| Backgroud:Chronic myelogenous leukemia (CML) is a hematological malignancy characterized by abnormal expansion of differentiated myelogenous cells. The Philadelphia (Ph) chromosome is generated from the reciprocal translocation of chromosome9and22, and has been found in approximately95%of patients with CML. The presence of Ph chromosome can lead to deregulated tyrosine kinase activi(?) of Bcr-Abl protein resulting in CML. Imatinib as the first tyrosine kinase inhibitor has improved the outcome of patients with CML. Imatinib has emerged as the standard of treatment for patients with chronic phase (CP) CML with an8year overall survival of85%-93%. However, nearly35%of patients develop drug resistance during treatment of CML. Despite the high efficiency of tyrosine kinase inhibitor in CP CML, patients with T3151mutation and those with blastic phase (BP) CML remain incurable. What is worse, quiescent CML leukemia stem cells (LSC), which represent the potential source of relapse, have been still retained in patients achieving remission with tyrosine kinase inhibitors treatment. Recent evidence shows that the survival of CML stem cells is independent of Bcr-Abl kinase activity. Therefore, it is challenging to identify a more effective therapy of CML.Some natural herbal products have been used as effective treatments of cancers. Matrine, an alkaloid extracted from Sophora flavescens, has exhibited many pharmacological activities, such as anti-tumor, anti-inflammation, anti-fibrosis, anti-arrhythmia. Recently, intensive studies have suggested that matrine can inhibit proliferation and induce apoptosis of cancer cells. In the present study, we investigated the effect of matrine on CML cells with T3151mutation and CML LSC.The study was divided into two parts:Part Ⅰ:Effects of matrine treatment on proliferation, colony growth and apoptosis in CML cells;Part Ⅱ:The role of β-catenin/c-Myc signaling pathway in CML cells.Part Ⅰ:Effects of matrine treatment on proliferation, colony growth and apoptosis in CML cellsObject:The aim of this part was to determine the inhibition and apoptosis of matrine on CML cells in vitro and in vivo.Methods:Cell growth of K562cells treated by alkaloids extracted from Sophora flavescens was measured by MTT assay; Cell growth of CML cell lines and L02cells treated by matrine was examined by MTT assay; Colony growth, cell cycle and apoptosis of CML cell lines treated by matrine were measured by colony formation assay and flow cytometry; Cell growth and apoptosis of primary CML cells and normal bone marrow cells were detected by MTT assay and flow cytometry; CD34+cells were isolated by magnetic activated cell sorting separation; Cell growth of CML CD34+cells treated by matrine was measured by MTT assay; Colony growth and apoptosis of CML CD34+cells treated by matrine were examined by colony formation assay and flow cytometry; Furthermore, we investigated the effect of matrine on tumor growth in vivo.Results:(1) K562cells were more sensitive to matrine compared with sophocarpine, sophoridine and oxymatrine with IC50at24h of1.34mM;(2) Matrine inhibited cell growth of K562, K562/G,32Dp210and32Dp210-T315I cells while no effect was observed on L02cells; There were statistically significant differences (K562vs L02, p=0.017; K562/G vs L02, p=0.031;32Dp210vs L02, p=0.048;32Dp210-T315I vs L02, p=0.013);(3) Matrine inhibited colony growth of K562,32Dp210-T315I cells;(4)Matrine did not influence cell cycle regulation of K562cells;(5) Matrine induced apoptosis of CML cell lines including K562, K562/G,32Dp210and32Dp210-T315I;(6) Matrine inhibited cell growth of CP CML cells, BP CML cells and CML ceils with T315I mutation while only a slight effect was observed on normal cells; There were statistically significant differences (CP CML cells vs normal cells, p=0.017; BP CML cells vs normal cells, p=0.044; CML cells with T315I mutation vs normal cells, p=0.030);(7) Matrine induced significantly apoptosis of CP CML cells compared with CML cells with T315I mutation(CP CML cells vs normal cells, p=0.006; CML