title: EHD1 confers resistance to cisplatin in non-small cell lung cancer by regulating intracellular cisplatin concentrations.
abstract: Non-small cell lung cancer (NSCLC) is one of the most aggressive types of cancer. However, resistance to cisplatin (CDDP) remains a major challenge in NSCLC treatment. The purpose of this study was to investigate the ability of EHD1 [Eps15 homology (EH) domain - containing protein 1] to confer CDDP resistance in NSCLC cells and to investigate mechanisms of this resistance.
  
  
  
  The associations between EHD1 expression in NSCLC specimens and clinicopathological features, including prognosis, were assessed by immunohistochemistry (IHC). Using DNA microarrays, we performed a genome-wide analysis of cisplatin-resistant NSCLC cells to identify the involvement of the EHD1 gene in this resistance. We overexpressed and knocked down EHD1 in cell lines to investigate the effect of this gene on proliferation and apoptosis. A quantitative analytical method for assessing CDDP in cells was developed. High-performance liquid chromatography was used to measure the concentration of cisplatin in cells.
  
  
  
  The immunohistochemistry assay showed that adjuvant chemotherapy-treated NSCLC patients expressing EHD1 exhibited reduced OS compared with patients who did not express EHD1 (P = 0.01). Moreover, DNA microarrays indicated that the EHD1 gene was upregulated in CDDP- resistant NSCLC cells. The IC50 value of CDDP in cells that overexpressed EHD1 was 3.3-fold greater than that in the A549-control line, and the IC50 value of EHD1 knockdown cells was at least 5.2-fold lower than that of the control cells, as evidenced by a CCK-8 assay. We found that the percentage of early apoptotic cells was significantly decreased in A549-EHD1 cells, but the rates of early apoptosis were higher in the EHD1 knockdown cell line than in the A549/DDP control line, as indicated by a flow cytometry analysis. High-performance liquid chromatography (HPLC) showed that the total platinum level was lower in A549-EHD1 cells than in control cells, and the concentration of CDDP was higher in the EHD1 knockdown cells than in the A549/DDP control cells.
  
  
  
  We conclude that EHD1 is required for tumour growth and that it is a regulator of CDDP accumulation and cytotoxicity. The selective knockdown of EHD1 in tumours offers a strategy for enhancing the efficacy of CDDP.
authors.affiliation: The Fourth Department of Medicine Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, 150040, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Gao
authors.affiliation: The Fourth Department of Medicine Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, 150040, China.
authors.forename: Qingwei
authors.initials: Q
authors.lastname: Meng
authors.affiliation: The Fourth Department of Medicine Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, 150040, China.
authors.forename: Yanbin
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: The Fourth Department of Medicine Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, 150040, China.
authors.forename: Xuesong
authors.initials: X
authors.lastname: Chen
authors.affiliation: The Fourth Department of Medicine Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, 150040, China. caili@ems.hrbmu.edu.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Cai
mesh_terms: D000072283:A549 Cells; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000401:mortality / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D017024:Chemotherapy, Adjuvant; D002851:Chromatography, High Pressure Liquid; D002945:Cisplatin / Q000494:pharmacology* / Q000627:therapeutic use; D003593:Cytoplasm; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D055785:Gene Knockdown Techniques; D006801:Humans; D007150:Immunohistochemistry; D020128:Inhibitory Concentration 50; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000401:mortality / Q000473:pathology; D008297:Male; D008875:Middle Aged; D011379:Prognosis; D015996:Survival Rate; D046888:Tissue Array Analysis; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; C120700:EHD1 protein, human; D033921:Vesicular Transport Proteins; D002945:Cisplatin
keywords: CDDP-resistant; EHD1; Intracellular concentrations; NSCLC