Growth Of HL-60 Research Articles

A112, a tamibarotene dimethylaminoethyl ester, may inhibit human leukemia cell growth more potently than tamibarotene

A112 is a tamibarotene dimethylaminoethyl ester considered a candidate compound for the treatment of acute promyelocytic leukemia (APL) and acute myeloid leukemia (AML). Our goal in this study was to evaluate the efficacy of anti-cancer activity, beginning by studying its inhibitory effects on leukemia cells and then comparing it to tamibarotene. A112 effectively inhibited the growth of HL-60 and NB4 cells as estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The inhibitory effect of A112 was confirmed in mice in which A112 delayed the growth of HL-60 xenografts after 3 weeks’ injection. The efficacy of A112 on leukemia cell growth was stronger than that of tamibarotene at the same dosage. The detection of A112 and tamibarotene in plasma of rats showed that A112 might sustain release of its hydrolysate tamibarotene, and the concentration was maintained at a higher level and for a longer time than that of tamibarotene injection. We studied the differentiation morphologies of leukemic cells exposed to A112 or tamibarotene. The number of differentiated NB4 cells was increased, suggesting that A112 possessed differentiation activity in the inhibition of leukemia growth. Further studies showed that the expression of CD11b, a marker of terminal granulocyte differentiation, was increased as estimated by flow cytometry with a direct immunofluorescence assay. A112 was found to induce the activation of CCAAT/enhancer-binding protein β (C/EBPβ) and cyclin-dependent kinase (CDK) inhibitors p21Waf1/cip1 and p27Kip1 while cell growth was inhibited. These activities of A112 were greater than those of tamibarotene. The higher efficacy of A112 was also evidenced by induction of apoptosis in leukemia cells. A112 induced a greater number of annexin V-positive cells than did tamibarotene as measured by flow cytometry analysis. Treatment of mice with A112 resulted in stronger terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in HL-60 xenografts. Western blot analysis revealed that A112 increased the expression of caspase-3, caspase-9 and cleaved poly(ADP-ribose) polymerase (PARP) in leukemia cells both in vitro and in vivo, indicating that induction of apoptosis was involved in the inhibition of leukemia growth. Taken together, these results suggest that A112 is a highly effective derivative of trans retinoic acid and a potential candidate compound for the treatment of leukemia.

Synthesis and Structure‐Activity Relationships of A Novel Class of Dithiocarbamic Acid Esters as Anticancer Agent

Based on a novel lead compound 4-methylpiperazine-1-carbodithioic acid 3-cyano-3,3-diphenylpropyl ester 1, the systematic structural modification was carried out. All the synthesized compounds were evaluated for their in-vitro anticancer activities on four to six different cell lines at three different concentrations. Most of the tested compounds could selectively inhibit the growth of HL-60 and Bel-7402 cell lines at a medium concentration. Four compounds (3f, 3g, 3n, and 5) were selected for the IC(50) test, and the results revealed that three compounds (3g, 3n, and 5) showed almost the same or a slightly weaker activity than compound 1 against HL-60, and three compounds (3f, 3g, and 3n) showed >2-fold higher potency than compound 1 against Bel-7402. The in-vivo efficacy of 3n · HCl was evaluated with transplanted hepatocyte carcinoma 22 as an in-vivo test model. It was found that 3n · HCl could inhibit significantly the growth of tumor, and that this effect was dose-dependent. Meanwhile, the compound 3n · HCl showed low toxicity compared with compound 1 · HCl as evidenced by the little body-weight loss. These results confirmed that compound 3n · HCl is more potent than the lead compound 1 · HCl. Preliminary structure-activity relationships indicated that: a) Both nitrile group and the cyclic amine containing at least two nitrogens were indispensable moieties to keep the activity; b) substitution of the piperazine ring is unfavorable for the improvement of activity; c) the suitable linker joining the piperazinyl dithiocarboxyl and diphenylacetonitril group should be ethylene; d) a non-coplanar arrangement of the two benzene rings appears to be essential for activity.

