Proliferation Of Human Leukemia Cells Research Articles

Design and application of hybrid cyclic-linear peptide-doxorubicin conjugates as a strategy to overcome doxorubicin resistance and toxicity

Doxorubicin (Dox) is used for breast cancer, leukemia, and lymphoma treatment as an effective chemotherapeutic agent. However, Dox use is restricted due to inherent and acquired resistance and an 8-fold increase in the risk of potentially fatal cardiotoxicity. Hybrid cyclic-linear peptide [R5K]W7A and linear peptide R5KW7A were conjugated with Dox through a glutarate linker to afford [R5K]W7A-Dox and R5KW7A-Dox conjugates to generate Dox derivatives. Alternatively, [R5K]W7C was conjugated with Dox via a disulfide linker to generate [R5K]W7C–S–S-Dox conjugate, where S–S is a disulfide bond. Comparative antiproliferative assays between conjugates [R5K]W7A-Dox, [R5K]W7C–S–S-Dox, linear R5KW7A-Dox, the corresponding physical mixtures of the peptides, and Dox were performed in normal and cancer cells. [R5K]W7A-Dox conjugate was 2-fold more efficient than R5KW7A-Dox, and [R5K]W7C–S–S-Dox conjugates in inhibiting the cell proliferation of human leukemia cells (CCRF-CEM). Therefore, hybrid cyclic-linear [R5K]W7A-Dox conjugate was selected for further studies and inhibited the cell viability of CCRF-CEM (84%), ovarian adenocarcinoma (SK-OV-3, 39%), and gastric carcinoma (AGS, 73%) at a concentration of 5 μM after 72 h of incubation, which was comparable to Dox (5 μM) efficacy (CCRF-CEM (85%), SK-OV-3 (33%), and AGS (87%)). While [R5K]W7A-Dox had a significant effect on the viability of cancer cells, it exhibited minimal cytotoxicity to normal kidney (LLC-PK1, 5–7%) and heart cells (H9C2, <9%) at concentrations of 5–10 μM (compared to free Dox at 5 μM that reduced the viability of kidney and heart cells by 85% and 44%, respectively). The fluorescence microscopy images were consistent with the cytotoxicity studies, indicating minimal uptake of the cyclic-linear [R5K]W7A-Dox (5 μM) in H9C2 cells. In comparison, Dox (5 μM) showed significant uptake, reduced cell viability, and changed the morphology of the cells after 24 h. [R5K]W7A-Dox showed 16-fold and 9.5-fold higher activity against Dox-resistant cells MDA231R and MES-SA/MX2 (lethal dose for 50% cell death or LC50 of 2.3 and 4.3 μM, respectively) compared to free Dox (LC50 of 36–41 μM, respectively). These data, along with the results obtained from the cell viability tests, indicate comparable efficiency of [R5K]W7A-Dox to free Dox in leukemia, ovarian, and gastric cancer cells, significantly reduced toxicity in normal kidney LLC-PK1 and heart H9C2 cells, and significantly higher efficiency in Dox-resistant cells. A number of endocytosis inhibitors did not affect the cellular uptake of [R5K]W7A-Dox.

Tanshinone I, a new EZH2 inhibitor restricts normal and malignant hematopoiesis through upregulation of MMP9 and ABCG2

Rationale: Tanshinone, a type of diterpenes derived from salvia miltiorrhiza, is a particularly promising herbal medicine compound for the treatment of cancers including acute myeloid leukemia (AML). However, the therapeutic function and the underlying mechanism of Tanshinone in AML are not clear, and the toxic effect of Tanshinone limits its clinical application.Methods: Our work utilizes human leukemia cell lines, zebrafish transgenics and xenograft models to study the cellular and molecular mechanisms of how Tanshinone affects normal and abnormal hematopoiesis. WISH, Sudan Black and O-Dianisidine Staining were used to determine the expression of hematopoietic genes on zebrafish embryos. RNA-seq analysis showed that differential expression genes and enrichment gene signature with Tan I treatment. The surface plasmon resonance (SPR) method was used with a BIAcore T200 (GE Healthcare) to measure the binding affinities of Tan I. In vitro methyltransferase assay was performed to verify Tan I inhibits the histone enzymatic activity of the PRC2 complex. ChIP-qPCR assay was used to determine the H3K27me3 level of EZH2 target genes.Results: We found that Tanshinone I (Tan I), one of the Tanshinones, can inhibit the proliferation of human leukemia cells in vitro and in the xenograft zebrafish model, as well as the normal and malignant definitive hematopoiesis in zebrafish. Mechanistic studies illustrate that Tan I regulates normal and malignant hematopoiesis through direct binding to EZH2, a well-known histone H3K27 methyltransferase, and inhibiting PRC2 enzymatic activity. Furthermore, we identified MMP9 and ABCG2 as two possible downstream genes of Tan I's effects on EZH2.Conclusions: Together, this study confirmed that Tan I is a novel EZH2 inhibitor and suggested MMP9 and ABCG2 as two potential therapeutic targets for myeloid malignant diseases.

