Cell Line CCRF-CEM Research Articles

Anti-cancer activity of carbamate derivatives of melampomagnolide B

Melampomagnolide B (MMB) is a natural sesquiterpene structurally related to parthenolide (PTL). We have shown that MMB exhibits anti-leukemic properties similar to PTL. Unlike PTL, the presence of a primary hydroxyl group in the MMB molecule allows the opportunity for examining the biological activity of a variety of conjugated analogs of MMB. We have now synthesized a series of carbamate analogs of MMB and evaluated these derivatives for anti-cancer activity against a panel of sixty human cancer cell lines. Analogs 6a and 6e exhibited promising anti-leukemic activity against human leukemia cell line CCRF-CEM with GI50 values of 680 and 620nM, respectively. Analog 6a also showed GI50 values of 1.98 and 1.38μM respectively, against RPMI-8226 and SR leukemia cell lines and GI50 values of 460 and 570nM against MDA-MB-435 melanoma and MDA-MB-468 breast cancer cell lines, respectively. Analog 6e had GI50 values of 650 and 900nM against HOP-92 non-small cell lung and RXF 393 renal cancer cell lines.

Inhibition of NO Production by Grindelia argentina and Isolation of Three New Cytotoxic Saponins

A bioassay-guided phytochemical analysis of the ethanolic extract of Grindelia argentina Deble & Oliveira-Deble (Asteraceae) allowed the isolation of a known flavone, hispidulin, and three new oleanane-type saponins, 3-O-β-D-xylopyranosyl-(1→3)-β-D-glucopyranosyl-2β,3β,16α,23-tetrahydroxyolean-12-en-28-oic acid 28-O-β-D-xylopyranosyl-(1→2)-β-D-apiofuranosyl-(1→3)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl ester (2), 3-O-β-D-glucopyranosyl-2β,3β,23-trihydroxyolean-12-en-28-oic acid 28-O-β-D-xylopyranosyl-(1→2)-β-D-apiofuranosyl-(1→3)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl ester, (3) and 3-O-β-D-xylopyranosyl-(1→3)-β-D-glucopyranosyl-2β,3β,23-trihydroxyolean-12-en-28-oic acid 28-O-β-D-xylopyranosyl-(1→2)-β-D-apiofuranosyl-(1→3)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl ester (4), named grindeliosides A-C, respectively. Their structures were determined by extensive 1D- and 2D-NMR experiments along with mass spectrometry and chemical evidence. The isolated compounds were evaluated for their inhibitory activities against LPS/IFN-γ-induced NO production in RAW 264.7 macrophages and for their cytotoxic activities against the human leukemic cell line CCRF-CEM and MRC-5 lung fibroblasts. Hispidulin markedly reduced LPS/IFN-γ-induced NO production (IC50 51.4 μM), while grindeliosides A-C were found to be cytotoxic, with grindelioside C being the most active against both CCRF-CEM (IC50 4.2±0.1 μM) and MRC-5 (IC50 4.5±0.1 μM) cell lines.

Cytotoxic Clerodane Diterpenes from Zuelania guidonia

The leaves of Zuelania guidonia yielded eight new clerodane diterpenes, namely, zuelaguidins A-H (1-8), and the known clerodane diterpene esculentin A (9). Some of these structures contained a 3,6-dihydro-1,2-dioxin moiety. The new compounds were isolated and identified using 1D- and 2D-NMR experiments. All compounds were evaluated for cytotoxicity against the CCRF-CEM (human acute lymphocytic leukemia), CEM-ADR5000 (human acute lymphocytic leukemia resistant to doxorubicin), and MIA-PaCa-2 (human pancreatic carcinoma) cell lines as well as for their selectivity against peripheral blood mononuclear cells from healthy human subjects. Zuelaguidins B, C, and E were the most potent compounds against the CCRF-CEM cell line, with IC50 values ranging from 1.6 to 2.5 μM.

Cytotoxicity, mode of action and antibacterial activities of selected Saudi Arabian medicinal plants

