[This retracts the article DOI: 10.3892/etm.2015.2209.].

目的通过血红素加氧酶-1（HO-1）诱导剂Hemin及抑制剂ZNPP IX调控HO-1,并联合阿霉素逆转K562A02细胞化疗耐药机制的研究,为慢性髓系白血病（CML）的逆转耐药提供新的策略。方法培养K562及K562A02细胞,采用荧光原位杂交（FISH）法检测K562A02细胞中bcr-abl融合基因表达。分别用HO-1诱导剂Hemin及抑制剂ZNPP IX调控HO-1基因表达联合阿霉素处理K562A02细胞后;流式细胞术检测药物诱导细胞凋亡情况。Western blot检测耐药相关基因及凋亡基因蛋白表达水平。结果 K562A02细胞中bcr-abl融合基因阳性细胞占94%。阿霉素处理细胞后,随着阿霉素浓度的增加,HO-1表达下降,耐药相关基因MDR1、NF-κB（P65）、MRP1、TopoⅡα、ABCD2表达亦降低;用HO-1诱导剂Hemin、抑制剂ZNPP IX、阿霉素单药分别及联合处理K562A02细胞后,显示HO-1高表达后耐药相关基因表达升高,细胞凋亡率下降。而降低HO-1表达,耐药相关基因表达下降,细胞凋亡率增加。结论 HO-1可作为逆转耐药的靶基因,可以使K562A02对阿霉素重新敏感,起到增敏效应。

The aim of this study was to investigate the in vivo and in vitro effects of targeted heme oxygenase-1 (HO-1) silencing on the proliferation and apoptosis of human acute myelocytic leukemia (AML)-M2 cells. Bone marrow mononuclear cells (BMMNCs) were infected by pRNAi-siHO-1-GFP. The viability of the BMMNCs was determined by cell counting kit-8 (CCK-8) assay following daunorubicin (DNR) treatment. The apoptotic rate was detected by flow cytometry. The expression levels of HO-1 and apoptosis-related genes were detected by quantitative polymerase chain reaction (qPCR) and western blot analysis. An AML-M2 xenograft mouse model was established. The tumor formation outcomes and survival were observed. The leukocyte and platelet counts and hemoglobin levels were monitored, and the copy numbers of AML1/ETO fusion gene were detected by qPCR. pRNAi-siHO-1-GFP silenced the expression of HO-1. DNR inhibited cell viability in a time- and dose-dependent manner. The survival rate of the cells was significantly reduced by infection with pRNAi-siHO-1-GFP. HO-1 expression in the BMMNCs infected with pRNAi-siHO-1-GFP was downregulated, whereas caspase-3, -8 and -9 expression was upregulated compared with that in control BMMNCs. Kasumi-1 cells were successfully inoculated into nude mice. The rats inoculated with pRNAi-siHO-1-GFP-transfected Kasumi-1 cells succumbed to tumors more slowly and survived longer than those inoculated with untransfected Kasumi-1 cells. Furthermore, the leukocyte and platelet counts and hemoglobin levels were higher and the copy numbers of AML1/ETO fusion gene were lower in the former group. HO-1 gene silencing may promote the apoptosis of human M2 leukemic cells by inhibiting a caspase-dependent apoptotic pathway. Targeted silencing of HO-1 is able to inhibit the proliferation and infiltration of leukemic cells in nude mice and thus prolong their survival. The findings provide valuable experimental evidence for the molecular targeted therapy of M2 leukemia.

OBJECTIVE:To study protective effects of glutamine (Gln) on cardiac muscle cell in septic model rats. METH-ODS:Rats were randomly divided into sham operation group (normal saline),model group (normal saline) and Gln low-dose, medium-dose and high-dose groups(0.5,0.75,1.0 g/kg)with 10 rats in each group. In these groups,septic rat model was induced by cecal ligation and puncture except sham operation group received sham operation. They were given relevant medicine intrave-nously 10 min after operation,and the characteristics and apoptosis of cardiac muscle cell were observed 12 h after operation. The serum contents of CK,LDH and TnⅠ,and the expression of Bcl-2 and p53 mRNA were all detected. RESULTS:Compared with sham operation group,myocardial necrosis of model group was found,and the serum content of CK,LDH and TnⅠ and apoptotic index increased,and mRNA expression of Bcl-2 in cardiac muscle cell decreased while that of p53 increased,with statistical signifi-cance(P<0.05). Compared with model group,myocardial injury relieved significantly in Gln high-dose and medium-dose groups, and serum contents of CK,LDH and TnⅠ and apoptotic index decreased;mRNA expression of Bcl-2 increased in cardiac muscle cell while that of p53 decreased,with statistical significance (P<0.05). CONCLUSIONS:Gln can improve myocardial injury of septic model rats significantly,by a possible mechanism of down-regulating the expression of p53 gene and up-regulating the ex-pression of Bcl-2 gene.