cells with T315I mutation vs normal cells, p=0.007);(8) Matrine inhibited cell growth of CML CD34+cells with IC50at24h of3.38mM;(9) Colonies were eliminated from CML CD34+cells compared with normal CD34+cells (p=0.002);(10) Matrine resulted in increased apoptosis of CP CML CD34+cells and CML CD34+cells compared with normal CD34+cells(CP CML CD34+cells vs normal CD34+cells, p=0.004; CML CD34+cells with T315I mutation vs normal CD34+cells, p=0.013);(11)50mg/kg and100mg/kg matrine resulted in significant tumor grow delay compared with control in vivo (Matrine(50mg/kg) vs control, p=0.000; Matrine(100mg/kg) vs control, p=0.000).Conclusions: (1) Matrine inhibited cell growth, colony growth and induced apoptosis of CML cell lines while no effect was observed on L02;(2) Matrine inhibited cell growth and induced apoptosis of CML cell lines while only a slight effect was observed on normal cells;(3) Matrine inhibited significantly cell growth, colony growth of CML CD34+cells compared with normal CD34+cells;(4) Matrine has a significant anti-leukemic effect in vivo.(5) Matrine inhibited cell growth and induced apoptosis of CML cells with T315I mutation and CML LSC with little toxicity on normal cells. Part II:The role of β-catenin/c-Myc signaling pathway in CML cellsObject:The aim of this part was to explore the role of β-catenin/c-Myc signaling pathway in CML cells.Methods:Total proteins from K562,32Dp210-T315I cells treated by matrine were analyzed by western blotting for Bcr-Abl and downstream proteins; Total proteins from K562,32Dp210-T315I cells treated by matrine were measured by western blotting for (3-catenin, c-Myc and apoptotic-related proteins; Real time PCR analysis was performed for detection of c-Myc transcripts from K562,32Dp210-T315I cells and CML CD34+cells; The expression of c-Myc protein in xenograft tumor was detected by immunohistochemistry; while c-Myc was inhibited by10058-F4, cell growth and apoptosis of K562,32Dp210-T315I cells and CML CD34+cells treated by matrine were measured by MTT assay and flow cytometry; Silencing c-Myc gene of THP-1cells by small interfering RNA (siRNA), cell growth and apoptosis of THP-1cells treated by matrine were measured by MTT assay and flow cytometry; Effects of caspase inhibitor z-VAD-fmk on cell growth of K562,32Dp210-T315I cells and CML CD34+cells treated by matrine were measured by MTT assay.Results:(1) Matrine did not regulate the protein levels of p-Bcr-Abl, Bcr-Abl, PI3K, p-Akt, p-STATS and p-ERK;(2) Matrine treatment resulted in reduction in total β-catenin and c-Myc levels in K562,32Dp210-T315I cells and CML CD34+cells treated by matrine, accompanied with the activation of caspase-9, caspase-3and PARP;(3) Real time PCR confirmed downregulation of c-Myc gene in K562,32Dp210-T315I cells and CML CD34+cells treated by matrine;(4)50mg/kg and100mg/kg matrine could decrease the expression of c-Myc protein in xenograft tumor compared with control in vivo;(5)10058-F4could enhance inhibition rate of matrine on K562(p=0.007),32Dp210-T315I (p=0.001) cells and CML CD34+cells (p=0.003);10058-F4could also increase apoptosis of matrine on K562(p=0.002) and CML CD34+cells (p=0.008);(6) While c-Myc gene of THP-1cells was silenced siRNA, inhibition rate of THP-1treated by matrine was obviously higher compared with negative control (p=0.001); apoptosis of THP-1treated by matrine increased significantly compared with negative control (p<0.05);(7) The inhibition rates of K562(p=0.003),32Dp210-T315I (p=0.021) cells and CML CD34+cells (p=0.002) treated by matrine were partially diminished by the addition of z-VAD-fmk.Conclusions:(1) Matrine downregulated β-catenin/c-Myc pathway resulting in apoptosis of CML cells;(2) Matrine decreased the expression of c-Myc protein in xenograft tumor in vivo;(3) c-Myc inhibitor could enhance the effect of matrine on CML CD34+cells;(4) The silence of c-Myc gene could enhance the effect of matrine on THP-1cells;(5) Matrine-induced apoptosis in CML cells was partially dependent on the activation of caspase. |
PDF Full Text Request