Résumé
@# Objective：To investigate the role and mechanism of chromosomal region maintenance 1 (CRM1) inhibitor sulforaphene (LFS-01) in killing triple negative breast cancer (TNBC) cells by inhibiting signal transducer and activator of transcription 3 (STAT3) signaling pathways. Methods: Whether LFS-01 could combine with the NES pocket of CRM1 was verified by molecular dynamics simulation techniques. The killing activity of LFS-01 on four different breast cancer cell lines was detected by CCK-8 method. TNBC MDA-MB-468 and MDA-MB-231 cells were treated with different concentrations of LFS-01, and the intracellular localization of CRM1 cargo protein STAT3 and protein with NES sequence was detected by immunofluorescence; WB was used to detect the effect of LFS-01 on the expression of STAT-3 signaling pathway and its downstream proteins; WB, cellular immunofluorescence and transmission electron microscopy were adopted to detect the occurrence of autophagy; the effect of LFS-01 on cell cycle and apoptosis was detected by flow cytometry. Results: Molecular dynamics simulations showed that LFS-01 can bind to the NES pocket of CRM1, indicating that it may structurally affect the latter's protein transport function. LFS-01 could specifically kill TNBC MDA-MB-468 and MDA-MB-231 cells. STAT3 and NES-tagged proteins were mainly blocked in the nucleus of TNBC cells after the treatment with 10 μmol/L LFS-01, while they were evenly distributed in the cytoplasm in the control group. The expressions of phosphorylated STAT3 protein, Bcl-xL and Cylin D1 were decreased in MDA-MB-468 and MDA-MB-231 cells with the increase of LFS-01 dose and the prolongation of treatment time; the expression of autophagy marker protein LC3B increased, and highdensity, multi-layered autophagosomes appeared at the same time; cell cycle arrest was observed in S phase and apoptosis rate was significantly increased (P<0.05 or P<0.01). Conclusion: LFS-01 blocks the export of CRM1 carrier protein, thereby inhibiting the activation of STAT3 signaling pathway and promoting autophagy, cell cycle arrest and apoptosis in TNBC MDA-MB-468 and MDAMB-231 cells.
Résumé
Objective: To investigate the expression level of chromosomal region maintenance 1 (CRM1) in mantle cell lymphoma, and to explore the possible mechanism of naturally derived sulforaphene LFS-01 inhibiting the proliferation of mantle cell lymphoma cells. Methods: The expression of CRM1 in mantle cell lymphoma was analyzed by Oncomine data mining. After treatment with LFS-01, the viability of mantle cell lymphoma JeKo-1 cells was detected by CCK-8 assay, the nuclear transport function of CRM1 protein was measured by laser confocal microscopy, the expression of CRM1 protein was detected by Western blotting, the cell cycle arrest and apoptosis were analyzed by FCM and transmission electron microscopy, the expression change of apoptosis pathway-related proteins was detected by Western blotting, and the impact of caspase inhibitor z-VAD-FAM on the effects of LFS-01 was detected by CCK-8 assay and FCM. Lentiviral infection was used to establish a stable JeKo-1 cells expressing mutant CRM1 (C528S), then the effects of LFS-01 on the nuclear transport function of CRM1 and the proliferation of JeKo-1 cells were detected by laser confocal microscopy and CCK-8, respectively. The transcriptional level in JeKo-1 cells after LFS-01 treatment was detected by RNAseq, and the viability of JeKo-1 cells treated with Toll-like receptor (TLR) inhibitor TAK-242 and LFS-01 was measured by CCK-8 assay. Results: CRM1 was overexpressed in mantle cell lymphoma (P < 0.05). LFS-01 inhibited the proliferation of JeKo-1 cells, and the 50% inhibitory concentration (IC50) at 24 h and 48 h were 5.81 μmol/L and 9.09 μmol/L, respectively. 20.0 μmol/L LFS-01 inhibited the nuclear transport function of CRM1 (P < 0.05), 6.0 μmol/L LFS-01 down-regulated the expression level of CRM1 (P < 0.05), 10.0 μmol/L LFS-01 induced cell cycle arrest at G2/M phase and induced apoptosis (both P < 0.05), and 4 μmol/L LFS-01 up-regulated the expression levels of poly ADP-ribose polymerase (PARP), cleaved caspase-3 and cleaved caspase-9 (all P < 0.01). The caspase inhibitor z-VAD-FAM reversed the apoptosis-induction effect of LFS-01 (P < 0.01). CRM 1 mutation eliminated the effects of LFS-01 on CRM1 function and cell proliferation (both P < 0.05). LFS-01 inhibited the cell proliferation-related pathways (P < 0.01), and TLR inhibitor TAK-242 combined with LFS-01 had synergetic inhibitory effect on JeKo-1 cells (P < 0.01). Conclusion: LFS-01 can inhibit the growth of mantle cell lymphoma cells through inducing cell cycle arrest and apoptosis, and CRM1 is essential in this process. In addition, LFS-01 and TLR inhibitor TAK-242 have synergetic inhibitory effect on mantle cell lymphoma cells.
Résumé
The function of the herpes simplex virus type 1(HSV-1)UL4 protein is still elusive. Our objective is to investigate the subcellular transport mechanism of the UL4 protein. In this study,fluorescence microscopy was employed to investigate the subcellular localization of UL4 and characterize the transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein(EYFP),the nuclear export signals(NES)of UL4 were for the first time mapped to amino acid residues 178 to 186. In addition,the N-terminal 19 amino acids are identified to be required for the granule-like cytoplasmic pattern of UL4.Furthermore,the UL4 protein was demonstrated to be exported to the cytoplasm through the NES in a chromosomal region maintenance 1(CRM l)-dependent manner involving RanGTP hydrolysis.