Mitogen-activated protein kinase kinase 3 (MKK3) [1]
Mitogen-activated protein kinase kinase 6 (MKK6) [1]
It directly binds to MKK3 and MKK6, inhibiting their kinase activity. Arginine-61 (Arg61) in MKK6 is critical for binding with gossypetin. [1]

Gossypetin (20–60 μM; 48 hours; KYSE30, KYSE450, and KYSE510 cells) therapy dramatically and dose-dependently reduced the development of anchorage-dependent esophageal cancer cells. Gossypetin significantly prevents esophageal cancer cells from growing anchorage-independently [1]. The administration of 60 μM gossypetin for three hours to KYSE30 and KYSE410 cells significantly reduced p38 activity in a way that was dependent on dose, confirming that gossypetin directly inhibits MKK3 or MKK6 activity [1]. Treatment with gossypetin (20–40 μM; 48 hours; KYSE450 and KYSE510 cells) shortens the S phase and causes dose-dependent G2 phase cell cycle arrest [1]. Treatment of esophageal cancer cells with gossypetin (20–40 μM; 72 hours) causes intrinsic apoptosis in these cells [1].

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Gossypetin inhibits anchorage-dependent growth of esophageal squamous cell carcinoma (ESCC) cell lines KYSE30, KYSE410, KYSE450, and KYSE510 in a dose-dependent manner after 48 hours of treatment, as measured by MTT assay. [1]
Gossypetin strongly suppresses anchorage-independent growth of KYSE30 and KYSE510 cells in soft agar after 2 weeks of treatment. [1]
In KYSE30 cells, treatment with 60 μM gossypetin for 3 hours strongly suppresses the phosphorylation of p38 MAPK, but has little effect on the phosphorylation of AKT, GSK3β, ERKs, JNKs, HER2, EGFR, or JAK1. [1]
In in vitro kinase assays, gossypetin directly suppresses the activity of both MKK3 and MKK6 in a dose-dependent manner (0, 10, 20, 40, 60 μM), as measured by the phosphorylation of their substrate, inactive p38α. [1]
Pull-down assays using gossypetin-conjugated Sepharose 4B beads confirm that gossypetin directly binds to MKK3 and MKK6 in KYSE30 cell lysates and to recombinant MKK3 and MKK6 proteins, but not to AKT1, ERK2, p38, or EGFR. [1]
Site-directed mutagenesis and pull-down assays show that the R61K mutant of MKK6 has the most reduced binding affinity with gossypetin, indicating Arg61 is a critical binding residue. [1]
Gossypetin induces G2 phase cell cycle arrest in KYSE30 and KYSE510 cells in a dose-dependent manner (0, 20, 40, 60 μM) after 48 hours treatment, as determined by flow cytometry. [1]
Gossypetin treatment (60 μM for 48 h) strongly increases the expression of the CDKN1B (p27) protein in KYSE30 and KYSE510 cells. [1]
Gossypetin induces apoptosis in KYSE30 and KYSE510 cells in a dose-dependent manner (0, 20, 40, 60 μM) after 72 hours treatment, as shown by increased number of suspended cells and Annexin V/PI staining. [1]
Gossypetin treatment (60 μM for 72 h) increases the protein levels of cleaved caspase 3, cleaved caspase 7, BAX, and cytochrome c, while decreasing BCL2 levels in KYSE30 and KYSE510 cells. [1]
The inhibition of cell growth by gossypetin is dependent on the expression of MKK3 and MKK6. Cells expressing siMKK3/6 are resistant to gossypetin, while cells overexpressing MKK3/6 are more sensitive. [1]

Treatment with gossypetin (100 mg/kg; oral; 5 times weekly; for 21 days; severe combined immunodeficient (SCID) female mice) significantly reduced the size of esophageal tumor growth without significant loss of body weight. Gossypetin significantly reduced Ki67 expression. There were no obvious morphological differences between treated or untreated mouse tissues. In the Gossypetin-treated group, the phosphorylation of p38, the direct downstream protein of MKK3/6, was strongly inhibited [1].