The effect of Phyllanthus niruri L extracts on human leukemic cell proliferation and apoptosis induction

Objective : To investigate the effect of Phyllanthus niruri Linn (Euphorbiaceae) in the proliferation of human leukemic cells (MOLT-4 and K562). Methods: Phyllanthus niruri L ( P.niruri ) was macerated by using various solvents to obtain the crude extracts. Cytotoxicity of the extracts against MOLT-4 and K562 cells was tested using MTT assay to find the IC 50 value. To analyse cell cycle progression, cellular DNA was measured using propidium iodide (PI) staining. Apoptosis induction was evaluated using Annexin V-FITC and PI staining and analysed using FACSVerse flow cytometry. Finally, the expression of p53 on MOLT-4 and K562 cell lysate was measured by western blotting, to identify the possible mode of action of the anticancer activity. Results: P. niruri crude extracts demonstrated a potential anti-cancer effect towards MOLT-4 cells (IC 50 range was 42.21 ± 4.98 to 97.06 ± 18.29 µg/ml). However, against K562 cells, P.niruri extracts exhibited a lower inhibitory potency (the IC 50 was 120.19 ± 8.48 to 256.55 ± 26.22 µg/ml). The results showed the selectivity of the toxic effect of the extracts against MOLT-4 and K562. To evaluate the possible mechanism of action the anticancer effect, we evaluated P. niruri extract action in apoptosis induction and p53 expression. The results showed that methanol and hexane extract inhibited MOLT-4 cell progression from G1 to S-phase, indicating G1 cell arrest. Moreover, apoptotic cell population following treatment of MOLT-4 and K562 cells with methanol extract was markedly increased, showing morphological signs of apoptosis including membrane degradation and chromatin condensation. Furthermore, we found that there was an increase in p53 expression following MOLT-4 treatment with methanol extract , suggesting that p53 induction may be involved in cell apoptosis. Conclusions: The results indicated the involvement of p53 pathway in the mechanism of anti-cancer activity exerted by P. niruri extract on MOLT-4 cells . However, for cancer cells lacking P53 expression, such as K562 cells, apoptosis might take place via other pathways.

Effects of PKM2 Knockdown on Proliferation and Apoptosis of Human Leukemia Cells and Its Potential Mechanism

Effects of PKM2 Knockdown on Proliferation and Apoptosis of Human Leukemia Cells and Its Potential Mechanism

A novel synthesized 3’, 5’-diprenylated chalcone mediates the proliferation of human leukemia cells by regulating apoptosis and autophagy pathways

Leukemia is a hematologic malignancy with poor prognosis in humans and chemotherapy is the main strategy for treating leukemia patients. Novel drugs with better selectivity and lower toxicity are required for the treatment of patients. A novel 3’,5’-diprenylated chalcone, (E)-1-(2-hydroxy-4-methoxy-3,5-diprenyl) phenyl-3-(3- pyridinyl)-propene-1-one (C10) is a potential new anti-leukemia agent. In this study, we investigated the molecular mechanisms of the anti-leukemia effects of C10 on different leukemia cells in vitro. C10 showed strong inhibition of proliferation of the human erythroleukemia cell line HEL and human myeloid leukemia cell line K562, and several cell and flow cytometer assays showed that inhibition by C10 was due to the regulation of gene expression or phosphorylation in the apoptosis and autophagy pathways. The results showed that C10 regulated the expression of Bax, c-Myc, Bcl-2, P38/AMPK and ERK 1/2, activated the expression of Caspase-3, -8, and PARP at the protein level in the apoptosis pathway of the two leukemia cell types, and inhibited the expression of erythroleukemia carcinogene Fli-1 in the human erythroleukemia cell line HEL. Additionally,treatment with the compound induced a time-dependent increase in expression of LC 3A/B via inhibiting the AKT-mTOR pathway, which is associated with cell autophagy. Taken together, the above results suggest that the novel synthesized 3’,5’-diprenylated chalcone can prevent the growth of leukemia cells by inducing apoptosis and autophagy.

Corrigendum to: Ginkgetin inhibits proliferation of human leukemia cells via the TNF-α signaling pathway.

Corrigendum to: Ginkgetin inhibits proliferation of human leukemia cells via the TNF-α signaling pathway.

