Target: SIRT3 (mitochondrial deacetylase) – no IC50/Ki/EC50/DC50 values reported. [1]
Cyclophilin D (CypD) – acetylated CypD is deacetylated via SIRT3. [1]
14-3-3 protein family (six isoforms: ζ/δ, β/α, θ, σ, γ, ε) – predicted to bind Ganoderic acid D; confirmed for 14-3-3 ζ with equilibrium dissociation constant (K_D) = (4.04 ± 0.32) × 10⁻⁶ M (from SPR analysis). [2]
Annexin A5 – predicted direct binding (ligand-protein interaction energy: -39.2). [2]
Aminopeptidase B – predicted direct binding (interaction energy: -49.3). [2]

The growth of several cancer cell lines can be inhibited by ganoderic acid D, which has an IC50 of 17.3 mM against the human cervical cancer cell HeLa[2]. Cell viability is lowered by ganoderic acid D (1–50 μM; 24-72 hours) in a dose- and time-dependent way [2]. G2/M phase arrest is induced by ganoderic acid D (10, 50 μM; 24, 48 hours) [2]. HeLa cell apoptosis can be induced with ganoderic acid D (10, 50 μM; 24, 48 hours) to produce the characteristic morphological alterations [2]. 10 μM; 48 hours) of ganoderic acid D upregulates PRDX3 and 14-3-3E[2].

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Ganoderic acid D (24 h treatment) inhibited glucose uptake, lactate production, pyruvate production, and acetyl-CoA production in HT29 and SW620 colon cancer cells in a dose-dependent manner (0, 50, 100, 200 μmol/L); the most beneficial effect was observed at 200 μmol/L. [1]
Ganoderic acid D did not regulate Glut1 and HK1 protein expression but upregulated SIRT3 protein expression in a dose-dependent manner in HT29 and SW620 cells; at 200 μmol/L for 24 h, SIRT3 expression increased 2.5-fold compared to control. [1]
SIRT3 knockdown by PGC-shSIRT3 plasmid (transfection efficiency ~87%) reversed the inhibitory effects of Ganoderic acid D (200 μmol/L, 24 h) on glucose uptake, lactate production, pyruvate, and acetyl-CoA in HT29 and SW620 cells. [1]
Ganoderic acid D (200 μmol/L, 24 h) significantly inhibited the acetylated level of cyclophilin D (CypD); SIRT3 shRNA reversed this effect. Co-immunoprecipitation showed that Ganoderic acid D induced the combination of SIRT3 and CypD, while PGC-shSIRT3 blocked this effect. [1]
Ganoderic acid D treatment for 48 h inhibited proliferation of HeLa human cervical carcinoma cells with an IC₅₀ value of 17.3 ± 0.3 μM. [2]
Flow cytometric analysis showed that Ganoderic acid D (10 and 50 μM) induced G2/M phase arrest and apoptosis in HeLa cells. After 24 h treatment with 10 μM GAD, G2/M population increased from 16.7% (control) to 28.8%; apoptosis rate increased from 0.7% (control) to 7.9%. After 48 h treatment with 10 μM GAD, G2/M population was 0.30% (vs 18.2% control) and apoptosis rate was 17.7% (vs 3.8% control). [2]
Morphological changes typical of apoptosis (nuclear condensation and fragmentation) were observed in HeLa cells treated with Ganoderic acid D (10 and 50 μM for 48 h) by DAPI staining. [2]
DNA fragmentation (DNA ladder) was observed in HeLa cells treated with 50 μM Ganoderic acid D for 48 h. [2]
Proteomics analysis (2-DE and MALDI-TOF MS/MS) identified 21 differentially expressed proteins in HeLa cells after 48 h treatment with 10 μM Ganoderic acid D: 7 down-regulated and 14 up-regulated. These included eIF5A (down), 14-3-3E (up), PRDX3 (up), EB1 (down), annexin A5 (up), spermidine synthase (up), EphA7 (up), cytokeratin 19 (down), etc. Western blotting confirmed the changes for eIF5A, 14-3-3E, PRDX3, and EB1. [2]