Essential Oil Composition, Antimicrobial and Cytotoxic Activities of Two Endemic Stachys Cretica Subspecies (Lamiaceae) from Turkey

The chemical compositions of the water-distilled essential oil of Stachys cretica ssp. lesbiaca Rech. fil. and S. cretica ssp. trapezuntica Rech. fil. were determined by GC and GC-MS. Altogether 63 compounds were identified. The sesquiterpene hydrocarbon, germacrene D (20.3% and 12.9% respectively) was the main component identified in both oils. Furthermore, ethanol, light petroleum, dichloromethane, ethyl acetate and n-butanol extracts prepared from the aerial parts of the plants were tested for their antimicrobial activities against six bacterial strains and the yeast Candida albicans. The extracts exhibited no antibacterial activity, but the light petroleum and n-butanolic fractions showed low antifungal activities. Crude ethanolic extracts of the two subspecies were tested for their ability to inhibit the growth of HL-60 and Ishikawa human tumor cell lines. The IC50 values were 100 microg/mL for the HL-60 cell line and 200 microg/mL for the Ishikawa cell line.

Analysis of Inhibitory Activity and Antineoplastic Effect of Wild Type rBTI and Its Mutants*

To examine the active site of recombinant buckwheat trypsin inhibitor (rBTI), two mutants (R45A-aBTI, R45F-fBTI) were generated through site-directed mutagenesis. Activity analysis found that the aBTI and fBTI had lost trypsin inhibitory activity. However, aBTI and fBTI showed new inhibitory activities against elastase and chymotrypsin, respectively. This result suggested that Arg45 is the active site of rBTI. These inhibitors showed remarkable stability to heat and pH. The possible effects of aBTI and fBTI on the proliferation of human HL-60 and EC9706 cell lines were investigated by MTT assays. It indicated that aBTI and fBTI could specifically inhibit the growth of HL-60 and EC9706 cells in a dose- and time-dependent manner. The active site of rBTI was determined and two new inhibitors were obtained in this research. Knowledge about the active site is useful for further clarifying the physiological mechanism of rBTI and other protease inhibitors. New inhibitory activities of aBTI and fBTI are potentially useful in the fields of health and medicine.

Abstract 1615: The redox modulator NOV-002 inhibits proliferation of ovarian tumor cells but increases proliferation of myeloid cells

Abstract Clinical trials in solid tumor indications have shown that NOV-002 (a formulation of glutathione disulfide) combined with standard chemotherapeutic regimens results in increased efficacy and improved tolerance of standard chemotherapy (e.g. enhanced hematological recovery, immune stimulation). Non-clinical studies have interrogated the myeloproliferative and antitumor activity of the drug. NOV-002 alters redox homeostasis, at the cell surface and intracellularly, through kinase signaling culminating in differential effects on cell proliferation/survival in myeloid verses tumor cells. Thus, we previously showed that NOV-002 has positive growth effects on human myeloid lineage cells (HL60) and yet the drug leads to cell cycle arrest and apoptosis in human ovarian (SKOV3) cancer cells. In the present study, we sought to understand differences in redox-mediated signaling events that govern the opposing pharmacological properties of NOV-002. While the redox-signaling events are quite similar, the cellular consequences are distinct. Chronic treatment of cells with NOV-002 leads to enhanced proliferation in HL60 cells and growth arrest in SKOV3 cells. The alteration in growth rate occurs in both myeloid and tumor cell types in parallel with stress-induced S-glutathionylation and activation of MAP kinase pathways. In myeloid lineage cells, activation of MAP- and JAK/STAT-kinases leads to proliferation whereas activation of this pathway in SKOV3 cells leads to apoptosis. NOV-002 treatment also results in changes in plasma and mitochondrial membrane potentials in both cell models. These changes were concurrent with time- and dose-dependent increases in the accumulation of intracellular Ca2+. Interestingly, dose- and time- dependent increases in nitric oxide (NO) generation were observed in HL60 cells and shown to be mediated through eNOS. SKOV 3 cells do not express eNOS and, consequently, NO generation was not detected. Finally, redox-modulation of cell surface thiols (via S-glutathionylation) occurs in both cell types but to different extents. NOV-002 treatment leads to a ∼75% decrease in free sulfhydryls in myeloid cells whereas ovarian cancer cell surface free sulfhydryls were reduced by only ∼5%. It remains to be determined whether these observed differences in cell surface protein glutathionylation and/or in mechanism of Ca2+ flux can be linked to the opposite effects of NOV-002 on proliferation/survival in myeloid and tumor cells. Regardless of the basis of this difference, it can be speculated that the differential effects of NOV-002 on myeloid and tumor cells in vitro may relate to the unique clinical profile that NOV-002 has demonstrated to date– increased survival and anti-tumor efficacy combined with mitigation of chemotherapy-induced hematological toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1615.

