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)

In a dose-dependent manner, LY294002 hydrochloride (0-75 μM; 24 and 48 hours) significantly reduces human nasopharyngeal cancer CNE-2Z cells[4]. LY294002 hydrochloride (0-75 μM; 24 and 48 hours) dose-dependently increases the rate of apoptosis in CNE-2Z cells[4]. In CNE-2Z cells, LY294002 hydrochloride (10–75 μM) dramatically reduces p-Akt (S473) expression levels and increases caspase-9 activity. There is no variation in the total Akt protein level across varied concentrations [4]. Treatment with LY294002 hydrochloride (5, 10, 100 µM; for 2 hours) partially inhibits the nuclear translocation of YAP produced by Lysophosphatidic acid (LPA) (20 µM; for 4 hours), which is followed by a decrease in p-AKT levels[6].

In a dose-dependent manner, LY294002 hydrochloride (10, 25, 50, 75 mg/kg; ip; twice weekly; for 4 weeks) considerably lowers the mean NPC tumor burden. The effectiveness of LY294002 (10, 25 mg/kg) in reducing tumor burden is lower[4]. In Sprague-Dawley rats, LY294002 hydrochloride (1.2 mg/kg ip; i.p.) for 14 days inhibits the deleterious effects of leptin (60 ug/kg) on spermatozoa[5].

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.

Cell Proliferation Assay
Cell Types: CNE-2Z cells[4]
Tested Concentrations: 0 μM, 10 μM, 25 μM, 50 μM, and 75 μM
Incubation Duration: 24 hrs (hours) and 48 hrs (hours)
Experimental Results: diminished CNE-2Z cells in a dose-dependent fashion.

---

Apoptosis Analysis
Cell Types: CNE-2Z cells[4]
Tested Concentrations: 0 μM, 10 μM, 25 μM, 50 μM, and 75 μM
Incubation Duration: 24 hrs (hours) and 48 hrs (hours)
Experimental Results: Induced apoptosis rate in a dose-dependent manner.

---

Western Blot Analysis
Cell Types: CNE-2Z cells[4]
Tested Concentrations: 0 μM, 10 μM, 25 μM, 50 μM, and 75 μM
Incubation Duration: 24 hrs (hours) and 48 hrs (hours)
Experimental Results: diminished phosphorylated Akt (S473) expression levels were Dramatically, up-regulated caspase-9 activity in CNE-2Z cells in treated group.

Animal/Disease Models: Athymic nude mice (6-8 weeks) with CNE-2Z xenograft[4]
Doses: 10 mg/kg, 25 mg/kg, 50 mg/kg, and 75 mg/kg
Route of Administration: IP; twice weekly, for 4 weeks
Experimental Results: Mean Nasopharyngeal carcinoma (NPC) tumor burden was remarkably decreased in a dose-dependent manner.

[1]. The effect of PI3K inhibitor LY294002 and gemcitabine hydrochloride combined with ionizing radiation on the formation of vasculogenic mimicry of Panc-1 cells in vitro and in vivo. Neoplasma. 2016;63(1):80-92.

[2]. Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling. Biochem J. 2007 Jun 15;404(3):449-58.

[3]. Exploring the specificity of the PI3K family inhibitor LY294002. Biochem J. 2007 May 15;404(1):15-21.

[4]. Phosphatidylinositol 3-kinase inhibitor(LY294002) induces apoptosis of human nasopharyngeal carcinoma invitro and in vivo. J Exp Clin Cancer Res. 2010 Apr 22;29:34.

[5]. LY294002, a PI3K pathway inhibitor, prevents leptin-induced adverse effects on spermatozoa in Sprague-Dawley rats. Andrologia. 2019 Apr;51(3):e13196.

[6]. Lysophosphatidic acid induces YAP-promoted proliferation of human corneal endothelial cells via PI3K and ROCK pathways. Mol Ther Methods Clin Dev. 2015 Apr 29;2:15014.

