In HepG2 cells, aflatoxilactone dose-dependently promotes apoptosis. According to studies, this apoptosis caused by alanolactone is linked to several factors, including increased Bax/Bcl-2 ratio, caspase-3 activation, ROS production, GSH depletion, and suppression of STAT3 activation [1]. The expression of STAT3 target genes, DNA binding, and translocation to the nucleus are all decreased by aminolactone. Though this impact is not mediated by inhibition of STAT3 upstream kinases, amyloidolactone dramatically reduces STAT3 activation and has minor effects on MAPK and NF-κB transcription [2]. In HCT-8 human colon cancer cells, alantolactone triggers activin/SMAD3 signaling. By preventing Cripto-1 and type IIA activin receptors from interacting in the activin signaling pathway, alantolactone has anti-tumor properties [4]. The growth of colon adenocarcinoma HCT-8 cells is inhibited by aflatolactone (5 μg/mL, 24 h) [4].

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Alantolactone inhibited the growth of HepG2 cells in a dose-dependent manner after 12 h treatment, with an IC50 value of 33 μM. [1]

Treatment with 40 μM alantolactone induced severe morphological changes characteristic of cell death, including cell rounding and shrinkage, in a time-dependent manner (3, 6, 12 h). Pretreatment with 3 mM N-acetyl-L-cysteine (NAC) completely protected cells from this cytotoxic effect. Live/dead assay using calcein AM and propidium iodide showed that cell viability decreased to 74.33%, 51.6%, and 27% after 3, 6, and 12 h of 40 μM alantolactone treatment, respectively, compared to 98.5% in control. NAC pretreatment reversed the cytotoxic effect. [1]

Flow cytometry with Annexin V-FITC/PI staining demonstrated that alantolactone (40 μM) induced apoptosis in a time-dependent manner. Early apoptosis increased at 3 and 6 h, while late apoptosis was also observed at 12 h. NAC (3 mM) reversed the apoptotic effect. [1]

Intracellular ROS generation measured by DCFH-DA flow cytometry increased to 25%, 42%, and 54% after 3, 6, and 12 h of 40 μM alantolactone treatment, respectively, compared to 15% in control. [1]

Mitochondrial membrane potential (MMP) assessed by Rhodamine 123 flow cytometry was reduced to 85%, 79%, and 62% after 3, 6, and 12 h of 40 μM alantolactone treatment, respectively, versus 98% in control. [1]

Intracellular GSH levels decreased significantly from 3 h of treatment and further over time, while GSSG levels remained unchanged. No GSH or GSSG was detectable in the culture medium. Pretreatment with PEG-catalase, PEG-SOD, or methionine (GSH carrier inhibitor) did not prevent GSH depletion. NAC pretreatment completely inhibited GSH depletion. Glutathione reductase (GR) expression slightly increased in alantolactone-treated cells by Western blot. mRNA expression of γ-glutamyl cysteine synthetase (γ-GCS) remained unchanged by RT-PCR. HPLC analysis showed that incubating 1 mM alantolactone with increasing concentrations of GSH (0, 5, 15, 30 mM) decreased the amount of alantolactone in a dose-dependent manner, indicating direct conjugation. [1]

Western blot analysis showed that alantolactone (40 μM, 6 and 12 h) decreased the expression of pTyr705 STAT3 and anti-apoptotic Bcl-2, increased the expression of pro-apoptotic Bax, and stimulated cleavage of caspase-3 (appearance of 32 kDa and 17 kDa fragments) in a time-dependent manner. [1]

When compared to control mice, the average tumor volume in mice treated with alanylactone was shown to be roughly 2.17 times smaller. Nonetheless, during the trial period, the administration of alanolactone had no effect on total body weight, suggesting no appreciable harm. Furthermore, compared to control mice, the average tumor weight of mice treated with alanylactone was considerably lower. More significantly, cyclin D1 and p-STAT3 expression in tumor tissues were significantly reduced after injection of alantolactone [2].

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In a xenograft model, athymic nude mice subcutaneously injected with MDA-MB-231 cells were administered alantolactone at 2.5 mg/kg body weight (i.p. injection) every 2 days for 14 days. The average tumor volume in alantolactone-treated mice was approximately 2.17-fold lower compared to control mice. Average tumor weight was significantly reduced. Administration did not affect overall body weight during the experimental period. Immunohistochemistry of tumor tissues showed significant decrease in p-STAT3 and Ki-67 protein expression in alantolactone-treated mice compared to controls. [2]