Andrographolide inhibits growth of acute promyelocytic leukaemia cells by inducing retinoic acid receptor-independent cell differentiation and apoptosis

Abstract Objectives The growth inhibiting potential of andrographolide was evaluated in three acute promyelocytic leukaemia cell line models (HL-60, NB4 and all-trans retinoic acid (ATRA)-resistant NB4-R2). Methods In elucidating the mechanisms of growth inhibition, a special emphasis was placed on assessing the induction of differentiation and apoptosis by andrographolide in the primary acute promyelocytic leukaemia NB4 cells. Key findings The compound was 2- and 3-fold more active in inhibiting the growth of HL-60 and NB4-R2 cells compared with NB4 cells, respectively. At IC50 (concentration at which growth of 50% of the cells (compared with medium only treated control cells) is inhibited; 4.5 μM) the compound exhibited strong cell-differentiating activity in NB4 cells, similar to ATRA (IC50 1.5 μM). In the presence of a pure retinoic acid receptor antagonist AGN193109, the growth inhibition of NB4 cells by ATRA was reversed, whereas the activity of andrographolide was not affected. This clearly suggested that andrographolide's cell differentiating activity to induce growth inhibition of NB4 cells most likely occurred via a retinoic acid receptor-independent pathway. At higher concentration (2 × IC50), andrographolide was an efficient inducer of apoptosis in NB4 cells. Conclusions Taken together, these results suggest andrographolide and its derivatives, apparently with a novel cell differentiating mechanism and with ability to induce apoptosis, might be beneficial in the treatment of primary and ATRA-resistant acute promyelocytic leukaemia.

Anticancer Effects of Alpinia pricei Hayata Roots

The leaves and roots of Alpinia pricei Hayata are used as a traditional wrapping for food and as a cooking substitute for fresh ginger. Our previous study showed that ethanol extracts from the roots of A. pricei Hayata (EEAP) and its phenolic compounds have anti-inflammatory effects. The aims of this work were to further study the in vitro anticancer activity of EEAP and its active compounds with respect to various cancer cells. The results from an MTT assay demonstrated that EEAP decreased the cell population growth of CH27, HL-60, and A549 cells. Flow cytometric analysis of HL-60 cells exposed to EEAP showed that the number of apoptotic cells increased in a time- and dose-dependent manner. Western blot data revealed that EEAP stimulated an increase in the level of protein expression of Fas, FasL, caspase-8, and tBid. Moreover, the ratio of the expression levels of pro- and anti-apoptotic Bcl-2 family members was changed after treatment with EEAP. EEAP-induced apoptosis involved the release of mitochondrial cytochrome c and subsequently induced the activation of caspase-9 and caspase-3, which were followed by the cleavage of poly(ADP-ribose) polymerase (PARP). The results also demonstrated that phenolic compounds (caffeic acid, apigenin, curcumin, and pinocembrin) from EEAP decreased the rate of population growth of HL-60 cells. Treatment of HL-60 cells with these phenolic compounds caused the loss of mitochondrial membrane potential. Our finding could provide critical information regarding the chemopreventive potential of ethanol extracts from A. pricei Hayata. These results also demonstrate that the EEAP-induced apoptotic ability in HL-60 cells might be related to the phenolic compounds.

Abstract B186: Preclinical antileukemic activity of F14512, a novel targeted cytotoxic agent

