The histone-deacetylase inhibitor Trichostatin A blocks proliferation and triggers apoptotic programs in hepatoma cells

BACKGROUND/AIMS: Effective treatment for hepatocellular carcinoma is urgently needed. The histone-deacetylase inhibitor Trichostatin A (TSA) was shown to induce apoptosis in non-hepatic cells at submicromolar concentrations. However, the effect of TSA on hepatoma cells is unknown. METHODS: The hepatoma cells HepG2, MH1C1, Hepa1-6 and Hep1B as well as human fibroblasts (control cells) were exposed to TSA (10(-6) to 10(-9)M). Cell proliferation was assessed by measuring DNA-synthesis and cell numbers. Apoptosis was quantified by flow cytometry and by the TdT-mediated dUTP nick-end labeling method. Expression patterns of cell cycle- and/or apoptosis-associated p27, p21(cip/waf), bax, bcl-2, cyclin A and (pro)-caspase 3 were studied using quantitative Western blotting. Activation of caspase 3 was analyzed via a colorimetric assay. RESULTS: 10(-6)M TSA inhibited DNA-synthesis by 46% (HepG2) to 64% (MH1C1) after 24h, inducing a G(2)/M-phase arrest and apoptosis. TSA increased activation of caspase 3 and expression of cyclin A, p2l(cip/waf), bax and (pro)-caspase 3, while bcl-2 was downregulated. Human fibroblasts remained unaffected. CONCLUSIONS: TSA inhibits hepatoma cell growth in vitro, which are otherwise particularly resistant to chemotherapy. Its anti-proliferative activity is paralleled by a comparable rate of apoptosis. TSA may be a promising agent for treatment of hepatocellular carcinoma in vivo.     

Testosterone suppression of ornithine decarboxylase activity in rat Sertoli cells

The effect of testosterone on the induction of Sertoli cell ornithine decarboxylase (ODC) activity was investigated in highly purified cultures derived from testes of 20-day-old rats. Sertoli cell cultures were maintained in serum-free Ham's F-10 medium, with refeeding on days 2 and 4. Before refeeding on day 4, ODC activity was 7.4 U (1 U = 1 pmol 14CO2 released/mg protein.60 min at 37 C). After a medium change, ODC activity increased (41.6 U at 10 h; 180.0 U at 24 h) and then returned to near-basal activity (26.3 U) after 48 h. Simultaneous addition of testosterone (150 ng/ml; 5.2 X 10(-7) M) with the day 4 medium change suppressed the increase in ODC induction (7.53 U at 10 h, 91.6 U at 24 h). Addition of testosterone 24 h before refeeding resulted in greater inhibition of ODC induction (6.9 U at 10 h; 45.4 U at 24 h) than when added simultaneously. Other androgens, 5 alpha-dihydrotestosterone, androsterone, and 5 alpha-androstane-3 alpha,17 beta-diol, also suppressed induction of ODC, whereas 17 beta-estradiol was ineffective, illustrating the steroid specificity of the effect. Coadministration of the antiandrogens hydroxyflutamide or cyproterone acetate partially blocked the testosterone effect. Induction of ODC activity by ovine FSH (10 ng/ml) was also suppressed when Sertoli cells were cultured in the presence of testosterone (150 ng/ml) from the time of isolation. However, induction of ODC activity by (BU)2cAMP was uncompromised by testosterone. These results suggest that testosterone may repress ODC activity and, hence, polyamine biosynthesis in the Sertoli cell, but that cAMP, acting through the trophic hormone FSH, can overcome this suppression of putrescine biosynthesis. Intratesticular and hypophyseal modulation of polyamine biosynthesis may influence not only cellular processes in the Sertoli cell itself but also its role in spermatogenesis.     

Epidermal growth factor stimulation of ornithine decarboxylase activity in a human hepatoma cell line.

