Retinoic Acid Receptor-β Research Articles

Inhibition of farnesoid X receptor controls esophageal cancer cell growth in vitro and in nude mouse xenografts.

Gastroesophageal reflux is a risk factor for esophageal adenocarcinoma, and bile acid and its farnesoid X receptor (FXR) have been implicated in esophageal tumorigenesis. The authors investigated the role of FXR expression and activity in esophageal cancer initiation and growth. FXR expression in esophageal adenocarcinoma tissues was assessed by immunohistochemistry. Knockdown of FXR expression in esophageal cancer cells in vitro and in nude mice xenografts was suppressed by FXR small hairpin RNA (shRNA) and guggulsterone (a natural FXR inhibitor). Esophageal cancer cells were treated with bile acids to demonstrate their effects on growth-promoting genes. FXR was expressed in 48 of 59 esophageal adenocarcinoma tissues (81.3%), and this overexpression was associated with higher tumor grade, larger tumor size, and lymph node metastasis; however, was inversely associated with retinoic acid receptor-β2 (RAR-β2 ) expression. Knockdown of FXR expression suppressed tumor cell growth in vitro and in nude mouse xenografts. Guggulsterone reduced the viability of esophageal cancer cells in a time-dependent and dose-dependent manner, whereas this effect was diminished after knockdown of FXR expression. Guggulsterone induced apoptosis through activation of caspase-8, caspase-9, and caspase-3 in tumor cells. FXR mediated bile acid-induced alterations of gene expression, eg, RAR-β2 and cyclooxygenase-2 (COX-2). Inhibition of FXR by FXR shRNA or guggulsterone suppressed tumor cell viability and induced apoptosis in vitro, and it reduced tumor formation and growth in nude mouse xenografts. FXR also mediated bile acid-induced alterations of cell growth-related genes in esophageal cancer cells.

Combination of acyclic retinoid with branched‐chain amino acids inhibits xenograft growth of human hepatoma cells in nude mice

Combination chemoprevention is a promising strategy to improve the prognosis of hepatocellular carcinoma (HCC). A malfunction of retinoid X receptor-α (RXR-α) due to phosphorylation by Ras/mitogen-activated protein kinase is closely associated with liver carcinogenesis and acyclic retinoid (ACR) can prevent HCC development by inhibiting RXR-α phosphorylation. The present study examined the possible combined effects of ACR plus branched-chain amino acids (BCAA), which can also prevent the development of HCC in obese patients with liver cirrhosis, in human HCC xenografts in nude mice. This study examined the effects of the combination of ACR plus BCAA on the growth of Huh7 human HCC xenografts in nude mice. The effects of the combination on the phosphorylation of RXR-α, extracellular signal-regulated kinase (ERK), Akt and insulin-like growth factor-1 receptor (IGF-1R) proteins, and on the expression levels of retinoic acid receptor-β (RAR-β) and p21(CIP1) mRNA, were also examined by western blot and real-time reverse transcription polymerase chain reaction analyses, respectively. The combined treatment with ACR plus BCAA significantly inhibited the growth of Huh7 xenografts. The combination of these agents caused a marked inhibition of the phosphorylation of RXR-α, ERK, Akt and IGF-1R proteins in the xenografts. In addition, the expression levels of RAR-β and p21(CIP1) mRNA significantly increased by these agents. The combination of ACR and BCAA restores the function of RXR-α by inhibiting its phosphorylation and increasing the level of RAR-β, a heterodimeric partner for RXR-α, and thus suppresses the growth of HCC xenografts. Therefore, this combination might be an effective regimen for the treatment and, probably, chemoprevention of HCC.

