Human Chronic Myeloid Leukemia Cells Research Articles

Proteome changes induced by c-myb silencing in human chronic myeloid leukemia cells suggest molecular mechanisms and putative biomarkers of hematopoietic malignancies

To shed light on the molecular mechanisms associated with aberrant accumulation of c-Myb in chronic myeloid leukemia, comparative proteomic analysis was performed on c-myb RNAi-specifically silenced K562 cells, sampled on a time-course basis. 2D-DIGE technology highlighted 37 differentially-represented proteins that were further characterized by nLC-ESI-LIT-MS/MS and validated by western blotting and qRT-PCR analysis. Most of the deregulated proteins were related to protein folding, energy/primary metabolism, transcription/translation regulation and oxidative stress response. Protein network analysis suggested that glycolysis, gluconeogenesis and protein ubiquitination biosynthesis pathways were highly represented, confirming also the pivotal role of c-Myc. A specific reduced representation was observed for glyceraldehyde-3-phosphate-dehydrogenase and α-enolase, suggesting a possible role of c-Myb in the activation of aerobic glycolysis. A reduced amount was also observed for stress responsive heat shock 70kDa protein and 78kDa glucose-regulated protein, previously identified as direct targets of c-Myb. Among over-represented proteins, worth mentioning is the chromatin modifier chromobox protein homolog 3 that contributes to silencing of E2F- and Myc-responsive genes in quiescent G0 cells. Data here presented, while providing novel insights onto the molecular mechanisms underlying c-Myb activity, indicate potential protein biomarkers for monitoring the progression of chronic myeloid leukemia. Myeloid leukemia is a malignant disease of the hematopoietic system in which cells of myeloid lineages accumulate to an undifferentiated state. In particular, it was shown that an aberrant accumulation of the c-Myb transcriptional factor is associated with the suppression of normal differentiation processes promoting the development of the hematopoietic malignancies. Many efforts have been recently made to identify novel genes directly targeted by c-Myb at a transcriptome level. In this work, we originally describe a differential proteomic approach to facilitate the comprehension of the regulation of the protein networks exerted by c-Myb. Our study reveals a complex network of proteins regulated by c-Myb. The functional heterogeneity of these proteins emphasizes the pleiotropic role of c-Myb as a regulator of genes that are crucial for energy production and stress response in leukemia. In fact, variations in glyceraldehyde-3-phosphate-dehydrogenase and α-enolase suggest a possible role of c-Myb in the activation of aerobic glycolysis. Moreover, significant differences were found for heat shock 70kDa protein and 78kDa glucose-regulated protein known as direct c-Myb targets. This work highlights potential protein biomarkers to look into disease progression and to develop translational medicine approaches in myeloid leukemia.

DNA interaction of [Cu(dmp)(phen-dion)] (dmp = 4,7 and 2,9 dimethyl phenanthroline, phen-dion = 1,10-phenanthroline-5,6-dion) complexes and DNA-based electrochemical biosensor using chitosan–carbon nanotubes composite film

The interaction of two new water-soluble [Cu(4,7-dmp)(phen-dione)Cl]Cl (1) and [Cu(2,9-dmp)(phen-dione)Cl]Cl (2) which dmp is dimethyl-1,10-phenanthroline and phen-dion represents 1,10-phenanthroline-5,6-dion, with DNA in solution and immobilized DNA on a chitosan–carbon nanotubes composite modified glassy carbon electrode were investigated by cyclic voltammetry and UV–Vis spectroscopy techniques. In solution interactions, spectroscopic and electrochemical evidences indicate outside binding of these complexes. To clarify the binding mode of complexes, it was done competition studies with Hoechst and Neutral red as groove binder and intercalative probes, respectively. All these results indicating that, these two complexes (1) and (2) interact with DNA via groove binding and partially intercalative modes, respectively. The electrochemical characterization experiments showed that the nanocomposite film of chitosan–carbon nanotubes could effectively immobilize DNA and greatly improve the electron-transfer reactions of the electroactive molecules that latter finding is the result of strong interactions between captured DNA and Cu complexes. This result indicates that these complexes could be noble candidates as hybridization indicators in further studies. At the end, these new complexes showed excellent antitumor activity against K562 (human chronic myeloid leukemia) cell lines.

