OBJECTIVE:To investigate the effect of retrovirus mediated heme oxygenase (HO)-1 gene on chronic myeloid leukemia (CML) resistance cell apoptosis induced by nilotinib (AMN107).METHODS:High titer viral particles of pQCXIP-EGFP-HO-1 were prepared, and K562/A02 cells stably transfected with HO-1 gene was established. The expression of HO-1 in K562/A02 cells was detected by RT-PCR. After treated with AMN107 for 24 h, HO-1 mRNA and protein expression by RT-PCR and Western blot, respectively; Cell proliferation by MTT assay; bcr-abl fusion gene by RQ-PCR, and the apoptosis and cell cycle by flow cytometry.RESULTS:Recombinant retrovirus vector was constructed successfully and K562/A02/HO-1 cells were successfully set up. The expression of HO-1 in K562/A02 cells was expressed clearly. After three groups cells treated with AMN107 for 24 h, the expression of HO-1 mRNA and protein was significantly higher in gene-transfected group than in either empty vector or no-transfected group. The difference was statistically significant (P < 0.05). The cell proliferation ofs was inhibited, but the cell viability was significantly higher in gene-transfected group than in other two groups. The difference was statistically significant(P < 0.05); After treated with 10 µmol/L AMN107 for 24 h, the CT values of bcr-abl fusion gene were (18.15 ± 0.18) in K562/A02/HO-1 group, being significantly higher than that in K562/A02/LXSN (20.32 ± 0.20) and K562/A02 (20.51 ± 0.21) group, the difference was statistically significant (P < 0.05); the apoptosis rate were (17.26 ± 0.23)%, (39.47 ± 0.17)%, and (41.84 ± 0.09)%, respectively in three groups, and were (3.74 ± 0.03)%, (5.91 ± 0.08)% in K563/A02/HO-1 untreated with drug and K562/A02 untreated with drug group. The number of G(0)/G(1) phase and S phase cells markedly decreased. The cells were arrested in G(2)/M phase. But cell cycle in gene-transfected group did not change significantly.CONCLUSION:AMN107 inhibits proliferation of CML resistance cells and induces cell apoptosis. HO-1 gene can protect CML resistance cells to apoptosis. There was a relationship between HO-1 gene and the growth of CML resistance cells.

目的 研究逆转录病毒介导的血红素加氧酶-1(HO-1)基因在尼洛替尼(Nilotinib,AMN107)诱导慢性髓性白血病(CML)耐药细胞凋亡中的作用.方法 制备高病毒滴度的逆转录病毒载体pQCXIP-EGFP-HO-1,建立稳定转染HO-1基因的K562/A02细胞.采用RT-PCR鉴定K562/A02细胞中HO-1基因的表达.RT-PCR和Western blot法检测AMNl07作用24 h后K562/A02细胞中HO-1 mRNA和蛋白的表达,采用MTT法观察细胞增殖变化,通过实时荧光定量PCR(RQ-PCR)法检测bcr-abl融合基凶的表达.通过流式细胞术检测细胞凋亡和细胞周期分布.结果 成功构建重组逆转录病毒载体,并建立稳定转染HO-1基因的K562/A02细胞系;证实转基冈组细胞HO-1 mRNA显著表达.AMN107作用3组细胞后,转基因组细胞HO-1 mRNA和蛋白的表达明显高于转空载体组和未转染组,差异有统计学意义(P<0.05);MTT法检测显示AMN107处理后细胞增殖受抑.而转基因组细胞存活率明显高于转空载体组和未转染组,差异有统计学意义(P<0.05);RQ-PCR法检测结果显示,10 ILLmol/L AMNl07抑制bcr-abl基因的表达,但转基因组的bcr-abl基因表达水平(Ct值18.15±0.18)高于转空载体组(20.32±0.20)和未转染组(20.51±0.21),差异有统计学意义(P<0.05);流式细胞术检测结果显示10 μmol/L AMNl07作用24 h后,转基因组、转空载体组、未转染组细胞凋亡率分别为(17.26±0.23)%、(39.47±0.17)%、(41.84±0.09)%.未加药转基因组、未加药未转染组细胞凋亡率分别为(3.74±0.03)%、(5.91±0.08)%;细胞周期分析结果显示经AMNl07处理后,Go/G1期和S期细胞明显减少,细胞阻滞在G2/M期,而转基因细胞组细胞周期改变不如转空载体组、未转染组明显.结论 AMN107可抑制CML耐药细胞增殖且诱导细胞凋亡,HO-1基因在CML耐药细胞凋亡过程中起保护作用,与促进CML耐药细胞的生长有关.

