Cell Line CCRF-CEM Research Articles

Abstract 2073: Changes in microRNA expression in response to short-term chemotherapeutic exposure is associated with transient anticancer drug resistance

Abstract MicroRNAs (miRNAs) are small, noncoding RNAs that act as posttranscriptional repressors of gene expression by binding the 3’ untranslated region of target genes. There is emerging evidence showing that miRNAs regulate diverse biologic processes including multidrug resistance (MDR). Our previous profiling study revealed that certain miRNAs are differentially expressed in multidrug resistant human T-cell leukemia cell line CEM/VM-1-5 compared to drug-sensitive parental cell line CCRF-CEM. To investigate whether these expression-altered miRNAs have a role in MDR, we ectopically expressed three miRNAs that are highly upregulated in CEM/VM-1-5 cells (hsa-miR-135b, −146b, and −196b) in CCRF-CEM cells with the use of lentiviral vectors. We found that overexpression of these miRNAs, individually or in combination, resulted in only slight increases in resistance of CCRF-CEM cells to teniposide, the agent used to select CEM/VM-1-5 cells. Moreover, we were unable to sensitize CEM/VM-1-5 cells to etoposide by suppression of hsa-miR-135b expression using LNA antisense oligonucleotide. Our data implied that these miRNAs do not confer drug resistance directly. However, it is still not clear whether these miRNAs play any role in the development of MDR. Therefore, we asked whether the expression of these miRNAs changed in CCRF-CEM cells after short exposure (3 to 48 hours) to various chemotherapeutic drugs at concentrations below their IC50-values. We observed substantial increases in hsa-miR-135b and −196b expression in cells treated with etoposide, doxorubicin and topotecan, but not in cells treated with vinblastine. These results suggested that the upregulation of these two miRNAs might be the consequence of DNA damage. Time-course and dose-response studies showed that the elevation of hsa-miR-135b and −196b correlated positively with the drug exposure time and drug concentration. Furthermore, the levels of miRNAs gradually decreased after the withdrawal of etoposide from the media following 48 h exposure to etoposide. Strikingly, the elevated expression of the miRNAs accompanied increased drug resistance. CCRF-CEM cells exposed to etoposide for 48 h were over 50-fold more resistant than control cells. The acquired drug resistance, like elevated expression of miRNAs, did not appear to be stable and became attenuated with increasing duration of the drug-free incubation. Together, our results suggest that miRNAs might play a significant role in cellular response to genotoxic agents and could be involved in the development of cancer drug resistance, but their roles in conferring resistance remain to be determined. That said, targeting miRNA expression could be a potential approach to prevent multidrug resistance. (Supported in part by 5RO1CA040570 from NCI to WTB, and in part by UIC) 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 2073.

Identification of potential HLA class I and class II epitope precursors associated with heat shock protein 70 (HSPA)

Heat shock protein 70 (HSPA) is a molecular chaperone which has been suggested to shuttle human leukocyte antigen (HLA) epitope precursors from the proteasome to the transporter associated with antigen processing. Despite the reported observations that peptides chaperoned by HSPA are an effective source of antigens for cross-priming, little is known about the peptides involved in the process. In this study, we investigated the possible involvement of HSPA in HLA class I or class II antigen presentation and analysed the antigenic potential of the associated peptides. HSPA was purified from CCRF-CEM and K562 cell lines, and using mass spectrometry techniques, we identified 44 different peptides which were co-purified with HSPA. The affinity of the identified peptides to two HSPA isoforms, HSPA1A and HSPA8, was confirmed using a peptide array. Four of the HSPA-associated peptides were matched with 13 previously reported HLA epitopes. Of these 13 peptides, nine were HLA class I and four were HLA class II epitopes. These results demonstrate the association of HSPA with HLA class I and class II epitopes, therefore providing further evidence for the involvement of HSPA in the antigen presentation process.