Surface plasmon resonance (SPR) biosensor analysis was performed to determine the binding affinity of Ganoderic acid D to 14-3-3 ζ protein. Human recombinant GST-14-3-3 ζ and GST (control) were immobilized onto a sensor chip via standard primary amine-coupling procedures using N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. Ganoderic acid D was serially diluted in running buffer (HBS-EP containing 0.5% DMSO) at concentrations of 10, 7, 4.9, 3.43, 2.40, and 1.68 μM. Samples were injected at a flow rate of 30 μL/min for 60 seconds, and dissociation was monitored for more than 120 seconds. The association rate constant (k_on) was (5.02 ± 0.24)×10³ M⁻¹s⁻¹, dissociation rate constant (k_off) was (2.03 ± 0.17)×10⁻² s⁻¹, and equilibrium dissociation constant (K_D) was (4.04 ± 0.32)×10⁻⁶ M. The χ² value was 0.32. Non-specific binding to GST was also measured (K_D = (8.06 ± 0.37)×10⁻⁶ M). [2]

Cell viability assay [2]
Cell Types: HeLa human cervical cancer cell line (CCL-2)
Tested Concentrations: 1, 5, 10, 20, 50 μM
Incubation Duration: 24, 48, 72 hrs (hours)
Experimental Results: diminished cell survival rate Dosage and Time-dependent, the IC50 value for 48 hrs (hours) treatment was 17.3 μM.

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Cell cycle analysis [2]
Cell Types: HeLa human cervical cancer cell line (CCL-2)
Tested Concentrations: 10, 50 μM
Incubation Duration: 24, 48 hrs (hours)
Experimental Results: Induced G2/M phase arrest. After 24 hrs (hours) of treatment with 10 μM, a cell cycle profile is shown with an increase in the G2/M cell population.

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Apoptosis analysis [2]
Cell Types: HeLa human cervical cancer cell line (CCL-2)
Tested Concentrations: 10, 50 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: HeLa cells were induced to show typical apoptotic morphological changes.

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Western Blot Analysis[2]
Cell Types: HeLa human cervical cancer cell line (CCL-2)
Tested Concentrations: 10 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: 14-3-3E and PRDX3 were upregulated.

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For glucose consumption and lactate production measurements, cells were treated with Ganoderic acid D for 48 h, then culture media were collected. Glucose uptake was measured using an Amplex Red Glucose/Glucose Oxidase assay, and lactate production was measured using a lactate assay kit. Results were normalized to total cellular protein. [1]
For pyruvate assay, cells treated with Ganoderic acid D were collected and dissolved in pyruvate assay buffer. A 50 μL sample was mixed with 50 μL reaction mixture and incubated at room temperature for 30 min. Absorbance was measured at 570 nm and pyruvate concentration was calculated relative to a standard curve (10-0.1 mmol per well), then normalized to cellular protein. [1]
For acetyl-coenzyme A assay, cells treated with Ganoderic acid D were harvested, washed with PBS, and dissolved in washing buffer. Ice-cold 3 M HClO₄ was added and incubated on ice for 30 min. After centrifugation, the supernatant was neutralized with saturated KHCO₃ and centrifuged again. Acetyl-CoA level was quantified using an acetyl-CoA assay kit with fluorescence measurement (Ex/Em = 535/589 nm), and concentration was calculated based on a standard curve, normalized to total cellular protein. [1]
For Western blotting, proteins were separated on 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBS/T buffer, then incubated with primary antibodies (anti-hexokinase II, anti-Glut1, anti-SIRT3, anti-acetylated CypD, anti-GAPDH) at 4°C overnight, followed by HRP-conjugated secondary antibodies for 60 min at room temperature. Signal was detected with ECL reagent. [1]
For plasmid transfection, PGC-shNegative and PGC-shSIRT3 plasmids were transfected into HT29 and SW620 cells using FuGENE HD reagent. Cells were used for experiments 48 h after transfection. [1]
For co-immunoprecipitation, cells were lysed in NP40 buffer. Protein lysates were precleared with agarose beads. Each IP reaction used 600 μg total protein and 4 μg SIRT3 antibody, rotated overnight at 4°C, then 30 μg beads were added and rotated for another 2 h. After centrifugation, the pellet was washed twice in NP40 buffer, and complexes were eluted in SDS lysis buffer. [1]
For cytotoxicity assay (MTT), HeLa cells were plated in 96-well plates at 1×10³ cells/well and incubated overnight. Media were replaced with fresh media containing various concentrations of Ganoderic acid D (1, 5, 10, 20, 50 μM) for 24, 48, or 72 h. Then 20 μL of MTT (5 mg/mL) was added and incubated for 3 h at 37°C. Then 100 μL lysis buffer was added and incubated overnight. Optical density was measured at 570 nm. IC₅₀ was calculated by the Logit method. [2]
For flow cytometric analysis of cell cycle, HeLa cells treated with Ganoderic acid D (10 or 50 μM) were harvested, washed with PBS, fixed in ice-cold 70% ethanol for 2 h at 4°C, centrifuged, and resuspended in propidium iodide stain buffer (0.1% Triton X-100, 10 μg/mL RNase A, 50 μg/mL propidium iodide in PBS) for 30 min in the dark. Analysis was conducted using a flow cytometer. [2]
For nuclear staining (apoptosis morphology), HeLa cells treated with Ganoderic acid D (10 or 50 μM for 48 h) were washed with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, stained with 0.5 mg/mL DAPI in PBS for 10 min, washed, and photographed under fluorescence microscope. [2]
For DNA fragmentation assay, genomic DNA was extracted from HeLa cells treated with Ganoderic acid D (10 or 50 μM for 48 h) using DNAzol, loaded on 2% agarose gels, electrophoresed, stained with ethidium bromide (0.5 mg/L), and photographed under UV illumination. [2]
For 2-DE analysis, HeLa cells were treated with 10 μM Ganoderic acid D or 0.1% DMSO (control) for 48 h. Cells were lysed in buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 1% DTT, 0.8% Pharmalyte, and protease inhibitor. Proteins (150 μg) were applied to IPG strips (pH 4–7, 17 cm) for IEF, then equilibrated and separated on 12% SDS-PAGE gels. Gels were silver-stained, scanned, and analyzed with PDQuest software. Protein spots with ≥2-fold change (p<0.05) were excised and identified by MALDI-TOF MS/MS. [2]

