Dafna Lotan

Expression of galectins on microvessel endothelial cells and their involvement in tumour cell adhesion.

Lactoside-binding lectins (galectins) with molecular weights of about 14.5 kDa (galectin-1) and 29–35 kDa (galectin-3) bind preferentially to polylactosaminoglycan-containing glycoconjugates and have been found on the surface of tumour cells and implicated in cell-cell and cell-extracellular matrix adhesion and metastasis. We have demonstrated by immunoblotting that both galectin-1 and galectin-3 are present in extracts of endothelial cells cultured from bovine aorta, rat lung, mouse lung and mouse brain microvessels, whereas mouse hepatic sinusoidal endothelial cells expressed primarily galectin-1. These galectins were also localized by indirect immunofluorescent labelling on the surface of the different endothelial cells in culture and by immunohistochemical staining in human tissuesin vivo. Anti-galectin-1 antibodies inhibited the adhesion of liver-preferring murine RAW117-H10 large-cell lymphoma cells to hepatic sinusoidal endothelial cells or lung microvessel endothelial cellsin vitro. The data indicate that galectin-1 is expressed on the extracellular surface of endothelial cells and can mediate in part the adhesion of RAW117-H10 cells to liver microvessel endothelial cells.

Hypermethylation of the Retinoic Acid Receptor-β2 Gene in Head and Neck Carcinogenesis

Purpose: Retinoic acid receptor-β2 (RAR-β2) expression is suppressed in oral premalignant lesions and head and neck squamous cell carcinomas (HNSCCs). This study was conducted to determine whether RAR-β2 gene expression in such lesions can be silenced by promoter methylation. Experimental Design: RAR-β2 methylation was analyzed in DNA samples from 22 pairs of primary HNSCC and adjacent normal epithelium, 124 samples of oral leukoplakia, and 18 HNSCC cell lines using methylation-specific PCR. RAR-β2 promoter was methylated in 67, 56, and 53% of HNSCC tumors, HNSCC cell lines, and microdissected oral leukoplakia specimens, respectively. RAR-β2 hypermethylation was confirmed by sodium bisulfite-PCR combined with restriction enzyme digestion analysis and by random cloning and sequencing of bisulfite-treated DNA isolates. Results: Significantly higher RAR-β2 hypermethylation levels were found in tumor tissue compared with adjacent normal tissue (P = 0.002). RAR-β2 methylation in the cell lines was correlated with loss of RAR-β2 expression (P = 0.013) and inversely related to the presence of mutated p53 (P = 0.025). The demethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR) restored RAR-β2 inducibility by all-trans-retinoic acid (ATRA) in some of the cell lines, which posses a methylated RAR-β2 promoter. In some cell lines, this effect was associated with increased growth inhibition after combined treatment with 5-aza-CdR and ATRA. Conclusions: RAR-β2 silencing by methylation is an early event in head and neck carcinogenesis; 5-Aza-CdR can restore RAR-β2 inducibility by ATRA in most cell lines, and the combination of 5-aza-CdR and ATRA is more effective in growth inhibition than single agents.

Retinoic acid-induced modifications in the growth and cell surface components of a human carcinoma (HeLa) cell line☆

Abstract The addition of retinoic acid to cultures of HeLa-S3 cells caused a reduction in cell proliferation rate which became apparent after 72 h and was linearly dependent on retinoic acid concentration in the range 10−9–10−5 M. After 72 h of exposure to retinoic acid, the cells assumed a flattened appearance and no longer formed multilayers. These changes were reversed within 48 h after removal of retinoic acid from the medium. Structural analogs of retinoic acid with a free COOH group at C-15 were usually more potent in growth inhibition than compounds with an alcohol, aldehyde, ether or ester group. A cellular retinoic acid-binding protein was detected in cell homogenates, and the binding of [3H]retinoic acid to the binding protein was inhibited by most, but not all, analogs possessing a free terminal COOH group. For example, the 4-oxo analog of retinoic acid, while capable of inhibiting cellular proliferation, failed to bind to the retinoic acid-binding protein. Analysis of cell surface and cellular glycoproteins by lactoperoxidase-catalysed 125I iodination and by metabolic labeling with [3H]glucosamine revealed that a 190000 D glycoprotein which was labeled by both methods and a 230000 D glycoprotein which was labeled only with [3H]glucosamine were labeled more intensely in retinoic acid-treated cells compared with untreated cells. The electrophoretic mobility of the 230000 D glycoprotein could be modified by treatment of intact cells with either neuraminidase or proteolytic enzymes, suggesting that this glycoprotein is also exposed on the cell surface. The cell surface alterations were detected much earlier than the onset of growth inhibition and appeared as early as 24 h after exposure to retinoic acid. The possible relationship between retinoic acid-induced changes in cell membrane structure, cell morphology, and cell proliferation is discussed.