Platinum and Palladium‐triazole Complexes as Highly Potential Antitumor Agents

The palladium complexes [(dppe)Pd(L)(2)PdCl(2)], [(dppe)Pd(L)(2)PtCl(2)], [(dppp)Pd(L)(2)PdCl(2)], [(dppm) Pd(L)(2)NiCl(2)], and [(dppm)Pd(L)(2)SnCl(4)] 15-19 were prepared. The antiproliferative activity of the newly synthesized complexes as well as their previously prepared analogues 3-14 and 20-26 were screened against a large panel of human cancer cell lines derived from haematological CD4(+) human T-cells containing an integrated HTLV-1 genome (MT-4). The complex 12a, b exhibited remarkable antiproliferative activity against MT-4, CD4(+) human acute T-lymphoblastic leukemia (CCRF-CEM), human splenic B-lymphoblastoid cells (WIL-2NS), human acute B-lymphoblastic leukemia (CCRF-SB), skin melanoma (SK-MEL-28), and prostate carcinoma (DU145) cell lines (CC(50 )= 0.5 microM, 0.4 +/- 0.05 microM, 0.6 +/- 0.05 microM, 0.4 +/- 0.1 microM, and 0.8 +/- 0.2 microM, respectively), meanwhile, 9a, b, 14a, b, and 23 showed significant activity against the CCRF-SB cell lines (CC(50) = 0.6 +/- 0.06 microM, 0.7 +/- 0.05 microM, 0.6 +/- 0.05 microM, and 0.8 +/- 0.15 microM, respectively). Further, 19 exhibited activity against the CCRF-CEM cell line (CC(50 )= 0.4 +/- 0.05 microM).

Chemical composition and biological activity of the essential oil obtained from Bupleurum marginatum (Apiaceae)

Abstract Objectives Bupleurum marginatum is a herb indigenous to the southern and southwestern part of China. It is widely used in many Chinese prescriptions. We aimed to investigate the chemical composition, antioxidant, anti-inflammatory, antimicrobial and in-vitro cytotoxic activity of the hydrodistilled and extracted essential oil from B. marginatum to validate some of its ethnopharmacologial uses. Methods The essential oil of the aerial parts of B. marginatum was analysed by capillary gas chromatography (GLC/FID) and gas chromatography-mass spectrometry (GLC/MS). The ability of the oil to reduce diphenylpicrylhydrazine (DPPH•) and to prevent the degradation of deoxyribose were used to evaluate the antioxidant activity. Inhibition of both prostaglandin E2 production and lipoxygenase were used to assess the anti-inflammatory activity. Antimicrobial activity was studied in vitro against a range of bacteria and fungi. The in-vitro cytotoxicity of the essential oil on six human cancer cell lines (HepG2, Caco-2, CCRF-CEM, HeLa, MiaPaCa-2 and MCF-7) was examined using the MTT assay. Key findings Seventy-two components, comprising almost 94.29% of the total peak area, were identified in the analysis. The main components were tridecane (13.18%), undecane (10.42%), pentadecane (8.71%), β-caryophyllene (5.53%) and β-caryophyllene oxide (5.29%). The ability of the oil to reduce diphenylpicrylhydrazine (DPPH•) and to prevent the degradation of deoxyribose were used to evaluate the antioxidant activity and the corresponding IC50 values (drug concentration which resulted in a 50% reduction in inhibition of the activity) were found to be 3.66 mg/ml and 17.4 μg/ml, respectively. Inhibition of both prostaglandin E2 production and lipoxygenase were used to assess the anti-inflammatory activity (IC50 of 63.64 μg/ml for lipoxygenase, 26.04% inhibition of prostaglandin E2 at 25 μg/ml dose). The oil also showed a significant in-vitro antimicrobial activity against Gram positive pathogens (Streptococcus pyogenes and Streptococcus agalactiae) with minimum inhibitory concentration (MIC) values ranging from 0.125 up to 4.00 mg/ml. The in-vitro cytotoxicity of the essential oil on six human cancer cell lines (HepG2, Caco-2, CCRF-CEM, HeLa, MiaPaCa-2 and MCF-7) examined using the MTT assay revealed the highest activity to be in the CCRF-CEM cell line with an IC50 (concentration which resulted in a 50% reduction in cell viability) of 46.01 μg/ml after 24 h treatment. Conclusions The essential oil of B. marginatum exhibited a promising anti-inflammatory activity along with strong cytotoxicity against many cancer cells (CCRF-CEM and HepG2) mediated through induction of apoptosis, and this in-vitro activity make its local traditional uses rational. However, its limited antimicrobial activity indicates that a combination with other drugs is essential for effective use. Further selectivity testing is required to evaluate the effect of the oil against normal cells.

Glyoxyl analogs of indole phytoalexins: Synthesis and anticancer activity

Glyoxyl analogs of indole phytoalexins brassinin, 1-methoxybrassinin, brassitin, 1-methoxybrassitin and 1-methoxybrassenin B were prepared, using (1H-indol-3-yl)-, (1-methoxyindol-3-yl)- and [1-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)indol-3-yl]glyoxyl chlorides as starting compounds. Synthesized products were examined for their antiproliferative activity against human cancer cell lines Jurkat (T-cell acute lymphoblastic leukemia), MCF-7 (breast adenocarcinoma, estrogen receptor-positive), MDA-MB-231 (breast adenocarcinoma, estrogen receptor-negative), HeLa (cervical adenocarcinoma), CCRF-CEM cell line (T-cell acute lymphoblastic leukemia) and A-549 cell line (lung adenocarcinoma), and their activity compared with natural phytoalexins and corresponding (1H-indol-3-yl)acetic acid derivatives. The highest potency with IC50 3.3–66.1 μmol l–1 was found for glyoxyl analogs of 1-methoxybrassenin B.

