Masahiko Matsuo

Effects of FK228, a novel histone deacetylase inhibitor, on human lymphoma U-937 cells in vitro and in vivo.

FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone; FR901228, depsipeptide] is a novel histone deacetylase inhibitor that shows therapeutic efficacy in Phase I trials of patients with malignant lymphoma. However, its mechanism of action has not been characterized. In this study, we examined the in vitro and in vivo effects of FK228 on human lymphoma U-937 cells. FK228 very strongly inhibited the growth of U-937 cells with an IC(50) value of 5.92 nM. In a scid mouse lymphoma model, mice treated with FK228 once or twice a week survived longer than control mice, with median survival times of 30.5 (0.56 mg/kg) and 33 days (0.32 mg/kg), respectively (vs. 20 days in control mice). Remarkably, 2 out of 12 mice treated with FK228 (0.56 mg/kg once or twice a week) survived past the observation period of 60 days. The apoptotic population of U-937 cells time-dependently increased to 37.7% after 48 hr of treatment with FK228. In addition, FK228 induced G1 and G2/M arrest and the differentiation of U-937 cells to the CD11b(+)/CD14(+) phenotype. Expression of p21(WAF1/Cip1) and gelsolin mRNA increased up to 654- and 152-fold, respectively, after 24hr of treatment with FK228. FK228 caused histone acetylation in p21(WAF1/Cip1) promoter regions, including the Sp1-binding sites. In conclusion, (i) FK228 prolonged the survival time of scid mice in a lymphoma model, and (ii) the beneficial effects of FK228 on human lymphoma may be exerted through the induction of apoptosis, cell cycle arrest, and differentiation via the modulation of gene expression by histone acetylation.

Antitumor efficacy of FK228, a novel histone deacetylase inhibitor, depends on the effect on expression of angiogenesis factors

UNLABELLED It has been recently demonstrated that histone deacetylase inhibitors inhibit angiogenesis, but their mechanism of action has not been characterized well. In this study, we examined the in vitro and in vivo effects of FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone; FR901228, depsipeptide], an HDAC inhibitor, on the expression of angiogenesis factors in FK228-sensitive PC-3 prostate and FK228-resistant ACHN renal cancer cells. FK228 suppressed the expression of VEGF mRNA in PC-3 cells, but not in ACHN cells. FK228 also suppressed the expression of basic fibroblast growth factor (bFGF) mRNA in both PC-3 and ACHN cells. Under conditions of hypoxia, FK228 suppressed the expression of VEGF mRNA without modulating the expression of hypoxia-inducible factor-1 alpha mRNA in PC-3 cells. FK228 induced the highest acetylation of histone H3 and H4 in the P2 region of the VEGF promoter, which includes the hypoxia-inducible factor-1 alpha binding site that plays an important role in regulating the expression of VEGF gene. Moreover, FK228 reduced the amount of VEGF and bFGF protein, and their mRNA levels in PC-3 xenograft implanted in nude mice, but did not reduce them in ACHN xenograft. IN CONCLUSION (i) FK228 showed a suppressive effect on the expression of angiogenesis factors, such as VEGF and bFGF, in PC-3 xenograft but not in ACHN xenograft, which suggests that the effect on the expression of angiogenesis factors is important for the antitumor efficacy of FK228; (ii) FK228 caused histone acetylation of the VEGF promoter regions, which may contribute to the suppression of VEGF gene expression.

Lesser reduction in bone mineral density by the immunosuppressant, FK506, compared with cyclosporine in rats.

Background. Posttransplantation osteopenia leading to osteoporosis induced commonly by treatment with immunosuppressants including cyclosporine (CsA) is a severe complication and results in lowering the quality of life in patients receiving organ transplantation. FK506 is a newly developed immunosuppressant and is currently being used for the prevention of rejection after organ transplantation. In this study, to investigate whether FK506 as well as CsA would cause osteopenia or not, we evaluated the effect of FK506 on bone mineral density and several parameters relevant to bone metabolism in comparison with that of CsA using normal rats. Methods. Ten-week-old male Sprague-Dawley rats were treated with FK506 (vehicle, 1 mg/kg, and 3.2 mg/kg) or CsA (vehicle, 10 mg/kg, and 32 mg/kg) by daily oral gavage for 28 days. Bone mineral density of the femur, plasma insulin-like growth factor I (IGF-I), and urinary deoxypyridinoline were determined by peripheral quantitative computerized tomography, radioimmunoassay, and enzyme-linked immunosorbent assay, respectively. Results. The reduction in bone mineral density of the femur was observed in both FK506- and CsA-treated rats. The reduction in CsA-treated rats, however, was statistically significant and strikingly severe, whereas that in FK506-treated rats was much less severe than CsA. Plasma IGF-I levels were significantly elevated in FK506-treated rats but not in CsA-treated rats. Urinary deoxypyridinoline levels were unchanged in FK506-treated rats but elevated in CsA-treated rats. Conclusions. Compared with CsA, FK506 does not appear to induce severe osteopenia by high-turnover bone metabolism in the rat by mediating via IGF-I induction in part. The results suggest that FK506 may exert favorable effects on bone metabolism in patients with organ transplantation compared with CsA. To assess this idea, further clinical investigations focused on bone metabolism will be required.

