In vitro treatment with retinoids decreases bcl-2 protein expression and enhances dexamethasone-induced cytotoxicity and apoptosis in multiple myeloma cells

All-trans retinoic acid (ATRA) has been shown to inhibit in vitro growth of multiple myeloma (MM) cells, and this effect can be further potentiated by the addition of Dexamethasone (DEX). We here extended this study by testing the activity of 9-cis retinoic acid (9-cis RA) and 13-cis retinoic acid (13-cis RA), both alone and in combination with DEX, in two MM cell lines, U266 and RPMI 8226. Furthermore, we aimed at investigating the mechanisms involved in the interactions of retinoids and DEX in this setting. 9-cis RA appeared to be the most active agent in U266 cell line (IC50 = 1.2 mumol/l vs 10.5 and 9.8 mumol/l obtained with ATRA and 13-cis RA, respectively) while, in RPMI 8226 cell line, 9-cis RA and 13-cis RA were almost equally cytotoxic (IC50 = 1 and 0.8 mumol/l) and ATRA was less effective. Co-incubation with DEX resulted in a synergistic cytotoxic activity in both the cell lines except for the combinations DEX + 9-cis RA in U266 cell line and DEX + 13-cis RA in RPMI 8226 cell line, where the effect was merely additive. A synergistic cytotoxic effect of retinoids and DEX was also observed on fresh MM cells obtained from 7 patients. Both retinoids and DEX are known to be inducers of apoptosis; we verified that the combined inhibitory activity of retinoids and DEX could be attributed to an increased induction of apoptosis. This effect may be mediated by a reduced intracellular expression of BCL-2 protein, which indeed observed after prolonged in vitro treatment with retinoids. It has been described recently that an enhanced expression of BCL-2 protein can contribute to the occurrence of early chemoresistance; the downregulation of BCL-2 protein induced by retinoids could thus be exploited, by means of novel chemotherapy plus retinoids combinations, in order to improve the efficacy of conventional chemotherapy in MM.     

Luteinizing hormone receptor, steroidogenesis acute regulatory protein, and steroidogenic enzyme messenger ribonucleic acids are overexpressed in thecal and granulosa cells from polycystic ovaries

Recent data suggest that steroidogenic enzyme messenger ribonucleic acids (mRNAs) may be overexpressed in thecal cells, and LH receptors may be prematurely expressed in granulosa cells in women with polycystic ovaries. The purpose of this study was to determine whether there is abnormal gene expression in thecal and granulosa cells from polycystic ovaries. Ovarian tissue specimens were obtained from 12 women with PCOS and 24 regularly cycling control women. The granulosa cells and the theca interna were microdissected from individual follicles. LH receptor, steroidogenesis acute regulatory protein (StAR), cholesterol side-chain cleavage cytochrome P450 (CYP11A), and 17alpha-hydroxylase/C(17-20) lyase cytochrome P450 (CYP17) mRNAs were measured by RT-PCR. There was no difference between 3- to 7-mm control follicles and dominant follicles with respect to LH receptor mRNA expression in either thecal or granulosa cells. CYP11A and CYP17 mRNAs were higher in thecal cells from 3- to 7-mm follicles than in dominant follicles, but StAR expression was not different. In granulosa cells, StAR and CYP11A mRNA expression was higher in dominant follicles than in 3- to 7-mm follicles. The mean levels of LH receptor, StAR, CYP11A, and CYP17 mRNA expression were higher in thecal cells from PCOS follicles than in size-matched control follicles. In granulosa cells, the mean levels of LH receptor and CYP11A, but not StAR, mRNA expression were higher in PCOS than in control follicles. These data demonstrate that regulatory protein and steroidogenic enzyme mRNAs are overexpressed in thecal and granulosa cells from polycystic ovaries and support the conclusions that the thecal cells are hyperstimulated and the granulosa cells may be prematurely luteinizing.     