A new high-performance liquid chromatography method determines low production of 9-β-D-arabinofuranosylguanine triphosphate, an active metabolite of nelarabine, in adult T-cell leukemia cells

The 9-beta-D-arabinofuranosylguanine (ara-G), an active compound of nelarabine, demonstrates potent cytotoxicity specifically on T-cell malignancies. In cells, ara-G is phosphorylated to ara-G triphosphate (ara-GTP), which is subsequently incorporated into DNA, thereby inhibiting DNA synthesis. Because ara-GTP is crucial to ara-G's cytotoxicity, the determination of ara-GTP production in cancer cells is informative for optimizing nelarabine administration. Here, we developed a new, sensitive isocratic-elution HPLC method for quantifying ara-GTP. Samples were eluted isocratically by using phosphate buffer at a constant flow rate. Ara-GTP was clearly separated from other nucleotides by using an anion-exchange column and it was quantitated by its peak area at 254 nm. The standard curve was linear with low variability and a sensitive detection limit (10 pmol). Furthermore, due to ara-G's specificity to T-cells we hypothesized that nelarabine might be effective against adult T-cell leukemia (ATL). The ara-GTP production was compared between T-lymphoblastic leukemia CCRF-CEM and ATL cell lines in vitro. When CEM cells were incubated with ara-G, the ara-GTP production increased in a concentration- and time-dependent manner. In contrast, 5 ATL cell lines accumulated lower ara-GTP in the same condition. While ara-G inhibited the growth of CEM cells with a 50% growth inhibition concentration of 2 microM, the inhibitory-concentration values were >1 mM in 8 of the 12 ATL cell lines. This ineffectiveness appeared to correspond with the low ara-GTP production. The present study is the first to evaluate the potential of ara-G against ATL cells; our results suggest that nelarabine would not be effective against ATL.

Abstract C222: Voreloxin is synergistic in in vitro combination with cytarabine and additive in combination with azacitidine, decitabine, and clofarabine

Abstract Voreloxin is a first-in-class anticancer quinolone derivative that intercalates DNA and poisons topoisomerase II (Stockett et al. and Hawtin et al., AACR 2008). This leads to replication-dependent, site-selective DNA double strand breaks (DSB) targeting G/C-rich sequences that are characteristic sites of quinolone-induced DNA cleavage (Noble et al., 2003; Richter et al., 2007; Stockett et al., AACR 2008). A consequence of this DNA damage is G2 arrest, and cell death by apoptosis. Voreloxin is under clinical investigation in acute myeloid leukemia (AML) and ovarian cancer. Clinical responses have been observed in these indications (Lancet et al., ASCO 2009; Maris et al., ASCO 2009; McGuire et al., SGO 2008), as well as in NSCLC and SCLC (Burris et al., ECCO 2007). The current analysis was performed in support of a Phase 1b/2 clinical study (SPO-0012) of voreloxin in combination with cytarabine in relapsed or refractory AML, and to investigate the feasibility for combining voreloxin with other agents currently in clinical use for the treatment of AML. The cytotoxicity of of voreloxin in combination with cytarabine was evaluated in 3 acute leukemic cell lines (HL-60 acute promyelocytic (APL), MV4-11 Flt-3 ITD positive AML and CCRF-CEM acute lymphoblastic leukemia (ALL)). The cytotoxicity of voreloxin in combination with the nucleoside analog hypomethylating agents azacitidine, decitabine (pyrimidine analogs), and clofarabine (purine analog) was also studied in the HL-60 APL cell line. Cytotoxicity was assessed by proliferation inhibition. The combined activity of voreloxin and cytarabine was evaluated using the combination index (CI) analysis method, in which each drug is serially diluted based on either 10X or 1X single agent IC50 values. The combination was synergistic in the AML cell lines MV4-11 and HL-60, as demonstrated by a leftward shift in the voreloxin IC50 curves and calculated CI less than 0.85. In the ALL cell line CCRF-CEM, an additive increase in cytotoxicity was observed with CI of ≤ 0.99. Voreloxin in combination with azacitidine, decitabine or clofarabine was evaluated after simultaneous or sequential addition of the drugs. The combinations were analyzed by: 1) serial-dilution of voreloxin combined with a fixed dose (IC10) of the second compound, and 2) fixed dose (IC10) of voreloxin combined with a serial dilution of the second compound. No significant change in activity was observed when compared with the single-agent activity of each compound. Sequential dosing of the agents did not alter the cytotoxicity of the reagents in combination. These data support the ongoing phase 1b/2 clinical study of voreloxin in combination with cytarabine in relapsed or refractory AML and demonstrate the feasibility for combining voreloxin with other DNA damaging agents currently in clinical use for the treatment of AML. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C222.

