We have previously demonstrated that the small molecule octadecenyl thiophosphate (OTP), a synthetic mimic of the growth factor-like mediator lysophosphatidic acid (LPA), showed radioprotective activity in a mouse model of total-body irradiation (TBI) when given orally or intraperitoneally 30 min before exposure to 9 Gy γ radiation. In the current study, we evaluated the effects of OTP, delivered subcutaneously, for radioprotection or radiomitigation from -24 h before to up to +72 h postirradiation using a mouse TBI model with therapeutic doses at around 1 mg/kg. OTP was injected at 10 mg/kg without observable toxic side effects in mice, providing a comfortable safety margin. Treatment of C57BL/6 mice with a single dose of OTP over the time period from -12 h before to +26 h after a lethal dose of TBI reduced mortality by 50%. When administered at +48 h to +72 h postirradiation (LD50/30 to LD100/30), OTP reduced mortality by ≥34%. OTP administered at +24 h postirradiation significantly elevated peripheral white blood cell and platelet counts, increased crypt survival in the jejunum, enhanced intestinal glucose absorption and reduced endotoxin seepage into the blood. In the 6.4-8.6 Gy TBI range using LD50/10 as the end point, OTP yielded a dose modification factor of 1.2. The current data indicate that OTP is a potent radioprotector and radiomitigator ameliorating the mortality and tissue injury of acute hematopoietic as well as acute gastrointestinal radiation syndrome.

Radiomitigators attenuate radiation injury when administered after irradiation. Lysophosphatidic acid (LPA) a lipid mediator rescues apoptotically condemned cells. LPA protects IEC‐6 intestinal epithelial cells in vitro and intestinal crypts in vivo from radiation‐induced apoptosis. Using KO mice and cells we established that the LPA2 receptor is required for radiomitigation. Through its C‐terminal PDZ and LIM‐binding motifs LPA2 forms agonist‐dependent macromolecular signaling complexes. LPA2 interacts with the proapoptotic protein Siva‐1 and targets it for proteasomal degradation. Full activation of the NFkB and ERK1/2 prosurvival pathway requires formation of an LPA2, NHERF2, and TRIP‐6 ternary macromolecular complex. These signals inhibit the mitochondrial apoptosis pathway. Reconstitution of the LPA2 receptor into MEFs derived from LPA1/2 KO mice restores radioprotection. Conditioned medium from irradiated U937 cells induces bystander apoptosis in non‐irradiated IEC‐6 cultures and LPA2‐transfected MEFs. Using in silico drug discovery we found a novel LPA2‐selective agonist GRI977143 that protects cells from radiation‐ and chemotherapy‐induced apoptosis. GRI977143 also increases the survival of mice exposed to lethal levels of radiation. Supported by AI08405.

Lysophosphatidic acid (LPA) is a growth factor‐like lysophospholipis that rescues apoptotically condemned cells. LPA protects IEC‐6 intestinal epithelial cells in vitro and intestinal crypts in vivo from radiation‐induced apoptosis. Using LPA receptor KO mice we established that the LPA2 receptor subtype plays an essential role in the antiapoptotic mechanism. Through its C‐terminal PDZ and LIM‐binding motifs LPA2 forms agonist‐dependent macromolecular signaling complexes. LPA2 interacts with the proapoptotic protein Siva‐1 and targets it for proteasomal degradation, which in turn arrests DNA‐damage‐activated apoptosis. Full activation of the NFkB and ERK1/2 prosurvival pathway requires formation of an LPA2, NHERF2, and TRIP‐6 ternary macromolecular complex. These signals inhibit the intrinsic mitochondrial apoptosis pathway. Furthermore, transfection of the LPA2 receptor into mouse embryonic fibroblasts derived from LPA1/2 double knockout mice restores LPA‐dependent radioprotection. Conditioned medium from gamma‐irradiated U937 cells induces bystander apoptosis in non‐irradiated IEC‐6 cultures and LPA2‐transfected MEFs. Using a validated computational model of the LPA2 receptor we developed a pharmacophore model and used it for in silico drug discovery of non‐lipid LPA2 agonists. We found a novel LPA2‐selective agonist GRI977143, which elicits an antiapoptotic effect and protects IEC‐6 cells from radiation‐ and chemotherapy‐induced apoptosis. GRI977143 also increases the survival of mice exposed to lethal levels of radiation. Supported by CA92160 and AI08405.