This study was aimed to investigate the effect of AMN107 (nilotinib) combined with heme oxygenase-1 (HO-1) inhibitor zinc protoporphyrin IX (ZnPPIX) on chronic myeloid leukemia (CML) cells and its mechanism. Proliferative rate of cells treated with AMN107 (10 µmol/L) and ZnPPIX (10 µmol/L) alone or both for different time was observed by MTT and trypan blue methods; the expression of HO-1 in the control group, ZnPPIX (10 µmol/L) group, AMN107 (10 µmol/L) group, AMN107 (10 µmol/L) combined with ZnPPIX (10 µmol/L) group was evaluated by semi-quantitative RT-PCR and Western blot at 48 h. Cell apoptosis was detected by flow cytometry with Annexin V/PI double staining at 48 h. The results showed that the strongest inhibition of cell proliferation was detected in combined group, and in a time-dependent manner; the expression level of HO-1 was lowest in combined group; the cell apoptosis rates were (11.38 ± 0.02)%, (17.44 ± 0.08)%, (39.81 ± 0.07)% and (56.46 ± 0.19)% in the control group, ZnPPIX group, AMN107 group, AMN107 combined with ZnPPIX group at 48 h respectively. It is concluded that the second-generation tyrosine kinase inhibitor AMN107 can induce the apoptosis in CML cells. Inhibition of HO-1 expression can enhance the killing effect of AMN107 on CML cells, which provides experimental evidence to further improve the clinical efficacy of CML treatment.

OBJECTIVETo establish a bcr-abl(+) cell line resistance to nilotinib, and to investigate the possible mechanisms of resistance.METHODSK562 cells were treated with gradually increasing concentrations of nilotinib to generate resistance cell line K562-RN. The folder of drug-resistance was evaluated by MTT assay. Cells apoptosis rate was detected by flow cytometry, the mRNA level of bcr-abl fusion gene by FISH, and the expression of apoptosis relative gene mRNA and protein (such as bcr-abl, HO-1, mdr1, Bcl-2 and caspase-3) by RQ-PCR and western blot.RESULTSThe resistant cell line K562-RN was successfully established, with 2.01 fold resistant to nilotinib compared with K562 cell line \[the IC(50) value of nilotinib to K562 and K562-RN were (12.320 ± 1.720) µmol/L and (24.742 ± 2.310) µmol/L, respectively\]. It also had the cross resistance to adriamycin, homoharringtonine, etoposide and imatinib. Treated with different concentrations of nilotinib, cell apoptosis rate of K562-RN was significantly lower than that of K562 cells. The rate of bcr-abl gene positive cells was 92% in K562-RN by FISH assay. The mRNA and protein levels of bcr-abl, HO-1 and mdr1 expression up-regulated in K562-RN cells, while those of caspase-3 expression down-regulated, being significantly statistical difference when compared with K562 cells (P < 0.05).CONCLUSIONHuman leukemic cell line resistance to nilotinib, K562-RN is established successfully by gradually increasing concentrations of drug. The mechanisms of resistance in K562-RN is probably associated with increasing expression of bcr-abl, HO-1, mdr1 and decreasing expression of caspase-3 mRNA and protein levels.

