P450-dependent enzymes as targets for prostate cancer therapy

Metastatic prostate adenocarcinoma is a leading cause of cancer-related deaths among men. First line treatment is primarily aimed at blocking the synthesis and action of androgens. As primary endocrine treatment, androgen deprivation is usually achieved by orchidectomy or LHRH analogues, frequently combined with androgen receptor antagonists in order to block the residual adrenal androgens. However, nearly all the patients will eventually relapse. Available or potential second line therapies include, among others, alternative endocrine manipulations and chemotherapy.

Cytochrome P450-dependent enzymes are involved in the synthesis and/or degradation of many endogenous compounds, such as steroids and retinoic acid. Some of these enzymes represent suitable targets for the treatment of prostate cancer.

In first line therapy, inhibitors of the P450-dependent 17,20-lyase may achieve a maximal androgen ablation with a single drug treatment. Ketoconazole at high dose blocks both testicular and adrenal androgen biosynthesis but its side-effects, mainly gastric discomfort, limit its widespread use. A series of newly synthesized, more selective, steroidal 17,20-lyase inhibitors related to 17-(3-pyridyl)androsta-5,16-dien-3β-ol, may open new perspectives in this field.

In prostate cancer patients who relapse after surgical or medical castration, therapies aiming at suppressing the remaining adrenal androgen biosynthesis (ketoconazole) or producing a medical adrenalectomy (aminoglutethimide + hydrocortisone) have been used, but are becoming obsolete with the generalization of maximal androgen blockade in first line treatment. The role of inhibition of aromatase in prostate cancer therapy, which was postulated for aminoglutethimide, could not be confirmed by the use of more selective aromatase inhibitors, such as formestane.

An alternative approach is represented by liarozole fumarate (LIA), a compound that blocks the P450-dependent catabolism of retinoic acid (RA). In vitro, it enhances the antiproliferative and differentiation effects of RA in cell lines that express RA metabolism, such as F9 teratocarcinoma and MCF-7 breast carcinoma cells. In vivo, monotherapy with LIA increases RA plasma levels and, to a greater extent, endogenous tissue RA levels leading to retinoid-mimetic effects. In the rat Dunning prostate cancer models, it inhibits the growth of androgen-independent as well as androgen-dependent carcinomas relapsing after castration. Concurrently, changes in the pattern of cytokeratins characteristic of increased differentiation were observed. Early clinical trials show that LIA, in second or third line therapy in metastatic prostate cancer, induces PSA responses in about 30% of unselected patients. In some patients regression of soft tissue metastasis has been observed. In a subgroup of patients, an important relief of metastatic bone pain was also noted.     

Regulation of retinoic acid signaling in the embryonic nervous system: a master differentiation factor

This review describes some of the properties of retinoic acid (RA) in its functions as a locally synthesized differentiation factor for the developing nervous system. The emphasis is on the characterization of the metabolic enzymes that synthesize and inactivate RA, and which determine local RA concentrations. These enzymes create regions of autocrine and paracrine RA signaling in the embryo. One mechanism by which RA can act as a differentiation agent is through the induction of growth factors and their receptors. Induction of growth factor receptors in neural progenitor cells can lead to growth factor dependency, and the consequent developmental fate of the cell will depend on the local availability of growth factors. Because RA activates the early events of cell differentiation, which then induce context-specific differentiation programs, RA may be called a master differentiation factor.     

Retinoic acid and ascorbic acid act synergistically in inhibiting human breast cancer cell proliferation

BACKGROUND: Breast cancer is an increasingly common malignancy. Several vitamins such as retinoic acid (RA), ascorbic acid (AA), vitamin D and vitamin E are known to prevent the development and progression of breast cancer. OBJECTIVE: We sought to determine whether RA and AA together (RA+AA) acted synergistically in blocking the proliferation of human breast cancer cells. To elucidate the mechanism by which RA+AA inhibited breast carcinoma proliferation, we then evaluated the gene expression profiles of the treated and untreated cells by radioactive cDNA microarray analysis. METHODS: We cultured the human breast cancer cell line MCF-7 for 3 days with 100 nM RA and/or 1 mM AA, counted the cell numbers and harvested the total RNAs for cDNA microarray analysis. RESULTS: RA, AA and RA+AA reduced MCF-7 cell proliferation by 20.7%, 23.3% and 75.7% relative to the untreated cell proliferation, respectively. The synergistic ratio of RA and AA was 1.72. The MCF-7 gene expression profiles showed that 29 genes were up-regulated and 38 genes were down-regulated after RA+AA treatment. The nature of these genes suggests that the mechanism by which RA and AA act synergistically in inhibiting human breast cancer cell proliferation may involve the expression of genes that induce differentiation and block proliferation, and the up-regulation of antioxidant enzymes and proteins involved in apoptosis, cell cycle regulation and DNA repair. CONCLUSION: Combined treatment with RA and AA inhibits the proliferation of human breast cancer cells by altering their gene expression related to antioxidation processes as well as the proliferation inhibitory pathway.     