Tumor suppressor gene methylation in tissue and urine specimens of bladder transitional cell carcinoma

Objective To investigate abnormal promoter methylation status of death-associated protein kinase(DAPK),retinoic acid receptorβ(RARβ),Hyperplastic polyps protein(HPP1) and integrin alpha-4 precursor (ITGA4) genes,and the diagnostic value for bladder transitional cell carcinoma (BTCC).Methods Methylation-specific polymerase chain reaction (MSP) was used to detect the methylation status of DAPK,RARβ,HPP1 and ITGA4 genes in tissue and urine specimens of 112 patients with BTCC.Results The methylation rate of DAPK,RARβ,HPP1 and ITGA4 in BTCC tissue was 29.4％,91.0％,75.0％ and 48.2％ respectively,and that in urine sediment cells was 26.7％,87.5％,69.6％ and 27.6％ respectively.Aberrant methylation rate of DAPK,HPP1 and RARβ genes in BTCC was significantly different from that in the glandular cystitis (P ＜ 0.05).There was no significant difference in the methylation rate of ITGA4 between BTCC and and cystitis glandularis (P ＞ 0.05).Conclusion Aberrant methylation status of HPP1,DAPK1,and RARβ is closely related with incidence of bladder cancer. Key words: Bladder carcinoma; Urine sediment cells; Tumor suppressor gene; Methylation

Intracrine prostaglandin E2 signalling regulates hypoxia-inducible factor-1α expression through retinoic acid receptor-β

We have previously found in human renal proximal tubular HK-2 cells that hypoxia- and all-trans retinoic acid-induced hypoxia-inducible factor-1α up-regulation is accompanied by retinoic acid receptor-β up-regulation. Here we first investigated whether hypoxia-inducible factor-1α expression is dependent on retinoic acid receptor-β and our results confirmed it since (i) hypoxia-inducible factor-1α-inducing agents hypoxia, hypoxia-mimetic agent desferrioxamine, all-trans retinoic acid and interleukin-1β increased retinoic acid receptor-β expression, (ii) hypoxia-inducible factor-1α up-regulation was prevented by retinoic acid receptor-β antagonist LE-135 or siRNA retinoic acid receptor-β and (iii) there was direct binding of retinoic acid receptor-β to the retinoic acid response element in hypoxia-inducible factor-1α promoter upon treatment with all-trans retinoic acid and 16,16-dimethyl-prostaglandin E2. Since intracellular prostaglandin E2 mediates hypoxia-inducible factor-1α up-regulation in normoxia in HK-2 cells, we next investigated and confirmed, its role in the up-regulation of retinoic acid receptor-β in normoxia by hypoxia-inducible factor-1α-inducing agents all-trans retinoic acid, interleukin-1β and 16,16-dimethyl-prostaglandin E2 by inhibiting cyclooxygenases, prostaglandin influx transporter or EP receptors. Interestingly, the hypoxia-induced increase in retinoic acid receptor-β expression and accumulation of hypoxia-inducible factor-1α was also blocked by the inhibitors tested. This is the first time, to our knowledge, that retinoic acid receptor-β signalling is involved in the control of the expression of transcription factor hypoxia-inducible factor-1α in both normoxia and hypoxia and that retinoic acid receptor-β expression is found to be strictly regulated by intracellular prostaglandin E2. Given the relevance of hypoxia-inducible factor-1α in the kidney in terms of tumorigenesis, progressive renal failure, production of erythropoietin and protection in several models of renal disease, our results open new therapeutic opportunities on the control of hypoxia-inducible factor-1α based upon the pharmacological modulation of retinoic acid receptor-β, either directly or through the control of intracellular prostaglandin E2 levels/signalling.

All-trans retinoic acid activates E-cadherin expression via promoter hypomethylation in the human colon carcinoma HCT116 cells

All-trans retinoic acid (ATRA) inhibits the invasive and metastatic potentials of various cancer cells; however, the underlying mechanism is unclear. Here, we show that ATRA activated E-cadherin expression via promoter hypomethylation to facilitate Sp1 binding on its recognition sites in human colon carcinoma HCT116 cells. This effect was mediated by retinoic acid receptor-β2, as demonstrated by knock-down experiments using a specific siRNA. As a result, ATRA increased cell-to-cell interactions, reduced cell migration, and downregulated levels of Vimentin and Fibronectin in HCT116 cells. The present study thus provides the mechanism for the beneficial effects of ATRA in the treatment of metastatic human carcinomas.