Wnt5a Induces Association of ROR1 with 14-3-3ζ to Enhance Chemotaxis and Proliferation in Chronic Lymphocytic Leukemia

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen that is expressed on CLL cells, but not on normal postpartum tissues. We found that ROR1 was a receptor for Wnt5a, which could activate Rho GTPases (e.g. RhoA and Rac1) in CLL cells by inducing the recruitment to ROR1 of guanine exchange factors (GEFs), notably ARHGEF2. How ARHGEF2 can complex with ROR1 was not known. We performed mass spectrometry-based proteomics to interrogate immune-precipitates of Wnt5a-activated ROR1 and identified 14-3-3ζ, a highly conserved, cytoplasmic-protein member of the tetratricopeptide repeat-like superfamily. 14-3-3ζ plays a critical role in cell-signaling pathways, which promote proliferation, adhesion, and survival in a variety of human cancers. We validated the Wnt5a-induced interaction of ROR1 with 14-3-3ζ in primary CLL cells using co-immunoprecipitation studies and immunoblot analyses. We found the capacity of Wnt5a to induce ROR1 to associate with 14-3-3ζ could be blocked by cirmtuzumab, a first-in-class humanized mAb specific for a functional epitope in the ROR1-extracellular domain; this mAb is undergoing clinical testing in patients with CLL. Furthermore, we found that 14-3-3ζ could interact with ARHGEF2 in CLL cells. Silencing 14-3-3ζ via RNAi impaired the capacity of Wnt5a to: (1) induce recruitment of ARHGEF2 to ROR1, (2) enhance the in vitro exchange activity of ARHGEF2 for RhoA and Rac1, and (3) induce activation of RhoA and Rac1 in primary CLL cells. Consistent with these findings, we found that Difopein, an inhibitor of 14-3-3ζ, also could inhibit Wnt5a-enhanced chemokine-directed migration and proliferation of primary CLL cells in vitro, at low concentrations that did not result in leukemia-cell apoptosis. To examine structure-function relationships, we employed the MEC1 cell line, which was derived from human CLL. Prior studies found MEC1 cells expressed Wnt5a, which induced activation of RhoA and Rac1 in MEC1 cells made to express ROR1 (MEC1-ROR1), but not in parental MEC1 cells, which lacked ROR1. Similar to work on primary CLL cells, we detected 14-3-3ζ in anti-ROR1 immune precipitates via mass spectrometry and immunoblot analyses. We identified a 14-3-3ζ binding motif (RSPS857SAS) in the cytoplasmic domain of ROR1; site directed mutagenesis and transfection of MEC1 with mutant forms of ROR1 determined that serine-857 was required for the recruitment of 14-3-3ζ and ARHGEF2-dependent activation of RhoA and Rac1, respectively. In addition, we used CRISPR/Cas9 technology to delete 14-3-3ζ (Δ14-3-3ζ) in MEC1 and MEC1-ROR1 cells. We found that MEC1-ROR1 had significantly higher rates of proliferation than MEC1-ROR1-Δ14-3-3ζ cells, which in turn had rates of proliferation comparable to those of MEC1 cells or MEC1-Δ14-3-3ζ lacking expression of ROR1. MEC1-ROR1 also had a significantly greater capacity to migrate in response to chemokine (CCL21) than did MEC1-ROR1-Δ14-3-3ζ cells, which migrated in response to CCL21 as well as MEC1-Δ14-3-3ζ or MEC1 cells lacking expression of ROR1. To examine whether such differences affected leukemia-cell growth in vivo, we engrafted Rag2−/−γc−/− mice each with equal numbers of MEC1, MEC1-Δ14-3-3ζ, MEC1-ROR1, or MEC1-ROR1-Δ14-3-3ζ cells. We found that mice that received MEC1-ROR1 cells had significantly higher levels of engraftment and human leukemia-cell proliferation than did mice that received MEC1, MEC1-Δ14-3-3ζ, or ROR1-Δ14-3-3ζ cells, which had comparable levels of engraftment and proliferation, indicating that 14-3-3ζ was necessary for the engraftment/growth advantage of MEC1-ROR1 over MEC1 cells in vivo. Collectively, this study reveals that 14-3-3ζ plays a critical role in Wnt5a/ROR1-dependent-signaling leading to enhanced migration and proliferation of CLL cells in vitro and in vivo. DisclosuresKipps:Roche: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding.

TIGAR cooperated with glycolysis to inhibit the apoptosis of leukemia cells and associated with poor prognosis in patients with cytogenetically normal acute myeloid leukemia.