Anticancer effect of altersolanol A, a metabolite produced by the endophytic fungus Stemphylium globuliferum, mediated by its pro-apoptotic and anti-invasive potential via the inhibition of NF-κB activity

Altersolanol A, a natural product from the endophytic fungus Stemphylium globuliferum isolated from the medicinal plant Mentha pulegium (Lamiaceae) growing in Morocco, shows cytotoxic, cytostatic, anti-inflammatory and anti-migrative activity against human chronic myeloid K562 leukemia and A549 lung cancer cells in a dose dependent manner without affecting the viability of non cancerous cells. Altersolanol A induces cell death by apoptosis through the cleavage of caspase-3 and -9 and through the decrease of anti-apoptotic protein expression. Moreover, we report here the importance of the distinct structural features of altersolanol A by testing other related anthracene derivatives in order to identify preliminary structure–activity relationships. Acetylation of altersolanol A did not improve activity where other derivatives such as tetrahydroaltersolanol B and ampelanol that differ from altersolanol A by reduction of one of a carbonyl group and removal of hydroxyl substituents were inactive in comparison. Altogether our results suggest that altersolanol A may be considered as an interesting lead for further development of chemotherapeutic agents.

Aclacinomycin A Sensitizes K562 Chronic Myeloid Leukemia Cells to Imatinib through p38MAPK-Mediated Erythroid Differentiation

Expression of oncogenic Bcr-Abl inhibits cell differentiation of hematopoietic stem/progenitor cells in chronic myeloid leukemia (CML). Differentiation therapy is considered to be a new strategy for treating this type of leukemia. Aclacinomycin A (ACM) is an antitumor antibiotic. Previous studies have shown that ACM induced erythroid differentiation of CML cells. In this study, we investigate the effect of ACM on the sensitivity of human CML cell line K562 to Bcr-Abl specific inhibitor imatinib (STI571, Gleevec). We first determined the optimal concentration of ACM for erythroid differentiation but not growth inhibition and apoptosis in K562 cells. Then, pretreatment with this optimal concentration of ACM followed by a minimally toxic concentration of imatinib strongly induced growth inhibition and apoptosis compared to that with simultaneous co-treatment, indicating that ACM-induced erythroid differentiation sensitizes K562 cells to imatinib. Sequential treatment with ACM and imatinib induced Bcr-Abl down-regulation, cytochrome c release into the cytosol, and caspase-3 activation, as well as decreased Mcl-1 and Bcl-xL expressions, but did not affect Fas ligand/Fas death receptor and procaspase-8 expressions. ACM/imatinib sequential treatment-induced apoptosis was suppressed by a caspase-9 inhibitor and a caspase-3 inhibitor, indicating that the caspase cascade is involved in this apoptosis. Furthermore, we demonstrated that ACM induced erythroid differentiation through the p38 mitogen-activated protein kinase (MAPK) pathway. The inhibition of erythroid differentiation by p38MAPK inhibitor SB202190, p38MAPK dominant negative mutant or p38MAPK shRNA knockdown, reduced the ACM/imatinib sequential treatment-mediated growth inhibition and apoptosis. These results suggest that differentiated K562 cells induced by ACM-mediated p38MAPK pathway become more sensitive to imatinib and result in down-regulations of Bcr-Abl and anti-apoptotic proteins, growth inhibition and apoptosis. These results provided a potential management by which ACM might have a crucial impact on increasing sensitivity of CML cells to imatinib in the differentiation therapeutic approaches.

Abstract 2170: Development of a "dual-hit" combination therapy for CML.