BACKGROUND & AIMS We recently identified lysophosphatidic acid (LPA) as a potent antiapoptotic agent for the intestinal epithelium. The objective of the present study was to evaluate the effect of octadecenyl thiophosphate (OTP), a novel rationally designed, metabolically stabilized LPA mimic, on radiation-induced apoptosis of intestinal epithelial cells in vitro and in vivo. METHODS The receptors and signaling pathways activated by OTP were examined in IEC-6 and RH7777 cell lines and wild-type and LPA(1) and LPA(2) knockout mice exposed to different apoptotic stimuli. RESULTS OTP was more efficacious than LPA in reducing gamma irradiation-, camptothecin-, or tumor necrosis factor alpha/cycloheximide-induced apoptosis and caspase-3-8, and caspase-9 activity in the IEC-6 cell line. In RH7777 cells lacking LPA receptors, OTP selectively protected LPA(2) but not LPA(1) and LPA(3) transfectants. In C57BL/6 and LPA(1) knockout mice exposed to 15 Gy gamma irradiation, orally applied OTP reduced the number of apoptotic bodies and activated caspase-3-positive cells but was ineffective in LPA(2) knockout mice. OTP, with higher efficacy than LPA, enhanced intestinal crypt survival in C57BL/6 mice but was without any effect in LPA(2) knockout mice. Intraperitoneally administered OTP reduced death caused by lethal dose (LD)(100/30) radiation by 50%. CONCLUSIONS Our data indicate that OTP is a highly effective antiapoptotic agent that engages similar prosurvival pathways to LPA through the LPA(2) receptor subtype.

Background & Aims: We recently identified lysophosphatidic acid (LPA) as a potent antiapoptotic agent for the intestinal epithelium. The objective of the present study was to evaluate the effect of octadecenyl thiophosphate (OTP), a novel rationally designed, metabolically stabilized LPA mimic, on radiation-induced apoptosis of intestinal epithelial cells in vitro and in vivo. Methods: The receptors and signaling pathways activated by OTP were examined in IEC-6 and RH7777 cell lines and wild-type and LPA(1) and LPA(2) knockout mice exposed to different apoptotic stimuli. Results: OTP was more efficacious than LPA in reducing gamma irradiation-, camptothecin-, or tumor necrosis factor alpha/cycloheximide-induced apoptosis and caspase-3-8, and caspase-9 activity in the IEC-6 cell line. In RH7777 cells lacking LPA receptors, OTP selectively protected LPA(2) but not LPA(1) and LPA(3) transfectants. In C57BL/6 and LPA(1) knockout mice exposed to 15 Gy gamma irradiation, orally applied OTP reduced the number of apoptotic bodies and activated caspase-3-positive cells but was ineffective in LPA(2) knockout mice. OTP, with higher efficacy than LPA, enhanced intestinal crypt survival in C57BL/6 mice but was without any effect in LPA(2) knockout mice. Intraperitoneally administered OTP reduced death caused by lethal dose (LD)(100/30) radiation by 50%. Conclusions: Our data indicate that OTP is a highly effective antiapoptotic agent that engages similar prosurvival pathways to LPA through the LPA(2) receptor subtype.

Our group has previously shown that polyamine depletion delays apoptosis in rat intestinal epithelial (IEC-6) cells (Ray RM, Viar MJ, Yuan Q, and Johnson LR, Am J Physiol Cell Physiol 278: C480-C489, 2000). Here, we demonstrate that polyamine depletion inhibits gamma-irradiation-induced apoptosis in vitro and in vivo. Pretreatment of IEC-6 cells with 5 mM alpha-difluoromethylornithine (DFMO) for 4 days significantly reduced radiation-induced caspase-3 activity and DNA fragmentation. This protective effect was prevented by the addition of 10 muM exogenous putrescine. Radiation exposure to mice resulted in a high frequency of apoptosis over cells positioned fourth to seventh in crypt-villus units. Pretreatment of mice with 2% DFMO in drinking water significantly reduced apoptotic cells from approximately 2.75 to 1.61 per crypt-villus unit, accompanied by significant decreases in caspase-3 levels. Further examination showed that DFMO pretreatment inhibited the radiation-induced increase in the proapoptotic protein Bax. Moreover, DFMO pretreatment significantly enhanced the intestinal crypt survival rate by 2.1-fold subsequent to radiation and ameliorated mucosal structural damage. We conclude that polyamine depletion by DFMO inhibits gamma-irradiation-induced apoptosis of intestinal epithelial cells both in vitro and in vivo through inhibition of Bax and caspase-3 activity, which leads to attenuation of radiation-inflicted intestinal injury. These data indicate that DFMO may be therapeutically useful to counteract the gastrointestinal toxicity caused by chemoradiotherapy. This is the first demonstration that polyamines are required for apoptosis in vivo.

