STAT3 (signal transducer and activator of transcription 3) [1]

Prosapogenin A (PSA) significantly inhibited the growth of human cervical carcinoma HeLa, hepatocellular carcinoma HepG2, and breast adenocarcinoma MCF-7 cells in a time- and concentration-dependent manner. At 10 μM, PSA significantly reduced cancer cell survival over 24, 48, and 72 h, while it did not significantly affect the survival of normal human hepatocyte 7701 and 293 cells. The IC50 values for PSA after 48 h treatment were 8.41 μM (HepG2), 9.36 μM (MCF-7), and 9.27 μM (HeLa), which were significantly lower than those of the positive control Rh2. [1]
PSA induced apoptosis in HeLa, HepG2, and MCF-7 cells as detected by Hoechst 33342 and propidium iodide (PI) double staining. After 48 h treatment with 5 or 10 μM PSA, a significant increase in apoptotic bodies was observed compared to untreated groups. The mean fluorescence intensity (MFI) of PI increased significantly in a concentration-dependent manner. The apoptotic effect of 10 μM PSA was stronger than that of 10 μM Rh2. [1]
PSA induced cell cycle arrest at the G2/M phase in HepG2 cells. After 48 h treatment with 5 μM and 10 μM PSA, the percentage of cells in G2/M phase increased to 19.19% and 22.80%, respectively, compared to 12.73% in untreated cells. [1]
PSA inhibited STAT3 mRNA expression and STAT3 phosphorylation (pSTAT3). RT-PCR showed that 5 μM PSA for 48 h decreased STAT3 mRNA levels by approximately 42% in HepG2, 19% in MCF-7, and 10% in HeLa cells. Western blot analysis showed that 5 μM PSA for 48 h did not significantly affect total STAT3 protein levels but significantly decreased pSTAT3 levels. [1]
PSA modulated the expression of STAT3 target genes as determined by a human STAT3-regulated cDNA plate array. In HepG2 cells treated with 5 μM PSA for 48 h, PSA downregulated the ratio of Bcl-2/Bax and the expression of survivin and glycoprotein 130 (gp130), and upregulated the expression of c-myc, C-reactive protein (CRP), cyclin E, glycogen synthase kinase 3β (GSK-3β), IL-10, oncostatin M (OSM), p21, and p27, subsequently promoting cell apoptosis. [1]
PSA regulated the expression of glycometabolism-related genes. In MCF-7 cells treated with 5 μM PSA for 48 h, RT-PCR showed reduced mRNA expression of glucose transporter 1 (GLUT1), hexokinase (HK), and phosphofructokinase (PFKL). [1]

For cell viability assay, cells (HeLa, HepG2, MCF-7, 7701, 293) were cultured in RPMI-1640 supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/ml) at 37°C in 5% CO2. Cells were exposed to PSA at concentrations ranging from 0 to 15 μM for 24, 48, and 72 h. Cell viability was determined by MTT assay. The IC50 values were calculated. [1]
For apoptosis assay, cells were treated with 0, 5, or 10 μM PSA or 10 μM Rh2 for 48 h in 96-well plates. Cells were then incubated with 5 mg/l Hoechst 33342 for 10 min and 5 mg/l propidium iodide (PI) for another 1 h in the dark, washed twice with ice-cold PBS, and then subjected to Array Scan VTIHCS600 High-Contents system to record red fluorescence (PI). Apoptotic bodies were quantified. [1]
For cell cycle analysis, cells treated with 0, 5, or 10 μM PSA for 48 h were fixed with 70% ethanol at 4°C for 12 h, then stained in PI solution (50 μg/ml PI, 100 μg/ml RNase A, and 0.2% v/v Triton X-100) for 20 min at 37°C. DNA content was analyzed by flow cytometry using a FACScalibur flow cytometer and Summit software. [1]
For RT-PCR analysis, total RNA was isolated using TRIzol reagent. Oligo(dT)-primed RNA (1 μg) was reverse-transcribed, and cDNA was used to determine mRNA levels of STAT3, GLUT1, HK, and PFKL. β-actin was used as internal control. Primers for STAT3 (forward: 5′-CCAAGGAGGAGGGCATTGC-3′, reverse: 5′-ACATCGGGCAGGTCAAATGG-3′, product size 147 bp); β-actin (forward: 5′-CTTCTACAATGAGCTGCGTG-3′, reverse: 5′-TCATGAGGTAGTCAGTCAGG-3′, 305 bp); GLUT1 (forward: 5′-CAACGCTGTCTTCTATTACTC-3′, reverse: 5′-GCCACGATGCTCAGATAG-3′, 252 bp); HK (forward: 5′-CCAGAAGGCTCAGAAGTC-3′, reverse: 5′-ATGCTTGTCCAGGAAGTC-3′, 216 bp); PFKL (forward: 5′-TCCGCATCTATGGTATTTCAC-3′, reverse: 5′-GTCTTTCATCTTCTCCGTCAT-3′, 400 bp). PCR products were analyzed. [1]
For Western blotting, tumor cells were lysed with RIPA buffer and centrifuged at 12,000 × g for 10 min. Protein concentration was determined. Equal amounts of protein per lysate were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked in Tris-buffered saline containing 3% skimmed dry milk, then incubated with primary rabbit anti-human STAT3, pSTAT3, and β-actin monoclonal antibodies (diluted 1:1,000 in PBS) at 4°C overnight, followed by HRP-conjugated secondary antibody (goat anti-rabbit IgG). Detection was done by enhanced chemiluminescence. [1]
For STAT3-regulated cDNA plate array, total RNA was reverse-transcribed into cDNA in the presence of biotin-dUTP. Targeted genes were captured onto individual wells of a plate via pre-coated gene-specific oligonucleotides. Captured cDNAs were detected with streptavidin-HRP, and luminescence was measured on a microplate luminometer. Expression levels were proportional to luminescence intensity. HepG2 cells were treated with or without 5 μM PSA for 48 h before RNA extraction. [1]

