Over‐expression of pemt2 into rat hepatoma cells contributes to the mitochondrial apoptotic pathway

We previously established a line of phosphatidylethanolamine N‐methyltransferase 2 (pemt2) ‐stably transfected CBRH‐7919 hepatoma cells, and showed that pemt2 over‐expression inhibited cell proliferation and induced apoptosis. This study was aimed to further elucidate the cellular mechanisms leading to this apoptosis in these cells. Fatty acid compositions of phosphatidylcholine (PC) in pemt2 over‐expressed cells and control cells, and the location of PC synthesized by PEMT2 pathway were analyzed with lipid extraction, high‐performance thin layer chromatography, high‐performance gas chromatography (HPGC), and [3H]‐ethanolamine tracing. The effects of pemt2 over‐expression on the mitochondrial membrane fluidity, the release of cytochrome C from mitochondria, and the activity of caspases were determined by Western blot. Newly synthesized PC by PEMT2 contained more acyl groups of oleic acid (P < 0.01) and was mainly located in mitochondria; pemt2 over‐expression increased the mitochondrial membrane fluidity and the release of cytochrome C from the mitochondria into the cytoplasma, which in turn activated caspase‐9 and caspase‐3, the key molecules in the mitochondrial apoptotic pathway. We demonstrated that, in rat hepatoma cells, PEMT2‐induced apoptosis proceeds through mitochondria. © 2009 IUBMB IUBMB Life, 61(8): 846–852, 2009

[1]  J. Belizário,et al.  A mechanistic view of mitochondrial death decision pores. , 2007, Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas.

[2]  S. Grimm,et al.  The permeability transition pore in cell death , 2007, Apoptosis.

[3]  B. Antonsson,et al.  A tale of two mitochondrial channels, MAC and PTP, in apoptosis , 2007, Apoptosis.

[4]  L. Galluzzi,et al.  Mechanisms of cytochrome c release from mitochondria , 2006, Cell Death and Differentiation.

[5]  B. Zhivotovsky,et al.  Multiple pathways of cytochrome c release from mitochondria in apoptosis. , 2006, Biochimica et biophysica acta.

[6]  C. Zazueta,et al.  Agaric acid induces mitochondrial permeability transition through its interaction with the adenine nucleotide translocase. Its dependence on membrane fluidity. , 2005, Mitochondrion.

[7]  K. Kinnally,et al.  The Role of the Mitochondrial Apoptosis Induced Channel MAC in Cytochrome c Release , 2005, Journal of bioenergetics and biomembranes.

[8]  G. Daum,et al.  Contribution of different pathways to the supply of phosphatidylethanolamine and phosphatidylcholine to mitochondrial membranes of the yeast Saccharomyces cerevisiae. , 2004, Biochimica et biophysica acta.

[9]  G. Daum,et al.  Contribution of different biosynthetic pathways to species selectivity of aminoglycerophospholipids assembled into mitochondrial membranes of the yeast Saccharomyces cerevisiae. , 2004, Biochimica et biophysica acta.

[10]  A. Heck,et al.  The Yeast Phospholipid N-Methyltransferases Catalyzing the Synthesis of Phosphatidylcholine Preferentially Convert Di-C16:1 Substrates Both in Vivo and in Vitro* , 2004, Journal of Biological Chemistry.

[11]  A. Colell,et al.  Cholesterol Impairs the Adenine Nucleotide Translocator-mediated Mitochondrial Permeability Transition through Altered Membrane Fluidity* , 2003, Journal of Biological Chemistry.

[12]  P. Oliveira,et al.  Chenodeoxycholate induction of mitochondrial permeability transition pore is associated with increased membrane fluidity and cytochrome c release: protective role of carvedilol. , 2003, Mitochondrion.

[13]  A. Heck,et al.  The two biosynthetic routes leading to phosphatidylcholine in yeast produce different sets of molecular species. Evidence for lipid remodeling. , 2003, Biochemistry.

[14]  Zhao-Yu Li,et al.  Overexpression of PEMT2 downregulates the PI3K/Akt signaling pathway in rat hepatoma cells. , 2002, Biochimica et biophysica acta.

[15]  A. Postle,et al.  Highly Saturated Endonuclear Phosphatidylcholine Is Synthesizedin Situ and Colocated with CDP-choline Pathway Enzymes* , 2001, The Journal of Biological Chemistry.

[16]  M. Wieckowski,et al.  Long‐chain fatty acids promote opening of the reconstituted mitochondrial permeability transition pore , 2000, FEBS letters.

[17]  M. Gordon,et al.  Preferential distribution of long chain polyunsaturated fatty acids in phospatidyl ethanolamine fraction of guinea pig alveolar apical membranes. , 2000, Prostaglandins, leukotrienes, and essential fatty acids.

[18]  G. Kroemer,et al.  Mitochondrial Membrane Permeabilization during the Apoptotic Process , 1999, Annals of the New York Academy of Sciences.

[19]  Michael J. Thomas,et al.  Molecular Distinction of Phosphatidylcholine Synthesis between the CDP-Choline Pathway and Phosphatidylethanolamine Methylation Pathway* , 1999, The Journal of Biological Chemistry.

[20]  L. Tessitore,et al.  Expression of phosphatidylethanolamine N-methyltransferase in Yoshida ascites hepatoma cells and the livers of host rats. , 1999, Carcinogenesis.

[21]  C. Zazueta,et al.  Carboxyatractyloside increases the effect of oleate on mitochondrial permeability transition , 1999, FEBS letters.

[22]  C. Kent.  CTP:phosphocholine cytidylyltransferase. , 1997, Biochimica et biophysica acta.

[23]  L. Tessitore,et al.  Induction of hepatocyte proliferation after partial hepatectomy is accompanied by a markedly reduced expression of phosphatidylethanolamine N-methyltransferase-2. , 1997, Biochimica et biophysica acta.

[24]  D. Vance,et al.  Inverse correlation between expression of phosphatidylethanolamine N-methyltransferase-2 and growth rate of perinatal rat livers. , 1997, Biochimica et biophysica acta.

[25]  Y. Mély,et al.  A comparison of the fluorescence properties of TMA-DPH as a probe for plasma membrane and for endocytic membrane. , 1995, Biochimica et biophysica acta.

[26]  D. Vance,et al.  Cloning and expression of a novel phosphatidylethanolamine N-methyltransferase. A specific biochemical and cytological marker for a unique membrane fraction in rat liver. , 1993, The Journal of biological chemistry.

[27]  D. Vance,et al.  Specificity of rat hepatic phosphatidylethanolamine N-methyltransferase for molecular species of diacyl phosphatidylethanolamine. , 1988, The Journal of biological chemistry.

[28]  M. Roberfroid,et al.  Subcellular fractionation of isolated rat hepatocytes. A comparison with liver homogenate. , 1980, Biochimica et biophysica acta.

[29]  J. MacGee,et al.  Preparation of methyl esters from the saponifiable fatty acids in small biological specimens for gas-liquid chromatographic analysis. , 1974, Journal of chromatography.

[30]  Zou Wei.  Transfection of pemt2-cDNA Inhibits c-Met and Bcl-2 Expression and Induces Apoptosis in Rat CBRH-7919 Hepatoma Cells , 2001 .

[31]  L. Tessitore,et al.  Diminished expression of phosphatidylethanolamine N-methyltransferase 2 during hepatocarcinogenesis. , 1999, The Biochemical journal.