Decreasing serum concentrations of all-trans, 13-cis retinoic acids and retinol during fasting and caloric restriction

OBJECTIVES: To investigate the effects of caloric restriction on the serum concentrations of retinoids in man. DESIGN: Samples were drawn before and during caloric restriction by fasting or 4-6 weeks after gastric surgery. SUBJECTS: The fasting group included 17 healthy subjects (11 women and six men) and 16 obese patients (10 women and six men) who underwent bariatric surgery (vertical banded gastroplasty). MAIN OUTCOME MEASURES: Serum concentrations of all-trans, 13-cis, 4-oxo-13-cis retinoic acids and retinol. RESULTS: The serum concentrations of retinol, all-trans and 13-cis retinoic acids decreased by about 20% after 5 days of fasting. After gastroplasty, the serum concentration of retinol, all-trans, 13-cis retinoic acids, retinol-binding protein and transthyretin also decreased to a similar extent after 1 month. In both groups we found a correlation between the delta values of 13-cis retinoic acid and its metabolite 4-oxo-13-cis retinoic acid. In all subjects there were also correlations between the delta values of the retinoids. However, these correlations were comparatively weak (e.g. r2 = 0.36 for retinol--all-trans retinoic acid). The change in retinoid concentrations did not correlate to the change of weight or body mass index. CONCLUSION: Our results support the hypothesis that serum retinol is one of the determinants of serum concentrations of all-trans and 13-cis retinoic acid and that the catabolism of 13-cis retinoic acid is not affected by fasting. However, in the individual case, S-Retinol is a poor predictor of S-All-trans retinoic acid.     

Retinoic acid-induced tissue transglutaminase and apoptosis in vascular smooth muscle cells

Retinoids exert antiproliferative and prodifferentiating effects in vascular smooth muscle cells (SMCs) and reduce neointimal mass in balloon-injured blood vessels. The mechanisms through which retinoids carry out these effects are unknown but likely involve retinoid receptor-mediated changes in gene expression. Here we report the cloning, chromosomal mapping, and biological activity of the retinoid-response gene rat tissue transglutaminase (tTG). Northern blotting studies showed that tTG is rapidly and dose-dependently induced in a protein synthesis-independent manner after stimulation with the natural retinoid all-trans retinoic acid (atRA). The induction of tTG was selective for atRA and its stereoisomers 9-cis and 13-cis RA, because little or no elevation in mRNA expression was observed with a panel of growth factors. Western blotting and immunofluorescence confocal microscopy showed an accumulation of cytosolic tTG protein after atRA stimulation. Radiolabeled cross-linking studies revealed a corresponding elevation in in vitro tTG activity. The increase in tTG activity was reduced in the presence of 2 distinct inhibitors of tTG (monodansylcadaverine and cystamine). atRA-induced tTG mRNA and protein expression were followed by a significant elevation in SMC apoptosis. Such retinoid-induced programmed cell death could be partially inhibited with each tTG inhibitor and was completely blocked when both inhibitors were used simultaneously. These results establish a role for atRA in the sequential stimulation of tTG and apoptosis in cultured SMCs. atRA-mediated apoptosis in SMCs seems to require the participation of active tTG, suggesting a potential mechanistic link between this retinoid-inducible gene and programmed cell death.     

Increased androgen response to follicle-stimulating hormone administration in women with polycystic ovary syndrome

CONTEXT: In women with polycystic ovary syndrome (PCOS), excess ovarian androgen production is driven by increased LH secretion. Studies conducted in animals suggest that the granulosa cell may influence LH-stimulated theca cell androgen production. OBJECTIVE: The objective of this study was to determine whether FSH enhances androgen production in women with PCOS compared with that of normal women. DESIGN: A prospective study was conducted to compare androgen production in response to FSH in two groups of women. SETTING: The study was conducted in a General Clinical Research Center in a tertiary academic medical center. PATIENTS: Women with PCOS, 18-35 yr (n = 20), and normal ovulatory controls, 18-35 yr (n = 10), were recruited for study. INTERVENTIONS: Serial blood samples were obtained over a 24-h period after an iv injection of recombinant human FSH (150 IU). MAIN OUTCOME MEASURES: The main outcome measures were serum 17-hydroxyprogesterone (17-OHP), androstenedione (A), dehydroepiandrosterone (DHEA), testosterone (T), and inhibin B (Inh B) responses after FSH administration. RESULTS: Basal serum 17-OHP, A, and T levels were markedly increased in women with PCOS compared with that observed in normal women. Basal DHEA and Inh B levels were similar to those of normal controls. After FSH injection, PCOS women demonstrated enhanced production of 17-OHP, A, DHEA, and Inh B, whereas in normal women no increases were observed. T levels declined slightly in both groups. CONCLUSIONS: These findings provide evidence that, in PCOS women, theca cell androgen production is enhanced by FSH administration and suggest a granulosa-theca cell paracrine mechanism.     

