Antisense Oligonucleotides Targeting XIAP Induce Apoptosis And Reverse Drug Resistance Of Human Promyelocytic Leukemia Cells (HL-60)

In the first section of the paper, we investigated the effect of XIAP (X-linked inhibitor of apoptosis protein) down-regulation by antisense oligonucleotides (AS ODNs) on the growth of human promyelocytic leukemia cell line (HL-60) in vitro. Fully phosphorothioated AS ODNs was synthesized and transiently transfected into HL-60 cells with Lipofectamine^TM2000 in the form of liposome-ODN complexes. Growth inhibition of HL-60 cells was determined by the colorimetric CCK-8 cell viability/proliferation assay. XIAP mRNA and protein were measured by Reverse transcription-PCR and Western blot, respectively. Mitochondrial membrane potential was assessed using JC-1.Cellular caspase-3 activities were measured by colorimetric method. Apoptosis was observed by flow cytometry with Annexin-V/PI dual staining. The results showed that (1) AS ODNs targeting XIAP displayed a strong growth inhibitory effect on HL-60 cells in a dose- and time-dependent manner.(2)AS ODNs targeting XIAP down-regulated the expression of XIAP in both mRNA and protein levels. It down-regulated XIAP mRNA by 77.78% and protein levels up to 73.33%, respectively. (3) The apoptosis rate of AS ODNs was 52.73%, which was significantly higher than that of mismatch oligonucleotides(11.99%)( P <0.05).(4) XIAP down-regulation resulted in caspase-3 activation. (5) Mitochondrial membrane potential of HL-60 cells was decreased by AS ODNs, as indicated by a fluorescence emission shift from red to green. (6) AS ODNs sensitized HL-60 cells to doxorubicin. When antisense oligonucleotides (900 nM) was combined with doxorubicin(0.5 μg/ml), approximately 86.57% of the cells were killed which was significantly higher than either oligonucleotide or doxorubicin used alone (56.59% and 63.05% respectively, P <0.05).The results indicated that XIAP was involved in the proliferation and apoptosis of leukemic cells. Down-regulation of XIAP might provide a new strategy for inhibiting the proliferation and inducing apoptosis of leukemic cells and enhancing the sensitivity of leukemic cells to chemotherapy.In the second section of the paper, we explored the role of XIAP in the fibronectin-adhesion mediated drug resistance of HL-60 cells. And we investigated whether XIAP down-regulation by AS ODNs could reverse drug resistance. Immunosorp plates were coated with fibronectin (FN) and plates coated with bovineserum albumin (BSA) were used as control. The colorimetric CCK-8 cell viability/proliferation assay was used to determine the effects of FN on the cytotoxicity of DNR to HL-60 cells. Intracellular DNR accumulation was assayed with flow cytometry. Reverse transcription-PCR and Western blot were applied to examine the expression of XIAP, BCL-2, MRP and MDR1 of mRNA and protein levels, respectively. Fully phosphorothioated AS ODNs and MS ODNs of XIAP were delivered into HL-60 cells cocultured with fibronectin with Lipofectamine^TM2000 in the form of liposome-ODN complexes. IC50 was calculated by linear regression of percent survival versus drug concentration. Reverse transcription-PCR and Western blot were applied to examine the expression of XIAP of mRNA and protein levels, respectively. The results showed that HL-60 cells adhered to FN-coated plates have a significant survival advantage over those grown on BSA and in suspension when exposed to DNR. The IC50 of FN group was significantly higher than that of BSA group and suspension group (5.26umol/L vs 1.32umol/L, 1.23umol/L, respectively, P<0.05). Flow cytometry analysis with Annexin-V/PI dual staining showed that the apoptotic cell ratio of FN group was significantly lower than that of BSA group and suspension group (53.71% vs 81.68%, 85.90%, respectively, P <0.05). Reverse transcription-PCR and Western blot showed that XIAP was up-regulated significantly in FN group when compared with BSA group and suspension group(P <0.05),whereas there was no significant difference in the expression of BCL-2, MRP and MDR1 among the three groups (P >0.05) . A flow cytometry-based intracellular drug accumulation assay revealed that the concentration of DNR in FN-adhered HL-60 cells was similar to that in BSA group and suspension group (P <0.05). AS ODNs of XIAP down-regulated the expression of XIAP both of mRNA and protein levels in FN-adhered HL-60 cells. In addition, AS ODNs sensitized HL-60 cells to the cytotoxic effects of doxorubicin. The results indicated that increased XIAP protein levels induced by adhesion to FN contributed to drug resistance. And the results above might provide the foundation for reversal drug resistance mediated by FN with AS ODNs of XIAP.In the third section of the paper, to explore the mechanisms of drug resistance of HL-60/ADR cells, we compared the reversal effects of AS ODNs of XIAP and AS ODNs of MRP used alone and in combination. Reverse transcription-PCR and Western blot were applied to examine the expression of XIAP, BCL-2, MRP and MDR1 of mRNA and protein levels in HL-60 cells and HL-60/ADR cells,respectively. Fully phosphorothioated AS ODNs of XIAP and MRP was delivered into HL-60/ADR cells with Lipofectamine^TM 2000 in the form of liposome-ODNs complexes alone or in combination. CCK-8 cell viability assay was used to determine the effect of AS ODNs of XIAP and MRP used alone or in combination on the chemotherapy sensitivity of HL-60/ADR cells to DNR. Reverse transcription-PCR and Western blot were applied to examine the changes of XIAP and MRP of mRNA and protein levels, respectively. The results showed that MRP and XIAP were both significantly higher in HL-60/ADR cells than that in HL-60 cells. AS ODNs of XIAP and MRP could down-regulate the expression of XIAP and MRP in HL-60/ADR cells and increase the sensitivity of HL-60/ADR cells to DNR, respectively. AS ODNs of XIAP+MRP did not enhance the inhibition expression of XIAP of HL-60/ADR cells but increased the sensitivity of HL-60/ADR cells to DNR significantly compared with AS ODNs of XIAP (P <0.05). AS ODNs of XIAP+MRP did not increase the concentration of DNR or enhance the inhibition expression of MRP of HL-60/ADR cells but increased the sensitivity of HL-60/ADR cells to DNR significantly (P <0.05) compared with AS ODNs of MRP. The results indicated that both XIAP and MRP were involved in the drug resistance mechanisms of HL-60/ADR cells. Drug-resistance of HL-60/ADR cells could be reversed significantly when antisense phosphorothioate oligonucleotides of XIAP and MRP used in combination.