Abstract Despite advances in the therapy of acute myeloid leukemia (AML), the majority of patients die from their disease. Therefore, the lack of effective therapy mandates the development of novel compounds to improve the outcome of patients with relapsed and refractory leukemias. F14512 is a potent spermine-epipodophyllotoxin conjugate exploiting the polyamine transport system for tumor cell delivery. In this study, we report the in vivo antitumor activity of F14512 against experimental models of AML cell lines and of patient AML samples. F14512 markedly reduced the growth of HL-60 and U937 cell lines in an in vivo xenotransplantation model, resulting in a highly significant increase of survival of leukemia-bearing mice. Etoposide evaluated concurrently demonstrated only moderate in vivo activity against these models. F14512 induced in vivo apoptosis of HL-60 cells, as shown by caspase-3 activation and PARP cleavage. In an effort to mimic the human disease, we injected approximately 106 AML cells collected from a patient into NOD/SCID mice and allowed them to establish as xenografts for 8 weeks. Subsequent treatment with F14512 was carried out for 2 or 3 weeks followed by the analysis at the end of treatment and 1 week after the end of treatment. Two human AML samples were analyzed. Multiple i.v. administrations of F14512 at 0.32 mg/kg, induced an extensive reduction of the number of leukemic cells in mouse bone marrow and blood (97–99%), assessed by flow cytometry analysis, quantitative RT-PCR and histology. To identify leukemic cells expressing an active polyamine transport system, we developed a functional method based on the measurement of the cellular uptake of a nitrobenzoxadiazole fluorescent probe (F96982) combining the same spermine moiety as F14512. The level of fluorescence emitted by the probe F96982 was high in HL-60 cells as well in the 2 patient AML samples that proved to be sensitive to F14512 in vivo. Collectively, these results demonstrated that F14512 exhibits a marked in vivo antileukemic activity, supporting its clinical development. Phase I clinical trials in onco-hematology are now initiated with this novel promising drug candidate. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B186.

Expression and Role of APP Gene in Acute Myeloid Leukemia.

Abstract 4691 IntroductionAmyloid precursor protein(APP) gene was increasingly expressed in solid tumors, promoted the proliferation of tumor cells and the overexpression of APP was a bad prognostic factor to oral squamous cell carcinoma. However, little has been known about the clinical significance and role of APP in acute myeloid leukemia(AML). MethodsThe expressions of APP mRNA in 85 AML patients and 20 nonmalignant hematological diseases that worked as control were measured by real-time PCR and the expressions of APP in AML cell lines were examined by real-time PCR and western blot. Small interfering RNAs(siRNAs) targeting APP gene were synthesized and transfected into HL60 cell by lipofectamine2000, after RNAi 24h, 48h and 72h, cell growth of HL60 was measured by trypan blue dye exclusion method and MTT, differentiation was observed by Wright-Giemsa staining, cell cycle was examined by PI/RNase staining, apoptosis induction was analyzed by Annexin V/PI and Hoechst33342 staining; apoptosis-related proteins NF-κB, bcl-2 and Caspase-3 were detected by Western blot after RNAi 48h; sensitivity of HL60 to adrimycin was measured by MTT. ResultsThe expression of APP mRNA among AML subtypes was significantly different(P=0.019), M2 with t(8;21) was the highest expression subtype and M5b was the lowest. APP expression had no significant effect on AML clinical characteristic excepting AML subtypes. kasumi-1 was the highest expression cell in AML cell lines and U937 was the lowest(P<0.05), and the expression of APP in HL60/ADM was significantly lower than HL60(P<0.05). The APP expressions in AML cell lines was in agreement with its expressions in primary AML subtypes. After RNAi 24h, 48h, and 72h, no significant differences in proliferation, differentiation, apoptosis, cell cycle and sensitivity of HL60 to adriamycin were detected between interfering group and control groups. ConclusionsThe APP mRNA expression in M2 with t(8;21) was high and M5b was low. Down-regulation of APP expression had no significant effect on biological behaviour of HL60 and APP was not tightly related to pathogenesis of AML. Disclosures:No relevant conflicts of interest to declare.

Wogonin, an active compound in Scutellaria baicalensis, induces apoptosis and reduces telomerase activity in the HL-60 leukemia cells