BackgroundThe flora of Saudi Arabia is one of the richest biodiversity areas in the Arabian Peninsula and comprises very important genetic resources of crop and medicinal plants. The present study was designed to investigate the cytotoxicity and the antibacterial activities of the organic extracts from twenty six Saudi Arabian medicinal plants. The study was also extended to the investigation of the effects of the extracts from the four best plants, Ononis serrata (SY160), Haplophyllum tuberculatum (SY177), Pulicaria crispa (SY179), and Achillea beiberstenii (SY-200) on cell cycle distribution, apoptosis, caspases activities and mitochondrial function in leukemia CCRF-CEM cell line.MethodsA resazurin assay was used to assess the cytotoxicity of the extracts on a panel of human cancer cell lines whilst the microbroth dilution was used to determine the minimal inhibitory concentration (MIC) of the samples against twelve bacterial strains belonging to four species, Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa.ResultsThe best activity on leukemia cell lines were recorded with SY177 (IC50 of 9.94 μg/mL) and SY179 (IC50 of 1.81 μg/mL) against CCRF-CEM as well as Ach-b (IC50 of 9.30 μg/mL) and SY160 (IC50 of 5.06 μg/mL) against HL60 cells. The extracts from SY177 and SY179 were also toxic against the seven solid cancer cell lines studied with the highest IC50 values of 31.64 μg/mL (SY177 against Hep-G2 cells). SY177 and Ach-b induced cell cycle arrest in G0/G1 and S phases whilst SY160 and SY179 induced arrest in G0/G1 phase. All the four plant extracts induced apoptosis in CCRF-CEM cells with the alteration of the mitochondrial membrane potential. In the antibacterial assays, only Ach-b displayed moderate antibacterial activities against E. coli and E. aerogenes ATCC strains (MIC of 256 μg/mL), AG100ATeT and K. pneumoniae ATCC strains (MIC of 128 μg/mL).ConclusionsFinally, the results of the present investigation provided supportive data for the possible use of some Saudi Arabian plants investigated herein, and mostly Haplophyllum tuberculatum, Pulicaria crispa, Ononis serrata and Achillea beiberstenii in the control of cancer diseases.

2′-Deoxy-5-Azacytidine Enhances Cytotoxic Effect Of Other Anti-Leukemic Agents Synergistically In Acute Lymphoblastic Leukemia Cell Line

IntroductionAdvances in chemotherapy have improved the outcome of childhood acute lymphoblastic leukemia (ALL). However, leukemia cells in refractory ALL are often resistant to anti-leukemic agents. Although recent studies have focused on the epigenetic changes in refractory leukemia, the relationship between the demethylating agent 2′-deoxy-5-azacytidine (decitabine, DAC) and ALL remains unclear. Here, we examine the combined effects of DAC and anti-leukemic agents such as clofarabine (CLO) and etoposide (ETO) on the ALL cell line CCRF-CEM. Methods and resultsIn vitro drug sensitivity was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. We cultured CCRF-CEM cells for 72 hours with or without DAC, and then removed DAC (when present) prior to culturing CCRF-CEM cells for 48 hours with ETO or CLO, or without chemotherapeutic drugs. After culturing for 48 hours, we removed the chemotherapeutic drugs and measured in vitro drug sensitivity using MTT assay. The MTT assay was performed in triplicate. We then evaluated the inhibitory concentration at 50% (IC50). IC50 for ETO, ETO+DAC, CLO, and CLO+DAC was 3.36, 0.625, 4.96, and 1.92, respectively.The combination Index (CI) was produced with Calcusyn® software, which uses the methodology of Chou and Talalay to perform formal synergy analyses. A CI < 1 indicated a synergistic effect. The CI was 0.026 for ETO+DAC and 0.431 for CLO+DAC.We assayed with Annexin-V, PI staining, and caspase-3/7 to detect apoptosis. We observed apoptosis rates of 31.6%, 53.3%, 31.2%, and 52.6% for ETO, ETO+DAC, CLO, and CLO+DAC, respectively. We observed greater caspase-3/7 activity with DAC+CLO and DAC+ETO than with CLO and ETO.Using real-time reverse transcription polymerase chain reaction (RT-qPCR) in CCRF-CEM cells, we examined mRNA expression levels for the pro-apoptotic genes BAK, BID, BAX, BAD, BIM, PUMA, ATM, TP53, and NOXA, as well as those for the anti-apoptotic genes BCL2, BCL2L1, and XIAP. The expression level of each target gene was calculated by normalizing it to the housekeeping gene GAPDH. The RT-qPCR was performed in triplicate. We used Student's t test to compare the data. We observed DAC increased mRNA expression levels of BAX and NOXA, but decreased those for BAK, BID, PUMA, BCL2L1, ATM, TP53, and XIAP.We then analyzed the methylation status of pro- and anti-apoptotic genes after 48 hours incubation with or without DAC. Methylation status of BAK, NOXA, BCL2L1 and XIAP incubation with DAC was 1.3%, 3.3%, 2.5% and 72.9%, respectively. Methylation status of BAK, NOXA, BCL2L1 and XIAP incubation without DAC was 1.9%, 3.6%, 0.7% and 92.3%, respectively. There was no significant difference. DiscussionOur results showed that DAC synergistically enhances CLO and ETO cytotoxicity, and this cytotoxic effect depends on caspase-3/7 activity. We examined mRNA expression levels of pro- and anti-apoptotic genes. We hypothesized that DAC would increase mRNA expression levels of most pro-apoptotic genes, and decrease mRNA levels of most anti-apoptotic genes. We found that DAC decreased some pro-apoptotic genes, such as BAK, BID, PUMA, ATM, and TP53, which disproves our hypothesis. Our present findings are similar to those of Shin et al., who reported that DAC decreased BID mRNA expression levels. However, they provided no explanation for this activity. Our results show that DAC did not demethylate the CpG of BAK, NOXA, BCL2L1, or XIAP. Thus, DAC must demethylate the CpG of other genes. Nevertheless, many genes are involved in apoptosis, and it remains unclear which genes are demethylated by DAC. Disclosures:Sakaguchi:Yakult Honsha Company: Research Funding; Japan Leukemia Research Fund: Research Funding; Japan Society for the Promotion of Science: Research Funding; Sanofi: Research Funding; Teijin Pharma: Research Funding.