BackgroundCancer cells show increased glycolysis and take advantage of this metabolic pathway to generate ATP. The TP53-induced glycolysis and apoptosis regulator (TIGAR) inhibits aerobic glycolysis and protects tumor cells from intracellular reactive oxygen species (ROS)-associated apoptosis. However, the function of TIGAR in glycolysis and survival of acute myeloid leukemia cells remains unclear.MethodsWe analyzed TIGAR expression in cytogenetically normal (CN-) AML patients and the correlations with clinical and biological parameters. In vivo and in vitro, we tested whether glycolysis may induce TIGAR expression and evaluated the combination effect of glycolysis inhibitor and TIGAR knockdown on human leukemia cell proliferation.ResultsHigh TIGAR expression was an independent predictor of poor survival and high incidence of relapse in adult patients with CN-AML. TIGAR also showed high expression in multiple human leukemia cell lines and knockdown of TIGAR activated glycolysis through PFKFB3 upregulation in human leukemia cells. Knockdown of TIGAR inhibited the proliferation of human leukemia cells and sensitized leukemia cells to glycolysis inhibitor both in vitro and in vivo. Furthermore, TIGAR knockdown in combination with glycolysis inhibitor 2-DG led leukemia cells to apoptosis. In addition, the p53 activator Nutlin-3α showed a significant combinational effect with TIGAR knockdown in leukemia cells. However, TIGAR expression and its anti-apoptotic effects were uncoupled from overexpression of exogenous p53 in leukemia cells.ConclusionsTIGAR might be a predictor of poor survival and high incidence of relapse in AML patients, and the combination of TIGAR inhibitors with anti-glycolytic agents may be novel therapies for the future clinical use in AML patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0360-4) contains supplementary material, which is available to authorized users.

Abstract 5348: The extracellular domain of thrombomodulin induces erythroid differentiation and hampers proliferation of human leukemia cells in vitro and in vivo

Abstract Thrombomodulin (TM) is a glycosylated type I transmembrane molecule with multiple domains that include an NH2-terminal lectin-like region, six tandem epidermal growth factor (EGF)-like structures, an O-glycosylation site-rich domain, a transmembrane domain, and a cytoplasmic tail domain. TM is ubiquitously expressed on endothelial cells and acts as an anticoagulant via generation of activated protein C. Recombinant human soluble thrombomodulin (rTM) comprises the active, extracellular domain of TM, and inactivates coagulation. Use of rTM for the treatment of disseminated intravascular coagulation was approved in Japan in 2008. We previous showed that exposure of acute promyelocytic leukemia or acute myelomonocytic leukemia cells to rTM induced myeloid differentiation and growth arrest in vitro (Exp Hemat. 2012, Leuk Res. 2012). The present study found that exposure of K562 and KU812 cell lines that were derived from chronic myeloid leukemia cells to rTM increased expression of cell surface glycophorin CD235a in association with upregulation of GATA1 mRNA, indicating a differentiation of leukemia cells toward erythroid lineage. Interestingly, treatment of K562 bearing immunodeficient mice with rTM significantly decreased number of K562 cells infiltrating in liver and prolonged their survival compared to mice who received control diluent. These observations shed light on a novel function of TM in hematopoietic cells. Note: This abstract was not presented at the meeting. Citation Format: Takayuki Ikezoe, Jing Yang, Chie Nishioka, Goichi Honda, Akihito Yokoyama. The extracellular domain of thrombomodulin induces erythroid differentiation and hampers proliferation of human leukemia cells in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5348. doi:10.1158/1538-7445.AM2015-5348

Naringenin suppresses K562 human leukemia cell proliferation and ameliorates Adriamycin-induced oxidative damage in polymorphonuclear leukocytes.

Treatments for leukemia remain unsatisfactory. Conventional chemotherapy agents that aim to kill tumor cells may also damage normal cells and thus result in severe side-effects. Naringenin, a natural polyphenolic compound with antioxidant effects, has been revealed to have significant antitumor effects with low toxicity in preliminary studies. Thus, it is considered as one of the most promising flavonoids in the treatment of leukemia. In the present study, the effects of naringenin on the K562 human leukemia cell line and the underlying mechanisms were explored in vitro. In addition, human peripheral blood polymorphonuclear leukocytes (PMNs) were used as a normal control in order to evaluate the effects of naringenin on normal granulocytes and in the mediation of Adriamycin (ADM)-induced oxidative damage. The results revealed that K562 proliferation was significantly inhibited by naringenin in a time- and concentration-dependent manner; however, minimal cytotoxic effects were observed in PMNs when naringenin was used at concentrations <400 μmol/l. Morphological changes indicative of apoptosis were observed in naringenin-treated K562 cells. Flow cytometric analysis indicated that the K562 cells were arrested in the G0/G1 phase of the cell cycle with a significantly upregulated rate of apoptosis. Furthermore, in the naringenin-treated K562 cells, the labeling index of proliferating cell nuclear antigen was observed to be increased by immunochemical staining, the mRNA and protein expression levels of p21/WAF1 were strongly upregulated in reverse transcription-polymerase chain reaction and western blot analyses, whereas p53 gene expression was not significantly changed. In PMNs to which naringenin (50~80 μmol/l) was added 1 h subsequent to ADM, the cell damage induced by ADM was significantly reduced, coincident with reductions in the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and increases in the activity of superoxide dismutase and glutathione peroxidase. However, the cytotoxic effect of ADM in K562 cells was not significantly altered by naringenin, and the oxidative stress indices in K562 cells remained stable. In conclusion, the present study revealed the promising value of naringenin in leukemia treatment. Naringenin demonstrated a significant inhibitory effect on the growth of K562 cells but not on normal PMNs. Furthermore, naringenin protected PMNs from ADM-induced oxidative damage at low concentrations. Cell cycle arrest and apoptosis-inducing effects, achieved through p53-independent p21/WAF1 upregulation, are likely to be the mechanism of the antileukemic effects of naringenin, and the protective effect against ADM chemotherapy-induced damage in PMNs may be due to the antioxidant capability of this agent at low concentrations.