Lysophosphatidic acid (LPA)-elicited transphosphorylation of receptor tyrosine kinases has been implicated in mediating extracellular signal-regulated kinase (ERK) 1/2 activation, which is necessary for LPA-induced cell proliferation, migration, and survival. B82L cells lack epidermal growth factor receptor (EGFR) but express LPA1–3, platelet-derived growth factor (PDGF), ErbB2, and insulin-like growth factor receptor transcripts, yet LPA caused no detectable transphosphorylation of these receptor tyrosine kinases. LPA equally protected B82L cells, or transfectants expressing EGFR, the kinase dead EGFRK721A, EGFRY5F receptor mutant, which lacks five autophosphorylation sites, or EGFRY845F, which lacks the Src phosphorylation site from tumor necrosis factor-α-induced apoptosis. In contrast, LPA-elicited DNA synthesis and migration were augmented in cells expressing EGFR, EGFRK721A, or EGFRY845F, but not EGFRY5F, although the PDGF responses were indistinguishable. LPA-induced transphosphorylation of the EGFR, ErbB2, or PDGF receptor was not required for its antiapoptotic effect. EGFR with or without intrinsic kinase activity or without the Src-phosphorylation site augmented, but was not required for, LPA-elicited cell proliferation or migration. In B82L cells, augmentation of these two LPA responses required intact autophosphorylation sites because among the four EGFR mutants, only cells expressing the EGFRY5F mutant showed no enhancement. In EGFRY5F-expressing cells, LPA failed to elicit tyrosine phosphorylation of Src homologous and collagen protein (SHC) and caused only a modest increase in ERK1/2 phosphorylation similar to that in wild-type B82L cells. The present data pinpoint the lack of importance of the intrinsic kinase activity in contrast to the importance of autophosphorylation sites of the EGFR for SHC phosphorylation in the enhancement of select ERK1/2-dependent LPA responses.

We previously showed (Gastroenterology 123: 206-216, 2002) that lysophosphatidic acid (LPA) protects and rescues rat intestinal epithelial cells (IEC-6) from apoptosis. Here, we provide evidence for the LPA-elicited inhibition of the mitochondrial apoptotic pathway leading to attenuation of caspase-3 activation. Pretreatment of IEC-6 cells with LPA inhibited campothecin-induced caspase-9 and caspase-3 activation and DNA fragmentation. A caspase-9 inhibitor peptide mimicked the LPA-elicited antiapoptotic activity. LPA elicited ERK1/ERK2 and PKB/Akt phosphorylation. The LPA-elicited antiapoptotic activity and inhibition of caspase-9 activity were abrogated by pertussis toxin, PD 98059, wortmannin, and LY 294002. LPA reduced cytochrome c release from mitochondria and prevented activation of caspase-9. LPA prevented translocation of Bax from cytosol to mitochondria and increased the expression of the antiapoptotic Bcl-2 mRNA and protein. LPA had no effect on Bcl-xl, Bad, and Bak mRNA or protein expression. These data indicate that LPA protects IEC-6 cells from camptothecin-induced apoptosis through G(i)-coupled inhibition of caspase-3 activation mediated by the attenuation of caspase-9 activation due to diminished cytochrome c release, involving upregulation of Bcl-2 protein expression and prevention of Bax translocation.

BACKGROUND & AIMS:We have investigated whether the phospholipid growth factor lysophosphatidic acid (LPA) could prevent intestinal epithelial cells-6 (IEC-6) from apoptosis elicited by 4 different mechanisms. The antiapoptotic effect of LPA was also tested in a mouse model of radiation-induced apoptosis. METHODS:Apoptosis was elicited by serum withdrawal, exposure to camptothecin, gamma-irradiation, or rat tumor necrosis factor alpha and evaluated by DNA fragmentation enzyme-linked immunosorbent assay (ELISA) and annexin V staining. Caspase-3/CPP32 activity and activation was measured by ELISA and Western blotting, respectively. Reverse-transcription polymerase chain reaction (RT-PCR) was applied to examine the expression of LPA-receptor transcripts. Mice were treated with 250 microL of 1 mmol/L LPA and exposed to whole-body gamma-irradiation with a dose of 12 or 15 Gy and the number and localization of apoptotic bodies along the crypt were recorded. RESULTS:LPA pretreatment reduced DNA fragmentation induced in all models of apoptosis. LPA rescued cells from apoptosis when applied up to 1 hour after camptothecin treatment or 2 hours after irradiation. LPA inhibited the activation of caspase-3/CPP32 and attenuated its activity. Blocking LPA1 receptors by pertussis toxin and the inhibition of epithelial growth factor receptor tyrosine kinase significantly attenuated the protective effect. In irradiated mice, oral LPA significantly reduced the number of apoptotic bodies in the crypt. CONCLUSIONS:(1) LPA prevents and rescues IEC-6 from apoptosis elicited by 4 different mechanisms. (2) This antiapoptotic activity is mediated through LPA1 and LPA2 receptors through the inhibition of caspase-3/CPP32 activation. (3) LPA protects enterocytes against radiation-induced apoptosis. This study suggests that in patients undergoing cancer therapy, dietary LPA might have therapeutically useful antiapoptotic capacity in the intestinal epithelium.