CHARACTERIZATION OF RETINOIC ACID‐INDUCED ALTERATIONS IN THE PROLIFERATION AND DIFFERENTIATION OF A MURINE AND A HUMAN MELANOMA CELL LINE IN CULTURE*

We employ the murine S91 and the human Hs939 melanoma cell lines for the characterization of various biochemical changes induced by retinoids. Retinoic acid (RA) causes a time-dependent, and reversible reduction in cell proliferation rate in liquid medium and inhibits growth in agar. The proportion of cells in the G1 phase of the cell cycle increases in RA-treated cells, and the uptake of TdR, UdR and Leu decreases. The growth inhibitory effect of RA is apparently not mediated via labilization of lysosomes, increase in cAMP or changes in the synthesis of prostaglandins or polyamines. Exposure to RA stimulates tyrosinase activity and increases melanin content severalfold over the levels found in untreated cells. Various retinoids exhibit the activities of RA; however, their potencies vary depending on their structure. Those possessing a free -COOH at C-15 are usually more effective than those with a different group or with a derivatized carboxyl. A positive correlation exists between the ability of retinoids with a free -COOH in C-15 to inhibit growth and to bind to an RA-binding protein found in the S91 melanoma cells. Future studies will explore recently discovered changes in the glycosylation of cell surface components and their relationship to the phenomena described here.

Knockout of the Tumor Suppressor Gene Gprc5a in Mice Leads to NF-κB Activation in Airway Epithelium and Promotes Lung Inflammation and Tumorigenesis

Mouse models can be useful for increasing the understanding of lung tumorigenesis and assessing the potential of chemopreventive agents. We explored the role of inflammation in lung tumor development in mice with knockout of the tumor suppressor Gprc5a. Examination of normal lung tissue and tumors from 51 Gprc5a+/+ (adenoma incidence, 9.8%; adenocarcinoma, 0%) and 38 Gprc5a−/− mice (adenoma, 63%; adenocarcinoma, 21%) revealed macrophage infiltration into lungs of 45% of the Gprc5a−/− mice and 8% of Gprc5a+/+ mice and the direct association of macrophages with 42% of adenomas and 88% of adenocarcinomas in the knockout mice. Gprc5a−/− mouse lungs contained higher constitutive levels of proinflammatory cytokines and chemokines and were more sensitive than lungs of Gprc5a+/+ mice to stimulation of NF-κB activation by lipopolysaccharide in vivo. Studies with epithelial cells cultured from tracheas of Gprc5a−/− and Gprc5a+/+ mice revealed that Gprc5a loss is associated with increased cell proliferation, resistance to cell death in suspension, and increased basal, tumor necrosis factor α–induced, and lipopolysaccharide-induced NF-κB activation, which were reversed partially in Gprc5a−/− adenocarcinoma cells by reexpression of Gprc5a. Compared with Gprc5a+/+ cells, the Gprc5a−/− cells produced higher levels of chemokines and cytokines and their conditioned medium induced more extensive macrophage migration. Silencing Gprc5a and the p65 subunit of NF-κB in Gprc5a+/+ and Gprc5a−/− cells, respectively, reversed these effects. Thus, Gprc5a loss enhances NF-κB activation in lung epithelial cells, leading to increased autocrine and paracrine interactions, cell autonomy, and enhanced inflammation, which may synergize in the creation of a tumor-promoting microenvironment. Cancer Prev Res; 3(4); 424–37. ©2010 AACR.