DNA methylation status of RARβ2 and p16(INK4α) in peripheral blood and tumor tissue in patients with esophageal squamous cell carcinoma

To study the methylation status of retinoic acid receptor β2 (RARβ2) and p16(INK4α) genes in peripheral blood and tumor tissues and the perioperative dynamic changes of free RARβ2 and p16(INK4α) in the peripheral blood, and to investigate the relationship between RARβ2 and p16(INK4α) methylation in peripheral blood and clinicopathological characteristics of esophageal squamous cell carcinoma (ESCC) and their value in evaluating the completeness of surgical resection. Real-time methylation specific polymerase chain reaction (real-time MSP) technique was used to detect the methylation status of RARβ2 and p16(INK4α) in tumor tissue, adjacent normal tissue and peripheral blood perioperatively in 76 cases of ESCC. Sixty age-matched healthy volunteers were randomly selected as a control. RARβ2 and p16(INK4α) hypermethylation presented in both tumor tissue [72.4% (55/76) and 86.8% (66/76)] and peripheral blood [63.2% (48/76) and 71.1% (54/76)] in the ESCC patients, showing a good agreement between them. RARβ2 and p16(INK4α) hypermethylation was significantly related with pathological stage, lymph node metastasis, and invasion of nerves and vessels (P < 0.05). The DNA methylation rate in peripheral blood was increasing first and then decreasing in the preoperative, intraoperative and postoperative periods. Moreover, the RARβ2 methylation in peripheral blood was shown to be significantly associated with family history of cancer (P = 0.023). RARβ2 and p16(INK4α) methylation in the peripheral blood in ESCC patients may reflect the tumor-bearing status in the body, and may serve as a valuable marker in assessment of the degree of completeness of surgical resection in ESCC patients.

Epstein–Barr Virus latent membrane protein 1 overcomes all-trans retinoic acid-induced apoptosis by inhibiting retinoic acid receptor-β2 expression

Nasopharyngeal carcinoma is closely associated with infection with Epstein–Barr Virus (EBV); however, the mechanism is still unclear. Here, we report that the EBV oncoprotein, latent membrane protein 1 (LMP1), suppresses apoptotic cell death provoked by all-trans retinoic acid (ATRA) in NPC cells. For this purpose, LMP1 downregulated levels of Bak whilst it upregulated levels of Bcl2, lowering the ratio of Bak to Bcl2. In addition, LMP1 suppressed ATRA-mediated activation of Caspase 9, Caspase 3, and PARP but not Caspase 8 in Ad-AH cells, suggesting that LMP1 acts by blocking the activation of intrinsic apoptosis pathway by ATRA. These effects were almost completely abolished when levels of retinoic acid receptor-β2 (RAR-β2) in the LMP1-expressing cells were recovered by either exogenous gene expression or treatment with a universal DNMT inhibitor, 5-Aza-2′dC, indicating that LMP1 executes its antiapoptotic effects by downregulating levels of RAR-β2 via DNA methylation.

Clinical Significance of Promoter Hypermethylation of ERβ and RARβ2 in Tumor and Serum DNA in Indian Breast Cancer Patients

To determine concordance of promoter hypermethylation of ERβ (estrogen receptor β) and RARβ2 (retinoic acid receptor β2) in tumor and circulating DNA of Indian breast cancer patients and their association with clinicopathologic parameters and disease prognosis. ERβ and RARβ2 methylation was analyzed by methylation-specific PCR in the tumors and circulating DNA of 100 patients with invasive ductal breast carcinoma. Promoter hypermethylation was associated with the expression of the encoded protein in tumors by immunohistochemistry, and their prognostic utility was explored in a follow-up study. Significant correlation was observed between promoter hypermethylation of ERβ (r = + 0.77; p ≤ 0.001) and RARβ2 (r = + 0.85; p ≤ 0.001) in tumors and paired sera. No association was found between ERβ and RARβ2 promoter hypermethylation and loss of protein expression. Kaplan-Meier survival curve showed loss of ERβ expression, and RARβ2 promoter hypermethylation was associated with poor overall survival (OS) (p = 0.03, p = 0.001). Breast cancer patients showing concurrent hypermethylation of ERβ and RARβ2 had a significantly shorter median OS (p = 0.02), underscoring that hypermethylation of these two genes may serve as an adverse prognosticator for breast carcinoma. Methylation status of ERβ and RARβ2 in serum could potentially be used to predict invasive ductal breast carcinoma. Furthermore, concurrent ERβ and RARβ2 methylation as well as loss of ERβ expression may serve as a good prognostic marker.

