Comparison of the mechanism of induction of apoptosis in ovarian carcinoma cells by the conformationally restricted synthetic retinoids CD437 and 4-HPR

All-trans-retinoic acid (ATRA) has been shown to inhibit the growth of a number of ovarian tumor cell lines while others have been found to be resistant to retinoid suppression of growth. Interestingly, two synthetic retinoids, CD437 and 4-HPR, inhibit the growth of both ATRA-sensitive (CA-OV-3) and ATRA-resistant (SK-OV-3) ovarian tumor cells. However, in contrast to ATRA, both induce apoptosis. Our goal was to elucidate the mechanism by which these two synthetic retinoids induce apoptosis in ovarian tumor cells. Since it has been documented that apoptosis induction is often mediated by the activation of a cascade of proteases known as caspases, we initially studied the role of caspases in induction of apoptosis by CD437 and 4-HPR. We found that both retinoids induced caspase-3 and caspase-9 enzyme activity. Furthermore, using caspase specific inhibitors we determined that caspase-3 and caspase-9 activity was essential for the induction of apoptosis by these synthetic retinoids since these inhibitors completely blocked CD437 and 4-HPR induced apoptosis. Interestingly, we found that treatment with bongkriekic acid (BA), a mitochondrial membrane depolarization inhibitor, blocked apoptosis, caspase-9 activation and caspase-3 activation induced by both retinoids. Finally, we were able to determine that CD437 treatment induced the translocation of TR3, a nuclear orphan receptor, whereas, 4-HPR did not. Our results suggest that CD437 and 4-HPR initially activate separate pathways to induce mitochondrial depolarization but both utilize mitochondrial depolarization, caspase-9 activation, and caspase-3 activation in the later stages of apoptosis induction.     

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.     

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.     

Retinoic acid stimulates growth hormone synthesis in human somatotrophic adenoma cells: Characterization of its nuclear receptors

In order to gain a better understanding on the possible role of retinoic acid (RA) on human GH secretion, we have characterized the expression of its nuclear receptors in somatotropic adenoma cell extracts. By immunoblotting with rabbit polyclonal antibodies directed against RARα, β, and γ and RXRα and β, we could only detect the presence of RARα and RXRα proteins. The predominant expression of RXRα was confirmed at the mRNA level by Northern and slot-blot analysis. When then investigated the effect of RA on GH synthesis in cell culture of adenomatous somatotrophs. In cultured cells, RA (1 μM) stimulated GH secretion, increased intracellular GH content and GH mRNA levels within 72 h, suggesting a modulation of GH synthesis by RA. J. Cell. Biochem 65:25–31. © 1997 Wiley-Liss, Inc.     

Retinoids in biological control and cancer

More than 80 years ago, Wolbach and Howe provided the first evidence suggesting a link between alterations within human cells that lead to malignancies and vitamin A deficiencies (Wolbach and Howe 1925 Nutr. Rev. 36: 16-19). Since that time, epidemiological, preclinical and clinical studies have established a causative relationship between vitamin A deficiency and cancer. Laboratory research has provided insight into the intracellular targets, various signaling cascades and physiological effects of the biologically-active natural and synthetic derivatives of vitamin A, known as retinoids. Collectively, this body of research supports the concept of retinoids as chemopreventive and chemotherapeutic agents that can prevent epithelial cell tumorigenesis by directing the cells to either differentiate, growth arrest, or undergo apoptosis, thus preventing or reversing neoplasia. Continued refinement of the retinoid signaling pathway is essential to establishing their use as effective therapeutics for tumor subtypes whose oncogenic intracellular signaling pathways can be blocked or reversed by treatment with retinoids.     

