ITR­284 modulates cell differentiation in human chronic myelogenous leukemia K562 cells.

ITR­284 is a carboxamide analog that can inhibit proliferation in human promyelocytic leukemia HL-60 cells. To understand the effects and molecular mechanisms of ITR­284 in human erythromyeloblastoid leukemia, we treated K562 cells with different concentrations of ITR­284 (0, 2, 4, 6, 8 and 10 nM) and all-trans retinoic acid (ATRA) (0, 0.1, 0.5, 1, 5 and 10 µM) for 24 h. The IC50 of ITR­284 was ~10 nM in K562 cells treated for 24 h as determined by MTT assay. May-Grünwald-Giemsa staining and nitro blue tetrazolium (NBT) assays were used to determine cell morphology changes and differentiation after ITR­284 and ATRA treatment. In addition, mRNA expression levels of hematopoietic factors, including GATA­1, NF-E2 and GATA­2, were elevated, while expression levels of BCR­ABL were downregulated in K562 cells after 24 h of treatment with ITR­284 as determined by quantitative reverse transcription polymerase chain reaction. In addition, western blot analyses showed that FOXM1, GLI 1 and c-MYC protein levels were decreased by ITR­284. Taken together, our data show that ITR­284 induced K562 cell differentiation, which led to decreased tumorigenesis. Our findings suggest that ITR­284 could be a potential candidate for treating chronic myelogenous leukemia.

Carboxamide analog ITR-284 evokes apoptosis and inhibits migration ability in human lung adenocarcinoma A549 cells.

Lung adenocarcinoma is the most common type of lung cancer and found in both smokers and non-smokers, but the treatment of lung cancer is limited. ITR-284 has been shown to be a potent carboxamide-derived anticancer agent and to induce apoptosis in leukemia and colon cancer cells. However, little is known whether ITR-284 has anticancer activity in human lung adenocarcinoma cells through induction of apoptosis and suppression of migration in vitro. We showed that ITR-284 inhibited human lung cancer A549 cells using the thiazolyl blue tetrazolium bromide (MTT) assay and evoked apoptosis via the cell cycle distribution at S phase arrest. After treatment with 20 nM ITR-284 for 24 h, apoptotic cells were induced and detected by Annexin V-FITC/PI staining. The production of reactive oxygen species (ROS) was dose-dependently increased in A549 cells caused by ITR-284. The results from immunoblotting analysis showed an elevation of protein levels of p53 and phosphorylation of p53 in A549 cells prior to ITR-284 exposure. Additionally, apoptosis-associated proteins such as Bax, cleaved caspase-3 and cleaved PARP were upregulated after ITR-284 treatment. By wound healing assay, low concentrations (1-5 nM) of ITR-284 exerted a greater effect on inhibition of A549 cell migration. The protein levels of E-cadherin and vimentin, which are the epithelial-mesenchymal transition (EMT) markers, were modulated in ITR-284-treated cells assessed by western blot analysis. Taken together, our data suggest that ITR-284 may be an effective anticancer agent for treating lung adenocarcinoma.

Design and synthesis of new 2-arylnaphthyridin-4-ones as potent antitumor agents targeting tumorigenic cell lines.

To develop new anticancer drug candidates from 2-arylnaphthyridin-4-one (AN), we have designed and synthesized a series of 3'-hydroxy and 6-hydroxy derivatives of AN. The results of cytotoxicity screening indicated that the replacement of the 3'-methoxy moiety on the C-ring phenyl group of AN (6a-e) with 3'-hydroxy (7a-e) made no significant effect on the inhibitory activity against HL-60, Hep3B and NCI-H460 cancer cell lines. On the other hand, replacing the 6-methoxy group on the A-ring of AN (6g-i) with a 6-hydroxy group (7g-i) resulted in reduced inhibitory activity against the above three cancer cell lines. Among the above-mentioned target compounds, 2-(3-hydroxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (7a) demonstrated the greatest potency and the best selectivity toward tumorigenic cancer cell lines. In a 7a preliminary mechanism of action study in Hep3B hepatoma cells, 7a showed the effects on microtubules followed by cell cycle arrest and sequentially led to apoptosis. In addition, a phosphate prodrug (11) of 7a exhibited significant antitumor activity when tested in a Hep3B xenograft nude mice model. Since compound 11 has demonstrated good development potential, it is recommended for further preclinical studies.