Rapamycin Induced Autophagy and Suppression in Human Leukemia Cells Proliferation

To investigate the effect of autophagy induced by rapamycin (Rap) on proliferation of Human Leukemia Cells (HL60). HL60 cells are treated with rapamycin to induce autophagy. Then, Western blot is performed to examine the expression of Beclin1 and LC3. MTT assay is used to evaluate cell proliferation. The cell cycle is analyzed by flow cytometry (FCM). After treatment with rapamycin for 24 hours, LC3 is dramatically increased at protein level and autophagic activity is significantly increased. And MTT assay indicated that cell proliferation is inhibited by rapamycin. Compared with control group, more cells are arrested at G0/ G1 phases. We conclude that rapamycin can induce autophagy and suppress proliferation in HL60 cells.

Gö6976, a FLT3 kinase inhibitor, exerts potent cytotoxic activity against acute leukemia via inhibition of survivin and MCL-1

Mutations of the FMS-like tyrosine kinase 3 (FLT3) have been reported in about a third of patients with acute myeloid leukemia (AML). The presence of FLT3 mutations confers a poor prognosis. Thus, pharmacological inhibitors of FLT3 are of therapeutic interest for AML. Gö6976 is an indolocarbazole with a similar structural backbone to staurosporine. In the present study, we demonstrated that Gö6976 displays a potent inhibitory activity against recombinant FLT3 using an in vitro kinase assay, with an IC50 value of 0.7nM. Gö6976 markedly inhibited the proliferation of human leukemia cells having FLT3-ITD such as MV4-11 and MOLM13. We also observed that Gö6976 showed minimal toxicity for human normal CD34(+) cells. Gö6976 suppressed the phosphorylation of FLT3 and downstream signaling molecules such as STAT3/5, Erk1/2, and Akt in MV4-11 and MOLM13 cells. Interestingly, induction of apoptosis by Gö6976 was associated with rapid and pronounced down-regulation of the anti-apoptotic protein survivin and MCL-1. Suppression of survivin protein expression by Gö6976 was due to the inhibition of transcription via the suppression of STAT3/5. On the other hand, Gö6976 induced proteasome-mediated degradation of MCL-1. Previously described FLT3 inhibitors such as PKC412 are bound by the human plasma protein, α1-acid glycoprotein, resulting in diminished inhibitory activity against FLT3. In contrast, we found that Gö6976 potently inhibited phosphorylation of FLT3 and exerted cytotoxicity in the presence of human serum. In conclusion, Gö6976 is a potent FLT3 inhibitor that displays a significant antiproliferative activity against leukemia cells with FLT3-ITD through the profound down-regulation of survivin and MCL-1.

Go6976, a Potent FLT3 Kinase Inhibitor, Exerts Antiproliferative Activity Against Acute Myeloid Leukemia Via Inhibition Of Survivin and Mcl-1 In The Presence Of Human Serum

