fenretinide and Liver-Neoplasms

N-(4-hydroxyphenyl)retinamide (4-HPR or fenretinide), which is a synthetic analog of all‑trans retinoic acid (ATRA), effectively inhibits the growth of several types of tumor cells; however, its molecular mechanism remains unclear. We found that 4‑HPR altered the morphology of human liver cancer HepG2 cells and also inhibited their proliferation and suppressed the colony formation in a dose‑ and time‑dependent manner. A wound healing assay revealed that 4‑HPR significantly hindered HepG2 cell migration, and that this was accompanied by the phosphorylation of p38‑MAPK (mitogen‑activated protein kinase). Mechanistically, the MAPK‑specific inhibitor SB203580 attenuated the inhibitory effects of 4‑HPR on the migration of HepG2 cells. Moreover, we also observed that 4‑HPR inhibited the activation and expression of myosin light chain kinase (MLCK) in HepG2 cells. Simultaneously, 4‑HPR lowered the expression of F‑actin and promoted the expression of E‑cadherin. ML‑7, a selective inhibitor of MLCK, significantly inhibited the migration of HepG2 cells while increasing the phosphorylation of p38‑MAPK and the expression of E‑cadherin, and decreasing the activation of MLCK and the expression of F‑actin. In conclusion, 4‑HPR inhibited the proliferation and migration of HepG2 cells, and p38‑MAPK plays an important role in regulating these 4‑HPR effects by reducing the activation of MLCK. The present study suggests that 4‑HPR may be a potent antimetastatic agent.

Topics: Apoptosis; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Fenretinide; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Imidazoles; Liver Neoplasms; Myosin-Light-Chain Kinase; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase Inhibitors; Pyridines; Signal Transduction

Fenretinide is significantly more effective in inducing apoptosis in cancer cells than all-trans retinoic acid (ATRA). The current study uses a genome-wide approach to understand the differential role fenretinide and ATRA have in inducing apoptosis in Huh7 cells. Fenretinide and ATRA-induced gene expressions and DNA bindings were profiled using microarray and chromatin immunoprecipitation with anti-RXRα antibody. The data showed that fenretinide was not a strong transcription regulator. Fenretinide only changed the expressions of 1 093 genes, approximately three times less than the number of genes regulated by ATRA (2 811). Biological function annotation demonstrated that both fenretinide and ATRA participated in pathways that determine cell fate and metabolic processes. However, fenretinide specifically induced Fas/TNFα-mediated apoptosis by increasing the expression of pro-apoptotic genes i.e., DEDD2, CASP8, CASP4, and HSPA1A/B; whereas, ATRA induced the expression of BIRC3 and TNFAIP3, which inhibit apoptosis by interacting with TRAF2. In addition, fenretinide inhibited the expression of the genes involved in RAS/RAF/ERK-mediated survival pathway. In contrast, ATRA increased the expression of SOSC2, BRAF, MEK, and ERK genes. Most genes regulated by fenretinide and ATRA were bound by RXRα, suggesting a direct effect. This study revealed that by regulating fewer genes, the effects of fenretinide become more specific and thus has fewer side effects than ATRA. The data also suggested that fenretinide induces apoptosis via death receptor effector and by inhibiting the RAS/RAF/ERK pathway. It provides insight on how retinoid efficacy can be improved and how side effects in cancer therapy can be reduced.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; DNA; Extracellular Signal-Regulated MAP Kinases; Fenretinide; Gene Expression Profiling; Gene Expression Regulation; Humans; Liver Neoplasms; raf Kinases; ras Proteins; Retinoid X Receptor alpha; Signal Transduction; Transcriptome; Tretinoin

