p110α (IC50 = 0.5 μM); p110δ (IC50 = 0.57 μM); p110β (IC50 = 0.97 μM); human CK2 (IC50 = 98 nM); human CK2α2 (IC50 = 3.869 μM); DNA-PK (IC50 = 1.4 μM)

LY294002 is not only selective for PI3Ks; it may also act on other lipid kinases and other, seemingly unrelated proteins. Additionally to mTOR and DNA-PK, LY294002 has been demonstrated to inhibit CK2 (casein kinase 2) and Pim-1, as well as other protein kinases. Apoptosis is triggered and cell proliferation is inhibited as a result of LY294002's inactivation of Akt/PKB. In these colon cancer cell lines, LY294002 exhibits a striking growth-inhibitory and apoptosis-inducing effect, along with decreased expression of phosphorylated Akt (Ser473). [2] In tumor cells, LY294002 significantly increases nuclear pyknosis and reduces cytoplasmic volume. As a result, LY294002 significantly reduces the proliferation of ovarian cancer cells in culture. LY294002 induces specific G1 arrest in cell growth, leading to almost complete inhibition of melanoma cell proliferation and partial inhibition of MG-63 (osteosarcoma cell line) proliferation. The impact of LY294002 on cell cycle progression may shed light on a potential connection between the PI3K activation pathway and the control of the cancer cell cycle. [3]

LY294002 exhibits remarkable effectiveness in the mouse peritonitis carcinomatosa model because it also induces apoptosis and suppresses tumor growth, particularly in LoVo tumors. [2] Ovarian carcinoma growth and ascites formation are markedly reduced by LY294002.[3] LY294002 significantly slows down the development of ascites and ovarian carcinoma.[3]

PI3K inhibition by LY294002 is determined in a radiometric assay using purified, recombinant enzymes with 1μM ATP. At room temperature (24oC), the kinase reaction lasts for an hour before being stopped by the addition of PBS. Then, IC50 values are calculated by fitting a variable slope sigmoidal dose-response curve. Kinase selectivity screening is used to determine the inhibition of CK2 and GSK3β (glycogen synthase kinase 3β). In 10μM ATP, LY294002 is evaluated against the Upstate panel of kinases.

Inoculated into 96-well microtiter plates are 1.0×105 cells (100 μL volume/well). After adding LY294002, triplicate wells are cultured at 37 °C for 0 – 48 hours. Following treatment, 10 μL of Premix WST-1 is added to each microculture well, and the plates are then incubated for 60 minutes at 37 °C. At this point, an absorbance measurement at 450 nm is made using a microplate reader.

Two groups of athymic nude mice (5–7 weeks) are inoculated i.p. with OVCAR-3 cells
0–100 mg/kg
Administered via i.p.

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Athymic mice were inoculated i.p. with the ovarian cancer cell line OVCAR-3. Seven days after inoculation, mice were treated with or without LY294002 (100 mg/kg of body weight) for 3 weeks. Body weight and abdominal circumference were measured twice weekly. At the end of the experiment, mice were sacrificed, ascites volume was measured, and tumors were excised. Mean tumor burden in the LY294002-treated group was reduced by approximately 65% versus controls. Virtually no ascites developed in the treatment group; mean volume of ascites in controls was 3.3 +/- 0.38 ml. OVCAR-3 cells also were cultured in vitro without and with LY294002 (1, 5, and 10 microM) for 24 h. The number of cells in 1, 5, and 10 microM LY294002-treated wells was reduced by 27, 56, and 75%, respectively, versus controls. In vivo and in vitro morphological studies demonstrated that LY294002 induced marked nuclear pyknosis and diminished cytoplasmic volume in the tumor cells, confirmed as apoptosis. Thus, LY294002 significantly inhibits growth and ascites formation of ovarian carcinoma in vivo and markedly inhibits ovarian cancer cell proliferation in vitro, suggesting an important role of PI3-K inhibitors as a potentially useful treatment for women with ovarian carcinoma.[3]

[1]. Biochem J. 2007 Jun 15;404(3):449-58.

[2]. Clin Cancer Res. 2002 Jun;8(6):1957-63.

[3]. Clin Cancer Res. 2000 Mar;6(3):880-6.

LY294002 is a chromone with a phenyl group substituted at position 8 and a morpholino group substituted at position 2. It exhibits EC 2.7.1.137 (phosphatidylinositol 3-kinase) inhibitor, autophagy inhibitor, and anti-aging activity. It belongs to the chromone class, morpholino class, and organochlorine compound family. It is a phosphatidylinositol 3-kinase specific inhibitor. 2-(4-morpholino)-8-phenyl-4H-1-benzopyran-4-one has been reported to exist in honeybees (Hexagonia apiaria) and Dietes bicolor, and relevant data are available. LY294002 is a morpholino phosphatidylinositol 3-kinase (PI3K) and bromodomain-terminal domain (BET) protein family inhibitor with potential antitumor activity. Following administration, the PI3K/BET inhibitor LY294002 specifically targets and binds to the acetylated lysine recognition motif in the bromine domain of PI3K and BET proteins. Inhibition of PI3K activity suppresses the PI3K/AKT kinase signaling pathway. This may lead to inhibition of growth and survival of tumor cells with overactivated PI3K-mediated signaling pathways. Inhibition of BET protein prevents its interaction with acetylated histones, disrupts chromatin remodeling, and inhibits the expression of oncogene drivers crucial for cell proliferation and survival; these factors combined may lead to inhibition of the proliferation of BET-overexpressing tumor cells. Activation of the PI3K signaling pathway is commonly associated with tumorigenesis. The BET protein, composed of BRD2, BRD3, BRD4, and BRDT, is a transcriptional regulator that plays an important role in development and cell growth. In tumor cells, the BET protein plays a key role in the regulation of oncogene transcription and tumor cell proliferation.