The FMS-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase involved in hematopoietic progenitor cell development. Mutations of FLT3 have been reported in about a third of patients with acute myeloid leukemia (AML). The presence of FLT3 mutations confers a poor prognosis, and thus recent studies are directed at developing and testing novel FLT3 inhibitors for the treatment of AML. Go6976 is an indolocarbazole with a simillar structural backbone to staurosporine. In the present study, we demonstrated that Go6976 displays a potent inhibitory activity against recombinant FLT3 using in vitro kinase assay. Its IC50 value is 0.7nM. We also tested the effect of Go6976 on several kinds of other kinases. Go6976 inhibited the kinase activity of Aurora-A, Aurora-B and JAK2 with IC50 values of 118.2 nM, 77.7 nM and 92.7 nM, respectively. Go6976 did not show the inhibitory activity against the FGFR3 even at 1 microM. These data indicated Go6976 preferentially and potently inhibit the FLT3. Go6976 significantly inhibited the proliferation of human leukemia cells having FLT3-ITD. The IC50 values of Go6976 against MV4-11 and MOLM-13 were 0.044 and 0.008 microM, respectively. In contrast, human leukemia HL-60 and U937 cells which lack FLT3-ITD showed strong resistance to Go6976 treatment. Furthermore, we observed that Go6976 shows minimal toxicity for purified human normal CD34(+) cells. Go6976 suppressed the phosphorylation of FLT3 in MV4-11 and MOLM13 cells. Consistent with FLT3 inhibition, Go6976 potently inhibited phosphorylation of constitutively activated STAT3/5, Erk1/2, and Akt. Western blotting analysis revealed that MV4-11 and MOLM13 cells possess abundant survivin and Mcl-1 protein. We hypothesized that the expression of survivin and Mcl-1 may be regulated by constitutive activation of FLT3. In order to test this hypothesis, the effect of siRNA for FLT3-ITD was examined. Indeed, we observed that siRNA-induced down-regulation of FLT3 decreased survivin and Mcl-1 expression in MOLM13 cells, suggesting that up-regulated survivin and Mcl-1 may be closely associated with FLT3 signaling. Interestingly, we found that both survivin mRNA and protein were rapidly downregulated by Go6976 treatment in MOLM13 and MV4-11 cells. It was also observed that Go6976 significantly suppressed Mcl-1 mRNA and protein. It has been reported that STAT-3 and STAT-5 signaling play a pivotal role in the regulation of survivin and Mcl-1, respectively. Thus, inhibitory effects of Go6976 on the expression Survivin and Mcl-1 may be a consequence of the suppression of phosphorylation of STAT-3 and STAT-5 by Go6976 in FLT3-ITD cells. This inhibition of anti-apoptotic proteins by Go6976 may be critical for its antiproliferative effect in FLT3-ITD cells. It has been known that previous FLT3 inhibitors such as PKC412 and CEP-701 bind to the human plasma protein, alpha1-acid glycoprotein, resulting in significantly diminished inhibitory activity against FLT3. Indeed, inhibitory effect of PKC412 on FLT3 was significantly decreased, when MOLM13 cells were treated with PKC412 in the presence of human serum. In contrast, we found that Go6976 potently inhibits phosphorylation of FLT3 and exerts cytotoxicity even in the presence of human serum or human alpha1-acid glycoprotein. In conclusion, our data indicate that Go6976 may have a unique therapeutic potential for FLT3-driven acute myeloid leukemia. Disclosures:No relevant conflicts of interest to declare.

Olean-12-Eno[2,3-c] [1,2,5]Oxadiazol-28-Oic Acid (OEOA) Induces G1 Cell Cycle Arrest and Differentiation in Human Leukemia Cell Lines

Oleanolic acid (3β-hydroxy-olea-12-en-28-oic acid) is a natural pentacyclic triterpenoic acid found in many fruits, herbs and medicinal plants. In the past decade, increasing evidence has suggested that oleanolic acid exhibits inhibitory activities against different types of cancer including skin cancer and colon cancer, but not leukemia. We report here that a derivative of oleanolic acid, olean-12-eno[2,3-c] [1], [2], [5]oxadiazol-28-oic acid (designated OEOA) effectively blocks the proliferation of human leukemia cells. OEOA significantly reduces cell proliferation without inducing cell death in three types of leukemia cell lines, including K562, HEL and Jurket. Moreover, exposure of K562 cells to OEOA results in G1 cell cycle arrest, with a concomitant induction of cyclin-dependent kinase inhibitor p27 and downregulation of cyclins and Cdks that are essential for cell cycle progression. Interestingly, OEOA also enhances erythroid differentiation in K562 cells through suppressing the expression of Bcr-Abl and phosphorylation of Erk1/2. These findings identify a novel chemical entity for further development as therapeutics against leukemia.

Properties of Mechano-Transduction via Simulated Microgravity and its Effects on Intracellular Trafficking of VEGFR's

This study emphasizes the dynamical properties of mechanical loading via simulated microgravity, its effect on acute myeloid leukemia proliferation and hematopoietic stem cell (HSPC) growth and its implications in the area of tissue regeneration. Data presented illustrates that mechanical transduction changes the expression of humoral factors by facilitating paracrine/autocrine signalling, therefore modulating intracellular trafficking of tyrosine kinase receptors. Understanding mechano-transduction in the context of cell and tissue morphogenesis is the major focus of this study. The effects of external physiological stresses, such as blood flow, on several cellular subtypes seem to produce very intricate cellular responses. It is well accepted that mechanical loading plays an intrinsic and extrinsic influence on cell survival. This study shows how microgravity effects hematopoietic stem cells, and human leukemic cell proliferation and expression of its receptors that control cell survival, such as the tyrosine kinase vascular endothelial growth factor receptor-1, receptor-2 and receptor-3.

