Matrine is a quinolizindine alkaloid with strong anticancer property towards various types of tumors. Han et al. reported that matrine inhibited human multiple myeloma RPMI8226 and U266 cells for 48 h with IC50 of 4.55 mM and 5.36 mM, respectively . Ma et al. showed that matrine suppressed the growth of human chronic myeloid leukemia K562 cells with an IC50 of 2 mM for 48 h treatment . Our previous study showed that matrine inhibited human NKTCL NK92 cells for 48 h with IC50 of 1.96 mM . In current study, matrine inhibited the growth of DLBCL cell lines SU-DHL-16 and OCI-LY3 cells in a dose and time dependent manner. It displayed anti-proliferative activities against SU-DHL-16 and OCI-LY3 cells at 48 h with IC50 of 1.76 ± 0.05 mM and 4.10 ± 0.05 mM, respectively. Matrine was more effective for SU-DHL-16 cells than that for OCI-LY3 cells, which meant matrine is better for germinal center B-cell lymphoma subtype than that for active B-cell lymphoma subtype. Vindesine is an alkaloid for the clinic treatment of DLBCL . It inhibited the growth of SU-DHL-16 and OCI-LY3 cells with IC50 of 1.33 ± 0.02 nM and 0.52 ± 0.02 nM for 72 h treatment, respectively. Considering the IC50 of matrine is much higher than that of vindesine, the influence of matrine on the normal lymphocytes is of much concern. Our previous study showed that matrine with lower than 2 mM did not significantly influence the proliferation of healthy human peripheral blood mononuclear cells (PBMCs) induced by PHA for 72 h . Han et al. disclosed that lower concentrations of matrine (1, 2, 4, 6 mM) had no influence on the PBMCs proliferation and higher concentrations of matrine (8, 12, 20 mM) inhibited the proliferation of PBMCs for 72 h . They also demonstrated that 2, 6, 12 and 20 mM matrine had no effects on the apoptosis of PBMCs for 48 h . Our data and previous literature strongly support that matrine with lower than 6 mM has no harmful effects on the normal PBMCs, which will be beneficial for DLBCL patients.
Our previous study showed that matrine induced caspase-dependent apoptosis in NKTCL cells . In present study, matrine induced apoptosis of DLBCL cell lines SU-DHL-16 and OCI-LY3 cells in a dose dependent manner from 2.8 ± 0.4% and 2.4 ± 0.2% to 46.2 ± 2.3% and 35.6 ± 1.2% after matrine treatment at different concentrations for 48 h, respectively (Fig. 2). It is interesting to notice that apoptosis caused by 2 mM matrine in SU-DHL-16 cells was less than 14% even though this dose was more than IC50 (1.76 mM). The apoptosis induced by 4 mM matrine in OCI-LY3 cells was near to the control group although this dose was close to IC50 (4.1 mM). These data suggests matrine does not preferentially induce apoptosis. The growth inhibition of DLBCL cells induced by matrine was partially caused by apoptosis. Furthermore, matrine did not induce the products of activated cleaved-Caspase-3 and cleaved PARP even with 2 fold of IC50 in DLBCL cells (Fig. 2). The execution of apoptosis comprises both caspase-dependent and caspase-independent pathways. Apoptosis inducing factor (AIF) was critical for caspase-independent cell death by direct interaction with DNA . Our finding indicated that matrine induces apoptosis of DLBCL cells through the activation of caspase-independent pathway. The detailed molecular mechanism of caspase-independent cell death induced by matrine in DLBCL cells need to be addressed in the future.
Deregulation of cell cycle is an important process in malignant transformation. Zhao et al. reported that matrine inhibited the growth of human retinoblastoma cells and induced cell cycle arrest at G0/G1 phase . Jin et al. showed that matrine suppressed melanoma M21 cells proliferation by promoting G0/G1 cell cycle arrest . Ma et al. reported that matrine inhibited human chronic myeloid leukemia K562 cells proliferation through promoting G0/G1 arrest . Our data showed that matrine induced the accumulation of SU-DHL-16 and OCI-LY3 cells in the G0/G1 phase, which finally led to the cell growth inhibition (Fig. 3). G0/G1 cell cycle arrest is the major mechanism for the matrine induced growth inhibition in DLBCL cells.
