Volume 123, Issue 1, Pages 206-216 (July 2002) Lysophosphatidic acid protects and rescues intestinal epithelial cells from radiation- and chemotherapy-induced apoptosis  Wenlin Deng, Louisa Balazs, De–An Wang, Lester Van Middlesworth, Gabor Tigyi, Leonard R. Johnson  Gastroenterology  Volume 123, Issue 1, Pages 206-216 (July 2002) DOI: 10.1053/gast.2002.34209 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 LPA protects IEC-6 cells against DNA fragmentation induced by serum withdrawal, camptothecin, γ-irradiation, and rat TNF-α. IEC-6 cells were washed twice and starved in serum-free DMEM overnight. LPA (25 μmol/L) was applied 15 minutes before apoptotic stimuli or after serum withdrawal. DNA fragmentation was measured 36 hours after continued withdrawal of serum, 6 hours after 20 μmol/L camptothecin treatment, 16 hours after 25-Gy γ-irradiation, or 12 hours after 100 ng/mL rat TNF-α. □, Control without apoptotic challenge; ■, apoptotic challenge; ▨, LPA treatment plus apoptotic challenge. *P < 0.001 (Student t test) compared with apoptotic stimuli alone. Data shown are mean ± SEM of 3 experiments. Gastroenterology 2002 123, 206-216DOI: (10.1053/gast.2002.34209) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 LPA protects against DNA fragmentation in a dose-dependent fashion. IEC-6 cells were washed twice and incubated overnight in DMEM without serum. LPA (ranging from 0.001–50 μmol/L) was added 15 minutes before apoptotic stimuli. DNA fragmentation was measured 36 hours after serum withdrawal (○), 6 hours after 20 μmol/L camptothecin treatment (●), 16 hours after 25-Gy γ-irradiation (▴), or 12 hours after 100 ng/mL rat TNF-α (■). Data shown are mean ± SEM of 3 independent results. Gastroenterology 2002 123, 206-216DOI: (10.1053/gast.2002.34209) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 LPA reduces DNA fragmentation and caspase-3/CPP32 activity induced by camptothecin and γ-irradiation. Serum-starved IEC-6 cells were treated with LPA (25 μmol/L) or vehicle at 1 hour, 15 minutes, and 0 minutes before, and 1 hour after 20 μmol/L camptothecin treatment or 2 hours after 20-Gy γ-irradiation. (A, C) DNA fragmentation and (B, D) caspase-3/CPP32 activity were measured 6 hours after camptothecin treatment and 16 hours after γ irradiation. Bars represent the mean ± SEM of 3 experiments. *P < 0.001 (Student t test) as compared with apoptotic stimuli alone. □, Negative control without apoptotic challenge; ■, LPA without apoptotic challenge; ▩, (A and B) camptothecin treatment, (C and D) γ-irradiation; ▨, (A and B) camptothecin plus LPA; and (C and D) γ-irradiation plus LPA. Gastroenterology 2002 123, 206-216DOI: (10.1053/gast.2002.34209) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 LPA inhibits caspase-3/CPP32 activation induced by camptothecin and γ-irradiation. Serum-starved IEC-6 cells were treated with LPA or vehicle 15 minutes before 20 μmol/L camptothecin treatment or 25-Gy γ-irradiation. (A) Caspase-3/CPP32 activity 6 hours after camptothecin (■) treatment and 16 hours after γ-irradiation (□). Shown are mean ± SEM of 3 experiments. *P < 0.001 (Student t test) as compared with apoptotic stimuli alone. (B) Caspase-3/CPP32 activation by Western blot analysis of procaspase to caspase cleavage. A total of 20 μg cytosolic protein was loaded for each lane. Similar results were obtained from 3 independent experiments. Data are representative of 3 experiments. Gastroenterology 2002 123, 206-216DOI: (10.1053/gast.2002.34209) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Involvement of LPA receptors in the antiapoptotic effect. RT-PCR was used for analysis of LPA receptor and lipid phosphate phosphatase 1 messenger RNA expression in IEC-6 cells. Serum-starved IEC-6 cells treated with 10 μmol/L LPA plus 60 μmol/L DGPP 15 minutes before 20 μmol/L camptothecin (CAM) treatment. (B) DNA fragmentation and (C) caspase-3/CPP32 activity were measured at 6 hours. Data are the mean ± SEM of 3 experiments. Based on Student t test, *P < 0.001 as compared with CAM alone. #P < 0.001 as compared with CAM and LPA. Gastroenterology 2002 123, 206-216DOI: (10.1053/gast.2002.34209) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 PTX but not U-73122 or toxin A inhibits LPA-elicited antiapoptotic effects. IEC-6 cells were washed twice and incubated overnight in DMEM without serum plus either 25 ng/mL PTX, 2 μmol/L U-73122, or 50 ng/mL toxin A, then 25 μmol/L LPA was added 15 minutes before 20 μmol/L camptothecin (CAM) treatment. (A) DNA fragmentation and (B) caspase-3/CPP32 activity were measured 6 hours after camptothecin treatment. (C) Procaspase-3/CPP32 and its active 20-kilodalton form were detected by Western blot. A total of 20 μg cytosolic protein was loaded in each lane. Data are the mean ± SEM of 3 experiments. *P < 0.001 (Student t test) as compared with CAM alone. Gastroenterology 2002 123, 206-216DOI: (10.1053/gast.2002.34209) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 LPA transactivates EGFR phosphorylation in a PTX-insensitive manner. IEC-6 cells were serum starved in the presence or absence of 25 ng/mL PTX overnight. A total of 20 μmol/L AG1478 was added 1 hour before treatment with 25 μmol/L LPA for 5 minutes or 50 ng/mL EGF for 2 minutes. (A) Tyrosine phosphorylation of the EGFR. Membrane was first probed by an antiphosphotyrosine monoclonal antibody, then stripped and reprobed with an antibody to EGFR. The picture is representative of 3 experiments. (B) DNA fragmentation after AG1478 or AG1296 pretreatment for 1 hour, followed by addition of 50 ng/mL EGF, or 50 ng/mL PDGF, or 25 μmol/L LPA 15 minutes before 20 μmol/L camptothecin. DNA fragmentation was measured 6 hours after camptothecin treatment. Data are mean ± SEM of 3 experiments. *P < 0.001 (Student t test) compared with camptothecin alone or AG1296 plus camptothecin. Gastroenterology 2002 123, 206-216DOI: (10.1053/gast.2002.34209) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 LPA protects against radiation-induced PCD in vivo. ICR mice were exposed to 15 or 18.75 Gy WBI, 2 hours after injection of 250 μL 1 mmol/L LPA or vehicle into the stomach. (A) The number of apoptotic bodies along 25 fully viewable crypt-villus units selected at random from 4 cross-sections of the jejunum showed a significant reduction in the LPA-treated animals as compared with controls that received vehicle only (ANOVA *P < 0.05). (B) The highest incidence for apoptotic bodies colocalized with the position of stem cells in the crypt. LPA treatment (■) reduced the apoptotic bodies significantly (*P < 0.05) in crypt cell positions 4–6 that correspond to the putative stem cells as compared with controls (□). Gastroenterology 2002 123, 206-216DOI: (10.1053/gast.2002.34209) Copyright © 2002 American Gastroenterological Association Terms and Conditions