Pharmacologic Profiling of Phosphoinositide 3-Kinase Inhibitors as Mitigators of Ionizing Radiation–Induced Cell Death

Abstract

Ionizing radiation (IR) induces genotoxic stress that triggers adaptive cellular responses, such as activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade. Pluripotent cells are the most important population affected by IR because they are required for cellular replenishment. Despite the clear danger to large population centers, we still lack safe and effective therapies to abrogate the life-threatening effects of any accidental or intentional IR exposure. Therefore, we computationally analyzed the chemical structural similarity of previously published small molecules that, when given after IR, mitigate cell death and found a chemical cluster that was populated with PI3K inhibitors. Subsequently, we evaluated structurally diverse PI3K inhibitors. It is remarkable that 9 of 14 PI3K inhibitors mitigated γIR-induced death in pluripotent NCCIT cells as measured by caspase 3/7 activation. A single intraperitoneal dose of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], administered to mice at 4 or 24 hours, or PX-867 [(4S,4aR,5R,6aS,9aR,Z)-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-2,7,10-trioxo-1-(pyrrolidin-1-ylmethylene)-1,2,4,4a,5,6,6a,7,8,9,9a,10-dodecahydroindeno[4,5-H]isochromen-5-yl acetate (CID24798773)], administered 4 hours after a lethal dose of γIR, statistically significantly (P < 0.02) enhanced in vivo survival. Because cell cycle checkpoints are important regulators of cell survival after IR, we examined cell cycle distribution in NCCIT cells after γIR and PI3K inhibitor treatment. LY294002 and PX-867 treatment of nonirradiated cells produced a marked decrease in S phase cells with a concomitant increase in the G1 population. In irradiated cells, LY294002 and PX-867 treatment also decreased S phase and increased the G1 and G2 populations. Treatment with LY294002 or PX-867 decreased γIR-induced DNA damage as measured by γH2AX, suggesting reduced DNA damage. These results indicate pharmacologic inhibition of PI3K after IR abrogated cell death.

A Small Molecule Screen Exposes mTOR Signaling Pathway Involvement in Radiation-Induced Apoptosis

“…9, 16 Interestingly, most of the PI3K inhibitors with radiation mitigative effects also inhibit mTOR, which is a master regulator of cell growth and metabolism and points to a potential role for mTOR in mitigating the effects of IR in NCCIT cells. This hypothesis was supported with the identification of rapamycin in the current screen.…”

“…9, 16 Our previously described caspase 3/7 assay 9, 16 was adapted to an automated screening format. Briefly, we seeded 3500 NCCIT cells/well in 384 well microtiter plates after 0 or 4 Gy IR using a JL Shepard Mark I Model 68 cesium source (75 cGy/min).…”

A review of radiation countermeasures focusing on injury-specific medicinals and regulatory approval status: part III. Countermeasures under early stages of development along with ‘standard of care’ medicinal and procedures not requiring regulatory approval for use

“…The effects of PI3K inhibitor treatments on cell cycle status following c-irradiation were studied, and it was found that both LY294002 and PX-867 treatments in vitro of human pluripotent embryonal carcinoma cells reduced the proportion of cells in S phase, while increasing the proportion of cells in G1. Similarly, treatments with LY294002 and PX-867 after irradiation also enhanced the G2 population as well as the G1 population, while decreasing the fraction of cells in S phase while increasing the extent of DNA damage as assessed by c-H2AX (Lazo et al 2013). Although quite interesting, these observations clearly suggest the need for more rational drug design based on specific pharmacological 'targeting' for radiomitigation purposes.…”

“…Single doses of LY294002 (CID3973) or PX-867 (CID24798773), PI3K inhibitors (30 mg/kg, ip), administered as injury-mitigators 4 h after 9.25 Gy c-irradiation, significantly increased survival in C57BL/6NTac female mice (Lazo et al 2013). In another study, LY294002 enhanced C57BL/6NTac female mice survival when administered ip at a dose of 30 mg/kg at 10 min, 4 h, or 24 h after 9.25 Gy TBI (Zellefrow et al 2012).…”

Radiation countermeasure agents: an update (2011 – 2014)

“…Ionizing radiation induces genotoxic stress that triggers adaptive cellular responses, such as activation of the phosphoinisitide-3 kinase (PI3K)/Akt signaling cascade. A single dose of LY294002 (CID3973) or PX-867 (CID24798773) administered after a lethal dose of γ-irradiation significantly enhanced mouse survival [ 149 ]. Cell cycle checkpoints are important regulators of cell survival after radiation exposure; cell cycles after γ-irradiation and PI3K inhibitor treatment was investigated [ 149 ].…”