Multi-Agent Chemotherapy Overcomes Steroid Resistance Conferred by a BIM Deletion Polymorphism in Pediatric Acute Lymphoblastic Leukemia (ALL)

BackgroundWe have shown previously that a novel BIM intronic deletion polymorphism was significantly associated with resistance to tyrosine kinase inhibitors (TKIs) in a chronic myeloid leukemia (CML) cohort (OR=2.94, 95% CI = 1.17-7.43, p=0.02) (Ng et al. Nature Medicine, 18: 521-528, 2012). BIM is a potent pro-apoptotic protein that binds pro-survival BCL-2 family members via its BH3 domain, and is required for apoptosis induced by a number of stimuli, including TKIs. Mechanistically, the deletion favors the generation of non-apoptotic exon 3-containing BIM splice variants that lack the BH3 domain found in exon 4. Furthermore, we found that introducing the deletion into a TKI-sensitive CML cell line, K562, was sufficient to confer resistance, and that substituting BIM function with a BH3 mimetic drug could restore sensitivity.Because BIM has also been implicated in steroid-induced apoptosis, we hypothesized that the polymorphism would contribute to response heterogeneity in ALL, where response to steroid treatment is an important prognostic factor. Hence, we used ALL as a model to study the effects of the deletion on steroid resistance and treatment outcome. ResultsFirst, we employed zinc finger nucleases to recreate the 2.9kb intronic deletion in subclones of the ALL cell line CCRF-CEM. Using qPCR, we confirmed that the BIM exon 3: exon 4 ratio was increased in the heterozygous and homozygous clones in a polymorphism dose-dependent manner. When treated with dexamethasone, the deletion-containing clones expressed lower pro-apoptotic BIM isoforms, and were rendered resistant to steroid-induced apoptosis, as determined by MTT assays and PARP and caspase 3 cleavage. These data demonstrate that the polymorphism is sufficient to confer resistance to dexamethasone, and also suggest that it might predict for inferior responses in ALL patients treated with steroid-containing regimens.To test this prediction, we genotyped 411 pediatric ALL patients from the Ma-Spore ALL 2003 study for the BIM polymorphism (Yeoh et al. J Clin Oncol. 30: 2384-92, 2012), and correlated genotype with clinical outcome. These patients were uniformly treated with a modified Berlin-Frankfurt-Münster protocol, which consisted of steroids as well as multi-agent chemotherapy including L-asparaginase and vincristine. When we compared outcome between patients with (n=50) and without (n=361) the polymorphism, there was no significant difference in either event-free survival or overall survival. Further, there was no significant enrichment in the intermediate and high Day 33 minimal residual disease group for patients with the polymorphism.To account for the lack of clinical predictive value, we hypothesized that the chemotherapy agents administered in the study overcame steroid resistance conferred by the polymorphism. Indeed, we found that either L-asparaginase or vincristine resensitized the deletion-containing clones to steroid-induced apoptosis, and that this was associated with MCL-1 downregulation, but not BIM induction. Together, our data demonstrate that BIM deletion-mediated steroid resistance can be overcome by co-administration of drugs that induce cell death via a BIM-independent manner. Importantly, such drugs include standard chemotherapy agents that lower the cellular apoptotic threshold by decreasing pro-survival molecules like MCL-1. Conclusions1. The BIM deletion polymorphism confers steroid resistance to ALL cells in vitro.2. In vitro resistance can be overcome with L-asparaginase or vincristine at clinically relevant doses.3. The BIM deletion polymorphism does not predict for poorer clinical outcome in patients treated with standard protocols, and this is likely to be because co-administration of L-asparaginase or vincristine results in BIM-independent cell death.4. Our findings suggest that selected chemotherapy agents may be used to overcome BIM deletion-associated inferior responses in cancer.5. Finally, our experience highlights the difficulties of identifying associations between genetic variants and clinical outcome in cancers treated with multiple agents, since these may employ different mechanisms of killing and cause confounding. Our earlier success in identifying the polymorphism, and its role in mediating TKI resistance in CML, is likely attributable to the fact that CML is treated with TKI mono-therapy. Disclosures:No relevant conflicts of interest to declare.