This study aimed to investigate the presence of angiogenic mimicry (VMs) in the three-dimensional matrix of cultured Panc-1 cells and in vivo orthotopic Panc-1 xenografts, and to verify whether the combination of the PI3K inhibitor LY294002 and gemcitabine hydrochloride with ionizing radiation (IR) could provide significant therapeutic benefits for pancreatic cancer. We first investigated the presence of VMs in the three-dimensional matrix of cultured Panc-1 cells and in vivo orthotopic Panc-1 xenografts. Subsequently, we investigated the activation of the PI3K/MMPs/Ln-5γ2 signaling pathway under IR irradiation. Finally, we evaluated the radiosensitizing effects of LY294002 and gemcitabine hydrochloride alone and in combination. The results showed that VMs existed in both the three-dimensional matrix of cultured Panc-1 cells and in vivo orthotopic Panc-1 xenografts. The expression of p-Akt and MMP-2 increased after ionizing radiation (IR) irradiation. LY294002 and gemcitabine hydrochloride combined with IR irradiation better inhibited the migration, angiogenesis mimicry (VM) formation, and MMP-2 mRNA expression of cultured Panc-1 cells in vitro. We also demonstrated that this novel treatment regimen better inhibited the growth, metastasis, and VM formation of orthotopic Panc-1 xenografts by inhibiting the PI3K/MMPs/Ln-5γ2 signaling pathway in vivo. This study is one of the first studies to confirm VM formation in orthotopic Panc-1 xenografts. In addition, our current study is also one of the first studies to provide preliminary evidence that the novel treatment regimen LY294002 combined with gemcitabine hydrochloride and ionizing radiation can be used to treat pancreatic cancer. [1]

---

PI3K (phosphatidylinositol 3-kinase) regulates cell signaling networks that are involved in cell survival, growth, proliferation, metabolism, and specialized differentiation. Abnormalities in this network are common in cancer and are also associated with inflammatory diseases. The elucidation of PI3K physiological function has primarily come from pharmacological studies using enzyme inhibitors such as Wojtmanin and LY294002, as well as PI3K gene knockout models, to investigate the effects of PI3K loss of function. Some reports indicate that LY294002 is not a specific inhibitor of PI3K and may actually act on other lipid kinases and other seemingly unrelated proteins. Since this inhibitor remains the drug of choice in numerous PI3K studies (over 500 last year), determining the precise specificity of this compound is crucial. This article reports a chemical proteomics strategy in which the LY294002 analog PI828 was immobilized on epoxy-activated agarose beads. This affinity material was then used as bait to screen for potential protein targets from cell extracts. Proteins with high affinity for immobilized PI828 were separated by one-dimensional gel electrophoresis and identified using liquid chromatography-tandem mass spectrometry. This study reveals that LY294002 can bind not only to class I PI3K and other PI3K-related kinases, but also to some novel targets that seem unrelated to the PI3K family. [3]

---

Background: To assess whether the PI3K/Akt pathway affects apoptosis in nasopharyngeal carcinoma cells and its mechanism. Methods: Using the PI3K inhibitor LY294002, the activation of the PI3K/Akt pathway and its effects on CNE-2Z cells in vivo and in vitro were studied by MTT assay, flow cytometry, Western blot, ELISA, terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) and immunohistochemical analysis. Results: The results showed that LY294002 inhibited Akt (S473) phosphorylation and cell proliferation, and induced apoptosis in CNE-2Z cells. However, our experimental results also showed that LY294002-induced apoptosis was directly regulated by the caspase-9 activation pathway. Conclusion: These data suggest that the PI3K inhibitor LY294002 induces apoptosis through caspase-9 activation pathway and may be a potential target for therapeutic intervention in nasopharyngeal carcinoma patients. [4]

C19H17NO3.HCL

343.80412

343.098

C, 66.38; H, 5.28; Cl, 10.31; N, 4.07; O, 13.96

934389-88-5

LY294002;154447-36-6

11957589

Solid powder

4.163

1

4

2

24

463

0

Cl.O=C1C2C=CC=C(C=2OC(N2CCOCC2)=C1)C1C=CC=CC=1

OQZQSRICUOWBLW-UHFFFAOYSA-N

InChI=1S/C19H17NO3.ClH/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;1H

2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride

LY-294002 HCl; LY294002 HCl;LY294002 hydrochloride; LY 294002 hydrochloride; LY 294002 HCl

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)

Typically soluble in DMSO (e.g. > 10 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)