| The induction of apoptosis by chemical agents in cancer cells would be desirable. It has been recognized that the majority of current chemotherapeutic agents induce tumour cell apoptosis, and scientists are testing agents signalling pathway to programmed cell death for the purpose of finding more effective anticancer compounds.There are many reports about fatty acids , including the butyric acid (a short-chain fatty acid), Arachidonic acid(AA), eicosapentaenoic acid(EPA), docosahexaenoic acid(DHA) (polyunsaturated fatty acids), and analogues of retinoic acid(fatty acid-containing), which could induce apoptosis. 13-Methyltetradecanoic Acid(13-MTD) which we studied in this report is a terminally branched-chain saturated fatty acid, with a main-chain contains 14 carbons. The chemical structure is unique among the death inducing fatty acids. By the year 2000, Yang primarily reported 13-MTD could induce apoptotic cell death of several huamn cell lines in vitro, and growth inhibition of human prostate carcinoma cell lines DU145 and hepatocarcinoma LCI-D35 in orthotopic mouse, models. In this report , we studied the initiation of growth arrest and apoptosis of HL60 human promyelocytic leukemia cells by 13-MTD, and the signal pathway of apoptosis.1. Induction of HL60 Cell apoptosis by 13-MTD13-MTD induced growth arrest in a time and concentration-dependent manner in HL60 cells, 13-MTD at 10 u.g/ml inhibits the cell growth, 13-MTD at 20 ug/ml and 30 ug/ml were found to be cytotoxic for the cell line, and the IC50 is about 20.5 ug/m at 24 h drug treatment. The induction of cell death by 13-MTD was affected by bovine albumine protein in a low fetal bovine serum(2.5%) culture medium, the 13-MTD effects was inhibited by the bovine albumine protein fraction V in a dose-dependent manner.The morphological changes of treated cells were directly analyzed under the fluorescence microscope, using dual staining with acridine orange and ethidium bromide. With 20ug/ml 13-MTD treatment for 2-4 h , cells broke up into small bodies which were negative staining against EB. Staining with Giemsa , blebling of cell membrane, condenced chromatin and cytoplasmic vacuoli were observed in most drug treatment cells.To determine whether apoptosis occurred during growth inhibituon, DNA fragmentation analysis and cell cycle analysis were performed. Agarose gel electrophoresis demonstrated DNA laddering, typical of apoptosis, following 12 h and 24 h of 13-MTD treatment at a concentration of 20ug/ml. Using flow cytometfy cell cycle analysis, following 4 h, 8 h and 12 h of 13-MTD treatment, revealed the increasing presence of a sub-G| apoptotic peak. Western blotting analysis showed the executioner caspase-3 protein decreased after 6 h treatment of 13-MTD, and the active subunit was detectable at 12 h.To study the relationship between the chemical structure and cell death effect, we synthesized a series of analogues of 13-MTD. The results suggested that the terminal methyl branch was necessary to induce cell death, and the effect diminished when the main chain was more than 18carbons.2. Effects of 13-MTD on Bax, BcI-2 expression and the mitochondria! functionApoptotic cell suicide can be initiated by a plenty of stimuli that generally feed into one of the two well known cell death signaling pathways. One pathway include the activation of the CD95 system, agents triggering the CD95 receptor(CD95,also called Fas or Apol) results in the protelytic activation of initiator caspase-8 and then the protelytic activation of various downstream caspase(caspase3,6), ultimatly resulting in DNA fragmentation. Another pathway feeds cell death signals through the mitochondria. Diverse apoptotic stimuli, including UVB, etoposide(VP-16), retinoic acid, staurosporine and polyunsaturated fatty acids induce intermembranous cytochrome C(Cyt-c)and other apoptotic induing factors release. Bcl-2 family members proteins regulate the Cyt-c release, and cytosolic Cyt-c binds to adaptor protein Apaf-1 (apoptotic protease act...
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