Chemotherapeutic Sensitization of Leptomycin B Resistant Lung Cancer Cells by Pretreatment with Doxorubicin

Abstract

The development of novel targeted therapies has become an important research focus for lung cancer treatment. Our previous study has shown leptomycin B (LMB) significantly inhibited proliferation of lung cancer cells; however, p53 wild type lung cancer cells were resistant to LMB. Therefore, the objective of this study was to develop and evaluate a novel therapeutic strategy to sensitize LMB-resistant lung cancer cells by combining LMB and doxorubicin (DOX). Among the different treatment regimens, pretreatment with DOX (pre-DOX) and subsequent treatment with LMB to A549 cells significantly decreased the 50% inhibitory concentration (IC50) as compared to that of LMB alone (4.4 nM vs. 10.6 nM, P<0.05). Analysis of cell cycle and apoptosis by flow cytometry further confirmed the cytotoxic data. To investigate molecular mechanisms for this drug combination effects, p53 pathways were analyzed by Western blot, and nuclear proteome was evaluated by two dimensional-difference gel electrophoresis (2D-DIGE) and mass spectrometry. In comparison with control groups, the levels of p53, phospho-p53 (ser15), and p21 proteins were significantly increased while phospho-p53 (Thr55) and survivin were significantly decreased after treatments of pre-DOX and LMB (P<0.05). The 2D-DIGE/MS analysis identified that sequestosome 1 (SQSTM1/p62) had a significant increase in pre-DOX and LMB-treated cells (P<0.05). In conclusion, our results suggest that drug-resistant lung cancer cells with p53 wild type could be sensitized to cell death by scheduled combination treatment of DOX and LMB through activating and restoring p53 as well as potentially other signaling pathway(s) involving sequestosome 1.

Leptomycin B reduces primary and acquired resistance of gefitinib in lung cancer cells

“…Cell viability was evaluated by the MTT assay as described previously (Shao et al , 2011; Lu et al , 2012; Gao et al , 2015). Briefly, based on the cytotoxicity of LMB observed in this study and our previous reports (Shao et al , 2011; Lu et al , 2012; Gao et al , 2015), 0.5 nM LMB was selected for co-treatment.…”

“…Briefly, based on the cytotoxicity of LMB observed in this study and our previous reports (Shao et al , 2011; Lu et al , 2012; Gao et al , 2015), 0.5 nM LMB was selected for co-treatment. The synergistic effect of gefitinib and LMB was evaluated by comparing to vehicle controls for gefitinib treatments or LMB (0.5 nM) for gefitinib+LMB treatments as described in our previous studies (Gao et al , 2015; Lu et al , 2015).…”

“…Cells were stained with Guava Cell Cycle Reagent (Millipore, Billerica, MA) and run on a Guava EasyCyte ™ Flow Cytometer (Millipore) as previously described (Lu et al , 2012). Each sample was run in triplicate and each experiment was repeated three times.…”

“…Chromosome region maintenance 1 (CRM1), also known as exportin 1 (XPO1), is a nuclear export receptor responsible for transporting a variety of cancer related proteins from nucleus to cytoplasm including p53, p21, p27, pRB, FOXO, and EGFR (Lo et al , 2006; Lu et al , 2012; Lu et al , 2015). Our previous studies have demonstrated that CRM1 is overexpressed in lung cancer and CRM1 inhibitor, leptomycin B (LMB) could serve as an effective adjuvant regimen for lung cancer treatment (Gao et al , 2015; Lu et al , 2015).…”

Serum amyloid A is increased in children with abusive head trauma: a gel-based proteomic analysis

“…Spots of interest were isolated using a spot picker, and placed into a 0.5 ml Eppendorf tube for trypsin digestion as described previously (21,43). Trypsinized peptides were analyzed by nano LC/MS/MS on a Thermo Fisher LTQ Orbitrap XL (Thermo Scientific, Waltham, MA).…”