Ganoderic acid D at 200 μmol/L for 24 h inhibited HT29 and SW620 cellular viability by no more than 20%, indicating low cytotoxic effect compared to standard cytotoxic anticancer medicines. [1]

[1]. Effect of ganoderic acid D on colon cancer Warburg effect: Role of SIRT3/cyclophilin D. Eur J Pharmacol. 2018 Apr 5;824:72-77.

[2]. Proteomics characterization of the cytotoxicity mechanism of ganoderic acid D and computer-automated estimation of the possible drug target network. Mol Cell Proteomics. 2008 May;7(5):949-61.

Ganoderic acid D is a triterpenoid compound. It has been reported that ganoderic acid D exists in Ganoderma applanatum, and relevant data are available for reference.

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Ganoderic acid D is a highly oxygenated tetracyclic triterpenoid from Ganoderma lucidum (Lingzhi), a mushroom used in traditional Chinese medicine for over 2000 years to preserve vitality and promote longevity. [2]
Ganoderic acid D inhibits the Warburg effect (aerobic glycolysis) in colon cancer cells, which is a reprogrammed energy metabolism hallmark of cancer. This effect is mediated by upregulation of SIRT3 and subsequent deacetylation of cyclophilin D. [1]
In HeLa cervical carcinoma cells, Ganoderic acid D induces G2/M cell cycle arrest and apoptosis, as shown by flow cytometry and DNA fragmentation. [2]
Proteomics and inverse docking analysis suggested that six isoforms of the 14-3-3 protein family are possible direct targets of Ganoderic acid D, and these proteins play central roles in the cytotoxicity mechanism. [2]

C30H42O7

514.6503

514.293

108340-60-9

14109406

White to off-white solid powder

1.2±0.1 g/cm3

688.3±55.0 °C at 760 mmHg

218 - 220 °C

384.1±28.0 °C

0.0±4.9 mmHg at 25°C

1.559

2.43

2

7

6

37

1120

7

C[C@H](CC(=O)CC(C)C(=O)O)[C@H]1CC(=O)[C@@]2([C@@]1(CC(=O)C3=C2[C@H](C[C@@H]4[C@@]3(CCC(=O)C4(C)C)C)O)C)C

YTVGSCZIHGRVAV-IYAQLQCNSA-N

InChI=1S/C30H42O7/c1-15(10-17(31)11-16(2)26(36)37)18-12-23(35)30(7)25-19(32)13-21-27(3,4)22(34)8-9-28(21,5)24(25)20(33)14-29(18,30)6/h15-16,18-19,21,32H,8-14H2,1-7H3,(H,36,37)/t15-,16?,18-,19+,21+,28+,29-,30+/m1/s1

(6R)-6-[(5R,7S,10S,13R,14R,17R)-7-hydroxy-4,4,10,13,14-pentamethyl-3,11,15-trioxo-1,2,5,6,7,12,16,17-octahydrocyclopenta[a]phenanthren-17-yl]-2-methyl-4-oxoheptanoic acid

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 : ~100 mg/mL (~194.31 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.86 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 (4.86 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 (4.86 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.)