Leptomycin B

CRM1 (chromosome maintenance region 1, Exportin 1) binds to nuclear export signals and is required for nucleocytoplasmic transport of a large variety of proteins and RNP complexes. Leptomycin B (LMB), the first specific inhibitor of CRM1 identified, binds covalently to cysteine 528 in the nuclear export signal binding region of CRM1 leading to the inhibition of protein nuclear export. Although the biochemical mechanisms of action of CRM1 inhibitors such as LMB are well studied, the subcellular effects of inhibition on CRM1 are unknown. We have found that LMB causes CRM1 to redistribute from the nucleus to the cytoplasm in A549 cells. A significant decrease in nuclear CRM1 coupled with an increase in cytoplasmic CRM1 was sustained for up to 4 h, while there was no change in total CRM1 protein in fractionated cells. Cells expressing an LMB insensitive HA-tagged CRM1-C528S protein were unaffected by LMB treatment, whereas HA-tagged wildtype CRM1 redistributed from the nucleus to the cytoplasm with LMB treatment, similar to endogenous CRM1. GFP-tagged CRM1 protein microinjected into the cytoplasm of A549 cells distributed throughout the cell in untreated cells remained primarily cytoplasmic in LMB-treated cells. Upon nuclear microinjection, GFP-CRM1 translocated to and accumulated in the cytoplasm of LMB-treated cells. Thus, LMB binds to CRM1 and causes its redistribution to the cytoplasm by inhibiting its nuclear import. Decreasing the nuclear availability of CRM1 likely contributes to the accumulation of CRM1 cargo proteins in the nucleus, suggesting a new mechanism of action for LMB.

The blood-brain barrier (BBB) disruption during brain insults leads to vasogenic edema as one of the primary steps in the epileptogenic process. However, the signaling pathway concerning vasogenic edema formation has not been clarified. In the present study, status epilepticus (SE) resulted in vascular endothelial growth factor (VEGF) over-expression accompanied by loss of BBB integrity in the rat piriform cortex. Leptomycin B (LMB, an inhibitor of chromosome region maintenance 1) attenuated SE-induced vasogenic edema formation. This anti-edema effect of LMB was relevant to inhibitions of VEGF over-expression as well as p38 mitogen-activated protein kinase (MAPK) phosphorylation. Furthermore, SB202190 (a p38 MAPK inhibitor) ameliorated vasogenic edema and VEGF over-expression induced by SE. These findings indicate that p38 MAPK/VEGF signaling pathway may be involved in BBB disruption following SE. Thus, we suggest that p38 MAPK/VEGF axis may be one of therapeutic targets for vasogenic edema in various neurological diseases.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) gefitinib has demonstrated dramatic clinical efficacy in non-small cell lung cancer (NSCLC) patients. However, its therapeutic efficacy is ultimately limited by the development of acquired drug resistance. The aim of this study was to explore the potential utility of chromosome region maintenance 1 (CRM1) inhibitor leptomycin B (LMB) in combination with gefitinib to overcome primary and acquired gefitinib resistance in NSCLC cells. The combinative effects of gefitinib and LMB were evaluated by MTT and its underlining mechanism was assessed by flow cytometry and Western blot. LMB displayed a synergistic effect on gefitinib-induced cytotoxicity in A549 (IC50: 25.0±2.1μM of gefitinib+LMB vs. 32.0±2.5μM of gefitinib alone, p<0.05). Gefitinib+LMB caused a significantly different cell cycle distribution and signaling pathways involved in EGFR/survivin/p21 compared with gefitinib. A549 cells then were treated with progressively increased concentrations of gefitinib (A549GR) or in combination with LMB (A549GLR) over 10months to generate gefitinib resistance. IC50 of gefitinib in A549GLR (37.0±2.8μM) was significantly lower than that in A549GR (53.0±3.0μM, p<0.05), which indicates that LMB could reverse gefitinib-induced resistance in A549. Further mechanism investigation revealed that the expression patterns of EGFR pathway and epithelial-mesenchymal transition (EMT) markers in A549, A549GR, and A549GLR were significantly different. In conclusion, LMB at a very low concentration (0.5nM) combined with gefitinib showed synergistic therapeutic effects and ameliorated the development of gefitinib-induced resistance in lung cancer cells.