L-Carnitine Is an Endogenous HDAC Inhibitor Selectively Inhibiting Cancer Cell Growth In Vivo and In Vitro

L-carnitine (LC) is generally believed to transport long-chain acyl groups from fatty acids into the mitochondrial matrix for ATP generation via the citric acid cycle. Based on Warburg's theory that most cancer cells mainly depend on glycolysis for ATP generation, we hypothesize that, LC treatment would lead to disturbance of cellular metabolism and cytotoxicity in cancer cells. In this study, Human hepatoma HepG2, SMMC-7721 cell lines, primary cultured thymocytes and mice bearing HepG2 tumor were used. ATP content was detected by HPLC assay. Cell cycle, cell death and cell viability were assayed by flow cytometry and MTS respectively. Gene, mRNA expression and protein level were detected by gene microarray, Real-time PCR and Western blot respectively. HDAC activities and histone acetylation were detected both in test tube and in cultured cells. A molecular docking study was carried out with CDOCKER protocol of Discovery Studio 2.0 to predict the molecular interaction between L-carnitine and HDAC. Here we found that (1) LC treatment selectively inhibited cancer cell growth in vivo and in vitro; (2) LC treatment selectively induces the expression of p21cip1 gene, mRNA and protein in cancer cells but not p27kip1; (4) LC increases histone acetylation and induces accumulation of acetylated histones both in normal thymocytes and cancer cells; (5) LC directly inhibits HDAC I/II activities via binding to the active sites of HDAC and induces histone acetylation and lysine-acetylation accumulation in vitro; (6) LC treatment induces accumulation of acetylated histones in chromatin associated with the p21cip1 gene but not p27kip1 detected by ChIP assay. These data support that LC, besides transporting acyl group, works as an endogenous HDAC inhibitor in the cell, which would be of physiological and pathological importance.

[1]  S. Grant,et al.  Induction of apoptosis in U937 human leukemia cells by suberoylanilide hydroxamic acid (SAHA) proceeds through pathways that are regulated by Bcl-2/Bcl-XL, c-Jun, and p21CIP1, but independent of p53 , 1999, Oncogene.

[2]  S. Weinhouse On respiratory impairment in cancer cells. , 1956, Science.

[3]  Muyang Li,et al.  Acetylation of p53 Inhibits Its Ubiquitination by Mdm2* , 2002, The Journal of Biological Chemistry.

[4]  G. T. Huang Dental pulp and dentin tissue engineering and regeneration: advancement and challenge. , 2011, Frontiers in bioscience.

[5]  O. Warburg On respiratory impairment in cancer cells. , 1956, Science.

[6]  M. Inoue,et al.  L‐carnitine inhibits hepatocarcinogenesis via protection of mitochondria , 2005, International journal of cancer.

[7]  M. Mann,et al.  Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions , 2009, Science.

[8]  P. Nicotera,et al.  Intracellular Adenosine Triphosphate (ATP) Concentration: A Switch in the Decision Between Apoptosis and Necrosis , 1997, The Journal of experimental medicine.

[9]  J. Girard,et al.  Rat Liver Carnitine Palmitoyltransferase 1 Forms an Oligomeric Complex within the Outer Mitochondrial Membrane* , 2007, Journal of Biological Chemistry.

[10]  R. Gillies,et al.  Why do cancers have high aerobic glycolysis? , 2004, Nature Reviews Cancer.

[11]  H. Handa,et al.  Mediator requirement for both recruitment and postrecruitment steps in transcription initiation. , 2005, Molecular cell.

[12]  R. Portenoy,et al.  Safety, tolerability and symptom outcomes associated with L-carnitine supplementation in patients with cancer, fatigue, and carnitine deficiency: a phase I/II study. , 2006, Journal of pain and symptom management.

[13]  L. Girard,et al.  Differential response of cancer cells to HDAC inhibitors trichostatin A and depsipeptide , 2011, British Journal of Cancer.

[14]  Di Chen,et al.  Gambogic acid enhances proteasome inhibitor-induced anticancer activity. , 2011, Cancer letters.

[15]  P. Marks,et al.  Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Yixue Li,et al.  Regulation of Cellular Metabolism by Protein Lysine Acetylation , 2010, Science.