Urinary retinoic acid receptor-β2 gene promoter methylation and hyaluronidase activity as noninvasive tests for diagnosis of bladder cancer

We evaluated the significance of urinary retinoic acid receptor-β2 (RAR-β2) gene promoter methylation and hyaluronidase activity in comparison with voided urine cytology (VUC) in diagnosis of bladder cancer. This study included 100 patients diagnosed with bladder cancer, 65 patients with benign urological disorders and 51 healthy volunteers. Urine supernatant was used for determining hyaluronidase activity by zymography while urine sediment was used for cytology and detection of methylated RAR-β2 gene promoter by methylation specific nested PCR. The sensitivity and specificity were 53% and 90.5% for VUC, 65% and 89.7% for percent methylation fraction of RAR-β2 gene promoter, and 89% and 90.5% for hyaluronidase activity; combination of the three parameters increased sensitivity to 95%. A significant association was observed between investigated markers and advanced grade tumor. Combined use of RAR-β2 gene promoter methylation, hyaluronidase activity and VUC is promising non-invasive tool for bladder cancer detection.

Retinoid dependent growth arrest and differentiation in melanoma

Retinoids are very effectively used for treatment of cancer like lung cancer, breast cancer, melanoma cells, etc. The pathway by which retinoic acid and its derivatives accomplish this work is still not elucidating. The present paper gives a hypothetic approach of retinoic acid dependent pathway of growth arrest in malignant cells. Besides retinoic acid receptor-b, the immediate target gene of RA is TBX2, which is also implicated in tumorigenesis. TBX2 is a target of microphthalmia-associated transcription factor (MITF) which is also induced by Alltrans retinoic acid. This sophisticated role of TBX2 concomitant with MITF is mediated by targeting other genes, which are responsible for growth arrest. The interaction of these genes is shown in the form of an optical coupler as a hypothesis to elucidate the comprehensive action of retinoic acid induced TBX2 expression and growth arrest. The simulation is done in MATLAB Simulink and the results are relevant to the early-proposed model of Niles et al. (2003).

Cloning and expression of a retinoic acid receptor β2 subtype from the adult newt: Evidence for an early role in tail and caudal spinal cord regeneration

Retinoic acid receptor beta 2 (RARβ2) has been proposed as an important receptor mediating retinoid-induced axonal growth and regeneration in developing mammalian spinal cord and brain. In urodele amphibians, organisms capable of extensive central nervous system (CNS) regeneration as adults, this receptor had not been isolated, nor had its function been characterized. We have cloned a full-length RARβ2 cDNA from adult newt CNS. This receptor, NvRARβ2, is expressed in various adult organs capable of regeneration, including the spinal cord. Interestingly, both the NvRARβ2 mRNA and protein are up-regulated during the first 2 weeks after amputation of the tail, primarily in the ependymoglial and meningeal tissues near the rostral cut surface of the cord. Treatment with LE135, a RARβ-selective antagonist, caused a significant inhibition of ependymal outgrowth and a decrease in tail regenerate length. These data support an early role for this receptor in caudal spinal cord and tail regeneration in this amphibian.

Research on retinoic acid signals in retina of guinea pig eyes with different monochromatic illumination

