The roles of bioactive sphingolipids in resveratrol-induced apoptosis in HL60: acute myeloid leukemia cells.
OBJECTIVE
Acute promyelocytic leukemia results from a translocation between 15 and 17 chromosomes that produce PML/RARα fusion protein. PML/RARα inhibits differentiation of myeloid precursor cells at stem cell level. Resveratrol is a phytoalexin that exerts cytotoxic effects on cancer cells. Ceramides have crucial roles in cell growth, proliferation, differentiation, drug resistance, and apoptosis. In this study, we examined the possible cytotoxic effects of resveratrol on acute myeloid leukemia cells and determined the roles of ceramide-metabolizing genes in resveratrol-induced apoptosis, in addition to investigating the possibility of increasing the sensitivity of HL60 cells to resveratrol by manipulating sphingolipids.


METHODS
Cytotoxic effects of resveratrol, C8:ceramide, PDMP, and SK-1 inhibitor were determined by XTT cell proliferation assay. Changes in caspase-3 enzyme activity and mitochondrial membrane potential (MMP) were measured using caspase-3 colorimetric assay and JC-1 MMP detection kit. Expression levels of ceramide-metabolizing genes were examined by RT-PCR.


RESULTS
The results revealed that manipulations of ceramide metabolism toward generation or accumulation of apoptotic ceramides increased apoptotic effects of resveratrol in HL60 cells, synergistically. More importantly, gene expression analyses revealed that resveratrol-induced apoptosis via increasing expression levels of ceramide-generating genes and decreasing expression levels of antiapoptotic sphingosine kinase-1 and glucosylceramide synthase genes.


CONCLUSIONS
These results showed for the first time that increasing intracellular levels of ceramides by biochemical approaches has also increased sensitivity of HL60 cells to resveratrol. We also showed that resveratrol induces apoptosis through manipulating ceramide-metabolizing genes that resulted in the accumulation of ceramides in HL60 cells.
Cakir
Zeynep
Z
Department of Molecular Biology and Genetics, Faculty of Science, Izmir Institute of Technology, Gulbahce Campus, 35430 Urla, Izmir, Turkey.
Saydam
Guray
G
Sahin
Fahri
F
Baran
Yusuf
Y
D000605:Amino Alcohols / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002518:Ceramides / Q000494:pharmacology; D003124:Colorimetry; D004357:Drug Synergism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D018922:HL-60 Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy* / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000077185:Resveratrol; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013107:Sphingolipids / Q000378:metabolism*; D013267:Stilbenes / Q000494:pharmacology*; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D000605:Amino Alcohols; D000972:Antineoplastic Agents, Phytogenic; D000975:Antioxidants; D002518:Ceramides; D008565:Membrane Proteins; D009025:Morpholines; C544832:N-methyl-5-(4'-pentylphenyl)-2-aminopent-4-ene-1,3-diol; D009363:Neoplasm Proteins; D013107:Sphingolipids; D013267:Stilbenes; D025521:Tumor Suppressor Proteins; C033110:RV 538; D053148:Caspase 3; D000077185:Resveratrol
FEBS Lett. 2006 Oct 9;580(23):5467-76
16970943
Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):4840-4
1317574
Leukemia. 2000 Aug;14(8):1362-70
10942230
Int J Cancer. 2001 Oct 15;94(2):157-65
11668492
Cell Death Differ. 2007 Oct;14(10):1780-91
17627285
Biochim Biophys Acta. 2006 Dec;1758(12):2096-103
17010304
Biochem J. 2005 Aug 15;390(Pt 1):263-71
15823095
Methods Enzymol. 2000;322:378-82
10914032
IUBMB Life. 2008 May;60(5):323-32
18421779
Nan Fang Yi Ke Da Xue Xue Bao. 2008 Aug;28(9):1727-30
18819906
J Biol Chem. 2000 Mar 24;275(12):9078-84
10722759
Cell Mol Biol (Noisy-le-grand). 2003 Nov;49(7):1017-23
14682383
J Biol Chem. 2003 Oct 31;278(44):43452-9
12912983
J Pharmacol Exp Ther. 2006 Jun;317(3):1188-99
16510697
Bioorg Med Chem. 2006 Nov 1;14(21):7083-104
16919460
Int J Mol Med. 2005 Nov;16(5):905-10
16211262
Cancer Cell. 2006 Mar;9(3):225-38
16530706
Leuk Res. 2008 Mar;32(3):475-80
17709137
J Biol Chem. 2007 Apr 13;282(15):10922-34
17303574
J Biol Chem. 1997 Apr 11;272(15):9609-12
9092485
Nat Rev Drug Discov. 2006 Jun;5(6):493-506
16732220
Hematology. 2009 Jun;14(3):139-44
19490758
Bioorg Med Chem. 2008 Jan 15;16(2):1032-45
17881234
J Cancer Res Clin Oncol. 2009 Jan;135(1):81-90
18560890
Turk J Haematol. 2007 Sep 5;24(3):102-9
27263767
J Biol Chem. 1999 Jan 8;274(2):1140-6
9873062
J Agric Food Chem. 2008 Aug 27;56(16):7572-7
18656932
J Pharmacol Exp Ther. 2006 Aug;318(2):596-603
16632640
Int J Biochem Cell Biol. 2007;39(6):1063-70
17468032
J Biol Chem. 1994 Jun 3;269(22):15451-9
8195187
Arch Biochem Biophys. 2007 Jun 15;462(2):171-5
17467652
Nat Rev Cancer. 2004 Aug;4(8):604-16
15286740
J Lipid Res. 2005 Jun;46(6):1229-38
15772421
J Natl Cancer Inst. 2001 Mar 7;93(5):347-57
11238696
J Biol Chem. 2002 Sep 20;277(38):35642-9
12105227
Hematology Am Soc Hematol Educ Program. 2006;:156-61, 514
17124055
J Biochem Mol Biol. 2006 Mar 31;39(2):113-31
16584625
J Biol Chem. 2006 Sep 1;281(35):25001-5
16793762
Biochim Biophys Acta. 2003 Sep 22;1633(3):161-9
14499735
J Biol Chem. 1998 May 1;273(18):11025-31
9556584
Science. 1997 Jan 10;275(5297):218-20
8985016
J Pharmacol Exp Ther. 2004 May;309(2):523-32
14742741