Fausto Andreola

Retinoids in chemoprevention and differentiation therapy

Retinoids are essential for the maintenance of epithelial differentiation. As such, they play a fundamental role in chemoprevention of epithelial carcinogenesis and in differentiation therapy. Physiological retinoic acid is obtained through two oxidation steps from dietary retinol, i.e. retinol-->retinal-->retinoic acid. The latter retinal-->retinoic acid step is irreversible and eventually marks disposal of this essential nutrient, through cytochrome P450-dependent oxidative steps. Mutant mice deficient in aryl hydrocarbon receptor (AHR) accumulate retinyl palmitate, retinol and retinoic acid. This suggests a direct connection between the AHR and retinoid homeostasis. Retinoids control gene expression through the nuclear retinoic acid receptors (RARs) alpha, beta and gamma and 9-cis-retinoic acid receptors alpha, beta and gamma, which bind with high affinity the natural ligands all-trans-retinoic acid and 9-cis-retinoic acid, respectively. Retinoids are effective chemopreventive agents against skin, head and neck, breast, liver and other forms of cancer. Differentiation therapy of acute promyelocytic leukemia (APL) is based on the ability of retinoic acid to induce differentiation of leukemic promyelocytes. Patients with relapsed, retinoid-resistant APL are now being treated with arsenic oxide, which results in apoptosis of the leukemic cells. Interestingly, induction of differentiation in promyelocytes and consequent remission of APL following retinoid therapy depends on expression of a chimeric PML-RAR alpha fusion protein resulting from a t(15;17) chromosomal translocation. This protein functions as a dominant negative against the function of both PML and RARs and its overexpression is able to recreate the phenotypes of the disease in transgenic mice. The development of new, more effective and less toxic retinoids, alone or in combination with other drugs, may provide additional avenues for cancer chemoprevention and differentiation therapy.

Reversal of liver fibrosis in aryl hydrocarbon receptor null mice by dietary vitamin A depletion

Aryl hydrocarbon receptor (AHR)‐null mice display a liver fibrosis phenotype that is associated with a concomitant increase in liver retinoid concentration, tissue transglutaminase type II (TGaseII) activity, transforming growth factorβ (TGFβ) overexpression, and accumulation of collagen. To test the hypothesis that this phenotype might be triggered by the observed increase in liver retinoid content, we induced the condition of retinoid depletion by feeding AHR‐null mice a vitamin A‐ deficient diet with the purpose to reverse the phenotype. Liver retinoid content decreased sharply within the first few weeks on the retinoid‐deficient diet. Analysis of TGFβ1, TGFβ2, and TGFβ3 expression revealed a reduction to control levels in the AHR ‐/‐ mice accompanied by parallel changes in TGaseII protein levels. In addition, we observed an increase in the TGFβ receptors, TGFβ RI and TGFβ RII, as well as in Smad4, and their reduction to wild‐type mouse liver levels in AHR ‐/‐ mice fed the retinoid‐deficient diet. Reduction of peroxisomal proliferator‐activated receptor γ (PPARγ) messenger RNA (mRNA) and protein levels in AHR ‐/‐ mice was consistent with the presence of hepatic stellate cell (HSC) activation and liver fibrosis. Vitamin A deficiency normalized PPARγ expression in AHR ‐/‐ mice. In conclusion, livers from AHR ‐/‐ mice fed the vitamin A‐deficient diet showed a decrease in collagen deposition, consistent with the absence of liver fibrosis. (HEPATOLOGY 2004;39:157–166.)

