5 microM to 20 microM inhibited tert-butyl hydroperoxide-induced lipid peroxidation dose-dependently in isolated hepatocytes as indicated by the level of malondialdehyde. These results suggest that the protective action of dihydroisotanshinone I against menadione-induced hepatotoxicity is attributed to its antioxidant properties including the free radical scavenging activity and inhibition of lipid peroxidation. Abbreviations. DTD:DT-diaphorase GSH:glutathione LDH:lactate dehydrogenase MDA:malondialdehyde MTT:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide TBHP: tert-butyl hydroperoxide[2].

Cell migration assays were performed as described previously. For the migration of monoculture of human prostate cancer cells, the human prostate cancer cell lines (DU145 cells, PC-3 cells, or 22Rv1 cells) (1 × 105 cells/well) were plated in the upper chambers of Transwell plates with 8-μm pore polycarbonate membrane inserts in a medium without FBS. A medium with FBS was plated in the lower chambers. After treatment with or without DMSO and with indicated concentrations of DT for 16–24 hours, the cells that have migrated into the bottom were fixed and stained using 1% toluidine blue, and the numbers of migratory cells were averaged after counting 6 randomly selected fields. For the prostate cancer recruitment assay, RAW264.7 cells or THP-1 cells (1 × 105 cells/well) were treated with or without DMSO and with an indicated concentration of DT for 24 hours. The conditioned medium or control medium was then collected and plated in the lower chambers. The indicated parental human prostate cancer cells (1 × 105 cells/well) were plated in the upper chambers in the medium without FBS. After incubation for 24 hours, the cells that have migrated into the bottom were fixed and stained using 1% toluidine blue, and the numbers of migratory cells were averaged after counting 6 randomly selected fields. For a prostate cancer cell and macrophage direct mixed co-culture system, the indicated prostate cancer cell line (DU145 cells, PC-3 cells, or 22Rv1 cells) (1 × 105 cells/dish) was plated in a 6-mm dish overnight. After becoming adherent, the indicated macrophages (RAW264.7 cells or THP-1 cells) (1 × 105 cells/dish) were plated in the same dish. After becoming adherent, the direct mixed co-culture system was treated with or without DMSO and with indicated concentrations of DT for 24 hours. Either the conditioned medium or the control medium was then plated in the lower chambers. The indicated parental human prostate cancer cells (1 × 105 cells/well) were plated in the upper chambers in the medium without FBS. After incubation for 24 hours, the cells that have migrated into the bottom were fixed and stained using 1% toluidine blue, and the numbers of migratory cells were averaged after counting 6 randomly selected fields. Each sample was assayed in triplicate, and each experiment was repeated at least twice[1].

[1]. Anti-cancer effect of danshen and dihydroisotanshinone I on prostate cancer: targeting the crosstalk between macrophages and cancer cells via inhibition of the STAT3/CCL2 signaling pathway. Oncotarget. 2017 Jun 20;8(25):40246-40263.
[2]. Dihydroisotanshinone I protects against menadione-induced toxicity in a primary culture of rat hepatocytes. Planta Med. 2002 Dec;68(12):1077-81.

In this study, we examined the protective effects of danshen and its compounds against prostate cancer. First, to investigate these effects in vivo, we analyzed the survival rate of prostate cancer patients by using data obtained from the NHIRD. In vitro, we observed the effects of DT on the interaction between macrophages and prostate cancer cells by interrupting the CCL2 pathway. We determined that DT inhibited the protein expression of p-STAT3 and blocked the translocation of STAT3 into the chromatin, as well as suppressed tumor epithelial–mesenchymal transition (EMT) gene expression.[1]

C18H14O3

278.3020

278.094

20958-18-3

89406

Typically exists as solid at room temperature

1.3±0.1 g/cm3

479.2±45.0 °C at 760 mmHg

214.9±28.8 °C

0.0±1.2 mmHg at 25°C

1.671

3.9

0

3

0

21

533

0

O1C2C(C3C4=C([H])C([H])=C([H])C(C([H])([H])[H])=C4C([H])=C([H])C=3C(C=2C([H])(C([H])([H])[H])C1([H])[H])=O)=O

KXNYCALHDXGJSF-UHFFFAOYSA-N

InChI=1S/C18H14O3/c1-9-4-3-5-12-11(9)6-7-13-15(12)17(20)18-14(16(13)19)10(2)8-21-18/h3-7,10H,8H2,1-2H3

4,8-dimethyl-8,9-dihydronaphtho[2,1-f][1]benzofuran-7,11-dione

20958-18-3; Dihydroisotanshinone I; Phenanthro[3,2-b]furan-7,11-dione, 8,9-dihydro-4,8-dimethyl-; 4,8-dimethyl-8,9-dihydronaphtho[2,1-f][1]benzofuran-7,11-dione; 4,8-Dimethyl-8,9-dihydrophenanthro[3,2-b]furan-7,11-dione; Phenanthro(3,2-b)furan-7,11-dione, 8,9-dihydro-4,8-dimethyl-; SCHEMBL13568194; DTXSID60943247;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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