Induction of G1 Arrest and Apoptosis in Human Cancer Cells by Crebanine, an Alkaloid from &lt;i&gt;Stephania venosa&lt;/i&gt;

In this study, we focused the effects of crebanine, an alkaloid isolated from the tuber of Stephania venosa, on various human cancer cells. Crebanine treatment was found to significantly inhibit the proliferation of human leukemic cells (HL-60, U937 and K562), human fibrosarcoma cells (HT1080) and cervix cancer cell lines (KB-3-1 and KB-V1), of which HL-60 cells were the most sensitive to its treatment. In contrast, crebanine caused much less toxicity in human normal fibroblast cells. Our results demonstrated that crebanine mediated cell cycle arrest at G0/G1 phase and this was associated with down-regulation of cyclins A and D. In addition, crebanine induced apoptosis, which was detected by observation of the membrane phospholipid exposure in flow cytometry. Its induction of apoptosis was accompanied by an increase in cleavage of caspase-3, -8, -9 and poly(ADP-ribose) polymerase (PARP), and was attributable to the augmentation of Bax/Bcl proteins level. Crebanine also decreased mitochondrial membrane potential. Taken together, crebanine exerts anti-proliferative effects on human cancer cells through the induction of cell cycle arrest at the G1 phases and apoptosis. Our results suggest that crebanine is a promising new candidate as a chemotherapeutic agent for cancer therapy.

Abstract C71: Overcoming mechanism(s) of resistance to active-site TOR inhibitors in leukemia and lymphoma.

Abstract Introduction: The PI3K/AKT/TOR signaling network has been the focus of intense research in the cancer field as its activity is commonly elevated across cancers including leukemia and lymphoma. As an integral effector of the network, the target of rapamycin (TOR) functions as the catalytic subunit of two distinct protein complexes, TORC1 and TORC2. Both complexes promote survival of hematological malignancies through protein translation (TORC1) and AKT activation (TORC2). Unlike the classical allosteric TOR inhibitors (e.g. rapamycin), a new class of active-site TOR inhibitors (asTORi) more completely inhibits TOR enzymatic function in both TORC −1 and −2 complexes. TORC2 inhibition correlates with accumulation of FOXO transcription factors in the nucleus. Our lab showed that asTORi kill mouse leukemia and inhibit proliferation of human leukemia cells in vitro and in vivo. Here we investigated mechanisms of resistance to asTORi-mediated cell death in a human leukemia as well as a panel of human lymphoma cell lines. We show that asTORi induce nuclear localization of FOXO and transcription of cytostatic (i.e. p27) but not pro-apoptotic (ex. TRAIL) FOXO target genes. Combining asTORi with pharmacologically achievable concentrations of the histone deacetylase inhibitor (HDACi) upregulates transcription of TRAIL and dramatically enhances death of human leukemia cells in vitro. Similarly, asTORi potentiate a small molecule antagonist of the anti-apoptotic Bcl-2 family proteins whose expression is commonly elevated in lymphoma. Material and Methods: Cell Lines and reagents: The mouse p190 and human SUP-B15 cell lines are Ph+ B precursor acute lymphoblastic leukemia cells. The human lymphoma cell lines OCI-LY1, SUDHL-4, SUDHL-6 and RCK8 are germinal-center diffuse large-B-cell non-Hodgkins lymphoma (DLBCL). The HDACi (Vorinostat) and BCL-2 antagonist (ABT-263) were purchased from Merck &amp; Co and Selleck. The active-site TOR inhibitors were generously provided by Intellikine Inc. Cell cycle and death analysis: Flow cytometry was used in conjunction with 7-amino actinomycin D or annexin V staining along with forward scatter profile to assay cell viability. Flow cytometry and propidium iodide staining was used to measure DNA content for cell cycle analysis. qPCR: RNA was isolated using trizol method and analyzed by real time PCR. Subcellular localization: SUP-B15 and OCI-LY1 cell lines were fractionated into cytosolic and nuclear fraction then probed with anti-FOXO1 antibody. Anti -actin and -histone antibodies were used to confirm the purity of the fractions. Conclusion:The human leukemia cell line SUP-B15 and DLBCL cell lines survive inhibition of TOR by asTORi.asTORi promotes nuclear localization of FOXO and transcription of the cytostatic effector and FOXO target gene, p27.Combining HDACi with asTORi promotes transcription of TRAIL, a pro-apoptotic FOXO target gene, and markedly enhances apoptosis in SUP-B15 cells.asTORi potentiates cytotoxic effects of the BCL-2 inhibitor in DLBCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C71.