Modulation of mammalian cell growth by a choline analog,N-isopropylethanolamine

Summary The choline analog,N-isopropylethanolamine (IPE), inhibits the growth of both Chinese hamster ovary CHO-K₁ and mouse L-M cells by two kinetically distinct mechanisms; I, a reversible and concentration-dependent reduction in the logarithmic population doubling rate and the saturation density of cultures by low IPE levels in the media; and II, an irreversible and time-dependent killing of cells by high IPE concentrations. Both types of inhibition are independent of media depletion, cell density, or the time of treatment after cell plating; however, the actual IPE concentration that is necessary to elicit Type I or Type II inhibition in each cell line is dependent on the choline level of the media. Ethanolamine, methionine, or betaine have no effect on IPE-induced growth inhibition. From a mutagenized population of CHO-K₁ cells we isolated variant cell strains that are resistant to the lethal effect of IPE. It was determined that with both the wild type and variant strains the sensitivity of cells to growth inhibition by IPE (both Type I and Type II) was proportional to the degree by which choline uptake was inhibited by the analog. Retinoic acid, which inhibits the growth of some fibroblast and epithelial cell lines by a concentration-dependent reduction in population doubling rate and saturation density, behaves synergistically with IPE to inhibit the growth of CHO-K₁ cells. Dibutyryl cyclic AMP, on the other hand, causes only an additive increase in the growth inhibition of CHO-K₁ populations that also are treated with IPE. This article is based on work supported by contract DE-AC05-760R00033 from the U.S. Department of Energy to Oak Ridge Associated Universities.     

Metabolic shifts toward glutamine regulate tumor growth,invasion and bioenergetics in ovarian cancer

Glutamine can play a critical role in cellular growth in multiple cancers. Glutamine‐addicted cancer cells are dependent on glutamine for viability, and their metabolism is reprogrammed for glutamine utilization through the tricarboxylic acid (TCA) cycle. Here, we have uncovered a missing link between cancer invasiveness and glutamine dependence. Using isotope tracer and bioenergetic analysis, we found that low‐invasive ovarian cancer (OVCA) cells are glutamine independent, whereas high‐invasive OVCA cells are markedly glutamine dependent. Consistent with our findings, OVCA patients’ microarray data suggest that glutaminolysis correlates with poor survival. Notably, the ratio of gene expression associated with glutamine anabolism versus catabolism has emerged as a novel biomarker for patient prognosis. Significantly, we found that glutamine regulates the activation of STAT3, a mediator of signaling pathways which regulates cancer hallmarks in invasive OVCA cells. Our findings suggest that a combined approach of targeting high‐invasive OVCA cells by blocking glutamine's entry into the TCA cycle, along with targeting low‐invasive OVCA cells by inhibiting glutamine synthesis and STAT3 may lead to potential therapeutic approaches for treating OVCAs.     

Differential regulation of tissue transglutaminase in rat hepatoma cell lines McA-RH7777 and McA-RH8994: Relation to growth rate and cell death

Close correlation between tissue transglutaminase (tTG) induction and growth regulation and/or cell death processes has been suggested in many cell lineages. In this study, the regulation of the tTG levels by various growth and differentiation factors and its relation to growth rate and cell death processes were investigated in two rat hepatoma cell lines, McA-RH7777 and McA-RH8994, using a monoclonal antibody against liver tTG. Transforming growth factor-β1 (TGF-β1) and retinoic acid (RA) each increased tTG to the level of 8- to 32-fold above that of control cultures in both cell lines after 72-h treatment. Dexamethasone (DEX) induced a 16- to 32-fold of tTG in McA-RH8994 cells while it did not change the enzyme level in McA-RH7777 cells. Simultaneous addition of DEX and RA increased the tTG level to more than 50-fold in McA-RH7777 cells as well as McA-RH8994 cells. Other factors, such as TGF-α, hepatocyte growth factor, dimethyl sulfoxide, and protein kinase C activator, did not show significant increases of the tTG levels. Although tTG induction by TGF-β1 or DEX appeared to be correlated with their growth suppressive effects, RA increased the tTG level without suppressing the growth rate of hepatoma cells. TGF-β1 was also shown to induce cell death in both cell lines. Our results demonstrate that RA and DEX are capable of modulating the TGF-β1-induced cell death processes independent of the tTG levels. We present evidence here that tTG induction by itself is not the direct cause of growth suppression and cell death in these hepatoma cells.     

