AKT/mTOR signaling pathway (inhibition of p-AKT^T308 and p-AKT^S473). No IC50/Ki/EC50 values reported. [1]

Conobafagin (30–300 nM, 24 hours) stimulates OCM1 cells to exhibit and exhibit associated changes in surface activity while inhibiting the G1 phase of the cell cycle in a concentration-inhibitory manner [1]. In uveal melanoma OCM1 cells, conobafagin (30-300 nM, 7 days) exhibits strong anticancer activity in a dose-dependent manner [1]. In pleomorphic astroblastoma U87MG-EGFR and U87MG-PTEN cells, conobafagin (0.01-1 μM, 6 hours) burns growth factor receptor (EGFR) phosphorylation, causing cellular anemia and cytotoxicity [2]. Cinobupsin (0.4, 0.7, 1.0 μM, 24-48 hours) initiates the G2/M phase of the cell life cycle, which results in

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Cinobufagin (0, 0.6, 1.2, 2.5, 5, 10, 20 μM) inhibited cell viability in four NSCLC cell lines (A549, H1299, H460, SK-MES-1) in a dose-dependent manner after 24 h, with 40-50% inhibition at <2 μM, showing higher efficacy than cisplatin, gemcitabine, docetaxel, and paclitaxel at the same concentrations. [1]
Cinobufagin (1 or 2 μM) induced cell cycle arrest: after serum starvation for 48 h and release, >70% of CB-treated cells were arrested at G0/G1 phase at 24 h, with time-dependent and cell-type differences. [1]
Cinobufagin (0.5-2 μM for 24 h) induced apoptosis in a dose-dependent manner in A549, H1299, H460, and SK-MES-1 cells, as measured by Annexin V/PI flow cytometry. Time-dependent apoptosis was also observed with 1 μM CB for up to 48 h. CB-induced apoptosis was significantly blocked by pretreatment with 5 mM N-acetylcysteine (NAC). [1]
Cinobufagin (1-2 μM for 24 h) increased reactive oxygen species (ROS) levels in A549 and H1299 cells (from 2.72% to 9.92% and 65.27% in A549; from 6.05% to 17.90% and 29.66% in H1299), and decreased mitochondrial membrane potential (MMP) (from 98.55% to 89.63% and 53.61% in A549; from 99.29% to 77.60% and 69.25% in H1299). Both effects were blocked by NAC. [1]
Cinobufagin (0.5-2 μM for 24 h) reduced expression of BCL-2, BCL-XL, and MCL-1, increased BAX, and increased cleaved-caspase-3 in H1299 cells. [1]
Cinobufagin (0.5-2 μM for 24 h) decreased p-MEK1/2 but not p-c-RAF or p-ERK1/2; dramatically downregulated p-AKT^T308 and p-AKT^S473 in a time-dependent manner (1 μM CB for 1-6 h) without changing total AKT, and also downregulated p-4EBP1 (mTOR pathway) but not p-GSK-3β (Ser9) in H1299 and A549 cells. [1]

In an induced xenograft model, cinobufonin (5 mg/kg intraperitoneally, once day for 10 days) inhibits cytochromes, hence suppressing xenograft growth [1]. In subcutaneous and intracranial U87MG-EGFR xenograft mouse models, cinobufen (5 mg/kg intraperitoneally, once day for 10 days) slows tumor growth and increases the median survival of nude mice with intracranial U87MGEGFR tumors. rate [2].
Cinobufagin (1.5, 5.0, 10.0 mg/kg/day, intraperitoneal, every 5 days) inhibited tumor growth in A549 NSG xenograft mouse models. Low dose (1.5 mg/kg) showed no change; middle dose (5 mg/kg) significantly inhibited tumor growth compared to effective doses of platinum drugs (cisplatin 5 mg/kg, gemcitabine 50 mg/kg, docetaxel 5 mg/kg, paclitaxel 5 mg/kg, all every 5 days); high dose (10 mg/kg) dramatically inhibited tumor growth. Body weight temporarily lost 5-10% at one week after administration; middle dose caused <5% weight loss. [1]
Cinobufagin at 0.5, 1, and 2 μM for 24 h did not significantly change viability of rat splenocytes (normal cells). [1]

Cell Proliferation Assay[1]
Cell Types: OCM1 cell
Tested Concentrations: 30,100,300 nM
Incubation Duration: 7 days
Experimental Results: Yes OCM1 cells produced potent cytotoxicity with an IC50 of 8.023 nM.