The Novel Immunoproteasome Inhibitor PR-924: Anti-Leukemic Activity and Mechanisms Of Resistance

Proteasome inhibition with bortezomib (BTZ) is an effective treatment for hematological malignancies and an emerging treatment strategy for acute leukemia. However, the side-effects of BTZ and emergence of BTZ-resistance call for alternative therapeutic approaches. The immunoproteasome may serve as a candidate since its expression is higher than constitutive proteasome expression in cells of hematological malignancies, including acute lymphocytic leukemia (ALL). To this end, the new immunoproteasome inhibitor PR-924 may elicit anti-leukemic activity, since it bears greater specificity for the β5i subunit of the immunoproteasome compared to other proteasome inhibitors including BTZ and carfilzomib. The aim of the current study was to examine the proteasome inhibition capacity and cell growth inhibitory impact of PR-924 in acute leukemia cells and sublines with acquired resistance to BTZ. We further determined whether PR-924 itself would be prone to resistance development and if so, to explore the underlying molecular basis. To assess the anti-leukemic activity of PR-924, its cytotoxicity was determined in two human cell lines of hematological origin: the T-ALL cell line CCRF-CEM and the acute myeloid leukemia cell line THP1, and their 150-fold BTZ-resistant sublines CEM/BTZ200, and THP1/BTZ200, respectively. Parental CEM and THP1 cells displayed similar sensitivity to PR-924 (IC50 CEM: 1.8 µM ± 0.4, THP1: 1.5 µM ± 0.2), whereas their BTZ-resistant lines displayed a moderate 10-12 fold cross-resistance to PR-924 (IC50 CEM/BTZ200: 21.1 µM ± 0.5, THP1/BTZ200: 15.6 µM ± 1.6). Flow cytometric analysis revealed that PR-924-induced cell death was mediated by induction of apoptosis. Moreover, PR-924 exposure resulted in a (up to 50%) reduction of cell surface expression of HLA Class I.To determine whether PR-924 activity relies on specific inhibition of β5i, proteasome activity inhibition experiments were performed over a range of 10 nM – 10 µM PR-924. Notably, in parental cells, β5i activity was already inhibited by 20% at 10 nM PR-924 and > 90% inhibition was achieved at 100 nM PR-924. At higher PR-924 concentrations of 1-10 µM, inhibition of both β5 and β1i activities was observed, thus indicating that PR-924 blocks the β5i activity far below concentrations that exert anti-proliferative activity (1.5 µM), consistent with earlier studies of PR-924 (Parlati et al. Blood, 2009).Next, acquired resistance to PR-924 was provoked in CEM and THP1 cells by step-wise increasing PR-924 concentrations in cell culture, starting at the IC50 concentrations. Following this process, CEM cells resistant to 20 µM PR-924 (CEM/PR20) and THP1 cells resistant to 12 mM PR-924 (THP1/PR12) exhibited IC50 values of 22.1 µM PR-924 (resistance factor 13) and 14.3 µM PR-924 (resistance factor 10), respectively. In addition, these PR-924 resistant cells displayed 10-fold cross-resistance to BTZ. To explore mechanisms of PR-924 resistance, we first sequenced exon 2/3 of the PSMB8 gene (encoding β5i), a similar functioning coding region that was previously reported to harbor PSMB5 mutations in BTZ-resistant leukemia cells (Franke et al. Leukemia, 2012). However, no mutations were found in PSMB8 exon 2/3. Remarkably, we did identify mutations in exon 2 of the PSMB5 gene (encoding the S1 pocket of β5) in both CEM/PR20 (Met45Ile) and of THP1/PR12 (Ala49Thr); these amino acid substitutions are similar to those observed in BTZ-resistant sublines. Finally, we characterized proteasome subunit expression by ProCISE analysis in the PR-924 resistant cell lines. This method is a subunit-specific active-site ELISA assay, which utilizes a purified proteasome standard curve to calculate ng of subunit per µg total protein. Expression of constitutive proteasome subunits in CEM/PR20 and THP1/PR8 cells were upregulated up to 2.5-fold, compared to their parental counterparts, whereas immunoproteasome subunit expression was moderately decreased (up to 2-fold) in CEM/PR20 and increased in THP1/PR8 (up to 1.6-fold).In conclusion, PR-924 displayed significant anti-leukemic activity. Although there was cross-resistance to BTZ, PR-924 retained activity in BTZ-resistant leukemia cells. Despite PR-924 specificity to the β5i subunit of the proteasome, its anti-leukemic effect requires concentrations that block both β5 and β5i subunits. This notion is underscored by emergence of acquired mutations in PSMB5 rather than in PSMB8. Disclosures:Kirk:Onyx Pharmaceuticals: Employment, Equity Ownership. Anderl:Onyx: Employment.

Nitensidine A, a guanidine alkaloid from Pterogyne nitens, is a novel substrate for human ABC transporter ABCB1