The role of vitamin A in differentiation and skin carcinogenesis

Abstract The field of vitamin A research has witnessed a remarkable surge in interest since the late 1980s, when the retinoid receptors were discovered and their genes cloned. Heterodimeric interactions between the retinoid X receptors (RXRs α, β, and γ), which bind 9-cis-retinoic acid, and other hormone receptors, including the retinoic acid receptors (RARs α, β, and γ), the thyroid hormone receptor (TR), the vitamin D receptor (VDR), the peroxisomal proliferator activated receptor (PPAR), and others make hormone action dependent on retinoid homeostasis. Retinoid response elements (RAREs) are present in the promoter and/or enhancer regions of several genes, including some of the homeobox genes, which control development and differentiation. The interaction between hormones and retinoids is added additional orders of complexity by the diversity of the RAREs including the spacer length, their 5′ or 3′ position, and their coexistence in composite sequences with other hormone response elements (e.g., an estrogen response element in the lactablbumin gene promoter, see Table 2). Control of normal epithelial differentiation is a fundamental function of retinoids. The histogenesis of squamous metaplasia caused by vitamin A deficiency is a stepwise process, which permits the gradual transition of phenotypes from simple-columnar, typical of the endocervical epithelium, to pseudostratified, to stratified-squamous and, eventually, to keratinizing. Conversely, the maintenance of the squamous keratinizing differentiation in the ectocervix and vagina requires estrogen. In the absence of this hormone, the squamous stratified ectocervical epithelium retrogrades to a simpler, two or three cell layer morphology, with the topmost layer expressing keratin K8, typical of the endocervical epithelium and mucous cells. Retinoid and estrogen receptor transcript expression is governed by dietary retinoid status and by estrogen availability, with squamous cells mostly expressing RARγ and estrogen receptor transcripts, and columnar cells mostly expressing RARβ. RXR transcripts appear mostly expressed in proliferating cells. The relevance of the retinoid receptors to carcinogenesis is highlighted in the work on acute promyelocytic leukemia. This work has demonstrated that the fusion gene PML-RARα, resulting from the t(15;17) chromosomal translocation, is etiologically connected with the disease and with complete remission after oral retinoid administration. Developments in retinoid metabolism, including the cloning of the cytochrome P450RAI and the connection between RA metabolism and cell growth inhibition, have recently taken place. Recent work has also shown that pharmacological dietary retinoic acid specifically inhibits malignant conversion in the mouse two-stage carcinogenesis system. Because RA upregulates retinoid receptor expression, it seems that retinoid receptors function as tumor suppressors. p]This field should serve as a paradigm for things to come for other essential nutrients, and spells out the notion that nutritional sciences are indeed fundamentally important, because they can contribute significantly to our understanding of different diseases and provide effective therapeutic approaches.

Differences in uptake and metabolism of retinoic acid between estrogen receptor-positive and -negative human breast cancer cells.

Purpose: Our previous work had shown that retinoic acid (RA) inhibits cell growth and induces apoptosis in estrogen receptor-positive (ER-positive) MCF-7 and T-47D human breast carcinoma cells, but not in ER-negative human breast carcinoma cells MB-231 and MB-453. The purpose of this work was to determine whether these differences might be due to differences in uptake and metabolism of the drug between ER-positive and ER-negative cells. Methods: We measured RA uptake in cultured human breast cancer cells and determined its metabolism by high-pressure liquid chromatographic analysis. Results: The two ER-positive cell lines reached maximum RA uptake at about 2 h, followed by a sharp decline, so that most RA had disappeared from the cells and from the medium by 24 h and was found as oxidation products in the culture medium. In contrast, the two ER-negative cell lines showed a pattern of lower accumulation without the sharp increase and subsequent steep decline, so that by 24 h there was more RA in these cells and their culture medium than in the RA-responsive ER-positive cells, even though at 2 h the ER-negative cells had taken up less RA than the ER-positive cells. Kinetic analysis of the uptake of RA in MCF-7 cells was consistent with rapid movement across the cell membranes and the actual rate determined by diffusion of albumin-bound retinoid to the cells. Conclusions: This study is the first to demonstrate profound differences in RA accumulation and confirms previous results on different rates of RA metabolism between ER-positive and ER-negative human breast cancer cells. The findings reported here, therefore, may introduce additional elements to be considered in the design of new drugs for cancer chemoprevention and therapy.

Cell death markers in patients with cirrhosis and acute decompensation

The aims of this study were to determine the role of cell death in patients with cirrhosis and acute decompensation (AD) and acute on chronic liver failure (ACLF) using plasma‐based biomarkers. The patients studied were part of the CANONIC (CLIF Acute‐on‐Chronic Liver Failure in Cirrhosis) study (N = 337; AD, 258; ACLF, 79); additional cohorts included healthy volunteers, stable patients with cirrhosis, and a group of 16 AD patients for histological studies. Caspase‐cleaved keratin 18 (cK18) and keratin 18 (K18), which reflect apoptotic and total cell death, respectively, and cK18:K18 ratio (apoptotic index) were measured in plasma by enzyme‐linked immunosorbent assay. The concentrations of cK18 and K18 increased and the cK18:K18 ratio decreased with increasing severity of AD and ACLF (P < 0.001, respectively). Alcohol etiology, no previous decompensation, and alcohol abuse were associated with increased cell death markers whereas underlying infection was not. Close correlation was observed between the cell death markers and, markers of systemic inflammation, hepatic failure, alanine aminotransferase, and bilirubin, but not with markers of extrahepatic organ injury. Terminal deoxynucleotidyl transferase dUTP nick‐end labeling staining confirmed evidence of greater hepatic cell death in patients with ACLF as opposed to AD. Inclusion of cK18 and K18 improved the performance of the CLIF‐C AD score in prediction of progression from AD to ACLF (P < 0.05). Conclusion: Cell death, likely hepatic, is an important feature of AD and ACLF and its magnitude correlates with clinical severity. Nonapoptotic forms of cell death predominate with increasing severity of AD and ACLF. The data suggests that ACLF is a heterogeneous entity and shows that the importance of cell death in its pathophysiology is dependent on predisposing factors, precipitating illness, response to injury, and type of organ failure. (Hepatology 2018;67:989–1002)