Effects of FK228, a novel histone deacetylase inhibitor, on tumor growth and expression of p21 and c-myc genes in vivo

In this study, we examined the effects of FK228 (FR901228, depsipeptide) on tumor growth and expression of p21 and c-myc genes in vivo. FK228 induced the expression of p21 mRNA and decreased c-myc mRNA in tumor xenograft sensitive to FK228. However, FK228 did not sufficiently modulate the expression of p21 mRNA and increased the expression of c-myc in tumor xenograft less sensitive to FK228. The modulation of p21 and/or c-myc genes may be critical for the marked antitumor activity of FK228 in vivo.

Effect of FR145237, a novel ACAT inhibitor, on atherogenesis in cholesterol-fed and WHHL rabbits. Evidence for a direct effect on the arterial wall

The hypocholesterolemic and antiatherosclerotic activities of FR145237, a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, were evaluated in cholesterol-fed and Watanabe heritable hyperlipidemic (WHHL) rabbits. In the first experiment, rabbits were fed a high cholesterol (1% cholesterol) diet for 2 weeks and further fed a high cholesterol diet containing FR145237 for 8 weeks. FR145237 (0.1, 0.32 and 1.0 mg/kg) dose-dependently lowered the plasma total cholestrol levels by 80%, 96% and 97%, respectively. and reduced aortic atherosclerosis by 44%, 90% and 90%, respectively. To clarify a direct effect of FR145237 at the aortic wall, a second experiment was performed. Rabbits were fed a high-cholesterol diet for 8 weeks to establish aortic atherosclerosis and then fed a normal diet with or without FR145237 for 8 weeks. Cholesterol content in the aorta and the liver was significantly reduced in the FR145237 group (10 mg/kg) by 50% and 43%, respectively, though plasma total cholesterol level did not differ from that in the control group. In the WHHL rabbits, FR145237 (10 mg/kg) did not affect plasma cholesterol level but significantly reduced the atherosclerotic lesion in the coronary arteries by 61%. These results suggest that FR145237 potently lowers the plasma cholesterol level in hypercholesterolemia induced by dietary cholesterol but not that by LDL receptor deficiency, and that FR145237 has a direct antiatherosclerotic activity on the arterial wall independent of its hypocholesterolemic activity.

Resiniferatoxin antagonizes cisplatin-induced emesis in dogs and ferrets

We evaluated the antiemetic activity of resiniferatoxin, an ultrapotent capsaicin analogue, on cisplatin- and apomorphine-induced emesis in dogs, and on cisplatin-induced acute and delayed emesis in ferrets. In the dog, resiniferatoxin (10 microg/kg, s.c.) 30 min before the injection of cisplatin markedly prevented acute emesis induced by cisplatin. When animals were given resiniferatoxin (10 microg/kg, s.c.) 24 h prior to cisplatin, the emesis was still inhibited, but not significantly. Resiniferatoxin (10 microg/kg, s.c.) 30 min before the administration of apomorphine also significantly reduced the emetic responses induced by apomorphine in dogs. In the ferret, resiniferatoxin (10 microg/kg, s.c.) 30 min prior to cisplatin completely inhibited acute emesis caused by cisplatin (10 mg/kg, i.p.). When ferrets were given resiniferatoxin (10 microg/kg, s.c.) 16 h prior to cisplatin, the emesis was still significantly inhibited. Cisplatin (5 mg/kg, i.p.) induced both acute (0-24 h) and delayed (24-72 h) phase emesis, and a single injection of resiniferatoxin (10 microg/kg, s.c.) at 36 h after cisplatin significantly reduced subsequent emetic responses during the 36-72 h period. These results suggest that resiniferatoxin-related vanilloids may be useful drugs against both acute and delayed emesis induced by cancer chemotherapy.

Effect of FR194738, a potent inhibitor of squalene epoxidase, on cholesterol metabolism in HepG2 cells.