Fenretinide, a synthetic retinoid, is a promising anticancer agent based on many in vitro, animal, and chemoprevention clinical trial studies. However, cells such as HepG2 human liver cancer cells are resistant to the apoptotic effect of fenretinide. Previously, we have shown that fenretinide-induced apoptosis is Nur77 dependent, and the sensitivity of the cancer cells to fenretinide-induced apoptosis is positively associated with cytoplasmic enrichment of Nur77. The goal of current study was to identify means to modulate nuclear export of Nur77 in order to improve the efficacy of fenretinide. Fenretinide treatment deactivated ERK1/2 in Huh7 cells, but activated ERK1/2 in HepG2 cells, which was positively associated with the sensitivity of cells to the apoptotic effect of fenretinide. Neither fenretinide nor ERK1/2 inhibitor PD98059 alone could affect the survival of HepG2 cells, but the combination of both induced cell death and increased caspase 3/7 activity. In fenretinide sensitive Huh7 cells, activation of ERK1/2 by epidermal growth factor (EGF) prevented fenretinide-induced cell death and caspase 3/7 induction. In addition, modulation of ERK1/2 changed the intracellular localization of Nur77. Fenretinide/PD98059-induced cell death of HepG2 cell was positively associated with induction and cytoplasmic location as well as mitochondria enrichment of Nur77. The effect was specific for ERK1/2 because other mitogen activated protein kinases such as P38, Akt, and JNK did not have correlated changes in their phosphorylation levels. Taken together, the current study demonstrates that ERK1/2-modulated Nur77 intracellular location dictates the efficacy of fenretinide-induced apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Line, Tumor; Cytoplasm; Fenretinide; Humans; Liver Neoplasms; Microscopy, Confocal; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nuclear Receptor Subfamily 4, Group A, Member 1

The synthetic retinoid fenretinide is one of the most promising clinically tested retinoids. Previously, we have shown that fenretinide induces apoptosis of Huh7 cells, but HepG2 cells are relatively resistant to fenretinide-induced apoptosis. This study examines the interactive role of fenretinide and histone deacetylase inhibitors (HDACi) in inducing apoptosis of human hepatocellular carcinoma (HCC) cells and the underlying mechanism. Trichostatin A and scriptaid can either enhance fenretinide-induced apoptosis in the fenretinide sensitive HCC cells (Huh7 and Hep3B) or sensitize the fenretinide resistant cells (HepG2) to become sensitive to the apoptotic effect of fenretinide in a cancer cell-specific manner. The sensitivity of cells to fenretinide-induced apoptosis was not associated with reactive oxygen species production nor with antioxidant gene expression. However, the level of retinoic acid receptor β (RARβ) and Nur77 (NR4A1) was important for inducing apoptosis. Upon fenretinide and HDACi treatment, the expression of RARβ and Nur77 were induced and colocalized in the cytosol. The induction of Nur77 protein level, but not the messenger RNA level, was RARβ-dependent. In addition, RARβ interacted with Nur77. Nur77 was essential for fenretinide-induced and HDACi-induced apoptosis of Huh7 cells. Induction of the expression, the interaction, and the nuclear export of RARβ and Nur77 mediate fenretinide-induced and HDACi-induced apoptosis.. Our findings suggest that targeting Nur77 and RARβ simultaneously provides an effective way to induce HCC cell death.

Topics: Antioxidants; Apoptosis; Carcinoma, Hepatocellular; Fenretinide; Hep G2 Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Hydroxylamines; Liver Neoplasms; Nuclear Receptor Subfamily 4, Group A, Member 1; Quinolines; Reactive Oxygen Species; Receptors, Retinoic Acid; RNA, Messenger

