Mechanisms Of The Antiapoptosis Of Plasma Membrane-Associated Sialidase Neu3 And MDR Of Tumor Cells

Sialidase is a kind of glycosidase,which can hydrolyzing sialic acid from non-deoxidized end glycogen and glycolipid。Human sialidases are divided three kinds, among which human membrane-associated membrane (Neu3) is more important. Neu3 is unique in specifically hydrolyzing gangliosides, thought to participate in cell differentiation and transmembrane signaling, thereby playing crucial roles in the regulation of cell surface functions. Many recent studies indicate that high expression of Neu3 in cancer cells leads to protection against programmed cell death, probably modulation of gangliosides. Therefore further investigation may provide a possible sialidase target for diagnosis and therapy of prostate cancer.[Objective]1. PC-3-C1-Neu3 cell line stablely transfected with Neu3 cDNA was exposed to DNR, IL-6 and NeuAC2en and induced apoptosis. We could identify the effects of Neu3 upon apoptosis of prostate cancer from the series of experiments.2. To investigate the impossible mechanism of the impact of Neu3 on apoptosis, some factors and protein concerned with signal transduction pathway of mitochondria apoptosis would be detected.3. The correlation between Neu3 and MDR would be detected.[Methods] 1. The mRNA and protein level of Neu3 of PC-3, PC-3-C1 and PC-3-C1-Neu3 cells was investigated through semi-quantitative RT-PCR analysis and western blot.2. The survival rate of PC-3, PC-3-C1 and PC-3-C1-Neu3 cells treated by drugs was detected by MTT assay.3. The activity of Neu3 of PC-3, PC-3-C1 and PC-3-C1-Neu3 cells treated by drugs was analyzed with the specific substrate reaction.4. Apoptotic rate was determined of PC-3-C1 and PC-3-C1-Neu3 treated by drugs by flow cytometry, DNA ladder and LDH assay. Neu3 of PC-3, PC-3-C1 and PC-3-C1-Neu3 cells was investigated through semi–quantitative RT-PCR analysis and western blot.5. The expression mRNA and /or protein level of Bcl-2, Bcl-xl, Bax, Capase-3, Caspase-9, Neu3, MDR, MRP and p65 of PC-3-C1 and PC-3-C1-Neu3 cells treated by drugs was investigated through semi-quantitative RT-PCR analysis and western blot.6. The level of intracellular ROS of PC-3, PC-3-C1 and PC-3-C1-Neu3 cells treated by drugs was examined with DCFH-DA staining by confocal microscopy and flow cytometry.7. The mitochondria membrane potential change of PC-3-C1 and PC-3-C1-Neu3 cells treated by drugs was investigated with JC-1 staining by flow cytometry.8. The level of Bcl-xl and Bax in cytosome and motochondria of PC-3-C1 and PC-3-C1-Neu3 cells treated by drugs was examined respectively through Western blot.9. The distribution and level of GD3 of PC-3 cells treated by drugs was analyzed by immunofluresence.10. The survival rate of K562 and k562/ADM treated by drugs was detected by MTT assay.11. The activity of Neu3 of of k562 and k562/ADM cells treated by drugs was analyzed with the specific substrate reaction.12. The mRNA expression level of MDR1,MRP,GMGT,GST,Neu3 of k562 and k562/ADM cells treated by drugs was analyzed by semi-quantitative RT-PCR .The level of protein of P-gp and Neu3 was analyzed by Western blotting.[Results]1. The mRNA and protein level of Neu3 increased obviously in PC-3-C1 cells, compared with PC-3 and PC-3-C1 cells.2. DNR inhibited the proliferation of PC-3,PC-3-C1 and PC-3-C1-Neu3 cells(P<0.05); IL-6 and NeuAC2en applying alone had no obvious effects on cell growth(P>0.05); The synergism between NeuAC2en and DNR was confirmed, and IL-6 could counteract the effecets of DNR; The survival rate of PC-3-C1-Neu3 cells was higher than that of PC-3-C1 cells under identical treatment condition.3. The activity of Neu3 of PC-3-C1-Neu3 cells was higher than that of PC-3 cells and PC-3-C1 cells under identical treatment condition(P<0.05); The activity of Neu3 of three kinds of cells decreased obviously treated by NeuAC2en and/or DNR compared with respective control group(.P<0.05). The synergism between NeuAC2en and DNR was confirmed, and IL-6 could counteract the effects of DNR;4. Apoptosis of PC-3-C1和PC-3-C1-Neu3 cells could be induced by DNR. (P<0.05); IL-6 and NeuAC2en applying alone had no obvious effects on cell growth(P>0.05); The synergism between NeuAC2en and DNR was confirmed, and IL-6 could counteract the effects of DNR; The apoptosis rate of PC-3-C1-Neu3 cells was higher than that of PC-3-C1 cells under identical treatment condition, but cell necrosis rate have no obvious difference (P>0.05).5. The mRNA level of bcl-xl decreased and bax increased in two kinds of cells with treated by DNR; Compared with PC-3-C1 cells, the mRNA level of P65, MDR1, Bcl-xl and bcl-2 of PC-3-C1-Neu3 increased, at the same time the mRNA expression of caspase-9, caspase-3 and bax decreased under identical treatment condition; NeuAC2en can enforce DNR and IL-6 inhibit it.6. PC-3 cells treated by DNR demonstrated stronger green fluorescence contrasting control group, and PC-3 cells treated by IL-6 and NeuAC2en alone. Strongest green fluorescence was produced through DNR and NeuAC2en coordination. Under identical treatment condition, fluorescence intensity of PC-3-C1-Neu3 was lower than that of PC-3-C1 Cells.7. The mitochondrial potential of PC-3-C1和PC-3-C1-Neu3 cells treated by DNR was reduced than that of respective control group. Mitochondria membrane potential of PC-3-C1-Neu3 cells was higher than PC-3-C1 cells Under identical treatment conditions. NeuAC2en can enforce DNR effects.8. Compared with control group respectively, the protein expression of Bcl-xl and Bax decreased in cytoplasm and increased in mitochondria of PC-3-C1和PC-3-C1-Neu3 treated by DNR. The level of Bcl-xl protein was higher and the level of Bax protein was lower in both cytoplasm and mitochondria of PC-3-C1-Neu3 cells than those of PC-3-C1 cells under identical treatment conditions.9. PC-3 cell membrane presented green fluorescence ;The cells treated by IL-6 expressing green fluorescence was less; Green fluorescence of PC-3 cells treated by DNR and/or NeuAC2en expressed increase; TNF-αcould induce GD3 translocate.10. The survival rate of k562/ADM cells is higher than that of k562 cells(P<0.05);Neu5AC2en can enforce DNR.11. The activity of the sialidase of k562/ADM is higher than control group;12. Compared with K562 cells, the mRNA level of MDR1 and Neu3 under identical treatment conditions.[Conclusion]1. Neu3 could resist prostate cancer cell apoptosis induced by DNR through regulating the ratio Bcl-xl/bax and Bcl-2/bax and reduce the level of caspases. It suggested that mitochondria apoptosis pathway could be involved in the mechanism of Neu3 anti- apoptosis.2. Neu3 could stabilize the mitochondrial membrane potential, repress the burst of ROS and reduce the proportion of bcl-xl/bax to resist apoptosis, but Neu3 have no effect on translocation of bax and bcl-xl.3. Neu3 could reduce GD3 expression.Neu3 have no effect on translocation of GD3 when apoptosis was induced.4. Neu3 may be related with MDR of k562 and k562/ADM...