Teruhiro Utsugi

Antitumor Activity of a Novel Quinoline Derivative, TAS‐103, with Inhibitory Effects on Topoisomerases I and II

A novel quinoline derivative, TAS‐103 (6‐[[2‐(dimethyIamino)ethyl]amino]‐3‐hydroxy‐7H‐indeno[2,l‐c]quinolin‐7‐one dihydrochloride), was developed as an anticancer agent targeting topoisomerases (topo) I and II, with marked efficacy in solid tumors. TAS‐103 inhibited topo I and II (IC50: 2 μM, 6.5 μM) at a concentration similar to or lower than those of previous agents, and had a strong cytotoxic effect on P388 and KB cells (IC50,: 0.0011 μM, 0.0096 μM). TAS‐103 stabilized topo I and II‐DNA cleavable complexes in KB cells, generating a similar amount of topo II‐DNA complex to that induced by etoposide (VP‐16) but a smaller amount of topo I‐DNA complex than that produced by camptothecin (CPT). In the in vivo study, intermittent i.v. administration was markedly effective against s.c.‐implanted murine tumors. Furthermore, TAS‐103 had marked efficacy against various lung metastatic tumors, and a broad antitumor spectrum in human tumor xenografts (derived from lung, colon, stomach, breast, and pancreatic cancer). The efficacy of TAS‐103 was generally greater than that of irinotecan (CPT‐11), VP‐16, or cis‐diamminedichloroplatinum (CDDP).

Pleural Mesothelioma Instigates Tumor-Associated Fibroblasts To Promote Progression via a Malignant Cytokine Network

The tumor microenvironment is crucial to the progression of various malignancies. Malignant pleural mesothelioma (MPM), which originates from the pleura, grows aggressively in the thoracic cavity. Here we describe an orthotopic implantation SCID mouse model of MPM and demonstrate that α-SMA-positive fibroblast-like cells accumulate in the tumors produced by the human MPM cell lines MSTO-211H and Y-Meso-14. We assessed the interaction between MPM cells and their microenvironments, focusing on tumor-associated fibroblasts. MSTO-211H and Y-Meso-14 cells produced fibroblast growth factor-2 (FGF-2) and/or platelet-derived growth factor-AA (PDGF-AA); they also enhanced growth, migration, and production of hepatocyte growth factor (HGF) by human lung fibroblast MRC-5 cells. MRC-5 cells stimulated HGF-mediated growth and migration of MSTO-211H and Y-Meso-14 cells in an in vitro coculture system. In the orthotopic model, tumor formation by MSTO-211H and Y-Meso-14 cells was significantly inhibited by TSU-68, an inhibitor of FGF, VEGF, and PDGF receptors; imatinib, an inhibitor of PDGF receptors; and NK4, an antagonist of HGF. Histological analyses of clinical specimens from 51 MPM patients revealed considerable tumor-associated fibroblasts infiltration and expression of HGF, together with FGF-2 or PDGF-AA, in tumors. These findings indicate that MPM instigates tumor-associated fibroblasts, promoting tumor progression via a malignant cytokine network. Regulation of this cytokine network may be therapeutically useful for controlling MPM.

In vitro Antitumor Activity of TAS-103, a Novel Quinoline Derivative That Targets Topoisomerases I and II