Crude extract of Scutellaria baicalensis (S. baicalensis) has cytotoxic effect on human myelogenous leukemia cells (HL-60). We invesigated which compound from the crude extract is responsible for the cytotoxic effect on HL-60 cells. We identified 29 compounds from the crude extract using high performance liquid chromatography mass spectrometry (HPLC/MS). Two of the compounds, baicalin and wogonoside, are converted to baicalein and wogonin, respectively, after treatment with beta-glucuronidase. We observed a dose-dependent reduction in cell viability when cells with either wogonin or aqueous extract of S. baicalensis. Several of the apoptotic features including deoxyribonucleic acid (DNA) fragmentation and increased caspase-3 activity were found in cells treated with wogonin and aqueous extract. The changes were associated with down-regulation of Bcl-2, and not Bax. Furthermore, treatment of HL-60 cells with wogonin or S. baicalensis led to the inhibition of human telomerase reverse transcriptase (hTERT), human telomerase-associated protein 1 (hTP1) and c-myc messenger ribonucleic acid (m-RNA) expression. Wogonin and S. baicaleisis down-regulated the telomerase activity. Our findings suggest that wogonin may be the major compound in S. baicalensis responsible for HL-60 growth inhibition in vitro. The inhibition of HL-60 cell growth is mediated partly through the induction of Bax/Bcl-2 apoptosis and by telomerase inhibition through suppression of c-myc, which is a promoter of hTERT.

APX3330 inhibition of the redox function of ape-1/ref-1 (Ref-1) in promyelocytic leukemia cells enhances retinoic acid (ATRA) induced myeloid differentiation and limits cell proliferation as an approach to the prevention of the retinoic acid syndrome (RAS)

e14613 Background: ATRA + chemotherapy has improved the treatment of promyelocytic leukemia(APL). However, 25% of ATRA treated APL patients experience toxicities that comprise the RAS (life-threatening respiratory distress, edema, renal failure, hypotension, coagulopathy and rising blast count). One approach to prevent RAS is to limit blast proliferation and enhance myeloid differentiation. Ref-1 is a DNA repair protein that functions in redox regulation of cellular proteins, such as Fos, Jun, p53, and NFkB. HL60 myeloid leukemia cells are promyeloblasts that respond to ATRA with granulocytic differentiation/growth arrest. Prior studies suggest Ref-1 redox control is integral to ATRA-induced differentiation. To define the role of the redox function of Ref-1, we used the Ref-1 specific drug, APX3330, to block Ref-1 redox function and examined the response of HL60 cells to ATRA. Methods: Cell growth assessed using trypan blue. Differentiation was evaluated by morphology and expression of CD11b by flow cytometry. Apoptosis was assayed by annexin-PI staining on flow cytometry and cell cycle analysis assayed with propidium iodide flow cytometry. To assess activation of the MAPK pathway, BLR-1 expression was determined by real time PCR. Results: 1) APX3330 blockade of Ref-1 redox function resulted in limited cell growth yet a profound increase in differentiation and a moderate increase in apoptosis. 2) dose dependent studies with ATRA showed a similar degree of differentiation in cells treated with 10 μM ATRA to cells treated with APX3330 + 0.01 μM ATRA; allowing HL60 cells + APX3330 to give a similar response to a 1000 fold lower dose of ATRA. APX3330 alone did not induce differentiation and induced only minimal apoptosis but in combination with ATRA, increased the number of cells in G1/G0 phase significantly. 3) APX3330 + ATRA increased BLR-1 expression significantly by real time PCR suggesting enhanced activation of the MAPK pathway. Conclusions: APX3330 + ATRA limits HL60 growth and dramatically enhances terminal granulocytic differentiation. These finding may provide a therapeutic approach for prevention of the RAS. No significant financial relationships to disclose.

Copper(II) and uranyl(II) complexes with acylthiosemicarbazide: Synthesis, characterization, antibacterial activity and effects on the growth of promyelocytic leukemia cells HL-60

New chelates of N 1-[4-(4-X-phenylsulfonyl)benzoyl]- N 4-butyl-thiosemicarbazide (X = H, Cl, Br) with Cu 2+ and UO 2 2+ have been prepared and characterized by analytical and physico-chemical techniques such as magnetic susceptibility measurements, elemental and thermal analyses, electronic, ESR and IR spectral studies. Room temperature ESR spectra of Cu(II) complexes yield { g} values characteristic of distorted octahedral and pseudo-tetrahedral geometry. Infrared spectra indicate that complexes contain six-coordinate uranium atom with the ligand atoms arranged in an equatorial plane around the linear uranyl group. Effects of these complexes on the growth of human promyelocytic leukemia cells HL-60 and their antibacterial activity (against Staphylococcus epidermidis ATCC 14990, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 14579, Pseudomonas aeruginosa ATCC 9027 and Escherichia coli ATCC 11775 strains) were studied comparatively with that of free ligands.