Abstract Functioning as a constitutively active tyrosine kinase, the oncoprotein Bcr-Abl, the causative agent of chronic myeloid leukemia (CML), requires homo-oligomerization via a coiled-coil domain present on the N-terminus of the Bcr portion of the protein. Currently, small molecule therapeutics that target the tyrosine kinase domain (tyrosine kinase inhibitors, or TKIs) are administered as the gold standard treatment for CML. However, evidence shows that the acquisition of mutations in the tyrosine kinase domain against these small molecules is inevitable, rendering many of the therapeutics useless against mutated forms of Bcr-Abl. The most recently discovered TKI, ponatinib (AP24534), has shown to combat nearly all of the commonly seen mutations, including the previously uncontrollable T315I mutation. But, ponatinib is not without limitations, as it has been shown to cause dose-dependent pancreatitis and thrombocytopenia as adverse side effects. While many small molecules have been created to target the tyrosine kinase domain, our recent work has shown that disruption of Bcr-Abl oligomerization can also induce apoptosis and decrease the oncogenic potential of the disease. This work was done using a modified coiled-coil domain, termed CCmut3, which was rationally designed and mutated to bind to the endogenous Bcr-Abl coiled-coil domain, inhibiting oligomerization and thus preventing Bcr-Abl signaling. The purpose of this work is to exploit a “dual-hit” approach to inhibit Bcr-Abl using two therapeutics with different mechanisms of action. Here, we show that combining CCmut3 with ponatinib not only increases the potency of the therapy but also lowers the required therapeutic dose of ponatinib in vitro using K562 cells (Bcr-Abl-positive, human CML cells). Western blot analysis shows that both Bcr-Abl phosphorylation and phosphorylation of downstream signaling targets STAT5 and CrkL can be decreased using the combination at a ponatinib dose of 100 pM. In addition, transformative ability (via colony forming assay) very significantly declines when using a 1 nM dose in combination as opposed to simply administering 1 nM ponatinib only. Finally, caspase 3/7, 7AAD, and DNA segmentation assays were performed to show enhanced apoptosis induction upon combination treatment when compared with either CCmut3 or ponatinib treatment alone. Currently, this combination is being tested in a Bcr-Abl-positive Ba/F3 cell line containing the T315I mutation in the Abl tyrosine kinase domain. Lastly, attempts to truncate and cap the CCmut3 helix to provide stability and more simple therapeutic delivery are underway, in addition to elucidating the individual intracellular mechanisms of action of both CCmut3 and ponatinib. Citation Format: Geoffrey D. Miller, David W. Woessner, Carol S. Lim. Development of a "dual-hit" combination therapy for CML. [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 2170. doi:10.1158/1538-7445.AM2013-2170

Abstract 1671: A genome-wide RNA interference screen identifies autophagy mediators with therapeutic implications in chronic myeloid leukemia.

Abstract Autophagy plays a critical role in cancer formation and therapeutic resistance. However, little is known about how autophagy is regulated in cancer and how it mediates therapeutic resistance. Here we elect to use chronic myeloid leukemia (CML) as a cancer model to study autophagy in that it is driven by a single onco-protein BCR-ABL, whose activity can be selectively blocked by imatinib a front-line treatment for CML. Moreover, imatinib resistance frequently occurs in CML patients. Thus, unraveling autophagy regulation in CML and its role in overcoming imatinib resistance has substantial therapeutic benefits not only for CML but also for other cancers that can be treated by imatinib. In this report, we performed a genome-wide RNA interference screen in K562 human CML cells using monodansylcadaverine (MDC) that marks autolysosomes followed by fluorescence-activated cell sorting to label and isolate autophagic cells. We have identified 336 candidate genes, knockdown of which significantly increased MDC fluorescence. Our further validation utilized Cyto-IDTM Green dye that selectively stains autophagy vacuoles and quantitative RT-PCR that measures knockdown efficiency of shRNAs. We have uncovered a set of genes acting as autophagy mediators in K562 cells. To study their role in imatinib resistance, we measured the effect of imatinib in combination with the knocking down of autophagy mediators. Our results showed that the shRNA of VAMP7, a pivotal factor in vesicle transportation, substantially sensitized K562 cells to imatinib. Intriguingly, our further work revealed that depletion of VAMP7 blocked the activity of key autophagy suppressors AKT and mTOR, suggesting that VAMP7 and perhaps other autophagy mediators regulate imatinib sensitivity through signaling pathways that inhibit autophagy. In conclusion, a set of genes revealed by a genome-wide RNA interference screen mediates autophagy in CML cells and, more importantly, some of these genes render CML cells resistant to imatinib thereby becoming appealing drug targets. Citation Format: Zhi Sheng, Sujuan Guo, Susan Murphy, Hanne Varmark, Amy Virbasius, Michael Green. A genome-wide RNA interference screen identifies autophagy mediators with therapeutic implications in chronic myeloid leukemia. [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 1671. doi:10.1158/1538-7445.AM2013-1671

An anti-tumor protein produced by Trichinella spiralis induces apoptosis in human hepatoma H7402 cells

Trichinella spiralis infection confers effective resistance to tumor cell expansion. In this study, a T7 phage cDNA display library was constructed to express genes encoded by T. spiralis. Organic phase multi-cell screening was used to sort through candidate proteins in a transfected human chronic myeloid leukemia cell line (K562) and a human hepatoma cell line (H7402) using the display library. The protein encoded by the A200711 gene was identified and analyzed using protein analysis software. To test the antitumor effects of A200711, variations in cell proliferation and apoptosis were monitored after recombinant pEGFP-N1-A200711 was transfected into H7402 cells. The results show that the expressed target gene successfully induced apoptosis in H7402 cells as measured by Hoechst-PI staining, MTT assay (p<0.05). This study warrants further investigation into the therapeutic use of A200711 for anti-hepatocellular carcinomas.

