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The hydroxyl functional group of N-(4-hydroxyphenyl)retinamide mediates cellular uptake and cytotoxicity in premalignant and malignant human epithelial cells.

ABSTRACT: In a previous study, we demonstrated that the anticancer synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) redox cycles at the mitochondrial enzyme dihydroorotate dehydrogenase to trigger anomalous reactive oxygen species (ROS) production and attendant apoptosis in transformed human epithelial cells. Furthermore, we speculated that the hydroxyl functional group of 4HPR was required for this pro-oxidant property. In this study, we investigated the role of the hydroxyl functional group in the in vitro cytotoxicity of 4HPR. Using 4HPR, its primary in vivo metabolite N-(4-methoxyphenyl)retinamide (4MPR), and the synthetic derivative N-(4-trifluoromethylphenyl)retinamide (4TPR), we examined the pro-oxidant and apoptotic effects, as well as the cellular uptake, of these three N-(4-substituted-phenyl)retinamides in premalignant and malignant human skin, prostate, and breast epithelial cells. Compared to 4HPR, both 4MPR and 4TPR were ineffective in promoting conspicuous cellular ROS production, mitochondrial disruption, or DNA fragmentation in these transformed cells. Interestingly, both 4MPR and 4TPR were not particularly cell permeative relative to 4HPR in skin or breast epithelial cells, which implied an additional role for the hydroxyl functional group in the cellular uptake of 4HPR. Moreover, the short-term uptake of 4HPR was directly proportional to cell size, but this characteristic, in obvious contrast to cellular bioenergetic status and/or dihydroorotate dehydrogenase expression, was not fundamentally influential in the overall sensitivity to the promotion of cellular ROS production and apoptosis induction by this agent. Together, these results strongly implicate the hydroxyl functional group in the cytotoxic effects of 4HPR.

SUBMITTER: Hail N

PROVIDER: S-EPMC3005946 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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The hydroxyl functional group of N-(4-hydroxyphenyl)retinamide mediates cellular uptake and cytotoxicity in premalignant and malignant human epithelial cells.

Hail Numsen N Chen Ping P Wempe Michael F MF

Free radical biology & medicine 20101023 12

In a previous study, we demonstrated that the anticancer synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) redox cycles at the mitochondrial enzyme dihydroorotate dehydrogenase to trigger anomalous reactive oxygen species (ROS) production and attendant apoptosis in transformed human epithelial cells. Furthermore, we speculated that the hydroxyl functional group of 4HPR was required for this pro-oxidant property. In this study, we investigated the role of the hydroxyl functional group in th ...[more]

PMID: 20923701

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Project description:BackgroundThe retinoid 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) is a polar metabolite of fenretinide (4-HPR) very effective in killing cancer cells of different histotypes, able to inhibit 4-HPR-resistant cell growth and to act synergistically in combination with the parent drug. Unlike 4-HPR and other retinoids, 4-oxo-4-HPR inhibits tubulin polymerization, leading to multipolar spindle formation and mitotic arrest. Here we investigated whether 4-oxo-4-HPR, like 4-HPR, triggered cell death also via reactive oxygen species (ROS) generation and whether its antimicrotubule activity was related to a ROS-dependent mechanism in ovarian (A2780), breast (T47D), cervical (HeLa) and neuroblastoma (SK-N-BE) cancer cell lines.Methodology/principal findingsWe provided evidence that 4-oxo-4-HPR, besides acting as an antimicrotubule agent, induced apoptosis through a signaling cascade starting from ROS generation and involving endoplasmic reticulum (ER) stress response, Jun N-terminal Kinase (JNK) activation, and upregulation of the proapoptotic PLAcental Bone morphogenetic protein (PLAB). Through time-course analysis and inhibition of the ROS-related signaling pathway (upstream by vitamin C and downstream by PLAB silencing), we demonstrated that the antimitotic activity of 4-oxo-4-HPR was independent from the oxidative stress induced by the retinoid. In fact, ROS generation occurred earlier than mitotic arrest (within 30 minutes and 2 hours, respectively) and abrogation of the ROS-related signaling pathway did not prevent the 4-oxo-4-HPR-induced mitotic arrest.Conclusions/significanceThese data indicate that 4-oxo-4-HPR anticancer activity is due to at least two independent mechanisms and provide an explanation of the ability of 4-oxo-4-HPR to be more potent than the parent drug and to be effective also in 4-HPR-resistant cell lines. In addition, the double mechanism of action could allow 4-oxo-4-HPR to efficiently target tumour and to eventually counteract the development of drug resistance.

Project description:PurposeMicroRNAs (miRNAs) are important regulators of many cellular functions due to their ability to target mRNAs for degradation or translational inhibition. Previous studies have reported that the expression of microRNA-9 (miR-9) is regulated by retinoic acid and reactive oxygen species (ROS). We have previously shown that N-(4-hydroxyphenyl)-retinamide (4HPR), a retinoic acid derivative, induces ROS generation and apoptosis in cultured human retinal pigment epithelial (RPE) cells, known as ARPE-19 cells. The aim of the present study was to investigate the expression of miR-9 in ARPE-19 cells in response to 4HPR treatment, and to identify other miRNAs normally expressed in these cells.MethodsARPE-19 cells in culture were treated with 4HPR, the total RNA fractions were isolated, and the expression of various miRNAs and mRNAs was analyzed using real-time PCR. The miRNA expression profile of ARPE-19 cells was analyzed using microarray hybridization.ResultsTreatment of ARPE-19 cells with 4HPR resulted in apoptosis characterized by the increased expression of HMOX1 and GADD153 genes. A twofold increase in the expression of miR-9 was also observed during this response. Potential binding sites for the transcription factors encoded by CEBPA and CEBPB genes were found to be present in the putative promoter regions of all three genes encoding miR-9. 4HPR-induced miR-9 expression was associated with parallel increases in the expression of these transcription factor genes. 5-Aza-2'-deoxycytidine, a methyl transferase inhibitor, also increased the expression of miR-9 in ARPE-19 cells. Microarray hybridization analysis identified let-7b, let-7a, miR-125b, miR-24, miR-320, miR-23b, let-7e, and let-7d as the most abundant miRNAs normally expressed in ARPE-19 cells. These miRNAs are known to regulate cell growth, differentiation or development. The 4HPR treatment increased the expression of miR-16, miR-26b, miR-23a, and miR-15b in ARPE-19 cells, although these increases were modest when compared to the increase in the expression of miR-9.ConclusionsOur studies demonstrate that miR-9 is expressed in the RPE cell line ARPE-19, and its expression is increased by a retinoic acid derivative and by an inhibitor of promoter hypermethylation. Several miRNAs with inherent ability to regulate cell growth, differentiation and development are also normally expressed in ARPE-19 cells. Thus, miR-9 and other miRNAs could be important in maintaining RPE cell function.

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The hydroxyl functional group of N-(4-hydroxyphenyl)retinamide mediates cellular uptake and cytotoxicity in premalignant and malignant human epithelial cells.

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The hydroxyl functional group of N-(4-hydroxyphenyl)retinamide mediates cellular uptake and cytotoxicity in premalignant and malignant human epithelial cells.

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