Department of Dermatology, the First Affiliated Hospital of Nanjing Medical University.
Department of Dermatology, The Affiliated Hospital of Nanjing University of Chinese Medicine.
Life Sci. 2018 Oct 1;210:150-157. doi: 10.1016/j.lfs.2018.08.068. Epub 2018 Aug 31.
This study aimed to identify the mechanism of how MG-132 stimulates cell death in SEB-1 sebocytes.
TUNEL staining and annexin-FITC/PI flow cytometry were utilized to examine the apoptotic cell number of SEB-1 sebocytes and HaCaT keratinocytes upon MG-132 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment. MTT assay and CCK-8 assay monitored the proliferative rate and viability of both cell lines with different treatment. Western blotting (WB) and qPCR were performed to detect the expression of TRAIL and members of Bcl-2 family at protein and gene level. Additionally, RNA interfering was used to knockdown the mRNA transcription of TRAIL and BIK gene.
MG-132 treatment enhanced cell death in SEB-1 sebocytes but not in HaCaT keratinocytes. Meanwhile, TRAIL concentrations in SEB-1 sebocytes treated with MG-132 were markedly elevated. Furthermore, treatment with TRAIL or the TRAIL receptor-specific monoclonal antibody AY4 at various doses stimulated cell death in SEB-1 sebocytes in a time- and dose-dependent manner. Silencing of TRAIL restored the cell viability of SEB-1 cells to a normal level after MG-132 treatment. Combined treatment of SEB-1 sebocytes with TRAIL and MG-132 synergistically triggered cell death, suppressed cell proliferation and survival, and promoted BIK expression. Furthermore, BCL2 Interacting Killer (BIK) knockdown via RNA interference participated in the recovery of cell survival reduced by treatment with TRAIL and MG-132.
These findings suggest that treatment with the selective proteasome suppressor MG-132 and TRAIL induces cell death in sebocytes through upregulation of BIK, a member of the Bcl-2 family.
本研究旨在确定 MG-132 刺激 SEB-1 皮脂腺细胞死亡的机制。
采用 TUNEL 染色和 Annexin-FITC/PI 流式细胞术检测 MG-132 和肿瘤坏死因子相关凋亡诱导配体（TRAIL）处理后 SEB-1 皮脂腺细胞和 HaCaT 角质形成细胞的凋亡细胞数。MTT 法和 CCK-8 法检测不同处理方式下两种细胞系的增殖率和活力。Western blot（WB）和 qPCR 检测 TRAIL 和 Bcl-2 家族成员在蛋白和基因水平的表达。此外，采用 RNA 干扰技术敲低 TRAIL 和 BIK 基因的 mRNA 转录。
MG-132 处理增强了 SEB-1 皮脂腺细胞的细胞死亡，但对 HaCaT 角质形成细胞没有影响。同时，MG-132 处理后的 SEB-1 皮脂腺细胞中 TRAIL 浓度明显升高。此外，用不同剂量的 TRAIL 或 TRAIL 受体特异性单克隆抗体 AY4 处理 SEB-1 皮脂腺细胞可呈时间和剂量依赖性地刺激细胞死亡。沉默 TRAIL 后，MG-132 处理后的 SEB-1 细胞活力恢复正常水平。TRAIL 和 MG-132 联合处理 SEB-1 皮脂腺细胞可协同触发细胞死亡，抑制细胞增殖和存活，并促进 BIK 表达。此外，通过 RNA 干扰敲低 BCL2 相互作用杀伤因子（BIK）可参与 TRAIL 和 MG-132 处理降低细胞存活的恢复。
这些发现表明，用选择性蛋白酶体抑制剂 MG-132 和 TRAIL 联合处理可通过上调 Bcl-2 家族成员 BIK 诱导皮脂腺细胞死亡。