Removal of LIF (leukemia inhibitory factor) results in increased vitamin A (retinol) metabolism to 4-oxoretinol in embryonic stem cells

Retinoids, vitamin A (retinol) and its metabolic derivatives, are required for normal vertebrate development. In murine embryonic stem (ES) cells, which remain undifferentiated when cultured in the presence of LIF (leukemia inhibitory factor), little metabolism of exogenously added retinol takes place. After LIF removal, ES cells metabolize exogenously added retinol to 4-hydroxyretinol and 4-oxoretinol and concomitantly differentiate. The conversion of retinol to 4-oxoretinol is a high-capacity reaction because most of the exogenous retinol is metabolized rapidly, even when cells are exposed to physiological ( approximately 1 microM) concentrations of retinol in the medium. No retinoic acid or 4-oxoRA synthesis from retinol was detected in ES cells cultured with or without LIF. The cytochrome P450 enzyme CYP26 (retinoic acid hydroxylase) is responsible for the metabolism of retinol to 4-oxoretinol, and CYP26 mRNA is greatly induced (>15-fold) after LIF removal. Concomitant with the expression of CYP26, differentiating ES cells grown in the absence of LIF activate the expression of the differentiation marker gene FGF-5 whereas the expression of the stem cell marker gene FGF-4 decreases. The strong correlation between the production of polar metabolites of retinol and the differentiation of ES cells upon removal of LIF suggests that one important action of LIF in these cells is to prevent retinol metabolism to biologically active, polar metabolites such as 4-oxoretinol.     

Resistin stimulation of 17alpha-hydroxylase activity in ovarian theca cells in vitro: relevance to polycystic ovary syndrome

CONTEXT: A newly discovered hormone resistin has been shown to be increased in women with polycystic ovary syndrome (PCOS). OBJECTIVE: The purpose of this study was to confirm increased resistin concentrations in women with PCOS and to test the direct effect of resistin on human theca cell androgen production. DESIGN: Resistin was measured in fasting serum samples by RIA. To test the direct effects of resistin on ovarian androgen biosynthesis, human theca cells were cultured with resistin for 3 d in the presence and absence of forskolin and insulin. PATIENTS: Fasting serum samples were obtained from 45 women with PCOS and 74 regularly cycling premenopausal control women in the follicular phase of their menstrual cycles, and ovarian theca cell cultures were established from two control women. RESULTS: The mean serum resistin concentration was increased (40%) in women with PCOS. Serum resistin concentrations correlated positively with body mass index and testosterone in PCOS women but not in controls. There were no significant correlations between resistin and fasting insulin or indicators of insulin resistance when corrected for body mass index. In cultured human theca cells, basal 17alpha-hydroxylase activity was unchanged by resistin alone, but resistin enhanced 17alpha-hydroxylase activity in the presence of forskolin or a combination of forskolin plus insulin. Resistin (> or =1 ng/ml) augmented forskolin and forskolin plus insulin stimulation of CYP17 mRNA expression in a concentration-dependent manner. CONCLUSION: These data indicate that abnormal resistin secretion in PCOS may play a role in causing ovarian hyperandrogenism.     

Retinoids (all-trans and 9-cis retinoic acid) stimulate production of macrophage colony-stimulating factor and granulocyte-macrophage colony- stimulating factor by human bone marrow stromal cells

Retinoic acids (RAs) exert pleiotropic effects on cellular growth and differentiation. All-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), a stereoisomer of ATRA, induce differentiation of leukemic cell lines and cells from patients with acute myelogenous leukemia (AML) in vitro. Despite information on the effects of RAs on hematopoietic cells, little is known about how RAs act on the hematopoietic microenvironment, especially on bone marrow stromal cells. Based on recent observations that various cytokines produced mainly by bone marrow stromal cells regulate hematopoiesis, we analyzed the effects of RAs on cytokine production by these cells. ATRA or 9-cis RA treatment of human bone marrow stromal cell line KM101, which produces macrophage colony-stimulating factor (M-CSF) and granulocyte- macrophage colony-stimulating factor (GM-CSF) constitutively, enhanced mRNA levels of both cytokines in a dose-dependent manner. Both RAs also stimulated M-CSF production from primary cultures of human bone marrow stromal cells. Both retinoic acid receptor (RAR)-alpha and retinoid X receptor (RXR)-alpha were expressed constitutively in KM101 cells. ATRA did not affect the expression of either receptor, whereas 9-cis RA increased RXR-alpha mRNA expression in a dose-dependent manner, but did not affect levels of RAR-alpha mRNA. These findings may have important biologic implications for both the role of RAs in hematopoiesis and the therapeutic effects of ATRA on the hematopoietic microenvironment in patients with acute promyelocytic leukemia (APL).     