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In a patient-derived esophageal tumor xenograft (PDX) mouse model, oral administration of gossypetin (100 mg/kg body weight, 5 times per week for 21 days) significantly decreases esophageal tumor volume by over 60% compared to the vehicle-treated group. [1]
Gossypetin treatment in the PDX model significantly decreases the expression of the Ki67 proliferation marker in tumor tissues, as shown by immunohistochemistry. [1]
Western blot analysis of PDX tumor samples shows that gossypetin treatment inhibits the phosphorylation of MKK3/6 and their downstream target p38 in vivo. [1]

In vitro kinase assay for MKK3 and MKK6: Active recombinant MKK3 (200 ng) or MKK6 (200 ng) protein was mixed with various doses of gossypetin (0, 10, 20, 40, 60 μM) in buffer and kept at room temperature for 15 minutes. Then, inactive p38α recombinant protein, ATP, and buffer were added and incubated at 30°C for 30 minutes. The reaction was stopped with protein loading buffer and separated by SDS-PAGE. MKK3 or MKK6 activity was detected by Western blotting using an antibody that specifically recognizes phosphorylated p38 (at T180/Y182). [1]

Cell proliferation experiment [1]
Cell Types: KYSE30, KYSE450, KYSE510 Cell
Tested Concentrations: 20 μM, 40 μM, 60 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: The growth of anchorage-dependent esophageal cancer cells was Dramatically inhibited.

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Western Blot Analysis[1]
Cell Types: KYSE30 and KYSE410 ccells
Tested Concentrations: 60 μM
Incubation Duration: 3 hrs (hours)
Experimental Results: p38 activity was strongly inhibited in a dose-dependent manner.

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Cell cycle analysis[1]
Cell Types: KYSE450 and KYSE510 Cell
Tested Concentrations: 20 μM, 40 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: S phase shortened and G2 phase cell cycle arrest was induced in a dose-dependent manner.

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Apoptosis analysis [1]
Cell Types: Esophageal cancer cells
Tested Concentrations: 20 μM, 40 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Induced apoptosis of esophageal cancer cells.

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Cell proliferation (MTT assay): Cells (1.2-2.5 × 10³ per well) were seeded in 96-well plates, incubated for 24 h, then treated with various concentrations of gossypetin in complete growth medium. After 48 h, MTT solution was added, incubated for 2 h, and the formazan crystals were dissolved in DMSO. Absorbance was measured at 570 nm. [1]
Anchorage-independent growth (Soft agar assay): Cells (8 × 10³ per well) suspended in complete growth medium with 0.3% agar and various concentrations of gossypetin were layered over a base layer of 0.6% agar with or without gossypetin. Cultures were maintained at 37°C for 2 weeks. Colonies were counted under a microscope using Image-Pro Plus software. [1]
Pull-down assay: Gossypetin-conjugated Sepharose 4B beads (or Sepharose 4B only as a control) were incubated with total cell lysates (1 mg) or recombinant proteins (300 ng) in reaction buffer overnight at 4°C with gentle rocking. Beads were washed, and binding was visualized by Western blotting. [1]
Cell cycle analysis: Cells were plated, synchronized by serum starvation for 24 h, and then treated with gossypetin for 48 h in 10% serum-supplemented medium. Cells were collected, fixed in 70% cold ethanol, digested with RNase, and stained with propidium iodide. Stained cells were analyzed by flow cytometry. [1]
Apoptosis assay (Annexin V/PI): Cells were treated with gossypetin for 72 h in 10% serum-supplemented medium. Cells were collected, stained with Annexin V and propidium iodide, and analyzed by flow cytometry. [1]
Western blotting: Total cell or tissue lysates were separated by SDS-PAGE and transferred to membranes. Membranes were probed with specific primary antibodies (e.g., against phosphorylated and total signaling proteins, apoptotic markers) followed by HRP-conjugated secondary antibodies. Protein bands were visualized and quantified. [1]

Animal/Disease Models: Severe combined immunodeficiency (SCID) female mice (6-9 weeks old) esophageal cancer tissue injection [1]
Doses: 100 mg/kg
Route of Administration: po (po (oral gavage)) 5 times per week; for 21 days
Experimental Results: Inhibited the growth of patient-derived esophageal xenograft tumors in an in vivo mouse model.

