bioactive compound
In HCT-116 colon cancer cells, Lobetyolin targets Alanine-serine-cysteine transporter 2 (ASCT2) and its effect is governed by p53. [2]

Lobetyolin, lobetyol, and methyl linoleate inhibited the gene expression of MUC5AC mucin induced by PMA; lobetyolin did not affect PMA-induced MUC5AC mucin production. However, lobetyol and methyl linoleate inhibited the production of MUC5AC mucin; lobetyolin and lobetyol did not significantly affect PMA-induced MUC5AC mucin secretion from NCI-H292 cells. However, methyl linoleate decreased the MUC5AC mucin secretion.[1]

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In NCI-H292 human pulmonary mucoepidermoid cells, Lobetyolin (1, 10, and 100 μM) inhibited phorbol 12-myristate 13-acetate (PMA, 10 ng/mL)-induced MUC5AC gene expression. [1]
In NCI-H292 cells, Lobetyolin (1, 10, and 100 μM) did not significantly affect PMA-induced MUC5AC mucin production or secretion. [1]
In HCT-116 colorectal cancer cells, Lobetyolin (10, 20, 40 μmol/L for 24 hours) significantly inhibited cell viability in a concentration-dependent manner. [2]
In NCM460 normal colon mucosal epithelial cells, Lobetyolin (10, 20, 40 μmol/L) did not significantly influence cell viability. [2]
In HCT-116 cells, Lobetyolin (20 and 40 μmol/L) notably increased the number of TUNEL-positive cells, indicating increased apoptosis. Lobetyolin (10, 20, 40 μmol/L) also remarkably increased the population of early apoptosis, late apoptosis, and necrosis as measured by Annexin V-FITC/PI staining. [2]
In HCT-116 cells, Western blot analysis showed that Lobetyolin (10, 20, 40 μmol/L) dramatically elevated the protein levels of cleaved-caspase-3, caspase-3, cleaved-caspase-7, caspase-7, cleaved-PARP, and PARP, while down-regulating the expression of survivin. [2]
In HCT-116 cells, Lobetyolin (10, 20, 40 μmol/L) evidently reduced the concentrations of glutamine and α-ketoglutarate. It (20, 40 μmol/L) also decreased glutamic acid, ATP, and GSH levels. [2]
In HCT-116 cells, Lobetyolin (10, 20, 40 μmol/L) prominently reduced ASCT2 mRNA and protein expression and inhibited ASCT2 expression measured by immunofluorescence. [2]
In HCT-116 cells, Lobetyolin (40 μmol/L) augmented the mRNA levels of Bax and p21, while significantly inhibiting Bcl-2 mRNA expression. [2]
In HCT-116 cells, Lobetyolin (10, 20, 40 μmol/L) caused p53 protein to be down-regulated in the nucleus and up-regulated in the cytoplasm, promoting the translocation of p53 from cytoplasm to nucleus. [2]
In HCT-116 cells, treatment with Lobetyolin (40 μmol/L) combined with the ASCT2 inhibitor Benser had a slightly more potent effect on reducing ATP and GSH levels and promoting apoptosis-related protein expressions (cleaved-caspase-3, caspase-3, cleaved-caspase-7) than Lobetyolin alone. [2]
In HCT-116 cells, the p53 inhibitor Pifithrin-α blocked the Lobetyolin-mediated down-regulation of ASCT2 mRNA and protein, glutamine metabolism biomarkers (α-ketoglutarate, ATP, GSH), and apoptosis protein overexpressions. [2]

In a nude mouse xenograft model bearing HCT-116 cells, Lobetyolin (10, 20, 40 mg/kg, intraperitoneal injection for 2 weeks) obviously inhibited tumor volume compared to the control group. Lobetyolin also markedly decreased ASCT2 mRNA and protein expression in the tumor tissues. [2]

Confluent NCI-H292 cells were pretreated with lobetyolin, lobetyol, or methyl linoleate for 30 minutes and then stimulated with phorbol 12-myristate 13-acetate (PMA) for 24 hours. The MUC5AC mucin gene expression, and mucin protein production and secretion were measured by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively.[1]