Retinoic acid (RA) is the only extrinsic biochemical candidate that could act as a growth controller, the present study was designed to evaluate the retinoic acid signals in retina of guinea pig eyes with different monochromatic illumination. Ninety 2-week-old guinea pigs were divided into 3 groups randomly and were raised under green (530 nm) (n = 30), blue (430 nm) (n = 30) or white lighting (5000K) (n = 30) conditions. The axial length was measured with Cine scan A/B ultrasonography before, 2 weeks, 4 weeks, 8 weeks during experiment. After the biometrical measurement finished, 9 guinea pigs were sacrificed randomly in each group and retina was dissected. The RA level in retina was detected by High Performance Liquid Chromatography (HPLC). The expressions of retinoic acid-binding proteins I (CRABP-I) and retinoic acid receptor-β (RAR-β) protein and mRNA were assayed by Western-blot and Real-time PCR, respectively. The experiment and use of animals followed the Standard of Association for Research in Vision and Ophthalmology. 1. The axial length among the three groups showed a significant difference at 4 weeks during experiment (F = 3.946; P < 0.05), the green lighting group showed the fastest growing, the axial length of the three groups are (8.36 ± 0.11) mm (green), (7.88 ± 0.49) mm (blue), 7.98 ± 0.32 mm (white) respectively at 8 weeks during experiment; 2. The RA levels demonstrated a same trend with the axial length, at 8 weeks during experiment the RA levels are 4.846 ± 0.56 µg/g (green), 3.583 ± 0.26 µg/g (blue), 4.419 ± 0.36 µg/g (white) respectively; 3. Changes of the expression of CRABP-I and RAR-β occurred at different times, a significant difference of RAR-β among three groups appeared earlier, the green lighting group showed a highest level. The different levels of retinoic acid signals were showed in retina of guinea pig eyes under different monochromatic illumination. The eye growth was significantly faster in green group than in blue group and white group, meanwhile, the RA level, the expressions of CRABP-I and RAR-β in the retina were higher in green group than the other two groups. These findings suggest that RA may act as a messenger in eye growth promoted by different monochromatic illumination.

The fate of Notch-deficient nephrogenic progenitor cells during metanephric kidney development

To determine which nephron segments require Notch signals for development, we conditionally deleted Rbpj, a transcription factor required for canonical Notch signaling, in nephrogenic progenitors (NPs) of the metanephric mesenchyme. The retinoic acid receptor-β2 (Rarb2) promoter efficiently directed Cre-recombinase (Cre) activity to these progenitors. Conditional knockout of Rbpj in mice (Rarb2Cre(+)/Rbpj (f/-)) caused severe renal hypoplasia, as indicated by a 70-95% reduction in nephron number and the development of tubular cysts. To track the fate of NPs following Rarb2Cre expression, we labeled them with membrane-associated enhanced green fluorescent protein (GFP). In TomatoGFP(+)/Rarb2Cre(+) control mice, NPs differentiated into epithelia of all nephron segments, except into collecting ducts. In TomatoGFP(+)/Rarb2Cre(+)/Rbpj (f/-) conditional knockout mice, NPs developed into podocytes or distal tubular epithelia, indicating that canonical Notch signals were not required for mesenchymal-to-epithelial transition or for the specification of these nephron segments. Conversely, the few proximal tubules and associated cysts that developed in these mice were derived from the 5-10% of NPs that had failed to express Cre and, therefore, had intact Notch signaling. Thus, our fate mapping studies establish that the profound effect of Notch signaling on nephrogenesis is due to the specification of proximal but not distal tubules or podocytes.

Expressions of cellular retinoic acid binding proteins I and retinoic acid receptor-β in the guinea pig eyes with experimental myopia.

All-trans retinoic acid (RA) is the only extrinsic biochemical candidate known to date that could act as a growth controller, the aim of this study was to investigate the expression cellular retinoic acid binding proteins I (CRABP-I) and retinoic acid receptor-β (RAR-β) in retina of the guinea pig eyes with experimental myopia. Ninety guinea pigs aged 14 days were equally and randomly divided into three groups: form deprivation (FD), -5D lens, and control. The diffusers for FD were white translucent hemispheres, and -5D lenses were used to introduce hyperopic defocus. Refraction was measured with streak retinoscopy after cycloplegia, and axial length was calculated with Cinescan A/B ultrasonography. Retina harvested at different time points were used to measure RA level with HPLC and expressions of cellular retinoic acid binding proteins I (CRABP-I) and RA receptor-β (RAR-β) were assayed with Western blot and Real-time PCR. SPSS13.0 software was used for statistical analysis. Up-regulations of CRABP-I and RAR-β in ocular tissues correlated with changes in the refractive status and growth rate of the guinea pig eye (P<0.05). 14 days of monocular form-deprivation led to -5.14D myopia and a 0.281mm axial elongation; 14 days of monocular defocus produced -3.64D myopia and a 0.163 mm axial elongation. The level of retinal RA started to elevate in 7 days (P<0.05) after visual manipulation in both FD and -5D lens groups and became more prominent by 14 days (P<0.01) . The expressions of CRABP-I and RAR-β increased by 14 days after visual manipulation (P<0.05), the mRNA level of RAR-β, however, increased by 7 days after visual manipulation (P<0.05), which suggested that changes of expressions of CRABP-I and RAR-β might lag behind the change of RA. The levels of CRABP-I and RAR-β were elevated in retina of the guinea pig eye with experimental myopia. During the progression of experimental myopia, the retinal RA level increased rapidly, and there might be a positive feedback between the increase of RA and up-regulation of RAR-β.