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Objective: Phosphatidylinositol 3-kinase (PI3K) and Akt/protein kinase B (PKB) enable various human cancer cells to evade apoptosis. We hypothesized that the PI3K inhibitor 2-(4-morpholino)-8-phenylchromone (LY294002) could inactivate Akt/PKB, thereby inhibiting cell proliferation and inducing apoptosis in vitro and in vivo. Experimental Design: This study used human colon cancer cell lines (DLD-1, LoVo, HCT15, and Colo205) and their mouse xenografts (DLD-1 and LoVo). Immunoblotting and immunohistochemistry were used to detect the expression of phosphorylated Akt (Ser(473)) and apoptosis in cancer cells. To assess the activity of caspase-3 in cultured cells, we also performed caspase colorimetric assays. Results: LY294002 showed significant growth inhibition and apoptosis induction in these colon cancer cell lines, accompanied by a decrease in the expression of phosphorylated Akt (Ser(473)). However, there was a significant difference between the sensitivity of LY294002 and the expression level of phosphorylated Akt. Although LoVo and Colo205 cells showed high sensitivity to LY294002 and increased apoptosis, DLD-1 and HCT15 cells did not show rapid apoptosis induction. Caspase-3 activity was significantly increased in LoVo cells, while caspase-3 activity was not significant in DLD-1 cells. In mouse xenograft tumor experiments, we found that in vivo administration of LY294002 could also inhibit tumor growth and induce apoptosis, especially in LoVo tumors, thus showing significant efficacy in mouse peritoneal cancer models. Conclusion: PI3K-Akt/PKB plays an important role in the development and progression of colon cancer, and it helps promote cell growth and enable cells to escape apoptosis. These results suggest that LY294002 is a promising anti-tumor drug for the treatment of colorectal cancer. [2] Phosphatidylinositol 3-kinase (PI3-K) can induce cell mitosis, cell growth and cell transformation. Gene amplification encoding the P110α subunit may be an important event in the progression of ovarian cancer, and PI3-K inhibitors may be potential drugs for the treatment of this disease. We evaluated the effects of the potent PI3-K inhibitor LY294002 on in vitro and in vivo ovarian cancer growth and ascites formation. Ovarian cancer cell line OVCAR-3 was intraperitoneally injected into athymic mice. Seven days after inoculation, mice were treated with or without LY294002 (100 mg/kg body weight) for 3 weeks. Body weight and abdominal circumference were measured twice weekly. At the end of the experiment, mice were sacrificed, ascites volume was measured, and tumors were excised. The mean tumor burden in the LY294002 treatment group was reduced by approximately 65% compared to the control group. Ascites was almost nonexistent in the treatment group; the mean ascites volume in the control group was 3.3 ± 0.38 ml. OVCAR-3 cells were treated in vitro with LY294002 (1, 5, and 10 μM) for 24 hours, followed by culture without LY294002 treatment. Compared to the control group, the cell numbers in the 1, 5, and 10 μM LY294002 treatment groups were reduced by 27%, 56%, and 75%, respectively. In vivo and in vitro morphological studies have shown that LY294002 can induce significant nuclear pyknosis and cytoplasmic shrinkage in tumor cells, confirming apoptosis. Therefore, LY294002 significantly inhibits the growth of ovarian cancer and ascites formation in vivo, and significantly inhibits the proliferation of ovarian cancer cells in vitro, suggesting that PI3-K inhibitors may be an effective means of treating ovarian cancer. [3]

C19H17NO3

307.3432

307.12

C, 74.25; H, 5.58; N, 4.56; O, 15.62

154447-36-6

LY294002 hydrochloride;934389-88-5

3973

White to yellow solid powder

1.3±0.1 g/cm3

494.6±45.0 °C at 760 mmHg

182-184ºC

253.0±28.7 °C

0.0±1.3 mmHg at 25°C

1.627

3.82

0

4

2

23

463

0

O1CCN(C2=CC(=O)C3C=CC=C(C4C=CC=CC=4)C=3O2)CC1

CZQHHVNHHHRRDU-UHFFFAOYSA-N

InChI=1S/C19H17NO3/c21-17-13-18(20-9-11-22-12-10-20)23-19-15(7-4-8-16(17)19)14-5-2-1-3-6-14/h1-8,13H,9-12H2

2-morpholin-4-yl-8-phenylchromen-4-one

LY-294002; LY 294002; 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; LY-294002; 2-morpholino-8-phenyl-4H-chromen-4-one; 2-morpholin-4-yl-8-phenyl-4H-chromen-4-one; 2-morpholin-4-yl-8-phenylchromen-4-one; LY294002

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: ~36 mg/mL (117.1 mM)
Water: <1 mg/mL (slightly soluble or insoluble)
Ethanol: ~21 mg/mL (~68.3 mM)

Solubility in Formulation 1: 2.87 mg/mL (9.34 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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: ≥ 2.25 mg/mL (7.32 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 22.5 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 3: ≥ 2.25 mg/mL (7.32 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 22.5 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 4: ≥ 2.25 mg/mL (7.32 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 22.5 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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Solubility in Formulation 5: 4%DMSO+30%PEG 300+5%Tween 80+ddH2O: 5mg/mL

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Solubility in Formulation 6: 15.71 mg/mL (51.12 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.)