Cellular retinol binding protein 1 transfection reduces proliferation and AKT-related gene expression in H460 non-small lung cancer cells

Abstract

In recent years, new treatments with novel action mechanisms have been explored for advanced non-small cell lung cancer (NSCLC). Retinoids promote cancer cell differentiation and death and their trafficking and action is mediated from specific cytoplasmic and nuclear receptors, respectively. The purpose of this study was to investigate the effect of Cellular retinol binding protein-1 (CRBP-1) transfection in H460 human NSCLC cell line, normally not expressing CRBP-1. H460 cells were transfected by using a vector pTargeT Mammalian expression system carrying the whole sequence of CRBP-1 gene. For proliferation and apoptosis studies, cells were treated with different concentrations of all-trans Retinoic Acid (atRA) and retinol. AKT-related gene expression was analyzed by using western blot and Signosis array and results analysed by one-way analysis of variance (ANOVA) or by t-student test. CRBP-1⁺ showed reduced proliferation and viability in basal condition and after atRA treatment when compared to empty-transfected H460 cells. Reduced proliferation in CRBP-1⁺ H460 cells associated to the down-regulation of pAKT/pERK/pEGFR-related genes. In particular, gene array documented the down-regulation of AKT and Stat-3-related genes, including M-Tor, Akt1, Akt2, Akt3, Foxo1, p27, Jun. Restoration of CRBP-1 expression in H460 cells reduced proliferation and viability in both basal condition and after atRA treatment, likely by down-regulating AKT-related gene level. Further studies are needed to better clarify how those CRBP-1-related intracellular pathways contribute to counteract NSCLC progression in order to suggest a potential tool to improve efficacy of retinoid anti lung cancer adjuvant therapy.

Keywords: AKT pathway; CRBP-1; Non-small cell lung cancer; Retinoids.

Fig. 1

CRBP-1 transfection reduces viability and retinoid-related survival of H460 cells. a RT-PCR and b representative blots of protein expression in CRBP-1-transfected in H460 cell line. Hek-293 cells as positive control. c CRBP-1⁺ H460 cells growth increased compared to empty-transfected cells. d MTT assay shows reduced viability of CRBP-1⁺ compared to empty-transfected H460 cells after 2 days of atRA treatment in the presence of 0.1% FBS. e, f CRBP-1⁺ maintained with 10% and 0.1% of FBS and after 48 h of 5 µM atRA treatment showed a poor ability to form colonies compared to empty-transfected H460 cells. Values expressed as means ± SD of three different experiments: *p < 0.05, **p < 0.005, ***p < 0.001

Fig. 2

CRBP-1 transfection influences transcriptional pathways and differentiation of H460 cells. a, b Bar graph of gene array showed gene modulation of Akt and Stat-3 pathways in CRBP-1⁺ compared to empty-transfected H460 cells

Fig. 3

CRBP-1 transfection modulates cytokeratins and Akt-related genes and protein expression in H460 cells. a Cytokeratins mRNA levels by real-time PCR; representative blot and bar graph of cytokeratins (b), RARs/RXR (c), pAKT/AKT, pERK1/2 and pEGFR/EGFR (d) protein expression in CRBP-1⁺ compared to empty-tranfected H460 cells maintained with 10% FBS; e–g Bar graphs of pAKT/AKT, pEGFR/EGFR and pERK1/2 protein expression in CRBP-1⁺ compared to empty-tranfected H460 cells after 48 h of treatment with 5 µM atRA and ROL. *p < 0.05, **p < 0.005 and ***p < 0.001. RLU relative light unit, ADU arbitrary densitometric units

Fig. 4

CRBP-1expression associates to modulation of AKT-related genes in NSCLC tissue. Bar graph of gene array showed the modulation of Akt-related gene in CRBP-1 positive compared to CRBP-1 negative NSCLC tissue samples