Modulation of galectin-1 content in human head and neck squamous carcinoma cells by sodium butyrate

Galectin‐1 and galectin‐3 are β‐galactoside‐binding proteins thought to be important for cellular interactions, growth regulation and differentiation. Alterations in cellular content of galectins have been associated with differentiation, transformation and malignant progression. We examined the modulation of galectin‐1 and galectin‐3 expression in head and neck squamous cell carcinoma (HNSCC) cell lines by treatment with sodium butyrate, a known differentiation‐modulating agent, and identified potential mechanisms of butyrate regulation of galectin‐1 levels in one of the cell lines. Sodium butyrate effected an increase in galectin‐1 protein concentration in 5 of 8 cell lines. One cell line, MDA‐886LN, showed a marked time‐ and dose‐dependent increase from barely detectable amounts with butyrate treatment. Concurrently with increased galectin‐1 expression, butyrate treatment promoted morphologic changes, induced growth inhibition and inhibited soft agar colony formation in MDA‐886LN cells. Butyrate‐treated MDA‐886LN cells demonstrated increased galectin‐1 mRNA content, suggesting a role for butyrate in transcriptional regulation of galectin‐1 expression. Treatment with other inhibitors of histone deacetylase also induced an increase in galectin‐1 expression. Together, our results indicate that butyrate treatment can modulate galectin‐1 content in MDA‐886LN HNSCC cells as well as induce morphologic changes and growth inhibition. This action may involve a combination of transcriptional regulation and inhibition of histone deacetylation. Int. J. Cancer 75:217–224, 1998.

Induction of endoplasmic reticulum stress by the pro-apoptotic retinoid N-(4-Hydroxyphenyl)retinamide via a reactive oxygen species-dependent mechanism in human head and neck cancer cells

N-(4-hydroxyphenyl)retinamide (4HPR), which has shown efficacy in cancer chemoprevention and therapy, induces the mitochondrial apoptosis pathway via increased generation of reactive oxygen species (ROS). ROS is also known to be able to induce an endoplasmic reticulum (ER) stress response, which can contribute to apoptosis but may also antagonize it. Therefore, we used human head and neck squamous carcinoma (HNSCC) cells to determine whether 4HPR affects ER stress. Different experimental approaches have indicated that 4HPR induces ER stress response: electron microscopy, which showed extensive ER dilation; splicing of the X-box binding protein 1 (XBP-1), a marker of unfolded protein response (UPR) activation; and quantitative real-time PCR and immunoblotting, which revealed the up-regulation of several ER-stress associated mRNAs and proteins, including the chaperone heat shock protein HSPA1A. Most of these effects of 4HPR were abrogated by co-treatment of cells with the antioxidant 3-tert-butyl-4-hydroxyanisole (BHA) indicating that they were downstream of the increase in ROS. Furthermore, siRNA-mediated silencing and chemical inhibition of HSPA1A, which exerts either pro- or anti-apoptotic effects, decreased 4HPR-induced apoptosis. These results demonstrate that 4HPR induces ER stress and uncovered a pro-apoptotic role for HSPA1A in 4HPR-induced apoptosis.

Gprc5a Deletion Enhances the Transformed Phenotype in Normal and Malignant Lung Epithelial Cells by Eliciting Persistent Stat3 Signaling Induced by Autocrine Leukemia Inhibitory Factor

