p53: Ginsenoside F2 induced the phosphorylation and accumulation of p53, suggesting its activation.[1]

Antiproliferative Activity in Breast Cancer Stem Cells (CSCs): Ginsenoside F2 inhibited the proliferation of breast CSCs in a dose- and time-dependent manner. After 24 hours of treatment, the IC50 value was 97.48 ± 4.66 μM. Treatment with 120 μM F2 for 24 hours resulted in a significant decrease in cell viability. [1]
- Induction of Apoptosis: F2 induced apoptosis in breast CSCs, as evidenced by nuclear fragmentation and condensation (observed with Hoechst staining), an increase in the sub-G1 cell cycle fraction (from 4.04% in control to 60.45% at 120 μM F2), and an increase in the percentage of annexin V-positive/PI-negative cells. This was accompanied by a dose-dependent loss of mitochondrial membrane potential (ΔΨm), as shown by JC-1 staining. [1]
- Modulation of Apoptosis-Related Proteins: Western blot analysis showed that F2 treatment increased the pro-apoptotic Bax/Bcl-2 ratio, elevated the levels of PUMA, cleaved caspase 9, and cleaved PARP. It also increased the levels of total p53 and p53 phosphorylated at Ser15. [1]
- Induction of Autophagy: F2 induced autophagy in breast CSCs, characterized by the formation of acidic vesicular organelles (AVOs) (observed with acridine orange staining), the recruitment of GFP-LC3-II to autophagosomes, and an increase in the expression of autophagy-related proteins Atg-7, Beclin-1, and LC3B-II. The percentage of AVO-positive cells increased to 35.8% at 100 μM F2. [1]
- Role of Autophagy: The autophagy inhibitor chloroquine (CQ) enhanced F2-induced cell death, indicating that F2-induced autophagy was protective. Co-treatment with F2 and CQ increased the percentage of apoptotic cells (52.92%) and the sub-G1 fraction (55.75%) compared to F2 alone (33.46% and 35.22%, respectively). [1]
- Role of p53: The p53 inhibitor pifithrin-α (PFT) partially reversed F2-induced cell death and apoptosis, restoring cell viability and reducing the sub-G1 fraction. Co-treatment with F2 and PFT reduced the percentage of apoptotic cells to 20.07%. [1]

Cell Culture and Treatment: Breast cancer stem cells (CSCs) were isolated and enriched. They were cultured in serum-free DMEM with supplements (BSA, insulin, bFGF, EGF, B-27) in low cell-binding dishes to form mammospheres. For experiments, cells were trypsinized, seeded in DMEM with 10% FBS for 24 hours, and then treated with various concentrations of F2, chloroquine (CQ), or pifithrin-α (PFT) for 1 to 48 hours. [1]
- Cytotoxicity (MTT Assay): Cells (5 x 10⁴ cells/mL) were plated in 96-well plates. After 24 hours, they were treated with different concentrations of F2, CQ, or PFT for 24 or 48 hours. Then, 20 μL of MTT solution (5 mg/mL) was added to each well and incubated for 3-4 hours. The supernatant was removed, and 150 μL of DMSO was added to dissolve the formazan crystals. Absorbance was measured at 570 nm. [1]
- Nuclear Morphology (Hoechst Staining): Cells were stained with 10 μM Hoechst 33342 for 10-15 minutes and observed under a fluorescence microscope to assess nuclear fragmentation and condensation. [1]
- Apoptosis Analysis (Annexin V/PI Staining): Treated cells were harvested, washed with PBS, and stained with Annexin V-FITC and propidium iodide (PI) in binding buffer for 15 minutes at room temperature. Apoptotic cells were quantified by flow cytometry. [1]
- Cell Cycle Analysis (PI Staining): Cells were fixed in 70% ethanol, rehydrated in EDTA-PBS, treated with RNase A (25 μg/mL), and stained with PI (40 μg/mL). The DNA content was analyzed by flow cytometry to determine the sub-G1, G1, S, and G2/M phase distribution. [1]
- Mitochondrial Membrane Potential (JC-1 Staining): Cells were stained with JC-1 for 10-15 minutes at 37°C, washed, and analyzed by flow cytometry. A decrease in the red/green fluorescence ratio (FL2/FL1) indicates a loss of mitochondrial membrane potential. [1]
- Autophagy Detection (Acridine Orange Staining): To detect acidic vesicular organelles (AVOs), cells were stained with 10 μM acridine orange for 5-10 minutes and then analyzed either by fluorescence microscopy or flow cytometry. [1]
- LC3-GFP Transfection and Imaging: Cells were transiently transfected with a pEGFP-LC3B vector. After 24 hours, they were treated with F2 for another 24 hours. The formation of GFP-LC3 puncta (autophagosomes) was observed under a fluorescence microscope. [1]
- Western Blot Analysis: Cells were lysed in TNN buffer with protease inhibitors. Protein concentrations were determined, and 30-50 μg of protein per sample were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk and probed with primary antibodies (e.g., anti-Bcl-2, -Bax, -cleaved PARP, -p53, -p-p53, -Atg-7, -Beclin-1, -LC3B, -β-actin) and then with HRP-conjugated secondary antibodies. Protein bands were visualized using a chemiluminescence detection system. [1]