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Apoptosis analysis[1]
Cell Types: OCM1 Cell
Tested Concentrations: 30,100,300 nM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induction of apoptosis and upregulation of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase (PARP) and cleaved caspase Expression level -9. Activation of the intrinsic mitochondrial apoptotic pathway, manifested by increased apoptosis, increased expression of Bad and Bax, diminished expression of Bcl-2 and Bcl-xl, and diminished mitochondrial membrane potential (MMP).

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Cell viability (MTT) assay: NSCLC cells were treated with various concentrations of Cinobufagin or platinum drugs for 24 h. MTT reagent (100 μl/ml) was added and incubated at 37°C for 4 h, then 150 μl DMSO added to dissolve formazan crystals. Absorbance read at 570 nm. Inhibitory ratio = (A[control] - A[sample])/A[control] × 100%. [1]
Cell cycle assay: Cells were serum-starved for 48 h, then treated with 0, 1, or 2 μM Cinobufagin for 12, 24, 36, 48 h. Cells harvested, fixed in 70% ice-cold ethanol at 4°C for 2 h, washed, incubated with RNase A and propidium iodide (PI) staining solution, analyzed by flow cytometry. [1]
Apoptosis detection (Annexin V/PI): Cells treated with Cinobufagin at various concentrations and times, collected, stained with Annexin V-FITC and PI using kit, analyzed by flow cytometry. For Hoechst staining, cells treated with CB (0,1,2 μM for 24 h), collected, dried on slides, stained with Hoechst 33342 for 10 min, observed under fluorescence microscope for fragmented/condensed nuclei. [1]
ROS measurement (DCFH-DA): Cells treated with CB (0,1,2 μM) ± 5 mM NAC for 24 h, collected, incubated with 10 mM DCF-DA at 37°C for 15 min, analyzed by flow cytometry. [1]
MMP measurement (Rh-123): Cells treated with CB (0,1,2 μM) ± 5 mM NAC for 24 h, collected, incubated with 10 μM Rh-123 at 37°C for 20 min, analyzed by flow cytometry. [1]
Western blot: Cells treated with CB (0,0.5,1,2 μM for 24 h; or 1 μM for 1,3,6 h), lysed in RIPA buffer, protein quantified by BCA, separated by 10% SDS-PAGE, transferred to PVDF membrane, probed with antibodies against BAX, BCL-2, BCL-XL, MCL-1, AKT, p-AKT^T308, p-AKT^S473, GSK-3β, p-GSK-3β(Ser9), p-c-RAF(Ser259), MEK1/2, p-MEK1/2(S217/221), ERK1/2, p-ERK1/2, TSC1, TSC2, 4EBP1, p-4EBP1, pro-caspase-3, cleaved-caspase-3, and β-actin, followed by HRP-conjugated secondary antibodies and chemiluminescence detection. [1]

Animal/Disease Models: OCM1 cell tumor Nu/Nu nude mouse xenograft [1]
Doses: 5 mg/kg
Route of Administration: intraperitoneal (ip) injection, one time/day for 10 days.
Experimental Results: The tumor growth rate is higher than that of intraperitoneal (ip) injection of normal saline or no Treated tumors grow more slowly. The expression of caspase-3 and PARP increased in tumor tissue, the expression of Bcl-2 and Bcl-xl diminished, and the expression of Bad and Bax increased in mouse tumor tissue.

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Animal/Disease Models: U87MG-EGFR subcutaneousand intracranial xenograft model [2]
Doses: 5 mg/kg
Route of Administration: intraperitoneal (ip) injection, one time/day for 10 days.
Experimental Results: The luminescence intensity of brain tumors diminished by approximately 70%. Levels of p-EGFR, p-STAT3, and p-Akt were diminished in intracranial tumors compared with vehicle. Intracranial tumors had diminished immunostaining for Ki67 and active caspase-3.