Effects of retinoids and juvenoids on moult and on phenoloxidase activity in the blood-sucking insect Rhodnius prolixus

Retinoic acid and insect juvenile hormone (JH) are structurally related terpenoids which are widespread in nature and are involved in many biological events such as morphogenesis, embryogenesis and cellular differentiation. Here, we investigated the effects of the retinoids 9-cis retinoic acid (9cisRA), all trans retinol (atROH), all trans retinoic acid (atRA) and the juvenoids methoprene (Met) and JH injection on moult and on phenoloxidase activity in the blood-sucking insect Rhodnius prolixus. Overall, we observed that injection of retinoids or juvenoids (120 pmols) in the hemocoel of 4th instar nymphs reduced the percentage of insects which appeared normal in morphology upon moult. Noteworthy, insects exposed to 9cisRA or JH underwent profound morphological changes upon moult, generating abnormal 5th instar nymphs and also markedly increased the death of insects during the moulting process. In addition, reduction in the percentage of insects that moult without any morphological alteration, induced by retinoids or juvenoids treatment, was negatively correlated with insects that both display abnormal moult and those that die during moult. Hemolymphatic phenoloxidase activity in adult male insects injected with 9cisRA, Met and JH were significantly reduced after a bacterial challenge. Together, these results indicate that not only juvenoids but also retinoids play an important role on morphogenesis and on immune response in R. prolixus, suggesting that the molecular mechanisms involved in these events recognize the terpenoid backbone as an important structural determinant in insects.     

Lack of difference between retinoic acid and retinol in stimulating progesterone production by luteinizing granulosa cells in vitro

Receptors for retinoids in the immature rat ovary and the effects of retinol and retinoic acid on luteinizing granulosa cells were studied. Radioreceptor assay demonstrated the presence of specific cellular retinol-binding protein and cellular retinoic acid-binding protein in the ovaries of rats injected with PMSG alone or PMSG and hCG. In addition, when luteinizing granulosa cell from PMSG/hCG-injected immature rats were cultured with or without retinoic acid, the morphology, viability, number of cells in culture, and progesterone (P) accumulation were not affected by up to 10 microM retinoic acid. Beyond 10 microM, the cells began to round up, which was associated with a decrease in cell viability. Surprisingly, the deleterious concentrations of retinoic acid increased progesterone accumulation significantly higher than the medium control value. This increase in progesterone, however, was not accompanied by an increase in cAMP. When cells preincubated for 2 days with 1 microM of either retinoic acid or retinol were subsequently incubated in retinoid-free medium containing various substrates for steroidogenesis, the following results were obtained. Basal progesterone and its accumulation in response to human low density lipoprotein were significantly higher in cells preincubated with retinoids than in the control cells. However, no difference was seen in the degree of stimulation between retinol and retinoic acid pretreatments. Both 25-hydroxycholesterol, a substrate for side-chain cleavage enzyme, and pregnenolone, a substrate for 3 beta-hydroxysteroid dehydrogenase, significantly stimulated the accumulation of progesterone in cells preincubated with retinoids over the control value. Again, no appreciable difference was observed between retinol and retinoic acid pretreatments. Our results suggest that receptors for retinoids are present in gonadotropin-primed immature rat ovaries, retinoids increase luteal cell progesterone accumulation, and no difference exists between retinol and retinoic acid in their ability to increase the accumulation of progesterone by these cells.     

All-trans retinoic acid at low concentration directly stimulates normal adult megakaryocytopoiesis in the presence of thrombopoietin or combined cytokines.

In order to investigate the direct effects of retinoids on normal adult hematopoietic progenitors, purified CD34+ cells were seeded in serum-free cultures in the presence of pharmacological (10(-6)) M or physiological (10(-12)) M concentrations of all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA) plus combinations of specific cytokines. 10(-6) M ATRA and 9-cis RA significantly decreased the number of granulomacrophagic, erythroid and megakaryocytic (CFU-meg) progenitors. On the other hand, 10(-12) M ATRA significantly promoted the growth of CFU-meg, in the presence either of thrombopoietin or of IL-3+ GM-CSF, and induced a reproducible stimulation of the immature CD34+DR- subset. In conclusion, our findings suggest that retinoic acids probably play a direct role in normal adult hematopoietic development at both physiological and pharmacological concentrations. The stimulatory effect on megakaryocytopoiesis should be considered in the perspective of a potential use of low-dose ATRA, combined with thrombopoietin or other cytokines, in pathological conditions where the megakaryocytic compartment is impaired and the stimulation of megakaryocytopoiesis is requested.     