Reversible upregulation of tropomyosin-related kinase receptor B by geranylgeranoic acid in human neuroblastoma SH-SY5Y cells

All-trans retinoic acid (ATRA) plays crucial roles in cell survival and differentiation of neuroblastoma cells. In the present study, we investigated the effects of geranylgeranoic acid (GGA), an acyclic retinoid, on differentiation and tropomyosin-related kinase receptorB (TrkB) gene expression in SH-SY5Y human neuroblastoma cells in comparison with ATRA. GGA induced growth suppression and neural differentiation to the same extent as ATRA. Two variants (145 and 95 kD) of the TrkB protein were dramatically increased by GGA treatment, comparable to the effect of ATRA. Following 6- to 8-day GGA treatment, the effect of GGA on TrkB was reversed after 2-4 days of its removal, whereas the effect of ATRA was irreversible under the same conditions. Both GGA and ATRA upregulated the cellular levels of three major TrkB messenger RNA splice variants in a time-dependent manner. Time-dependent induction of cell cycle-related genes, such as cyclinD1 and retinoblastoma protein, and amplification of the neural progenitor cell marker, brain lipid binding protein, were suppressed by GGA treatment and were completely abolished by ATRA. ATRA and GGA induced retinoic acid receptorβ (RARβ) expression, whereas the time-dependent expression of both RARα and RARγ was abolished by ATRA, but not by GGA. Our results suggest that GGA may be able to restore neuronal properties of SH-SY5Y human neuroblastoma cells in a similar but not identical way to ATRA.

The role of retinoic acid receptor inhibitor LE135 on the osteochondral differentiation of human bone marrow mesenchymal stem cells

The present study aimed to investigate the role of a retinoic acid receptor-β (RARβ) inhibitor LE135 on TGF-β induced chondrogenesis of human bone marrow mesenchymal stem cells (hMSCs). Pellet culture with exogenous transforming growth factor-β (TGF-β), and a mechanically loaded scaffold system were used to provide two culture models. All samples were cultured for 8 days and changes in early gene expression were determined. Glycosaminoglycan and mRNA expression data showed that LE135 itself did not induce any chondrogenic response in either pellet culture or scaffold culture of hMSCs. LE135 actually inhibited the chondrogenic response caused by exogenous TGF-β, or endogenous TGF-β induced by mechanical load, while the expression of genes normally associated with osteogenesis was not affected. This suggests that the inhibitor LE135 affects the osteochondral differentiation pathway at a different stage, inhibiting chondrogenic gene expression while having no effect on genes normally associated with the osteogenic phenotype. Alternatively, it might be that different cells were proceeding down different lineages. Some cells were undergoing chondrogenesis and this was affected by LE135, while other cells underwent osteogenic differentiation and were not affected by LE135.

Relationship between promoter methylation of p16, DAPK and RAR beta genes and the clinical data of non-small cell lung cancer

To investigate the effects of promoter methylation of p16, death-associated protein kinase (DAPK) and retinoic acid receptor-beta (RAR beta) genes on clinical data in non-small cell lung cancers, and to study the effect of smoking on the risk of gene methylation. The promoter methylation of p16, DAPK and RAR beta genes in 200 primary non-small cell lung cancers and the corresponding nonmalignant lung tissues were determined by methylation-specific PCR. Methylation in the tumor tissues was detected in 51.0% for p16, 60.0% for DAPK, and 58.0% for RAR beta gene, with significant differences (P < 0.05) when compared with those in the corresponding nonmalignant tissues(12.5%, 11.5% and 15.0%) respectively. p16 gene methylation in tumor tissue was associated with age significantly in unconditional logistic regression analysis (P < 0.01) and histologic type (P < 0.05). DAPK gene methylation in tumor tissue was associated significantly with age (P < 0.05), gender (P < 0.05) and clinical type (P < 0.05). RAR beta gene methylation in tumor tissue was associated with clinical type (P < 0.05) and tumor stage (P < 0.05) significantly. The interaction odds ratio (OR) for the gene-gene interaction in tumor tissue between p16 and DAPK was 1.987 (95%CI:1.055-3.743). The results of the gene-smoking analyses revealed that a relationship existed between cigarette smoking and p16 gene methylation (OR = 3.139, 95%CI: 1.046-9.419), the OR for the relationship of DAPK gene methylation and cigarette smoking was 3.585(95%CI: 1.270-10.123) in tumor tissue. The RAR beta gene methylation did not differ based on the smoking status of patients in tumor tissue. The p16, DAPK and RAR beta genes methylation are strongly associated with clinical data of non-small cell lung cancer, and methylation of p16 and DAPK genes are associated with tobacco smoking.

