Chronic Myelogenous Leukemia Cells Research Articles

New Thalidomide-Resembling Dicarboximides Target ABC50 Protein and Show Antileukemic and Immunomodulatory Activities.

We identified novel dicarboximides that were selectively cytotoxic towards human leukemia cells. Using chemical and biological methods, we characterized the biological activity, identified cellular protein targets and defined the mechanism of action of the test dicarboximides. The reported IC50 values (concentration required to reduce cell survival fraction to 50% of control) of selected dicarboximides were similar or lower than IC50 of registered anticancer drugs, for example cytarabine, sorafenib, irinotecan. Test compounds induced apoptosis in chronic myelogenous (K562) and acute lymphoblastic (MOLT-4) leukemia cells by activation of receptor and mitochondrial apoptotic pathways and increased the expression of proapoptotic genes (BAX, NOXA, HTRA2, TNFRSF10B, ESRRBL1). Selected dicarboximides displayed immunomodulatory activity and downregulated IKZF1 and IKZF3 transcription factors in K562 and MOLT-4 leukemia cells. ATP-binding cassette protein 50 (ABC50) was identified as a target for dicarboximides. Cancer cells with knocked down ABC50 showed increased resistance to dicarboximides. Based on the structure of dicarboximides and thalidomide, novel proteolysis-targeting chimeras (PROTACs) were synthesized and used as tools to downregulate ABC50 in leukemia cells.

Improvement of the Anticancer Activity of Chlorambucil and Ibuprofen via Calix[4]arene Conjugates

One of the possible ways of improving the activity and selectivity profile of anticancer agents is to design drug carrier systems employing nanomolecules. Calix[4]arene derivatives and chlorambucil and ibuprofen are important compounds that exhibit interesting anticancer properties. The objective of this article is the synthesis of new calix[4]arene-derivative conjugates of chlorambucil or ibuprofen with potential anticancer activity. Cytotoxicity assays were determined using the protein-binding dye sulforhodamine B (SRB) in microculture to measure cell growth as described [19, 20]. Conjugates of chlorambucil and resorcinarene-dendrimers were prepared in 2% DMSO and added into the culture medium immediately before use. Control cells were treated with 2% DMSO. Thus, calix[4]arene-derivative conjugates of chlorambucil or ibuprofen showed good stability of the chemical link between drug and spacer. Evaluation of the cytotoxicity of the calix[4]arene chlorambucil or ibuprofen conjugates employing a sulforhodamine B (SRB) assay in K-562 (human chronic myelogenous leukemia cells) and U-251 (human glioblastoma cells) demonstrated that the conjugate was more potent as an antiproliferative agent than free chlorambucil and ibuprofen. The conjugates did not show any activity against the COS-7 African green monkey kidney fibroblast cell line. In the paper, we report the synthesis and spectroscopic analyses of new calix[4]arene derivative conjugates of chlorambucil or ibuprofen. Cytotoxicity assays revealed that at 10 μM, the conjugates were very active against K-562 (human chronic myelogenous leukemia cells) and U- 251 (human glioblastoma cells) cancer cells' proliferation. In order to explain the molecular mechanisms involved in the anticancer activity of calix[4]arene chlorambucil or ibuprofen conjugates, our research will be continued.

A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia.

Noncoding RNAs are increasingly being accredited with key roles in gene regulation during development and disease. Here we report the discovery and characterization of a novel long noncoding RNA, Hmrhl, which shares synteny and partial sequence similarity with the mouse lncRNA, Mrhl. The human homolog, Hmrhl, transcribed from intron 14 of phkb gene, is 5.5 kb in size, expressed in all tissues examined and is associated with chromatin. Analysis of Hmrhl locus using ENCODE database revealed that it exhibits hallmarks of enhancers like the open chromatin configuration, binding of transcription factors, enhancer specific histone signature etc. in the K562 Chronic Myelogenous Leukemia (CML) cells. We compared the expression of Hmrhl in the normal lymphoblast cell line, GM12878, with that of K562 cells and lymphoma samples and show that it is highly upregulated in leukemia as well as several cases of lymphoma. Further, we validated the enhancer properties of Hmrhl locus in K562 cells with the help of ChIP-qPCR and Luciferase assay. Moreover, siRNA mediated down-regulation of Hmrhl in K562 cells leads to a concomitant down regulation of its parent gene, phkb, showing that Hmrhl functions as an enhancer RNA and positively regulates its host gene, phkb, in chronic myelogenous leukemia. This study is significant in view of the fact that a better understanding of mechanism of gene regulation under normal conditions and its perturbation in cancer could in turn help in its therapeutic intervention through molecular medicine/RNA based drug discovery.