The novel carboxamide analog ITR-284 induces caspase-dependent apoptotic cell death in human hepatocellular and colorectal cancer cells.

We have previously reported that ITR-284, a potent carboxamide-derived anticancer agent, induced apoptosis in leukemia cells. However, there are no reports showing that ITR-284 inhibits human hepatocellular and colorectal cancer cells. In this study, we investigated the antiproliferative effects and apoptotic induction of ITR-284 on various types of human hepatocellular and colorectal cancer cells in vitro. The growth inhibition effect of ITR-284 on cancer cells was evaluated by thiazolyl blue tetrazolium bromide (MTT) assay. Cell morphology was examined under a phase-contrast microscope. The activities of caspase-3, -8 and -9 were determined by caspase colorimetric assay. ITR-284 reduced the cell viability in human hepatocellular cancer cells (Hep G2, Hep 3B, SK-HEP-1 and J5) and colorectal cancer cells (HT 29, COLO 205, HCT 116 and SW 620). ITR-284 had highly selective effects on Hep 3B and COLO 205 cells. ITR-284 stimulated morphological changes of Hep 3B and COLO 205 cells. The activation of caspase-3, -8 and -9 contributed to ITR-284-induced apoptosis. ITR-284-triggered growth inhibition was significantly attenuated by the inhibitors of caspase-3, -8 and -9 in Hep 3B and COLO 205 cells. ITR-284 induced apoptosis in Hep 3B and COLO 205 cells through the caspase cascade-dependent signaling pathway.

Arsenic trioxide (As2O3) inhibits murine WEHI-3 leukemia in BALB/c mice in vivo.

Arsenic trioxide (As2O3) is used clinically to treat acute promyelocytic leukemia (APL) and has activity in vitro for induction of apoptosis in several solid tumor cell lines. To investigate the potential therapeutic application of As2O3 for leukemia, we analyzed the effects of As2O3 on the WEHI-3 cells-induced orthotopic leukemia animal model in vivo in this study. We established the WEHI-3 cells leukemia mice through the injection of murine WEHI-3 cells into BALB/c mice, and they were then treated with As2O3 (0.9 and 4.5 mg kg⁻¹ ; p.o.) and/or combined with all-trans-retinoic acid (ATRA), (30 mg kg⁻¹ ; i.p.). The results indicated that (1) As2O3 alone or As2O3 combined with ATRA promoted the total survival rate of leukemia mice and these effects are dose-dependent; (2) As2O3 did not affect the body weight but decreased the spleen weight; however, it did not affect liver weight; (3) As2O3 alone or As2O3 combined with ATRA increased the levels of CD3 and CD19, indicating that the differentiation of T and B cells were promoted; and (4) As2O3 alone or As2O3 combined with ATRA did not change the levels of Mac-3 and CD11b markers, indicating that the differentiation of the precursor of macrophage were not inhibited. Based on these observations, As2O3 alone or As2O3 combined with ATRA have efficacious antileukemia activity in WEHI-3 cells leukemia in vivo.

Novel azulene-based derivatives as potent multi-receptor tyrosine kinase inhibitors.

A series of azulene-based derivatives were synthesized as potent inhibitors for receptor tyrosine kinases such as FMS-like tyrosine kinase 3 (FLT-3). Systematic side chain modification of prototype 1a was carried out through SAR studies. Analogue 22 was identified from this series and found to be one of the most potent FLT-3 inhibitors, with good pharmaceutical properties, superior efficacy, and tolerability in a tumor xenograft model.

Investigation of anti-leukemia molecular mechanism of ITR-284, a carboxamide analog, in leukemia cells and its effects in WEHI-3 leukemia mice.