New candidate proteins for Benzo(a)pyrene-induced spatial learning and memory deficits

Spatial learning and memory (LM) is a property of central importance in the nervous system, yet many of the molecular mechanisms for benzo(a)pyrene[B(a)P]-induced LM deficits remain enshrouded in mystery. In this study, influence of exposure to B(a)P on LM deficits in adult male Sprague-Dawley rats was evaluated by Morris water maze. Then morphological changes in the ultramicrostructure of hippocampal neurons were observed by transmission electron microscopy. Furthermore, to better understand the molecular changes that occur in B(a)P induced LM deficits, antibody-based protein microarrays was used to analyze protein expression changes in rats submitted to sub-chronic oral gavage of B(a)P (2 mg/kg for 90 days). Results suggested that rats in the B(a)P-treated groups have significantly impaired Morris water maze performance when compared to controls. Meanwhile, the B(a)P-induced neuronal damage was also found in the hippocampus under transmission electron microscopy. Our results demonstrate that LM deficits associated protein expression signatures could be identified from tissue proteomes, as well as potential biomarkers such as retinoic acid receptor b (RARb), synaptotagmin iosfomrs 1 (Syt1) and Brain-derived neurotrophic factor (BDNF), previously not found. This study, therefore, identifies, for the first time, multiple novel proteins that are dysregulated by B(a)P, which both enhance our understanding of B(a)P induced LM deficits and represent targets of novel therapeutics.

Association of Cigarette Smoking with Aberrant Methylation of the Tumor Suppressor Gene RAR?2 in Papillary Thyroid Cancer

Aberrant gene methylation is often seen in thyroid cancer, a common endocrine malignancy. Tobacco smoking has been shown to be associated with aberrant gene methylation in several cancers, but its relationship with gene methylation in thyroid cancer has not been examined. In the present study, we investigated the relationship between smoking of patients and aberrant methylation of tumor suppressor genes for TIMP3, SLC5A8, death-associated protein kinase, and retinoic acid receptor β2 (RARβ2) in papillary thyroid cancer (PTC), the most common type of thyroid cancer. The promoter methylation status of these genes was analyzed using quantitative real-time methylation-specific PCR on bisulfite-treated genomic DNA isolated from tumor tissues and correlated with smoking history of the patients. Among the four genes, methylation of the RARβ2 gene was significantly associated with smoking and other three genes showed a trend of association. Specifically, among the 138 patients investigated, 13/42 (31.0%) ever smokers vs. 10/96 (10.4%) never smokers harbored methylation of the RARβ2 gene (P = 0.003). This association was highly significant also in the subset of conventional variant PTC (P = 0.005) and marginally significant in follicular variant PTC (P = 0.06). The results demonstrate that smoking-associated aberrant methylation of the RARβ2 gene is a specific molecular event that may represent an important mechanism in thyroid tumorigenesis in smokers.