Identification of key candidate targets and pathways for the targeted treatment of leukemia stem cells of chronic myelogenous leukemia using bioinformatics analysis.

BackgroundChronic myelogenous leukemia (CML) is a myeloproliferative neoplasm that arises from the acquisition of constitutively active BCR‐ABL tyrosine kinase in hematopoietic stem cells. The persistence of bone marrow leukemia stem cells (LSCs) is the main cause of TKI resistance and CML relapse. Therefore, finding a key target or pathway to selectively target LSCs is of great significance for the thorough treatment of CML.MethodsIn this study, we aimed to identify key microRNAs, microRNA targets and pathways for the treatment of CML LSCs by integrating analyses of three microarray data profiles. We identified 51 differentially expressed microRNAs through integrated analysis of GSE90773 and performed functional gene predictions for microRNAs. Then, GSE11889 and GSE11675 were integrated to obtain differentially expressed genes (DEGs), and the overlapping DEGs were used as models to identify predictive functional genes. Finally, we identified 116 predictive functional genes. Clustering and significant enrichment analysis of 116 genes was based on function and signaling pathways. Subsequently, a protein interaction network was constructed, and module analysis and topology analysis were performed on the network.ResultsA total of 11 key candidate targets and 33 corresponding microRNAs were identified. The key pathways were mainly concentrated on the PI3K/AKT, Ras, JAK/STAT, FoxO and Notch signaling pathways. We also found that LSCs negatively regulated endogenous and exogenous apoptotic pathways to escape from apoptosis.ConclusionWe identified key candidate targets and pathways for CML LSCs through bioinformatics methods, which improves our understanding of the molecular mechanisms of CML LSCs. These candidate genes and pathways may be therapeutic targets for CML LSCs.

Homoharringtonine enhances the effect of imatinib on chronic myelogenous leukemia cells by downregulating ZFX.

Homoharringtonine (HHT) and imatinib have a synergistic effect in the clinical treatment of chronic myeloid leukemia (CML). The purpose of the present study was to explore the underlying mechanisms by which HHT enhanced imatinib sensitivity. K562 CML cells were treated with HHT and imatinib separately or in combination. Cell viability was detected by Cell Counting Kit-8 assay; apoptotic rates and protein expression levels of phosphorylated-tyrosine (p-Tyr) and p-CRK like proto-oncogene, adaptor protein (p-Crkl) were analyzed by flow cytometry; zinc-finger protein, X-linked (ZFX) overexpression plasmid was transfected to cells using electroporation; western blotting was used to detect the protein expression levels of PI3K, AKT, p-AKT and ZFX; and reverse transcription-quantitative PCR was used to measure ZFX mRNA expression levels. The results demonstrated that HHT and imatinib co-treatment had significant effects of proliferation inhibition and apoptosis induction on K562 CML cells compared with imatinib alone. Co-treatment also significantly downregulated the expression levels of p-Tyr, p-Crkl, PI3K and p-Akt compared with imatinib or HHT treatment. In addition, HHT downregulated ZFX mRNA and protein expression. ZFX overexpression reversed cell sensitivity to imatinib and HHT and also reduced the HHT-induced imatinib sensitization by increasing p-Akt expression. In conclusion, HHT may enhance the effect of imatinib on CML cells by downregulating ZFX.

