An Exopolysaccharide From Antarctic Marine Bacteria Pseudoaltermonas Sp.s-5and Its Apoptotic Activity On Human Leukemia Cells

Objective:To observe the effect of exopolysaccharide from Antarctic bacteriaPseudoaltermonas sp.S-5on the proliferation and the possible apoptosis mechanism inhuman leukemia cells.Methods:1. The effect of PEP (exopolysaccharide) has been evaluated in humanleukemia cells.The K562cells were treated with increasing concentration of PEP for24,48,72h and tested cell proliferation in vitro by SRB (Sulforhodamine B) assay.2. To observe whether the K562cells start to have some changes in nuclear stainingafter exposed to PEP. Nuclear morphology was observed and photographed under afluorescence microscope.3. Apoptosis rate were analyzed by flow cytometer after double-labeled fluorescentstaining (Annexin V, PI).4. Membrane potential changes were examined by flow cytometer after JC-1labeled.5. To evaluate the expression of caspase-3and caspase-9, the caspase testing kit wasused.6. To measure the changes in intracellular calciumion in K562cells after exposed toPEP for24h,the fluorescence microscope and the flow cytometer was used after labeledwith Fluo-3AM. 7. Expression of apoptosis-related proteins was detected by Western Blotting method.Results:1. PEP showed in vitro inhibition of K562cells proliferations in aconcentration-dependent compared with control cells. It was obviously that the growthof K562cells was significantly inhibited, with the growth inhibition ratio of11.49-46.16%after treated with increasing concentration of PEP for72h. Treatmentwith0.6mg/mL PEP for24h、48h、72h, the inhibition rates were18.78%、39.34%and46.16%respectively.2. PEP-treated cells showed significant changes in cell nucleus. The nucleus appeared tobe slightly smaller and looked brighter than the control.3. Annexin V and PI double staining results showed that PEP can promote apoptosis ofhuman leukemia K562cells.4. The mitochondrial membrane potential in K562cells was declined after treated withPEP for24h.5. The activity of caspase-3and caspase-9were significantly enhanced compared withthe control group.6. Intracellular calcium was increased compared with the control group after exposed toPEP for24h.7. Western Blotting results showed that the expression of pro-apoptotic protein (Bax)was upregulated and the protein of inhibiting apoptotic (Bcl-2) was down.Conclusion: PEP possessed significant tumor inhibition effect on K562cells in aconcentration-dependent manner. PEP could inhibit the growth of K562cells byinduction of apoptosis. The most probably mechanisms of PEP induced apoptosis inK562cells involved in the mitochondria pathway.