Design, Synthesis, and In vitro Antitumor Evaluation of Novel Phenylaminopyrimidine Derivatives

Two series of novel phenylaminopyrimidine derivatives were designed and synthesized. All target compounds were determined against the human acute monocytic leukemia cell line U937 and the human chronic myeloid leukemia cell line K562 in vitro. Some of the target compounds demonstrated significant inhibitory activity against both cell lines. Compared with the control drug VX-680, 8a, 8e, 8g, 8h, 8j, and 8k demonstrated more potent antitumor activity. The structures of all target compounds were identified by 1H-NMR, 13C-NMR, IR, MS, and EA.

Notch2 inhibits proliferation of chronic myeloid leukemia cells.

The Notch signaling pathway has been shown to be involved in the progression of chronic myeloid leukemia (CML). The aim of this study was to investigate the effects of exogenous Notch2 overexpression on cell proliferation and possible mechanisms in the human CML cell line K562. When exogenous intracellular fragment of Notch2 (ICN2) was transfected into K562 cells with Lipofectamine™ 2000, the expression of Notch2 mRNA and protein were upregulated. Cell numbers decreased and the proliferation was inhibited significantly after transfection with ICN2. G1 phase cells increased and S phase cells decreased 48 h after transfection. Finally, the expression of Numb, Bcl-2, NF-κB and TGF-β1 was detected. It was found that the expression of NF-κB and TGF-β1 mRNA was increased, while Bcl-2 was downregulated, with Numb expression unchanged. Our study indicates that the Notch pathway is activated in K562 cells after ICN2 transfection. It inhibited the proliferation of K562 cells, likely by upregulating the expression of NF-κB and TGF-β1 mRNA and downregulating the expression of Bcl-2.

A comparative study of tellurite toxicity in normal and cancer cells

Data regarding tellurium (Te) toxicity are scarce but metabolic studies in bacteria highlight a major role of reactive oxygen species (ROS). Concentration- and time-dependent cell death has been reported in cancer cells exposed to inorganic tellurite. However, the potential in vitro effect that tellurite could generates in non-transformed human cells is still unknown. Therefore, we have studied the toxicity of inorganic tellurite (K2TeO3) in both freshly isolated peripheral blood leukocytes and in human chronic myeloid leukemia cells (K562 cells). Interestingly, we observed higher increases of ROS in leukocytes after treatment with tellurite, as compared to K562 cells. Given the high reactivity of tellurite with glutathione (GSH), a mechanism that leads to ROS formation (and mainly superoxide anion), we postulate that such a difference between cancer and normal cells is likely due to the higher GSH contents found in leukocytes versus leukemia cells. Taken together, our data point out the major differences that can be observed between cancer and corresponding normal cells in studies looking for in vitro toxicity.

Mechanisms responsible for nilotinib resistance in human chronic myeloid leukemia cells and reversal of resistance

Multidrug resistance remains a significant obstacle to successful chemotherapy. The ability to determine the possible resistance mechanisms and surmount the resistance is likely to improve chemotherapy. Nilotinib is a very effective drug in the treatment of imatinib-sensitive or -resistant patients. Although very successful hematologic and cytogenetic responses have been obtained in nilotinib-treated patients, in recent years cases showing resistance to nilotinib have been observed. We aimed to examine the mechanisms underlying nilotinib resistance and to provide new targets for the treatment of chronic myeloid leukemia (CML). There was an up-regulation of antiapoptotic BCR/ABL, GCS and SK-1 genes and MRP1 transporter gene and down-regulation of apoptotic Bax and CerS1 genes in nilotinib-resistant cells. There was no mutation in the nilotinib-binding region of BCR/ABL in resistant cells. Inhibiton of GCS and SK-1 restored nilotinib sensitivity. Targeting the proteins that are involved in nilotinib resistance in addition to the inhibition of BCR/ABL could be a better method of treatment in CML.

Metastasis Suppressor 1 Is Downregulated in CML Stem Cells and Overexpression Impairs Early Leukemic Cell Propagation.