Inhibiting the platelet derived growth factor receptor increases signs of retinoic acid syndrome in myeloid differentiated HL-60 cells

PDGFR inhibitors are successfully used in a number of cancer treatments. The standard treatment for acute promyelocytic leukemia (APL) involves differentiation therapy with retinoic acid (RA). However, the relapse rates are significant. In the present work we evaluated the effects of RA therapy in the presence of PDGFR inhibitor, AG1296. Adding AG1296 with RA increased secretion of TNF-alpha, IL-8, and MMP-9 expression. This treatment induced higher levels of ICAM-1 endothelial cell expression, and increased cellular mobility. Inhibiting PDGFR enhanced RA-induced expression of integrin. Integrin ligand increased differentiation markers CD11b, inducible oxidative metabolism and PDGFR-beta phosphorylation. While the neutrophil-endothelial cell interactions are strengthened by the combined treatment, the endothelium-substratum interactions are weakened, a situation common in RAS.     

Recovery from Parkinsonism with N-acetylcysteine-differentiated neurons

The upregulation of dopaminergic neuronal differentiation is necessary for stem cell therapy in Parkinson’s disease (PD). In this study, neuronal differentiation efficiency increased by more than 2 times in P19 embryonic stem cells (ESCs) induced by N-acetylcysteine (NAC) and retinoic acid (RA) as compared to RA alone, with suppressed glial differentiation. The majority of NAC-treated stem cells grafted into brains of PD mice differentiated into dopaminergic neurons and persisted well for 6 weeks. Parkinsonism was also greatly improved after grafting NAC-treated cells in comparison to cells treated with only RA. Our results strongly suggest that NAC treatment may be an effective strategy for generating stem cells fated to become dopaminergic neurons for PD clinical therapy.     

Inhibition of Retinoic Acid Metabolising Enzymes by 2-(4-aminophenylmethyl)-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and Related Compounds

In a search for inhibitors of all-trans retinoic acid (RA)-metabolising enzymes as potential agents for the treatment of skin conditions and cancer we have examined 2-(4-aminophenylmethyl)-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one (5). Compound (5) is a moderate inhibitor of RA-metabolising enzymes in mammalian cadaverous tissue microsomes and homogenates as well as RA-induced enzymes in cultured human genital fibroblasts and HaCat cells. Overall (5) was more potent than or equipotent with ketoconazole, a standard inhibitor, in the cadaverous systems but less active towards the RA-induced cell culture systems. Examination of the data suggests that RA-induction generates metabolising enzymes not present in the cadaverous systems, which are more susceptible to inhibition by ketoconazole than (5).     

Inhibition of retinoic acid metabolising enzymes by 2-(4-aminophenylmethyl)-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and related compounds

In a search for inhibitors of all-trans retinoic acid (RA)-metabolising enzymes as potential agents for the treatment of skin conditions and cancer we have examined 2-(4-aminophenylmethyl)-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one (5). Compound (5) is a moderate inhibitor of RA-metabolising enzymes in mammalian cadaverous tissue microsomes and homogenates as well as RA-induced enzymes in cultured human genital fibroblasts and HaCat cells. Overall (5) was more potent than or equipotent with ketoconazole, a standard inhibitor, in the cadaverous systems but less active towards the RA-induced cell culture systems. Examination of the data suggests that RA-induction generates metabolising enzymes not present in the cadaverous systems, which are more susceptible to inhibition by ketoconazole than (5).     

Cytoskeletal organization and cell motility correlates with metastatic potential and state of differentiation in prostate cancer.

The actin cytoskeleton is the key cellular machinery responsible for cellular movement. Changes in the organization and distribution of actin and actin binding protein are necessary for several cellular processes such as focal adhesion formation, cell motility and cell invasion. Here we examined differences in cytoskeletal protein distribution, cell morphometry and cell motility of metastatic and non-metastatic cells. Correlations were found between metastatic potential phenotypic properties such as cell motility, cell spreading and cytoskeletal organization in prostate cancer. As a cell progresses from a normal state to a malignant state, it loses its ability to function normally and also become poorly differentiated. Differentiation therapy is concerned with the redirection of malignant cells toward a terminal, non-dividing state using non-cytotoxic agents. Two well acknowledged differentiation agents, retinoic acid (RA) and diflouromethylomithine (DFMO) were examined for their ability to alter cellular phenotypes associated with metastatic potential in rat prostate cancer cell lines. The results of these studies indicate that there are sub-cellular differences between non-metastatic and highly metastatic cells relative to cytoskeletal organization. We also show that treatment of highly metastatic cells with either RA or DFMO significantly alters cell morphology, cell morphometry and motility to states similar to non-metastatic cells.     