The N-acetylgalactosamine-transferase11 (GALNT11) activation in leukemia and the role of cyclophosphamide and cytarabine in expression of this enzyme

Background: We investigated the activation of N-acetylgalactosamine-transferase11 (GALNT11) and the role of cyclophosphamide and cytarabine in expression of this enzyme in AML and CML patients. Methods: GALNT11 activity and expression levels were evaluated in human chronic myelogenous leukemia cells (K562) and human acute monocytic leukemia cells (THP1) after incubation with different concentrations of cyclophosphamide (Cyclo) and cytarabine (Cyta). Anti-proliferative effects of Cyclo and Cyta were examined by MTT assay. Real time-PCR and ELISA assays were applied to investigate the expression and activity of GALNT11, respectively. Results: The IC50 values for Cyclo were 34.73±0.12 μM for K562 and 36.16±0.23 µM for THP1 cells. These valuses for Cyta were also 0.38±0.15 μM for K562 and 0.52±0.18 µM for THP1 cells . Conclusion: The activation and expression of GALNT11 increased in acute and chronic leukemia as glycosylation disorders. Also cyclophosphamide and specially cytarabine cause significant increase in activation and expression of GALNT11 which inhibits glycosylation and tumorogenesis dependent signaling pathway.

Abstract 5239: Cisplatin disrupts MDM2-DAXX-HAUSP complex, p53 accumulation, cell cycle regulation, and apoptosis in acute leukemia cells

Abstract Cisplatin (CDDP) is an antitumor drug successfully used in the treatment of various types of human malignancies. It enhances toxicity of arsenic trioxide (ATO) in head and neck squamous cell carcinoma cell, small cell lung cancer, ovarian cancer cells, and oral squamous cell carcinoma cells (, and chronic myelogenous leukemia cells. CDDP induces cytotoxicity, DNA damage, oxidative stress, and apoptosis in acute promyelocytes leukemia (APL) cells. However, the molecular mechanisms of CDDP-induced P53 activation, cell cycle arrest, and apoptosis in APL cells remain poorly understood. We hypothesized that CDDP inhibits proliferation of APL cells through MDM2-DAXX-HAUSP complex disruption, p53 activation leading to cell cycle arrest and apoptosis. To test hypothesis, we used three APL cell lines and mice model of APL and investigated CDDP effects on cells growth, complex disruption, cell cycle progression, and apoptosis by applying western blotting, IP, gene knockdown techniques, immune/histochemistry, and confocal imaging. We found that CDDP - induced stress signal transmitted by protein kinase (ATM, ATR) and its downstream targets (CHK1 & CHK2) phosphorylation leading to down-regulation of complex molecules expression and accumulation of p53. CDDP-induced p53 expression modulated cell cycle progression and apoptosis in APL cells, and stimulated the formation of more promyelocytes with dense granules through reduction of MDM2 expression in bone marrow cells. CDDP also activated p53 in APL mice liver tissues. This novel mechanism of action of CDDP in APL cells may help in the design of new anti-leukemic drugs for treatment of APL patients. Acknowledgements: This research was supported by National Institutes of Health NIMHD [grant # G12MD007581], through the RCMI-Center for Environmental Health at Jackson State University. Citation Format: Sanjay Kumar, Paul Tchounwou. Cisplatin disrupts MDM2-DAXX-HAUSP complex, p53 accumulation, cell cycle regulation, and apoptosis in acute leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5239.