ITR-284, a potent anti-leukemia agent of carboxamide derivative, has been shown to inhibit the proliferation of leukemia cells. In this study, the underlying molecular mechanisms in vitro and anti-leukemia activity in vivo of ITR-284 were investigated. ITR-284 reduced the cell viability and induced apoptosis in HL-60 and WEHI-3 leukemia cells. Following exposure of cells to 30 nM of ITR-284, there is a time-dependent decrease in the mitochondrial membrane potential (DeltaPsi(m)) and an increase in the reactive oxygen species (ROS). ITR-284 treatment also caused a time-dependent increase of Fas/CD95, cytosolic cytochrome c, cytosolic active form of caspase-8/-9/-3, cytosolic Apaf-1 and Bax, and the decrease of Bcl-2. However, the ITR-284-induced caspase-8/-9 and -3 activities can be blocked by pan-caspase inhibitor (Z-VAD-FMK). In addition, the anti-leukemia effects of ITR-284 in vivo were further evaluated in BALB/c mice inoculated with WEHI-3 cells. Orally treatment with ITR-284 (2 and 10mg/kg/alternate day for 7 times) increased the survival rate and prevented the loss of body weight in leukemia mice. The enlargement of spleen and infiltration of immature myeloblastic cells into spleen red pulp were significantly reduced in ITR-284-treated mice compared with control mice. Moreover, ITR-284 application can enhance the anti-leukemia effect of all-trans retinoic acid (ATRA). These results revealed that ITR-284 acted against both HL-60 and WEHI-3 in vitrovia both intrinsic and extrinsic apoptotic signaling pathways, and exhibited an anti-leukemic effect in a WEHI-3 orthotopic mice model of leukemia.

2-(1-Hydroxethyl)-4,8-dihydrobenzo[1,2-b:5,4-b']dithiophene-4,8-dione (BTP-11) enhances the ATRA-induced differentiation in human leukemia HL-60 cells.

2-(1-Hydroxethyl)-4,8-dihydrobenzo[1,2-b:5,4-b']dithiophene-4,8-dione (BTP-11) is a potent enhancer for all-trans retinoic acid (ATRA)-induced differentiation in HL-60 cells. Combination of BTP-11 and ATRA cut down the concentration of ATRA significantly, and that BTP-11 promoted the progression of ATRA-induced into the terminal granulocytic differentiation. Further, Western blot analysis revealed that combination of BTP-11 and ATRA decreased cyclin D/CDK4 and increased C/EBPvarepsilon protein expression to arrest the cells into G0/G1 phase leading to granulocytic maturation. These results confirmed that BTP-11 is a potent enhancer for ATRA-induced differentiation of HL-60 cells, and the great developmental potential of BTP-11 will be expected.

Cell differentiation enhancement by hydrophilic derivatives of 4,8-dihydrobenzo[1,2-b:5,4-b']dithiophene-4,8-diones in HL-60 leukemia cells.

Among five carboxamide derivatives (13-17), N-(2-dimethylaminoethyl)-4,8-dihydrobenzo[1,2-b:5,4-b']dithiophene-4,8-dione-2-carboxamide (13) showed the greatest enhancement of all-trans retinoid acid (ATRA)-induced differentiation in HL-60 cells, inducing nearly complete differentiation at a concentration of 0.02microM. On the other hand, 2-hydroxymethyl-4,8-dihydrobenzo[1,2-b:5,4-b']dithiophene-4,8-dione (2) and 2-(1-hydroxylethyl)-4,8-dihydrobenzo[1,2-b:5,4-b']dithiophene-4,8-dione (18) exhibited excellent and equally potent differentiation effects on HL-60 cells. To improve their water solubility, ester-type hydrophilic prodrugs (23-26) were also synthesized. Compounds 13 and 23-26 are identified in this paper as new anti-leukemic drug candidates.

A Highly diastereoselective synthesis of (1R)-(+)-camphor-based chiral allenes and their asymmetric hydroboration-oxidation reactions.

Synthesis of camphor derived chiral allenes and their hydroboration-oxidation reactions are described. Reaction of (1R)-(+)-camphor with alkynyllithium followed by the reduction of the resulted propargyl alcohol derivatives using AlH3 furnished chiral allenes 2a-g in excellent yields with high diastereoselectivity. Reduction of the propargyl alcohols with aluminum hydride proceeded through selective intermolecular anti-addition of hydride ion. The stereochemistry of the chiral allenes 2 was assigned based on lanthanide shift studies and chemical correlations. Diastereoselectivity was observed in the hydroboration-oxidation of 2 which produced a mixture of (E,R) and (E,S) stereoisomers in a ratio of 6:1 to 18:1.