OBJECTIVE:To investigate the effect of heme oxygenase-1 (HO-1) expression on cell growth and apoptosis in imatinib resistant chronic myeloid leukemia (CML) cells (K562/A02-IM), and explore the relationship between HO-1 gene and CML.METHODS:The expression of HO-1 in 20 drug-resistant CML patients was detected by RT-PCR. Different concentrations of hemin were used to induce HO-1 expression of K562/A02-IM, HO-1 expression at different time was detected by RT-PCR and Western blot analysis. Cell apoptosis was detected by Annexin V/PI staining, and MTT assay was used to detect viability of K562/A02-IM cells after induction or inhibition of HO-1 gene by hemin and zinc protoporphyrin (ZPP).RESULTS:RT-PCR showed that HO-1 was expressed in the bone marrow mononuclear cells (BMMNCs). When treated with hemin at different concentrations (0, 10, 20, 40 µmol/L) for 16 h, the expression of HO-1 in K562/A02-IM was increased in a dose-dependent manner, and peaked at 20 µmol/L of hemin for 16 h. The apoptosis rates were (17.61 ± 0.01)%, (12.13 ± 0.11)%, (7.94 ± 0.03)% and (4.62 ± 0.15)% at 0,10, 20 and 40 µmol/L of hemin respectively for 16 h and were (14.7 ± 0.05)%, (8.1 ± 0.07)% and (16.3 ± 0.13)% at 20 µmol/L of hemin treatment for 8,16, and 24 h respectively. Hemin induced apoptosis of K562/A02-IM cells in a dose-dependent manner. The expression of HO-1 was induced in K562/A02-IM cells in a dose-dependent manner, and the survival of K562/A02-IM cells was significantly increased as compared to that of control group. When HO-1 was inhibited by ZPP, the cells survival was sharply decreased compared to that of the control group (P < 0.05).CONCLUSION:HO-1 was expressed in the BMMNCs. It is a kind of molecules whose expression can be induced and can promote the growth of drug-resistant cells. Inhibition of HO-1 expression probably be used for the treatment of drug-resistant CML.

OBJECTIVE: To investigate the effect of nilotinib on HO-1 gene expression and induction of apoptosis in K562/A02 cells. METHODS: BCR-ABL in K562/A02 cell was detected by Fluorescence in situ hybridization (FISH). K562/A02 cells were treated with AMN107 of different concentrations for 24 h. The expression of BCR-ABL gene on mRNA level was detected by RQ-PCR. Cell proliferation was observed by MTT. Cell apoptosis and cell cycle were inspected by Annexin V/PI double staining method. The expression of HO-1 gene was determined using RT-PCR and Western Blot. RESULTS: Ninety-eight percent of K562/A02 cells was detected with BCR-ABL fusion gene by FISH. RQ-PCR results showed that BCR-ABL fusion gene had a dose-dependent relationship with AMN107 in K562/A02 cells treated by AMN107 (0, 5, 10 and 20 μmol L -1) for 24 h. MTT and Annexin V/PI tests showed that survival rate of treated cells was negatively associated with the concentrations of AMN107, while the apoptosis rate was positively associated. Cell cycle analysis showed that the number of cells in G 0/G 1 phase decreased sharply and the cell cycle arrested at G 2/M checkpoint in AMN107 treated cells. RT-PCR and Western blot tests indicated that the expression of HO-1 gene decreased negatively with increasing concentration of AMN107. CONCLUSION: AMN107 can inhibit the expression of BCR-ABL and HO-1, which may be the mechanism underlying AMN107-induced apoptosis of drug-resistant CML cells. The results indicats that HO-1 is a key factor related with the growth of CML cells and the existence of BCR-ABL gene, and it may be a novel target for overcoming resistance of CML.

Abstract Abstract 4716 Aim: Heme oxygenase-1(HO-1) is well characterized survival factor that inhibits apoptosis in tumor cells. Chronic myeloid leukemia (CML) cells constitutively express HO-1 in chronic phase, but express highly in accelerated phase (AP) and blast phase (BP). However, resistance against tyrosine kinase inhibitor (TKI) can occur during therapy with TKI, particularly in AP and BP. This study was designed to confirm the relationship between HO-1 and drug resistant in CML, and seek ways to reversal of TKI resistant. Method: HO-1 gene was cloned from human liver by RT-PCR. And the lentiviral vector pLenti6-GFP-HO-1 was constructed. K562 cells which was expressed HO-1 highly was seemed as gene-transfected group. At the same time, we set the empty vector transfected group and untransfected group. K562 cells was cultivated with dasatinib in gene-transfected, empty vector transfected and untransfected groups. Expression of HO-1 mRNA was demonstrable by RT-PCR