IC50: apoptosis
Polyporenic acid C activates caspase-8 and caspase-3, but not caspase-9; it suppresses PI3-kinase/Akt signaling (reduces Akt phosphorylation), enhances p53 activation (increases p53 Ser15 phosphorylation), and activates JNK (though JNK is not required for apoptosis). [1]

- Polyporenic acid C inhibited A549 cell proliferation in a dose- and time-dependent manner. After 72 h treatment, cell viability was reduced to 84% at 6 μM, to below 50% at 60 μM, and by more than 90% at 200 μM (MTT assay). [1]

- PPAC induced apoptosis as shown by sub-G1 analysis: treatment with 60 μM PPAC for 48 h increased the sub-G1 population from 2% to 47%; dose-dependent increase from 5% (20 μM) to 51% (80 μM) after 48 h. [1]

- Annexin V-FITC staining revealed early and late apoptosis: after 24 h, apoptotic cells increased from 5.6% (control) to 20.8% (60 μM) and 33.8% (100 μM); after 48 h, to 30.3% (60 μM) and 93.8% (100 μM). [1]

- Western blot analysis showed that PPAC (60 μM, 24 h) induced cleavage of procaspase-8, procaspase-3, and PARP, but not procaspase-9. [1]

- PPAC-induced apoptosis was blocked by pan-caspase inhibitor (z-VAD-fmk) and caspase-8 inhibitor (z-IETD-fmk), but not by caspase-9 inhibitor (z-LEHD-fmk). PARP cleavage was also prevented by caspase-8 inhibitor but not by caspase-9 inhibitor. [1]

- PPAC did not disrupt mitochondrial membrane potential (ΔΨm) at 20, 60, or 100 μM for 24 or 48 h (DiOC6(3) staining). In contrast, pachymic acid (a related triterpenoid) caused a dose- and time-dependent decrease in ΔΨm. [1]

- PPAC induced sustained JNK activation, but the JNK-specific inhibitor SP600125 (20 μM) did not prevent PARP cleavage or apoptosis, indicating JNK is not required. [1]

- PPAC treatment (60 μM, 24 h) dramatically reduced Akt phosphorylation (Ser473) without changing total Akt expression; increased PTEN phosphorylation; and enhanced p53 phosphorylation at Ser15. [1]

- Cell culture and treatment: A549 human non-small cell lung cancer cells were cultured in F12K medium supplemented with 10% fetal bovine serum, 10 mM HEPES, and antibiotics (100 U/mL penicillin G and 100 μg/mL streptomycin) at 37°C in 5% CO2. Polyporenic acid C was dissolved in DMSO as a stock solution (20 mg/mL) and further diluted with DMSO to working concentrations; final DMSO concentration ≤0.5% in all experiments (no significant effect on cell growth). [1]

- Cell proliferation assay (MTT): A549 cells (5×10^3 per well) were plated in 96-well plates, incubated overnight, then treated with fresh medium containing various concentrations of PPAC (0–200 μM) for indicated times. Absorbance was measured at 590 nm using a microplate reader. Cell viability was expressed as percentage relative to vehicle-treated control (set at 100%). [1]

- Propidium iodide (PI) staining for sub-G1 analysis: After treatment, floating and trypsinized adherent cells were collected, washed with PBS, fixed in ice-cold ethanol at -20°C for ≥30 min, then incubated with PI staining solution (0.1% Triton X-100 in PBS containing 200 μg/mL RNase and 20 μg/mL PI) for 30 min at room temperature. Cell cycle distribution was analyzed by flow cytometry; cells with sub-G1 DNA content were classified as apoptotic. [1]