Novel retinoic acid, 9-cis retinoic acid, in combination with all-trans retinoic acid is an effective inducer of differentiation of retinoic acid-resistant HL-60 cells

Recent studies have shown that a high proportion of patients with acute promyelocytic leukemia (APL) achieve complete remission after treatment with all-trans retinoic acid (RA). Nevertheless, despite an initial good response, most patients that received continuous treatment with all-trans RA relapse and develop RA-resistant disease. The 9-cis RA is a high-affinity ligand for retinoid X receptors (RXRs) and also binds efficiently to retinoic acid receptors (RARs); all-trans RA is a ligand for RARs. Both alone are able to induce differentiation of wild-type HL- 60 cells. We found that neither all-trans RA nor 9-cis RA (< 2 x 10(-6) mol/L) induced differentiation of RA-resistant HL-60 cells into either mature granulocytes or monocytes. However, morphologic differentiation of the RA-resistant HL-60 cells was induced by 10(-6) mol/L all-trans RA combined with various concentrations (10(-12) to 10(-6) mol/L) of 9- cis RA. Electron microscopic examination also confirmed that the combination of both retinoids induced RA-resistant HL-60 cells to differentiate to mature granulocytes. Functional analysis of differentiation (NBT reduction activity) confirmed the necessity of both analogs to induce differentiation. Also, expression of myeloid- specific differentiation antigens (CD11b and CD14) as well as migration inhibitory factor-related protein (MRP)-8/14 mRNAs were upregulated only in the presence of both retinoids in a dose-dependent manner. In these conditions 3H-thymidine incorporation was inhibited and numbers of viable cells were decreased, suggesting that all-trans RA with 9-cis RA may inhibit cell growth and induce differentiation of RA-resistant HL-60 cells into mature granulocytes. These studies suggest that 9-cis RA in combination with all-trans RA is an effective inducer of RA- resistant HL-60 cells and may have implications for both the biology of retinoids and clinical treatment of RA-resistant acute myelogenous leukemia, including APL patients.     

Localization and expression of low-density lipoprotein receptor, steroidogenic acute regulatory protein, cytochrome P450 side-chain cleavage and P450 17-alpha-hydroxylase/C17-20 lyase in developing swine follicles: in situ molecular hybridization and immunocytochemical studies

The present study utilizes in situ molecular hybridization and immunocytochemistry to investigate the follicular localization and expression of four thematically related sterol-metabolizing genes; low-density lipoprotein (LDL) receptor, steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (CYP11A) enzyme, and cytochrome P450 17alpha-hydroxylase/C(17-20) lyase (CYP17). To this end, semiquantitative analyses were applied to individual nonatretic follicles (N=54) harvested from cycling gilts slaughtered on days 1, 3, 5, and 7 (N=3 per day) following withdrawal of the progesterone agonist, altrenogest. In situ and immunocytochemical signal intensities were assigned numerical values of 0-3 corresponding to the degree of expression of each mRNA and its corresponding protein. LDL receptor mRNA and protein content was undectable in theca and granulosa cells on days 1, 3, and 5, and then increased to low levels in theca cells on day 7. StAR message rose progressively in theca cells with follicular maturation, reaching a maximum on day 5, and then declining slightly on day 7 after the LH surge. In granulosa cells, small amounts of StAR mRNA and protein were detected on days 5 and 7. The amounts of CYP11A mRNA and protein were high in theca cells, and increased at each time point studied. Granulosa cells exhibited minimal CYP11A message on days 3, 5, and 7, while protein became detectable at low levels on day 7 only. Expression of CYP17 was localized exclusively in theca cells with protein and message content increasing unidirectionally to maxima on days 5 (RNA) and 7 (protein), respectively. Follicular fluid concentrations of androstenedione, and progesterone in contralateral ovaries correlated strongly and positively with accumulation of CYP17, and CYP11A proteins. In summary, these analyses demonstrate that preovulatory follicular development proceeds with the coordinate induction of pivotal genes and proteins that mediate the selective uptake, delivery and utilization of sterol substrate in granulosa and theca-cell steroidogenesis.