Chemotherapeutic Drug-Induced ABCG2 Promoter Demethylation as a Novel Mechanism of Acquired Multidrug Resistance

ABCG2 is an efflux transporter conferring multidrug resistance (MDR) on cancer cells. However, the initial molecular events leading to its up-regulation in MDR tumor cells are poorly understood. Herein, we explored the impact of drug treatment on the methylation status of the ABCG2 promoter and consequent reactivation of ABCG2 gene expression in parental tumor cell lines and their MDR sublines. We demonstrate that ABCG2 promoter methylation is common in T-cell acute lymphoblastic leukemia (T-ALL) lines, also present in primary T-ALL lymphoblast specimens. Furthermore, drug selection with sulfasalazine and topotecan induced a complete demethylation of the ABCG2 promoter in the T-ALL and ovarian carcinoma model cell lines CCRF-CEM and IGROV1, respectively. This resulted in a dramatic induction of ABCG2 messenger RNA levels (235- and 743-fold, respectively) and consequent acquisition of an ABCG2-dependent MDR phenotype. Quantitative genomic polymerase chain reaction and ABCG2 promoter-luciferase reporter assay did not reveal ABCG2 gene amplification or differential transcriptional trans-activation, which could account for ABCG2 up-regulation in these MDR cells. Remarkably, mimicking cytotoxic bolus drug treatment through 12- to 24-hour pulse exposure of ABCG2-silenced leukemia cells, to clinically relevant concentrations of the chemotherapeutic agents daunorubicin and mitoxantrone, resulted in a marked transcriptional up-regulation of ABCG2. Our findings establish that antitumor drug-induced epigenetic reactivation of ABCG2 gene expression in cancer cells is an early molecular event leading to MDR. These findings have important implications for the emergence, clonal selection, and expansion of malignant cells with the MDR phenotype during chemotherapy.

Chemical composition and biological activity of the essential oil obtained from <I>Bupleurum marginatum</I> (Apiaceae)

Bupleurum marginatum is a herb indigenous to the southern and southwestern part of China. It is widely used in many Chinese prescriptions. We aimed to investigate the chemical composition, antioxidant, anti-inflammatory, antimicrobial and in-vitro cytotoxic activity of the hydrodistilled and extracted essential oil from B. marginatum to validate some of its ethnopharmacologial uses. The essential oil of the aerial parts of B. marginatum was analysed by capillary gas chromatography (GLC/FID) and gas chromatography-mass spectrometry (GLC/MS). The ability of the oil to reduce diphenylpicrylhydrazine (DPPH(.)) and to prevent the degradation of deoxyribose were used to evaluate the antioxidant activity. Inhibition of both prostaglandin E(2) production and lipoxygenase were used to assess the anti-inflammatory activity. Antimicrobial activity was studied in vitro against a range of bacteria and fungi. The in-vitro cytotoxicity of the essential oil on six human cancer cell lines (HepG2, Caco-2, CCRF-CEM, HeLa, MiaPaCa-2 and MCF-7) was examined using the MTT assay. Seventy-two components, comprising almost 94.29% of the total peak area, were identified in the analysis. The main components were tridecane (13.18%), undecane (10.42%), pentadecane (8.71%), beta-caryophyllene (5.53%) and beta-caryophyllene oxide (5.29%). The ability of the oil to reduce diphenylpicrylhydrazine (DPPH(.)) and to prevent the degradation of deoxyribose were used to evaluate the antioxidant activity and the corresponding IC50 values (drug concentration which resulted in a 50% reduction in inhibition of the activity) were found to be 3.66 mg/ml and 17.4 microg/ml, respectively. Inhibition of both prostaglandin E(2) production and lipoxygenase were used to assess the anti-inflammatory activity (IC50 of 63.64 microg/ml for lipoxygenase, 26.04% inhibition of prostaglandin E(2) at 25 microg/ml dose). The oil also showed a significant in-vitro antimicrobial activity against Gram positive pathogens (Streptococcus pyogenes and Streptococcus agalactiae) with minimum inhibitory concentration (MIC) values ranging from 0.125 up to 4.00 mg/ml. The in-vitro cytotoxicity of the essential oil on six human cancer cell lines (HepG2, Caco-2, CCRF-CEM, HeLa, MiaPaCa-2 and MCF-7) examined using the MTT assay revealed the highest activity to be in the CCRF-CEM cell line with an IC50 (concentration which resulted in a 50% reduction in cell viability) of 46.01 microg/ml after 24 h treatment. The essential oil of B. marginatum exhibited a promising anti-inflammatory activity along with strong cytotoxicity against many cancer cells (CCRF-CEM and HepG2) mediated through induction of apoptosis, and this in-vitro activity make its local traditional uses rational. However, its limited antimicrobial activity indicates that a combination with other drugs is essential for effective use. Further selectivity testing is required to evaluate the effect of the oil against normal cells.