Combinations of Epigenetic Modifying Agents Accentuate Retinoid-Mediated Gene Expression Over Single Agent Therapies In Acute Myelogenous Leukemia Cells

AbstractAbstract 2497 Background.High cure rates seen in acute promyelocytic leukemia (APL) result from adding the retinoid all trans retinoic acid (ATRA) to chemotherapy. The addition of ATRA to chemotherapy has not consistently improved survival for other acute myelogenous leukemia (AML) subtypes. This likely reflects differences in the degree ATRA overcomes transcriptional repression imposed by the epigenetic mechanisms of histone acetylation and DNA methylation between AML and APL. Purpose To determine if differences in induction of retinoic acid receptor (RAR) β2 accounts for variations in ATRA-responses between APL and AML cells and to determine the effects epigenetic modifying agents have on the expression of RARβ2 and related gene sets in AML cells. Methods Primary AML samples, NB-4 (APL), and OCI/AML-2 (AML) cell lines were cultured in standard conditions. Treatments included ATRA 1μM, valproic acid (VPA) 0.6 mM, and 5-aza-2'-deoxycytidine (5-aza-2CDR) 400 ng/mL. Gene expression was measured by real-time PCR. Histone-acetylation was measured by chromatin immunoprecipitation (ChIP) and DNA methylation by EpiTYPER. Gene expression profiles were analyzed with Affymetrix U133A arrays followed by Gene Set Enrichment Analysis (GSEA) to identify RARβ2 related gene sets. Results Treatment of APL (NB-4 and primary samples) with ATRA induced expression of RARβ2 and expression remained attenuated in similarly treated AML (OCI/AML-2 and primary samples) cells. Histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) can potentially reverse epigenetic-mediated transcriptional repression. Treatment of OCI/AML-2 cells with the HDACi VPA and/or the DNMTi 5-aza-2CDR and treatment of primary AML samples with VPA restored ATRA-inducible RARβ2 expression. In accordance, DNA methylation and predominantly deacetylated histones were seen at the OCI/AML-2 RARβ promoter. Treatment of OCI/AML-2 cells with ATRA and VPA (HDACi) in the absence of 5-aza-2CDR (DNMTi) had little effect on DNA methylation at the RARβ promoter, however, 5-aza-2CDR treatments markedly decreased DNA methylation. An unexpected finding was VPA further reduced DNA methylation when added to 5-aza-2CDR treatment. Histone acetylation increased minimally at the RARβ promoter in ATRA-treated OCI/AML-2 cells. However, combining ATRA with VPA (HDACi) and/or 5-aza-2CDR (DNMTi) markedly increased histone acetylation that correlated with gene-induction. The greatest change in histone acetylation and DNA methylation was seen in OCI/AML-2 cells treated with the combination of ATRA + VPA + 5-aza-2CDR that correlated with the largest induction of RARβ2. Heterogeneous levels of DNA methylation were seen at the RARβ promoter in 26 primary AML samples compared to 4 normal bone marrow (NBM) samples. Levels of DNA methylation were similar to NBM in 13 AML samples (50%), decreased in 5 (19%), both increased and decreased in the same sample in 2 (8%), and increased in 6 (23%) primary AML samples. Next, we used Affymetrix gene arrays and GSEA to delineate differences in ATRA-mediated gene regulation between NB-4 (APL) and OCI/AML-2 (AML) cells and to determine whether treatment with VPA (HDACi) and 5-aza-2CDR (DNMTi) restored expression of gene sets that included RARβ2 signaling. ATRA-treatment induced twelve such gene sets in NB-4 cells and six gene sets in OCI/AML-2 cells. Treatment of OCI/AML-2 cells with VPA alone did not induce expression of RARβ2 associated gene sets, whereas treatment of OCI/AML-2 cells with 5-AZA-2CDR modulated expression of a further five gene sets that were modulated in ATRA-treated NB-4 (APL) cells. Treatment of OCI/AML-2 cells with all three agents ATRA + VPA + 5-aza-2CDR was the only treatment combination that modulated the ligand_dependent_nuclear_receptor_activity gene set. This gene set includes many genes regulating retinoid signaling and are engaged as early as 3 hours after ATRA treatment in NB-4 cells. Conclusions RARβ2 is a frequent target for transcriptional repression by epigenetic mechanisms in AML cells. Combinations of HDACi and DNMTi increase ATRA-mediated induction of RARβ2 and epigenetic modifications that favor transcription over individual treatments. This includes a possible demethylating effect of the HDACi VPA. These treatment combinations also restore modulation of a number of RARβ2 related gene sets in AML cells that are similarly regulated in ATRA-treated APL cells. Disclosures:No relevant conflicts of interest to declare.