AbstractAbstract 2776The implementation of Bcr-Abl tyrosine kinase inhibitors (TKIs) has greatly improved the outcome of patients with chronic myeloid leukemia (CML). However, discontinuation of TKI therapy often results in relapse suggesting that leukemic stem cells (LSCs) survive despite treatment. More detailed investigations utilizing patient samples and murine CML models have confirmed that the leukemia-initiating cell population is usually not eradicated by inhibiting Bcr-Abl activity and that this is due to a lack of oncogene addiction of LSCs, showing that further research is required aiming to fully understand LSC biology.To identify new Bcr-Abl targets that are involved in LSC persistence, we performed a microarray analysis of the leukemia-initiating cell population in an inducible transgenic SCLtTAxBcr-Abl CML mouse model in which we had previously shown that these cells are not oncogene-addicted (Schemionek et al., BLOOD 2010; Hamilton et al., BLOOD 2012). One of the most downregulated genes in CML vs. normal stem cells was Metastasis Suppressor 1 (Mtss1/MIM). Although the multidomain protein Mtss1 may be involved in carcinogenesis of several solid tumors, its exact physiological role is still unknown. Current findings suggest that Mtss1 interacts with multiple partners and is involved in various signalling pathways that regulate actin dynamics and cell motility. Interestingly, Rac and Src are Mtss1 interacting partners, and both proteins are known to be involved in Bcr-Abl mediated leukemogenesis.We have previously shown that Mtss1 is downregulated in mouse LSCs and demonstrated that this is a Bcr-Abl kinase mediated effect in various human and murine CML cell lines. Moreover, we have shown that Mtss1 overexpression in 32D-Bcr-Abl cells induces a defect in Bcr-Abl mediated migration in vitro and reduces the potential to form solid tumors in vivo.Here we show that Bcr-Abl kinase-dependent regulation of Mtss1 expression was also evident in mononuclear cells and CD34+ progenitor cells from patients with CML upon IM or dasatinib treatment in vitro. Moreover, cells from IM-treated patients with chronic phase CML showed elevated Mtss1 expression levels within one to three weeks of treatment. Increasing Mtss1 expression upon 5-aza-2′-deoxycytidine-treatment of K562 and 32D-Bcr-Abl cells suggested that methylation might be involved in Mtss1 regulation. To determine a potential leukemia suppressing effect of Mtss1 overexpression, we performed colony assays using lineage negative SCLtTAxBcr-Abl (dtg) bone marrow (BM) cells that had been retrovirally infected to overexpress Mtss1 (dtg::Mtss1) or empty-vector (dtg::ev). Successfully transduced BM cells were FACS-sorted via GFP-expression, encoded by the retroviral vector. Mtss1 overexpression led to a 2.3-fold decrease in CFU numbers. In a second set of experiments we transplanted 1.3×105 GFP-FACS-sorted dtg::Mtss1 or dtg::ev cells into 9 Gy-irradiated recipients. While dtg::ev recipients contained 66% (+/−8%) of GFP-positive cells in the BM, these cells were decreased in dtg::Mtss1 transplanted mice to 23% (+/−21%), 12 days after transplantation. A similar effect was evident in the spleen [dtg::ev recipients: 90% (+/− 3%) versus dtg::Mtss1 recipients 59% (+/−20%)] suggesting that Mtss1 confers a disadvantage to Bcr-Abl positive BM cells in the early steps of leukemic cell propagation, compared to Bcr-Abl cells alone.Since the multidomain Mtss1 protein contains a putative Abl-SH2-binding site, we performed co-immunoprecipitations using 32D-Bcr-Abl-Flag-Mtss1 cells. These experiments showed that both proteins were direct binding partners and that Mtss1 was not phosphorylated by Bcr-Abl.Taken together, our data show that Mtss1 is downregulated via a Bcr-Abl kinase mediated mechanism and this might involve methylation. Moreover, additional inhibition of Mtss1 activity might be mediated through direct binding by Bcr-Abl. Forced expression of the potential tumor suppressor in CML stem and progenitor cells reduces leukemic cell propagation in vivo and may thus provide a rationale to contribute to LSC elimination in patients with CML. Disclosures:Mhairi:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria. Koschmieder:Novartis / Novartis Foundation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Combination Effects of Nilotinib and Zoledronic Acid On Chronic Myeloid Leukemia Are Canceled in the Co-Culture System Which Mimic the Bone Marrow Microenvironment.