(E)-N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[2-methyl-2-(3-thienylmethoxy)propyloxy]benzylamine hydrochloride (FR194738) inhibited squalene epoxidase activity in HepG2 cell homogenates with an IC50 value of 9.8 nM. In the study using intact HepG2 cells, FR194738 inhibited cholesterol synthesis from [14C]acetate with an IC50 value of 4.9 nM, and induced intracellular [14C]squalene accumulation. On the other hand, the 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor simvastatin reduced both cholesterol and squalene synthesis from [14C]acetate. Incubation with simvastatin for 18 h produced increases in HMG-CoA reductase activity in HepG2 cells, which was related to the degree of reduction in cholesterol synthesis. The HMG-CoA reductase activity increased by 13- and 19-fold at the concentrations of simvastatin that inhibited cholesterol synthesis by 65% and 82%, respectively. In contrast, FR194738 did not increase HMG-CoA reductase activity at the concentrations that inhibited cholesterol synthesis by 24% and 69%, and moderate increase (4.6-fold) was observed at the concentration that inhibited cholesterol synthesis by 90%. These results suggest that non-sterol metabolite(s) derived from mevalonate prior to the squalene epoxidation step in the cholesterol synthetic cascade have a regulatory role in the suppression of HMG-CoA reductase activity. We speculate that FR194738 inhibits cholesterol synthesis with a minimal change of the regulator(s) and would be highly effective in the treatment of hypercholesterolemia.

Effects of FK317, a novel anti-cancer agent, on survival of mice bearing B16BL6 melanoma and Lewis lung carcinoma

The effects of FK317 (11-acetyl-8-carbamoyloxymethyl-4-formyl-6-methoxy-14- oxa-1,11-diazatraacylo[7.4.1.0(2.7).0(10.2)]-tetradeca-2,4,6-trien-9-yl acetate), a novel anti-cancer agent, and mitomycin C (MMC) on survival time of mice bearing B16BL6 melanoma and Lewis lung carcinoma (LLC), induced by intravenous inoculation of the tumor, were investigated. Treatment with FK317 resulted in a significant prolongation of survival time in both tumor models. Four of ten mice bearing B16BL6 were disease-free following FK317 treatment. In contrast, MMC was not effective in prolonging survival time. Overall, this study demonstrated that FK317 shows more potent survival extension in mice bearing B16BL6 and LLC than MMC, suggesting that FK317 may have therapeutic utility for cancer chemotherapy.

Mode of relaxing action of FK336, a new antianginal agent, in rabbit aorta.

1. FK336 (10(-6)-10(-4) M) inhibited contractile responses to norepinephrine (NE), KCl and Ca2+ in isolated rabbit aortas. 2. Relaxing effect of FK336 on KCl-response was inhibited by nitroglycerin (NG), but not by nifedipine or verapamil. 3. FK336 inhibited residual NE response and a subsequent Ca2+ response in Ca(2+)-free medium. FK336 did not affect the inositol monophosphate level. 4. Relaxing effect of FK336 on NE response was inhibited by methylene blue, NG, K(+)-channel inhibitors and acetylcholine (ACh), and potentiated by M&B 22,948 and theophylline. 8-Br cGMP and dibutyl cAMP had no effect. 5. FK336 increased cGMP level in rat aorta. 6. Potentiation of isoproterenol-relaxation by FK336 was inhibited by methylene blue. 7. The inhibitory effect of ACh on FK336-relaxation was eliminated by endothelium removal, nordihydroquaiaretic acid and guinacrine, but not by indomethacin. These treatments themselves did not affect FK336-relaxation. 8. The mode of vasorelaxing action of FK336 is discussed.

The inhibitory effect of quinacrine on contractile responses to norepinephrine in isolated rabbit aorta

1. In rabbit aorta, quinacrine, but not indomethacin nor nordihydroguaiaretic acid, inhibited contractile responses to norepinephrine and KCl. Amiloride and nifedipine did not affect the effect of quinacrine. 2. In Ca2(+)-free medium, quinacrine (3 x 10(-6)-10(-4) M) inhibited the norpinephrine response less than that to a subsequent addition of Ca2+. 3. M&B 22, 948, nitroglycerin and forskolin inhibited the Ca2+ response. The effect of quinacrine was inhibited by M&B 22,948, but not by forskolin and potentiated by nitroglycerin. 4. Quinacrine and M&B 22,948 potentiated the nitroglycerin-relaxation. The effect of quinacrine plus M&B 22,948 was not different from that of quinacrine. 5. These results indicate that the effect of quinacrine may be different from that of nifedipine but is related to cGMP.