Na,K-ATPase (α1β1, α2β1, α3β1 isoforms) – binding affinity KD: α1 42.5±6.5 nM, α2 45±8 nM, α3 40±15 nM; no isoform selectivity [1]

In NCI-H460 cells, bufalin (0, 1, 2, 4 μM) reduces cell viability for 48 hours[2]. Bufalin (2 μM) decreases the expression of GRP78 mRNA while increasing that of caspae-3, Endo G, and GADD153 mRNA[2].

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Bufalin at concentrations of 1, 2, and 4 μM for 48 h induced concentration-dependent cell morphological changes (cell shrinkage and debris appearance) and decreased the percentage of viable NCI-H460 cells as measured by flow cytometry [2]
- Bufalin (1, 2, 4 μM for 48 h) induced DNA condensation (an apoptotic characteristic) in a dose-dependent manner as shown by DAPI staining [2]
- Annexin V/PI double staining revealed that bufalin (1–4 μM for 48 h) increased the percentage of early apoptotic cells at all tested concentrations, and significantly increased late apoptotic cells at 2 and 4 μM [2]
- Bufalin (2 μM) increased reactive oxygen species (ROS) production in NCI-H460 cells at 12 h treatment compared to control; pretreatment with ROS scavenger N-acetyl-cysteine (NAC, 15 mM for 3 h) significantly reversed bufalin-induced cell death [2]
- Bufalin (2 μM) decreased mitochondrial membrane potential (ΔΨm) in NCI-H460 cells at 24–48 h treatment [2]
- Bufalin (2 μM for 24 h) significantly increased caspase-9 activity but did not significantly affect caspase-3 or caspase-8 activities in NCI-H460 cells [2]
- Western blot analysis showed that bufalin (2 μM for 0–48 h) increased protein expression of cytochrome c, Apaf-1, active caspase-3, Fas, Fas ligand, AIF, and endonuclease G, while it decreased protein levels of Bcl-2 and XIAP in NCI-H460 cells [2]
- Real-time PCR demonstrated that bufalin (2 μM for 24 or 48 h) increased mRNA levels of caspase-3, endonuclease G, and GADD153, but decreased mRNA expression of GRP78 in NCI-H460 cells [2]

Mice harboring NCI-H460 cells demonstrated notable antitumor activity when bufalin (0.1, 0.2, or 0.4 mg/kg, intraperitoneally injected once daily for 14 days) was administered [2].

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In a xenograft mouse model (BALB/c nu/nu mice implanted with NCI-H460 cells), intraperitoneal injection of bufalin at 0.1, 0.2, or 0.4 mg/kg once daily for 14 days dose-dependently suppressed tumor growth compared to vehicle control. Tumor weights and volumes were significantly reduced in bufalin-treated groups [2]
- Bufalin injection did not produce significant drug-related toxicity in experimental animals except at the high dose (0.4 mg/kg), which caused body weight loss [2]

Competitive displacement of [3H]ouabain binding was measured using P. pastoris membranes expressing human α1β1, α2β1, or α3β1 isoforms. Membranes (200-300 μg protein) were incubated at 37°C for 1 hour in a medium containing 10 mM MOPS-Tris pH 7.2, 3 mM MgCl2, 1 mM vanadate-Tris, and 1 mM EGTA-Tris. [3H]ouabain was used at 1-2 nM. For displacement experiments, varying concentrations of unlabeled bufalin were added. Bound and free ligand were separated, and KD values were calculated using a one-site competition model: B = Bmax[CG]/([CG] + KD). The KD for bufalin was calculated from K0.5 by accounting for ouabain-CG competition: KD_CG = K0.5 / (1 + [OU_H]/KD_OU). Experiments were performed 3-5 times independently. [1]
Bufalin was dissolved in DMSO as a stock solution, then diluted in 50% DMSO before addition to the reaction medium. The final DMSO concentration in the assay was 1%. [1]

Cell viability assay[2]
Cell Types: NCI-H460 Cell
Tested Concentrations: 0, 1, 2, 4 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: The viability of NCI-H460 cells was diminished in a dose-dependent manner.

