Screening of a novel CRM1 targeting inhibitor and its effects on the proliferation and growth of extranodal NK/T cell lymphoma / 中国药科大学学报

@#The purpose of this study was to screen out the novel chromosome maintenance protein 1(CRM1)covalent targeting inhibitors by computer-assisted drug design(CADD), and to study their effects on the proliferation of extranodal nature killer/T cell lymphoma(ENKTL). A novel CRM1 inhibitor LFS-829 was designed based on the molecular structure of LFS-01 by means of ADME/T and covalent docking. The target binding of LFS-829 with CRM1 was analyzed by MALDI-TOF mass spectrometry. The effects of LFS-829 on the proliferation of extranodal NK/T cell lymphoma SNK6 and HANK-1 cells were detected by CCK-8. The cell morphology was observed by live cell workstation. Immunofluorescence experiments were used to analyze the effect of LFS-829 on nuclear export function of CRM1. The changes of NF-κB signaling pathway under different concentrations of LFS-829 were analyzed by Western blot, dual luciferase reporter gene assay and enzyme-linked immunosorbent assay. Apoptosis was detected by flow cytometry, and the expression of proteins related to apoptosis pathway was detected by Western blot. Tests of peripheral blood mononuclear lymphocyte(PBMC)toxicity, platelet toxicity and mouse acute toxicity were done to make sure that it is not harmful to human. LFS-829 could bind covalently to the cysteine residue of the hydrophobic active pocket of CRM1. LFS-829 could selectively kill SNK6 and HANK-1 cells, with IC50 of 366 nmol/L and 158 nmol/L in 72 h, respectively, and cell morphology was significantly changed. LFS-829 at 800 nmol/L significantly inhibited the nuclear export function of CRM1, promoted nuclear assembly of IκB-α, down-regulated the transcriptional activity of NF-κB signaling pathway, significantly up-regulated the expression of apoptotic pathway protein p53, cleaved Caspase 3 and cleaved Caspase 9 and induced apoptosis, with no obvious killing effect on PBMC or platelets. It did not cause substantial tissue damage to mice at the high dose of 300 mg/kg, which shows its great prospect of future application.

Genetic variants of chromosome 9p21.3 region associated with coronary artery disease and premature coronary artery disease in an Asian Indian population

Introduction Asian Indians have a propensity for premature, severe, and diffuse coronary artery disease (CAD). Several single-nucleotide polymorphisms (SNPs) in the ‘core CAD’ region of the chromosomal region 9p21.3 are known to be strongly associated with CAD. Objectives We aimed to study SNPs in the 9p21.3 region associated with CAD and premature CAD and identify their association with demographic and clinical characteristics in an Asian Indian population. Methods SNP genotyping was performed for 30 SNPs of the 9p21.3 region using MassARRAY® technology. Along with demographic and SNP data analysis, we also performed multivariate logistic regression analysis and multifactor dimensionality reduction analysis to study SNP–SNP and SNP–demographic/clinical variable interactions. Results Our results suggest that females are at a higher risk of premature CAD. We found that SNPs rs1333045 (CC), rs16905599 (AA), rs2383206 (GG), rs2383208 (AG), and rs4977574 (GG) were significantly associated with premature CAD. When adjusted for covariates/confounders, we found that rs2383206 showed the strongest risk association with CAD followed by rs16905599 and rs2383208. Further, SNPs rs1333049 (CC) and rs4977574 (GG) were found to be exclusively associated with premature CAD cases, suggesting their potential as genetic markers for premature CAD in the local population. Upon gender-based stratification, it was found that rs10757272 (TT and TC) is significantly associated with eightfold to ninefold CAD risk specifically among females. SNP rs7865618 (GG) is significantly associated with more than 2.5-fold CAD risk specifically among males. Conclusion Our study suggests that SNPs at the 9p21 risk locus may be used to generate a reliable genetic risk score along with markers at other loci.

Sulforaphene kills triple negative breast cancer MDA-MB-468 and MDA-MA-231 cells by blocking STAT3 signaling pathway / 中国肿瘤生物治疗杂志

@# 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.

Naturally derived sulforaphene inhibits proliferation of mantle cell lymphoma by down-regulating CRM1 expression / 肿瘤

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.

Effect of integration loci of genome on heterologous gene expression in Saccharomyces cerevisiae / 生物工程学报

Chromosomal integration of heterologous genes or pathways is preferred over the use of episomal plasmids for its inherently stability and thus more desirable in the industrial setting. However, the position of integration of heterologous genes in the genome influences the expression levels. In combination of high throughput transformation of the Yeast Knock-out Collection (YKO) and FACS analysis, the position effect on heterologous reporter gene gfp was identified across the whole genome in yeast. In total 428 high-expressed sites and 444 low-expressed sites were spotted, providing massive data to analyze patterns and reasons for region dependency of gene expression on the genome-wide scale.

Molecular Determinants Responsible for the Subcellular Localization of HSV-1 UL4 Protein / 中国病毒学·英文版

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.