Study On The Apoptosis Of K562 Cells Treated With Antisense Oligonucleotide Targeting Survivin And Etoposide

Objective: To study the apoptosis of leukemia cells induced byantisense oligonucleotide targeting survivin, and investigate whetherantisense oligonucleotide can sensitize leukemia cells to chemotherapy. Method: 1. the expression of survivin in K562 cells after treatedwith antisense oligonucleotide. Oligonucleotides were delivered in the formof complexes with Lipofectin. K562 cells were divided into four groups:ASON group,NSON group,lipofectin group,and control group. Theexpression of survivin was examined by immunohistochemistry(SABC).2. The apoptosis of K562 cells effected by antisense oligonucleotide. TheK562 cells were also divided into four groups: ASON group,NSONgroup,lipofectin group,and control group. We analysised the expression ofcaspse-3 by immunohistochemistry(SABC) in the four groups, and then wedetected the apoptotic rate of each group with two methods: TUNELassay and Annexin V-FITC assay. 3. Detect the inhibition ratio ofK562 cells treated with etoposide(VP16). Analysis the inhibition ratio ofK562 cells exposed to etoposide in different concentration by MTT assay,to decide which concentration can be used to the next test: combinationwith antisense oligonucleotide targeting survivin and etoposide. 4. Toinvestigate whether down-regulation of survivin expression has thepotential to sensitize K562 cells to chemotherapy, a combination treatmentwith antisense oligonucleotide and etoposide was performed. K562 cellswere divided into four groups: combination group,ASON group, etoposidegroup,and control group. We also detected the apoptotic rate of each groupwith two methods: TUNEL and Annexin V-FITC. Result: 1.The protein of survivin is overexpress in K562 cells ,butafter treated with antisense oligonucleotide, the level of expression wasdown regulation. 2. 24h later, after the start of transfection. the expressionof caspase-3 of ASON group was upgrade contrast to other three controlgroups(p<0.05). And 48h later, that expression of ASON groups washigher than that of 24h. The apoptotic rates detected by TUNEL assay ofK562 cells in ASON group was 22% at 24 h and 32% at 48 h after the startof transfection ,those were higher than other three groups. We also usedAnnexin V-FITC assay to detected the apoptotic rates of all the fourgroups at 24 h after the start of transfection and rate in ASON group weobtained was about 19%, it was similar to that of TUNEL assay. 3. Theinhibition ratio of K562 cells by etoposide was concentration dependent,the concentration higher, the inhibition ratio higher. Combination withinhibition ratio and morphology, we found that the concentration ofetoposide 5ug/ml fit to the next step. 4. When K562 cells were exposed toASON and etoposide at the same time, the apoptotic rate was obviouslyhigher than exposed to ASON or etoposide alone(p<0.05), test by TUNELand Annexin V-FITC assay. Conclusion: 1. Antisense oligonucleotide targeting survivin can downregulate the protein expression of survivin. 2. Survivin gene is related tothe apoptosis of K562 cells, and ASON targeting survivin can induceK562 cells to apoptosis. 3. Etoposide can inhibit the growth of K562 cells,and it is concentration dependent. 4. ASON targeting survivin cansensitize K562 cells to chemotherapeutic agent etoposide.