Cellular Retinol Binding Protein I transfection in H460 non-small lung carcinoma cells reduces proliferation and AKT-related gene expression

Background In recent years, new treatments with novel action mechanisms have been explored for advancer lung cancer. Retinoids were shown to promote cancer cell differentiation and death of and their action is mediated from specific cytoplasmic and nuclear receptors.Objective The purpose of this study was to investigate the effect of Cellular retinol binding Protein I (CRBPI) transfection in H460 human non-small cell lung cancer cell line normally not expressing that receptor. Methods H460 cells were transfected by using a vector pTargeT Mammalian expression system carrying the whole sequence of CRBPI gene. For proliferation and apoptosis studies, cells were treated with different concentrations of all-trans retinoic acid ( at RA) and retinol. AKT-related gene expression was analyzed by using western blot and Signosis array. Results were analysed using d by one-way analysis of variance (ANOVA) followed from a Bonferroni post hoc test or with t-student test. Results CRBPI + showed a reduced proliferation and viability in basal condition and after at RA treatment when compared to empty-transfected H460 cells. Reduced proliferation in CRBPI + H460 cells associated to the down-regulation of pAKT/pERK/pEGFR genes. In particular, gene array documented in CRBPI + H460 cells the down-regulation of AKT and Stat-3-related genes, including M-Tor, Akt1, Akt2, Akt3, Foxo1, Foxo3, p21, p27, pTen, Jun. Conclusion Restoration of CRBPI expression in H460 cells reduced proliferation and viability in both basal condition and after at RA treatment, likely by down-regulating AKT-related gene level. Further studies are needed to better clarify how those


Abstract
Background In recent years, new treatments with novel action mechanisms have been explored for advancer lung cancer. Retinoids were shown to promote cancer cell differentiation and death of and their action is mediated from specific cytoplasmic and nuclear receptors.Objective The purpose of this study was to investigate the effect of Cellular retinol binding Protein I (CRBPI) transfection in H460 human non-small cell lung cancer cell line normally not expressing that receptor.
Methods H460 cells were transfected by using a vector pTargeT Mammalian expression system carrying the whole sequence of CRBPI gene. For proliferation and apoptosis studies, cells were treated with different concentrations of all-trans retinoic acid ( at RA) and retinol. AKT-related gene expression was analyzed by using western blot and Signosis array. Results were analysed using d by one-way analysis of variance (ANOVA) followed from a Bonferroni post hoc test or with tstudent test.
Results CRBPI + showed a reduced proliferation and viability in basal condition and after at RA treatment when compared to empty-transfected H460 cells. Reduced proliferation in CRBPI + H460 cells associated to the down-regulation of pAKT/pERK/pEGFR genes. In particular, gene array documented in CRBPI + H460 cells the down-regulation of AKT and Stat-3-related genes, including M-Tor, Akt1, Akt2, Akt3, Foxo1, Foxo3, p21, p27, pTen, Jun.
Conclusion Restoration of CRBPI expression in H460 cells reduced proliferation and viability in both basal condition and after at RA treatment, likely by down-regulating AKT-related gene level. Further studies are needed to better clarify how those CRBPI-related intracellular pathways contribute to counteract non-small cell lung carcinoma progression in order to suggest a potential tool to improve efficacy of retioid anti lung cancer adjuvant therapy.

Background
Lung cancer is the first cause of neoplastic death worldwide both in man and women [1]. Nevertheless the recent innovative treatment for non-small lung cancer (NSLCC), the prognosis remains very poor. Lung cancer are largely classified into non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma, with NSCLC accounting for about 80% of all cases and only 18% all patients are alive for 5 or more years after diagnosis [2]. In lung cancer, Vitamin A deficiency has been associated with bronchial metaplasia and increased lung cancer development with many other factors [3][4][5]. Retinoids were shown to promote differentiation and cell death of cancer cells in a number of experimental systems, including lung [5][6][7]. In recent years, new treatments with novel action mechanisms have been explored for advancer lung cancer, including retinoids administration [3,7,8]. Biological activity of retinoids, in particular all-trans retinoic acid (atRA) is normally mediated by specific cytoplasmatic and nuclear receptors [8][9][10]. Cellular retinol binding protein type I (CRBPI) is a 15 kDa cytosolic binding protein crucial for uptake and subsequent esterification of retinol, by regulating its bioavailability [8,11]. CRBPI is indispensable for embryonic development and growth of vertebrates and, in the lung parenchyma, CRBPI expressing cells observed during development and prenatal alveolus formation [12]. Defects in CRBPI gene expression has been linked to oncogenic process in breast, prostatic, renal, lung and endometrial cancer [13][14][15].
Retinoids exert their pleiotropic and transcriptional effects through the binding to nuclear receptors, namely the retinoic acid receptors RAR , , and and retinoid X receptors RXR , , and [5,8,10,16]. The latter may form omo/heterodimers among them [17,18] or with other receptors as thyroid hormone receptors, vitamin D3 receptors, peroxisome proliferator active receptors (PPARs) and several orphan receptors, contributing to starting alternative signalling pathways [19]. For example, RAR and RXR in normal respiratory epithelium, binding PPAR with other cofactors, ensures cyclin D1 mediated cell cycle inhibition hence favouring apoptosis or differentiation [5,18]. Down regulation of RAR combined with AP-1 up-regulation triggers tumor progression and proliferation of NSCL cells [20].
Cuncurrently, the inability of RXR to form heretodimers with PPAR enables an AP-1/CRB-dependent up regulation of Cox2, resulting in inhibition of apoptosis [16,21].
The loss of RAR mRNA expression has been observed in many lung cancer cells line and its expression is contingent on intracellular concentration of retinoids, mediated by CRBPI and II [20]. Moreover, retinoids can activate several pathway, including AKT/ERK signalling in lung cancer cells through a transcritional independentmechanism [3,22]. Retinol can induce cytokine-dependent activation of JAk2 and subsequently STATs transcription factor, while the exchange from RBP to intracellular CRBPI is mediated by Stra6 receptor [23].
We recently documented that high expression of CRBPI in lung adenocarcinoma in vivo was associated to a lower overall survival [24]. Napoli et al. proposed that CRBPI expression in NSLCC should be considered as marker of RAR down regulation [8]. In fact, authors suggested that CRBPI cytoplasmic accumulation represent a block of nuclear disponibility of retinoids.
In the present study, we investigated the effect of atRA in native and CRBP1transfected H460 lung cancer cells with particular reference also to the modulation of RAR/RXRs and pAKT/pERK/pEGFR gene signaling. dimethylthiazol-2-yl)-2,5diphenyl-tetrazolimbromide assay (MTT, Sigma-Aldrich) was performed [26]. For the clonogenic assay, cells were seeded and treated with 5µM atRA. Colonies arising from survival cells were fixed and stained with 1% methylene blue (Sigma-Aldrich) in 0.1% methanol and their percentages as plating efficiency (PE) calculated [24].