Epidermal growth factor (EGF) bound specifically to the human hepatoma cell line PLC/PRF/5. Treatment of these cells with nanomolar concentrations of EGF for 4-6 hr resulted in a 2- to 6-fold increase in ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity. 12-O-Tetradecanoylphorbol 13-acetate also produced an increase in enzyme activity in these cells and exhibited an additive effect with EGF. It did not inhibit the binding of 125I-labeled EGF to these cells. The stimulation of enzyme activity by EGF was not inhibited by cycloheximide or actinomycin D, although these agents did cause a significant decrease in enzyme levels when added without EGF. Also, colchicine, chloroquine, ammonium chloride, and methylamine, compounds that inhibit EGF degradation in various cells types, did not interfere with the ability of EGF to elevate enzyme levels in the human hepatoma cells.     

Bile acids, ornithine decarboxylase, and cell proliferation in colon cancer: a review

Cell proliferation has an important role in carcinogenesis. Increased ornithine decarboxylase (ODC) activity with resulting polyamine synthesis is a phenomenon common to cells undergoing rapid proliferation and to models of tumor promotion. In colon cancer, bile acids are considered to be important tumor promoters. Inferential data suggest that bile acids can increase ODC activity and stimulate or alter cell proliferation in the large bowel. These changes may underlie tumor promotion by bile acids. Confirmation of this relationship will solidify this concept and build a framework for understanding the mechanism of bile acid promotion in colon carcinogenesis.     

Immunolocalization of ornithine decarboxylase in rat heart atria

Mammalian atrial myocytes secrete a potent natriuretic and diuretic factor, atrial natriuretic peptide (ANP), in response to volume expansion or elevations of right atrial pressure. ANP is synthesized in the atrial atrial myocytes and stored in cytoplasmic secretory granules. Ornithine decarboxylase (ODC) is the initial rate-limiting enzyme in the biosynthesis of polyamines, organic cations which are essential for various aspects of DNA, RNA and protein synthesis, structure, and function. The enzyme has been reported to be localized predominantly in the cytoplasm. A polyclonal antibody elicited against ODC was used to analyse the intracellular distribution of the enzyme protein within atrial myocytes at the ultrastructural level through the use of a post-embedding immunogold technique. In control animals, the density of gold particles associated with the atrial granules was seven to 30-fold higher than that detected in association with other subcellular structures. Administration of Isoproterenol increased atrial of Isoproterenol increased atrial ODC activity 18-fold and the density of the immunolabelling of the atrial myocytes five-fold. Statistically significant increases in the density of labelling after stimulation occurred in association with the atrial granules and the nucleus. After isoproterenol stimulation, 60% of the immunodetectable ODC protein in the atrial myocyte was found in association with the atrial granules. The atrial granules were demonstrated to contain ANP by immunocytochemical analysis of adjacent sections.     

Hormonal regulation of renal ornithine decarboxylase activity in the rat.

The regulation of the activity of the renal enzyme ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was examined in the rat. In the intact animal adapted to a light/dark cycle of 14 hours and 10 hours, respectively, the level of renal ornithine decarboxylase activity was rhythmical and paralleled the diurnal rhythm in plasma corticosteroid concentration. Renal ornithine decarboxylase activity and plasma corticosterone were highest during the early hours of darkness and lowest during the hours of light. Following hypophysectomy, the level of renal ornithine decarboxylase activity declined rapidly and remained low and without a demonstrable diurnal rhythm. When pituitary hormone levels were temporarily restored in the hypophysectomized rat by the injection of pituitary extract, renal ornithine decarboxylase activity increased rapidly, reached a peak within 8 hours, and returned toward pre-injection levels by 12 hours. Exogenous growth hormone, ACTH and cortisol each increased renal ornithine decarboxylase activity in the hypophysectomized rat, with the highest levels of activity being achieved with growth hormone. Other pituitary hormones (FSH, LH, TSH and prolactin) were ineffective. After bilateral adrenalectomy, renal ornithine decarboxylase activity retained a rhythmical pattern similar to that observed in the intact rat, but the levels were increased. Growth hormone and cortisol increased renal ornitine decarboxylase activity in the adrenalectomized-hypophysectomized animal to the same extent as in the hypophysectomized animal, but ACTH was almost totally ineffective. These data suggest that the pituitary plays a major role in the regulation of renal ornithine decarboxylase activity in the rat, primarily through the rhythmical secretion of growth hormone and ACTH.     

