Anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL): Gossypol Acetate (as (-)-Gossypol, the active enantiomer) functions as a BH3 mimetic, competitively binding to the BH3-binding groove of Bcl-2 and Bcl-xL. In fluorescence polarization binding assays, its Ki values were ~1.2 μM for human recombinant Bcl-2 and ~0.8 μM for human recombinant Bcl-xL; it showed low affinity for Mcl-1 (Ki > 10 μM) [1]

One natural substance that has been investigated as an anticancer agent is gingsypol, which is extracted from cottonseeds and roots. Tested in multiple clinical trials, the racemic version of Gossypol [(±)-Gossypol] is well tolerated. -Gossypol) has a Ki of 0.5 to 0.6 μM when binding to the Bcl-xL protein. Additionally, (±)-Gossypol binds to Bcl-2 protein potently, with a Ki value of 0.2-0.3 mM. The (-)-Gossypol and (+)-Gossypol enantiomers are the two enantiomers of the natural racemic Gossypol. In 6-day MTT tests, the racemic form and all enantiomers of Gossypol are evaluated against UM-SCC-6 and UM-SCC-14A. shows higher growth inhibition in comparison to (+)-Gossypol compared to (±)-Gossypol in both evaluated cell lines (P<0.001). With (±)-Gossypol, an intermediate growth inhibitory impact is seen, however this effect is only noticeable at the higher dose (10 μM, P<0.0001)[1].

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Antiproliferative activity on head and neck squamous cell carcinoma (HNSCC) cells (SCC-15, SCC-25, FaDu):
- Cell viability inhibition: Using the sulforhodamine B (SRB) assay (72 h incubation), Gossypol Acetate dose-dependently inhibited HNSCC cell viability. The IC50 values were 1.5 μM (SCC-15), 1.8 μM (SCC-25), and 2.1 μM (FaDu). At 3 μM, cell viability was reduced by 70-75% in all three cell lines vs. vehicle controls [1]
- Apoptosis induction: Gossypol Acetate (1-3 μM) induced caspase-dependent apoptosis in SCC-15 cells. Annexin V-FITC/PI double staining showed that 2 μM treatment for 48 h increased the apoptotic rate (Annexin V⁺/PI⁺ + Annexin V⁺/PI⁻) from 5% (control) to 38%. Western blot analysis revealed upregulated cleaved caspase-3 (3.2-fold) and cleaved caspase-9 (2.8-fold), and downregulated pro-caspase-3 (0.3-fold) [1]
- Bcl-2 family interaction: Co-immunoprecipitation experiments showed that Gossypol Acetate (2 μM) disrupted the binding of Bax (pro-apoptotic protein) to Bcl-2/Bcl-xL in SCC-25 cells, increasing free Bax levels by 2.5-fold. No significant changes in total Bcl-2 or Bcl-xL protein expression were observed (Western blot) [1]
- Clonogenic survival inhibition: Gossypol Acetate (0.5-2 μM) reduced the clonogenic capacity of FaDu cells. At 1 μM, the number of colonies (>50 cells) decreased by 60% vs. control; at 2 μM, colony formation was almost completely inhibited (survival fraction <10%) [1]
- Selectivity for cancer cells: Gossypol Acetate showed lower cytotoxicity on normal human oral keratinocytes (OKF6 cells), with an IC50 of 8.5 μM (SRB assay, 72 h), ~4-5-fold higher than that in HNSCC cells [1]

Gossypol acetate is able to inhibit tumor growth in Wus1-bearing mice, but the survival of mice is not prolonged, and tumor grows rapidly after short inhibition. Gossypol has now been found to have inhibitory effects on proliferation or to induce apoptosis in ovarian cancer, endometrial cancer, adrenal cortical tumor, thyroid cancer, lung cancer, colon carcinoma, leukemia, pancreatic cancer, melanoma and lymphoma. In addition, gossypol can increase the sensitivity of drug-resistant tumor cells to chemotherapy and radiotherapy. Some clinical trials showed gossypol is well-tolerated, and partial responses are observed in some patients.