TAS‐103 is a novel anticancer agent targeting both topoisomerase (Topo) I and Topo II, that stabilizes cleavable complexes of Topo‐DNA at the cellular level. In this study, the in vitro antitumor effects of TAS‐103 were compared with those of other known Topo I and Topo II inhibitors. TAS‐103 inhibited DNA synthesis more strongly than RNA and protein synthesis, and induced an increase of cell population in the S‐G2/M phase. The cytotoxicity of TAS‐103 was strongest against S‐phase cells, but its cell cycle phase specificity was not clear, and depended on drug concentration and exposure time. The cytotoxicity of TAS‐103 (IC50: 0.0030–0.23 μM) against various tumor cell lines was much stronger than that of VP‐16 and comparable to that of SN‐38. The cytotoxicity of TAS‐103 seemed to be more related to the amount of protein‐DNA complexes than to the accumulation of TAS‐103 in the cells. P‐Glycoprotein (P‐gp)‐mediated MDR, CDDP‐resistant and 5‐FU‐resistant cell lines did not show cross‐resistance to TAS‐103. Although PC‐7/CPT cells bearing a Topo I gene mutation showed cross‐resistance to TAS‐103, the sensitivity of P388/CPT, HT‐29/ CPT and St‐4/CPT cells, showing decreased Topo I expression, was not changed. KB/VM4 and HT‐29/Etp cells, showing decreased Topo II expression, were slightly cross‐resistant to TAS‐103. These results suggest that TAS‐103 may act as an inhibitor of both Topo I and Topo II at the cellular level. This property may be responsible for its strong antitumor effect and broad‐spectrum, growth‐inhibitory effect on drug‐resistant cell lines.

Selective and Potent Akt Inhibition Triggers Anti-Myeloma Activities and Enhances Fatal Endoplasmic Reticulum Stress Induced by Proteasome Inhibition

The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we, therefore, examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patient MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of C/EBP homologous protein (CHOP), a fatal ER stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM.

Mechanism of action of the dual topoisomerase-I and -II inhibitor TAS-103 and activity against (multi)drug resistant cells.

Abstract TAS-103 is a recently developed dual inhibitor of topoisomerase-I (topo-I) and topoisomerase-II (topo-II). TAS-103 has documented cytotoxicity in vitro and antitumor activity against a variety of mouse, rat, and human xenografts in vivo. Purpose: To determine TAS-103 activity against (multi)drug resistant cells in vitro and to delineate its mechanism of action. Methods: TAS-103 was evaluated for activity against three human multidrug-resistant cell lines representing resistance mediated by P-glycoprotein (Pgp)-, multidrug resistance protein (MRP), and lung resistance protein (LRP) as well as one camptothecin-resistant cell line associated with a mutated topo-I enzyme. Drug sensitivity following short (2 h), intermediate (6–8 h) and long term (24 h) exposures were compared. The mechanism of action was studied by evaluating inhibition of topoisomerase-I and -II specific DNA relaxation assays, drug-induced DNA/protein cross-link formation, and competitive DNA intercalation with ethidium bromide. Results: Increasing the exposure time only modestly potentiated TAS-103 cytotoxicity (3–5 fold) demonstrating a lack of strong exposure duration dependency. TAS-103 cytotoxicity was not affected by the presence of any of the drug resistance mechanisms studied. TAS-103 inhibits topo-I and -II activity in DNA relaxation assays, but in our assay system TAS-103 was found to have only a weak ability to induce DNA-protein crosslinks. DNA migration patterns in agarose gel electrophoresis indicate that TAS-103 can interact directly with DNA. Also its ability to displace ethidium bromide which has intercalated into the DNA provides an indication on the nature of drug-DNA interaction. Conclusions: TAS-103 cytotoxicity is not affected by the presence of Pgp, MRP, LRP or mutations in the CAM binding region of the topo-I enzyme and its growth-inhibitory effect appears to be weakly dependent on exposure duration. The presented evidence suggest that the inhibitory effects of TAS-103 on topo-I and -II may in part be related to its DNA binding rather than primarily through stabilization of topo-I or -II intermediates with DNA through specific binding to the enzymes.

Comparative efficacy of liposomes containing synthetic bacterial cell wall analogues for tumoricidal activation of monocytes and macrophages.