The Pterogyne nitens (Fabaceae) tree, native to South America, has been found to produce guanidine alkaloids as well as bioactive flavonols such as kaempferol, quercetin, and rutin. In the present study, we examined the possibility of interaction between human ATP-binding cassette (ABC) transporter ABCB1 and four guanidine alkaloids isolated from P. nitens (i.e., galegine, nitensidine A, pterogynidine, and pterogynine) using human T cell lymphoblast-like leukemia cell line CCRF-CEM and its multi-drug resistant (MDR) counterpart CEM/ADR5000. In XTT assays, CEM/ADR5000 cells were resistant to the four guanidine alkaloids compared to CCRF-CEM cells, although the four guanidine alkaloids exhibited some level of cytotoxicity against both CCRF-CEM and CEM/ADR5000 cells. In ATPase assays, three of the four guanidine alkaloids were found to stimulate the ATPase activity of ABCB1. Notably, nitensidine A was clearly found to stimulate the ATPase activity of ABCB1 as strongly as the control drug, verapamil. Furthermore, the cytotoxic effect of nitensidine A on CEM/ADR5000 cells was synergistically enhanced by verapamil. Nitensidine A inhibited the extrusion of calcein by ABCB1. In the present study, the possibility of interaction between ABCB1 and two synthetic nitensidine A analogs (nitensidine AT and AU) were examined to gain insight into the mechanism by which nitensidine A stimulates the ATPase activity of ABCB1. The ABCB1-dependent ATPase activity stimulated by nitensidine A was greatly reduced by substituting sulfur (S) or oxygen (O) for the imino nitrogen atom (N) in nitensidine A. Molecular docking studies on human ABCB1 showed that, guanidine alkaloids from P. nitens dock to the same binding pocket as verapamil. Nitensidine A and its analogs exhibit similar binding energies to verapamil. Taken together, this research clearly indicates that nitensidine A is a novel substrate for ABCB1. The present results also suggest that the number, binding site, and polymerization degree of the isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their stimulation of ABCB1's ATPase activity.

DNA-based aptamer fails as a simultaneous cancer targeting agent and drug delivery vehicle for a phenanthroline-based platinum(II) complex

The sgc8c aptamer is a 41-base DNA oligonucleotide that binds to leukaemia cells with high affinity and specificity. In this work we examined the utility of this aptamer as both a delivery vehicle and an active targeting agent for an inert platinum complex [(1,10-phenathroline)(ethylenediamine)platinum(II)]2+. The aptamer forms a stem-and-loop confirmation as determined by circular dichroism. This conformation is adopted in both water and phosphate buffered saline solutions. The metal complex binds through intercalation into the aptamer's double helical stem with a binding constant of approximately 4.3×104M−1. Binding of the metal complex to the aptamer had a significant effect on the aptamer's global conformation, and increased its melting temperature by 28°C possibly through lengthening and stiffening of the aptamer stem. The effect of the aptamer on the metal complex's cytotoxicity and cellular uptake was determined using in vitro assays with the target leukaemia cell line CCRF-CEM and the off-target ovarian cancer cell lines A2780 and A2780cp70. The aptamer has little inherent cytotoxicity and when used to deliver the metal complex results in a significant decrease in the metal complex's cytotoxicity and uptake. The reason(s) for the poor uptake and activity may be due to the change in aptamer conformation which affects its ability to recognise leukaemia cells.

Ferrocenylvinyl-flavones: Synthesis, structure, anticancer and antibacterial activity studies

Four new ferrocenyl-flavone complexes were obtained via palladium-catalyzed Heck cross-coupling reactions; (E)-6-ferrocenylvinyl-chromen-4-one (4), (E)-6-ferrocenylvinyl-2-methyl-chromen-4-one (5), (E)-6-ferrocenylvinyl-2-phenyl-chromen-4-one (6) and (E)-6-ferrocenylvinyl-chromen-4-one-3-propionic acid (7). All compounds were characterized by 1H NMR, 13C NMR, IR spectroscopy, high resolution-MS, elemental analysis and cyclic voltammetry. The molecular structure of derivatives 4 and 6 was also confirmed by X-ray crystallography. The biological activity of the complexes was rationalized on the basis of their anticancer and antibacterial properties. The anticancer activity of ferrocenyl-flavones 4–7 against established human cell lines derived from hematological and solid tumors has been evaluated in vitro. The following cell lines were investigated: MCF-7 (estrogen receptor-responsive breast adenocarcinoma), MDA-MB-231 (estrogen receptor-negative breast adenocarcinoma), HepG2 (hepatocellular carcinoma) and CCRF-CEM (T lymphoblast-like polymorph cells). All investigated ferrocenyl-flavones show cytotoxicity against CCRF-CEM cell line. The antibacterial activity of the four ferrocenyl-flavones against Gram-positive methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA) and Staphylococcus epidermidis bacterial strains was determined. Our experiments show antibacterial activity for the carboxylic acid derivative 7 against all tested Gram-positive bacterial strains while no activity was detected for the ferrocene-free 6-bromo-chromen-4-one-3-propionic acid.

Abstract 2757: The dual PI3K/mTOR inhibitor NVP-BEZ235 enhances dexamethasone induced apoptosis in models of T-cell ALL with PTEN dysfunction and hyperactivated PI3K/Akt pathway.

