JAK2; Src; STAT3; PTEN
Ginkgolic acid C 17:1 targets the STAT3 signaling pathway. It inhibits constitutive activation of STAT3 by abrogating upstream JAK2 and Src kinases (but not JAK1). It also induces the expression of protein tyrosine phosphatases PTEN and SHP-1, which negatively regulate STAT3 activation. [1]

Ginkgolic acid C 17:1 (GAC 17:1) reduced the viability of U266 multiple myeloma cells in a concentration‑dependent manner with an IC50 value of approximately 64 μM after 24 h treatment, while it did not significantly affect the viability of normal human peripheral blood mononuclear cells (PBMCs) under the same conditions. [1]
GAC 17:1 (50 μM, 3 h) suppressed constitutive STAT3 phosphorylation at Tyr705 in U266 cells, whereas GAC 15:1 (50 μM) did not. The inhibition was dose‑dependent (30 or 50 μM for 3 h) and time‑dependent (50 μM for 1.5 or 3 h), without affecting total STAT3 protein levels. [1]
GAC 17:1 (50 μM) attenuated the constitutive phosphorylation of upstream kinases Src and JAK2 in a time‑dependent manner (1.5 and 3 h), but did not affect JAK1 phosphorylation in U266 cells. [1]
Immunocytochemistry showed that GAC 17:1 (50 μM, 3 h) abolished STAT3 translocation from the cytoplasm to the nucleus in U266 cells. [1]
Electrophoretic mobility shift assay (EMSA) revealed that GAC 17:1 (50 μM, 1.5 or 3 h) inhibited STAT3‑DNA binding activity in a time‑dependent manner in U266 cells. [1]
In MM.1S cells, pretreatment with GAC 17:1 (50 μM, 3 h) inhibited IL‑6‑induced (10 ng/mL, 15 min) STAT3 phosphorylation. [1]
The inhibitory effect of GAC 17:1 on STAT3 phosphorylation was reversible; removal of the compound restored STAT3 phosphorylation. [1]
Pretreatment with the broad‑acting protein tyrosine phosphatase inhibitor sodium pervanadate reversed the GAC 17:1‑induced suppression of STAT3 phosphorylation in U266 cells, indicating the involvement of a PTP. [1]
GAC 17:1 (50 μM, 1.5 or 3 h) induced the expression of PTEN and SHP‑1 at both protein (Western blot) and mRNA (RT‑PCR) levels in a time‑dependent manner in U266 cells. [1]
Transfection of U266 cells with PTEN‑specific or SHP‑1‑specific siRNA (50 nM, 24 h) abolished the GAC 17:1‑induced upregulation of PTEN and SHP‑1 and reversed the inhibitory effect of GAC 17:1 on STAT3 phosphorylation. [1]
Cell cycle analysis showed that GAC 17:1 (30 or 50 μM, 24 h) increased the sub‑G1 DNA content in U266 cells in a dose‑dependent manner. [1]
Annexin V assay demonstrated that GAC 17:1 (30 or 50 μM, 24 h) increased the percentage of Annexin V‑positive U266 cells in a dose‑dependent manner. [1]
TUNEL assay confirmed that GAC 17:1 (30 or 50 μM, 24 h) significantly induced apoptosis in U266 cells. [1]
Live/Dead assay showed increased cell death after GAC 17:1 (50 μM, 24 h) treatment in U266 cells. [1]
GAC 17:1 (30 or 50 μM, 24 h) caused loss of mitochondrial membrane potential in U266 cells as measured by TMRE staining and flow cytometry. [1]
GAC 17:1 (30 or 50 μM) suppressed the proliferation of U266 and MM.1S cells in a dose‑ and time‑dependent manner (assessed by MTT assay over indicated time intervals). [1]
Western blot analysis showed that GAC 17:1 (30 or 50 μM, 24 h) down‑regulated the expression of STAT3‑regulated gene products in U266 cells, including anti‑apoptotic proteins Bcl‑2, Bcl‑xL, Survivin, IAP‑1; proliferation‑related COX‑2 and Cyclin D1; angiogenic VEGF; and invasion‑related MMP‑9 and MMP‑2. [1]
Real‑time PCR confirmed that GAC 17:1 (30 or 50 μM, 24 h) reduced mRNA expression of Bcl‑2, Bcl‑xL, and Cyclin D1 in U266 cells. [1]
GAC 17:1 (30 or 50 μM, 24 h) induced cleavage of caspase‑3 and PARP in a dose‑dependent manner in U266 cells. [1]
In MEF cells transfected with pMXs‑STAT3C (constitutively active STAT3 mutant), GAC 17:1 (100 μM, 3 h) significantly inhibited STAT3 phosphorylation. Furthermore, GAC 17:1 (100 μM, 24 h) induced apoptosis in MEF cells, and this effect was abrogated by transfection with pMXs‑STAT3C. [1]