Multidrug Resistance Protein 4 (MRP4/ABCC4) Regulates cAMP Cellular Levels and Controls Human Leukemia Cell Proliferation and Differentiation

Increased intracellular cAMP concentration plays a well established role in leukemic cell maturation. We previously reported that U937 cells stimulated by H2 receptor agonists, despite a robust increase in cAMP, fail to mature because of rapid H2 receptor desensitization and phosphodiesterase (PDE) activation. Here we show that intracellular cAMP levels not only in U937 cells but also in other acute myeloid leukemia cell lines are also regulated by multidrug resistance-associated proteins (MRPs), particularly MRP4. U937, HL-60, and KG-1a cells, exposed to amthamine (H2-receptor agonist), augmented intracellular cAMP concentration with a concomitant increase in the efflux. Extrusion of cAMP was ATP-dependent and probenecid-sensitive, supporting that the transport was MRP-mediated. Cells exposed to amthamine and the PDE4 inhibitor showed enhanced cAMP extrusion, but this response was inhibited by MRP blockade. Amthamine stimulation, combined with PDE4 and MRP inhibition, induced maximal cell arrest proliferation. Knockdown strategy by shRNA revealed that this process was mediated by MRP4. Furthermore, blockade by probenecid or MRP4 knockdown showed that increased intracellular cAMP levels induce maturation in U937 cells. These findings confirm the key role of intracellular cAMP levels in leukemic cell maturation and provide the first evidence that MRP4 may represent a new potential target for leukemia differentiation therapy.

Metal-based biologically active agents: Synthesis, characterization, antibacterial and antileukemia activity evaluation of Cu(II), V(IV) and Ni(II) complexes with antipyrine-derived compounds

The paper presents the synthesis of complex combinations of Cu(II), V(IV) and Ni(II) with Schiff bases obtained through the condensation of 4-amino-1,5-dimethyl-2-phenyl-1H-3-pyrazol-3(2H)-one (antipyrine) with 2-hydroxybenzaldehyde, 4-hydroxy-5-methoxyisophthalaldehyde and 4,5-dihydroxyisophalaldehyde respectively. The characterization of newly formed complexes was done by 1H NMR, 13C NMR, UV–VIS, IR, EPR spectroscopies and molar electric conductibility studies. The effect of these complexes on proliferation of human leukemia cells (HL-60) and their antibacterial activity against Staphylococcus aureus var. Oxford 6538, Escherichia coli ATCC 10536 and Candida albicans ATCC 10231strains were studied and compared with those of free ligands.

Novel oligosaccharide has suppressive activity against human leukemia cell proliferation

Various oligosaccharides containing galactose(s) and one glucosamine (or N-acetylglucosamine) residues with beta1-4, alpha1-6 and beta1-6 glycosidic bond were synthesized; Galbeta1-4GlcNH(2), Galalpha1-6GlcNH(2), Galalpha1-6GlcNAc, Galbeta1-6GlcNH(2), Galbeta1-4Galbeta1-4GlcNH(2) and Galbeta1-4Galbeta1-4GlcNAc. Galalpha1-6GlcNH(2) (MelNH(2)) and glucosamine (GlcNH(2)) had a suppressive effect on the proliferation of K562 cells, but none of the other saccharides tested containing GlcNAc showed this effect. On the other hand, the proliferation of the human normal umbilical cord fibroblast was suppressed by none of the saccharides other than GlcNH(2). Adding Galalpha1-6GlcNH(2) or glucosamine to the culture of K562 cell, the cell number decreased strikingly after 72 h. Staining the remaining cells with Cellstain Hoechst 33258, chromatin aggregation was found in many cells, indicating the occurrence of cell death. Furthermore, all of the cells were stained with Galalpha1-6GlcNH-FITC (MelNH-FITC). Neither the control cells nor the cells incubated with glucosamine were stained. On the other hand, when GlcNH-FITC was also added to cell cultures, some of them incubated with Galalpha1-6GlcNH(2) were stained. The difference in the stainability of the K562 cells by Galalpha1-6GlcNH-FITC and GlcNH-FITC suggests that the intake of Galalpha1-6GlcNH(2) and the cell death induced by this saccharide is not same as those of glucosamine. The isolation of the Galalpha1-6GlcNH(2) binding protein was performed by affinity chromatography (melibiose-agarose) and LC-MS/MS, and we identified the human heterogeneous ribonucleoprotein (hnRNP) A1 (34.3 kDa) isoform protein (30.8 kDa). The hnRNP A1 protein was also detected from the eluate(s) of the MelNH-agarose column by the immunological method (anti-hnRNP-A1 and HRP-labeled anti-mouse IgG (gamma) antibodies).