Structure and Function of the Nicotinic Arm of Acetylcholine Regulatory Axis in Human Leukemic T Cells

Although acetylcholine (ACh) is widely known as a neurotransmitter, it also functions as a local humoral factor translating environmental stimuli into alterations in T cell development and function. The cholinergic components present in neurons are expressed in T cells where they constitute an independent cholinergic system. Both non-immunologic and immunologic stimulations can alter expression and function of cholinergic elements in T cells. Recent studies have convincingly demonstrated regulation of immune system by auto/paracrine ACh, which provides a basis for development of new immunomodulatory therapies with nicotinic agonists. The purpose of our research is to integrate information about the structure and activity of the ACh regulatory axis with the phenotypic and functional alterations of T cells during their development and commitment. In this study, we used the Ach producing human leukemic T cell line CCRF-CEM (CEM) to investigate auto/paracrine mechanisms of T cell regulation through the nicotinic class of ACh receptors (nAChRs). The intact CEM expressed alpha3, alpha5, alpha6, alpha7, alpha 9, beta2 and beta4 nAChR subunits. Stimulation of CEM with 10 microg/ml of phytohemagglutinin (PHA) for 16 h upregulated expression of the alpha3, alpha5, alpha7, alpha9 and beta2 and downregulated that of alpha6 and beta4 subunits, indicating that TCR activation leads to overexpression of high Ca2+-permeable ACh-gated ion channels. Activation of alpha7- and alpha3 AChRs predominantly abrogated PHA-dependent upregulation of the pro-inflammatory cytokine TNF-alpha and IFN-gamma receptors, respectively, at the mRNA and protein levels. Signaling through alpha7 and alpha3 nAChRs also significantly (p<0.05) altered expression of the cell state regulators p21 and Bcl-2, respectively, suggesting that downregulation of inflammation via nAChRs includes effects on the T cell cycle progression and apoptosis. These findings indicate that constant stimulation of alpha7 and alpha3 nAChRs by endogenously released ACh controls T cell activation and that signaling downstream of distinct nAChR subtypes targets specific inflammatory and cell cycle genes. Learning the cholinergic pharmacology of inflammation should allow to regulate specific types of immune reactions by selectively activating or blocking the types of nAChRs expressed by the immune cells mediating specific immune reactions.

Cytotoxic Activity of Isoliquiritigenin towards CCRF‐CEM Leukemia Cells and its Effect on DNA Damage

The cytotoxic activity of isoliquiritigenin (ISL) towards CCRF-CEM human T-cell leukemia cells and DNA damage induction were investigated in the present study. The anti-proliferative effect of ISL on CCRF-CEM cell line IN VITRO was analyzed. In order to further explore the underlying mechanism of cell growth inhibition of ISL towards CCRF-CEM cell line, the cell cycle distribution and mitochondrial membrane potential (DeltaPsim) disruption were measured. ISL exerted significant activity towards CCRF-CEM cells. The growth inhibitory ratio was concentration- and time-dependent with an IC(50) value of 18.38 microM. The cell cycle was arrested in the G2/M phase upon treatment with low doses of ISL. Mitochondrial membrane potential (DeltaPsim) disruption was observed at low ISL doses. The effect of ISL on DNA damage was analyzed by agarose gel electrophoresis and atomic force microscopy (AFM). Either ISL or Cu2+ failed to cause pBR322 DNA damage. However, plasmid DNA was damaged after treatment with ISL in the presence of Cu2+. Two forms of plasmid DNA closed circular and linear forms were visualized by AFM.

A Short Entry to α-Substituted γ-Alkylidene Pentenolides. Synthesis and Preliminary Biological Evaluation of Novel Gelastatin Analogues

Biologically interesting 2-substituted 4-alkylidene pentenolides were prepared with complete control of regio- and stereoselectivity from 2-iodo allylic alcohols via an array of Pd-catalyzed processes, including alkynylation with methyl propiolate, tributyltin hydride addition, and alpha-functionalization. Some of the compounds possess selective cytostatic activity against ovarian carcinoma HeLa S3 and leukemia CCRF-CEM cell lines.

