The Roles Of Nitidine Chloride,Wnt Signaling Receptor FZD7 In CML And Related Mechanisms

Section Ⅰ Novel agent Nitidine Chloride induces erythroid differentiation and apoptosis in CML cells through c-Myc-miRNAs axisBackground:Chronic myeloid leukemia (CML) is a hematopoietic stem/progenitor cell disorder, in which BCR-ABL oncoprotein leads to a progressive block of differentiation and increased genetic instability. Tyrosine kinase inhibitors (TKIs), specifically inhibiting BCR-ABL fusion protein and triggering apoptosis and differentiation of CML cells, are used as first-line treatment for CML. Although TKIs have revolutionized the treatment of CML, CML is rarely curative. Exploring novel differentiation inducer is considered an alternative strategy for CML therapy.The proto-oncogene c-Myc has been shown to play pivotal roles in cell cycle regulation, metabolism, apoptosis, differentiation, cell adhesion and tumorigenesis. Study showed that BCR-ABL indirectly activated c-Myc via either JAK2 pathway or MAPK pathway. C-Myc expression was elevated in CML blast crisis and correlated with poor response to imatinib (IM). c-Myc antagonized erythroid differentiation and apoptosis induced by imatinib or dasatinib, suggesting its vital roles in drug sensitivity. The vital functions of c-Myc in CML suggested that further mechanistic understanding of c-Myc and finding novel agents targeting c-Myc would be a promising strategy for the treatment of CML.Nitidine Chloride (NC), derived from Zanthoxylum nitidum, had been identified as a potential anti-tumor drug in several tumors, e. g. breast cancer, nasopharyngeal carcinoma, renal cancer and hepatocellular carcinoma. Recently, intensive studies have shown that NC possesses potent anti-tumor activities by inhibiting proliferation, inducing apoptosis, and modulating invasionin in these solid cancer cells. However, the function of NC in leukemia and the underlying molecular mechanisms are still unclear.Objectives:To investigate the effect and the underlying mechanism of NC induced erythroid differentiation and apoptosis in CML cells. To clarify the effect of NC on c-Myc-miRNAs regulatory axis in CML cells. To test the possibility of NC used as an anti-CML drug.Methods:1. The effects of NC on erythroid differentiation in K562 cells:Benzidine staining was performed to detect differentiation rate of K562 cells. Flow cytometry was used to determine the CD71 and CD235a positive cells. Real-time RT-PCR was used to analyze the expression of erythroid differentiation markers after NC (4 or 8μM) treatment for 2 days. Western blot was performed to detect the expression of globin γ.2. The effects of NC on apoptosis in K562 cells:MTT assay was performed to evaluate cell viability. Flow cytometry was used to detect apoptosis rate by annexin V/PI double staining. Western blot was performed to detect cleaved caspese-3 and Parp-13. The effects of NC on c-Myc protein level:Western blot and Real-time RT-PCR were performed to examine the expression level of c-Myc treated with indicated concentrations of NC for indicated time. CHX chase assay was used to detect the effects of NC on c-Myc degradation.4. The effects and related mechanisms of NC in the degradation of c-Myc:Western blot was performed to detect phosphorylation status of Thr58 and GSK3β protein level in K562 cells after NC treatment. After transfected with GFP-tagged wild-type or c-Myc T58A mutant plasmids, K562 cells were exposed to NC for 2hrs, and CHX chase assay was performed.5. The effects of NC on c-Myc activated miRNAs:Real-time RT-PCR was used to detect the relative level of c-Myc activated miRNAs in K562 cells treated by NC or stably transformed shc-Myc cell line. K562 cells infected with LV-miR-17 or LV-miR-20a lentiviral particles were treated with NC for additional 2 days. Western blot was performed to detect the expression of p21 and globin γ, and the apoptosis rate was detected by annexin V/PI double staining.6. The effects of NC on IM sensitivity of K562 and K562/G01 cells:Soft agar colony formation assay was performed using K562 cells after treatment with NC, IM or combination of NC and IM. K562 or K562/G01 cells were treated with various concentrations of NC or IM for 2 days and cell proliferation was measured by MTT assay. Apoptosis rate was detected by annexin V/PI double staining after K562 or K562/G01 cells were treated with NC, IM or NC+IM for 2 days.7. The effects of NC on primary CML cells:Bone marrow mononuclear cells were