1. Recombinant Protein Preparation: Human recombinant Bcl-2 and Bcl-xL proteins were expressed in Escherichia coli and purified via affinity chromatography. Protein concentration was determined using the Bradford assay, and purity (>90%) was confirmed by SDS-PAGE [1]
2. Reaction System Setup: Prepare 50 μL reaction mixtures containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% BSA, 20 nM recombinant Bcl-2/Bcl-xL protein, and 10 nM fluorescently labeled BH3 peptide (FAM-conjugated Bad BH3 peptide). Add Gossypol Acetate at concentrations ranging from 0.01 to 50 μM (vehicle: 0.1% DMSO) [1]
3. Incubation and Detection: Incubate the mixtures at room temperature for 1 h to reach binding equilibrium. Measure fluorescence polarization (FP) values using a fluorescence plate reader (excitation: 485 nm, emission: 535 nm). The FP value is proportional to the binding of the BH3 peptide to Bcl-2/Bcl-xL [1]
4. Data Calculation: Calculate the inhibition rate of Gossypol Acetate on peptide-protein binding using the formula: Inhibition rate = [(FPcontrol - FPtreatment)/FPcontrol] × 100%. Fit the dose-response curve using GraphPad Prism software to determine the Ki values (using the competitive binding model) [1]

1. Cell Viability Assay (SRB Method)
1. Cell Seeding: HNSCC cells (SCC-15, SCC-25, FaDu) and normal OKF6 cells were seeded into 96-well plates at a density of 5×10³ cells/well. Incubate the plates at 37℃, 5% CO₂ for 24 h to allow cell adhesion [1]
2. Drug Treatment: Replace the culture medium with fresh medium containing Gossypol Acetate (0.1, 0.5, 1, 2, 5, 10 μM) or vehicle (0.1% DMSO). Each concentration has 6 replicate wells. Continue incubation for 72 h [1]
3. Fixation and Staining: Add 50 μL of 50% trichloroacetic acid (TCA) to each well to fix cells (final TCA concentration: 10%), incubate at 4℃ for 1 h. Wash the plates 5 times with distilled water and air-dry. Add 100 μL of 0.4% SRB solution (dissolved in 1% acetic acid) to each well, stain at room temperature for 30 min. Wash 4 times with 1% acetic acid to remove unbound dye, air-dry [1]
4. Absorbance Measurement: Dissolve the bound SRB dye in 150 μL of 10 mM Tris base solution (pH 10.5). Measure the absorbance at 515 nm using a microplate reader. Calculate cell viability as (absorbance of treatment group / absorbance of control group) × 100%. IC50 values are derived from dose-response curves [1]
2. Apoptosis Assay (Annexin V-FITC/PI Double Staining)
1. Cell Preparation: Seed SCC-15 cells into 6-well plates at a density of 2×10⁵ cells/well. Incubate for 24 h, then treat with Gossypol Acetate (1, 2, 3 μM) or vehicle for 48 h [1]
2. Cell Collection and Washing: Collect floating and adherent cells by trypsinization (without EDTA), combine them, and wash twice with cold phosphate-buffered saline (PBS). Resuspend the cells in 1× binding buffer at a concentration of 1×10⁶ cells/mL [1]
3. Staining: Add 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) to 100 μL of the cell suspension. Incubate in the dark at room temperature for 15 min [1]
4. Flow Cytometry Analysis: Add 400 μL of 1× binding buffer to the stained cell suspension. Analyze the cells using a flow cytometer within 1 h. Quantify the percentage of early apoptotic cells (Annexin V⁺/PI⁻) and late apoptotic cells (Annexin V⁺/PI⁺) [1]
3. Western Blot Analysis
1. Protein Extraction: Treat SCC-25 cells with Gossypol Acetate (2 μM) for 0, 12, 24, 48 h. Collect cells, wash with cold PBS, and lyse in RIPA buffer containing protease and phosphatase inhibitors. Centrifuge at 12,000 × g for 15 min at 4℃ to collect the supernatant (total protein extract) [1]
2. Protein Quantification and Electrophoresis: Determine protein concentration using the BCA assay. Load 30 μg of protein per lane onto a 12% SDS-PAGE gel, run electrophoresis at 100 V for 2 h [1]
3. Transfer and Blocking: Transfer proteins from the gel to a polyvinylidene fluoride (PVDF) membrane using a semi-dry transfer system (15 V for 30 min). Block the membrane with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST) at room temperature for 1 h [1]
4. Antibody Incubation and Detection: Incubate the membrane with primary antibodies (anti-Bcl-2, anti-Bcl-xL, anti-cleaved caspase-3, anti-pro-caspase-9, anti-GAPDH) overnight at 4℃. Wash 3 times with TBST, then incubate with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. Wash 3 times with TBST, then detect protein bands using an enhanced chemiluminescence (ECL) kit. Quantify band intensity using ImageJ software [1]
### 4. Clonogenic Survival Assay
1. Cell Seeding: FaDu cells were seeded into 6-well plates at a density of 200 cells/well (adjusted based on drug concentration to ensure countable colonies). Incubate for 24 h [1]
2. Drug Treatment: Add Gossypol Acetate (0.5, 1, 2 μM) or vehicle to the wells. Incubate for 24 h, then replace the medium with fresh medium without drug [1]
3. Colony Formation and Counting: Incubate the plates for 14 days (until colonies containing >50 cells are visible). Fix cells with 4% paraformaldehyde for 15 min, stain with 0.1% crystal violet for 30 min. Wash with water, air-dry, and count colonies manually. Calculate the survival fraction as (number of colonies in treatment group / number of colonies in control group) × 100% [1]