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Patient-derived xenograft (PDX) model:** Female SCID mice (6-9 weeks old) were used. A human esophageal tumor specimen was cut into small pieces and inoculated subcutaneously into the back of the neck of each mouse. Mice were divided into two groups (n=10 per group): vehicle group (5% DMSO in 10% Tween 80) and gossypetin treatment group (100 mg/kg body weight). Compounds were administered orally 5 times per week. Tumor volume was measured and calculated using the formula: (length × width × height × 0.52). Mice were monitored until tumors reached 1.5 cm³, then euthanized. Tumors, liver, kidney, and spleen were collected for further analysis. [1]

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Patient-derived xenograft (PDX) model: Female SCID mice (6-9 weeks old) were used. A human esophageal tumor specimen was cut into small pieces and inoculated subcutaneously into the back of the neck of each mouse. Mice were divided into two groups (n=10 per group): vehicle group (5% DMSO in 10% Tween 80) and gossypetin treatment group (100 mg/kg body weight). Compounds were administered orally 5 times per week. Tumor volume was measured and calculated using the formula: (length × width × height × 0.52). Mice were monitored until tumors reached 1.5 cm³, then euthanized. Tumors, liver, kidney, and spleen were collected for further analysis. [1]

In the in vivo PDX study, mice treated with gossypetin (100 mg/kg, oral) tolerated the treatment well without significant loss of body weight compared to the vehicle-treated group. [1]
Hematoxylin and eosin (H&E) staining of liver, kidney, and spleen tissues from treated mice showed no obvious morphological differences compared to tissues from untreated mice, indicating no significant toxicity at the tested dose. [1]

[1]. Gossypetin is a novel MKK3 and MKK6 inhibitor that suppresses esophageal cancer growth in vitro and in vivo. Cancer Lett. 2019 Feb 1;442:126-136.

Gossypetin is a hexahydroxyflavonoid with hydroxyl groups located at positions 3, 3', 4', 5, 7, and 8. It is a plant metabolite. It is both a 7-hydroxyflavonol and a hexahydroxyflavonoid. It is the conjugate acid of Gossypetin-3-ol and Gossypetin(1-). Gossypetin has been reported in Sedum (Sinocrassula indica), Rhododendron latoucheae, and other organisms with relevant data. See also: Primula veris flower (partial); Larrea tridentata whole plant (partial).

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Gossypetin is a naturally occurring hexahydroxylated flavonoid found in many flowers and in Hibiscus sabdariffa (roselle). [1]
It exerts various pharmacological activities, including antioxidant, antibacterial, and anticancer activities. This is the first study reporting its chemopreventive properties in esophageal cancer. [1]
The study identifies gossypetin as a novel direct inhibitor of MKK3 and MKK6, suppressing the MKK3/6-p38 signaling pathway. [1]
Gossypetin inhibits esophageal cancer cell growth by inducing G2 phase cell cycle arrest and intrinsic apoptosis (through caspase 3/7 activation and BAX/cytochrome c upregulation). [1]
Its anticancer efficacy was validated in a clinically relevant patient-derived xenograft (PDX) model of esophageal cancer. [1]

C15H10O8

318.23

318.038

489-35-0

5280647

Light yellow to green yellow solid powder

1.912 g/cm3

679.3ºC at 760 mmHg

302-304ºC

260.6ºC

4.49E-19mmHg at 25°C

1.863

1.693

6

8

1

23

518

0

YRRAGUMVDQQZIY-UHFFFAOYSA-N

InChI=1S/C15H10O8/c16-6-2-1-5(3-7(6)17)14-13(22)12(21)10-8(18)4-9(19)11(20)15(10)23-14/h1-4,16-20,22H

2-(3,4-dihydroxyphenyl)-3,5,7,8-tetrahydroxychromen-4-one

Gossypetin C.I. 75750 8 hydroxy Quercetin 8-hydroxy QuercetinArticulatidin Equisporol

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.)