A New Sesquiterpene from Ximenia americanaLinn.

Abstractmagnified imageThe new sesquiterpene 1, named ximonicane, and the known stigmastane steroid 2 were isolated from the EtOH extract of the stems of Ximenia americana. Spectroscopic methods, including 2D‐NMR techniques, have been used to characterize 1 and assign all the signals of the 1H‐ and 13C‐NMR spectra of 1. Ximonicane did not inhibit the growth of HL‐60 (human leukemia), HTC‐8 (human colon), and MDA‐MB‐435 (human breast cancer) cell lines.

Andrographolide inhibits growth of acute promyelocytic leukaemia cells by inducing retinoic acid receptor-independent cell differentiation and apoptosis

The growth inhibiting potential of andrographolide was evaluated in three acute promyelocytic leukaemia cell line models (HL-60, NB4 and all-trans retinoic acid (ATRA)-resistant NB4-R2). In elucidating the mechanisms of growth inhibition, a special emphasis was placed on assessing the induction of differentiation and apoptosis by andrographolide in the primary acute promyelocytic leukaemia NB4 cells. The compound was 2- and 3-fold more active in inhibiting the growth of HL-60 and NB4-R2 cells compared with NB4 cells, respectively. At IC50 (concentration at which growth of 50% of the cells (compared with medium only treated control cells) is inhibited; 4.5 microM) the compound exhibited strong cell-differentiating activity in NB4 cells, similar to ATRA (IC50 1.5 microM). In the presence of a pure retinoic acid receptor antagonist AGN193109, the growth inhibition of NB4 cells by ATRA was reversed, whereas the activity of andrographolide was not affected. This clearly suggested that andrographolide's cell differentiating activity to induce growth inhibition of NB4 cells most likely occurred via a retinoic acid receptor-independent pathway. At higher concentration (2xIC50), andrographolide was an efficient inducer of apoptosis in NB4 cells. Taken together, these results suggest andrographolide and its derivatives, apparently with a novel cell differentiating mechanism and with ability to induce apoptosis, might be beneficial in the treatment of primary and ATRA-resistant acute promyelocytic leukaemia.

Energy Metabolism of Leukemia Cells: Glycolysis Vs Oxidative Phosphorylation

Cancer cells are more dependent on glycolysis than oxidative phosphorylation in the mitochondria for generation of ATP as energy source. By using 2-deoxy-D-glucose (2-DG: glycolysis inhibitor) and oligomycin (inhibitor of oxidative phosphorylation), we examined the energy metabolism of various leukemia cell lines. The growth of the cell lines was measured by MTS assay, which detects viable cells in proliferation. 2-DG suppressed the growth of all leukemia cell lines examined in dose-dependent manners. The IC50 of each cell line was as follows: Kasumi-1 0.5±0.1mM, KG-1a 1.8±0.6mM, HL-60 3.3±0.1mM, NB4 3.8±0.4mM, and THP-1 23.1±3.8mM. The concentration of lactic acid (the final product of glycolytic pathway) in the culture supernatant was greatly reduced by the treatment with 0.2mM 2-DG for 24 hours in Kasumi-1 (54.5% of the control), compared with THP-1 (92.2%). It is suggested that the growth of Kasumi-1 was strongly suppressed by 2-DG through inhibition of glycolysis, which is supposed to be a main metabolic pathway in this cell line. On the other hand, treatment with oligomycin (1μg/ml) for 48 hours potently suppressed the growth of THP-1 (44.7%), then Kasumi-1 (72.1%). The growth of NB4, KG-1a and HL-60 was minimally suppressed (more than 90%) by oligomycin. Cell cycle was analyzed after 24 hours treatment with 2-DG or oligomycin. Sub-G1 fraction (apoptosis) was greatly increased by 2-DG (5mM) in Kasumi-1 (56.5%) and NB4 (30.6%), compared with THP-1 (7.6%). The apoptosis inducing effect was confirmed by annexinV staining. Oligomycin treatment (1μg/ml) increased apoptosis (subG1) in THP-1 (35.8%), then Kasumi-1 (16.6%) and NB4 (12.2%). Oligomycin treatment also increased G1 population (G1 arrest) in THP-1 (35.9% to 69.4%). AMP-activated protein kinase (AMPK) is activated by an elevated AMP/ATP ratio, which means the energy-deprived status of the cell. Western blot analysis using phospho-AMPK α (Thr172) antibody revealed that treatment with 2-DG or oligomycin induced prompt (30 min) phosphorylation of AMPK in leukemia cell lines. The extent of AMPK phosphorylation was almost proportional to the suppression of the growth. Collectively, it is suggested that leukemia cells are dependent almost exclusively on either glycolysis or oxidative phosphorylation in the mitochondria for energy production. Then, inhibition of glycolysis by 2-DG or oxidative phosphorylation by oligomycin results in growth suppression by inducing apoptosis and/or cell cycle arrest through activation of AMPK. Our data clarified the characteristics of the energy metabolism of each leukemia cell, and showed the key to produce novel therapeutic approach targeting metabolic pathway.