STAT3‑DNA binding activity was analyzed by electrophoretic mobility shift assay (EMSA). Nuclear extracts were prepared from Ginkgolic acid C 17:1‑treated U266 cells (50 μM for 1.5 or 3 h) following a standard protocol. The nuclear extract protein was incubated with a 5′‑biotinylated STAT3 oligonucleotide probe (sequence: 5′‑GATCCTTCTGGGAATTCCTAGATC‑3′ and complementary strand). The protein‑oligonucleotide complex was separated on a 5% native polyacrylamide gel, transferred to a nylon membrane, and detected using a chemiluminescent EMSA kit according to the manufacturer’s instructions. [1]
For Western blot analysis of phosphorylated and total proteins, whole‑cell extracts were prepared using cell lysis buffer. Equal amounts of protein (50 μg) were resolved by SDS‑PAGE, transferred to nitrocellulose membranes, blocked, and probed with primary antibodies against p‑STAT3 (Tyr705), STAT3, p‑JAK1 (Tyr1022/1023), JAK1, p‑Src (Tyr416), Src, PTEN, SHP‑1, and other target proteins overnight at 4°C. After washing with TBST, membranes were incubated with HRP‑conjugated secondary antibodies for 1 h, and signals were detected using chemiluminescence substrate. [1]

Cell viability was measured by MTT assay. U266 or MM.1S cells (1‑2 × 10⁴ cells/well) were treated with various concentrations of Ginkgolic acid C 17:1 for indicated times (e.g., 24 h). Then, 30 μL of MTT solution (2 mg/mL) was added and incubated for an additional 2 h. The formed formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm using an automated spectrophotometric plate reader. Cell viability was normalized as relative percentages compared to untreated controls. [1]
For Western blot analysis, cells (5 × 10⁵ cells/well) were treated with GAC 17:1 at indicated concentrations and times. Whole‑cell extracts were prepared using cell lysis buffer, and 50 μg of protein was subjected to SDS‑PAGE, transferred to nitrocellulose membranes, blocked, and probed with specific primary antibodies overnight at 4°C, followed by HRP‑conjugated secondary antibodies and chemiluminescence detection. [1]
Immunocytochemistry for STAT3 localization: After GAC 17:1 treatment (50 μM, 3 h), U266 cells were fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.2% Triton X‑100, and blocked with 5% BSA for 1 h. Cells were incubated with anti‑STAT3 or anti‑p‑STAT3 antibody overnight at 4°C, then with FITC‑conjugated secondary antibody for 1 h at room temperature, and stained with DAPI (1 μg/mL). Images were captured using a confocal microscope with excitation at 405 nm (DAPI) and 488 nm (FITC). [1]
EMSA for STAT3‑DNA binding was performed as described in the Enzyme Assay section. [1]
RNA isolation and reverse transcription polymerase chain reaction (RT‑PCR): Total RNA was extracted using Trizol reagent. PTEN primers: forward 5′‑TTTCTAACCGTCGACGCTCTT‑3′, reverse 5′‑AGCTGTGGTGGGTTATTGGTCT‑3′; SHP‑1 primers: forward 5′‑GGCTTCTGGGAGGAGTTTGAG‑3′, reverse 5′‑CGGAGTTTGTATTCCGGTTGTG‑3′. RT‑PCR conditions were performed as described. [1]
Electroporation‑mediated transfection: U266 cells (1 × 10⁶) were suspended in 120 μL of resuspension buffer and mixed with 50 nM PTEN, SHP‑1, or scrambled siRNA, then transfected using a transfection system with two pulses at 1150 V. MEF cells were transfected with 2 μg of pMXs‑STAT3C (constitutively active STAT3 mutant) or pMXs‑gw control vector. After 24 h, cells were treated with GAC 17:1 (50 μM for U266, 100 μM for MEF) and whole‑cell extracts were prepared for Western blotting. [1]
Cell cycle analysis: U266 cells (1 × 10⁶) were treated with GAC 17:1 for 24 h, washed with PBS, fixed with 70% ethanol, then washed and incubated in PBS containing 0.1% RNase A at 37°C for 30 min, stained with propidium iodide, and analyzed by flow cytometry. [1]
Annexin V assay: U266 cells (1 × 10⁶) were treated with GAC 17:1 for 24 h, washed with PBS, stained with FITC‑conjugated Annexin V and propidium iodide, and analyzed by flow cytometry. [1]
TUNEL assay: U266 cells were treated with GAC 17:1 (50 μM) for 24 h, fixed with 4% paraformaldehyde for 30 min at room temperature, resuspended in TUNEL reaction solution, and incubated for 1 h in the dark. TUNEL‑positive cells were analyzed by flow cytometry. [1]
Mitochondrial membrane potential measurement: U266 cells were treated with GAC 17:1 (50 μM) for 24 h, washed with PBS, incubated with 5 μM TMRE (tetramethylrhodamine, ethyl ester) for 30 min, and analyzed by flow cytometry. [1]
Live/Dead assay: Cytotoxicity was assessed using a Live/Dead assay kit as described. [1]
Real‑time quantitative PCR: Total RNA was extracted using Trizol, and 1 μg RNA was converted to cDNA by M‑MLV reverse transcriptase. Primers for Bcl‑2 (forward 5′‑TCCCTCGGTGCACAAATACTC‑3′, reverse 5′‑GACGACCCGATGGCCATA‑3′), Bcl‑xL (forward 5′‑TACCAGCCTGACCAATATGGGC‑3′, reverse 5′‑TGGGTTCAAGTGATTCTCCTG‑3′), Cyclin D1 (forward 5′‑AGAAGCTGTGCATCTACACCGACA‑3′, reverse 5′‑AGAAGCTGTGCATCTACACCGACA‑3′), and GAPDH (forward 5′‑ACCTGACCTGCCGCTCTAGAAAA‑3′, reverse 5′‑ACGCCTGCTTCACCACCTT‑3′) were used. The 2⁻ΔΔCt value was determined. [1]

