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. 2020 Jul 8;20(1):214.
doi: 10.1186/s12906-020-03006-2.

Matrine inhibits the growth of natural killer/T-cell lymphoma cells by modulating CaMKIIγ-c-Myc signaling pathway

Jianyou Gu  1   2 , Yu Zhang  1 , Xiao Wang  1   2 , Jingjing Xiang  1 , Shu Deng  1 , Dijiong Wu  1 , Junfa Chen  1 , Lihong Yu  1 , Yan Zhou  1 , Yaokun Wang  3 , Jianping Shen  4
Affiliations

Matrine inhibits the growth of natural killer/T-cell lymphoma cells by modulating CaMKIIγ-c-Myc signaling pathway

Jianyou Gu et al. BMC Complement Med Ther. .

Abstract

Background: C-Myc overexpression is associated with poor prognosis and aggressive progression of natural killer/T-cell lymphoma (NKTCL). Matrine, a main alkaloid of the traditional Chinese herb Sophora flavescens Ait, has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells. The present study investigated the effects and possible mechanisms of matrine inhibiting the growth of natural killer/T-cell lymphoma cells.

Methods: The effects of matrine on the proliferation, apoptosis and expression of apoptotic molecules, STAT3, LMP1, RUNX3, EZH2 and activation of CaMKIIγ/c-Myc pathway were examined in cultured NKTCL cell line NK92 cells.

Results: In cultured NK92 cells, matrine inhibited the proliferation in a dose and time dependent manner. The IC50 value of matrine was 1.71 mM for 72 h post exposure in NK92 cells. Matrine induced apoptosis with decreased Bcl-2 expression and the proteasome-dependent degradation of c-Myc protein in NK92 cells. c-Myc protein half-life in NK92 was reduced from 80.7 min to 33.4 min after matrine treatment, which meant the stability of c-Myc was decreased after matrine exposure. Furthermore, we found that matrine downregulated c-Myc phosphorylation at Ser62 together with the inhibition of CaMKIIγ, a key regulator of c-Myc protein in NKTCL. The downregulation of c-Myc transcription by matrine was mediated through LMP1 inhibition. We also observed that anti-proliferative activity of matrine was irrelevant to STAT3, RUNX3 and EZH2.

Conclusions: The results of the present study indicated that matrine inhibits the growth of natural killer/T-cell lymphoma cells by modulating LMP1-c-Myc and CaMKIIγ-c-Myc signaling pathway.

Keywords: C-Myc; CaMKIIγ; LMP1; Matrine; NK/T-cell lymphoma; NK92 cell.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Anti-proliferation and apoptosis induction of matrine in NKTCL cells. a NK92 cells and PBMCs were treated with matrine and vindesine at different concentrations for different times. The total viable cells were determined by MTT assay. b NK92 cells were exposed to matrine at different concentrations for 48 h and then determined for apoptotic cells by annexin V and PI staining using flow cytometry. c Percentage (%) of apoptotic cells induced by matrine at various concentrations. Analyses in triplicates. (*p < 0.05, **p < 0.01 compare to 0 mM group)
Fig. 2
Fig. 2
Matrine induced apoptosis of NKTCL cells via activation of the mitochondrial pathway. a NK92 cells were treated with 1.96 mM matrine for 48 h, followed by western blot. GAPDH was used as a loading control. b The relative intensities of target proteins were normalized to those of GAPDH. Analyses in triplicates. (n.s., not significant; *p < 0.05)
Fig. 3
Fig. 3
Matrine inhibited NKTCL cells independent of JAK/STAT3 pathway. a NK92 cells were treated with 1.96 mM matrine for 48 h, followed by western blot for STAT3, p-STAT3 (Tyr705) antibodies. GAPDH was used as a loading control. b The relative intensities of target proteins were normalized to those of GAPDH. Analyses in triplicates. (*p < 0.05, **p < 0.01)
Fig. 4
Fig. 4
Decreased c-Myc protein induced by matrine and rescued by proteasome inhibitor. a Effect of matrine on c-Myc protein expression in NK92 cells. NK92 cells were treated with matrine at 1.96 mM for 48 h, and c-Myc protein levels were measured by western blot. b c-Myc mRNA levels in NK92 cells were determined by quantitative RT-PCR at 24 h after matrine treatment at 0, 1.2, 2.4, and 3.6 mM (*p < 0.05, **p < 0.01 compared to 0 mM group). c CHX chase assay for the half-time of c-Myc. NK92 cells were treated with or without 1.96 mM matrine for 12 h. Cells were then treated with CHX (100 μg/mL) for the indicated minutes, and western blotting was performed. d c-Myc levels were quantified relative to GAPDH levels and graphed as percent c-Myc protein remaining after CHX treatment. Half-lives of c-Myc were calculated from exponential line equations and shown for each treat. e c-Myc protein levels were determined at 6 h post-treatment of MG132 and/or matrine, and (f) the relative intensities of target proteins were normalized to those of GAPDH. Analyses in triplicates. (*p < 0.05, **p < 0.01)
Fig. 5
Fig. 5
Matrine inhibited NKTCL cells through CaMKIIγ/c-Myc pathway. a NK92 cells were treated with 1.96 mM matrine for 48 h, followed by western blot for c-Myc, p-c-Myc (Ser62), CaMKIIγ and LMP1 antibodies. GAPDH was used as loading control. b The relative intensities of target proteins were normalized to those of loading control (**p < 0.01). c LMP1, RUNX3, EZH2, miR-26a, miR-26b and miR-101 transcription levels in NK92 cells were determined by quantitative RT-PCR at 24 h after matrine treatment at 0, 1.2, 2.4, and 3.6 mM (*p < 0.05, **p < 0.01 compared to 0 mM group). Analyses in triplicates
Fig. 6
Fig. 6
Cartoon diagram of the mechanisms for growth suppression of NKTCL cells by matrine. Matrine inhibits the transcription of c-Myc through the downregulated LMP1 protein. Matrine downregulates c-Myc phosphorylation at Ser62 through CaMKIIγ inhibition, and then promotes the destabilization and degradation of c-Myc protein in a proteasome-dependent manner
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