JNK signaling pathway [2]

In prostate cancer PC3 and LNCap cells, Sitogluside (0-80 µM for 48h) inhibited cell proliferation in a dose-dependent manner as measured by CCK-8 assay. It induced significant G1 phase cell cycle arrest. The compound also promoted apoptosis, as shown by increased apoptotic rate via flow cytometry, and increased protein expression of cleaved caspase 3, cleaved caspase 9, and Bax, while decreasing Bcl-2 expression. Additionally, Sitogluside induced autophagy, evidenced by increased LC3II/LC3I ratio and Beclin 1 expression, and decreased p62 expression. The autophagy inhibitor 3-methyladenine (3-MA) attenuated Sitogluside-triggered autophagy, growth inhibition, and apoptosis, indicating that the apoptotic response was dependent on autophagy. Sitogluside treatment also increased phosphorylation of JNK, and a JNK-specific inhibitor (SP600125) abated Sitogluside-elicited autophagy and apoptotic cell death [2].
In human colon cancer HCT-116 cells, Sitogluside showed significant dose- and time-dependent cytotoxic effects with IC50 values of 26.6 µM (24h) and 47.3 µM (48h) by MTT assay. It inhibited cell migration in a dose-dependent manner: the percentage of migrated cells decreased from 99% (control) to 84.2%, 45.2%, 39.5%, and 14.4% at 0, 5, 50, 75, and 100 µM, respectively. Similarly, it inhibited cell invasion: the percentage of invading cells decreased from 99% (control) to 95.2%, 67%, 34.5%, and 18.7% at 0, 5, 50, 75, and 100 µM, respectively. Sitogluside induced apoptosis, with apoptotic cells increasing from 2.5% (control) to 23.6%, 46.9%, and 74.2% at 5, 50, and 100 µM, respectively (including early and late apoptosis). It also induced sub-G1 cell cycle arrest, with sub-G1 phase cells increasing from 3.32% (control) to 13.3%, 27.3%, and 49.8% at 5, 50, and 100 µM, respectively, accompanied by a decrease in G0/G1 phase cells. Western blot analysis showed that Sitogluside decreased Bcl-2 expression and increased expression of Bax, cytochrome c, caspase-3, and caspase-9 [4].

PC3 and LNCap human prostate cancer cells were seeded in 6-well plates (1×10^5 cells/well) and exposed to different concentrations of Sitogluside (0, 5, 10, 20, 40, and 80 µM) for 48h. For cell viability, CCK-8 solution (10 µl) was added and incubated for 2h at 37°C, and absorbance was measured at 450nm. For cell cycle analysis, cells were fixed in 75% ethanol, incubated with propidium iodide (50 µg/mL) containing RNase (40 µg/mL) for 30 min, and analyzed by flow cytometry. For apoptosis, cells were stained using annexin V-FITC/PI kit and analyzed by flow cytometry. For Western blot, cell lysates were separated on 10% SDS-PAGE gels, transferred to PVDF membranes, probed with primary antibodies (cleaved caspase 3, cleaved caspase 9, Bax, Bcl-2, LC3, Beclin 1, p62, p-JNK, JNK, and β-actin), then with peroxidase-conjugated secondary antibody, and detected by ECL system [2].
HCT-116 human colon cancer cells were cultured in RPMI-1640 with 10% FBS and antibiotics. For cell viability (MTT assay), cells (2×10^5/well) in 96-well plates were treated with Sitogluside (0, 5, 10, 25, 50, 75, 100 µM) for 24 and 48h, then MTT dye (10 µl) was added and incubated for 3h, followed by DMSO (200 µl) and absorbance measured at 570nm. For cell migration (wound healing assay), scratches were made in confluent monolayers in 12-well plates, cells were treated with Sitogluside (0, 5, 50, 75, 100 µM) for 48h, fixed, stained with crystal violet, and wound length measured by ImageJ software. For cell invasion assay, HCT-116 cells (2×10^5) pre-incubated with Sitogluside (0, 5, 50, 75, 100 µM) were seeded in Matrigel-coated upper chambers (24-well, 6 µm pore size) with chemoattractant in lower chamber; after 48h, filters were fixed, stained with crystal violet, and invading cells counted. For apoptosis (Annexin V-FITC assay), cells treated with Sitogluside (0, 5, 50, 100 µM) for 48h were stained with annexin V-FITC and propidium iodide, then analyzed by flow cytometry. For cell cycle analysis, cells treated with Sitogluside (0, 5, 50, 100 µM) for 48h were fixed in cold ethanol, stained with propidium iodide solution containing Triton X-100 and RNase, and analyzed by flow cytometry. For Western blot, proteins were extracted with RIPA lysis buffer containing cocktail and PMSF, quantified by BCA assay, resolved on 10% SDS-PAGE, transferred to PVDF membranes, blocked with 6% skimmed milk, incubated with primary antibodies (caspase-3, cleaved caspase-9, cytochrome c, Bax, Bcl-2) overnight at 4°C, then with HRP-conjugated secondary antibodies for 1h at room temperature, and visualized by ECL [4].