Ginkgolic acid C 17:1 exhibited minimal toxicity towards normal human peripheral blood mononuclear cells (PBMCs). In MTT assays, treatment with GAC 17:1 at concentrations up to 50 μM for 24 h did not significantly reduce PBMC viability compared to untreated controls (as shown in Figure 1C). The compound was comparatively less toxic towards normal cells than towards multiple myeloma cells. [1]

[1]. Molecules. 2017 Feb 13;22(2):276.

Ginkgolic acid C17-1 is a hydroxybenzoic acid. Functionally, it is related to salicylic acid. It has been reported that 2-(10-heptadecenyl)-6-hydroxybenzoic acid is present in Ephedra sinica, ginkgo, and Emblica emblica, and relevant data are available for reference.

---

Ginkgolic acid C 17:1 is one of the most abundant ginkgolic acids isolated from Ginkgo biloba leaves. It has been previously shown to inhibit proliferation of various tumor cell types (pancreatic, breast, lung, leukemia) and induce apoptosis through modulation of lipogenesis pathways, cell cycle arrest, and decrease of the Bcl‑2/Bax ratio. It can also inhibit SUMOylation. This study demonstrates for the first time that GAC 17:1 suppresses constitutive and inducible STAT3 signaling in multiple myeloma cells by abrogating upstream JAK2 and Src activation and by inducing the protein tyrosine phosphatases PTEN and SHP‑1. This leads to down‑regulation of STAT3‑regulated gene products (e.g., Bcl‑2, Bcl‑xL, survivin, cyclin D1, VEGF, MMP‑9) and induction of apoptosis. The study suggests that GAC 17:1 could be an effective anticancer agent against multiple myeloma with a favorable safety profile. [1]

C24H38O3

374.5567

374.282

111047-30-4

111047-30-4

5469634

White to off-white solid

1.0±0.1 g/cm3

499.1±33.0 °C at 760 mmHg

45 - 46 °C

269.7±21.9 °C

0.0±1.3 mmHg at 25°C

1.522

10.5

2

3

16

27

391

0

O([H])C1=C([H])C([H])=C([H])C(=C1C(=O)O[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C(/[H])=C(/[H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H]

MBYNDKVOZOAOIS-FPLPWBNLSA-N

InChI=1S/C24H38O3/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-18-21-19-17-20-22(25)23(21)24(26)27/h7-8,17,19-20,25H,2-6,9-16,18H2,1H3,(H,26,27)/b8-7-

2-[(Z)-heptadec-10-enyl]-6-hydroxybenzoic acid

Ginkgolic Acid C17-1; C17:1

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: 75~100 mg/mL (200.2~267 mM)
Ethanol: ~75 mg/mL(~200.2 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.67 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (6.67 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (6.67 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.

---

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