obtained from 5 CML patients, without any treatment with any TKIs. The CML patient 1,2 and 3 were subsequently proved to be IM responders. Patient 4 and 5 were IM nonresponders. Annexin V/PI double staining was used to detect apoptotic rate of primary CML cells treated with NC, IM or NC+IM for 2 days.Results:1. NC induced erythroid differentiation in K562 cells:Exposure to NC resulted in higher percentage of benzidine-positive cells in a dose and time dependent manner. Erythroid specific surface markers CD71 and CD235a were increased in K562 cells after NC treatment. mRNA expression levels of erythroid differentiation markers globin{globin a, s and y), as well as CD235a and a-hemoglobin stabilizing protein (AHSP) were significantly increased comparing with control group. The enhanced expression level of globin y after NC treatment was further confirmed by western blot analysis.2. NC induced apoptosis in CML cells:NC treatment for 24 or 48 hrs remarkably resulted in decreased viability of K562 cells. Treatment with 4 and 8 μM of NC for 48 hrs resulted in significant higher apoptotic rate by 10.56 ±1.47% and 23.78±5.38%, respectively. NC treatment significantly increased level of cleaved caspase-3 and Parp-1.3. NC downregulated c-Myc protein level by accelerating its degradation in K562 cells:Endogenous c-Myc was downregulated in a NC dosage or time dependent manner. NC treatment had little effect on the mRNA level of c-Myc, suggesting that NC elicited c-Myc protein attenuation could be mediated on the post-translational level. CHX chase assay showed NC treatment could markedly accelerate the degradation of c-Myc. Overexpression of c-Myc markedly reversed increased globin y level induced by NC, while knocking down of c-Myc enhanced up-regulated globin y protein level after NC treatment. Overexpressing c-Myc could partially rescued NC elicited apoptosis in K562 cells, while NC induced apoptosis was further triggered by c-Myc down-regulation. These findings provided strong evidences that c-Myc was intimately involved in NC induced differentiation and apoptosis of K562 cells.4. NC accelerated c-Myc degradation via enhanced Thr58 phosphorylation:The phosphorylated c-Myc at Thr58 was significantly increased normalized to total c-Myc. CHX chase assay showed that NC could markedly enhance degradation of wild type c-Myc. However, NC failed to promote degradation of c-Myc T58A mutant. The total proportion of GSK3β did not change significantly after NC treatment in K562 cells. GSK3β inhibitor LiCl or Bio rarely affected NC-induced c-Myc degradation.5. NC elicited erythroid differentiation and apoptosis were mediated by c-Myc-activated miRNAs:NC treatment decreased the relative levels of miR-17, miR-20a, miR-30a, miR-221, miR-222 and miR-378, among which miR-17 and miR-20a showed the sharpest decrement by 65.0±0.6% and 62.6±2.6%, respectively. We found NC could markedly upregulate p21, the target gene of miR-17/20a. Overexpression of miR-17 or miR-20a could remarkably weaken the incremental effects of NC on p21 and globin y.6. NC enhanced the biological effect of IM in K562 and K562/G01 cells:NC and IM caused significantly inhibition of colony growth, and co-treatment with NC and IM resulted in the slowest colony growth comparing with either agent alone. Unlike IM, which failed to inhibit viability of K562/G01 cells, NC decreased survival ability of both K562 and K562/G01 cells lines. K562 cells co-treated with NC and IM showed higher percentage of apoptosis (44.83±5.65%) than cells treated with either NC (10.56±1.47%) or IM (29.49±2.25%) separately. More importantly, NC could also induce apoptosis (9.89±1.38%) of K562/G01, an IM resistant cell line.7. NC enhanced the effect of imatinib on primary CML cells:NC could induce apoptosis in samples of IM resistant patient 4 and 5, and enhance IM sensitivity. NC treatment decreased the level of c-Myc, miR-17 and miR-20a in primary CML cells.Conclusion:1. NC could induce erythroid differentiation and apoptosis. These effects were associated with concomitant attenuation of c-Myc.2. NC promoted c-Myc degradation via enhanced phosphorylation of Thr58 residue, probably independent of GSK3β.3. NC downregulated a specific group of miRNAs (miR-17, miR-20a, miR-30a, miR-221, miR-222 and miR-378), which were activated by c-Myc.4. NC enhanced the effects of IM in K562 and primary CML cells, and even IM-resistant CML cell line (K562/G01) and CML primary cells exhibited high sensitivity to NC. miR-221, miR-222 