Acquisition of Mutations in the PSMB5 Gene during Chronic Exposure of Human Acute Lymphoblastic Leukemia CCRF-CEM Cells to the Proteasome Inhibitor Bortezomib

Despite the good prognosis of children suffering from acute lymphoblastic leukemia (ALL), approximately 25% of the patients develop a relapse. The proteasome inhibitor bortezomib (BTZ) is a representative of a novel generation of therapeutic drugs that revealed promising activity in patients with multiple myeloma (MM) and certain types of lymphomas, and is being studied in the treatment of relapsed ALL. Whereas most research on the mechanism of action of BTZ has focused on MM or lymphoma, the present study was designed to examine the activity of BTZ in an ALL model and in particular to assess the propensity to develop resistance to BTZ after prolonged drug exposure. Previously, we have shown (Franke et al., ASH 2007) that BTZ-resistant subclones of the human T-ALL cell line CCRF-CEM could emerge relatively rapid (within 2–3 months) after chronic exposure to stepwise increasing concentrations (0.1–200 nM) of BTZ. These BTZ-resistant cells (CEM/BTZ) showed cross-resistance against other proteasome inhibitors while remaining completely sensitive to a broad spectrum of other anticancer drugs. Subsequent analysis of these BTZ-resistant subclones showed an upregulation of the catalytic proteasomal β-subunits at both mRNA and protein level. In the present study we further elucidated the molecular basis for BTZ-resistance in a panel of CEM/BTZ cells selected at 7 nM BTZ (CEM/BTZ7) up to 200 nM BTZ (CEM/BTZ200). Since the proteasome β5 subunit is the primary target of BTZ, we screened whether mutations in the PSMB5 gene could be involved in conferring resistance to BTZ. Indeed, sequencing of exon 2 of the PSMB5 gene for CEM/BTZ7 cells revealed a single G to T nucleotide shift at position 332 of this gene. In the mature and functional β5 proteasome subunit, this mutation introduces a Cys to Phe substitution at amino acid 52, residing in a highly conserved substrate/inhibitor binding domain of the β5 subunit. In this context, it was of interest to note that CEM/BTZ7 cells already displayed a marked level (28-fold) of cross-resistance to 4A6, a 6-mer peptide that specifically targets the proteasome β5 catalytic subunit, thus suggesting that this mutation is responsible for diminished inhibitor binding. Upon screening for mutations in CEM cells with higher BTZ resistance levels, an additional mutation in the PSMB5 gene was revealed in CEM/BTZ30 cells. Herein, an additional C to T nucleotide shift at position 323 of the PSMB5 gene was identified, introducing an Ala to Val amino acid substitution at amino acid 49 of the mature and functional β5 subunit protein. This amino acid also resides in a highly conserved substrate/inhibitor binding domain of the β5 subunit. Both Cys52Phe and Ala49Val mutations were retained, and no others revealed, in BTZ-resistant CEM cells up to CEM/BTZ200 cells. Of note, recently we have identified an Ala49Thr mutation in the BTZ binding domain of the proteasome β5 subunit in a BTZ-resistant subclone of the human acute myeloid leukemic cell line THP-1 (Oerlemans & Franke, Blood 2008). Moreover, an Ala49Thr mutation was also recently described in a BTZ-resistant Jurkat cell line (Lü et al., J Pharmacol Exp Ther 2008). Together, this study demonstrates that mutations in the PSMB5 gene are provoked relatively fast in ALL cells, already at low levels of BTZ resistance. Furthermore, given the notion that independent studies in 3 different leukemia cell line models demonstrated that BTZ-resistance is associated with a mutation introducing amino acid substitutions at position Ala49 in the highly conserved substrate/inhibitor binding domain of the β5 subunit, this position may be considered as a hot spot for mutations in the PSMB5 gene through which acquired resistance to BTZ may be facilitated. Analysis of mutational status of the PSMB5 gene in future clinical BTZ studies in ALL patients should further confirm the clinical relevance of our finding, and might be exploited in the development of next generation proteasome inhibitors, designed to overcome resistance.