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For morphological changes and cell viability: NCI-H460 cells were plated at 2×10^5 cells/well in 12-well plates, grown for 24 h, then treated with bufalin (0, 1, 2, 4 μM) or DMSO (0.1% solvent control) for 48 h. Morphological shrinkage was observed under phase-contrast microscope at 24 and 48 h. Cell viability was estimated using flow cytometry [2]
- For DNA condensation (DAPI staining): Cells (2×10^5 cells/well) were seeded in 12-well plates and treated with bufalin (0, 1, 2, 4 μM) for 48 h. Cells were harvested, washed with PBS, fixed with 4% paraformaldehyde, stained with DAPI (1 μg/mL) for 30 min in the dark, then examined under a fluorescent microscope [2]
- For apoptotic cell death (Annexin V/PI double staining): Cells (2×10^5 cells/well) were treated with bufalin (0, 1, 2, 4 μM) for 48 h, harvested, washed, and stained with Annexin V-FITC/PI for 20 min in the dark. Stained cells were measured by flow cytometry [2]
- For ROS measurement: Cells (2×10^5 cells/well) were incubated with 2 μM bufalin for 1, 3, 6, and 12 h, harvested, washed, resuspended in 10 μM 2,7-dichlorodihydrofluorescein diacetate, incubated at 37°C for 30 min, and analyzed by flow cytometry. In separate experiments, cells were pretreated with or without 15 mM NAC for 3 h before adding 2 μM bufalin, then cell viability was measured by flow cytometry [2]
- For mitochondrial membrane potential (ΔΨm) detection: Cells (2×10^5 cells/well) were incubated with 2 μM bufalin for 12, 24, 36, and 48 h, harvested, washed, resuspended in 4 μmol/L 3,3′-dihexyloxacarbocyanine iodide, incubated at 37°C for 30 min, and analyzed by flow cytometry [2]
- For caspase-3, -8, and -9 activities: Cells were incubated with 2 μM bufalin for 24 h, harvested, washed, resuspended in 10 μM substrate solution (PhiPhiLux and Caspalux kit), incubated at 37°C for 60 min, washed again with PBS, and analyzed by flow cytometry [2]
- For Western blotting: Total proteins were collected from cells treated with 2 μM bufalin for 0, 12, 24, 36, and 48 h. Protein amounts were determined by SDS-PAGE and Western blot [2]
- For real-time PCR: Total RNA was extracted from cells after treatment with 2 μM bufalin for 24 and 48 h using a kit, reverse-transcribed, and then gene expression of caspase-3, endonuclease G, GADD153, and GRP78 was examined using SYBR Green PCR Master Mix on a real-time PCR system. Expression fold-changes were derived using the comparative CT method [2]

Animal/Disease Models: 40 male athymic BALB/c nu/nu (nude) mice (6-8 weeks old) [2]
Doses: 0.1, 0.2 or 0.4 mg/kg
Route of Administration: Daily intraperitoneal (ip) injection until 14 days
Experimental Results: Dose dependent Inhibit tumor growth.

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Male athymic BALB/c nu/nu mice (6–8 weeks old) were acclimated for 7 days. NCI-H460 cells (1×10^7 cells/mouse) were subcutaneously injected into one flank. Tumor size was measured every 3 days using calipers, and tumor volume calculated as V = L × W^2 / 2. When tumor volume exceeded 100 mm^3, mice were randomly divided into four groups (n=10 per group). They received intraperitoneal injections of either vehicle (0.1% DMSO) or bufalin at 0.1, 0.2, or 0.4 mg/kg once daily until day 14 after therapy initiation. At the end of the experiment, animals were anaesthetized with CO2 and sacrificed. Tumors were removed, photographed, measured, and weighed [2]