BACKGROUNDThere are limited eligible clinical markers at present to monitor the progress of chronic myeloid leukemia (CML). Heme oxygenase-1 (HO-1), as one of the most important oxidation-regulating enzymes in vivo, suggests the onset and progression of cancer when highly expressed. Furthermore, HO-1 level is related with the occurrence and development of hematological diseases. But the relationship between HO-1 expression and progression/relapse of CML has seldom been studied hitherto. This study aimed to investigate the relationship between them to find out a new molecular marker for prediction.METHODSA total of 60 peripheral blood and bone marrow (BM) samples from 25 CML patients in different phases were collected respectively to detect the expressions of HO-1 and bcr/abl using real-time PCR. Routine blood test was performed to detect the changes of leukocyte and platelet counts. The proportion of primitive cells in BM was detected by flow cytometry. The relationship between high HO-1 expression and CML progression and relapse was explored by the analysis of variance by Wilcoxon test and linear regression analysis. The diagnostic accuracy and cutoff values were determined by receiver operating characteristic curve.RESULTSRelative expression of HO-1 mRNA in CML patients peripheral blood was significantly higher than that of donors (P < 0.0001), which were 0.57±3.78 and (1.417±1.125)×10(-6), respectively. HO-1 expression level in CML patients was 0.061 5±0.062 4, which decreased to 0.009 4±0.006 7 upon CMoR, and remained remarkably higher 0.016 3±0.017 5 than that of normal donors (1.417±1.125)×10(-6), P < 0.001. When relapse occurred, HO-1 expression significantly increased from 0.020 6±0.021 0 to 3.852±10.285 in CMoR stage and undergoing relapse. According to progression of CML, HO-1 expression level in CML patients increased from CP (0.009 5±0.017 6) to AP (0.028 0±0.055 7) and then to BP (0.276 7 ± 0.447 0). And there was a linear correlation between HO-1 expression and proportion of primitive CML cells. The diagnostic accuracies and cutoff values of HO-1 expression for CML-CP, CML-AP, and CML-BP were 1.0, 0.748, and 0.965, respectively, as well as 0.000 070, 0.001 917, and 0.020 696, respectively.CONCLUSIONHO-1 may be a potential molecular indicator for the progression and relapse of CML.

Acute myeloid leukemia (AML) requires new therapies on the molecular level. Downregulation of heme oxygenase-1 (HO-1) by gene silencing improves the sensitivity of tumor cells to chemotherapy drugs and promotes apoptosis. For the first time, we verified that endoplasmic reticulum and mitochondrial apoptotic pathways were activated by small interfering RNA that targeted-silenced the expression of HO-1 in AML-M2 Kasumi-1 cells. Ca2+ was prone to accumulation and reactive oxygen species were easily generated, while mitochondrial transmembrane potential was reduced. Thus, cytochrome c was released from mitochondria to the cytoplasm and caspases were activated for the following cascade to facilitate apoptosis.

Phenethyl isothiocyanate (PEITC), a potential cancer chemopreventive constituent of cruciferous vegetables, including watercress, has been reported to inhibit cancer cell growth by arresting the cell cycle and inducing apoptosis in various human cancer cell models. However, the role of PEITC in the inhibition of human chronic myeloid leukemia (CML) K562 cell growth and its underlying mechanisms have yet to be elucidated. In the present study, PEITC was found to induce cell death through the induction of reactive oxygen species (ROS) stress and oxidative damage. Heme oxygenase‑1 (HO‑1), which participates in the development of numerous tumors and the sensitivity of these tumors to chemotherapeutic drugs, plays a protective role by modulating oxidative injury. Therefore, the present study assessed the inhibitory effect of PEITC on K562 cells and whether HO‑1 facilitated cell apoptosis and ROS generation. PEITC was found to suppress cell growth and cause apoptosis by promoting Fas and Fas ligand expression, increasing ROS generation and by the successive release of cytochrome c as well as the activation of caspase‑9 and caspase‑3. PEITC was also combined with the HO‑1 inhibitor zinc protoporphyrin IX and the inducer hemin to assess whether HO‑1 determines cell survival and ROS generation. The results of the present study suggest that PEITC may be a potential anti‑tumor compound for CML therapy, and that HO‑1 has a critical function in PEITC‑induced apoptosis and ROS generation.