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For mucin studies, NCI-H292 cells were cultured in RPMI 1640 with 10% FBS. For serum deprivation, confluent cells were washed with PBS and maintained in RPMI 1640 with 0.2% FBS for 24 hours. Cells were then pretreated with Lobetyolin (1, 10, and 100 μM) for 30 minutes and then stimulated with PMA (10 ng/mL) for 24 hours in serum-free RPMI 1640. After 24 hours, spent media were collected to measure MUC5AC protein secretion, cells were lysed to measure MUC5AC protein production, and total RNA was extracted to measure MUC5AC gene expression via RT-PCR. [1]
For the MUC5AC protein analysis (ELISA), spent media or cell lysates were diluted 1:10 in PBS, and 100 μL of each sample was incubated at 42°C in a 96-well plate until dry. Wells were washed, blocked with 2% BSA, and then incubated with a mouse monoclonal MUC5AC antibody. After washing, a horseradish peroxidase-goat anti-mouse IgG conjugate was added. Color reaction was developed with TMB peroxide solution and stopped with H2SO4, and absorbance was read at 450 nm. [1]
For RT-PCR, total RNA was isolated and reverse transcribed. 2 μg of total RNA was primed with 1 μg of oligo (dT) in a 30 μL reaction. 2 μL of RT reaction product was PCR amplified in a 20 μL reaction. Primers for MUC5AC and the housekeeping gene Rig/S15 rRNA were used. The PCR mixture was denatured at 94°C for 5 minutes, followed by 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, with a final extension at 72°C for 10 minutes. PCR products were subjected to 1% agarose gel electrophoresis and visualized with ethidium bromide. [1]
For cell viability and anti-cancer studies, HCT-116 and NCM460 cells were cultured in RPMI 1640 with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO2. For the MTT assay, 1x10^4 cells/mL HCT-116 cells were seeded in a 96-well plate for 24 hours. Cells were treated with various concentrations of Lobetyolin (1, 5, 10, 20, 40, 80 μmol/L) for 24 hours. Then, 20 μL of 5 mg/mL MTT was added for 4 hours. The medium was discarded, and formazan crystals were dissolved in DMSO. Optical density was measured at 570 nm. Cell viability (%) = (A_Treated / A_Control) x 100%. [2]
For Annexin V-FITC/PI staining, HCT-116 cells were washed with cold PBS, resuspended in binding buffer, and exposed to V-FITC/PE solution in the dark at 25°C for 10 minutes. The percentage of apoptotic cells was analyzed by flow cytometry. [2]
For TUNEL assay, HCT-116 cells were seeded on chamber slides and treated with Lobetyolin. After washing with PBS, TUNEL staining was performed. Anti-fluorescence quenching solution was used before visualization under a fluorescence microscope. [2]
For RT-qPCR, HCT-116 cells were seeded (2.5x10^5/mL, 2 mL) in a six-well plate for 24 hours, treated with compound for 24 hours, and then harvested. Total RNA was isolated using TRlzol reagent. 1 μg of total RNA was reverse transcribed to make cDNA. The reaction mixture consisted of supermix, total RNA, and DNase-free water. RNA was transcribed using M-MuLV Reverse Transcriptase. [2]
For Western blot, HCT-116 cells were seeded (2.5x10^5/mL, 2 mL) in a six-well plate for 24 hours, treated with Lobetyolin for 24 hours, and then collected. Cells were washed with cold PBS, immersed in RIPA lysis buffer, and centrifuged at 10,000g for 10 minutes at 4°C. Protein concentration was determined. Equal amounts of sample were separated by 8%-12% SDS-PAGE and transferred onto a PVDF membrane. The blots were blocked with 5% non-fat milk and incubated with primary antibodies at 4°C overnight, then with a secondary antibody conjugated with HRP for 1 hour. The membranes were incubated with an ECL detective system and visualized. [2]
For immunofluorescence staining, 5x10^5 cells were seeded on a six-well plate for 24 hours, then treated with Lobetyolin. After 24 hours, cells were fixed with 4% polyformaldehyde for 30 minutes. First antibodies diluted in BSA were incubated with the cells overnight at 4°C. The stained cells were then treated with a goat anti-rabbit Alexa Fluor secondary antibody in the dark for 1 hour and exposed to DAPI. After sealing with anti-fluorescence quenching solution, samples were observed by fluorescence microscope. [2]