c-Myc levels tightly correlate with cell proliferation. A major role for c-Myc in the proliferation of normal cells is to promote progression through G1 and into S phase of the cell cycle. A systematic study in 23 cell lines with short-hairpin-mediated depletion of c-Myc showed that arrest occurred at G0/G1 phase in normal cells and some tumor-derived cell lines . c-Myc genetic alternations are the characterized events in DLBCL, which confer a more aggressive clinical behavior with dismal prognosis . Our previous data showed that matrine inhibited the mRNA and protein expression of c-Myc in NKTCL NK92 cells . Our present data showed that c-Myc protein expression was inhibited by matrine in DLBCL cells while the gene transcription of c-Myc was not suppressed (Fig. 4). The degradation of c-Myc protein was accelerated by matrine treatment in DLBCL cells. c-Myc protein half-lives were much shorter after exposure to matrine in SU-DHL-16 and OCI-LY3 cells (Fig. 4), which meant the stabilities of c-Myc in matrine-treated DLBCL cells are decreased. The ectopic expression of c-Myc rescued the growth of matrine-treated SU-DHL-16 cells by recombinant adenovirus infection (Fig. 6), which verified that matrine inhibits the growth of SU-DHL-16 cells by c-Myc pathway.
Based on the classical cell cycle model, in the early G1 phase, mitogenic signals are first received and integrated by the expression of cyclin D that preferentially binds to and activates CDK4 and CDK6. c-Myc-induced cell proliferation is related to the increase of CDK4 and CDK6 activities to regulate G1/S progression . CDK4 and CDK6 were listed as transcriptional targets of c-Myc. Hermeking et al. reported that c-Myc induced a rapid increase of CDK4 mRNA levels through four highly conserved c-Myc binding sites within the CDK4 promoter among different cell models . Li et al. showed that c-Myc bound to CDK6 promoter in ChIP-on-chip analysis in the Burkitt's lymphoma Daudi cells . Our data showed that the expressions of CDK4 and CDK6 protein were significantly inhibited after matrine treatment in DLBCL cells (Fig. 5). The ectopic expression of c-Myc rescued the expression of CDK6, not CDK4, in the matrine treated SU-DHL-16 cells (Fig. 6), which identified that CDK6 is a bona fide c-Myc target gene in SU-DHL-16 cells. These results verified that matrine inhibits the growth of SU-DHL-16 cells through c-Myc-CDK6 pathway.
CaMKIIγ was reported to phosphorylate Ser62 of c-Myc and increased the stability of c-Myc in T cell lymphoma. Inhibition of CaMKIIγ ameliorated T cell lymphoma burden in mice . Our previous study showed that matrine inhibited the growth of NKTCL cells by modulating CaMKIIγ-c-Myc pathway . Present study showed that a positive correlation between CaMKIIγ and p-c-Myc (Ser62)/c-Myc was observed in DLBCL cells. The levels of phospho-c-Myc (Ser62) and CaMKIIγ in SU-DHL-16 and OCI-LY3 cells were together remarkably reduced after matrine treatment (Fig. 5). Our findings indicated that matrine suppresses cell growth of DLBCL by regulating CaMKIIγ/c-Myc pathway. The specific functions of CaMKIIγ in DLBCL are not fully known, our data support that CaMKIIγ inhibition may be a great way to treat c-Myc-driven DLBCL.
Indirect inhibition of c-Myc represents a great opportunity to cure associated cancers. The first small molecule bromodomain inhibitor, JQ1, inhibited the c-Myc function and its target genes in DLBCL . Alkaloids, such as matrine, berberine and vindesine, are strong therapeutic agents for cancers. Ma et al. reported that one of berberine derivations, quinolino-benzo-[5, 6]-dihydroisoquindolium compound, inhibited the c-Myc transcription by selectively binding G-quadruplex c-Myc DNA in leukemia cell line HL60 . Small molecule analog of berbamine, tosyl chloride-berbamine, inhibited CaMKIIγ expression to decrease c-Myc protein in c-Myc-positive leukemia cells . Our data confirmed that matrine accelerated c-Myc protein degradation via CaMKIIγ inhibition in DLBCL cells. CaMKIIγ/Myc axis represents a promising target in MYC-mediated DLBCL. Matrine will be helpful for those c-Myc-driven DLBCL patients.
Limitations of this study focus on the DLBCL cell lines. The particular mechanism of apoptosis induction by matrine in DLBCL needs further exploration. The effects of matrine on primary DLBCL cells and DLBCL in vivo need to be investigated in the future.