Aim To explore the mechanisms underlying the HO-1-regulated apoptosis and autophagy in human chronic myeloid leukemia (CML) K562 cell line in vitro. Methods K562 cells were devided into three groupsFK562(untreated),K562-H(infected by lenti-GFP-HO-1) and K562-siHO-1(infected by lenti-GFP-siHO-1). The protein expression of HO-1 were determined by Western blot, and its mRNA levels were obtained by real-time PCR.  Cell viability was assessed by MTT assay. Morphological changes of apoptosis and autophagy were observed by the application of transmission electron microscope. Annexin V-FITC/PI dual staining assays were used to measure apoptosis. Autophagy was analyzed by Western blot and immunofluorescence staining with an anti-LC3 antibody. DCFH-DA was used as the cell-permeate indicator for intracellular ROS measurement. Protein levels of active caspase 3, Beclin1,cyto-C and Bcl2 were determined by Western blot. Results The Expression of HO-1 on mRNA and protein level increased in K562-H group while reduced in K562-siHO-1, the differences showed statistically significance compared to K562-untreated group (p<0.05). Imatinib (0.20-1.60μmol/L) exerted inhibitory effects on cell proliferation in a concentration-dependent manner with IC(50) value of 1.0μmol/L, 0.7μmol/L and 0.4μmol/L, while viability of 63.41±1.46%, 41.24±2.01%, 29.49±1.87% in K562-H, K562 and K562-siHO-1( p<0.05 ), respectively. Exposure to imatinib (0.40 μmol/L) simultaneously induced mitochondrial-mediated apoptosis and Beclin1-dependent autophagy in all groups. Autophagy flux was found in K562-siHO-1 group while autophagosome formation decreased in K562-H group, which was significantly different from K562 group. The expressions of LC3 II and cyto-C were higher in K562-siHO-1 group than other two groups ( p<0.05 ). Both apoptosis and autophagy caused cell death in a time-dependent manner. However, apoptosis played a more effective role in cell death. After exposure to imatinib (0.40 μmol/L), both intracellular ROS generation and the protein expression of Beclin1 and caspase9 were increased, while Bcl2 protein level were reduced in a time-dependent manner. The combination of NAC (2.50 mmol/L) with imatinib (0.40 μmol/L) contributed to the blockade of ROS generation which abrogated apoptosis, autophagy and Beclin1 expression. 3-MA (3.0mmol/L) and HO-1 silencing resulting in the inhibition of autophagy enhanced the effect of apoptosis which was induced by imatinib (respectively 70.65±2.71% and 69.27±3.13%), while this phenomenon was accompanied by increased ROS generation (1.3 and 1.6 folds higher than K562 group). Conclusions Imatinib and HO-1 siRNA simultaneously induced apoptosis and autophagy in K562 cells, which was associated with the increasing of intracellular ROS generation, cyto-C and Beclin1 protein expression. Beclin1, an autophagy related protein induced by HO-1 silencing, could further induce apoptosis in K562 cells through the inhibition of Bcl2. The responsive cytoprotection to chemotherapeutics caused by autophagy decreased while sustaining up-regulation of HO-1 in K562 cells. However, the underlying mechanisms remained to be well elucidated. Disclosures: No relevant conflicts of interest to declare.