Electrophoretic mobility shift assay (EMSA) for STAT3 DNA-binding activity: Nuclear extracts were incubated with a 32P-end-labeled double-stranded STAT3 consensus oligonucleotide (sequence 5′-GATCCTTCTGGGATTTCCTAGATC-3′). For competition, 100-fold excess of unlabeled oligonucleotide was added 10 min before adding the labeled probe. DNA-protein complexes were separated on 6% polyacrylamide gel electrophoresis, and radioactive bands were analyzed using an image analyzer. [2]
Molecular docking simulation: The X-ray crystal structure of the STAT3 homodimer bound to DNA (PDB code: 1BG1) was used. Double-stranded DNA, crystallographic water, and PO43- buffer molecules were removed. STAT3 monomer was added with hydrogen atoms, assigned Kollman-Uni charge, and minimized using Sybyl software (version 6.5). The native pTyr peptide was extracted as a ligand. The centroid of the ligand was used as the center of a grid box (20×20×20 points, spacing 0.375 Å) covering the STAT3 SH2 domain–pTyr interaction region. Alantolactone was docked into the SH2 domain using AutoDock (version 4.0). Schematic representation was generated by Discovery Studio (version 3.0). [2]
Gelatin zymography for MMP-9 activity: Cells were seeded in serum-free DMEM and exposed to alantolactone for 24 h. Supernatants were subjected to 8% SDS-PAGE gels containing 0.1% gelatin. Gels were washed with 2.5% Triton X-100 and incubated in developing buffer (50 mM Tris-Cl, pH 7.6, 200 mM NaCl, 10 mM CaCl2) at 37°C for 24 h. Gelatinolytic activity was visualized by staining with 0.1% Coomassie blue R-250 in 45% methanol/10% acetic acid for 30 min and destaining with 45% methanol/10% acetic acid. [2]

Cell Viability Assay[4]
Cell Types: HCT-8 cells.
Tested Concentrations: 5 μg/mL (~21.6 μM).
Incubation Duration: 24 h.
Experimental Results: Activated the activin signaling pathway in HCT-8 cells.

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HepG2 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO2. Cells were treated with alantolactone dissolved in DMSO (final DMSO concentration 1%). DMSO-treated cells served as controls. [1]

Cell viability was determined by MTT assay. Cells were treated with different concentrations of alantolactone for 12 h, then MTT reagent (500 μg/mL) was added and incubated for 4 h at 37°C. Formazan crystals were dissolved in 150 μL DMSO, and absorbance was measured at 570 nm. Cell viability percentage was calculated relative to control. IC50 was calculated using GraphPad Prism 5. [1]

Morphological changes were observed by phase contrast microscopy after treatment with 40 μM alantolactone with or without NAC for 0, 3, 6, and 12 h. [1]

Live/dead assay: Cells were treated with 40 μM alantolactone with or without NAC for 0, 3, 6, 12 h, then stained with 2 μM calcein AM and 4 μM PI in PBS for 20 min at room temperature in the dark. Live and dead cells were counted microscopically (100 cells per sample). [1]

Apoptosis assay: Cells were treated with 40 μM alantolactone for 0, 3, 6, 12 h, harvested, washed with PBS, and resuspended in 500 μL binding buffer containing 5 μL Annexin V-FITC and 5 μL PI for 15 min in the dark, then analyzed by flow cytometry. [1]

ROS measurement: Cells were treated with 40 μM alantolactone for 0, 3, 6, 12 h, then incubated with 10 μM DCFH-DA for 30 min at 37°C, harvested, rinsed, resuspended in PBS, filtered, and analyzed for DCF fluorescence by flow cytometry. [1]

MMP measurement: Cells were treated with 40 μM alantolactone for 0, 3, 6, 12 h, then resuspended in 1 mL PBS containing 10 μg Rhodamine 123 for 30 min in the dark, centrifuged, rinsed, resuspended in PBS, filtered, and analyzed by flow cytometry. [1]

GSH and GSSG measurement: Cells were treated with 40 μM alantolactone for 0, 3, 6, 12 h, or with 40 μM alantolactone in the presence or absence of 2 mM methionine and 3 mM NAC for 6 h. Intracellular and extracellular GSH/GSSG were measured spectrophotometrically using a commercial kit. Values expressed as nmol GSH/mg protein. [1]

HPLC analysis: 1 mM alantolactone was incubated with 0, 5, 15, 30 mM GSH in DMEM without FBS for 30 min at 37°C. Samples were analyzed by HPLC using a C18 column with mobile phase acetonitrile/water (65:35) over 30 min, and elution profile was detected at 227 nm. [1]

RNA isolation and RT-PCR: Total RNA was isolated using a commercial kit. cDNA was reverse-transcribed from 500 ng total RNA. PCR was performed for 35 cycles (94°C 1 min, 52°C 30 sec, 72°C 1 min) with a final extension at 72°C for 10 min. Primers for γ-GCS and GAPDH were used. Products were visualized on 1% agarose gel with ethidium bromide. [1]