AbstractAbstract 2459Prognosis of chronic myeloid leukemia (CML) has been dramatically improved by the induction of tyrosine kinase inhibitors (TKIs) as a first-line treatment. However, cure is still difficult because of the existence of leukemic stem cell (LSC).Zoledronic acid (ZOL) is a 3rd-generation bisphosphonate that inhibits bone matrix resorption of osteoclasts (OCs), and inhibit the proliferation of several tumor cells by preventing posttranslational prenylation of Ras-related proteins. We have previously reported that ZOL possess anti-leukemic effect that augments synergistically the activity of imatinib mesylate (Kuroda, et al. Blood 2003), and OCs maintain leukemic cells in a quiescent state under newly developed co-culture system (Yokota, et al. Leuk Res 2010). Thus, we hypothesized that the treatment of ZOL combined with nilotinib (Nilo) may be effective to eliminate dormant leukemic cell population, called LSCs, by attacking the OCs. To test this hypothesis, we examined the combined effect of Nilo and ZOL on the human CML cell line BV173 and BaF3 cells expressing p190 wild type BCR-ABL (BaF3/BCR-ABL) in vitro.Firstly, modified MTT assay was performed with ordinary condition (without any other co-cultured cells), resulting that ZOL augmented synergistically in vitro effects of Nilo both on cell lines (combination index < 1 at Fa=0.5) (Figure 1). Next we investigated the anti-leukemic effects of Nilo and ZOL using our developed co-culture system including osteoblasts (OBs), OCs and bone slices (Yokota, et al. Leuk Res 2010). 104 BaF3/BCR-ABL cells were applied to transwell chambers in each well of the co-culture plate (thus, BaF3/BCR-ABL cells did not attach directly to OBs, OCs and bones). Next day, each well was exposed to saline, Nilo 10nM, ZOL 10uM or Nilo 10nM plus ZOL 10uM. 48 hours after treatment, the viable cell number of BaF3/BCR-ABL was counted by trypan blue staining. Cell numbers were as follows; control: 55.79×104/well, Nilo only: 36.17×104/well, ZOL only: 64.46×104/well, Nilo plus ZOL: 39.42×104/well, respectively (Figure 2). These results suggested that combination effect of Nilo plus ZOL in vitro was canceled in the co-culture system. In the same condition, 7-AAD/Annexin V double staining was also performed. The apoptotic rates were as follows; control: 38.05 ± 2.36 %, Nilo only: 29.49 ± 2.59 %, ZOL only: 37.67 ± 1.39 %, Nilo plus ZOL: 29.66 ± 5.67 %. The results indicated that co-culture system of mimic the bone marrow microenvironment changed the drug effect on BaF3/BCR-ABL to resistance.To reveal the mechanisms of this phenomenon, TGF-ƒÀ1 concentration in the co-culture supernatants was measured by ELISA. In the previous report, when BaF3/BCR-ABL were co-cultured with OBs, OCs and bone slices, their proliferation was significantly suppressed and TGF-ƒÀ1 was significantly higher in supernatants from the co-culture system consisted of OB+OC+bone than control. In the present study, TGF-ƒÀ1 ratios (TGF-ƒÀ1 concentration in each well administrated with each agent/that in the medium without any cells) were almost same among wells treated with each agent. These results suggested that TGF-ƒÀ1 concentrations maintained for a while even after OCs were destroyed by ZOL and the remained TGF-ƒÀ1 affected the effects of anti-cancer agents on the proliferation of CML cells.In conclusion, the present study suggested that the evaluation of combined effects in ordinary culture system without any other cells which consist of microenvironment might not be correctly reflected in more physiological system. Therefore, it may be important to utilize the co-culture system used here to evaluate the effects of anti-cancer agents. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Stalk Domain and the Glycosylation Status of the Activating Natural Killer Cell Receptor NKp30 Are Important for Ligand Binding

The natural cytotoxicity receptors are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The human natural cytotoxicity receptor family comprises the three type I membrane proteins NKp30, NKp44, and NKp46. Especially NKp30 is critical for the cytotoxicity of NK cells against different targets including tumor, virus-infected, and immature dendritic cells. Although the crystal structure of NKp30 was recently solved (Li, Y., Wang, Q., and Mariuzza, R. A. (2011) J. Exp. Med. 208, 703-714; Joyce, M. G., Tran, P., Zhuravleva, M. A., Jaw, J., Colonna, M., and Sun, P. D. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 6223-6228), a key question, how NKp30 recognizes several non-related ligands, remains unclear. Therefore, we investigated the parameters that impact ligand recognition of NKp30. Based on various NKp30-hIgG1-Fc fusion proteins, which were optimized for minimal background binding to cellular Fcγ receptors, we identified the flexible stalk region of NKp30 as an important but so far neglected module for ligand recognition and related signaling of the corresponding full-length receptor proteins. Moreover, we found that the ectodomain of NKp30 is N-linked glycosylated at three different sites. Mutational analyses revealed differential binding affinities and signaling capacities of mono-, di-, or triglycosylated NKp30, suggesting that the degree of glycosylation could provide a switch to modulate the ligand binding properties of NKp30 and NK cell cytotoxicity.