Gene expression analysis
Gene expression analysis was performed using Signosis array (Signosis, Inc. Santa Clara, CA, USA). Briefly, total RNA was extracted [27,28], reverse-transcribed into cDNA in the presence of biotin-dUTP and a profile of 24 genes for human AKT and Western blot analysis quantified as reported [29] in three independent experiments. AKT and EGFR activity was expressed as phospho/total protein ratio [30].

Statistical analysis
Results were analyzed as the arithmetical mean±SEM. Data were analyzed by oneway analysis of variance (ANOVA) followed from a Bonferroni post hoc test and using the Student t-test. The differences were considered statistically significant for p values <0.05. All the statistical analyses were performed with SSPS V20 (Stat Corp, College Station, Texas, USA). .

Results
Restoration of CRBPI expression reduces survival and clonogenicity of H460 cells As reported in figure 1A and B, empty-transfected H460 cells did not express CRBPI, similarly to native cells. To investigate the effect of CRBPI transfection on H460 cell survival, cell counting and MTT assay were performed. Our results showed that 10% FBS-cultured CRBPI + grew less than empty-transfected H460 cells starting from second day (p<0.05) ( Figure 1C). CRBPI + viability was also reduced more markedly  (Figure 2A and B). Complessively, CRBPI expression seems to reduce the expression of proliferative proteins and to increase that of differentiation markers. As concerning cytokeratins (CK), we observed the upregulation of CK1 and 10 (p<0.02 and p<0.01, respectively) and the downregulation of CK5 expression (p<0.01) in CRBPI + compared to empty-transfected H460 cells (Figure 3A and B). In CRBPI + H460 cells maintained with 10% of FBS, only RAR protein expression resulted down-regulated compared to empty-transfected cells, whereas CRBPI signaling did not significally influences other RAR/RXR receptor expression ( Figure 3C). As reported in Figure 2D It is well documented that chemio and radiotherapy resistance is mainly due to so called cancer stem cells selection, epigenetic mechanisms silencing physiological pathway (for example hypo or hypermethilation of genes) and finally acquisition of other mutations [32]. In fact, also with the promising tyrosine kinase inhibitors or the immunotherapy patients develop inevitably progression [28,33,34]. atRA is well known to have a dramatic effect on M3 subtype of acute myeloid leukemia [34].
However, in most other cancers this effect is not observed because of epigenetic silencing of retinoid pathway [35]. Low doses of atRA (20 mg/m2/day) in combination with chemotherapy showed remarkable activity as demonstrated in a randomized phase II trial of patients with advanced NSCLC [6]. Our results showed a reduced viability and the down-regulation of several genes and proteins involved in proliferative and transcriptional pathways in CRBPI + compared to empty-transfected H460 cells in both basal condition and after atRA treatment. AtRA was used in clinical trials to suppress the growth and progression of different cancer types [18,36]. However, its effectiveness is limited in some cancer, including lung cancer [6,37,38]. AtRA is an active metabolite of Vitamin A that regulates diverse cellular functions such differentiation, proliferation and apoptosis by binding with RAR/RXR receptors [8]. RAR is a tumor suppressor gene whose expression is significantly decrease in human cancers and increase with atRA treatment [39]. The loss of RAR mRNA expression has been observed in many lung cancer cells line and its expression is contingent on intracellular concentration of retinoids, mediated by CRBPI [20]. CRBPI loss may be responsible for intracellular retinoid deficiency, since CRBPI is required for retinol bioconversion [40][41][42]. Epidemiological studies suggest that the addition of atRA or synthetic retinoids to human cancer cell lines or human tumor xenografts in nude mice result in growth arrest, apoptosis or differentiation [43]. Expression of CRBPI may help to switch between proliferation and differentiation in response to oncogenetic stimuli [44].
It has been reported that in human mammary tumor cells the reintroduction of CRBPI reduces tumorigenicity in athymic mice [45]. Moreover, the inhibition of PI3K/Akt pathway was involved in the antitumor effect of CRBPI mediated from p85 regulatory and p110 catalytic subunit heterodimerization [46]. It is possible to hypothesize that derepression of PI3K/Akt signaling mediates CRBPI loss-induced cancer progression and down-regulation of RAR in a trascription-indipendent mechanism of atRA Several in vitro studies showed that atRA induces transcriptionindipendent activation of the PI3K/Akt pathway [18,24]. For these reasons, we  CRBPI transfection associates to modulation of AKT-related gene pathway in H460 cells. Bar