[1]. Daucosterol inhibits cancer cell proliferation by inducing autophagy through reactive oxygen species-dependent manner. Life Sci. 2015 Sep 15;137:37-43.

[2]. Daucosterol induces autophagic-dependent apoptosis in prostate cancer via JNK activation. Biosci Trends. 2019 May 12;13(2):160-167.

[3]. Daucosterol protects neurons against oxygen-glucose deprivation/reperfusion-mediated injury by activating IGF1 signaling pathway. J Steroid Biochem Mol Biol. 2015 Aug;152:45-52.

[4]. Daucosterol inhibits colon cancer growth by inducing apoptosis, inhibiting cell migration and invasion and targeting caspase signalling pathway. Bangladesh Journal of Pharmacolog, 2016, 11(2): 395-401.

Daucosterol is a steroidal saponin composed of a β-D-glucopyranose residue linked to a β-sitosterol residue at position 3 via a glycosidic bond. It has been isolated from Japanese ginseng (Panax japonicus var. major) and shrubby Breynia (Breynia fruticosa). It is a plant metabolite. It is a steroidal saponin, β-D-glucoside, and monosaccharide derivative. Its function is related to β-sitosterol. It is derived from the hydrogenation of stigmasterane. Citoglucoside has also been reported in Acanthus ilicifolius, Iris tectorum, and other organisms with relevant data.

---

Sitogluside (daucosterol) is a β-sitosterol glycoside, one of the major phytosterols in higher plants. It has been reported to display anti-inflammatory and immunomodulatory activities, neuroprotective action in ischemia models by activating IGF1 signaling, and promote neural stem cell proliferation. In cancer, it has been shown to induce apoptosis in human breast cancer via PTEN/PI3K/Akt pathway, repress proliferation/migration/invasion in hepatocellular carcinoma via Wnt/β-catenin signaling, and suppress cancer cell proliferation by triggering autophagy through ROS-dependent manner. The present studies demonstrate that Sitogluside blocks prostate cancer growth via inducing autophagic-dependent apoptosis through activating JNK signaling, and inhibits colon cancer growth by inducing apoptosis, inhibiting cell migration/invasion, and targeting caspase signaling pathway [2][4].

C35H60O6

576.859

576.438

474-58-8

5742590

White to off-white solid powder

1.1±0.1 g/cm3

673.6±55.0 °C at 760 mmHg

361.2±31.5 °C

0.0±4.7 mmHg at 25°C

1.554

8.78

4

6

9

41

920

14

CC[C@H](CC[C@@H](C)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2CC=C4[C@@]3(CC[C@@H](C4)O[C@H]5[C@@H]([C@H]([C@@H]([C@H](O5)CO)O)O)O)C)C)C(C)C

NPJICTMALKLTFW-VAWFKKCMSA-N

InChI=1S/C35H60O6/c1-7-22(20(2)3)9-8-21(4)26-12-13-27-25-11-10-23-18-24(14-16-34(23,5)28(25)15-17-35(26,27)6)40-33-32(39)31(38)30(37)29(19-36)41-33/h10,20-22,24-33,36-39H,7-9,11-19H2,1-6H3/t21-,22-,24-,25+,26-,27+,28+,29-,30-,31+,32-,33-,34+,35-/m1/s1

(3-beta)-Stigmast-5-en-3-yl-beta-D-glucopyranoside

NSC165962 NSC 165962 NSC-165962LyonisideDaucosterolEleutheroside AAlexandrinCoriandrinolbeta-Sitosterol glucosideSitoglusideDaucosterin

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)

DMSO : ~3.33 mg/mL (~5.77 mM)

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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