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Identification of the Genes Involved in Enhanced Fenretinide-Induced Apoptosis by Parthenolide in Human Hepatoma Cells

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Identification of the Genes Involved in Enhanced Fenretinide-Induced Apoptosis by Parthenolide in Human Hepatoma Cells Research Article Identification of the Genes Involved in Enhanced Fenretinide-Induced Apoptosis by Parthenolide in Human Hepatoma Cells Jeong-Hyang Park, Lan Liu, In-Hee Kim, Jong-Hyun Kim, Kyung-Ran You, and Dae-Ghon Kim Division of Gastroenterology and Hepatology, The Research Institute of Clinical Medicine, Department of Internal Medicine, Chonbuk National University Medical School and Hospital, Chonju, Chonbuk, Republic of Korea Abstract prostate cancer (6), bladder cancer (7), and a neuroblastoma (8) in Fenretinide (N-4-hydroxyphenyl retinamide, 4HPR) is a syn- preclinical experiments and in early clinical trials. It was previously thetic anticancer retinoid that is a well-known apoptosis- observed that fenretinide effectively induces apoptosis in hepato- ma cells (9, 10), and it was found that this ferentinide-induced inducing agent. Recently, we observed that the apoptosis n n induced by fenretinide could be effectively enhanced in apoptosis could be enhanced by the nuclear factor- B (NF- B) hepatoma cells by a concomitant treatment with parthenolide, inhibitor parthenolide. Fenretinide-induced cytotoxicity is retinoic which is a known inhibitor of nuclear factor-nnB (NF-nnB). acid receptor (RAR)–independent (11). However, fenretinide has Furthermore, treatment with fenretinide triggered the activa- been found to activate the RARs and several RAR-specific tion of NF-nnB during apoptosis, which could be substantially antagonists partially inhibited ferentinide-induced apoptosis (2). inhibited by parthenolide, suggesting that NF-nnB activation Therefore, both RAR-independent and RAR-dependent pathways are involved in the ferentinide-mediated apoptosis. Previously, it during fenretinide-induced apoptosis has an antiapoptotic effect. This study investigated the molecular mechanism of was observed that a lethal concentration of parthenolide could this apoptotic potentiation by NF-nnB inhibition. The genes induce apoptosis, which is associated with oxidative stress via involved in the enhanced fenretinide-induced apoptosis by glutathione depletion (12). In contrast, sublethal concentrations of parthenolide were identified using the differential display- parthenolide could potentiate ferentinide-induced apoptosis, which seems to in part correlate with NF-nB inhibition. PCR method and subsequent Northern blot or semiquantita- n tive reverse transcriptase PCR analysis. This study identified NF- B activation in cancer cells correlates with tumor resistance to the induction of apoptosis. This activation usually requires 35 apoptosis-related genes including 12 unknown genes that n were either up- or down-regulated by parthenolide. Interest- phosphorylation of I B at two specific serine residues at the NH2- n a 32/36 n h 19/23 ingly, one up-regulated gene (HA1A2) was isolated and cloned terminus of I B (Ser )orIB (Ser ), which triggers the from the liver cDNA, and was found to be identical to ANKRD1, polyubiquitination and subsequent proteasome-dependent degra- n which is also referred to as the CARP gene. Compared with dation of the inhibitors (13, 14). This leaves free NF- B, which controls treated with an empty vector or with antisense cDNA, translocates into the nucleus and binds the DNA to activate the n the ectopic expression of ANKRD1 led to reduced colony transcription of the responsive genes. Thus, inhibition of NF- B formation and to enhanced apoptotic cell death in hepatoma sensitizes cells to death by various stimuli including tumor necrosis cells. These results suggest that ANKRD1 and the other genes, factor-a and cancer therapy drugs (15–17). Recently, proteasome n whose expressions were substantially modulated by the inhibitors have been used to block NF- B nuclear localization via n parthenolide-mediated inhibition of NF-nnB activation, play the inhibition of I Ba degradation and the processing of the p105 n roles in the enhanced drug-induced apoptosis. In addition, NF- B precursor (18–20). Other molecules have been shown to n this study suggests that those identified genes may be useful inhibit NF- B, including a peptide that blocks the interaction of n n in anticancer strategies against hepatoma. (Cancer Res 2005; I B kinase-g with the catalytic core of I B kinase, the nonsteroidal 65(7): 2804-14) anti-inflammatory drug sulindac, cyclopentenone prostaglandins, arsenic trioxide, thalidomide, and a variety of antioxidants, natural Introduction products such as parthenolide and resveratrol, and pharmaceuti- cally developed synthetic small-molecule inhibitors of the InB Fenretinide is a synthetic retinoid with anticancer