Synthesis of [1,2,4]triazolo[1,5- a]pyridines of potential PGE 2 inhibitory properties

A variety of 5-amino-6,8-dicyano-1 H-[1,2,4]triazolo[1,5- a]pyridin-4-ium-2-thiolate containing compounds 3a– i, 5a– c were prepared via reaction of arylidenemalononitriles 1a– c, 4a and 4b with 2-[(substituted amino)thiocarbonyl]cyanoacetohydrazides 2a– d in refluxing ethanol in the presence of triethylamine. Anti-inflammatory activity screening of the synthesized compounds (at a dose of 50 mg/kg body weight) utilizing in vivo acute carrageenan-induced paw oedema standard method in rats exhibited that the prepared heterocycles possess considerable pharmacological properties especially, 3f, 3h, 5b and 5c which reveal remarkable activities relative to indomethacin (which was used as a reference standard at a dose of 10 mg/kg body weight). PGE 2 inhibitory properties of the highly promising synthesized anti-inflammatory active agents ( 3f, 3h, 5b and 5c) were determined by PGE 2 assay kit technique, which reveal remarkable activity coinciding greatly with the observed anti-inflammatory properties. Anti-tumor activity screening of 3b and 3e, as representative examples of the synthesized compounds, at a dose of 10 μM utilizing 59 different human tumor cell lines, representing leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate and kidney exhibited that the tested compounds reflect mild or no activity at all against most of the used human tumor cell lines. However, compound 3e reveals considerable anti-tumor properties against leukemia CCRF-CEM and HL-60(TB) cell line.

Synthesis and potent antileukemic activities of 10-benzyl-9(10H)-acridinones

A novel series of 10-benzyl-9(10H)-acridinones and 1-benzyl-4-piperidones were synthesized and tested for their in vitro antitumor activities against CCRF-CEM cells. Assay-based antiproliferative activity study using CCRF-CEM cell lines revealed that the acridone group and the substitution pattern on the benzene unit had significant effect on cytotoxicity of this series of compounds, among which 10-(3,5-dimethoxy)benzyl-9(10H)-acridinone ( 3b) was found to be the most active compound with IC 50 at about 0.7 μM. Compound 3b was also found to have antiproliferative activity against two other human leukemic cell lines K562 and HL60 using the MTT assay. The antitumor effect of 3b is believed to be due to the induction of apoptosis, which is further confirmed by PI (Propidium iodide) staining and Annexin V-FITC/PI staining assay using flow cytometry analysis.

Carmustine-Resistant Cancer Cells Are Sensitized to Temozolomide As A Result of Enhanced Mismatch Repair during the Development of Carmustine Resistance

The cytotoxicity of the monofunctional alkylator temozolomide (TMZ) is mediated by mismatch repair (MMR) triggered by O(6)-alkylguanine, whereas MMR protects cells against bifunctional alkylators, including carmustine (BCNU). Therefore, TMZ may be cytotoxic to BCNU-resistant cancer cells because MMR affects sensitivity to TMZ and BCNU in a converse way. We evaluated TMZ cytotoxicity on BCNU-resistant variant (CEM-R) compared with the parental CCRF-CEM cell line (CEM-S). The mechanisms of its BCNU-resistance involved DNA repairs including nucleotide excision repair, base excision repair, alkylguanine alkyltransferase, MMR, and apoptotic and survival pathways. In particular, transcript levels of MMR-related hMLH1 and hMSH2 were enhanced in CEM-R cells. CEM-R cells were 8-fold more BCNU-resistant but surprisingly 9-fold more TMZ-sensitive than were CEM-S cells. Although TMZ-induced adducts include N-alkylated purines and O(6)-alkylguaine, DNA excision repair was enhanced in CEM-R cells, suggesting the efficient repair of N-alkylation adducts. Cotreatment with methoxyamine, a base excision repair inhibitor, did not sensitize CEM-R cells to TMZ, suggesting little or no contribution of N-alkylation to TMZ-induced cytotoxicity. Cotreatment with O(6)-benzylguanine, an alkylguanine alkyltransferase inhibitor, further sensitized CEM-R cells to TMZ, confirming the cytotoxic impact of O(6)-alkylguanine. Cotreatment with cadmium chloride, an MMR inhibitor, disrupted the sensitivity of CEM-R cells to TMZ. The sensitivity to TMZ was reversed in the CEM-R variant clone that had been established by transfecting CEM-R cells with short hairpin hRNA against hMLH1, suggesting the critical role of MMR on sensitization to TMZ. In conclusion, BCNU-resistant CEM-R cells were sensitized to TMZ as a result of enhanced MMR during the development of BCNU resistance.