Fenretinide, a synthetic retinoid, is known to induce apoptosis in various cancer cells. However, the mechanism by which fenretinide induces apoptosis remains unclear. The current study examines the mechanisms of fenretinide-induced apoptosis in human hepatoma cells. The induction of Nur77 and the cytoplasmic distribution of Nur77 induced by fenretinide were positively correlated with the apoptotic effect of fenretinide in HCC cells. The sensitivity of Huh-7 cells was related to Nur77 translocation and targeting mitochondria, whereas the mechanism of resistance for HepG2 cells seemed due to Nur77 accumulating in the nucleus. The intracellular location of Nur77 was also associated with the differential capability of fenretinide-induced ROS generation in these two cell lines. In addition, the knockdown of Nur77 expression by siRNA greatly reduced fenretinide-induced apoptosis and cleaved caspase 3 in Huh-7 cells. Therefore, our findings demonstrate that fenretinide-induced apoptosis of HCC cells is Nur77 dependent and that the intracellular localization of Nur77 dictates the sensitivity of the HCC cells to fenretinide-induced apoptosis.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Fenretinide; Hep G2 Cells; Humans; Liver Neoplasms; Mitochondria; Nuclear Receptor Subfamily 4, Group A, Member 1; Reactive Oxygen Species; Receptors, Retinoic Acid; RNA, Messenger

N-(4-hydroxyphenyl)-retinamide (fenretinide) is a synthetic derivative of all-trans-retinoic acid and induces apoptosis in several cancer cell lines. We determined the anti-cancer activity of fenretinide using human hepatoma cell lines, Bel-7402, HepG2 and Smmc-7721. An in-vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that fenretinide exhibited growth inhibition in these cell lines, with IC50 values ranging from 13.1 to 15.5 micromol/l. In Bel-7402 cells, apoptosis with 15 micromol/l fenretinide for 0 and 48 h was 3 and 48%, respectively. In-vivo studies using the Bel-7402 xenografted athymic mouse model showed tumor inhibition rates ranging from 37.2 to 57.2%, with fenretinide administration once per 3 days at the rate of 25-100 mg/kg. Western blot analysis further showed down-regulation of procaspase-3, X-linked inhibitor of apoptosis protein and poly(ADP-ribose) polymerase cleavage in Bel-7402 cells treated with 15 mumol/l fenretinide for 48 h. Overexpression of p53 was observed in a time-dependent manner, along with a decrease in the Bcl-2/Bax ratio. Depolarized mitochondrial membranes were found in fenretinide-induced apoptotic cells, in a time-dependent manner. We conclude that fenretinide effectively inhibits the proliferation of Bel-7402, both in vitro and in vivo. Both procaspase-3 and p53-mediated apoptotic pathways are involved in its potent anti-cancer activity.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Fenretinide; Flow Cytometry; Humans; In Vitro Techniques; Liver Neoplasms; Membrane Potentials; Mice; Models, Biological; Tumor Cells, Cultured

Retinoids are used to treat several types of cancer; however, their effects on liver cancer have not been fully characterized. To investigate the therapeutic potential of retinoids on hepatocellular carcinoma (HCC), the present study evaluates the apoptotic effect of a panel of natural and synthetic retinoids in three human HCC cell lines as well as explores the underlying mechanisms.. Apoptosis was determined by caspase-3 cleavage using western blot, DNA double-strand breaks using TUNEL assay, and phosphatidylserine translocation using flow cytometry analysis. Gene expression of nuclear receptors was assessed by real-time PCR. Transactivation assay and chromatin immunoprecipitation (ChIP) were conducted to evaluate the activation of RXRalpha/RARbeta pathway by fenretinide. Knockdown of RARbeta mRNA expression was achieved by siRNA transfection.. Our data revealed that fenretinide effectively induces apoptosis in Huh-7 and Hep3B cells. Gene expression analysis of nuclear receptors revealed that the basal and inducibility of retinoic acid receptor beta (RARbeta) expression positively correlate with the susceptibility of HCC cells to fenretinide treatment. Furthermore, fenretinide transactivates the RXRalpha/RARbeta-mediated pathway and directly increases the transcriptional activity of RARbeta. Knockdown of RARbeta mRNA expression significantly impairs fenretinide-induced apoptosis in Huh-7 cells.. Our findings reveal that endogenous expression of retinoids receptor RARbeta gene determines the susceptibility of HCC cells to fenretinide-induced apoptosis. Our results also demonstrate fenretinide directly activates RARbeta and induces apoptosis in Huh-7 cells in a RARbeta-dependent manner. These findings suggest a novel role of RARbeta as a tumor suppressor by mediating the signals of certain chemotherapeutic agents.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Fenretinide; Gene Expression Regulation, Neoplastic; Humans; In Situ Nick-End Labeling; Liver Neoplasms; Phosphatidylserines; Receptors, Retinoic Acid; Transcriptional Activation

Cell-cycle deregulation is an early event of hepatocarcinogenesis. We evaluated the role of changes in activity of nuclear factor kappaB (NF-kappaB) and some related pathways in this alteration, and the interference of N-(4-hydroxyphenyl)retinamide (HPR), a retinoid chemopreventive for various cancer types, with these molecular mechanisms and the evolution of preneoplastic liver to cancer. Male F344 rats, initiated according to the 'resistant hepatocyte' model of liver carcinogenesis, received weekly 840 nmol of liposomal HPR (SL-HPR)/100 g body wt or empty liposomes, between 5 and 25 weeks after initiation. Inhibition of DNA synthesis and induction of apoptosis occurred in pre-cancerous lesions, 7-147 days after starting SL-HPR, and a decrease in carcinoma incidence and multiplicity was observed 25 weeks after arresting treatment. An increase in NF-kappaB expression and binding activity, and under-expression of the inhibitor kappaB-alpha (IkappaB-alpha) were found in preneoplastic liver and neoplastic nodules, 5 and 25 weeks after initiation, respectively. These lesions also showed low expression of Mat1A and low activity of methionine adenosyltransferase I/III, whose reaction product, S-adenosyl-l-methionine, enhances IkappaB-alpha expression. SL-HPR prevented these changes and induced a decrease in expression of iNos, c-myc, cyclin D1 and Vegf-A genes, that were over-expressed in preneoplastic liver and nodules, and a decrease in Bcl-2/Bax, Bcl-2/Bad and Bcl-xL/Bax mRNA ratios with respect to the lesions of control rats. Liposomes alone did not influence the parameters tested. These results indicate that signal transduction pathways controlled by NF-kappaB, nitric oxide and S-adenosyl-l-methionine are deregulated in pre-cancerous lesions. Recovery from these alterations by SL-HPR is associated with chemoprevention of hepatocarcinogenesis. Overall, these studies elucidate some molecular changes, in early stages of hepatocarcinogenesis, and underline their pathogenetic role. Moreover, they demonstrate a partially new mechanism of HPR chemopreventive effect and indicate the potential clinical relevance of this compound for prevention of hepatocellular carcinoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Transformation, Neoplastic; Cyclin D1; Fenretinide; I-kappa B Proteins; Liposomes; Liver; Liver Neoplasms; Male; Methionine Adenosyltransferase; NF-KappaB Inhibitor alpha; NF-kappaB-Inducing Kinase; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Rats; Rats, Inbred F344; S-Adenosylmethionine; Vascular Endothelial Growth Factor A

Fenretinide (N-4-hydroxyphenyl retinamide, 4HPR) is a synthetic anticancer retinoid that is a well-known apoptosis-inducing agent. Recently, we observed that the apoptosis induced by fenretinide could be effectively enhanced in hepatoma cells by a concomitant treatment with parthenolide, which is a known inhibitor of nuclear factor-kappaB (NF-kappaB). Furthermore, treatment with fenretinide triggered the activation of NF-kappaB during apoptosis, which could be substantially inhibited by parthenolide, suggesting that NF-kappaB activation during fenretinide-induced apoptosis has an antiapoptotic effect. This study investigated the molecular mechanism of this apoptotic potentiation by NF-kappaB inhibition. The genes involved in the enhanced fenretinide-induced apoptosis by parthenolide were identified using the differential display-PCR method and subsequent Northern blot or semiquantitative reverse transcriptase PCR analysis. This study identified 35 apoptosis-related genes including 12 unknown genes that were either up- or down-regulated by parthenolide. Interestingly, one up-regulated gene (HA1A2) was isolated and cloned from the liver cDNA, and was found to be identical to ANKRD1, which is also referred to as the CARP gene. Compared with controls treated with an empty vector or with antisense cDNA, the ectopic expression of ANKRD1 led to reduced colony formation and to enhanced apoptotic cell death in hepatoma cells. These results suggest that ANKRD1 and the other genes, whose expressions were substantially modulated by the parthenolide-mediated inhibition of NF-kappaB activation, play roles in the enhanced drug-induced apoptosis. In addition, this study suggests that those identified genes may be useful in anticancer strategies against hepatoma.

Topics: Amino Acid Sequence; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Base Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; Down-Regulation; Drug Synergism; Fenretinide; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Molecular Sequence Data; Muscle Proteins; NF-kappa B; Nuclear Proteins; Polymerase Chain Reaction; Repressor Proteins; RNA, Messenger; Sesquiterpenes; Up-Regulation

To investigate the effect of N-(4-hydrophenyl) retinamide (4-HPR), the derivative of retinoic acid, on inhibition of migration, invasion, cell growth, and induction of apoptosis in hepatocellular carcinoma cells (HCCs) and malignant melanoma cells.. 4-HPR was chemically synthesized. Cellular migration and invasion were assayed by Borden chamber experiment. Cell growth was assayed by MTT chromometry. Apoptosis effect was measured using Hoechst 32258 staining and flow cytometry. Gene transfection was performed with lipofectamine.. We observed that the migration of HCC and melanoma cells was significantly suppressed by 4-HPR and the migration cells were reduced to 58+/-5.03 (control 201+/-27.2, P<0.05, n = 4) in SMMC 7721-k3 HCC, and to 254+/-25.04 (control 302+/-30.1, P<0.05, n = 4) in melanoma cells after 6-h incubation with 4-HPR. The invasion through reconstituted basement membrane was also significantly reduced by 4-HPR treatment to 11.2+/-3.3 in SMMC 7721-k3 HCC (control 27+/-13.1), and to 24.3+/-3.2 in melanoma cells (control 67.5+/-10.1, P<0.05, n = 3). Cell growth, especially in melanoma cells, was also significantly inhibited. Furthermore, 3 micromol/L of 4-HPR induced apoptosis in B16 melanoma cells (37.11+/-0.94%) more significantly than all-trans retinoic acid (P<0.05), but it failed to induce apoptosis in SMMC 7721-k3 HCC. The mechanism for 4-HPR-induced apoptosis was not clear, but we observed that 4-HPR could regulate p27(kip1), and overexpression of cerebroside sulfotransferase (CST) diminished the apoptosis induced by 4-HPR in melanoma cells.. 4-HPR is a potent inhibitor of HCC migration and inducer of melanoma cell apoptosis. CST and p27(kip1) expression might be associated with 4-HPR-induced apoptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Movement; Fenretinide; Humans; Liver Neoplasms; Melanoma; Mice; Mice, Nude; Sulfotransferases

The transcription factor CHOP/GADD153 is reportedly induced by cellular stresses such as UV light, genotoxic agents, and protein misfolding in the endoplasmic reticulum. However, the mechanism whereby induction of the GADD153 gene is linked to a downstream pathway is still unclear. Previously, we observed that a synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) effectively impaired cell growth and survival (induction of growth arrest and apoptosis) in human hepatoma cells, which was accompanied by over expression of GADD153. Furthermore, GADD153-transfected Hep 3B cells were growth arrested and were sensitized to drug-induced apoptosis. Thus, in this study, we used suppression subtractive hybridization (SSH) to identify GADD153 target genes that were up-regulated or down-regulated in the GADD153 transfectants. We screened 614 sequence-verified clones by Northern blotting, of which 42 genes were scored as over expressed and 17 genes as under expressed in GADD153 transfectants compared with control vector transfectants. Of those genes, 49 corresponded to known genes in public databases. Among them, we further verified that the expression of transferrin (Tf), which is a negative acute-phase protein and is essential to cell survival as a growth factor, was highly modulated by drug-induced GADD153 over expression or by in vitro transfection. GADD153 significantly antagonized the C/EBP (C/EBP-alpha, -beta, and -delta)-mediated transcriptional activation of the Tf gene. In conclusion, Tf and other target genes identified may play a functional role in the downstream pathway of GADD153.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; CCAAT-Enhancer-Binding Proteins; Down-Regulation; Fenretinide; Gene Expression; Humans; Liver Neoplasms; Transcription Factor CHOP; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transferrin; Tumor Cells, Cultured

In this paper, a significantly effect of N-(4-hydrophenyl) retinamide (4-HPR), a derivative of retinoic acid, was observed on inhibition of migration, invasion, cell growth, and induction of apoptosis in hepatoma cells and B16 melanoma cells. The number of migratory hepatoma cells reduced significantly from the control 201 +/- 27.2 to 58 +/- 5.03 after 6-hour incubation with 4-HPR (p < 0.01, n = 4). The number of migratory B16 melanoma cells reduced from the control 302 +/- 30.1 to 254 +/- 25.04 (p < 0.05, n = 4). The invasive ability of these cells was also suppressed by 4-HPR treatment. Cells that penetrated the artificial membrane matrigel decreased from 27 +/- 13.1 to 11.2 +/- 3.3 in hepatoma cells, from 67.5 +/- 10.1 to 24.3 +/- 3.2 in B16 melanoma cells (p < 0.05, n = 3). Furthermore, cell growth was significantly inhibited especially in B16 melanoma cells and 37.11 +/- 0.94% cells were induced to apoptosis after 48-hour induction by 4-HPR, which was significantly higher than those by retinoic acid treatment (p < 0.05). Although the mechanism of 4-HPR effects was not very clear, over expression of CST, which was inhibited by 4-HPR in our previous study, could diminish the apoptosis--inducing effect by 4-HPR. We believe that 4-HPR has a strong inhibitory effect on melanoma and hepatocarcinoma cells and might become a potent therapeutic agent.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Movement; Fenretinide; Humans; Liver Neoplasms; Melanoma, Experimental; Mice; Sulfotransferases

N-(4-hydroxyphenyl)retinamide (4HPR), a chemopreventive and chemotherapeutic retinoid, induces apoptosis in various types of cells. Currently, oxidative mitochondrial damage is thought to cause 4HPR-induced apoptosis, although the exact mechanism has not yet been clarified. 4HPR effectively induces apoptosis in hepatoma cells although the susceptibility differs in a cell-specific manner. Hep-3B and PLC/PRF/5 cells were more susceptible to 4HPR than were Hep-G2 and SK-HEP-1 cells, and the resistance to 4HPR seems to be related to growth inhibition (G(1) arrest). We further observed that 4HPR specifically down-regulates cytochrome c oxidase subunit III (CO III) transcript levels through destabilization of its mRNA and thus decreases the activity of cytochrome c oxidase (complex IV). To explore the mechanism whereby the CO III transcript was decreased by 4HPR, we used adenine nucleotide translocator (ANT) ligands, which modulate mitochondrial transmembrane potential (deltapsi(m)) without altering CO III transcription. Intriguingly, bongkrekic acid, a specific ANT inhibitor, enhanced 4HPR-induced deltapsi(m) disruption, which in turn decreased the level of CO III transcripts, which was accompanied by increases in the generation of reactive oxygen species and in apoptosis. In contrast, atractyloside, an activator of ANT, inhibited those 4HPR-induced effects. Taken together, these results indicate that down-regulation of CO III, a molecular marker of oxidative stress, may result from upstream deltapsi(m) disruption and that ligands of ANT may be capable of modulating 4HPR-induced oxidative stress and apoptosis.