properties kinase complex (20–22). Interestingly, The RARg-selective agonists that has been shown to induce apoptosis in a variety of malignant (MX3350-1 and CD2325) and the retinoid antagonist, MX781, cell lines (1–3). It has been evaluated in humans in several clinical inhibit InB kinase via a retinoid receptor-independent pathway trials and seems to play a role in the chemoprevention of (23). During most types of chemotherapy, the inhibition of NF-nB malignancies in humans (4, 5). Furthermore, fenretinide, which is activation promotes cell death. Several agents, including arsenic a chemotherapeutic agent, is relatively nontoxic and effective in trioxide (24) and thalidomide (25), could inhibit the NF-nB function and are currently in clinical use as primary cancer therapeutics, whereas others are undergoing clinical development. Therefore, the Note: J-H. Park and L. Liu contributed equally to this work. n Requests for reprints: Dae-Ghon Kim, Division of Gastroenterology and role of NF- B in antiapoptotic signaling has also raised hopes that Hepatology, Department of Internal Medicine, Chonbuk National University inhibitors will enhance the efficacy of conventional chemothera- Medical School and Hospital, Keumam-dong 634-18, Dukjin-ku, Chonju, Chonbuk peutic drugs as a new adjuvant approach for cancer treatment. 561-712, Republic of Korea. Phone: 82-63-250-1681; Fax: 82-254-1609; E-mail: [email protected]. The aim of this study was to identify the gene(s) involved in the I2005 American Association for Cancer Research. enhancement of ferentinide-induced apoptosis by parthenolide in Cancer Res 2005; 65: (7). April 1, 2005 2804 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Apoptosis-Related Genes and Parthenolide Table 1. Gene-specific PCR primer sets for the genes in low abundance No. Clone ID Direction Sequence (5V!3V) Size of PCR Annealing Temperature Product (bp) (jC)/Cycles 1 HA50C1 F 5V-GGGTGGTATTGATGGAGT-3V 503 42/35 R5V-CTTGGGGGCATACAACTC-3V 2 HA52C2 F 5V-CCAAGGGGAACTTCCTCG-3V 307 60/35 R5V-TGGTCTGAGATGCCTGCC-3V 3 HA62A2 F 5V-CAGGCGCCTACTTAACCT-3V 424 47/35 R5V-ACATGGTACCACTGCCGT-3V 4 HA73C1 F 5V-AATGGAAAGTTGGGCAGG-3V 120 47/35 R5V-GGGTTTCACTGTGTTAGCC-3V 5 HC2C1 F 5V-ACACACTGCCAGTCTTCCC-3V 342 50/30 R5V-TGGCCCTTTCAGTACAGGC-3V 6 HC15C2 F 5V-AGGCTGAAGTGCAGCGAT-3V 262 50/30 R5V-AACTGATTTCGGCCGGGT-3V 7 HC26C1 F 5V-GGCTGGAACTTCAGGGTCAG-3V 473 60/35 R5V-GCTCCAGTGACTGTTGAGCC-3V 8 HC65A1 F 5V-CCACTCTCCAGATGCTAAGC-3V 402 55/35 R5V-GCGTTGCAAAGGACAGATCC-3V 9 HC71A1 F 5V-CTCCTGAAGGACAAAGTG-3V 240 42/35 R5V-CTAGTAACGGAGGAACAG-3V 10 HG33C1 F 5V-TCCATGCTCATGGGTAGGA-3V 162 50/35 R5V-GTCTTGGCAATATGGGCTC-3V order to elucidate the molecular and cellular mechanism(s) by which The terminal deoxynucleotidyl transferase enzyme mixture was added and parthenolide potentiates ferentinide-induced apoptosis. Using the then processed for the in situ detection of apoptotic cells. The quantitative differential display (DD)-PCR method, the apoptosis-related genes, measurements of apoptotic cells and the cell cycle distribution were done which are differentially expressed between vehicle-treated control using a modification of the techniques described elsewhere (10). Electrophoretic mobility shift assay. The nuclei from Hep 3B cells cells and cells treated with fenretinide, were identified and treated with the vehicle or with fenretinide were prepared according to the subsequently classified as being either up-regulated or down- modified procedure described by Dignam et al. (26). The nuclear proteins regulated genes according to the alteration of their mRNA were quantified using a bicinchoninic acid protein assay kit (Sigma, St. Louis, expression level by parthenolide. Among the genes identified, it MO), and were used to carry out electrophoretic mobility shift assay. The was first confirmed that ANKRD1, which is also known as CARP, oligonucleotide probes for NF-nB containing the IgG chain-binding site (5V- was transcriptionally up-regulated by parthenolide and its up- CCG GTT AAC AGA GGG GGC TTT CCG AG-3V; ref. 27) were used to measure regulation might be involved in the enhancement of drug-induced the activation of the transcription factors (including NF-nB). Two apoptosis. Furthermore, the other genes identified may also prove to complementary strands of the oligonucleotides were annealed and labeled 32 be valuable in dissecting the role of NF-nB inhibition in drug- with [a- P]dCTP using a random primer labeling kit (Rediprime II, A induced apoptosis and in determining the applicability of NF-nB Amersham Biosciences, United Kingdom). The nuclear extracts (5 g) were reacted with 2 to 5 ng of the radiolabeled NF-nB (50,000-100,000 cpm/ng). The inhibitors as an adjuvant approach to cancer therapy. reactions were done in the presence of 10 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, 1 mmol/L DTT, and 4% glycerol (final volume,
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