For the in vivo xenograft model, 6-week-old athymic nude (nu/nu) mice were subcutaneously inoculated with HCT-116 cells (2x10^6 cells) at the right flank. 24 hours after tumor transplantation, mice were randomly assigned into 4 groups (n=10): control (vehicle), Lobetyolin (10 mg/kg), Lobetyolin (20 mg/kg), and Lobetyolin (40 mg/kg). Mice were intraperitoneally injected with Lobetyolin or vehicle for 2 weeks. Afterwards, mice were killed, and tumors were removed, weighed, and applied for mRNA and Western blot analyses. [2]

In NCI-H292 cells, possible cytotoxicity of Lobetyolin (1, 10, and 100 μM) was checked by lactate dehydrogenase (LDH) assay, and there was no remarkable cytotoxic effect at the tested concentrations. [1]
In NCM460 normal colon mucosal epithelial cells, the MTT assay demonstrated that Lobetyolin (10, 20, 40 μmol/L) did not significantly influence cell viability. [2]

[1]. Effects of Lobetyolin, Lobetyol and Methyl linoleate on Secretion, Production and Gene Expression of MUC5AC Mucin from Airway Epithelial Cells. Tuberc Respir Dis (Seoul). 2014 Nov;77(5):203-8.

[2]. Lobetyolin induces apoptosis of colon cancer cells by inhibiting glutamine metabolism. J Cell Mol Med. 2020 Mar;24(6):3359-3369.

Lobetyolin is an O-acyl carbohydrate. It has been reported that (2R,3R,4S,5S,6R)-2-[(4E,12E)-1,7-dihydroxytetradec-4,12-diene-8,10-diyn-6-yl]oxy-6-(hydroxymethyl)oxacyclohexane-3,4,5-triol is found in Campanula sylvestris, Haloxylon ammodendron, and other organisms with relevant data. See also: Lobetyolin (note moved to).

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The inhibitory action of Lobetyolin on airway mucin secretion, production, and gene expression might explain, at least in part, the folk use of Codonopsis pilosula as an anti-inflammatory and anti-allergic agent for pulmonary inflammatory diseases in traditional folk medicine. [1]
In colon cancer HCT-116 cells, Lobetyolin induces apoptosis via the inhibition of ASCT2-mediated glutamine metabolism, which is possibly governed by p53. The study suggests that Lobetyolin could be a therapeutic candidate for colorectal tumor. [2]

C20H28O8

396.4315

396.178

136085-37-5

53486204

White to yellow solid

1.4±0.1 g/cm3

698.5±55.0 °C at 760 mmHg

376.2±31.5 °C

0.0±5.0 mmHg at 25°C

1.607

Codonopsis pilosula

2.02

6

8

9

28

646

5

O1[C@]([H])([C@@]([H])([C@]([H])([C@@]([H])([C@@]1([H])C([H])([H])O[H])O[H])O[H])O[H])OC([H])(/C(/[H])=C(\[H])/C([H])([H])C([H])([H])C([H])([H])O[H])C([H])(C#CC#C/C(/[H])=C(\[H])/C([H])([H])[H])O[H]

MMMUDYVKKPDZHS-UPPVCQNNSA-N

InChI=1S/C20H28O8/c1-2-3-4-5-7-10-14(23)15(11-8-6-9-12-21)27-20-19(26)18(25)17(24)16(13-22)28-20/h2-3,8,11,14-26H,6,9,12-13H2,1H3/b3-2+,11-8+/t14?,15?,16-,17-,18+,19-,20-/m1/s1

(2R,3R,4S,5S,6R)-2-[(4E,12E)-1,7-dihydroxytetradeca-4,12-dien-8,10-diyn-6-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol

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: ~50 mg/mL (126.1 mM)
H2O: ~50 mg/mL (126.1 mM)

≥ 2.5 mg/mL (6.3 mM) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline
≥ 2.5 mg/mL (6.3 mM) in 10% DMSO + 90% (20% SBE-β-CD in saline)
≥ 2.5 mg/mL (6.3 mM) in 10% DMSO + 90% Corn oil  (Please use freshly prepared in vivo formulations for optimal results.)