SummaryWe examined the activation to the tumoricidal state of normal mouse peritoneal exudate macrophages, bone marrow macrophages, and human blood monocytes by liposomes containing either lipophilic muramyl tripeptide (CGP 19 835) or a new synthetic analogue of lipoprotein from gram-negative bacteria outer wall, CGP 31 362, or combinations of the two. The superiority of liposomes containing the synthetic lipopeptide over liposomes containing lipophilic muramyl tripeptide for in vitro activation of monocytes and macrophages was demonstrated in several experiments. First, liposome-CGP-19 835 activated monocytes only in the presence of interferon-γ, whereas activation with liposome-CGP 31 362 was interferon-independent. Second, activation of both mouse macrophages and human blood monocytes by liposome-CGP 31 362 occurred at a lower liposomal concentration than that by liposome-CGP 19 835. Third, monocytes incubated with liposome-CGP 31 362 released both tumor necrosis factor (TNF) and interleukin-1 activities, whereas monocytes treated with liposome-CGP 19 835 (in the absence of interferon-γ) released only TNF activity. These data suggest that liposomes containing the synthetic lipopeptide CGP 31 362 are superior to liposomes containing CGP 19 835 for systemic activation of macrophages.

Optimization of the liposomes encapsulating a new lipopeptide cgp 31362 for efficient activation of tumoricidal properties in monocytes and macrophages

Summary The purpose of this study was to optimize a suitable liposomal carrier for CGP 31362, a new synthetic lipopeptide analogue of gram-negative bacterial cell walls. CGP 31362 was inserted into the membranes of different liposomes with different phospholipid composition. We determined the ability of these liposomes to activate tumoricidal properties in mouse peritoneal and bone marrow macrophages, and in human monocytes. The ideal liposome carrier for CGP 31362 consisted of phosphatidylcholine and phosphatidylserine in a 7:3 molar ratio. Subsequent to efficient binding and endocytosis, CGP 31362 in liposomes of this composition rendered mouse macrophages and human monocytes highly tumoricidal. Moreover, even in the absence of interferon-gamma, human monocytes released significant levels of tumor necrosis factor and interleukin-1. These data show that in a suitable liposomal carrier, the new synthetic lipopeptide in liposomes is a potent activator of tumoricidal properties in macrophages.

In situ activation of mouse macrophages and therapy of spontaneous renal cell cancer metastasis by liposomes containing the lipopeptide CGP 31362

SummaryWe determined whether the intravenous administration of multilamellar vesicle liposomes (MLV) containing a lipopeptide analogue of a fragment from the cell wall of gram-negative bacteria (CGP 31 362) can render BALB/c mouse alveolar macrophages tumoricidal in situ and reduce the incidence of spontaneous lung metastasis of syngeneic renal carcinoma (RENCA) cells. Alveolar macrophages (a) incubated in vitro with MLV containing CGP 31 362 (MLV-31 362) and (b) harvested from mice injected i.v. with MLV-31 362 were rendered cytotoxic against the RENCA cells. Maximum cytotoxic activity of the macrophages was induced by injecting 5 µmol MLV consisting of 250 mg phospholipids and 0.5 mg CGP 31 362. The single i.v. injection of 5 µmol MLV-31 362 produced activation of macrophages that lasted for up to 4 days. Repeated i. v. injections of MLV-31 362 produced a continuous antitumor activity in alveolar macrophages. To study the lipopeptides effects on metastasis, we injected the left kidneys of BALB/c mice with RENCA cells. The kidney with growing tumor was resected 10 days later and, after a further 2 days, groups of mice were injected i.v. with MLV-31 362 or with MLV-HBSS (twice weekly for 3 weeks). Treatment with MLV-31 362 significantly decreased the median number of spontaneous lung metastases. These data demonstrate that the systemic administration of MLV-31 362 can activate murine lung macrophages in situ and reduce the incidence of spontaneous RENCA lung metastases.

Abstract A270: TAS-120, a highly potent and selective irreversible FGFR inhibitor, is effective in tumors harboring various FGFR gene abnormalities.