[17]  J. Trepel,et al.  Transcriptional activation of p21(WAF1/CIP1) by apicidin, a novel histone deacetylase inhibitor. , 2001, Biochemical and biophysical research communications.

[18]  T. Hunter The age of crosstalk: phosphorylation, ubiquitination, and beyond. , 2007, Molecular cell.

[19]  M. Oren,et al.  Inhibition of p53 degradation by Mdm2 acetylation , 2004, FEBS letters.

[20]  P. Marks,et al.  Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors , 1999, Nature.

[21]  S. Pavey,et al.  Up-regulation of p21(WAF1/CIP1) by histone deacetylase inhibitors reduces their cytotoxicity. , 2001, Molecular pharmacology.

[22]  R A Rifkind,et al.  Suberoylanilide hydroxamic acid, an inhibitor of histone deacetylase, suppresses the growth of prostate cancer cells in vitro and in vivo. , 2000, Cancer research.

[23]  R. Ramsay,et al.  The mechanism of fatty acid uptake by heart mitochondria: An acylcarnitine‐carnitine exchange , 1975, FEBS letters.

[24]  R C Coombes,et al.  Trichostatin A is a histone deacetylase inhibitor with potent antitumor activity against breast cancer in vivo. , 2001, Clinical cancer research : an official journal of the American Association for Cancer Research.

[25]  M. Pagano,et al.  Degradation of cyclin A is regulated by acetylation , 2009, Oncogene.

[26]  E. Seto,et al.  Lysine acetylation: codified crosstalk with other posttranslational modifications. , 2008, Molecular cell.

[27]  Hao Jiang,et al.  Triple Layer Control: Phosphorylation, Acetylation and Ubiquitination of FOXO Proteins , 2005, Cell cycle.

[28]  A. Evdokiou,et al.  Involvement of p21(Waf1/Cip1) and its cleavage by DEVD-caspase during apoptosis of colorectal cancer cells induced by butyrate. , 2000, Carcinogenesis.

[29]  G. Mantovani,et al.  Efficacy of l-carnitine administration on fatigue, nutritional status, oxidative stress, and related quality of life in 12 advanced cancer patients undergoing anticancer therapy. , 2006, Nutrition.

[30]  P. Atadja HDAC inhibitors and cancer therapy. , 2011, Progress in drug research. Fortschritte der Arzneimittelforschung. Progres des recherches pharmaceutiques.

[31]  Q. Dou,et al.  Physiological levels of ATP negatively regulate proteasome function , 2010, Cell Research.

[32]  F. R. van der Leij,et al.  Molecular enzymology of carnitine transfer and transport. , 2001, Biochimica et biophysica acta.

[33]  V. Richon,et al.  Histone deacetylase inhibitors: a new class of potential therapeutic agents for cancer treatment. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[34]  U. Das,et al.  Essential fatty acids: biochemistry, physiology and pathology , 2006, Biotechnology journal.

[35]  H. Karlic,et al.  Supplementation of L-carnitine in athletes: does it make sense? , 2004, Nutrition.

[36]  Di Chen,et al.  Shikonin exerts antitumor activity via proteasome inhibition and cell death induction in vitro and in vivo , 2009, International journal of cancer.

[37]  Q. Dou,et al.  Sanggenon C decreases tumor cell viability associated with proteasome inhibition. , 2011, Frontiers in bioscience.

[38]  Peng Huang,et al.  Mitochondrial defects in cancer , 2002, Molecular Cancer.

[39]  R. Deberardinis Is cancer a disease of abnormal cellular metabolism? New angles on an old idea , 2008, Genetics in Medicine.

[40]  Chi V Dang,et al.  Cancer's molecular sweet tooth and the Warburg effect. , 2006, Cancer research.

[41]  E. Bonora,et al.  Defective oxidative phosphorylation in thyroid oncocytic carcinoma is associated with pathogenic mitochondrial DNA mutations affecting complexes I and III. , 2006, Cancer research.

[42]  M. Sayed-Ahmed,et al.  Progression of diethylnitrosamine-induced hepatic carcinogenesis in carnitine-depleted rats. , 2009, World journal of gastroenterology.