- Annexin V-FITC apoptosis assay: Treated cells were collected and stained with Alexa Fluor 488 annexin V and PI in Annexin Binding Buffer for 15 min at room temperature, then analyzed by flow cytometry. Early apoptotic cells (annexin V-positive, PI-negative) appear in the lower right quadrant; late apoptotic cells (double-positive) appear in the upper right quadrant. For inhibitor studies, cells were pretreated with inhibitors (pan-caspase inhibitor z-VAD-fmk, caspase-8 inhibitor z-IETD-fmk, or caspase-9 inhibitor z-LEHD-fmk at 50 μM) for 1 h before PPAC addition. [1]

- Mitochondrial membrane potential (ΔΨm) analysis: Cells were stained with 40 nM DiOC6(3) during the final 30 min of PPAC treatment, washed with PBS/0.1% BSA, and analyzed by flow cytometry. Decreased fluorescence indicates loss of ΔΨm. [1]

- Protein extraction and immunoblotting: After treatment, cells were lysed in ice-cold lysis buffer containing freshly added 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, 5 μg/mL pepstatin A, and 100 μM leupeptin. Lysates were sonicated briefly on ice, and protein concentration was quantified by bicinchoninic acid (BCA) assay. Protein (40 μg per sample) was resolved by 10% SDS-PAGE, transferred to PVDF membrane, blocked, and incubated with primary antibodies (specific for cleaved caspase-3, cleaved caspase-8, caspase-9, cleaved PARP, phospho-p53Ser15, phospho-AktSer473, phospho-JNK, and actin) followed by HRP-conjugated secondary antibodies. Protein complexes were visualized by enhanced chemiluminescence. [1]

[1]. Polyporenic acid C induces caspase-8-mediated apoptosis in human lung cancer A549 cells.Mol Carcinog. 2009 Jun;48(6):498-507.

According to reports, polyporic acid C is found in Daedalea dickinsii, Antrodia heteromorpha, and other organisms with relevant data.

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- Polyporenic acid C is a lanostane-type triterpenoid isolated from the sclerotia of Poria cocos, a fungus traditionally used in Chinese herbal medicine as a diuretic and sedative agent. This study is the first to demonstrate that PPAC induces apoptosis in non-small cell lung cancer A549 cells via the death receptor-mediated pathway (caspase-8-dependent) without involving mitochondrial disruption, distinguishing its mechanism from that of pachymic acid (another triterpenoid from P. cocos which acts via the mitochondrial pathway). [1]

- PPAC suppresses the PI3-kinase/Akt survival pathway (reduces Akt phosphorylation and increases PTEN phosphorylation) and enhances p53 activation (Ser15 phosphorylation), which may contribute to apoptosis induction. JNK activation is observed but not required for PPAC-induced apoptosis. [1]

- The study suggests that PPAC is a promising candidate for lung cancer therapy, warranting further investigation. [1]

C31H46O4

482.6946

482.34

465-18-9

9805290

White to off-white solid

6.744

2

4

6

35

1000

7

O([H])[C@]1([H])C([H])([H])[C@@]2(C([H])([H])[H])C3=C([H])C([H])([H])[C@@]4([H])C(C([H])([H])[H])(C([H])([H])[H])C(C([H])([H])C([H])([H])[C@]4(C([H])([H])[H])C3=C([H])C([H])([H])[C@]2(C([H])([H])[H])[C@@]1([H])[C@]([H])(C(=O)O[H])C([H])([H])C([H])([H])C(=C([H])[H])C([H])(C([H])([H])[H])C([H])([H])[H])=O

KPKYWYZPIVAHKU-WMNQUVFJSA-N

InChI=1S/C31H46O4/c1-18(2)19(3)9-10-20(27(34)35)26-23(32)17-31(8)22-11-12-24-28(4,5)25(33)14-15-29(24,6)21(22)13-16-30(26,31)7/h11,13,18,20,23-24,26,32H,3,9-10,12,14-17H2,1-2,4-8H3,(H,34,35)/t20-,23-,24+,26+,29-,30-,31+/m1/s1

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

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 : ~16.67 mg/mL (~34.54 mM)

Solubility in Formulation 1: 5 mg/mL (10.36 mM) in 50% PEG300 +50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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.)