In vivo hormonal induction of ornithine decarboxylase in rat kidney.

Single pharmacological doses of parathyroid hormone, calcitonin, vasopressin, d-aldosterone, or L-triiodothyronine produced a significant increase in the ornithine decarboxylase activity of rat kidney. The activity of kidney ornithine decarboxylase was also enhanced by other hormones, such as pentagastrin and serotonin, which, although they are not known to modify kidney physiology, are secreted by cells having close relationships to the calcitonin-secreting parafollicular cells. The induction of the enzyme was observed in hypophysectomized rats, with or without some other hormone-secreting glands remaining. However, the magnitude of the stimulation elicited by the hormones was somewhat diminished in animals still having the endocrine gland whose hormone was being tested. The maximal stimulation of kidney ornithine decarboxylase activity by parathyroid hormone, calcitonin, vasopressin, L-triiodothyronine, pentagastrin, and serotonin occurred at 4 h after the hormone injection. The enhancement in ornithine decarboxylase activity produced by d-aldosterone was maximal at 3 h after the injection of the hormone. The content of ornithine in the kidney was found to be virtually unchanged whatever the type of hormone treatment. No statistically significant increases in renal ornithine decarboxylase activity of hypophysectomized animals were observed after injection of melatonin or of vitamin D3. Since the stimulating hormones possess clearly different mechanisms of action, the role of cyclic AMP as a general mediator of ornithine decarboxylase induction is questioned.     

Regulation of thyroid ornithine ornithine decarboxylase (ODC) by thyrotropin. I. The rat.

We studied the effects of TSH on rat thyroid ornithine decarboxylase (ODC) activity. After 1 day of goitrogen treatment, there was an abrupt fall in serum triiodothyronine (T3) a rise in circulating TSH, and a dramatic increase in thyroid ODC activity. Despite the continued rise in TSH and progressive increase in thyroid gland size with further treatment, thyroid ODC activity declined on the third day and remained at submaximal levels. Thyroid ODC activity was also stimulated in a dose-related manner by administration of exogenous TSH. Little TSH effect was noted before 3 h. Maximal ODC activity occurred between 4 and 5 h. The TSH stimulation of ODC could be inhibited by pretreatment with actinomycin D or cycloheximide, suggesting that the increase in ODC activity requires new RNA and protein synthesis. Although pretreatment with agents that alter microtubule structure (e.g., colchicine and vinblastine) prevent stimulation of ODC activity by TSH, additional data suggest, but do not confirm, that hrmone secretion and ODC activation may be dissociable. Further studies were undertaken to determine whether cyclic AMP (cAMP) or prostaglandins played any role in the regulation of thyroidal ODC activity. Dibutyryl cAMP, alone, or together with aminophylline, did not stimulate thyroidal ODC activity in dosages which concomitantly stimulated adrenal enzyme activity. Likewise, prostaglandin E2 (PGE2) did not stimulate thyroidal ODC activity, but did stimulate adrenal enzyme activity in a dose-related manner. However, pre-treatment of rats with inhibitors of prostaglandin synthesis prevented the activation of thyroidal ODC BY TSH. One inhibitor, indomethacin, attenuated the TSH stimulation of enzyme activity in a dose-related manner. Indomethacin pretreatment also resulted in approximately a 10-fold decrease in thyroidal prostaglandin levels. Exogenous PGE9, in dosages as high as 500 pg, did not overcome the inhibitory effect of indomethacin on ODC activation. Although the precise role for endogenous prostaglandins remains to be defined, it does appear that a reduction in thyroidal prostaglandins prevents activation of the enzyme by TSH.     