Abstract Introduction: Glucocorticoid (GC) resistance is a negative prognostic factor in pediatric acute lymphoblastic leukemia (ALL) treatment. The tumor suppressor PTEN, when inactivated, may lead to increased GC resistance through aberrant activation of the PI3K/Akt pathway and inhibition of apoptosis. We investigated the effects of inhibiting the PI3K/Akt pathway with the dual PI3K/mTOR inhibitor NVP-BEZ235 on the enhancement of dexamethasone apoptosis activity in a panel of T-cell and B-cell ALL cell lines and the role PTEN plays in resistance to GCs. Methods: Cytotoxicity of NVP-BEZ235, dexamethasone, and the combination were evaluated using the fluorescence based microscopy system, DIMSCAN, in in vitro models of T- and B-cell ALL and combination indices (CI) were calculated using CalcuSyn. Apoptotic activity for each single agent and the combination was determined using the Annexin-V/PI staining method and measured by flow cytometry. Protein and mRNA expression were measured by immunoblotting and RT-PCR, respectively. Knockdown of PTEN was accomplished using siRNA. Results: The combination of dexamethasone plus NVP-BEZ235 showed strong synergistic activity (CI&amp;lt;0.3) in 6/6 T-cell ALL models tested, while only 1/6 B-cell ALL models, were characterized by such activity. In COG-LL-317h, a T-cell ALL cell line, the percentage of apoptotic cells were significantly greater in cells treated with the combination (17.3±1.0%) compared to vehicle control (4.0±1.7%, p=0.01), dexamethasone (4.5±0.4%, p=0.004), and NVP-BEZ235 (2.3±0.3%, p=0.002). In COG-LL-317h and another T-cell ALL cell line CCRF-CEM, the pro-apoptotic Bcl-2 family protein Bim was substantially increased at both the mRNA and protein levels when treated with the combination compared to either single agent alone, while the B-cell ALL models COG-LL-319h and RS4;11 did not show any enhancement with the combination. Knockdown of PTEN in dexamethasone-sensitive RS4;11 showed a significant reduction in dexamethasone cytotoxicity (non-targeting siRNA: 88.3±1.5% vs. PTEN siRNA: 82.6±2.6%; p&amp;lt;0.001), and activity was restored when dexamethasone was in combination with NVP-BEZ235 (non-targeting siRNA: 91.9±1.8% vs. PTEN siRNA: 92.1±1.6%, p=0.77). Conclusion: These data suggest PTEN dysfunction and subsequent PI3K/Akt activation play a significant role in altering GC responses and use of the combination may offset the negative consequences of PTEN loss by increasing Bim expression and apoptosis. Our results show that NVP-BEZ235 enhances dexamethasone apoptotic activity in models of T-cell ALL which have loss of PTEN function, and may provide a new adjunct to increase response in current T-cell ALL treatment protocols. Citation Format: Connor Hall, Min Kang. The dual PI3K/mTOR inhibitor NVP-BEZ235 enhances dexamethasone induced apoptosis in models of T-cell ALL with PTEN dysfunction and hyperactivated PI3K/Akt pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2757. doi:10.1158/1538-7445.AM2013-2757

Determination of the Gene Expression Profiles of JAK/STAT Cascade Components for the Potential Role of Capsaicin Induced Apoptosis of Acute T-Cell Lymphoblastic Leukemia Cells

Dr. Burcin Tezcanli Kaymaz, Ege Universitesi Tip Fakultesi Hastanesi, Bornova 35100 Izmir Turkiye, Tel. 0232 390 22 60 Email. bt1980@gmail.com Gelis Tarihi: 12.04.2013 • Kabul Tarihi: 18.07.2013 ABSTRACT AIM: Capsaicin, an ingredient of red chili pepper consumed as spice throughout the world is defi ned as “double-edged sword” agent for having possible tumorigenicity/genotoxicity properties, for being antagonist to carcinogens/mutagens for inducing tumor cell apoptosis. However, reasons of capsaicin induced leukemic cell apoptosis are investigated. In this study, following capsaicin treatment to acute lymphoblastic leukemia cell line CCRF-CEM, detecting apoptosis; determining gene expression profi les of JAK/STAT signaling pathway members STAT3, STAT5A,-5B, JAK2,IL-6 mRNA levels and clarifying possible molecular effect mechanism of capsaicin were aimed.

Core Transcriptional Regulatory Circuit Controlled by the TAL1 Complex in Human T Cell Acute Lymphoblastic Leukemia

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human Tcell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells.

Carotenoids reverse multidrug resistance in cancer cells by interfering with ABC-transporters