Excessive Retinoic Acid Impaired Proliferation and Differentiation of Human Fetal Palatal Chondrocytes (hFPCs)

Chondrocyte proliferation and differentiation is a fundamental process during hard palatogenesis. Excessive retinoic acid (RA), the biologically most active metabolite of vitamin A, has been reported to adversely affect chondrogenesis. The aim of this study was to investigate the mechanisms underlying RA‐induced chondrocyte differentiation by using human fetal palatal chondrocytes (hFPCs) aging about 9 weeks of amenorrhea. RA treatment inhibited proliferation and induced apoptosis in hFPCs. Alkaline phosphatase activity assay, quantitative alcian blue staining, and real‐time PCR analysis revealed that RA treatment stimulated hFPCs to undergo maturation and terminal differentiation, as demonstrated by decreased chondrogenic markers and increased osteogenic markers. Further studies demonstrated that RA treatment increased Wnt/β‐catenin signaling, as demonstrated by Wnt/β‐catenin target gene expression analysis and a luciferase‐based β‐catenin–activated reporter assay. To address the role of Wnt/β‐catenin signaling, we treated hFPCs with Dickkopf‐related protein 1, an extracellular inhibitor of Wnt/β‐catenin signaling, and the observed all‐trans retinoic acid–mediated increases in nuclear accumulation of β‐catenin, alkaline phosphatase activity, and type I collagen mRNA were attenuated, suggesting that RA modulated Wnt signaling at ligand–receptor level. In summary, excessive all‐trans retinoic acid inhibited proliferation and promoted ossification of hFPCs by upregulation of Wnt/β‐catenin signaling     

Leukemia: beneficial actions of retinoids and rexinoids

Acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia, is the prototype of a cancer that can be cured by differentiation therapy using combined retinoic acid (RA) and chemotherapy. Acute promyelocytic leukemia is caused by chromosomal translocations, which in the large majority of cases generate the prototypic promyelocytic leukemia-retinoic-acid receptor alpha (PML-RARalpha) an oncogenic fusion protein formed from the retinoic-acid receptor alpha and the so-called PML protein. The fusion protein leads to the deregulation of wild type PML and RARalpha function, thus inducing the differentiation block and an altered survival capacity of promyelocytes of affected patients. A plethora of studies have revealed molecular details that account for the oncogenic properties of acute promyelocytic leukemia fusion proteins and the events that contribute to the therapy-induced differentiation and apoptosis of patients' blasts. Illustrating the beneficial mechanisms of action of retinoids for acute promyelocytic leukemia patients this review goes on to discuss a plethora of recently recognized molecular paradigms by which retinoids and rexinoids, alone or in combination with other compounds, regulate growth, differentiation and apoptosis also in non-acute promyelocytic leukemia cells, highlighting their potential as drugs for cancer therapy and prevention.     

Interaction of tissue transglutaminase with nuclear transport protein importin-alpha3

Tissue transglutaminase is a multifunctional enzyme which has been involved in the regulation of cell growth, differentiation, and apoptosis. Recently, nuclear localization of tTG has been reported indicating the potential of active nuclear transport. In this study we use the yeast two-hybrid assay and co-immunoprecipitation to show that tTG interacts with the nuclear transport protein importin-alpha3. Using electron microscopy we demonstrate that nuclear expression of tTG in a non-small cell lung cancer cell line is induced by retinoic acid (RA). These data suggest that importin-alpha3 could mediate active nuclear transport of tTG which may be important for the regulation of critical cellular processes.     

The zebrafish retinol dehydrogenase, rdh1l, is essential for intestinal development and is regulated by the tumor suppressor adenomatous polyposis coli

Retinoic acid (RA) is a potent signaling molecule that plays important roles in multiple and diverse developmental processes. The contribution of retinoic acid to promoting the development and differentiation of the vertebrate intestine and the factors that regulate RA production in the gut remain poorly defined. Herein, we report that the novel retinol dehydrogenase, rdh1l, is required for proper gut development and differentiation. rdh1l is expressed ubiquitously during early development but becomes restricted to the gut by 3 days postfertilization. Knockdown of rdh1l results in a robust RA-deficient phenotype including lack of intestinal differentiation, which can be rescued by the addition of exogenous retinoic acid. We report that adenomatous polyposis coli (APC) mutant zebrafish harbor an RA-deficient phenotype including aberrant intestinal differentiation and that these mutants can be rescued by treatment with retinoic acid or injection of rdh1l mRNA. Further, we have found that although APC mutants are deficient in rdh1l expression, they harbor increased expression of raldh2 suggesting the control of RA production by APC is via retinol dehydrogenase activity. These results provide genetic evidence that retinoic acid is required for vertebrate gut development and that the tumor suppressor APC controls the production of RA in the gut by regulating the expression of the retinol dehydrogenase, rdh1l.     