Carbonyl Reductase 1 Offers a Novel Therapeutic Target to Enhance Leukemia Treatment by Arsenic Trioxide

Arsenic trioxide (As2O3) is used, in current practice, as an effective chemotherapeutic agent for acute promyelocytic leukemia (APL). However, the side effects and relatively low efficacy of As2O3 in treating other leukemias have limited its wider use in therapeutic applications. In the present study, we found that the expression of carbonyl reductase 1 (CBR1) affects the resistance to As2O3 in leukemias, including APL; As2O3 upregulated CBR1 expression at the transcriptional level by stimulating the activity of the transcription factor activator protein-1. Moreover, CBR1 overexpression was sufficient to protect cells against As2O3 through modulation of the generation of reactive oxygen species, whereas the attenuation of CBR1 was sufficient to sensitize cells to As2O3. A combination treatment with the specific CBR1 inhibitor hydroxy-PP-Me remarkably increased As2O3-induced apoptotic cell death compared with As2O3 alone, both in vitro and in vivo. These results were confirmed in primary cultured human acute and chronic myeloid leukemia cells, with no significant cell death observed in normal leukocytes. Taken together, our findings indicate that CBR1 contributes to the low efficacy of As2O3 and, therefore, is a rational target for the development of combination chemotherapy with As2O3 in diverse leukemias including APL.

Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors

Grb2-associated binder 2 (Gab2) serves as a critical amplifier in the signaling network of Bcr-Abl, the driver of chronic myeloid leukemia (CML). Despite the success of tyrosine kinase inhibitors (TKIs) in CML treatment, TKI resistance, caused by mutations in Bcr-Abl or aberrant activity of its network partners, remains a clinical problem. Using inducible expression and knockdown systems, we analyzed the role of Gab2 in Bcr-Abl signaling in human CML cells, especially with respect to TKI sensitivity. We show for the first time that Gab2 signaling protects CML cells from various Bcr-Abl inhibitors (imatinib, nilotinib, dasatinib and GNF-2), whereas Gab2 knockdown or haploinsufficiency leads to increased TKI sensitivity. We dissected the underlying molecular mechanism using various Gab2 mutants and kinase inhibitors and identified the Shp2/Ras/ERK and the PI3K/AKT/mTOR axes as the two critical signaling pathways. Gab2-mediated TKI resistance was associated with persistent phosphorylation of Gab2 Y452, a PI3K recruitment site, and consistent with this finding, the protective effect of Gab2 was completely abolished by the combination of dasatinib with the dual PI3K/mTOR inhibitor NVP-BEZ235. The identification of Gab2 as a novel modulator of TKI sensitivity in CML suggests that Gab2 could be exploited as a biomarker and therapeutic target in TKI-resistant disease.

Macromolecular Changes in Nilotinib Resistant K562 Cells; an In vitro Study by Fourier Transform Infrared Spectroscopy

Nilotinib is a second generation tyrosine kinase inhibitor which is used in both first and second line treatment of chronic myeloid leukemia (CML). In the present work, the effects of nilotinib resistance on K562 cells were investigated at the molecular level using Fourier transform infrared (FT-IR) spectroscopy. Human K562 CML cells were exposed to step-wise increasing concentrations of nilotinib, and sub-clones of K562 cells resistant to 50nM nilotinib were generated and referred to as K562/NIL-50 cells. Antiproliferative effects of nilotinib were determined by XTT cell proliferation assay. Changes in macromolecules in parental and resistant cells were studied by FT-IR spectroscopy. Nilotinib resistance caused significant changes which indicated increases in the level of glycogen and membrane/lipid order. The amount of unsaturated lipids increased in the nilotinib resistant cells indicating lipid peroxidation. The total amount of lipids did not change significantly but the relative proportion of cholesterol and triglycerides altered considerably. Moreover, the transcriptional status decreased but metabolic turn-over increased as revealed by the FT-IR spectra. In addition, changes in the proteome and structural changes in both proteins and the nucleus were observed in the K562/NIL-50 cells. Protein secondary structural analyses revealed that alpha helix structure and random coil structure decreased, however, anti-parallel beta sheet structure, beta sheet structure and turns structure increased. These results indicate that the FT-IR technique provides a method for analyzing drug resistance related structural changes in leukemia and other cancer types.