Proteins of the ATP-binding cassette superfamily, mainly P-glycoprotein (P-gp; MDR1), play an important role in the development of multidrug resistance (MDR) in cancer cells and thus in the potential failure of chemotherapy. A selection of carotenoids (β-carotene, crocin, retinoic acid, canthaxanthin, and fucoxanthin) was investigated whether they are substrates of P-gp, and if they can reverse MDR in resistant Caco-2 and CEM/ADR5000 cells as compared to the sensitive parent cell line CCRF-CEM. The activity of ABC transporter was determined in resistant and sensitive cells by spectrofluorometry and flow cytometry using the substrates doxorubicin, rhodamine 123, and calcein as fluorescent probes. The carotenoids increased accumulation of these P-gp substrates in a dose-dependent manner indicating that they themselves also function as substrates. Fucoxanthin and canthaxanthin (50–100μM) produced a 3–5-fold higher retention of the fluorescent probes than the known competitive inhibitor verapamil. Carotenoids showed a low cytotoxicity in cells with MDR with IC50 values between 100 and 200μM. The combination of carotenoids with eight structurally different cytotoxic agents synergistically enhanced their cytotoxicity in Caco-2 cells, probably by inhibiting the function of the ABC transporters. For example, fucoxanthin synergistically enhanced the cytotoxicity of 5-FU 53.37-fold, of vinblastine 51.01-fold, and of etoposide 12.47-fold. RT-PCR was applied to evaluate the mRNA levels of P-gp in Caco-2 cells after treatment with carotenoids. Fucoxanthin and canthaxanthin significantly decreased P-gp levels to 12% and 24%, respectively as compared to untreated control levels (p<0.001). This study implies that carotenoids may be utilised as chemosensitisers, especially as adjuvants in chemotherapy.

Prostaglandin E2 induces growth inhibition, apoptosis and differentiation in T and B cell-derived acute lymphoblastic leukemia cell lines (CCRF-CEM and Nalm-6)

Despite advances in the treatment of ALL, in most patients long-term survival rates remain unsatisfactory. The objective of the present study was to investigate the anti-cancer effects of Prostaglandin E2 (PGE2) in two different ALL cell lines (CCRF-CEM (T-ALL) and Nalm-6 (B-ALL)). The anti-leukemic effects of PGE2 were also compared with two epigenetic compounds (trichostatin A and 5-aza-2´-deoxycytidine). MTT assay was used to assess growth inhibition by anti-cancer drugs in these cells. All three compounds were shown to induce apoptosis in both ALL cell lines using flow cytometry and Western blotting. To evaluate the differentiation induction by these agents, the expressions of CD19 and CD38 markers on Nalm-6 cell line and CD7 marker on CCRF-CEM cell line were assayed. Surprisingly, the flow cytometric analysis showed a significant increase in CD markers expression in response to PGE2 treatments. We, for the first time, provide evidences that PGE2 has anti-leukemic effects and induces differentiation at micromolar ranges in both T- and B-cell derived ALL cell lines. Since T-ALL cells are insensitive to current chemotherapies, these findings may help the designing of new protocols for T-ALL differentiation therapy in the future.

Substituted thiazoles VI. Synthesis and antitumor activity of new 2-acetamido- and 2 or 3-propanamido-thiazole analogs

A novel series of 2-acetamido and 2 or 3-propanamido derivatives of 4- or 5-substituted-thiazoles was designed and synthesized. Structure elucidation of the new synthesized compounds was attained by the use of 1H &13C NMR, and Mass spectrometry. Compounds were subjected to NCI in vitro assessment for their antitumor activity, at a single dose of 10μM of test compounds. Compounds bearing straight chain substituent or 4-phenyl function proved to be more active than their branched or 4-methyl congeners. Compounds 37, 41 and 42 exhibited broad spectrum antitumor activity. Compounds 23 and 27 proved lethal while compounds 18, 21, 32 and 37 showed remarkable GI values of 75.5, 69.3, 96.2 and 92.7% to the Leukemia CCRF-CEM cell line, respectively.

Abstract 4258: In vivo anti-leukemia activity of novel C-ring modified prodigiosenes in a zebrafish xenograft model

Abstract Acute myeloid leukemia (AML) remains fatal for 40% of patients, both due to refractory disease and toxicity from traditional therapeutic agents. Zebrafish provide an efficient and robust leukemia model with the capacity for embryonic screens to test new drugs in vivo. Naturally-occurring Prodigiosin has demonstrated potential as an anti-cancer agent, but toxicity prevents its use as a chemotherapeutic. Here, we present four novel derivatives, prodigiosenes designated DSB-8, -31, -39, -50, synthesized using a multi-step sequence beginning with simple pyrroles. These synthetic compounds demonstrated anti-leukemia activity in vitro against cells in the NIH/NCI Developmental Therapeutics Program, including K562, CCRF-CEM, MOLT-4, RPMI-8226 and SR cell lines. We have previously xenografted human leukemia cells into 48 hour zebrafish embryos and developed a cell quantification assay to evaluate drug responses (Corkery et al BJH, 2011). To assess the anti-leukemia activity and toxicity of our novel prodigiosenes in vivo, we first conducted toxicity curves using 48-hour zebrafish embryos treated for 72 hours to determine an optimal dose (50% of maximum tolerated dose [MTD]). Prodigiosenes DSB-39 (1.0 uM) and DSB-50 (1.5 uM) were tolerated better than DSB-8 (0.2 uM) and DSB-31 (0.2 uM). Subsequently, 40-50 CM-DiI-labeled K562 chronic myelogenous leukemia (CML) cells were injected into the yolk sac of 48-hour zebrafish embryos, which were treated with prodigiosenes at 50% MTD. Engrafted embryos were followed using live cell microscopy. Embryos treated with 0.03% DMSO served as negative control, showing abundant K562 cell proliferation and entry of cells into circulation. Embryos treated with 20 µM imatinib mesylate (IM), a targeted inhibitor of the BCR-ABL1 oncoprotein in K562 cells, served as a positive control, demonstrating no cell proliferation or migration. By comparison, prodigiosenes inhibited K562 cell activity to a similar or greater degree than IM. Qualitatively, the effects of prodigiosenes were graduated: DSB-8 (n=6/7) was cytostatic, DSB-31 (n=5/8) and DSB-50 (n=6/9) were moderately cytotoxic, and DSB-39 (n=7/7) was strongly cytotoxic. To quantify this effect, we are applying a modified ex vivo proliferation assay. Embryos are dissociated to a single cell suspension at 24 and 72 hours post-injection followed by immunohistochemistry directed to human CD34. Our work extends the use of zebrafish xenografts to determine the in vivo sensitivity of human cancer cells to novel drugs and suggests that tailored prodigiosenes may represent novel therapeutic agents for leukemia with improved anti-cancer potency and reduced toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4258. doi:1538-7445.AM2012-4258