Background: FGFs (fibroblast growth factors) and their receptors (FGFRs) play crucial roles in regulation of cell proliferation, survival, migration and differentiation. The FGFRs are comprised of four subtypes (FGFR1∼4) and their gene abnormalities such as gene amplification, translocation and mutations have been reported in multiple cancers including breast, bladder, lung, gastric, endometrial and multiple myeloma. We have identified a highly potent and selective irreversible FGFR inhibitor, TAS-1201) which inhibits all 4 subtypes of FGFR. In this report, we present the in vitro efficacy of this compound in tumor cells harboring the various FGFR gene abnormalities. In addition, we present the antitumor efficacy and pharmacodynamic (PD) activity of in vivo xenograft models. Materials and Methods: For a growth inhibition assay, various tumor cells with FGFR gene abnormalities were treated with TAS-120 for 3 days, and living cells were determined by using CellTiter-Glo™ which measures cellular ATP. Cellular phosphorylation of FGFR and its inhibition by TAS-120 were assayed using ELISA (R&D Systems) or western blotting method. For analysis of in vivo antitumor efficacy, various tumor cell lines were subcutaneously implanted into the side flank of nude mice. Dosing of compound was started when transplanted tumor size reached > ∼ 200 mm3 and tumor size was measured with digital calipers for the entire treatment period. To confirm target engagement by TAS-120 in human tumor xenograft models, we determined FGFR phosphorylation in tumor as a pharmacodynamic marker. FGFR phoshorylation in tumor was determined by ELISA or western blotting. Results: In a cell proliferation assay, TAS-120 selectively inhibited growth of human cancer cell lines with FGFR gene abnormalities. Growth of cell lines without FGFR abnormalities were not inhibited by TAS-120 treatment. TAS-120 inhibited cellular phosphorylation of FGFR as well as intercellular signaling pathways downstream of FGFR in these cells. In addition, TAS-120 inhibited tumor growth in human tumor xenograft mouse models and FGFR phosphorylation in tumor in a dose-dependent manner. Conclusion: TAS-120 is a highly potent irreversible FGFR inhibitor. It selectively inhibited growth of human cancer cell lines selectively, in a FGFR gene abnormality-dependent manner. In addition, TAS-120 demonstrated tumor growth inhibition in mice xenograft models. PD assays suggested this compound inhibits FGFR activity in human tumor xenograft models. 1) 24th EORTC-NCI-AACR Symposium (2012) abstract #380 & #383 Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A270. Citation Format: Hiroaki Ochiiwa, Hidenori Fujita, Kimihiro Itoh, Hiroshi Sootome, Akihiro Hashimoto, Yayoi Fujioka, Yoko Nakatsuru, Nobuyuki Oda, Kazuhiko Yonekura, Hiroshi Hirai, Teruhiro Utsugi. TAS-120, a highly potent and selective irreversible FGFR inhibitor, is effective in tumors harboring various FGFR gene abnormalities. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A270.

A dual topoisomerase inhibitor, TAS-103, induces apoptosis in human cancer cells.

TAS‐103 (6‐[[2‐(dimethylamino)ethyl]amino]‐3‐hydroxy‐7H‐indeno[2,1‐c]quinolin‐7‐one dihydrochloride), a dual topoisomerase (topo) inhibitor, was developed as an anticancer agent by targeting topo I and topo II and has previously been shown to be effective against lung tumors. In this study, we investigated the cytotoxic activity of TAS‐103 in various human cancer cell lines (including gastric, colon, squamous, lung, and breast cancer cells) and the induction of apoptosis by TAS‐103. We next established stable transfectants of Bcl‐2 in the gastric cancer cell line AZ521 and found that Bcl‐2 blocked TAS‐103‐induced apoptosis. In addition, we demonstrated that the activities of ICE‐like and CPP32‐like proteases are involved in the signal transduction pathway of TAS‐103‐induced apoptosis. In summary, TAS‐103 is a novel type of anticancer agent with a unique mechanism and could be useful as a lead compound for development of new drugs.