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A549 xenograft NSG mouse model: 8-week-old male NSG mice received subcutaneous injection of 5×10^6 A549 cells in 100 μl of 50:50 Matrigel/collagen I on left flank. Ten days after injection, mice randomized into 7 groups (n=10/group). Treatments: vehicle control; cisplatin (5 mg/kg/day, i.p., every 5 days); gemcitabine (50 mg/kg/day, i.p., every 5 days); docetaxel (5 mg/kg/day, i.p., every 5 days); paclitaxel (5 mg/kg/day, i.p., every 5 days); Cinobufagin (1.5, 5.0, or 10.0 mg/kg/day, i.p., every 5 days). Tumor size measured daily with Vernier caliper, volume calculated as 0.5 × D1 × (D2)^2. Body weight measured every 5 days. Mice euthanized when tumor size reached 1500 mm³, ulceration, or up to 45 days. [1]
Rat splenocyte isolation: Splenocytes isolated from one-year-old rats, treated with Cinobufagin (0.5, 1, 2 μM) or platinum drugs for 24 h, cell viability determined by MTT assay. [1]

Toxicity Summary
Bufotalin blocks the sodium-potassium pump in myocardial tissue. (L1083, L1084)

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Cinobufagin at low doses (0.5-2 μM) showed no significant cytotoxicity to rat splenocytes. In vivo, body weight temporarily lost 5-10% at one week after administration of CB or platinum drugs; middle dose of CB (5 mg/kg/day) caused less than 5% body weight loss. [1]

[1]. Cinobufagin inhibits tumor growth by inducing intrinsic apoptosis through AKT signaling pathway in human nonsmall cell lung cancer cells. Oncotarget. 2016 Mar 3.

[2]. Immunomodulatory Effects of Cinobufagin on Murine Lymphocytes and Macrophages. Evid Based Complement Alternat Med. 2015;2015:835263.

[3]. Cinobufagin exerts anti-proliferative and pro-apoptotic effects through the modulation ROS-mediated MAPKs signaling pathway. Immunopharmacol Immunotoxicol. 2015 Jun;37(3):265-73.

Cinobufagin is a steroidal lactone with functions similar to bufotalin. It has been reported to exist in the African toad (Phrynoidis asper), the African toad (Bufo gargarizans), and other organisms with relevant data. Cinobufagin is a bufodiene lactone compound extracted from the dried venom secreted by the parotid glands of toads, and is also a glycoside found in the traditional Chinese medicine toad venom, possessing potential anti-tumor activity. Although the mechanism of action of Cinobufagin is still under investigation, it has been found to inhibit cancer cell proliferation and induce apoptosis in cancer cells through a series of apoptosis regulators, including mitochondrial Bax, cytoplasmic chromosome c, and caspases 3, 8, and 9. Possible upstream mediators of Cinobufagin-induced apoptosis include Fas and p53.

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Cinobufagin is a bufadienolide from Chan Su with anti-cancer activity. This study demonstrates that CB inhibits NSCLC cell growth and tumor growth by inducing intrinsic mitochondrial apoptosis through the AKT signaling pathway. CB increases ROS, decreases MMP, upregulates BAX, downregulates BCL-2, BCL-XL, and MCL-1, activates caspase-3, and inhibits p-AKT (T308 and S473) and p-4EBP1. CB may be a potential alternative anti-cancer drug to platinum drugs for anti-lung cancer therapy. [1]

C26H34O6

442.552

442.235

470-37-1

11969542

White to off-white solid powder

1.3±0.1 g/cm3

595.4±50.0 °C at 760 mmHg

222-223ºC

199.4±23.6 °C

0.0±3.8 mmHg at 25°C

1.595

2.43

1

6

3

32

923

10

CC(=O)O[C@@H]1[C@@H]([C@]2(CC[C@H]3[C@H]([C@@]24[C@@H]1O4)CC[C@H]5[C@@]3(CC[C@@H](C5)O)C)C)C6=COC(=O)C=C6

SCULJPGYOQQXTK-OLRINKBESA-N

InChI=1S/C26H34O6/c1-14(27)31-22-21(15-4-7-20(29)30-13-15)25(3)11-9-18-19(26(25)23(22)32-26)6-5-16-12-17(28)8-10-24(16,18)2/h4,7,13,16-19,21-23,28H,5-6,8-12H2,1-3H3/t16-,17+,18+,19-,21+,22-,23-,24+,25-,26-/m1/s1

[(1R,2S,4R,5R,6R,7R,10S,11S,14S,16R)-14-hydroxy-7,11-dimethyl-6-(6-oxopyran-3-yl)-3-oxapentacyclo[8.8.0.02,4.02,7.011,16]octadecan-5-yl] acetate

NSC90325 14,15β-Epoxy-3β,16β-dihydroxy-5β,20(22)-bufadienolide 16-acetateCinobufagine Cino-bufaginCinobufagin

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 (~225.97 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.65 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 (5.65 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 (5.65 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.)