3,3'-Diindolylmethane induces G1 arrest and apoptosis in human acute T-cell lymphoblastic leukemia cells.

Certain bioactive food components, including indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) from cruciferous vegetables, have been shown to target cellular pathways regulating carcinogenesis. Previously, our laboratory showed that dietary I3C is an effective transplacental chemopreventive agent in a dibenzo[def,p]chrysene (DBC)-dependent model of murine T-cell lymphoblastic lymphoma. The primary objective of the present study was to extend our chemoprevention studies in mice to an analogous human neoplasm in cell culture. Therefore, we tested the hypothesis that I3C or DIM may be chemotherapeutic in human T-cell acute lymphoblastic leukemia (T-ALL) cells. Treatment of the T-ALL cell lines CCRF-CEM, CCRF-HSB2, SUP-T1 and Jurkat with DIM in vitro significantly reduced cell proliferation and viability at concentrations 8- to 25-fold lower than the parent compound I3C. DIM (7.5 µM) arrested CEM and HSB2 cells at the G1 phase of the cell cycle and 15 µM DIM significantly increased the percentage of apoptotic cells in all T-ALL lines. In CEM cells, DIM reduced protein expression of cyclin dependent kinases 4 and 6 (CDK4, CDK6) and D-type cyclin 3 (CCND3); DIM also significantly altered expression of eight transcripts related to human apoptosis (BCL2L10, CD40LG, HRK, TNF, TNFRSF1A, TNFRSF25, TNFSF8, TRAF4). Similar anticancer effects of DIM were observed in vivo. Dietary exposure to 100 ppm DIM significantly decreased the rate of growth of human CEM xenografts in immunodeficient SCID mice, reduced final tumor size by 44% and increased the apoptotic index compared to control-fed mice. Taken together, our results demonstrate a potential for therapeutic application of DIM in T-ALL.

Microwave electromagnetic field regulates gene expression in T-lymphoblastoid leukemia CCRF-CEM cell line exposed to 900 MHz

Electric, magnetic, and electromagnetic fields are ubiquitous in our society, and concerns have been expressed regarding possible adverse effects of these exposures. Research on Extremely Low-Frequency (ELF) magnetic fields has been performed for more than two decades, and the methodology and quality of studies have improved over time. Studies have consistently shown increased risk for childhood leukemia associated with ELF magnetic fields. There are still inadequate data for other outcomes. More recently, focus has shifted toward Radio Frequencies (RF) exposures from mobile telephony. There are no persuasive data suggesting a health risk, but this research field is still immature with regard to the quantity and quality of available data. This technology is constantly changing and there is a need for continued research on this issue. To investigate whether exposure to high-frequency electromagnetic fields (EMF) could induce adverse health effects, we cultured acute T-lymphoblastoid leukemia cells (CCRF-CEM) in the presence of 900 MHz MW-EMF generated by a transverse electromagnetic (TEM) cell at short and long exposure times. We evaluated the effect of high-frequency EMF on gene expression and we identified functional pathways influenced by 900 MHz MW-EMF exposure.

Synthesis and antileukemic activity of 16E-[4-(2-carboxy)ethoxybenzylidene]-androstene amides

In order to determine the structural requirements for cytotoxicity against various tumor cell lines, a new series of 16E-arylidene androstene amides with varying degrees of unsaturation in ring A has been synthesized. Characterization and invitro cytotoxic studies of the newly synthesized compounds are discussed. The compounds on evaluation against various tumor cell lines exhibited significant growth inhibition on leukemia cell lines. 3-Chloro-16E-{[4-(4-methylpiperazin-1-yl)-2-oxoethoxy]benzylidene}androst-5-en-17-one (10) emerged as the most potent compound of the series with GI50 values of 3.94, 2.61, 6.90 and 1.79μM against CCRF-CEM, K-562, RPMI-8226 and SR leukemia cell lines, respectively.