Geraniin suppresses ovarian cancer growth through inhibition of NF‐κB activation and downregulation of Mcl‐1 expression

This study investigated the anticancer effects of geraniin on ovarian cancer cells and the signaling pathways involved. Ovarian cancer cells were treated with different concentrations of geraniin for 48 h and examined for viability, apoptosis, mitochondrial membrane depolarization, and gene expression. Xenograft tumor studies were performed to determine the anticancer activity of geraniin in vivo. Geraniin significantly decreased cancer cell viability in a concentration‐dependent fashion. Geraniin significantly triggered apoptosis, which was accompanied by loss of mitochondrial membrane potential and increased cytochrome c release and caspsase‐3 activity. Mechanistically, geraniin significantly downregulated Mcl‐1 and impaired NF‐κB p65 binding to the mcl‐1 promoter. Overexpression of Mcl‐1 significantly reversed geraniin‐induced apoptosis in OVCAR3 cells. In addition, geraniin retarded ovarian cancer growth and reduced expression of phospho‐p65 and Mcl‐1. Collectively, geraniin elicits growth suppression in ovarian cancer through inhibition of NF‐κB and Mcl‐1 and may provide therapeutic benefits for this malignancy.     

Osthole interacts with an ER‐mitochondria axis and facilitates tumor suppression in ovarian cancer

Osthole is a natural coumarin found in a variety of plants and has been reported to have diverse biological functions, including antimicrobial, antiviral, immunomodulatory, and anticancer effects. Here, we investigated the natural derivative osthole as a promising anticancer compound against ovarian cancer and evaluated its ability to suppress and abrogate tumor progression. In addition, we found the endoplasmic reticulum‐mitochondrial axis‐mediated anticancer mechanisms of osthole against ES2 and OV90 ovarian cancer cells and demonstrated its calcium‐dependent pharmacological potential. Mechanistically, osthole was found to target the phosphatidylinositol 3‐kinase/mitogen‐activated protein kinase signaling pathway to facilitate tumor suppression in ovarian cancer. Furthermore, we identified the effects of osthole in a three‐dimensional tumor‐formation model using the zebrafish xenograft assay, providing convincing evidence of the pharmacological effects of osthole within the anchorage‐independent tumor microenvironment. These findings suggest that osthole has strong potential as a pharmacological agent for targeting ovarian cancer.     

Retinoic acid modulates the level of nerve growth factor gene expression and proliferation of cultured human keratinocytes

Retinoic acid (RA) inhibited proliferation of cultured human keratinocytes. Southern blot analysis at 24 h showed that RA inhibited nerve growth factor (NGF) mRNA synthesis in a dose-dependent manner. However, RA stimulated the production of NGF protein up to 187% of control after 96 h and the treatment of cells with RA did not enhance apoptosis, either in the presence of low or high concentration of Ca²⁺, when compared to the control with DMSO.     

Therapeutic potency of pharmacological adenosine receptors agonist/antagonist on cancer cell apoptosis in tumor microenvironment,current status,and perspectives

The hypoxic niche of tumor leads to a tremendous increase in the extracellular adenosine concentration through alteration of adenosine metabolism in the tumor microenvironment (TME). This consequently affects cancer progression, local immune responses, and apoptosis of tumor cells. Regulatory effect of adenosine on apoptosis in TME depends on the cancer cell type, pharmacological characteristics of adenosine receptor subtypes, and the adenosine concentration in the tumor niche. Exploiting specific pharmacological adenosine receptor agonist and antagonist inducing apoptosis in cancer cells can be considered as a proper procedure to control cancer progression. This review summarizes the regulatory role of adenosine in cancer cell apoptosis for a better understanding, and hence better management of the disease.     