[1]. Prosapogenin A induces apoptosis in human cancer cells in vitro via inhibition of the STAT3 signaling pathway and glycolysis. Oncol Lett. 2013 Nov;6(5):1323-1328.

Prosapogenin A is a steroidal saponin. It has been reported that Prosapogenin A exists in Ipomoea sepium, Paris polyphylla, and other organisms with relevant data.

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Prosapogenin A (PSA) is a saponin extracted from Veratrum, a traditional Chinese medicine, using 70% ethanol, followed by separation, purification, and characterization. Veratrum has been shown to significantly inhibit the growth of human leukemia HL-60, human gastric carcinoma BGC-823, human liver carcinoma BEL-7402, and human nasopharyngeal carcinoma KB cells. PSA induces apoptosis in cancer cells via inhibition of the STAT3 signaling pathway and glycolysis (Warburg effect). It blocks STAT3 pathway and modulates glycometabolism-related genes, leading to reduced cell proliferation and increased apoptosis. The study suggests PSA as a potential anticancer agent with low toxicity to normal cells. [1]

C39H62O12

722.91

722.424

19057-67-1

11061578

White to off-white solid powder

1.3±0.1 g/cm3

838.8±65.0 °C at 760 mmHg

212℃

461.1±34.3 °C

0.0±0.6 mmHg at 25°C

1.605

6.9

6

12

5

51

1310

21

C[C@@H]1CC[C@@]2([C@H]([C@H]3[C@@H](O2)C[C@@H]4[C@@]3(CC[C@H]5[C@H]4CC=C6[C@@]5(CC[C@@H](C6)O[C@H]7[C@@H]([C@H]([C@@H]([C@H](O7)CO)O)O)O[C@H]8[C@@H]([C@@H]([C@H]([C@@H](O8)C)O)O)O)C)C)C)OC1

HDXIQHTUNGFJIC-FOAHKCLGSA-N

InChI=1S/C39H62O12/c1-18-8-13-39(46-17-18)19(2)28-26(51-39)15-25-23-7-6-21-14-22(9-11-37(21,4)24(23)10-12-38(25,28)5)48-36-34(32(44)30(42)27(16-40)49-36)50-35-33(45)31(43)29(41)20(3)47-35/h6,18-20,22-36,40-45H,7-17H2,1-5H3/t18-,19+,20+,22+,23-,24+,25+,26+,27-,28+,29+,30-,31-,32+,33-,34-,35+,36-,37+,38+,39-/m1/s1

(2S,3R,4R,5R,6S)-2-(((2R,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-(((4S,5'R,6aR,6bS,8aS,8bR,9S,10R,11aS,12aS,12bS)-5',6a,8a,9-tetramethyl-1,3,3',4,4',5,5',6,6a,6b,6',7,8,8a,8b,9,11a,12,12a,12b-icosahydrospiro[naphtho[2',1'

Prosapogenin A Paris saponin VSaponin Ta Lilioglycoside D

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 : ~100 mg/mL (~138.33 mM)

Solubility in Formulation 1: 2.5 mg/mL (3.46 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 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 2: ≥ 2.5 mg/mL (3.46 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 25.0 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (3.46 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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 (Please use freshly prepared in vivo formulations for optimal results.)