The Effects Of Calmodulin Antagonist Tetrandrine Citrate On The Growth In Vitro And In Vivo Of Chronic Myeloid Leukemia And Its Mechanisms

Background:Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by BCR-ABL, a chimeric gene generated as a result of a reciprocal translocation [t(9;22)(q34;q11), cytogenetically visible as the Philadelphia chromosome (Ph)] that places sequences from the ABL gene from chromosome9downstream of the BCR gene on chromosome22. The resultant oncogenic Bcr-Abl kinase is essential for the growth of CML cells and has become an attractive target for treatment of Ph+CML cases. Inhibition of Bcr-Abl with Abl tyrosine kinase inhibitors (TKIs), such as imatinib(IM), is highly effective in controlling CML. Targeted therapy with these TKIs has markedly improved the outcome of Ph+CML. However, resistance to imatinib has been observed especially in more advanced phases of the disease. Mutation or amplification of the Bcr-Abl, Pgp overexpression, clonal evolution are known to be involved in imatinib treatment failures. This is largely due to the inability of these kinase inhibitors to kill leukemia stem cell (LSC) responsible for tumor initiation, drug resistance and relapse in myeloid leukemia. Therefore, eradication of LSCs is required for developing curative therapies for CML, which relies on identification of specific targets in leukemia stem cells, and the insensitive of the TKIs to leukemia stem cells. Many different approaches to overcome clinical resistance to imatinib have been reported, such as dual Src-family kinase/ABL kinase inhibitory effects on imatinib-resistant leukemias. Though these novel drugs are known to inhibit the growth of resistant cells, obvious side effects or high cost limits their usefulness. So the major concern in CML treatment is to develop novel agents with more potent anti-tumor activity and low toxicity.Tetrandrine, a plant natural compound from Stephamia tetrandra S. Moore, is a bis-benzylisoquinoline alkaloid. It is a traditional Chinese medicine (TCM) that has been widely used to treat silicosis and arthritis for decades in China. Recent studies showed that tetrandrine inhibited the cell growth of a variety of human tumors such as HepG-2cells, A549cells, HT29cells,U937cells and HL-60cells. In this study, we investigated the effect and mechanism of tetrandrine on CML in vivo and in vitro.PartiAnti-proliferation Effect of tetrandrine citrate on CML CellsObjective:To investigate the effect of tetrandrine citrate on the growth of K562cells, Imatinib-resistant K562R cells and human primary CML cells.Methods:MTT assay was used to determine the inhibitory effect of tetrandrine citrate on the growth of K562cells, Imatinib-resistant K562R cells and human primary CML cells. Morphological analysis was used to detect apoptosis. Flow cytometry was performed to characterize cell cycle profile upon tetrandrine citrate treatment. Methylcellulose colony-forming assay with MethoCult GF H4434was used to measure leukemia colony forming cells according to the manufacturer's protocol. CD34+cells in chronic myeloid leukemia colony-forming units (CML CFUs) were identified by immunofluorescent staining. Briefly, CML CFUs in primary CML specimens were cultured in tetrandrine citrate using methylcellulose colony-forming assay for14days, and then stained with CD34antibody-R conjugated to phycoerithrin. Cells were counterstained with4',6-diamidino-2-phenylindole (DAPI), and imaged using fluorescence microscopy with a ZEISS fluorescent microscope. Colonies were scored via microscopy after10to14days of culture. Western blot was used to measure the BCR-ABL protein levels.Results:Cells were cultured in the presence of various concentrations of tetrandrine citrate for24hours,48hours and72hours and cell viability was measured by MTT assay. The inhibition concentration that inhibited50%of target cells (IC50) was calculated from OriginPro7.5. Proliferation of both of K562and K562R cells was significantly inhibited by tetrandrine citrate (48h IC50values were2.78μ.g/ml and3.5lμg/ml, respectively), indicating that tetrandrine citrate inhibited the proliferation of K562cells, K562R cells. Similarly, primary leukemia cells from five CML patients also showed a strong cytotoxic response to tetrandrine citrate. MTT assay revealed that tetrandrine citrate inhibited the proliferation of K562cells, K562R cells in a dose-and time-dependent manner. In contrast, normal hematopoietic cells from peripheral blood of six healthy donors showed less cytotoxic response to tetrandrine citrate. Thus, tetrandrine citrate is much more cytotoxic to CML cells than to