Signal transducers and activators of transcription 3 (Stat3) is activated by cytokines and growth factors in lung cancers and regulates expression of genes implicated in cell growth, survival, and transformation. Previously, we found that mice with a deletion of the G protein-coupled receptor, family C, group 5, member a (Gprc5a) gene develop lung tumors, indicating that Gprc5a is a tumor suppressor. Herein, we show that epithelial cells from Gprc5a knockout mouse lung (Gprc5a(-/-) cells) survive better in vitro in medium deprived of exogenous growth factors and form more colonies in semisolid medium than their counterparts from wild-type mice (Gprc5a(+/+) cells). Stat3 tyrosine 705 phosphorylation and expression of several Stat3-regulated antiapoptotic genes were higher in Gprc5a(-/-) than in Gprc5a(+/+) cells. Both cell types secreted leukemia inhibitory factor (Lif); however, whereas Stat3 activation was persistent in Gprc5a(-/-) cells, it was transient in Gprc5a(+/+) cells. Lung adenocarcinoma cells isolated from Gprc5a(-/-) mice also exhibited autocrine Lif-mediated Stat3 activation. The level of Socs3, the endogenous Stat3 inhibitory protein, was higher in Gprc5a(+/+) than in Gprc5a(-/-) cells, and expression of the tumor suppressor stabilized Socs3. Inhibition of Stat3 signaling in Gprc5a(-/-) normal and cancer cells by the Janus-activated kinase 2 inhibitor AG490 or by a dominant negative Stat3(Y705F) increased starvation-induced apoptosis and inhibited colony formation. These results show that persistent Stat3 activation is important for the survival and transformation of Gprc5a(-/-) lung cells and suggest that the tumor suppressive effects of Gprc5a are mediated, at least in part, by inhibition of Stat3 signaling through Socs3 stabilization.

Differential inhibition of protein translation machinery by curcumin in normal, immortalized, and malignant oral epithelial cells.

Curcumin has shown some promise in the prevention of oral carcinogenesis by mechanism(s) that are still not completely resolved. Messenger RNA translation is mediated in eukaryotes by the eIF4F complex composed of eukaryotic translation initiation factors eIF4E, eIF4G, and eIF4A. Overexpression of some of these components or the inactivation of initiation repressor proteins (4E-BP1) has been implicated in cancer development including oral carcinogenesis by affecting cell survival, angiogenesis, and tumor growth and invasion. In this study, we examined the possibility that curcumin affects the translational machinery differently in normal, immortalized normal, leukoplakia, and malignant cells. Curcumin treatment in vitro inhibited the growth of immortalized oral mucosa epithelial cells (NOM9-CT) and the leukoplakia cells (MSK-Leuk1s) as well as in the UMSCC22B and SCC4 cells derived from head and neck squamous cell carcinoma. Curcumin only exerted minor effects on the growth of normal oral epithelial cells (NOM9). In the immortalized, leukoplakia, and cancer cells, curcumin inhibited cap-dependent translation by suppressing the phosphorylation of 4E-BP1, eIF4G, eIF4B, and Mnk1, and also reduced the total levels of eIF4E and Mnk1. Our findings show that immortalized normal, leukoplakia, and malignant oral cells are more sensitive to curcumin and show greater modulation of protein translation machinery than the normal oral cells, indicating that targeting this process may be an important approach to chemoprevention in general and for curcumin in particular. Cancer Prev Res; 3(3); 331–8

Induction of GDF-15/NAG-1/MIC-1 in human lung carcinoma cells by retinoid-related molecules and assessment of its role in apoptosis

Growth and Differentiation Factor-15 (GDF-15, also known as NAG-1 and MIC-1) is induced by several apoptosis-inducing agents including the retinoid-related molecule (RRM) 6-[3-(1-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437). It has been suggested that GDF-15 may be involved in the induction of apoptosis by CD437 in H460 lung cancer cells. The present study was designed to probe this hypothesis more directly. Several RRMs (CD437, ST1926, and MX3350) but not the retinoids all-trans-retinoic acid and 4HPR were able to induce GDF-15 in H460 cells. A similar differential effect of these retinoids was observed for the induction of p53, which has been reported to regulate GDF-15 expression. In H460 cells transfected with a neo vector control (H460-Neo), treatment with RRMs but not ATRA or 4HPR resulted in increases in p53, GDF-15 and apoptosis evidenced by polyADP ribose polymerase (PARP) cleavage. In contrast, RRMs failed to increase p53 or induce apoptosis in H460 cells in which p53 was inactivated by transfection of the human papillomavirus E6-6 (H460-E6-6). The increase in GDF-15 by RRMs was also compromised in the H460-E6-6 cells. Because PARP cleavage was only evident when GDF-15 levels where elevated it appeared that GDF-15 was mediating the pro-apoptotic effects of RRMs. However, silencing of GDF-15 induction by RNA interference failed to decrease the ability of CD437 and ST1926 to induce apoptosis. These results demonstrate that GDF-15 is dispensable for the proapoptotic activity of CD437 and ST1926.