Synthesis and anticancer activity of novel thiopyrano[2,3-d]thiazole-based compounds containing norbornane moiety

The series of 9,14-disubstituted 3,7-dithia-5,14-diazapentacyclo[9.5.1.02, 10.04, 8.012, 16]heptadecen-4(8)-triones-6, 13, 15 were synthesized using hetero-Diels-Alder reactions starting from 4-thioxo-2-thiazolidinones and 5-norbornene-2,3-dicarboxylic acid imido-derivatives. Screening of anticancer activity in vitro yielded the most active compounds 5c, 5d, and 7b in micromolar concentrations at the GI50 level (LogGI50 is−6.40 to−4.02 for different cell lines, LogGI50 mean graph midpoint varies from−4.67 to−4.05); moreover, compounds 5c and 5d have a distinctive selectivity against leukemia. The highest sensitivity to compound 5d showed leukemia cell lines CCRF-CEM (LogGI50=−6.40) and SR (LogGI50=−6.06).

Synthesis and biological evaluation of novel symmetry bis-enediynes

A series of acyclic symmetry bis-enediynes have been synthesized successfully and their bioactivities were evaluated. Among them, 1,6-bis(4-((2-(pyridin-2-ylethynyl)phenyl)ethynyl)phenoxy)hexane 8g showed good inhibition activity against the CCRF-CEM (GI 50 = 0.04 μM) and HL-60 (GI 50 = 0.09 μM) cell lines of human leukemia. The cell cycle analysis shows that compound 8g arrests cell cycle via inhibiting Cyclin A and Cyclin B expressions in low concentration and induces a significant apoptosis progress in high concentration.

Modulation of ceramide metabolism in T-leukemia cell lines potentiates apoptosis induced by the cationic antimicrobial peptide bovine lactoferricin

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that selectively induces apoptosis in several different types of human cancer cells. However, the potential use of LfcinB as an anticancer agent is presently limited by the need for relatively high concentrations of the peptide to trigger apoptosis. Ceramide is a membrane sphingolipid that is believed to function as a second messenger during apoptosis. In this study, we investigated the role of ceramide in LfcinB-induced apoptosis in CCRF-CEM and Jurkat T-leukemia cell lines. Exposure to LfcinB caused nuclear condensation and fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation in CCRF-CEM and Jurkat T-cell acute lymphoblastic leukemia cell lines. Treatment with C6 ceramide, a cell-permeable, short-chain ceramide analog, also induced apoptotic nuclear morphology, PARP cleavage, and DNA fragmentation in T-leukemia cells. Although LfcinB treatment did not cause ceramide to accumulate in CCRF-CEM or Jurkat cells, the addition of C6 ceramide to LfcinB-treated T-leukemia cells resulted in increased DNA fragmentation. Furthermore, modulation of cellular ceramide metabolism either by inhibiting ceramidases with D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol or N-oleoylethanolamine, or by blocking glucosylceramide synthase activity with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, enhanced the ability of LfcinB to trigger apoptosis in both Jurkat and CCRF-CEM cells. In addition, LfcinB-induced apoptosis of T-leukemia cells was enhanced in the presence of the antiestrogen tamoxifen, which has multiple effects on cancer cells, including inhibition of glucosylceramide synthase activity. We conclude that manipulation of cellular ceramide levels in combination with LfcinB therapy warrants further investigation as a novel strategy for the treatment of T cell-derived leukemias.

Behavior of synthetic polymers immobilized on a cell membrane

We used three kinds of polymers that interact with living cells in different modes: poly(ethylene glycol)–conjugated phospholipid (PEG–lipid) and poly(vinyl alcohol) carrying alkyl side chains (PVA–alkyl), expected to anchor to the membrane lipid bilayer through hydrophobic interactions; N-hydroxysuccinimidyl–PEG (PEG–NHS), which covalently bonds with all kinds of membrane proteins having amino groups on cell surfaces; and polyelectrolytes, poly(ethylene imine) (PEI) and carboxylated PVA (PVA–COOH), which interact with cells electrostatically. CCRF-CEM (T-cell like) and HEK293 (adherent cell) cell lines were used. We followed the surface dynamics of fluorescently labeled polymers on living cells over time using confocal laser scanning microscopy and flow cytometry. PEI destroyed cells, while PVA–COOH did not interact with cells. PEG–lipid, PVA–alkyl, and PEG–NHS interacted with cells without cytotoxicity and existed on the cell surface even after cells were washed. PEG–lipid and PEG–NHS were rapidly excluded from the cell surface without cytoplasmic uptake, while PVA–alkyl assembled on the living cell surface was taken into the cytoplasm and then excluded. Most polymers were excluded within 24 h although exclusion routes seemed to be different between polymers, suggesting that cell transplant surface modifications are shorter than has been assumed. The short life of modified polymers on the cell surface should be a consideration for cell transplant surface modifications.