Abstract
BackgroundC-Myc aberrations confer a more aggressive clinic behavior in diffuse large B-cell lymphoma (DLBCL). Matrine is an alkaloid extracted from Sophora flavescens Ait. It possesses anti-cancer property through inhibiting the cell proliferation and inducing the apoptosis. The present study aimed to explore the underlying mechanisms of matrine in suppressing the cell growth of DLBCL.MethodsThe influence of matrine on the viability of cultured DLBCL cell lines SU-DHL-16 and OCI-LY3 cells were determined by CCK-8. Apoptosis and cell cycle were measured by flow cytometry after matrine exposure. Western blot was taken to investigate the expression of activated Caspase-3, cleaved PARP, c-Myc, phospho-c-Myc (Ser62), CaMKIIγ, phospho-CaMKIIγ (Thr287), CDK4 and CDK6 after matrine treatment. Cycloheximide chase analysis was used to determine the c-Myc protein half-lives before and after matrine treatment. Growth salvage analysis was taken by ectopic expression of c-Myc.ResultsIn cultured DLBCL cells, matrine suppressed cell viability in a concentration and time dependent fashion. Matrine treated SU-DHL-16 and OCI-LY3 cells for 48 h with IC50 value of 1.76 mM and 4.1 mM, respectively. Matrine induced apoptosis through a caspase-independent pathway and caused G0/G1 cell cycle arrest in a concentration dependent manner in DLBCL cells. The protein expression of c-Myc was inhibited while the transcription of c-Myc was not reduced by matrine. c-Myc protein half-lives were decreased from 30.4, 69.4 min to 16.6, 15.9 min after matrine treatment in SU-DHL-16 and OCI-LY3, respectively. As a critical protein kinase of c-Myc, CaMKIIγ phosphorylation at Thr287 was found to be down-regulated and c-Myc phosphorylation at Ser62 was reduced together after matrine treatment in DLBCL. The growth suppression of SU-DHL-16 cells induced by matrine was rescued by over-expression of c-Myc achieved by recombinant adenovirus infection. The decreased expression of CDK6, not CDK4, induced by matrine was rescued by ectopic expression of c-Myc protein.ConclusionsThis study has shown for the first time that matrine suppresses cell growth of DLBCL via inhibiting CaMKIIγ/c-Myc/CDK6 signaling pathway.
Highlights
C-Myc aberrations confer a more aggressive clinic behavior in diffuse large B-cell lymphoma (DLBCL)
Matrine inhibits the growth of DLBCL cells Human DLBCL cell lines SU-DHL-16 and OCI-LY3 were maintained in exponential phase with good morphology (Supplementary Figure 1)
Exposure to matrine for a longer time than 48 h was found to be more potent in inhibiting SU-DHL-16 and OCI-LY3 cell viability, it inclined to result in increased frequency of necrotic cells, so we Matrine induces apoptosis To explore whether the growth inhibition of DLBCL cells induced by matrine was caused by apoptosis, SUDHL-16 and OCI-LY3 cells were treated with the increasing concentrations of matrine for 48 h, and the occurrence of apoptosis was determined by the annexin V and PI staining (Fig. 2a and b)
Summary
C-Myc aberrations confer a more aggressive clinic behavior in diffuse large B-cell lymphoma (DLBCL). Diffuse large B-cell lymphoma (DLBCL) is the most common class of non-Hodgkin lymphomas with a highly heterogeneous group of diseases [1]. According to cell-oforigin determined by immunohistochemical expression of CD10, BCL6, and IRF4/MUM1, DLBCL can be assorted into two subtypes: germinal center B-cell lymphoma subtype and active B-cell/non-germinal center B-cell lymphoma [2]. The gold standard treatment for DLBCL patients is R-CHOP regimen (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) offering a 5-year overall survival of around 65%. About 35% patients develop relapsed/refractory disease after initial response, with 70% of the patients dying from lymphoma within the first 2 years after disease progression [3]. 10% of refractory/relapsed DLBCL patients can be cured by high-dose therapy with autologous stem cell transplantation [4]. The remaining 90% of patients are incurable, which demands new targeted agents for clinical trials
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