Wus1-bearing mice

Selective cytotoxicity: Gossypol acetate showed higher cytotoxicity (IC50: 1.5-2.1 μM) against head and neck squamous cell carcinoma (HNSCC) cells than against normal human oral keratinocytes (OKF6 cells, IC50: 8.5 μM), indicating that it has a good therapeutic index [1].

[1]. In vitro effects of the BH3 mimetic, (-)-Gossypol, on head and neck squamous cell carcinoma cells. Clin Cancer Res. 2004 Nov 15;10(22):7757-63.

Gossypol acetate is the natural acetic acid form of gossypol, an orally administered polyphenolic aldehyde primarily derived from cottonseed, possessing potential antitumor activity. The bioactivity of gossypol acetate is similar to that of gossypol, including inhibition of DNA replication, inhibition of tumor cell proliferation, and male contraceptive effects. (NCI04)
R-(-)-Gossypol acetate is an orally bioavailable solvate of the R-(-) enantiomer of gossypol with acetic acid, possessing potential antitumor activity. As a BH3 mimic, R-(-)-gossypol binds to the hydrophobic surface binding groove BH3 of the anti-apoptotic proteins Bcl-2 and Bcl-xL, blocking their heterodimerization with pro-apoptotic proteins of the Bcl-2 family (such as Bad, Bid, and Bim); this may lead to inhibition of tumor cell proliferation and induce tumor cell apoptosis. Racemic gossypol is a polyphenolic compound isolated from cottonseed.

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Gossypol acetate is a natural polyphenol compound extracted from cottonseed, and its (-)-enantiomer ((-)-gossypol) is the bioactive form with BH3 mimicry activity. It exerts its anticancer effect by targeting anti-apoptotic Bcl-2 family proteins, a mechanism different from traditional chemotherapeutic drugs that directly damage DNA [1].
In head and neck squamous cell carcinoma (HNSCC) cells, gossypol acetate does not downregulate the expression of Bcl-2 or Bcl-xL, but rather competitively binds to their BH3 binding grooves, releasing pro-apoptotic proteins (such as Bax) to trigger the intrinsic apoptotic pathway. This mechanism is consistent with its classification as a "BH3 mimic" [1]

C32H34O10

578.61

578.215

12542-36-8

Gossypol;303-45-7;(R)-(-)-Gossypol acetic acid;866541-93-7;(S)-Gossypol (acetic acid);1189561-66-7;(R)-(-)-Gossypol;90141-22-3

227456

Light yellow to yellow solid powder

707.9ºC at 760 mmHg

164-168ºC

395.9ºC

1.05E-20mmHg at 25°C

6.473

7

10

5

42

811

0

NIOHNDKHQHVLKA-UHFFFAOYSA-N

InChI=1S/C30H30O8.C2H4O2/c1-11(2)19-15-7-13(5)21(27(35)23(15)17(9-31)25(33)29(19)37)22-14(6)8-16-20(12(3)4)30(38)26(34)18(10-32)24(16)28(22)36;1-2(3)4/h7-12,33-38H,1-6H3;1H3,(H,3,4)

acetic acid;7-(8-formyl-1,6,7-trihydroxy-3-methyl-5-propan-2-ylnaphthalen-2-yl)-2,3,8-trihydroxy-6-methyl-4-propan-2-ylnaphthalene-1-carbaldehyde

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.32 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)