SU5416 is a potent inhibitor of hepatocyte growth factor receptor (c-Met) and blocks HGF-induced invasiveness of human HepG2 hepatoma cells

BACKGROUND/AIMS: SU5416 is a potent inhibitor of receptor tyrosine kinases, including those of the vascular endothelial growth factor receptor, stem cell factor receptor, and platelet-derived growth factor receptor. Because of the overwhelming evidence favoring the role of aberrant hepatocyte growth factor (HGF)/Met signaling in the pathogenesis of various human cancers, various inhibitor strategies have been employed to therapeutically target this receptor. METHODS: Cell proliferation was determined by incorporation of [(3)H] thymidine. Invasiveness was assayed in Boyden Chambers with 8 microm Matrigel coated filters. Phosphorylation of ERK1/2, Akt by HGF stimulation was detected by Western blotting. RESULTS: We found that SU5416 inhibited motility scattering and the invasive activity of a hepatocellular carcinoma cell line HepG2 in vitro and growth in primary cultured hepatocytes induced by HGF. Consequently, tyrosine autophosphorylation of the c-met induced by HGF was inhibited in these cells by SU5416 in a dose-dependent manner. Furthermore, ERK1/2 and Akt phosphorylation, the signaling events down-stream of c-met activation were reduced. Moreover, SU5416 caused reversion in NIH3T3 fibroblasts transformed by the oncogenic form of the receptor, Tpr-Met. CONCLUSIONS: Inhibition of various solid tumors growth and metastasis by SU5416 may be partially attributed to blocking activation of the hepatocyte growth factor receptor.     

Induction of a protein inhibitor to ornithine decarboxylase by the end products of its reaction.

Putrescine, the end-product of ornithine decarboxylase (ODC: L-ornithine carboxylyase, EC; 4.1.1.17) action, induces the synthesis of a protein(s), in L1210, neuroblastoma, and H-35 cells as well as in rat liver, which inhibits ODC activity. Spermidine and spermine, distal products of ODC activity, also induce the synthesis of a similar protein in H-35 cells. These ODC-inhibitors are heat-labile, trypsin-sensitive, and their induction is dependent upon protein synthesis. They have short half-lives which range from 18 to 66 min; these half-lives are similar to those of the ODC derived from the same source. They are noncompetitive inhibitors of ODC activity with an apparent molecular weight of 26,500. Each inhibitor crossreacts with the ODC's of the other cells and forms an enzyme-inhibitor complex which is stable during Sephadex chromatography; however, after treatment with ammonium sulfate, enzyme and inhibitor activities can be dissociated and recovered intact from the same column. We propose the name antizyme for proteins whose synthesis is induced by the proximal or distal products of the enzyme they inhibit.     

Ovarian ornithine decarboxylase regulation in the immature, the pubescent, and the pseudopregnant rat.

Ovarian ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) regulation was investigated in immature, pubescent, and pseudopregnant rats. After initial induction of the enzyme by injection of lutropin in the immature rat, continued daily injections resulted in a graded decrease in the activity of the enzyme. In the 32-day-old rat, the induction and subsequent decline in enzymic activity after a single injection of lutropin could only be partially reversed by injections of the hormone at 4-hr intervals. This decrease could not be attributed to a decrease in ovarian in vivo uptake of the hormone. In the pseudopregnant rat, ovarian ornithine decarboxylase is highly refractory to induction by either a single injection or repeated injections of lutropin. This refractoriness occurs despite a 2- to 3-fold increase in ovarian in vivo uptake of the hormone compared to that in the 32-day-old rat. It is suggested that the refractoriness observed with all three tissues is a function of cell differentiation.     

