AIM:To study the mechanism of oleanolic acid induced apoptosis and its influence on cell cycle in HL-60 cells in vitro.METHODS:The HL-60 cells were treated with different concentrations of oleanolic acid and then cultured for 12 h,24 h,48 h and 72 h,respectively.MTT assay was used to evaluate the inhibitory effect of oleanolic acid on HL-60 cells in vitro.The argarose gel electrophoresis was used to detect the chromatin DNA fragmentation.FACS was used to analyze the cell cycle of HL-60 cells.Western blotting was used to detect the activation of caspase-3 which has been confirmed the last execution of apoptosis pathway.RESULTS:MTT assay showed that oleanolic acid dramatically inhibited the growth of HL-60 cells in vitro,more than 50% HL-60 cells were inhibited when the cells were treated with 80 ?mol/L oleanolic acid for 48 h;the apparent DNA ladder was detected after exposure of HL-60 cells to oleanolic acid for 48 h.FACS analysis showed that cell cycle of HL-60 cells was arrested in G1 phase,the inhibition ratio of HL-60 cells achieved 63.24% and 67.90% after treated with oleanolic acid for 48 h and 72 h correspondingly.Western blotting detected the activation of caspase-3 after exposure of HL-60 cells to 80 ?mol/L oleanolic acid for 48 h.CONCLUSION:These results suggest that oleanolic acid induces apoptosis and the cell cycle of HL-60 cells is arrested in G1 phase.

Three long-chain polyunsaturated fatty acids, docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), are the most biologically active polyunsaturated fatty acids in the body. They are important in developing and maintaining the brain function, and in preventing and treating many diseases such as cardiovascular disease, inflammation and cancer. Although mammals can biosynthesize these long-chain polyunsaturated fatty acids, the efficiency is very low and dietary intake is needed to meet the requirement. In this study, a multiple-genes expression vector carrying mammalian A6/A5 fatty acid desaturases and multiple-genes expression vector carrying mammalian Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases coding genes was used to transfect HEK293T cells, then the overexpression of the target genes was detected. GC-MS analysis shows that the biosynthesis efficiency and level of DHA, EPA and ARA were significantly increased in cells transfected with the multiple-genes expression vector. Particularly, DHA level in these cells was 2.5 times higher than in the control cells. This study indicates mammal possess a certain mechanism for suppression of high level of biosynthesis of long chain polyunsaturated fatty acids, and the overexpression of Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases broke this suppression mechanism so that the level of DHA, EPA and ARA was significantly increased. This study also provides a basis for potential applications of this gene construct in transgenic animal to produce high level of these long-chain polyunsaturated fatty acid.
Acetyltransferases ; genetics ; metabolism ; Arachidonic Acid ; biosynthesis ; Docosahexaenoic Acids ; biosynthesis ; Eicosapentaenoic Acid ; biosynthesis ; Fatty Acid Desaturases ; genetics ; metabolism ; Fatty Acid Synthases ; genetics ; metabolism ; Fatty Acids, Unsaturated ; biosynthesis ; Genetic Vectors ; HEK293 Cells ; Humans ; Transfection

DHA (22:6n-3) is a Ω-3 polyunsaturated fatty acid with 22 carbon atoms and 6 double bonds, which has important biological functions in human body. Human and other mammals synthesize only limited amounts of DHA, more requirements must be satisfied from food resources. However, the natural resources of DHA (Mainly deep-sea fish and other marine products) are prone to depletion. New resources development is still insufficient to satisfy the growing market demand. Previous studies have revealed that the mammals can increase the synthesis of DHA and other long-chain polyunsaturated fatty acids after transgenic procedures. In this study, mammalian cells were transfected with Δ6, Δ5 desaturase, Δ6, Δ5 elongase, Δ15 desaturase (Isolated from nematode Caenorhabditis elegans) and Δ4 desaturase (Isolated from Euglena gracilis), simultaneously. Results show that the expression or overexpression of these 6 enzymes is capable of conversion of the o-6 linoleic acid (LA, 18:2n-6) in DHA (22:6n-3). DHA content has increased from 16.74% in the control group to 25.3% in the experimental group. The strategy and related technology in our research provided important data for future production the valuable DHA (22:6n-3) by using genetically modified animals.
Animals ; Caenorhabditis elegans ; enzymology ; Cells, Cultured ; Docosahexaenoic Acids ; chemistry ; Euglena gracilis ; enzymology ; Fatty Acid Desaturases ; biosynthesis ; Linoleic Acid ; chemistry ; Mammals ; Transfection