In a dose-dependent manner, crotonoside (0-200 μM) specifically reduces the survival of AML cell lines MV4-11, MOLM-13 (with FLT3-ITD mutant), and KG-1 (without FLT3-ITD mutant). IC50 Their respective values are 11.6 μM, 12.7 μM, and 17.2 μM [1]. Crotonin (0-100 μM; 7 hours) suppresses the phosphorylation of Akt/mTOR, FLT3 Erk1/2, and STAT5, with the highest concentration of 12.5 μM showing a concentration-dependent strong inhibition [1]. A dose-dependent rise in the proportion of cells in the G0/G1 phase and a dose-dependent decrease in the percentage of cells in the G2/M and S phases are observed when using crotonin (0-100 μM; 12 hours) [1]. The number of apoptotic MV4-11 cells varies concentration-dependently with crotonoside (0-100 μM; 24 hours), pro-caspase-3 levels drop with dose, and caspase-3 precursor levels increase with dose. Cleaved caspase-3 fragment levels [1].

In a dose-dependent manner, crotonoside (0-200 μM) specifically reduces the survival of AML cell lines MV4-11, MOLM-13 (with FLT3-ITD mutant), and KG-1 (without FLT3-ITD mutant). IC50 Their respective values are 11.6 μM, 12.7 μM, and 17.2 μM [1]. Crotonin (0-100 μM; 7 hours) suppresses the phosphorylation of Akt/mTOR, FLT3 Erk1/2, and STAT5, with the highest concentration of 12.5 μM showing a concentration-dependent strong inhibition [1]. A dose-dependent rise in the proportion of cells in the G0/G1 phase and a dose-dependent decrease in the percentage of cells in the G2/M and S phases are observed when using crotonin (0-100 μM; 12 hours) [1]. The number of apoptotic MV4-11 cells varies concentration-dependently with crotonoside (0-100 μM; 24 hours), pro-caspase-3 levels drop with dose, and caspase-3 precursor levels increase with dose. Cleaved caspase-3 fragment levels [1].

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Crotonoside selectively inhibits the viability of AML cell lines MV4-11 (IC50 = 11.6 ± 2.7 µM), MOLM-13 (IC50 = 12.7 ± 3.3 µM), and KG-1 (IC50 = 17.2 ± 4.6 µM) in a dose-dependent manner, while showing much lower cytotoxicity in normal cells (HEK293A, IC50 = 182.8 ± 34.9 µM; MCF-10A, IC50 = 167.3 ± 38.3 µM) and other cancer cell lines (IC50 > 200 µM for most). [1]
Crotonoside inhibits phosphorylation of FLT3 and its downstream signaling proteins (Erk1/2, Akt/mTOR, STAT5) in MV4-11 cells after 7 hours of treatment in a dose-dependent manner. [1]
Crotonoside decreases the expression of HDAC3 and HDAC6 (but not HDAC1, HDAC2, HDAC4) in MV4-11 and KG-1 cells after 20 hours of treatment at concentrations ≥25 µM. It also reduces expression of transcription factors NF-κB-p65 and c-Myc. [1]
Crotonoside induces G0/G1 phase cell cycle arrest and apoptosis in MV4-11 cells in a dose-dependent manner, as shown by flow cytometry and increased cleaved caspase-3 levels. [1]
The HDAC3-selective inhibitor RGFP966 and HDAC6-selective inhibitor HPOB also show selective growth inhibition in AML cells (MV4-11, MOLM-13, KG-1) with IC50 values in the low micromolar range. [1]
Crotonoside exhibits a "post-inhibition" effect where treated MV4-11 cells show delayed regrowth after drug removal, especially at higher concentrations (4×IC50, 8×IC50). This effect is enhanced in combinations with sunitinib, RGFP966, and HPOB. [1]

In comparison to vehicle treatment, crotonin (intraperitoneal and intravenous injection; 70 mg/kg, 35 mg/kg; once daily) generated considerable anticancer activity and reduced the growth of xenograft tumors [1].

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Crotonoside significantly inhibits the growth of MV4-11 tumor xenografts in NOD-SCID mice. Tumor inhibition rates were 93.5% at 70 mg/kg/day (i.p.), 73.6% at 35 mg/kg/day (i.p.), and 78.3% at 70 mg/kg every other day (i.v.). Sunitinib (10 mg/kg/day p.o.) showed 96.3% inhibition as a positive control. [1]
Treatment with crotonoside resulted in reduced Ki67 staining (proliferation marker) and increased TUNEL-positive cells (apoptosis marker) in tumor tissues compared to vehicle control. [1]
Body weight loss was less than 20% in the high-dose groups during the first 6 days, with no further loss by the end of the study. No severe toxicity (skin ulceration or lethality) was observed. [1]

Cell Viability Assay [1]
Cell Types: AML cell lines MV4-11, MOLM-13 and KG-1 Cell
Tested Concentrations: 0-200 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Inhibited AML cell growth than other cell lines tested.