and the HO-1 protein by Western blotting. Gene mutation was detected by high performance liquid chromatography (HPLC) analysis. HO-1, multi-drug resistance gene(MDR1), lung reristance-related protein (LRP), glutathione S-transferase-π (GST-π), topoisomerase-I (Topo-I) in mRNA and protein level were deteceted by RT-PCR and Western blot. Apoptosis and cell cycle were determined by flow cytomertry analysis after treated with dasatinib. The activity of HO-1 against dasatinib for CML cells in vivo was evaluated by using the nude mouse xenograft model. The virus was injected into mouse through tail vein. Result: HO-1 gene was cloned successfully from human liver. The sequences were confirmed by restriction enzyme digestion analysis and sequencing. The virus was packaged in 293 cells and titer of virus was tested by Real-Time PCR, 1.02×109v.p./mL. Following transfer the lentiviral vector into K562, 72 hours after transfection, it showed that HO-1 was expressed highest by fluorescence micrope. The expression of HO-1 was detected by RT-PCR and Western blotting. HPLC results showed that there were not gene mutation after transfection□GRT-PCR and Western blot showed that the expression of MDR1 was significantly higher than transfection (P<0.01), also LRP and Topo-I levels were higher than transfection (P<0.05), there was no obvious changes of GST-π after transfection. At 48 hours after treatment with 10umol/L dasatinib by flow cytometry, the survival rate in transfected group was lowest which is 7.2±0.9% (P<0.05). And in empty vector transfected and untransfected group the survival rate was 38.2±1.6% and 39.3±1.7%. Cell cycle results showed that the cell population in G0/G1 and S phases decreased significantly in empty vector transfected and untransfected group, cell cycle arrest at G2/M checkpoint when treated by dasatinib. Nude mouse xenegraft models bearing carcinoma were established successfully. HO-1 in nude mouse xenegraft models was associated with protection of tumor cell against dasatinib. Conclusion: Up-regulating of HO-1 in chronic myeloid leukemia cell is associated with cell growth and anti-apoptotic effect. At the same time, there was a close relationship between HO-1 and resistance gene. HO-1 expression was accompanied by the expression of resistance genes. Based on these data, it seems desirable to explore the value of the HO-1-targeting drug in clinical trials in patients with leukemia and solid tumors. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 5017 Aim Rapamycin(RAPA) is the inhibitor of m-TOR. The activation of m-TOR results in changes in multiple cellular processes, eg, catabolism, anabolism, proliferation, growth and apoptosis. Rapamycin, which directly inhibits the Akt downstream molecule mammalian target of rapamycin (mTOR), effectively inhibits survival and proliferation in the development of BCR- ABL–induced chronic myelogenous leukemia(CML) from PTENfl/fl. Nilotinib (AMN107), a BCR-ABL tyrosine kinase inhibitor, was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia positive chronic myelogenous leukemia. We aim to introduce the CML cell line (k562/A02) resistant to AMN107 and investigate the effect of RAPA reverse AMN107 resistance on k562/A02 cell line. Method K562/A02 be added in the culture medium final concentration 1μmol/ L of AMN107 drugs a week before the withdrawal. For all experiments, cells were on the logarithmic phase.K562/A02 cells were treated with 400nmol/L rapamycin(400nmol/L RAPA group),100nmol/L rapamycin(100nmol/L RAPA group), 20μmol/L nilotinib(20μmol/L AMN107 group), 400nmol/L rapamycin +20μmol/L nilotinib (400nmol/L RAPA+20μmol/L AMN107 group), 100nmol/L rapamycin +20μmol/L nilotinib (100nmol/L RAPA+20μmol/L AMN107 group). Tetrazolium—based colorimetric assay (MTT) was used to detect the inhibiton effect of cell growth. The expression of Survivin, m-TOR, Bcl-2, and Caspase-3 were detected by western blot; Cell apoptosis was inspected by Annexin V/PI double staining method; The expression of Survivin, m-TOR, Bcl-2 and Caspase-3 in mRNA level was determined using reverse transcriptase—polymerase chain reaction (RT-PCR). BCR-ABL gene was detected by Real Time-PCR when K562/AO2 cells in different groups were treated by drugs for 48h. Result K562/A02 cells were successfully introduced to resistant toAMN107. Combination of 400 and 100 nmol/L RAPA with AMN107 could significantly enhance the sensitivity of k562/A02 to AMN107. The reverse factor was 1.97 and 2.73 fold respectively. RT-PCR and