normal blood cells, suggesting that tetrandrine citrate preferentially inhibits the growth of CML cells. Morphological changes of cells undergoing apoptosis were observed under light microscope. Flow cytometric analysis revealed that apoptotic cells increased from various concentrations of tetrandrine citrate for48hours. To assess the survival of tetrandrine citrate-treated CML leukemic primitive stem/progenitor cells, we used methylcellulose colony assays. Four primary CML specimens were treated with tetrandrine citrate (2.0or6.0μg/ml;14days), and the leukemia colony-forming units (CFUs) were scored after14days of culture. The number of CML CFUs was dramatically reduced by tetrandrine citrate treatment. To further confirm ablation of the CML stem/progenitor cells, we investigated the effect of tetrandrine citrate on CML CD34+CFUs by immunofluorescent staining. CML CFUs were treated with tetrandrine citrate (2.0μg/ml;14days) and then stained with anti-CD34antibody, tetrandrine citrate treatment resulted in a dramatic inhibition of the CML CFUs and loss of CD34+cells. Since Bcr-Abl protein has been shown to play a critical role in CML treatment, we determined whether tetrandrine citrate can affect expression of p210Bcr-Abl protein in K562R cells using Western blotting. K562R cells were treated with tetrandrine citrate at various concentrations for indicated times, and total cellular proteins were extracted for Western blotting analysis of p210BcrAbl protein. We also simultaneously evaluated effect of imatinib on expression of p210BcrAbl protein in K562R cells. Experimental results showed that tetrandrine citrate treatment markedly decreased p210BcrAbl protein levels of K562R cells.Conclusion:Tetrandrine citrate inhibited the proliferation of K562cells, Imatinib-resistant K562R cells and human primary CML cells and induced apoptosis. Tetrandrine citrate treatment resulted in a dramatic inhibition of the CML CFUs and loss of CD34+cells. Experimental results showed that tetrandrine citrate treatment markedly decreased p210BcrAbl protein levels of K562R cells.Part2The effect of tetrandrine citrate on Imatinib-resistant K562R leukemia xenograft model in vivoObjective:To investigate the effect of tetrandrine citrate on Imatinib-resistant K562R leukemia xenograft model in vivo.Methods:All animal procedures were approved by the Institution's Ethics Committee. BABL/C nu/nu mice (nu-/-;6-7-weeks-old) were injected subcutaneously in the left or right flank with2×107log-phase K562R cells in a0.2-ml suspension. When the tumors reached volumes of50to100mm3, the mice were randomly assigned to two groups (treated group and control group), tetrandrine citrate or imatinib was administered orally at a dose of100mg/kg (in0.4ml) three times a day, for10consecutive days. The mice in the control group were given equal volumes of water. Mouse weight and tumor volumes were measured every five days. Tumor weight was measured at the end of experiments.Results:we evaluated the effects of this compound on Imatinib-resistant K562R cells xenografts in nude mice by oral administration. We investigated the in vivo anti-leukemia activity of against K562R cells in nude mice. The nude mice bearing Imatinib-resistant K562R xenografts were treated with imatinib for10consecutive days. Tumor weights in control and Imatinib-treated groups were2.44±0.25g and1.02±1.23g, respectively, while body weights were14.43±1.57g and15.00±3.78g, respectively. These results indicate that imatinib can partially inhibit the growth of the Imatinib-resistant K562R xenografts. All mice in the control group developed tumors, with an average tumor weight of2.20±0.77g at35days. Tumor growth was significantly inhibited when the mice were treated with tetrandrine citrate, their average tumor weight of0.135±0.32g at35days. Tetrandrine citrate toxicity was limited. The body weights in control and tetrandrine citrate-treated groups were15.09±1.23g and20.13±0.92g, respectively. The weights of tumor-bearing mice treated by tetrandrine citrate increased, but decreased in untreated mice with the development of tumors. No gross abnormalities were observed in tetrandrine citrate-treated groups at the end of experiments. These results suggest that tetrandrine citrate treatment is safe at least for mice. Experimental results showed that xenografts of Imatinib-resistant K562R cells were significantly inhibited and no obviously toxic or side effects were observed during experiments.Conclusion:Tetrandrine citrate can display an anti-chronic myeloid leukemia effect without obvious side effects in vivo. Part3Molecular mechanisms by which