Embryonic Carcinoma Cells Show Specific Dielectric Resistance Profiles during Induced Differentiation

Induction of differentiation in cancer stem cells by drug treatment represents an important approach for cancer therapy. The understanding of the mechanisms that regulate such a forced exit from malignant pluripotency is fundamental to enhance our knowledge of tumour stability. Certain nucleoside analogues, such as 2′-deoxy-5-azacytidine and 1β-arabinofuranosylcytosine, can induce the differentiation of the embryonic cancer stem cell line NTERA 2 D1 (NT2). Such induced differentiation is associated with drug-dependent DNA-damage, cellular stress and the proteolytic depletion of stem cell factors. In order to further elucidate the mode of action of these nucleoside drugs, we monitored differentiation-specific changes of the dielectric properties of growing NT2 cultures using electric cell-substrate impedance sensing (ECIS). We measured resistance values of untreated and retinoic acid treated NT2 cells in real-time and compared their impedance profiles to those of cell populations triggered to differentiate with several established substances, including nucleoside drugs. Here we show that treatment with retinoic acid and differentiation-inducing drugs can trigger specific, concentration-dependent changes in dielectric resistance of NT2 cultures, which can be observed as early as 24 hours after treatment. Further, low concentrations of nucleoside drugs induce differentiation-dependent impedance values comparable to those obtained after retinoic acid treatment, whereas higher concentrations induce proliferation defects. Finally, we show that impedance profiles of substance-induced NT2 cells and those triggered to differentiate by depletion of the stem cell factor OCT4 are very similar, suggesting that reduction of OCT4 levels has a dominant function for differentiation induced by nucleoside drugs and retinoic acid. The data presented show that NT2 cells have specific dielectric properties, which allow the early identification of differentiating cultures and real-time label-free monitoring of differentiation processes. This work might provide a basis for further analyses of drug candidates for differentiation therapy of cancers.     

Revealing a natural marine product as a novel agonist for retinoic acid receptors with a unique binding mode and inhibitory effects on cancer cells

Retinoids display anti-tumour activity on various cancer cells and therefore have been used as important therapeutic agents. However, adverse side effects and RA (retinoic acid) resistance limit further development and clinical application of retinoid-based therapeutic agents. We report in the present paper the identification of a natural marine product that activates RARs (RA receptors) with a chemical structure distinct from retinoids by high-throughput compound library screening. Luffariellolide was uncovered as a novel RAR agonist by inducing co-activator binding to these receptors in vitro, further inhibiting cell growth and regulating RAR target genes in various cancer cells. Structural and molecular studies unravelled a unique binding mode of this natural ligand to RARs with an unexpected covalent modification on the RAR. Functional characterization further revealed that luffariellolide displays chemotherapeutic potentials for overcoming RA resistance in colon cancer cells, suggesting that luffariellolide may represent a unique template for designing novel non-retinoid compounds with advantages over current RA drugs.     

Initiating meiosis: the case for retinoic acid

The requirement for vitamin A in reproduction and development was first determined from studies of nutritional deficiencies. Subsequent research has shown that embryonic development and both male and female reproduction are modulated by retinoic acid (RA), the active form of vitamin A. Because RA is active in multiple developmental systems, its synthesis, transport, and degradation are tightly regulated in different tissues. A growing body of evidence implicates RA as a requirement for the initiation of meiosis in both male and female mammals, resulting in a mechanistic model involving the interplay of RA, RA synthesis enzymes, RA receptors, and degradative cytochrome P450 enzymes in this system. Recently, that model has been challenged, prompting a review of the established paradigm. While it remains possible that additional molecules may be involved in regulating entry into meiosis, the weight of evidence supporting a key role for RA is incontrovertible.     

Retinoic acid synthesis in the prevertebrate amphioxus involves retinol oxidation

All- trans-retinoic acid (RA) contributes to the establishment of the anterior-posterior (AP) axis in chordates. In vertebrates, all- trans-retinol is oxidized to RA by two oxidative steps. However, the controversy about the enzymes responsible for retinol oxidation (ADH vs RDH) and the fact that some candidates are absent in cephalochordates questioned retinol oxidation in this lineage. Retinoid quantitation has revealed that Branchiostoma floridae adults contain both retinol and retinoic acid as well as retinal, the intermediate in the metabolic pathway. Furthermore, our data show that the developmental effects of retinol treatment are comparable to those reported for RA. SEM analysis revealed mouth and gill slit aberrations due to a posteriorization effect, also visualized by changes in the beta-galactosidase pattern. Overall, these findings support the idea that amphioxus metabolizes endogenous retinol to retinoic acid and suggest a common oxidative pathway for RA in the chordate phylum.