Toshitaka Manda

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.

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.

FK317: a novel substituted dihydrobenzoxazine with potent antitumor activity which does not induce vascular leak syndrome.

Purpose: FK973, a substituted dihydrobenzoxazine, is an antitumor antibiotic which has shown high therapeutic efficacy in a phase I study, but its development has been abandoned because of the side effect of vascular leak syndrome (VLS) in the clinical study. This study was performed to investigate whether or not FK317, a new benzmethoxy derivative of FK973, retains the antitumor activity of FK973 without the side effect of VLS. Methods: VLS was evaluated by the volume of pleural effusion in rats. Cytotoxic activities were determined by a tetrazolium-based colorimetric assay (MTT assay) against murine (B16, P388) and human (HeLa S3, KB) tumor cell lines. Antitumor activities against murine ascitic leukemia (P388, L1210), murine solid tumors (reticulum cell sarcoma M5076, Colon 38 carcinoma) and human xenografts (mammary carcinoma MX-1, lung carcinoma LX-1) were examined. Results: FK973 (1.8 mg/kg) given i.v. to rats induced pleural effusion, one of the elements of VLS, 36 days after the first dosing, but did not 28 days after dosing. This model reflects clinical VLS delayed-type effusion with high protein concentrations. In contrast, FK317 (1.0–3.2 mg/kg) did not induce pleural effusion at all. FK317 had stronger cytotoxic effects against in vitro cultured B16, P388, HeLa S3 and KB tumor cell lines, and in in vivo experiments, FK317 showed equivalent antitumor activity against P388, M5076 and MX-1, and more potent antitumor activity against L1210, Colon 38 and LX-1 compared with FK973. Conclusion: These results suggest that FK317 retains the antitumor activity of FK973 and does not induce VLS, and FK317 is a drug with high clinical potential for treating tumors in humans.

The efficacy of combined treatment with recombinant human tumor necrosis factor-α and 5-fluorouracil is dependent on the development of capillaries in tumor

The antitumor effects of recombinant human tumor necrosis factor-alpha (rTNF-alpha) and 5-fluorouracil (5-FU) in combination treatment were examined on Meth A fibrosarcoma implanted intradermally in mice. Growth of the tumor was inhibited when rTNF-alpha was given i.v. on day 7 or 11 after implantation, but the effect was countered when 5-FU was additionally given i.p. once a day on days 1-4 after implantation. Conversely, 5-FU given on days 5-8 after implantation augmented the antitumor effects of rTNF-alpha. Injection of carbon particles showed that fine capillaries did not develop in the tumors of mice treated with 5-FU on days 1-4 after implantation, but that a delicate network of capillaries developed in the tumors of both the mice treated with 5-FU on days 5-8 after implantation and the controls given saline. The results show that the timing of 5-FU treatment is important when attempting to enhance the antitumor effects of rTNF-alpha, and suggest that these effects are directly associated with newly formed fine capillaries in the tumor.

Anti‐cachectic Effect of FK317, a Novel Anti‐cancer Agent, in Colon26 and LX‐1 Models in Mice

The effects of FK317 (11‐acetyl‐8‐carbamoyloxymethyl‐4‐formyl‐6‐methoxy‐14‐oxa‐1,11‐diazatetra‐cyclo[7.4.1.02,7.010,2]tetradeca‐2,4,6‐trien‐9‐yl acetate), a novel anti‐cancer agent, on murine adenocarcinoma colon26‐ and human lung carcinoma LX‐1‐induced cachexia were investigated in mice. Mice bearing colon26 or LX‐1 s.c. lost weight and became cachectic, associated with tumor growth. FK317 and mitomycin C (MMC) inhibited the growth of both tumors. FK317 ameliorated the weight loss induced by the presence of colon26 or LX‐1, while MMC enhanced it. An attenuation of the reduction in the weights of epididymal fat, gastrocnemius muscle and carcass was observed in FK317‐treated tumor‐bearing mice in both cachexia models, but not in MMC‐treated mice. The decreases in the circulating levels of triglyceride, glucose and non‐esterified fatty acid, which were induced by the presence of colon26, was partially inhibited by treatment with FK317. Overall, this study revealed that FK317 is a potent anti‐cancer drug with anti‐cachectic activity, suggesting that FK317 has potential utility for the treatment of cancer.