tetrandrine citrate eliminates chronic myeloid leukemia cellsObjective:To evaluate effects of tetrandrine citrate on elimination of chronic myeloid leukemia and its mechanisms.Methods:Cellular protein was extracted using the Mammalian Protein Extraction Reagent for western blotting as previously described. CaMKⅡ γ, pCaMKⅡ γ and β-catenin protein level were determined with western blotting. To further assess the role of CaMKⅡ γ in leukemia cells, we established a stable CML cell line with high CaMKⅡ γ expression using human K562cells transfected with EGFP-tagged CaMKⅡ γ expression plasmid. we analyzed subcellular localization of this kinase and effect of CaMKⅡ γ on cell cycle of CML cells using flow cytometry.Results:CaMKII y kinase was examined in primary leukemia cells using western blot, and found to be expressed at significantly higher levels in blast crisis CML and weekly or undetectable in chronic phase CML and normal hematopoietic cells from healthy cord blood samples. To determine if this aberrantly upregulated expression of CaMKⅡ γ in tumor cells of CML in blast crisis is associated with leukemia stem cell of CML, we then examined expression of β-catenin. Western blot analysis revealed that expression of CaMKⅡ γ was well correlated with β-catenin in CML patient samples, suggesting that CaMKⅡ γ might play an important role in P-catenin-driven leukemia stem cell. We therefore further analyzed CaMKII y in CD34+CD38" CML cells sorted by FACS from CML patients. Western blot analysis showed that both total and phosphorylated CaMKⅡ γ proteins were highly expressed in the CD34+CD38-CML cells, but low in CD34-CML cells. Notably, we found that both total and phosphorylated CaMKⅡ γ proteins were weakly expressed or undetectable in CD34+ CD38" cells from healthy cord bloods. To further assess the role of CaMKⅡγ in leukemia cells, we established a stable CML cell line with high CaMKⅡ γ expression using human CML K562cells transfected with EGFP-tagged CaMKⅡ γ expression plasmid. we analyzed subcellular localization of this kinase, and found that CaMKⅡ γ protein level greatly differed in the cells at different phases of the cell cycle. In nonproliferating cells (Go phase), CaMKⅡ γ protein level was low. However, once cells enter proliferating status, CaMKⅡ γ protein expression level dramatically increased with cell-cycle progression, peaked in early S/G2phase cells, and then decreased. These results suggest that CaMKⅡ γ protein may play a role in initiating proliferation of CML cells. To substantiate these observations, we further analyzed effect of CaMKⅡ γ on cell cycle of pcDNA3.1-CaMKⅡ γ-EGFP K562cells using flow cytometry (FCM). Consistent with above results, FCM analysis indicated that ectopic expression of CaMKⅡ γ caused a significant increase of the number of cells in G2/M phase compared with mock. The percentages of the cells in the G2/M phase were increased by37.16%(compared with mock), suggesting that CaMKⅡ γ promotes S-G2/M transition of cell cycle progression, which is responsible for the growth promotion of CML cells by CaMKⅡ γ. Tetrandrine citrate is a calmodulin antagonist. CaMKⅡ γ and pCaMKII y protein level were determined with western blotting. The phosphorylation level of CaMKⅡ γ decreased prominently in the cells with the tetrandrine citrate treatment, while the total CaMKⅡ γ protein do not change. To gain an insight into the mechanism by which tetrandrine citrate inhibited CaMKⅡ γ activity. β-catenin in the Wnt/β-catenin signaling pathway, which is stabilized by the Bcr-Abl protein level, has been linked to the survival and self-renewal of leukemia stem cells. Therefore, we assessed the effect of tetrandrine citrate on the β-catenin protein levels of K562R cells. K562R cells were treated with tetrandrine citrate at various concentrations for indicated times, and total cellular proteins were extracted for Western blotting analysis of β-catenin protein. The Western blot results indicated that tetrandrine citrate treatment caused a significant decrease of β-catenin protein levels of leukemia cells in dose-and time-dependent manners. These results indicate that tetrandrine citrate can also reduce β-catenin protein levels in leukemia cells.Conclusion:CaMKⅡ γ kinase was expressed at significantly high levels in primary leukemia cells at blast crisis CML. Both total and phosphorylated CaMKⅡ γ proteins were highly expressed in the CD34+CD38-CML cells, but low in CD34-CML cells. CaMKⅡ γ protein expression level increased with cell-cycle progression. Tetrandrine citrate can inhibited CaMKⅡ γ activity and also reduce β-catenin protein levels in leukemia cells.