Normal Cell Toxicity: Ginsenoside F2 (up to 120 μM) showed no obvious cytotoxicity in normal human CCD-25Lu lung fibroblasts after 24 hours of treatment. [1]

[1]. Ginsenoside F2 induces apoptosis accompanied by protective autophagy in breast cancer stem cells. Cancer Lett. 2012 Aug 28;321(2):144-53.

Ginsenoside F2 is a ginsenoside found in plants of the genus Panax. Its structure is dammarane-type, with hydroxyl groups substituted at positions 3β, 12β, and 20 (pro-S position). The hydroxyl groups at positions 3 and 20 are converted to the corresponding β-D-glucopyranoside, and a double bond is introduced at positions 24-25. It possesses apoptosis-inducing, antitumor, and plant metabolite-like effects. It is a ginsenoside, a tetracyclic triterpenoid, a 12β-hydroxysteroid, and a β-D-glucopyranoside. It is derived from the hydride of dammarane. Ginsenoside F2 has been reported to exist in Panax notoginseng, ginseng, and Aralia elata, with relevant data available.

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Background: Ginsenoside F2 is a dammarene-type triterpene saponin and a metabolite of the ginsenoside Rb1, produced by human intestinal bacterial enzymes after oral ingestion. It has been studied for its anti-cancer properties. [1]
- Mechanism of Action in Breast CSCs: The study proposes a mechanism where F2 induces DNA damage, leading to p53 activation. This, in turn, triggers the intrinsic apoptotic pathway (via Bax, PUMA, and caspases) and simultaneously induces protective autophagy. The autophagy acts as a survival mechanism, and its inhibition (e.g., by chloroquine) enhances the apoptotic effect of F2. [1]

C42H72O13

785.02

784.497

62025-49-4

9918692

White to off-white solid powder

1.3±0.1 g/cm3

871.5±65.0 °C at 760 mmHg

185 °C

480.9±34.3 °C

0.0±0.6 mmHg at 25°C

1.594

3.53

9

13

10

55

1370

20

CC(=CCC[C@@](C)([C@H]1CC[C@@]2([C@@H]1[C@@H](C[C@H]3[C@]2(CC[C@@H]4[C@@]3(CC[C@@H](C4(C)C)O[C@H]5[C@@H]([C@H]([C@@H]([C@H](O5)CO)O)O)O)C)C)O)C)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O)C

SWIROVJVGRGSPO-JBVRGBGGSA-N

InChI=1S/C42H72O13/c1-21(2)10-9-14-42(8,55-37-35(51)33(49)31(47)25(20-44)53-37)22-11-16-41(7)29(22)23(45)18-27-39(5)15-13-28(38(3,4)26(39)12-17-40(27,41)6)54-36-34(50)32(48)30(46)24(19-43)52-36/h10,22-37,43-51H,9,11-20H2,1-8H3/t22-,23+,24+,25+,26-,27+,28-,29-,30+,31+,32-,33-,34+,35+,36-,37-,39-,40+,41+,42-/m0/s1

(2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[[(3S,5R,8R,9R,10R,12R,13R,14R,17S)-12-hydroxy-4,4,8,10,14-pentamethyl-17-[(2S)-6-methyl-2-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyhept-5-en-2-yl]-2,3,5,6,7,9,11,12,13,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl]oxy]oxane-3,4,5-triol

Ginsenoside F2 FT0686625 FT 0686625FT-0686625 N1568 N 1568 N-1568 X 1142 X1142 X-1142C20779 C 20779 C-20779 Q 100717 Q 100717 Q-100717

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 : ~50 mg/mL (~63.69 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.18 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 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 (3.18 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 (3.18 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.)