Western blot method indicated that the expression of Survivin and Bcl-2 was increased by RAPA+AMN107 group, but the MDR1 and Caspase-3 was decreased. The expression of BCR-ABL fusion gene in100 nmol/L RAPA group was higher than the blank groups, the copy number is 5.6×10−2. But the copy number in 400nmol/L RAPA +20μmol/L AMN107 group is1.64×10−2 that is lowest. Annexin V/PI showed that the apoptosis rates were (8.21±0.05)%, (32.07±0.03)%, (28.88±0.11)%, (16.57±0.05)%, (38.49±0.04)%, and(42.31±0.07)% respectively in control group, AMN107 group, 400nmol/L RAPA group, 100nmol/L RAPA group, 100nmol/L RAPA +20μmol/L AMN107 group, and 400nmol/L RAPA +20μmol/L AMN107 group. Conclusion We successfully introduced K562/A02 resistant toAMN107. Moreover, RAPA can enhance the sensitivity of AMN107 resistant 562/A02 cells. Combined of RAPA and AMN107 has significant synergistic growth inhibiting effect and apoptosis inducing effect on AMN107-resistant K562/A02 cells. The mechanism may be related at down-regulation of expression MDR1 and up-regulation of expression Survivin. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1231 Aim: Heme oxygenase-1(HO-1), also known as heat shock protein 32(Hsp32), has recently been identified as a stress-related survival molecule that acts anti-apoptotic and cytoprotective in inflammatory reactions. In the present study, we provide evidence that HO-1 is effective on AMN107(Nilotinib) in promoting K562/A02 apoptosis which is induced by STI571(Imatinib),and investigate AMN107 on the mechanism of resistance to STI571. Method: HO-1 gene was cloned from rat liver by RT-PCR. And the retrovirus vector pQCXIP-EGFP-C1 was constructed. K562/A02 cell which was expressed HO-1 highly was seemed as gene-transfected group. At the same time, we set the empty vector transfected group and untransfected group. K562/A02 cell was cultivated with AMN107 in gene-transfected, empty vector transfected and untransfected groups. Expression of HO-1 mRNA was demonstrable by RT-PCR, Real-time PCR, and the HO-1 protein by Western blotting. Constant MTT assay and cell number count were used to detect the proliferation of leukemia cells after treatment with AMN107. Apoptosis was determined by morphological observation and flow cytomertry analysis after AnnexinV/PI double labeling. Also we detected the intracellular drug concentration by HPLC after treatment with the same concentration of AMN107. Result: HO-1 gene was cloned from rat liver successfully. The sequences were confirmed by restriction enzyme digestion analysis and sequencing. The virus was packaged in 293T cells and titer of virus was tested by Real-Time PCR, 8.09×1010v.p./mL. Following transfer the retrovirus vector into K562/A02, 72 hours after transfection, it showed that HO-1 was expressed highest by fluorescence micrope. High expression of HO-1 was detected by RT-PCR, Real-Time PCR and Western blotting. After 10umol/L AMN107 treated, the expression of HO-1 was clearly lower in empty vector transfected group and untransfected group (p <0.05), but in transfected group there was no obvious change. MTT assay showed that after AMN107 treated, IC50 value in transfected group was 5.62174 mmol/L, significantly higher than empty vector transfected and untransfected group(P<0.05). Cell number count showed that after AMN107 treated the activity of cells were decreased sharply in empty vector transfected and untransfected group, but no significant change in transfected group. At 48 hours after treatment with 10umol/L AMN107 by flow cytometry, the survival rate in transfected group was lowest which is 8.3±1.2%(P>0.05). And in empty vector transfected and untransfected group the survival rate was 35.3±1.6% and 37.5±1.3%(P<0.01). HPLC analysis showed that intracellular drug concentration was lowest in transfected group which treatment with the same concentration of AMN107. Conclusion: STI571 resistance is a major cause of STI571 treatment failure in chronic myeloid leukemia(CML) patients. Our data showed that HO-1/Hsp32 is a novel survival factor and interesting target in K562/A02. In addition, it could predict the risk of resistance to STI571 therapy. Down-regulating of HO-1/Hsp32 in chronic myeloid leukemia cell is associated with reduced tumor cell growth and induction of apoptosis. Based on these data, it seems desirable to explore the value of the Hsp32-targeting drug in clinical trials in patients with leukemia and solid tumors. Disclosures: No relevant conflicts of interest to declare.