Synthesis, characterization, theoretical calculations and biological studies of nano Sodium tetrafluoroborate (III)

Synthesis, characterization, spectral and theoretical calculations of sodium tetrafluoroborate (III) (STFB) has been studied in this research. Sodium tetrafluoroborate (III) was synthesized by a sonochemical method and characterized by IR, UV/VIS, 11 B-NMR and Mass spectrometer techniques. The nano compound was characterized by scanning electron microscopy (SEM), X-ray powder diffraction (XRD) and IR spectroscopy. The structure of synthesized compound was optimized at the B3LYP/LANL2DZ level of theory and theoretical parameters such as structural data, molecular specifications, and infrared spectra were extracted by using Gaussian 03 program. Theoretical data show good agreement with the experimental results. Biological properties of this compound such as antitumor and antibacterial properties studied. This new complex showed excellent antitumor activity against one kind of cancer cells that is K742 (human chronic myeloid leukemia) cells. Also the compound was tested against the bacterial species Staphylococcus aureus, Escherichia coli, Staphylococcus Epidermidis, Estreptococo B and Shigella.

Cytotoxic and antioxidant effects of grape seed oil on the treatment of leukemia with methotrexate

Methotrexate (MTX), a widely used cytotoxic chemotherapeutic agent, is often limited by its severe toxicity. Regarding the mechanisms of its adverse effects, several hypotheses have been put forward, among which oxidative stress is highly noticeable. Additive effect of oxidative damage caused by MTX to oxidative stress induced by cancer makes the situation dramatically bad. In order to reduce the damage, several approaches have been suggested. Grape seed is one of the most significant prophylactic agents due to its antioxidant and bioflavonoids composition. The aim of this study was to investigate the protective effect of grape seed oil against MTX-induced oxidative stress in K-562 human chronic myeloid leukemia cell lines. Cells were divided into groups as following control, GSOH (tumor cells treated with 200 mg/mL of grape seed oil), GSOL (tumor cells treated with 100 mg/mL of grape seed oil), MTX (tumor cells treated with 50 nM methotrexate) and MTX + GSOH ( tumor cells treated with 200 mg/mL of grape seed oil and methotrexate). For antioxidant statue; superoxide dismutase (SOD), catalase (CAT), paraoxonase (PON) and aryl esterase (ARE) activities, for lipid peroxidation; malondialdehyde (MDA) level and also for cytotoxicity; cell viability were detected in 24 th and 48 th hours of the cell culture incubation. Based on the data, 200 mg/mL of grape seed oil indicates synergic effects with MTX on K562 regarding cytotoxicity especially in 48 th hour. In case of GSOH + MTX combined treatment for 24 hours, antioxidant system take part preventing lipid peroxidation and a possible oxidative damage. Upon 48 hour-GSOH treatment, antioxidant parameters show significant increase and hence, prevent lipid peroxidation in cancer cells. In conclusion, GSOH complimentary treatment may be suggested for leukemia therapy with MTX to reduce side effects and enhance the cytotoxicity of MTX.

Synthesis, characterization, theoretical calculations and biological studies of potassium trifluorothiocyanoborate (III)

Synthesis, characterization, spectral and theoretical calculations of potassium trifluorothiocya noborate (III) (PTFTCB) has been studied. Potassium trifluorothiocyanoborate (III) was synthesized and characterized by IR, UV/VIS, 11B-NMR and mass spectrometer techniques. The structure of synthesized compound was optimized at the B3LYP/LANL2DZ level of theory and theoretical parameters such as structural data, molecular specifications, and infrared spectra were extracted by using Gaussian 03 program. Theoretical data show good agreement with the experimental results. Biological properties of this compound such as antitumor and anti-bacterial properties studied. This new complex showed excellent antitumor activity against one kind of cancer cells that is K742 (human chronic myeloid leukemia) cells. Also, the compound was tested against the bacterial species Staphylococcus aureus, Escherichia coli,Staphylococcus epidermidis, Estreptococo B and Shigella. Key words: Potassium trifluorothiocyanoborate (III), new synthetic method, optimized, antitumor activity, K742 (human chronic myeloid leukemia) cells, anti bacterial activity.