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Male athymic BALB/c nu/nu mice (6–8 weeks old) were acclimated for 7 days. NCI-H460 cells (1×10^7 cells/mouse) were subcutaneously injected into one flank. Tumor size was measured every 3 days using calipers, and tumor volume calculated as V = L × W^2 / 2. When tumor volume exceeded 100 mm^3, mice were randomly divided into four groups (n=10 per group). They received intraperitoneal injections of either vehicle (0.1% DMSO) or bufalin at 0.1, 0.2, or 0.4 mg/kg once daily until day 14 after therapy initiation. At the end of the experiment, animals were anaesthetized with CO2 and sacrificed. Tumors were removed, photographed, measured, and weighed [2]

Bufalin injection did not produce significant drug-related toxicity in experimental animals except at the high dose (0.4 mg/kg), where tumor-bearing mice lost much of their body weight [2]

[1]. Selectivity of digitalis glycosides for isoforms of human Na,K-ATPase. J Biol Chem. 2010 Jun 18;285(25):19582-92.

[2]. Bufalin induces apoptosis in vitro and has Antitumor activity against human lung cancer xenografts in vivo. Environ Toxicol. 2017 Apr;32(4):1305-1317.

Bufalin is a 14β-hydroxy steroid with the structure bufotoxin-20,22-diene lactone, containing hydroxyl substituents at both the 5β- and 14β- positions. It has been isolated from the skin of the toad (Bufo bufo). Bufalin possesses antitumor, cardiotonic, anti-inflammatory, and animal metabolic effects. It is a 3β- and 14β-hydroxy steroid, functionally related to bufotoxin lactone. Bufalin has been reported to exist in the African toad (Phrynoidis asper), the Chinese toad (Bufo gargarizans), and other organisms with relevant data. Bufalin is one of the active ingredients of the traditional Chinese medicine toad venom, and is also a glycoside compound; it was originally isolated from the venom of the Chinese toad (Bufo gargarizans) as a bufodiene lactone toxin, possessing potential cardiotonic and antitumor activities. Although the pharmacological mechanism of bufalin is still under investigation, it is a specific Na+/K+-ATPase inhibitor that can activate transcription factor AP-1 through the mitogen-activated protein kinase (MAPK) pathway, thereby inducing apoptosis in cancer cell lines.

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Bufalin is a bufadienolide aglycone (steroid without sugar moiety). It showed essentially identical binding affinity for all three human Na,K-ATPase isoforms (α1, α2, α3), indicating no isoform selectivity. This supports the conclusion that aglycones cannot discriminate between isoforms, and the sugar moiety is essential for isoform selectivity. [1]

C24H34O4

386.53

386.245

465-21-4

9547215

White to off-white solid powder

1.2±0.1 g/cm3

556.6±50.0 °C at 760 mmHg

242 - 243ºC

189.0±23.6 °C

0.0±3.4 mmHg at 25°C

1.594

3.42

2

4

1

28

741

8

O([H])[C@]12C([H])([H])C([H])([H])[C@]([H])(C3=C([H])OC(C([H])=C3[H])=O)[C@@]1(C([H])([H])[H])C([H])([H])C([H])([H])[C@]1([H])[C@@]3(C([H])([H])[H])C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])[C@@]3([H])C([H])([H])C([H])([H])[C@@]21[H])O[H]

QEEBRPGZBVVINN-BMPKRDENSA-N

InChI=1S/C24H34O4/c1-22-10-7-17(25)13-16(22)4-5-20-19(22)8-11-23(2)18(9-12-24(20,23)27)15-3-6-21(26)28-14-15/h3,6,14,16-20,25,27H,4-5,7-13H2,1-2H3/t16-,17+,18-,19+,20-,22+,23-,24+/m1/s1

5-[(3S,5R,8R,9S,10S,13R,14S,17R)-3,14-dihydroxy-10,13-dimethyl-1,2,3,4,5,6,7,8,9,11,12,15,16,17-tetradecahydrocyclopenta[a]phenanthren-17-yl]pyran-2-one

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 (~258.72 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.47 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 2: ≥ 2.5 mg/mL (6.47 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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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.38 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 20.8 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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 (Please use freshly prepared in vivo formulations for optimal results.)