Topics: Apoptosis; Carcinoma, Hepatocellular; Electron Transport Complex IV; Fenretinide; Humans; Liver Neoplasms; Oxidative Stress; Tumor Cells, Cultured

We observed that N-(4-hydroxyphenyl)retinamide (4HPR), a chemopreventive and chemotherapeutic agent, effectively induced apoptosis in hepatoma cells. Interestingly, Fas-negative (Hep 3B and PLC/PRF/5) hepatoma cells were shown to be more susceptible to apoptosis induced by 4HPR than were Fas-positive (Hep G2 and SK-HEP-1) hepatoma cells. Thus, we explored the mechanisms underlying 4HPR-induced apoptosis in Fas-defective hepatoma cells. Hep 3B cells stably expressing the dominant-negative Fas-associated death domain (dnFADD) showed no alteration in 4HPR drug susceptibility, but when stably expressing E1B19K, Crm A, or dominant-negative FLICE (dnFLICE), Hep 3B cells were resistant, suggesting that 4HPR-induced apoptosis was mediated by caspase-8 activation. Furthermore, apoptosis could be completely blocked by Z-VAD-FMK (a general caspase inhibitor) or by IETD-CHO (a caspase-8 inhibitor), but was only partially blocked by Ac-DEVD-CMK (a caspase-3 inhibitor), by N-acetyl-L-cysteine (NAC) (an antioxidant), by N-acetyl-leucyl-leucyl-norleucinal (ALLN) (a calpain inhibitor I), or by Z-LEHD-FMK (a caspase-9 inhibitor). Time-sequence analysis of the induction of apoptosis by 4HPR revealed that an initial caspase-8 activation was followed by late mitochondrial cytochrome c release and minor caspase-9 activation, which suggested that caspase-8 activation is the primary upstream regulatory point. Activation of Bid or induction of proapoptotic Bax was not observed during apoptosis. In contrast, Bcl-xL expression was decreased during 4HPR-induced apoptosis. Taken together, these results indicate that 4HPR may be a potential chemotherapeutic drug, which is able to induce apoptosis in Fas-defective hepatoma cells through caspase-8 activation.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Carcinoma, Hepatocellular; Carrier Proteins; Caspase 8; Caspase 9; Caspases; Enzyme Activation; fas Receptor; Fas-Associated Death Domain Protein; Fenretinide; Humans; Liver Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

The synthetic retinoid 4-HPR has been shown to markedly lower the plasma concentration of both retinol and RBP in rats and humans. We have studied the effect of 4-HPR on the secretion of retinol-RBP from liver cells in vivo and in vitro. In rats maintained with a normal diet, a vitamin A-deficient diet or a normal diet supplemented with 4-HPR, chylomicrons [3H]retinyl esters were rapidly cleared from the plasma. The secretion of chylomicron-derived [3H]retinol from tissues to the circulation, however, was different. In control rats, the lymph-derived [3H]retinol peaked after about 2 hr, whereas 4-HPR treatment effectively reduced this peak of [3H]retinol. Our results suggest that 4-HPR inhibits secretion of retinol-RBP from the liver. Therefore, we decided to study the effect of 4-HPR on the secretion of RBP using the human hepatoma cell line HepG2. Retinol and 4-HPR were found to induce the secretion of RBP. The medium from cells treated with 4-HPR was immunoprecipitated with antibodies against human RBP. HPLC analysis of the precipitated RBP revealed the presence of 4-HPR. When the medium from cells incubated with either 4-HPR or retinol was applied to a TTR affinity column, we found that RBP from cells incubated with 4-HPR had a considerably reduced affinity for TTR. We conclude that 4-HPR binds RBP and thereby induces secretion of RBP in HepG2 cells, and that the secreted 4-HPR-RBP complex has a reduced affinity for TTR. This observation may explain the 4-HPR-induced reduction of plasma retinol and RBP observed in in vivo studies.

Topics: Animals; Carcinoma, Hepatocellular; Chromatography, High Pressure Liquid; Chylomicrons; Culture Media; Diet; Fenretinide; Humans; Liver; Liver Neoplasms; Male; Prealbumin; Protein Binding; Rats; Rats, Wistar; Retinol-Binding Proteins; Retinol-Binding Proteins, Plasma; Vitamin A Deficiency