Prevention of neonatal hyperbilirubinemia by tin protoporphyrin IX, a potent competitive inhibitor of heme oxidation.

The effects of various metalloporphyrins on hepatic heme oxygenase (EC 1.14.99.3) activity were examined in order to identify compounds that could inhibit heme degradation to bile pigment and might therefore be utilized to suppress the development of hyperbilirubinemia in the newborn. Among nine metal-protoporphyrin IX chelates (i.e., metal-hemes) studied, Sn-heme, Mn-heme, and Zn-heme substantially diminished heme oxygenase activity in vivo in the rat. These metalloporphyrins act as competitive inhibitory substrates in the heme oxygenase reaction but are not themselves oxidatively degraded. Sn-heme was the most potent enzyme inhibitor (Ki = 0.011 microM) in liver, spleen, kidney, and skin. Sn-heme administered to newborn animals within the first 72 hr after birth blocked the postnatal increase in heme oxygenase activity that occurs in various tissues. Its effect on the enzyme levels was prompt and protracted. Sn-heme administration also entirely prevented the development of hyperbilirubinemia that normally occurs postnatally. The effect of the metalloporphyrin in lowering the increased concentrations of serum bilirubin in neonates was prompt (within 1 day) and persisted throughout the 42 days after birth. No deleterious effects of Sn-heme treatment of the newborn were observed. This demonstrates that a synthetic metalloporphyrin that is a potent competitive inhibitor of heme oxidation can, when administered to the newborn, also prevent the hyperbilirubinemia that normally develops postnatally. The potential clinical implications of these findings are evident, and it is suggested that the pharmacological properties of Sn-heme and related synthetic metalloporphyrins merit further study.     

Cyclosporine inhibits prolactin induction of ornithine decarboxylase in rat tissues

Cyclosporine (CyA), formerly cyclosporin A, significantly inhibited the ability of prolactin (PRL) to elevate ornithine decarboxylase (ODC) activity in a variety of rat tissues. Administration of PRL to hypophysectomized rats also resulted in an induction of ODC activity which was inhibited markedly in all tissues studied in the presence of CyA. Transglutaminase ( TGase ) activity was not affected in any significant manner by PRL or CyA in most tissues studied. However, it was elevated in the adrenal by 10(-8) M PRL. Bromocryptine, which selectively antagonizes pituitary PRL release, decreased the kidney ODC basal levels to 30% of vehicle control and serum PRL level to 4.3 +/- 1.4 compared to 28 +/- 10 in controls, suggestive of PRL maintenance of steady-state ODC activity in the kidney. CyA administration did not affect the action of glucagon, a known cyclic AMP-mediated hormone, or 8-bromo-cyclic AMP on kidney ODC activity. The elevation of rat kidney ODC activity by dexamethasone and triiodothyronine (T3), compounds which elevated serum prolactin levels in all cases, was also blocked by administration of CyA. Epidermal growth factor (EGF), which did not induce rat kidney ODC activity by itself, was capable of producing a small increment in ODC activity in the presence of CyA. The marked effect of CyA to selectively block ODC induction by PRL may be due to the ability of CyA to interact with receptor-required phospholipids in membranes and thus to antagonize hormone-receptor interaction.     

The effect of isoprenaline and propranolol on rat myocardial ornithine decarboxylase.

The intraperitoneal injection of isoprenaline in rats caused an increase in myocardial ornithine decarboxylase activity which reached a maximum of about four times the control value one hour after the injection. The intraperitoneal injection of dl-propranolol had no effect on myocardial ornithine decarboxylase activity. The injection of dl-propranolol 30 min before the injection of isoprenaline almost completely prevented the effect of isoprenaline.     

Induction of ornithine decarboxylase activity in a temperature-sensitive cell cycle mutant of Chinese hamster cells.