Novel Gemini vitamin D 3 analogs have potent antitumor activity

The active form of vitamin D 3, 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3], modulates proliferation and induces differentiation of many cancer cells. A new class of analogs of vitamin D 3 has been synthesized, having two side-chains attached to carbon-20 (Gemini) and deuterium substituted on one side-chain. We have examined six of these analogs for their ability to inhibit growth of myeloid leukemia (HL-60), prostate (LNCaP, PC-3, DU145), lung (H520), colon (HT-29), and breast (MCF-7) cancer cell lines. Dose–response clonogenic studies showed that all six analogs had greater antiproliferative activities against cancer cells than 1,25(OH) 2D 3. Although they had similar potency, the most active of these analogs was BXL-01-0120. BXL-01-0120 was 529-fold more potent than 1,25(OH) 2D 3 in causing 50% clonal growth inhibition (ED 50) of HL-60 cells. Pulse-exposure studies demonstrated that exposure to BXL-01-120 (10 −9 M, 48 h) resulted in 85% clonal inhibition of HL-60 growth. BXL-01-0120 (10 −11 M, 4 days) induced the differentiation marker, CD11b. Also, morphologically differentiation was more prominent compared to 1,25(OH) 2D 3. Annexin V assay showed that BXL-01-0120 (10 −10 M, 4 days) induced significantly ( p < 0.05) more apoptosis than 1,25(OH) 2D 3. In summary, these analogs have a unique structure resulting in extremely potent inhibition of clonal proliferation of various types of cancer cells, especially HL-60 cells.

Identification of 6-Benzylthioinosine as a Myeloid Leukemia Differentiation–Inducing Compound

As the pathophysiology of acute myelogenous leukemia (AML) involves a block of myeloid maturation, a desirable therapeutic strategy is to induce leukemic cell maturation to increase the efficacy and to avoid the side effects of traditional chemotherapeutics. Through a compound library screen, 6-benzylthioinosine (6BT) was identified as a promising differentiation-inducing agent. 6BT induces monocytic differentiation of myeloid leukemia cell lines such as HL-60 and OCI-AML3, as well as primary patient samples as evidenced by morphology, immunophenotyping, and nitroblue tetrazolium reduction. Not only can 6BT induce differentiation but a subset of AML cell lines such as MV4-11 and HNT34 instead undergo 6BT-mediated cell death. Despite inducing cell death in some leukemic cells, 6BT exhibits extremely low toxicity on several nonmalignant cells such as fibroblasts, normal bone marrow, and endothelial cells. This toxicity profile may relate to the function of 6BT as an inhibitor of the nucleoside transporter, ent1, which is thought to prevent it from entering many cell types. In contrast, 6BT likely enters at least some leukemic cell lines as shown by its requirement for phosphorylation for its differentiation activity. 6BT is also able to synergize with currently used myeloid differentiation agents such as ATRA and decitabine. Early studies indicate that the mechanism of action of this compound may involve ATP depletion that leads to growth inhibition and subsequent differentiation. Besides in vitro activity, 6BT also shows the ability to impair HL-60 and MV4-11 tumor growth in nude mice. 6BT is a promising new monocytic differentiation agent with apparent leukemic cell-specific activity.

