Modulation of growth in human esophageal adenocarcinoma cells by group IIa secretory phospholipase A2 David Mauchley, MD, Xianzhong Meng, MD, PhD, Thomas.

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Modulation of growth in human esophageal adenocarcinoma cells by group IIa secretory phospholipase A2 David Mauchley, MD, Xianzhong Meng, MD, PhD, Thomas Johnson, PhD, David A. Fullerton, MD, Michael J. Weyant, MD The Journal of Thoracic and Cardiovascular Surgery Volume 139, Issue 3, Pages 591-599 (March 2010) DOI: 10.1016/j.jtcvs.2009.10.061 Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 Expression of group IIa secretory phospholipase A2(sPLA2) protein in esophageal adenocarcinoma cell lines. A, Immunofluorescence microscopy shows expression of group IIa secretory phospholipase A2(red) in esophageal adenocarcinoma cell lines FLO-1 and OE33. Nuclei are stained blue, and cell membranes are stained green. B, Reverse transcriptase polymerase chain reaction shows near equal expression of group IIa secretory phospholipase A2 in esophageal cancer cells. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; NS, not statistically significant. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 591-599DOI: (10.1016/j.jtcvs.2009.10.061) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 Effect of group IIa secretory phospholipase A2 inhibition on esophageal adenocarcinoma cell number. Relative cell numbers after 72 hours of treatment of FLO-1 cells (A) and OE33 cells (B) relative to controls, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Statistical significance relative to untreated controls and vehicle (dimethyl sulfoxide, DMSO) controls was reached with 10- and 20-μmol/L doses for FLO-1 cells and with 5-, 10-, and 20-μmol/L doses for OE33 cells. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 591-599DOI: (10.1016/j.jtcvs.2009.10.061) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Effect of group IIa secretory phospholipase A2 inhibition on esophageal adenocarcinoma cellular proliferation. Relative bromodeoxyuridine (BrdU) incorporation after 72 hours of treatment in FLO-1 cells (A) and OE33 cells (B) relative to controls. Statistical significance versus both untreated and vehicle (dimethyl sulfoxide, DMSO) controls was reached with 10- and 20-μmol/L doses in both cell types. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 591-599DOI: (10.1016/j.jtcvs.2009.10.061) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 Infection of esophageal adenocarcinoma cells with lentiviral particles. Both FLO-1 and OE33 cells are efficiently infected with lentiviral particles derived from lentiviral expression vector pLVUT-tTR-KRAB (LVUT) and pLKO.1 puro constructs, as shown by green fluorescent protein expression under ultraviolet (UV) light (green). Infection with lentiviral particles did not affect cell shape or size. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 591-599DOI: (10.1016/j.jtcvs.2009.10.061) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 Overexpression of group IIa secretory phospholipase A2(sPLA2) in esophageal adenocarcinoma cells augments cell growth. A, In quantitative reverse transcriptase polymerase chain reaction analysis of FLO-1 and OE33 cells infected with lentiviral expression vector pLVUT-tTR-KRAB (LVUT) with gene for secretory phospholipase A2, viral particles show significant overexpression of group IIa secretory phospholipase A2 relative to controls. B, Immunofluorescent microscopy of both FLO-1 and OE33 cells shows increased protein levels of group IIa secretory phospholipase A2(red) in cytoplasm of cells infected with viral particles derived from pLVUT-tTR-KRAB–secretory phospholipase A2 gene construct. C, Viable cell number is increased in both FLO-1 and OE33 cells that overexpress group IIa secretory phospholipase A2 relative to uninfected and pLVUT-tTR-KRAB–infected controls. D, FLO-1 and OE33 cells that overexpress group IIa secretory phospholipase A2 demonstrate greater cell proliferation than controls. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UT, untreated; BrdU, bromodeoxyuridine. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 591-599DOI: (10.1016/j.jtcvs.2009.10.061) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 6 Genetic knockdown of group IIa secretory phospholipase A2(sPLA2) attenuates growth of esophageal adenocarcinoma cells. A, Quantitative reverse transcriptase polymerase chain reaction analysis of FLO-1 and OE33 cells infected with group IIa secretory phospholipase A2 short hairpin RNA (shRNA) viral particles shows significant reduction in expression relative to nontargeting (NT) and untreated (UT) controls. B, Immunofluorescent microscopy of both FLO-1 and OE33 cells shows decreased protein levels of group IIa secretory phospholipase A2(red) in cells infected with secretory phospholipase A2 short hairpin RNA viral particles relative to nontargeting short hairpin RNA control. C, Viable cell number is diminished in both FLO-1 and OE33 cells that have genetic knockdown of group IIa secretory phospholipase A2 transcript relative to uninfected and nontargeting controls. D, Knockdown of group IIa secretory phospholipase A2 expression results in attenuated cell proliferation relative to uninfected and nontargeting short hairpin RNA controls. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BrdU, bromodeoxyuridine. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 591-599DOI: (10.1016/j.jtcvs.2009.10.061) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions