Evidence that retinoic acid receptor beta induction by retinoids is important for tumor cell growth inhibition

Retinoic acid receptor beta (RARbeta) is thought to be involved in suppressing cell growth and tumorigenicity. Many premalignant and malignant cells exhibit a reduced RARbeta expression. However, in some of these cells (e.g. H157 human squamous cell carcinoma cells), RARbeta can be induced by retinoids (e.g. all-trans-retinoic acid, ATRA) because its promoter contains a retinoic acid response element. To examine the hypothesis that RARbeta induction is important for inhibition of cell proliferation by retinoids, we blocked ATRA-induced RARbeta expression in H157 cells using a retroviral vector harboring multiple copies of antisense RARbeta2 sequences. Antisense RARbeta-transfected cells showed not only decreased expression of ATRA-induced RARbeta protein but also reduced ATRA-induced RARE binding activity and transactivation. Importantly, all antisense RARbeta transfectants of H157 cells were less responsive than vector-transfected cells to the growth inhibitory effects of the retinoids ATRA and Ch55 in vitro. These results demonstrate that RARbeta induction may play an important role in mediating growth inhibitory effects of retinoids in cancer cells.     

Effect of retinoids on growth factor-induced anchorage independent growth of human fibroblasts

Summary We have studied the effects of all-trans retinol, all-trans retinoic acid, and anhydroretinol, a biologically inactive retinoid, on anchorage-independent growth of human fibroblasts induced by purified growth factors. The anchorage-independence assay was, conducted in medium supplemented with serum that had had its peptide growth factors inactivated by treatment with dithiothreitol and iodoacetamide. Physiologic concentrations of either all-trans retinol (0.5 μM) or all-trans retinoic acid (1.0 nM) but not anhydroretinol (0.5 μM) reduced the frequency of anchorage-independent growth of normal human fibroblasts induced by platelet-derived growth fator (PDGF). All-trans retinol was also tested for its effect on the frequency of anchorage-independent growth induced by basic fibroblast growth factor (bFGF) and was found to decrease this growth. All-trans retinol also reduced the frequency of anchorage-independent growth of the human fibrosarcoma-derived cell, line, HT1080, which grew in semisolid medium without added growth factors. Inasmuch as these retinoids reduced the frequency of anchorage-independent growth induced by either PDGF or bFGF and because PDGF and bFGF bind to independent cell membrane receptors and are known to stimulate different pathways leading to DNA synthesis, the data suggest that physiologically active retinoids, have an effect on a step that is common to both signal pathways. This research was supported in part by Department of Energy, Washington, DC, grant DE-F602-87ER-60524 and by DHHS grants CA21289 and, CA32490 from the National Cancer Institute, Bethesda, MD.     

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.     

Gene expression profiling identifies retinoids as potent inducers of macrophage lipid efflux

Vitamin A and its naturally occurring derivatives 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (ATRA) exert a variety of biological effects including immunomodulation, growth, differentiation, and apoptosis of normal and neoblastic cells. In order to directly study the effects of these retinoids on macrophage gene expression and lipid metabolism, primary human monocytes and in vitro differentiated macrophages were stimulated with beta-carotene, 9-cis RA, and ATRA and global gene expression profiles were analyzed by Affymetrix DNA-microarrays and differentially regulated genes were verified by quantitative TaqMan RT-PCR. Among others, we have identified a strong up-regulation of a cluster of genes involved in cholesterol metabolism including apolipoproteins (apoC-I, apoC-II, apoC-IV, apoE), the scavenger receptor CD36, steroid-27-hydroxylase (CYP27A1), liver X receptor alpha (LXRalpha), and ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1). Since the CYP27A1 gene displayed the strongest up-regulation on the mRNA level, we cloned various deletion constructs of the promoter region and analyzed the response to retinoids in macrophages. Thereby, a novel retinoic acid-responsive element could be located within 191 bp of the proximal CYP27A1 promoter. To further assess the functional consequences of retinoid receptor action, we carried out phospholipid and cholesterol efflux assays. We observed a strong induction of apoA-I-dependent lipid efflux in stimulated macrophages, implicating an important role for retinoids in cellular functions of macrophages.     

Differential response to retinoic acid of Syrian hamster embryo fibroblasts expressing v-src or v-Ha-ras oncogenes.

It has been shown that treatment of many but not all tumor cell lines with retinoids affects cell proliferation and expression of the transformed phenotype. To determine whether the response of the tumor cell to retinoids is influenced by specific oncogenes activated in the cell, we studied the action of these agents in the immortal, nontumorigenic Syrian hamster embryo cell lines DES-4 and 10W transfected with either v-Ha-ras or v-src oncogenes. In this paper we show that in transformed DES-4 cells expressing v-src, retinoic acid inhibited anchorage-independent growth, reduced saturation density, and inhibited the induction of ornithine decarboxylase by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. In contrast, retinoic acid enhances the expression of the transformed phenotype in DES-4-derived cells that express v-Ha-ras. In these cells retinoic acid increases the number and the average size of colonies formed in soft agar. Moreover, retinoic acid enhances ornithine decarboxylase activity and acts in a synergistic fashion with 12-O-tetradecanoylphorbol-13-acetate. These results indicate that oncogenes activated in cells can indeed influence the response of cells to retinoids. Retinoic acid does not appear to alter the levels of pp60src or p21ras proteins in these cells, suggesting that retinoic acid does not affect the synthesis of these oncogene products. Furthermore, retinoic acid does not affect the protein kinase activity of pp60src. Transformed cell lines derived from 10W cells responded differently, indicating that the presence of a specific oncogene is not the only factor determining the response to retinoids. Possible mechanisms by which retinoic acid may interfere with the expression of the oncogene products are discussed.     

Growth and intestinal differentiation are independently regulated in HT29 colon cancer cells

The polar-planar compound hexamethylene bisacetamide (HMBA) can inhibit HT29 colon carcinoma cell growth and induce a more benign phenotype, as defined by decreased anchorage-independent clonogenicity, loss of a cell surface malignancy marker, and decreased in vivo tumorigenicity. The principle aim of this study was to determine whether HMBA's effects on HT29 cell growth and biologic behavior correlate with effects on intestinal differentiation. Parallel studies were performed with sodium butyrate (NaBT), a potent inducer of intestinal differentiation HT29 cell growth, proliferation, and markers of intestinal differentiation were assayed after short- and long-term treatment with HMBA, NaBT, or the combination. Both 5 mM HMBA and 5 mM NaBT were potent inhibitors of monolayer growth; in combination their effects were nearly additive. Inhibition of DNA synthesis was detectable within 6 h of treatment and was preceded by down-regulation of c-myc expression. Soft agar clonogenicity was also decreased by 90%, > 99%, and > 99% by HMBA, NaBT, and the combination, respectively. Despite these parallel effects on growth and in vitro markers of a benign phenotype, effects on intestinal differentiation were discordant. NaBT induced significant increases in membrane-associated alkaline phosphatase activity, cytosolic mucin content, PAS⁺/diastase-resistant cells, and ultrastructural evidence of intestinal cell differentiation. HMBA not only failed to induce markers of intestinal differentiation, but attenuated NaBT's effects when used in combination. These data suggest that growth and intestinal differentiation may be independently regulated in HT29 cells. They also suggest that expression of intestinal markers of differentiation is not a prerequisite for the acquisition of a more benign phenotype. © 1994 Wiley-Liss, Inc.     

Differential regulation of protein expression, growth and apoptosis by natural and synthetic retinoids

All-trans retinoic acid (ATRA) can down regulate the anti-apoptotic protein Bcl-2 and the cell cycle proteins cyclin D1 and cdk2 in estrogen receptor-positive breast cancer cells. We show here that retinoids can also reduce expression of the inhibitor of apoptosis protein, survivin. Here we have compared the regulation of these proteins in MCF-7 and ZR-75 breast cancer cells by natural and synthetic retinoids selective for the RA receptors (RARs) alpha, beta, and gamma then correlated these with growth inhibition, induction of apoptosis and chemosensitization to Taxol. In both cell lines ATRA and 9-cis RA induced the most profound decreases in cyclin D1 and cdk2 expression and also mediated the largest growth inhibition. The RARalpha agonist, Ro 40-6055 also strongly downregulated these proteins although did not produce an equivalent decrease in S-phase cells. Only ATRA induced RARbeta expression. ATRA, 9-cis RA and 4-HPR initiated the highest level of apoptosis as determined by mitochondrial Bax translocation, while only ATRA and 9-cis RA strongly reduced Bcl-2 and survivin protein expression. Enumeration of dead cells over 96 h correlated well with downregulation of both survivin and Bcl-2. Simultaneous retinoid-mediated reduction of both these proteins also predicted optimal Taxol sensitization. 4-HPR was much weaker than the natural retinoids with respect to Taxol sensitization, consistent with the proposed requirement for reduced Bcl-2 in this synergy. Neither the extent of cell cycle protein regulation nor AP-1 inhibition fully predicted the antiproliferative effect of the synthetic retinoids suggesting that growth inhibition requires regulation of a spectrum of RAR-regulated gene products in addition even to pivotal cell cycle proteins.     

Effects of olive oil polyphenols on fatty acid synthase gene expression and activity in human colorectal cancer cells

Oleuropein (OL) and hydroxytyrosol (HT), the main olive oil polyphenols, possess anti-proliferative effects in vitro. Fatty acid synthase, a key anabolic enzyme of biosynthesis of fatty acids, plays an important role in colon carcinoma development. Our aim was to investigate whether gene expression of FAS, as well as its enzymatic activity, is regulated by HT and OL in two human colon cancer cell lines, as HT-29 and SW620. In addition, we investigated the effects of these polyphenols on growth and apoptosis in these cells. FAS gene expression and activity in treated HT-29 and SW620 cells were evaluated by real-time PCR and radiochemical assay, respectively. Cell growth and apoptosis, after polyphenols treatment, were measured by MTT test and flow cytometry, respectively. The inhibition of proliferation, detected after HT treatment, was mediated by an inhibition of FAS expression and its enzymatic activity in SW620 cells, while the anti-proliferative effect in HT-29 cells seems to be independent from FAS. OL exerted an anti-proliferative effect only on SW620 cells with a mechanism which excluded FAS. Olive oil polyphenols used were able to induce apoptosis in both cell lines studied. The increase of apoptosis in these cells was accompanied by the block of cell cycle in the S phase. This study demonstrates that HT and OL may induce anti-proliferative and pro-apoptotic effects only in certain human colorectal cancer cell types. These effects are FAS mediated only in SW620 cells after treatment with HT.     

Evidence for species differences in the regulation of MMPs by all-trans retinoic acid in cytokine-stimulated chondrocytes

In inflammatory conditions, chondrocytes produce large amounts of matrix metalloproteases (MMP) and nitric oxide (NO) thought to contribute to joint degradation. We tested the ability of all-trans retinoic acid (ATRA, a retinoic acid receptor (RAR) agonist) to modulate these inflammatory genes in chondrocytes from humans or rats, chosen as representative of animal models of arthritis. All RAR subtypes and RXR-alpha or -beta were expressed at the mRNA level in both species, although IL-1beta (10 ng/ml) inhibited RAR subtypes more markedly in rat than in human cells. ATRA (300 or 1000 nM) inhibited IL-1-induced expression of iNOS and nitrites level in both species, although the NO pathway was induced maximally in rat cells. ATRA displayed controversial effects on MMPs between rat and human chondrocytes, especially for MMP-9 expression. The effects of ATRA were irrelevant to the nuclear translocation of AP-1. The present data underlines that retinoids have a species-dependent impact on IL-1-induced responses in chondrocytes, suggesting that extrapolation of their pharmacological properties from animal cells has a poor relevance to clinical situation.     

Celecoxib increases retinoid sensitivity in human colon cancer cell lines

Retinoid resistance has limited the clinical application of retinoids as differentiation-inducing and apoptosis-inducing drugs. This study was designed to investigate whether celecoxib, a selective COX-2 inhibitor, has effects on retinoid sensitivity in human colon cancer cell lines, and to determine the possible mechanism of said effects. Cell viability was measured using the MTT assay. Apoptosis was detected via Annexin-V/PI staining and the flow cytometry assay. PGE₂ production was measured with the ELISA assay. The expression of RARβ was assayed via western blotting. The results showed that celecoxib enhanced the inhibitory effect of ATRA in both COX-2 high-expressing HT-29 and COX-2 low-expressing SW480 cell lines. Further study showed the ATRA and celecoxib combination induced greater apoptosis, but that the addition of PGE₂ did not affect the enhanced growth-inhibitory and apoptosis-inducing effects of the combination. Moreover, NS398 (another selective COX-2 inhibitor) did not affect the inhibitory effects of ATRA in the two cell lines. Western blotting showed that the expression of RARβ in HT-29 cell lines was increased by celecoxib, but not by NS398, and that the addition of PGE₂ did not affect the celecoxib-induced expression of the retinoic acid receptor beta. In conclusion, celecoxib increased the expression of RARβ and the level of cellular ATRA sensitivity through COX-2-independent mechanisms. This finding may provide a potential strategy for combination therapy.     

血清对全反式视黄酸抑制肺癌细胞生长的影响

 研究不同浓度的血清对全反式视黄酸 (ATRA)抑制肺癌细胞生长的影响 .当细胞培养在 10 %血清中 ,ATRA不能抑制肺癌细胞生长 ,但是当细胞培养在 1%血清中 ,ATRA能够有效地抑制肺癌细胞生长 .视黄酸受体RARβ介导视黄酸的抗癌作用 .Northern印迹分析表明 ,在高浓度血清中AT RA不能诱导RARβ表达 ,但在低浓度血清中ATRA可以诱导RARβ表达 ,并且瞬时转染和CAT测定证实是通过激活RABβ启动子转录活性而诱导RARβ表达的 .孤生受体Nur77受到血清生长因子刺激后会大量表达 ,具有抗视黄酸活性的作用 .肺癌细胞培养在低浓度血清中 ,Nur77mRNA低水平表达和Nur77蛋白不表达 .然而在高浓度血清中 ,Nur77mRNA和蛋白高水平表达 .另外 ,在无血清条件下 ,EGF也可以诱导Nur77表达 .结果提示 ,血清中的生长因子可能拮抗ATRA抑制肺癌细胞生长的作用 ,其作用途径可能是通过刺激细胞中Nur77表达 ,或者通过下调RARβ启动子的转录活性而抑制RARβ的表达     

Retinoids Differentially Regulate the Proliferation of Colon Cancer Cell Lines

In this study, the proliferative effects of retinoids were examined in the MC-26 and LoVo colon adenocarcinoma cell lines. The proliferation of the LoVo cell line was not altered in the presence of the retinoidsall trans-retinoic acid (atRA) and 9-cis-retinoic acid (9-cis-RA). Both retinoids, however, stimulated the growth, as measured by cell proliferation, of MC-26 cells.atRA and 9-cis-RA were equipotent in increasing MC-26 cell proliferation, suggesting that the growth stimulation is mediated by one or more retinoic acid receptors (RARs). To determine the RAR which might be responsible for this growth stimulatory effect, we characterized the RAR subtypes which were present in both cell lines. mRNA for the RARα, RARβ, and RARγ were detected in the MC-26 cell. Of the RARs present in MC-26 cells, the RARα does not mediate the growth stimulatory effects of retinoids, for a selective RARα antagonist was unable to prevent the retinoid-induced increase in MC-26 cell growth. RARα, RARβ, and RARγ mRNA are also expressed in the LoVo cell line; the lack of growth-stimulation by retinoids in LoVo cells, therefore, does not seem to be due to the absence of RARs. The results obtained in these experiments demonstrate that the growth response elicited by retinoids can vary between colon cancer cells and that the differences in response may not be solely determined by the RAR subtypes which are expressed in a colon cancer cell line.     

Retinoic acid hydroxylase (CYP26) is a key enzyme in neuronal differentiation of embryonal carcinoma cells.

Besides nuclear retinoid receptors and cellular retinoid binding proteins also retinoic acid (RA)-synthesizing enzymes (using all-trans-retinal as substrate) and RA-catabolizing enzymes (producing hydroxylated products) may explain the specific effects of retinoids. In the past we have established an active role for 4-hydroxy-RA and 4-oxo-RA, which originally were considered to be inactive retinoids, but in fact are highly active modulators of positional specification in Xenopus development. Here we present evidence for a specific role of hydroxylated RA metabolites in the onset of neuronal differentiation. 4-Hydroxy- and 18-hydroxy-RA are products of the hydroxylation of RA by a novel cytochrome P450 (CYP)-type of enzyme, CYP26, expression of which is rapidly induced by RA. P19 embryonal carcinoma (EC) cell lines stably expressing hCYP26 undergo extensive and rapid neuronal differentiation in monolayer at already low concentrations of RA, while normally P19 cells under these conditions differentiate only in endoderm-like cells. Our results indicate that the effects on growth inhibition and RARbeta transactivation of P19 EC cells are mediated directly by RA, while the onset of neuronal differentiation and the subsequent expression of neuronal markers is mediated by hCYP26 via the conversion of RA to its hydroxylated products.