and miR-378), which were activated by c-Myc.4. NC enhanced the effects of IM in K562 and primary CML cells, and even IM-resistant CML cell line (K562/G01) and CML primary cells exhibited high sensitivity to NC.Section II FZD7 is elevated in CML and regulates BMSCs-mediated preservation of CML cellsBackground:Imatinib mesylate (IM) and other ABL tyrosine kinase inhibitors (TKIs) have made great progress in treatment of chronic myeloid leukemia (CML). However, a significant proportion of patients do not obtain expected effectiveness, while other CML patients become refractory to further treatment. Moreover, cessation of drug treatment leads to disease recurrence in most CML patients. Minimal residual disease (MRD), retained in patients even when they reach complete remission, may be the source of relapse in CML patients after TKIs discontinuation.Some evidences suggest that disease relapse and treatment resistance in CML patients are largely due to the protection of leukemia cells by various components of the bone marrow microenvironment, especially bone marrow mesenchymal stem cells (BMSCs). Indirect communication through extracellularly secreted growth factors and direct contact between leukemia cells and BMSCs appear to be essential to CML cells survival and chemoresistance. The interaction between CML cells and BMSCs may trigger molecular changes that lead CML cells to chemotherapy resistance.The Wnt/β-catenin signaling is initiated by binding of Wnt ligands to frizzled transmembrane receptors (FZD) and low-density lipoprotein receptor-related proteins (LRP). β-catenin, a central molecule of canonical Wnt signal, associates with T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors to activate target genes, such as CD44, cyclin Dl and c-Myc. Studies showed that BMSCs enhanced (3-catenin nuclear translocation and transcriptional activity in CML cells. However, the molecular basis that how Wnt signaling activity is regulated by BMSCs remains obscure. In this study, we found that BMSCs could increase the expression of FZD7 and subsequently activate Wnt/β-catenin signaling pathway in CML cells.Frizzled-7 (FZD7) is one of ten members of the frizzled receptor family. Recent studies shows that FZD7 acts as an oncogene in the development and progression of solid tumors. For example, the expression level of FZD7 is up-regulated in colorectal cancer, hepatocellular carcinoma, esophageal cancer, lung cancer and gastric cancer. FZD7 activates Wnt signaling and enhances the expression of target genes, including c-Myc, cyclin D1, c-Jun, RhoA and Rac, most of which play as oncogenes in CML LSCs. Moreover, FZD7 expression is reported to be positively regulated by SIRT1, which is overexpressed in CML LSCs, suggesting that FZD7 contributes to oncogenesis in CML. Therefore, it is intriguing to postulate that the FZD7 targeting could be a potential therapeutic strategy for developing efficient therapies against CML.Objectives:To investigate the expression of FZD7 in patients with CML. To study the effect of FZD7 overexpression on IM sensitivity and IM induced apoptosis in leukemic cells. To reveal the role of FZD7 in a co-cultured system with BMSCs and provide the basis for molecular targeted therapy.Methods:1. The expression of FZD7 in CML patients:CD34+cells were enriched using a human CD34 MicroBead Kit from bone marrow samples from IM-sensitive patients (n=9), IM-resistant patients (n=7) and healthy donors (n=6). qRT-PCR and western blot were used to analysis of expression levels of FZD7.2. The effects of BMSCs on the expression of FZD7 in CML cells:Real time RT-PCRand western blot were applied to detect the alteration of FZD7 and other Wnt/β-catenin signaling moleculars in K562 or primary CML cells after co-culture with BMSCs derived from both normal and CML bone marrow.3. The effects of FZD7 down-regulation on proliferation of CML cells:Real time RT-PCRand Western blot were applied to detect the effect of FZD7 shRNA lentiviral pariticles on FZD7 expression. Cell proliferation assays were performed by MTT assay. Cell cycle analysis by flow cytometry.4. The effects of FZD7 down-regulation on IM sensitivity of CML cells:Tranduced cells were treated with serial dilutions of IM for 48 hrs. MTT method was used to detect live cells. The apoptotic cells were analyzed by flow cytometry using Annexin V and PI.5. The effects of FZD7 down-regulation on IM resistance induced by BMSCs:K562 cells tranduced with FZD7 shRNA lentiviral pariticles