Immunoblotting: Proteins (40 μg) were electrophoresed on 12% SDS-PAGE and transferred to PVDF membrane. After blocking with 5% nonfat milk, membranes were incubated with primary antibodies against Bax (1:300), Bcl-2 (1:1000), caspase-3 (1:500), pTyr705 STAT3 (1:300), glutathione reductase (1:300), and β-actin (1:400) for 2 h at room temperature, then with HRP-conjugated secondary antibodies (1:5000) for 1 h. Signals were detected using ECL chemiluminescence kit on X-ray film. [1]

Animal/Disease Models: Female athymic BALB/c nude mice at the age of 6 weeks[2].
Doses: 2.5 mg/kg.
Route of Administration: IP injection every 2 days.
Experimental Results: demonstrated anti-cancer activity.

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Tumor xenograft study: Female athymic BALB/c nude mice (6 weeks old) were subcutaneously injected with MDA-MB-231 cells (5×10^6 cells/200 μL) into the right flank. Ten days after injection, mice were randomly divided into treatment and control groups (n=5). Alantolactone was suspended in 0.1% DMSO (v/v) in saline. The treatment group received alantolactone at 2.5 mg/kg body weight via intraperitoneal (i.p.) injection every 2 days for 14 days. Control animals received equal volume of vehicle (0.1% DMSO in saline, 100 μL i.p. injection). Tumor volume was measured using a caliper every 2 days according to formula: length × width^2 × π/6. After 14 days of treatment, mice were sacrificed, tumors removed, weighed, and fixed in 4% paraformaldehyde for immunohistochemical analysis. [2]
Immunohistochemistry: Tumor tissues were embedded in paraffin, sections incubated with antibodies against Ki-67 and p-STAT3 (1:200 dilution). Sections were developed using HRP EnVision System, and peroxidase binding sites detected by staining with 3,3′-diaminobenzidinetetrahydrochloride. Sections were counterstained with Mayer’s hematoxylin and mounted, then observed under a microscope. [2]

[1]. Alantolactone induces apoptosis in HepG2 cells through GSH depletion, inhibition of STAT3 activation, and mitochondrial dysfunction. Biomed Res Int. 2013;2013:719858.

[2]. Alantolactone selectively suppresses STAT3 activation and exhibits potent anticancer activity in MDA-MB-231 cells. Cancer Lett. 2015 Feb 1;357(1):393-403.

Arantoractone is a sesquiterpene lactone with the structure 3a,5,6,7,8,8a,9,9a-octahydronaphtho[2,3-b]furan-2-one, containing two methyl substituents at positions 5 and 8a, and one methylene substituent at position 3. It is a plant metabolite with apoptosis-inducing and antitumor activities. It is a sesquiterpene lactone, a naphthofuran compound, and an olefin compound. Arantoractone has been reported to be found in Inula grandis, Inula japonica, and other organisms with relevant data.

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Alantolactone is a sesquiterpene lactone with anti-inflammatory and anticancer effects. The study demonstrates for the first time that alantolactone depletes GSH in HepG2 cells via direct conjugation through its α-methylene-γ-lactone moiety. This GSH depletion leads to inhibition of STAT3 phosphorylation, oxidative stress, modulation of Bcl-2 family proteins (increased Bax/Bcl-2 ratio), mitochondrial membrane potential dissipation, and caspase-3 activation, ultimately inducing apoptosis. The compound has potential as a lead chemotherapeutic candidate for liver cancer treatment. [1]

C15H20O2

232.323

232.146

546-43-0

72724

White to off-white solid powder

1.1±0.1 g/cm3

275.0±0.0 °C at 760 mmHg

78-79ºC

111.5±18.2 °C

0.0±0.5 mmHg at 25°C

1.534

3.69

0

2

0

17

421

4

C[C@H]1CCC[C@]2(C1=C[C@H]3[C@@H](C2)OC(=O)C3=C)C

PXOYOCNNSUAQNS-AGNJHWRGSA-N

InChI=1S/C15H20O2/c1-9-5-4-6-15(3)8-13-11(7-12(9)15)10(2)14(16)17-13/h7,9,11,13H,2,4-6,8H2,1,3H3/t9-,11+,13+,15+/m0/s1

(3aR,5S,8aR,9aR)-5,8a-dimethyl-3-methylidene-5,6,7,8,9,9a-hexahydro-3aH-benzo[f][1]benzofuran-2-one

AI3-31147Alantolactone AI331147 AI3 31147

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 (~430.44 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.76 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 (10.76 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 (10.76 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.)