Apoptosis induced by progesterone in human ovarian cancer cell line SNU-840

Progesterone has been used as an ingredient of anticancer drug for patients with ovarian carcinoma. However, the mechanism of anticancer effects by progesterone has not been understood. In this study, the effects of progesterone on ovarian cancer cells, SNU-840, were investigated. After the incubation with progesterone, the viability of the cells was evaluated by MTT assay. As a result, 45% of the cells were viable after 48 h of incubation with 100 microM progesterone. In addition, [(3)H]thymidine incorporation assay showed that the proliferation of the cells was completely inhibited by progesterone after 48 h of incubation at 100 microM concentration. Colorimetric TUNEL assay revealed the fragmentation of the chromosomal DNA, suggesting that the process of the cell death was apoptosis. The level of the p53 mRNA was determined by northern blotting assay, since many apoptosis processes are mediated by up-regulation of the p53 expression. The level of the p53 mRNA reached its maximum at 12 h and decreased after 24 h of incubation with progesterone. In conclusion, progesterone inhibits the proliferation and elicits apoptosis of SNU-840 cells. Also, it up-regulates the p53 mRNA transiently.     

Over-expression of fibroblast activation protein alpha increases tumor growth in xenografts of ovarian cancer cells

Fibroblast activation protein alpha （FAPα） is a 95-kDa serine protease of post-prolyl peptidase family on cell surface. FAPoL is widely expressed in tumor microenviron- ment. The wide spread association of FAPα expression with cancer suggests that it has important functions in the disease. However, the nature of FAPα＇s roles in cancer cell activity is not well-determined. It has been showed that FAPα silencing in SKOV3 cells induces ovarian tumors but significantly reduces tumor growth in a xenograft mouse model. To further determine the role of FAPoL in epithelial ovarian cancer cells, SKOV3-FAPα and HO8910-FAPα cell lines, which over-expressed FAPα stably, were con- structed and then their biological behaviors were investi- gated. It was found that FAPoL promoted ovarian cancer cell proliferation, drug resistance, invasiveness, and migra- tion in vitro. Immunochemistry assay showed that FAPα significantly facilitated tumor growth in xenograft tumor tissues. These results suggested that FAPα might directly promote tumor growth and invasiveness in ovarian cancer cells.     

Liposome mediated delivery of retinoids and aromatic polyamidines: Effects on growth of tumor cell lines

Ceramic pieces composed of 99.5% Al₂O₃, 3 to 6 mm long, were found to be a good matrix for growth of the human embryonic lung diploid fibroblast, IMR-90 cells. The tissue plasminogen activator (t-PA) was secreted in DME medium containing proteose peptone as a t-PA inducer. In addition, production of t-PA was enhanced by increasing extracellular CaCl₂, from 3.6 to 5.4 mM. In order to eliminate negative feed-back control caused by t-PA produced and thus raise productivity, perfusion cultivation was performed using a ceramic-packed bed column, with a recirculating vessel. The recirculating vessel was used to mix fresh medium with spent medium, and to control dissolved oxygen concentrations in the extracellular environment by stirring. In continuous production using the packed bed column with 2 kg of ceramics (Ø=H=150 mm), increasing dilution rate to 0.5 day^-1 could reduce product inhibition at 3–4×10⁵cells/ml. Cellular productivity of 560 IU/10⁶cells/day was obtained over 40 days and corresponded to the volumetric productivity of 183 IU/ml/day.Abbreviations CBR Ceramic Bed Reactor - Lv Linear Velocity     

Cellular retinoic acid-binding protein and its relationship to the biological activity of four synthetic retinoids in hamster tracheal organ culture

Summary The mechanism of action of retinoid in reversing keratinization in hamster trachea is yet unknown. The purpose of this study was to determine if cellular retinoic acid binding protein (CRABP) is present in tracheal epithelium following incubation in serum-free, vitamin A-deficient culture medium for 10 days, and if the effectiveness of a retinoid in reversing keratinization in organ culture is correlated with its ability to compete for CRABP sites. The cytosol prepared from tracheal cultures contained CRABP at a concentration of 2.61 pmoles per mg protein. Of the four retinoids with carboxyl end group selected for the study, two of the biological active retinoids competed for the CRABP sites. However, no correlation was observed between the biological activity of the inactive retinoids and their ability to associate with the CRABP sites. These results indicate that even though the action of retinoid may be mediated by retinoid binding protein, it cannot be used as a sole predicator of retinoid response in hamster trachea. This investigation was supported by Contract N01-CP-31012 and U. S. P. H. Grants CA30512 and CA32428, which were awarded by the Division of Cancer Etiology, National Cancer Institute, DHHS. Editor's Statement Tracheal organ cultures provide a useful model for the study of epithelial differentiation and carcinogenesis. Much attention has been given to the action of retinoids in this process. Mehta et al. demonstrate a lack of correlation between biological activity and specific cytosolic binding of members of this class of compounds, pointing out the need for a more complete biochemical understanding of the mechanism of action and active forms of retinoids in this and other systems in vivo and in vitro. David W. Barnes     