Aim Using lentivirus-mediated HO-1 siRNA (lenti-siHO-1-GFP) to silence the HO-1 gene in Kasumi cells so as to explore the role and mechanism of HO-1 on cell apoptosis. Methods To infect Kasumi cells with lenti-siHO-1-GFP and check the infection efficiency by using fluorescence microscopy and flow cytometry (FCM). Experimental group was divided into three groups: untreated Kasumi (K), infected Kasumi by empty vector (lenti-GFP-K) and infected Kasumi by lentivirus-mediated HO-1 siRNA (lenti-siHO-1-K). The HO-1 expression of each group was detected by realtime PCR. Fluo3-AM method was used to detect the intracellular Ca2+ accumulation. DCFH-DA was used for the measurement of intracellular ROS. The change of mitochondrial membrane potential was evaluated by JC-1 stainning by using FCM. After being treated with various concentrations of daunorubicin for 24, 48, and 72 h respectively, cell viability was determined by MTT assay. Cell apoptosis was determined by FCM following with cells dual-stained with Annexin-V-FITC and propidium iodide (PI). The mRNA of HO-1 and apoptosis-related genes were analyzed by realtime PCR and, the expressions of their corresponding protein were determined by western blot. Additionally, After treating with 10mM Ca2+chelator BAPTA-AM and 0.5mM NAC for 12h, Ca2+ accumulation, ROS generation, the expression of HO-1 and apoptosis-related genes were detected respectively. Result presented in mean±sd manner. Results After lenti-siHO-1-GFP infection for 48h, we could observe the fluorescence clear, the fluorescent intensity was 95.87% after 72 hours. The HO-1 silencing efficiency of lenti-siHO-1-K was 77.00%. MTT result showed that daunorubicin exerted moderate inhibitory effects on cell proliferation in a dose and time dependent manner. With the same treating conditions, the cell viability of lenti-siHO-1-K group was significantly lower than the other two groups(e.g 49.20±1.30% survival in lenti-siHO-1-K group, 72.40±1.90% in K group and 74.10±2.10% in lenti-GFP-K group after being treated by 5ug/ml DNR,respectively, p =0.014), while the apoptosis rate was higher than the other two groups(e.g 75.77±3.41% in lenti-siHO-1-K group, 23.72±2.03% in K group and 26.10±1.95% in lenti-GFP-K group after being treated by 5ug/ml DNR,respectively, p =0.011). Compared with other two groups, the lenti-siHO-1-K group showed a downregulation in the mRNA and protein expression of HO-1. The mRNA and protein expressions of cyto-C, caspase3, caspase8, caspase9 and caspase12 in lenti-siHO-1-K group were upregulated after exposure to 5ug/ml daunorubicin for 24 hours. Compared with K and lenti-GFP-K groups, Ca2+ accumulation in lenti-siHO-1-K group was increased significantly(e.g 40.35±2.10% in lenti-siHO-1-K group, 17.30±1.81% in K group and 14.15±1.75% in lenti-GFP-K group,respectively, p =0.041). The ROS generation was higher than the other two groups(e.g 47.65±2.05% in lenti-siHO-1-K group, 21.30±1.94% in K group and19.90±2.01% in lenti-GFP-K group,respectively, p =0.037). The ratio of Green/Red fluorescence intensity increased significantly in lenti-siHO-1-K group(e.g 0.704±0.06 in lenti-siHO-1-K group, 0.57±0.09 in K group and 0.527±0.05 in lenti-GFP-K group, respectively, p =0.042). After exposure to 10mM BAPTA-AM and 0.1mM NAC alone or combined with, both the intracellular Ca2+accumulation and the ROS level in lenti-siHO-1-K group reduced(17.59±1.01% of Ca2+acumulation and 19.78±1.3% of ROS production after BAPTA-AM treatment alone, 23.42±1.97% of Ca2+and 15.47±1.14% of ROS after being treated by NAC alone, 16.52±1.23% of Ca2+and 14.37±1.21% of ROS after treatment by both agent) , while the mRNA and protein expressions of cyto-C, caspase3, caspase8, caspase9 and caspase12, decreased significantly. Conclusion HO-1 gene silencing played a role in pro-apoptosis in Kasumi cells. The mechanism may be related to the endoplasmic reticulum stress and abnormal accumulation of intracellular Ca2+, ROS generation, descending of the mitochondrial membrane potential and release cyto-C, then further activated the caspases cascade and promoted apoptosis. However, it tended to be initiated by crosstalk in Ca2+-ROS pathway. Disclosures: No relevant conflicts of interest to declare.

OBJECTIVE:To study the role of clinical pharmacists in the treatment for patients with allogeneic peripheral blood stem cell transplantation(allo-PBSCT).METHODS:Clinical pharmacists provided pharmaceutical care for 2 cases of allo-PBSCT,participated in the formulation of therapeutic plan,ward-round and case discussion,and carried out pharmaceutical care and medication education.RESULTS:11 days after allo-PBSCT,routine blood test of patient A reminded hematopoietic reconstruction;HCMV-DNA was negative;bone marrow inspection showed active bone marrow hyperplasia;the vital signs of patients were stable without fever and other ADR.11 days after allo-PBSCT,routine blood test of patient B reminded hematopoietic reconstruction;HCMV-DNA was negative;bone marrow inspection showed myeloid cell hyperplasia and low erythroid cell hyperplasia;no fever,erythra and other GVHD manifestation were found.CONCLUSION:Through clinical pharmacists participate in pharmaceutical care for allo-PBSCT patients,and collaborate with doctors on medical plan to improve the acceptance of doctors,nurses and patients for clinical pharmacists and realize the value of them.

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.