| Malignant tumor is one of the main causes of human beings’ death. The statistics report suggested that the cancer mortality has been raised over cerebrovascular disease. Cancer is the leading cause of death in China. The Leukemia is a malignant disease of blood cell-forming tissue, also known as "cancer of the blood". Chemotherapy combined with pharmaceutical medication are the typically strategy to treat leukemia. However, chemotherapy causes a number of undesirable side effects in the host. Therefore, new anti-cancer drugs with higher bioactivities and less or without side effects from nature appear to be a great significance.Recently, many polysaccharides have been isolated from fungi, yeasts, plants, lichens and algae. Amounts of attention have focused on the biological activities of polysaccharides due to their immunomodulatory and antioxidant effects in the biochemical and medical areas. Increasing lines of evidence demonstrate that the polysaccharides could induce cancer cells apoptosis, whereas less toxic to normal cells. Drugs promoting cancer cells apoptosis may be an effective and important strategy to counteract cancer. Therefore, it is great meaningful to find the polysaccharide which possesses the splendid activity of inducing apoptosis in oncotherapy and adjuvant therapy.Our group has isolated and purified the EPS-1(exopolysaccharide) and EPS-2from the fermentation broth of Trichoderma pseudokoningii. The average molecular weight of EPS-1was31930Da measured by gel permeation chromatography (GPC). The component sugars of EPS-1, determined by GC, were rhamnose, xylose, fucose, mannose, glucose, and galactose and with molar ratios of16.2:14.4:1:25.8:23.6:48.1. The average molecular weight of EPS-2was18325Da. The component sugars of EPS-2were rhamnose, galactose and galactose and with molar ratios of5.6:2.7:1.0. Based on our earlier finds, EPS-1and EPS-2were showed to enhance the immunity system. In the current research, we have investigated the anticancer activity of EPS-1and EPS-2against cancer cells, and possible apoptosis mechanism of K562 induced by EPS-1was also studied.1. To evaluate the growth inhibitory by EPS-1and EPS-2, the K562, HL-60, SGC-7901cells were treated with increasing concentrations of EPS-1and EPS-2for24,48,72h and cell viability was measured by SRB assay. The results showed that EPS-1and EPS-2inhibited the growth of those cells in a time-and concentration-dependent relative to control cells. K562cells were treated with1mg/mL EPS-1for72h, the inhibition rate approximately to41%, highest among the inhibition rates.2. The results showed the significant morphological changes induced by EPS-1in the nuclear chromatin, expressed cell shrinkage, chromatin compaction, condensation of cytoplasm and nuclear fragmentation. In the untreated groups, the nuclei have similarly sized, regularly shaped with control, and evenly stained (lightly stained). However, condensed chromatin (brightly stained) could be observed in treated cells, and some of them formed the structure of apoptotic bodies, which is one of the typical characteristics of apoptotic cells. The results indicated that EPS-1inhibited the growth of cancer cells by induced apoptosis. Further confirmation of EPS-1induced apoptosis was obtained by using flow cytometry with Annexin V-FITC/PI staining. The results showed that the population of pro-apoptotic cells (lower right) significantly elevated in EPS-1treated cells from0.1%in untreated cells to20.5%. The results indicated that PS extern alizati on and necrotic cells were evident in K562cells treated with EPS-1. These data provided support for EPS-1induced apoptosis contributes to the growth inhibition of K562cells.3. In order to investigate the apoptosis mechanism of K562induced by EPS-1, a fluorescent carbocyanine dye,5,5’,6,6’-tetrachloro-1,1’,3,3’tetraethylbenzimi-da zolocarbocyanine iodide, also called JC-1, was used as a probe to measure the mitochondrial membrane potential (△ψm) during the early stages of apoptosis. ROS was determined with2’,7’-dichlorofluorescein diacetate (DCFH-DA) and the concentration of Ca2’ was detected by Fluo-3/AM. The results showed that EPS-1reduced mitochondrial membrane potential, boosted generation of ROS and increased concentration of intracellular calcium from the intracellular Ca2+pool, which implied that EPS-1was an apoptotic stimulus and could impact the mitochondria of the cells.4. To gain further information on the mechanism of apoptosis induced by EPS-1, the expression of p53, Bcl-2, Bax, FasL and Fas mRNAs were examined in K562cells. By studying the results of RT-PCR, we observed that the expression of Bcl-2was down-regulated, but the level of p53and Bax mRNA were up-regulated, compared to the control. Therefore, the possibly mechanism of EPS-1induced apoptosis in K562cells by the increase of p53and the low ratio of Bcl-2to Bax modulated. The expression of FasL and Fas mRNAs were also up-regulated, compared to the control, which means the death-receptor apoptosis signaling pathways involved the apoptosis induced by EPS-1.The results suggested the mechanism of cell apoptosis induced by EPS-1was mainly mediated by the intrinsic mitochondnal apoptotic pathway and death-receptor apoptosis signaling pathway. All our findings provide a new clue for the drug therapy of human leukemia, provide data and theoretical support for application of EPS-1as a new adjuvant chemotherapeutic and chemo preventive strategy against human leukemia. |
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