FK317, a novel substituted dihydrobenzoxazine, exhibits potent antitumor activity against human tumor xenografts in nude mice.

The antitumor effects of FK317, a novel substituted dihydrobenzoxazine, were evaluated using human tumor xenografts (small cell lung cancer, non‐small cell lung cancer, stomach cancer, colon cancer, pancreatic cancer, breast cancer, cervical cancer and ovarian cancer). Tumor growth‐inhibitory effects and the effective dose‐range of FK317 were much stronger and broader, respectively, than those of reference drugs such as mitomycin C, adriamycin, cisplatin, taxol and irinotecan. Furthermore, the body weight decrease and myelosuppression in FK317‐treated mice were less than in the animals given any of the reference drugs. To explain this tumor selectivity, the distribution of FK317 was investigated after dosing tumor‐bearing mice with the 14C‐labelled compound. The concentration of FK317 in tumor tissues was relatively low, and long tumor retention was not observed. However, thin‐layer chromatographic separation revealed that the radioactivity in the tumor resided mainly in strongly cytotoxic metabolites, while that in other tissues resided mainly in non‐cytotoxic metabolites. These results suggest that FK317 shows strong antitumor activity without side effects, and one reason for this is its specific metabolite pattern. FK317 is now undergoing phase I clinical trials.

Cytotoxic Mechanisms of FK317, a New Class of Bioreductive Agent with Potent Antitumor Activity

FK317 is a member of a new class of bioreductive agents that exhibit strong cytotoxicity against various human cancer cells. The effect of FK317 was found to be stronger than that of mitomycin C (MMC), adriamycin (ADR) or cisplatin (CDDP). Alkaline elution analysis indicated that FK317 formed interstrand DNA‐DNA and DNA‐protein cross‐links in cells. On the other hand, no DNA single‐strand breaks were observed in the cells treated with FK317. In a cell‐free system the deacetylated metabolites produced cross‐linked DNA under reductive conditions, though FK317 itself did not form DNA‐DNA cross‐links. In order to elucidate the metabolic activation mechanisms, we established an FK317‐resistant subline from human non‐small cell lung cancer cells (Lu99) by stepwise and brief exposure (1 h) to FK317. The resistant subline (Lu99/317) showed cross‐resistance to MMC and carboquone (CQ), but not to ADR or CDDP. DT‐diaphorase, which is one of the activation enzymes of MMC and CQ, was deficient in Lu99/317 cells as determined by enzyme activity assay. However, the levels of NADPH:cytochrome P450 reductase, which is another activation enzyme for MMC and CQ, were comparable in resistant and parent cell lines. Treatment of the cells with dicumarol, an inhibitor of DT‐diaphorase, reduced the cytotoxicity of FK317 to Lu99 cells, but not to Lu99/317 cells. These results indicate that deacetylation of FK317 is necessary for its reductive activation, and deacetylated FK317 is reduced by DT‐diaphorase to form an active metabolite, which produces DNA‐DNA interstrand and DNA‐protein cross‐links that lead to cell death.

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.

Different Effects of FK317 on Multidrug‐resistant Tumor in vivo and in vitro

FK317, a novel substituted dihydrobenzoxazine, was examined for antitumor effects on multidrug‐resistant (MDR) tumor cells in vitro and in vivo. In nude mice, FK317 markedly inhibited the growth of s.c. implanted KB‐V1 vinblastine (VLB)‐resistant human epidermal carcinoma KB cells, as well as the parent cells (KB‐3‐1). However, KB‐V1 showed much greater resistance to FK317 than to VLB and adriamycin (ADM) in the in vitro study. This resistance was reversed by the addition of verapamil, whereby intracellular accumulation of FK317 in the KB‐V1 cells was also decreased. After incubation of FK317 in human and mouse blood, it was shown to be rapidly metabolized to a monodeacetylated form, and slowly metabolized further to a dideacetylated form. With the removal of the acetyl groups from FK317, resistance indexes in KB‐V1 and SBC‐3/ADM, ADM‐resistant human lung carcinoma, decreased. In addition, photolabeling of P‐glycoprotein with [3H]azidopine in KB‐V1 plasma membrane was completely inhibited by FK317, but not by the deacetylated metabolites. These results indicate that FK317 is metabolized to deacetylated forms, which do not bind to P‐glycoprotein and are incorporated into MDR cells, causing cytotoxic effects.