Conditionally immortal ovarian cell lines for investigating the influence of ovarian stroma on the estrogen sensitivity and tumorigenicity of ovarian surface epithelial cells

The tendency of the ovarian surface epithelium (OSE) to undergo metaplastic and morphogenetic changes during the life cycle, at variance with the adjacent peritoneal mesothelial cells, suggests that its biology may be regulated by underlying ovarian stromal cues. However, little is known about the role that the ovarian stroma plays in the pathobiology of the OSE, largely because of the lack of a suitable in vitro model. Here, we describe the establishment and characterization of conditionally immortalized ovarian stromal and surface epithelial cell lines from H-2K(b)-tsA58 transgenic mice that carry the thermolabile mutant of SV-40 large T antigen under the control of an interferon-gamma (IFN-gamma)-inducible promoter. These cells express functional T antigens, grow continuously under permissive conditions at 33 degrees C in the presence of IFN-gamma, and stop dividing when the activity and expression of the tumor antigen is suppressed by restrictive conditions without IFN-gamma at 39 degrees C. Morphological, immunohistochemical, and ultrastructural analyses show that conditionally immortal OSE cells form cobblestone-like monolayers, express cytokeratin and vimentin, contain several microvilli, and develop tight junctions, whereas stromal cells are spindle-like, express vimentin but not cytokeratin, and contain rare microvilli, thus exhibiting epithelial and stromal phenotypes, respectively. At variance with the reported behavior of rat epithelial cells, conditionally immortal mouse epithelial cells are not spontaneously transformed after continuous culture in vitro. More importantly, conditioned media from stromal cells cultured under permissive conditions increase the specific activity of the endogenous estrogen receptor in BG-1 human ovarian epithelial cancer cells and promote these cells' anchorage-independent growth, suggesting the paracrine influence of a stromal factor. In addition, stromal cells cultured under restrictive conditions retain this growth-stimulatory activity, which, therefore, appears to be independent of T antigen expression. These established cell lines should provide a useful in vitro model system for studying the role of cellular interactions in OSE cell growth and tumorigenesis.     

Effects of cell density and phorbol esters on the expression of epidermal growth factor receptors

Previously, it has been shown that the binding of epidermal growth factor (EGF) by a wide range of cells decreases as cell density increases. In this report, we demonstrate that KB cells treated chronically with phorbol esters continue to exhibit decreases in EGF receptor binding as cell density increases. This finding suggests that protein kinase-C may not be essential for density-induced down regulation of EGF receptors, since phorbol esters are known to down regulate protein kinase-C. We also report that short-term and long-term effects of phorbol esters on the binding of EGF are affected by density. As shown previously for several cell lines, the phorbol ester 12-0-tetradecanoylphorbol-13-acetate transiently reduces EGF binding. We now show that the magnitude of this reduction diminishes as cell density increases. In addition, we determined that long-term treatment of KB cells with phorbol ester increases EGF binding. Again, this effect is diminished at high cell densities. Finally, we report that the increases in EGF binding induced by long-term treatment with phorbol esters are due to increases in the number of EGF receptors.Abbreviations EGF epidermal growth factor - FGF fibroblast growth factor - PBS phosphate buffered saline - PDBu 4-phorbol-12,13-dibutyrate - PDGF platelet-derived growth factor - PK-C protein kinase-C - TGF- transforming growth factor- - TPA 12-0-tetradecanoylphorbol-13-acetate