were cocultured with or without BMSCs, and then were treated with IM for 48hrs. MTT method and apoptosis assays by flow cytometry were used to detect cells death.6. The effects of FZD7 down-regulation on the activation of Wnt/β-catenin signaling induced by BMSCs:After K562 cells tranduced with FZD7 shRNA or control lentiviral pariticles were cocultured with BMSCs, Real time RT-PCRand Western blot analysis were applied to detect the alteration of Wnt/β-catenin signaling moleculars K562 cells.Results:1. FZD7 was up-regulated in CML CD34+cells1.1 The expression of 10 FZD genes was detectable, with relatively high expression of FZD6 and FZD7.1.2 Several FZD genes were differentially expressed in CML CD34+cells compared to NBM CD34+cells, with the highest level detected for FZD71.3 FZD7 mRNA levels were up-regulated in pre-treatment CML CD34+cells from subsequent IM-resistant patients versus IM-sensitive patients. Western blot analysis further revealed that FZD7 protein levels were also significantly elevated in IM-resistant CML CD34+cells, compared to their counterparts.2. BMSCs up-regulated the expression of FZD7 in CML cells2.1 Western blot assay showed that protein levels of FZD7, P-catenin and MDR1 in K562 and primary CML cells were up-regulated by BMSCs derived from both normal and CML bone marrow. The CML BMSCs induced higher levels of FZD7, β-catenin, and MDR1 than normal BMSCs. 2.2 Real-time RT-PCR showed that protein levels of MDR1, c-Myc, Survivin, CD44, and Trib2 were up-regulated in K562 cells by BMSCs derived from both normal and CML bone marrow.3. Down-regulation of FZD7 suppressed proliferation and cell cycle progression of CML cells3.1 Real-time RT-PCR and Western blot assay showed that both ShFZD7-1 and ShFZD7-2 lentiviral pariticles could efficiently decrease FZD7 mRNA and protein level in K562 cells.3.2 Down-regulation of FZD7 suppressed cell growth compared with negative control in K562 cells.3.3 K562 transduced with shFZD7 lentiviral particles formed less colonies than negative control.3.4 Down-regulation of FZD7 significantly increased the ratio of cells in G0/G1 phase, and simultaneously reduced that of cells undergoing S phase.3.5 Down-regulation of FZD7 significantly increased P21 and P27, while downregulated cyclin D1 and CDK4 in K562 cells.4. Down-regulation of FZD7 sensitized CML cells to IM:4.1 Down-regulation of FZD7 enhanced the inhibitory effects of IM against K562 cells, and the IC50 values of ShFZD7-1, ShFZD7-2 and ShCtrl were 0.103 ±0.016,0.072±0.026 and 0.318±0.056μM, respectively (P< 0.01) in K562 cells.4.2 After transduced cells were cultured in 5μM IM for 48 hrs, apoptosis was assessed by annexin V-APC/PI labelling and flow cytometric analysis. The percentage of apoptotic cells were 37.6±8.09% and 40.9±7.48% in ShFZD7-1 and ShFZD7-2 transduced CML CD34+cells respectively, versus 22.3±6.40% in ShCtrl transduced cells (P< 0.01).4.3 The cleaved caspase-3 and cleavage of PARP-1 were significantly higher in K562 cells when FZD7 was knocked down.5. Down-regulation of FZD7 abrogated imatinib resistance induced by BMSCs5.1 The mean apoptotic rates of K562 cells transduced with ShFZD7-2 or ShCtrl and treated with IM in the presence of nornal BMSCs were 35.1±6.1% and 18.6 ±4.1%, respectively.5.2 The mean apoptotic rates of K562 cells transduced with ShFZD7-2 or ShCtrl and treated with IM in the presence of CML BMSCs were 32.9±6.1% and 13.1 ±3.9%, respectively. These results indicated that BMSCs protected CML cells from IM-induced apoptosis and FZD7 down-regulation reversed IM resistance induced by BMSCs.6. Down-regulation of FZD7 abrogated the activation of Wnt/β-catenin signaling induced by BMSCs6.1 Western blot assay showed that FZD7 shRNA lentiviral particles abrogated the up-regulation of β-catenin and MDR1 induced by BMSCs derived from both normal and CML bone marrow.6.2 Real-time RT-PCR revealed that FZD7 shRNA lentiviral particles reversed the up-regulation of FZD7, MDR1 and CD44 induced by BMSCs derived from both normal and CML bone marrow.Conclusion:1. Compared to normal CD34+cells, FZD7 was up-regulated in CML CD34+cells.2. BMSCs up-regulated levels of FZD7, β-catenin as well as Wnt downstream genes in CML cells.3. Down-regulation of FZD7 induced growth inhibition and enhanced imatinib sensitivity in K562 cells.4. FZD7, involved in Wnt/β-catenin signaling, played a critical role in the contact between BMSCs and the CML cells, indicating FZD7 could be a potential therapeutic target for CML.