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Western Blot Analysis[1]
Cell Types: MV4-11 Cell
Tested Concentrations: 0 μM, 12.5 μM, 25 μM, 50 μM
Incubation Duration: 7 hrs (hours)
Experimental Results: Inhibition of AML cell growth compared to other cell lines tested.

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Cell cycle analysis [1]
Cell Types: MV4-11 Cell
Tested Concentrations: 0 μM, 12.5 μM, 25 μM, 50 μM
Incubation Duration: 12 hrs (hours)
Experimental Results: Induced cell cycle arrest at G0/G1.

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Apoptosis analysis [1]
Cell Types: MV4-11 Cell
Tested Concentrations: 0 μM, 12.5 μM, 25 μM, 50 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induced apoptosis of MV4-11 cells.

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Cell viability was assessed using the MTT assay. Cells were seeded in 96-well plates, treated with crotonoside (3.12–200 µM) for 72 hours, followed by MTT addition and measurement of absorbance at 570 nm. [1]
For Western blot analysis, MV4-11 or KG-1 cells were treated with crotonoside for 7 or 20 hours, lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with specific antibodies against FLT3, phospho-FLT3, HDACs, NF-κB-p65, c-Myc, caspase-3, etc. [1]
Cell cycle analysis was performed by propidium iodide (PI) staining and flow cytometry after 12 hours of treatment. Apoptosis was assessed using Annexin V-FITC/PI staining and flow cytometry after 24 hours of treatment. [1]
Post-inhibition effect assay: MV4-11 cells were treated with drugs for 24 hours, then washed and re-cultured in fresh medium. Cell numbers were counted daily for 6 days to assess regrowth. [1]

Animal/Disease Models: MV4-11 cells NOD-SCID (severe combined immunodeficient) mouse [1]
Doses: 70 mg/kg, 35 mg/kg
Route of Administration: intraperitoneal (ip) injection, intravenous (iv) (iv)injection; 70 mg/kg, 35 mg/kg; one time/day
Experimental Results: Produced significant AML tumor inhibition rate of 93.5% at 70 mg/kg.

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MV4-11 cells (1×10⁷ cells/mouse) were injected subcutaneously into female NOD-SCID mice. When tumors reached ~200 mm³, mice were treated with crotonoside at 70 mg/kg/day intraperitoneally (i.p.), 35 mg/kg/day i.p., or 70 mg/kg every other day intravenously (i.v.) for 21 days. Vehicle control was 2% DMSO in normal saline. Sunitinib (10 mg/kg/day orally) was used as a positive control. Tumor volume and body weight were measured every 3 days. [1]
After sacrifice, tumors were collected, fixed, paraffin-embedded, and sectioned for immunohistochemical staining of Ki67 and TUNEL assay to assess proliferation and apoptosis. [1]

[1]. Crotonoside exhibits selective post-inhibition effect in AML cells via inhibition of FLT3 and HDAC3/6. Oncotarget. 2017 Sep 8;8(61):103087-103099.

Crotonoside is a purine nucleoside. Crotonoside has been found in croton (Croton tiglium) and there are related data reports. Crotonoside is a natural product extracted from the seeds of croton (Croton tiglium L., a traditional Chinese medicine). [1] It exhibits a multi-target mechanism against acute myeloid leukemia (AML) by inhibiting the FLT3 signaling pathway and selectively downregulating the expression of HDAC3 and HDAC6, which distinguishes it from classic FLT3 inhibitors such as sunitinib. [1] This study suggests that simultaneous inhibition of FLT3, HDAC3 and HDAC6 may provide a more effective treatment strategy for AML, especially in overcoming drug resistance. [1]

C10H13N5O5

283.2407

283.091

1818-71-9

65085

White to off-white solid powder

2.3±0.1 g/cm3

831ºC at 760 mmHg

456.4ºC

2.83E-29mmHg at 25°C

1.955

-3.42

5

7

2

20

544

4

C1=NC2=C(NC(=O)N=C2N1[C@H]3[C@@H]([C@@H]([C@H](O3)CO)O)O)N

MIKUYHXYGGJMLM-UUOKFMHZSA-N

InChI=1S/C10H13N5O5/c11-7-4-8(14-10(19)13-7)15(2-12-4)9-6(18)5(17)3(1-16)20-9/h2-3,5-6,9,16-18H,1H2,(H3,11,13,14,19)/t3-,5-,6-,9-/m1/s1

6-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-purin-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 : ~25 mg/mL (~88.26 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.34 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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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.34 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 20.8 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: ≥ 2.08 mg/mL (7.34 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 20.8 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.)