We have investigated the induction of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity in a temperature-sensitive cell cycle mutant of Chinese hamster fibroblasts. This activity is not induced at the nonpermissive temperature, although the synthesis of the majority of proteins is normal. From a combination of studies with inhibitors of mRNA synthesis and maturation (alpha-amanitin, and cordycepin) and of proteins synthesis (cycloheximide, diphtheria toxin, and emetine), we conclude that the temperature-sensitive block is at the level of translation of one or more specific mRNAs.     

alpha-Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, inhibits tumor promoter-induced polyamine accumulation and carcinogenesis in mouse skin.

The role of ornithine decarboxylase (OrnDCase, EC 4.1.1.17) and of the polyamines [putrescine (Put), spermidine (Spd), and spermine (Spm)] in mouse skin tumor promotion was investigated by the use of alpha-difluoromethylornithine (CHF2-Orn), an enzyme-activated irreversible inhibitor of OrnDCase. 12-O-Tetradecanoylphorbol 13-acetate (TPA), mezerein, and ethyl phenylpropiolate (EPP) were employed as complete, stage II specific, and nonpromoting agents, respectively. TPA and mezerein, but not EPP, provided for a dose-dependent increase in tissue Put accumulation. The Put level in papillomas developed by TPA (2 micrograms) treatment was approximately equal to 15-fold higher than that of the surrounding skin tissue; Spd accumulation was 2- to 3-fold greater in the papillomas. Put administered (intraperitoneally) with TPA greatly enhanced papilloma yield. CHF2-Orn, given orally or intraperitoneally, abolished the TPA-induced OrnDCase activity and Put accumulation in mouse epidermis. The reduction of polyamine accumulation by CHF2-Orn was directly proportional to reduction of tumor size. CHF2-Orn administered in a two-stage (TPA-mezerein) promotion protocol [Slaga, T. J., Fischer, S. M., Nelson, K. G. & Gleason, G. L. (1980) Proc. Natl. Acad. Sci. USA 77, 3659-3663; Slaga, T. J., Klein-Szanto, A. J. P., Fischer, S. M., Weeks, C. E., Nelson, K. & Major, S. (1980) Proc. Natl. Acad. Sci. USA 77, 2251-2254] reduced tumor size, inhibited by 65-70% the number of papillomas per mouse, and decreased by 40% the percentage of mice with tumors when given with the stage II agent mezerein. CHF2-Orn provided considerably less effect on tumorigenesis when administered with the TPA portion of the protocol, and CHF2-Orn did not inhibit the induction of dark basal keratinocytes by TPA. Based on our results with CHF2-Orn, we suggest that regulation of polyamine biosynthesis, particularly Put, is a critical factor in stage II promotion.     

Cyclic AMP-mediated induction of ornithine decarboxylase of glioma and neuroblastoma cells.

The activity of ornithine decarboxylase (EC 4.1.1.17; L-ornithine carboxy-lyase) of C6-BU-1 glioma and N115 neuroblastoma cells increases significantly when confluent cultures are treated with compounds that increase cellular cAMP levels. These include norepinephrine or isoproterenol, and prostaglandin E1 or adenosine, which stimulate ornithine decarboxylase activity in C6-BU-1 glioma and N115 neuroblastoma cells, respectively. Ornithine decarboxylase activity is also elevated in confluent C6-BU-1 glioma cells treated with dibutyrylcAMP and theophylline, or after the glioma cells are fed with a serum-depleted medium in the presence of catecholamines and inhibitors of cyclic nucleotide phosphodiesterase. The activity of the enzyme increases 500- to 1000-fold, 2-6 hr after stationary-phase N115 neuroblastoma cells are fed with a serum-free medium, supplemented with phosphodiesterase inhibitors, adenosine, or prostaglandin E1. This stimulation is antagonized by carbamoyl choline and is blocked by actinomycin D or cycloheximide. These results suggest that the synthesis of ornithine decarboxylase of C6-BU-1 glioma and N115 neuroblastoma cells is controlled by cAMP.