Chemical Constituents of Ximenia Americana

Two steroids (1 and 2), five pentacyclic triperpenoids (3–7) and a new furanosesquiterpene (8) were isolated from the stems of Ximenia americana, family Olacaceae. The structures of the compounds were established by spectral data; two-dimensional NMR spectroscopy has been used to assign all 1H and 13C chemical shifts of 8. Compound 8 did not inhibit the growth of HL-60 (human leukemia), HTC-8 (human colon), and MDA-MB-435 (human breast cancer) cell lines.

Mechanisms of Antileukemic Activity of the Novel Bcl-2 Homology Domain-3 Mimetic GX15-070 (Obatoclax)

In this study, we investigated the mechanism of apoptosis induction of obatoclax (GX15-070), a novel Bcl-2 homology domain-3 (BH3) mimetic, in acute myeloid leukemia (AML) cell lines and primary AML samples. Obatoclax inhibited cell growth of HL-60, U937, OCI-AML3, and KG-1 cell lines. Apoptosis induction contributed to the observed antiproliferative effects at concentrations of this agent that mirror its affinity for antiapoptotic Bcl-2 proteins. We show that obatoclax can promote the release of cytochrome c from isolated leukemia cell mitochondria and that apoptosis induced by this agent is preceded by the release of Bak from Mcl-1, liberation of Bim from both Bcl-2 and Mcl-1, and the formation of an active Bak/Bax complex. Notably, apoptosis was diminished, but not fully prevented, in the absence of Bak/Bax or Bim, suggesting that obatoclax has additional targets that contribute to its cytotoxicity. At growth inhibitory doses that did not induce apoptosis or decrease viability, obatoclax induced an S-G(2) cell-cycle block. Obatoclax induced apoptosis in AML CD34+ progenitor cells with an average IC(50) of 3.59 +/- 1.23 micromol/L although clonogenicity was inhibited at concentrations of 75 to 100 nmol/L. Obatoclax synergized with the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells and synergistically induced apoptosis in combination with AraC in leukemic cell lines and in primary AML samples. In conclusion, we show that obatoclax potently induces apoptosis and decreases leukemia cell proliferation and may be used in a novel therapeutic strategy for AML alone and in combination with other targeted agents and chemotherapeutics.

Novel Deuterated Gemini-Vitamin D3 Analogs Have Potent Antitumor Activity.

The active form of vitamin D3, 1,25(OH)2D3, is an important regulator of calcium and bone metabolism. Also, it inhibits proliferation and induces differentiation of a variety of malignant cells. Here, we synthesized a new class of vitamin D3 analogs, which have a C-20 methyl group and a deuterium substituted basic side chain [Deuterated Gemini (DG)]. Six DG analogs were evaluated for their ability to inhibit growth of myeloid leukemia (HL-60), prostate (LNCaP and PC-3), lung (H520) and breast (MCF-7) cancer cell lines. All 6 DG analogs inhibited growth in a dose-dependent manner and had very similar potency. Most effective DG, BXL-01-0120, was over 70-fold more potent than 1,25(OH)2D3 in inhibiting 50% clonal growth (ED50) of HL-60, LNCaP, PC-3, H520 and MCF-7 cells with ED50s ranging from 10−11 M to 10−13 M. Focusing on this analog for further analyses, pulse-exposure, wash and culture experiments showed that 48 hours exposure of HL-60 cells to BXL-01-0120 (10−9 M) had potent anti-proliferative activity (82% growth inhibition compared to diluent control cells). Cell cycle assays found that BXL-01-0120 (10−10 M, 4 days) increased the percent of cells in the G0/G1-phase (61%, diluent control 47%) associated with a decrease S-phase (18%, diluent control 42%). BXL-01-0120 (10−10 M, 4 days) caused apoptosis in approximately 15% of cells. 1,25(OH)2D3 at the same concentration and duration changed neither the cell cycle nor number of cells undergoing apoptosis compared to control HL-60 cells. BXL-01-0120 (10−10 M, 4 days) strongly induced expression of the CD11b and CD14 (monocyte / macrophage differentiation markers) on HL-60 cells (87%); in contrast, 1,25(OH)2D3(10−10 M) stimulated a mean 43% of the cells to express these cell surface proteins. In summary, DGs strongly inhibited clonal proliferation, induced differentiation and cell cycle arrest in several types of cancer cells, especially HL-60 cells, suggesting that further preclinical and clinical studies to explore their anticancer potential are warranted.