Abstract 1327: UM-9107: A selective wild-type and T315I Bcr-Abl inhibitor with in vivo activity against chronic myelogenous leukemia

Abstract Purpose: Long-term treatment of chronic myelogenous leukemia (CML) with the Bcr-Abl inhibitor imatinib, is initially successful but gives rise to several drug-resistant mutations. The most prevalent of these is the T315I ‘gatekeeper’ mutation that is sensitive to the second-line therapy, ponatinib. However, ponatinib causes serious vascular adverse events, including fatalities, in over 25% of patients, in part due to its target promiscuity. We have designed a novel Bcr-Abl inhibitor (UM-9107) that shows significant in vitro and in vivo anti-CML activity against wild-type and drug-resistant mutants, including the gatekeeper mutant. Furthermore, UM-9107 has excellent selectivity across the kinome and in vitro surrogate toxicity assessment models demonstrate a low likelihood of vascular adverse events as compared to ponatinib. Experimental Design: We designed a series of compounds to specifically interact with a specific kinked conformation of the phosphate-binding loop of Abl. Since only a small proportion of kinases display this kinked conformation, we hypothesized that our compounds would be selective for Abl. We have analyzed the compound series biochemically and in vitro. The most potent compound, UM-9107, has been comprehensively characterized including: biochemical inhibition, cellular target engagement, kinome-wide profiling, in vitro cell proliferation, pharmacokinetic studies, and in vivo activity in xenograft models of CML. Results: We demonstrate that UM-9107 potently inhibits all clinical mutants of Bcr-Abl, including the T315I gatekeeper mutant. UM-9107 shows robust activity against CML cell lines, comparable to ponatinib, with significantly fewer off-targets, as determined by kinome-wide profiling. Phenotypic profiling of UM-9107 in human primary cells shows low risk of vascular adverse events as indicated by specific biomarker readouts. UM-9107 demonstrates good oral bioavailability and in vivo activity in xenograft models of CML, with low toxicity. Conclusions: The success of imatinib has been revolutionary and has led to an increase in the number of patients on long-term therapy. This inevitably gives rise to drug resistance through point mutations in the Bcr-Abl protein. Our results suggest that UM-9107 is a safe and efficacious preclinical candidate for the treatment of imatinib-resistant CML. Citation Format: Sameer Phadke, Lluis Lopez-Barcons, Taylor K. Johnson, Eric J. Lachacz, Sofia D. Merajver, Matthew B. Soellner. UM-9107: A selective wild-type and T315I Bcr-Abl inhibitor with in vivo activity against chronic myelogenous leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1327.

LncRNA FENDRR attenuates adriamycin resistance via suppressing MDR1 expression through sponging HuR and miR-184 in chronic myelogenous leukaemia cells.

Chemotherapy is a major anticancer therapeutic modality, however, multidrug resistance (MDR) is frequently observed and hinders treatment efficacy. Here, we investigated the role and potential mechanism of the long noncoding RNA (lncRNA) FENDRR in adriamycin resistance of chronic myeloid leukaemia (CML) cells. FENDRR overexpression attenuates adriamycin resistance, as shown by increased Rhodamine 123 accumulation, promotion of cell apoptosis invitro and suppression of tumour growth invivo. Mechanistically, we identified that FENDRR reduces the interaction of the RNA-binding protein HuR with MDR1 via acting as a sponge, and miR-184 competitively binds to FENDRR with HuR. Thus, the HuR/FENDRR/miR-184 interaction contributes to MDR1 activity. These findings indicate that FENDRR is a potential target for reversing adriamycin resistance.

New Heteroleptic Ruthenium(II) Complexes with Sulfamethoxypyridazine and Diimines as Potential Antitumor Agents.

Two new complexes of Ru(II) with mixed ligands were prepared: [Ru(bpy)2smp](PF6) (1) and [Ru(phen)2smp](PF6) (2), in which smp = sulfamethoxypyridazine; bpy = 2,2′-bipyridine; phen = 1,10-phenanthroline. The complexes have been characterized by elemental and conductivity analyses; infrared, NMR, and electrospray ionization mass spectroscopies; and X-ray diffraction of single crystal. Structural analyses reveal a distorted octahedral geometry around Ru(II) that is bound to two bpy (in 1) or two phen (in 2) via their two heterocyclic nitrogens and to two nitrogen atoms from sulfamethoxypyridazine—one of the methoxypyridazine ring and the sulfonamidic nitrogen, which is deprotonated. Both complexes inhibit the growth of chronic myelogenous leukemia cells. The interaction of the complexes with bovine serum albumin and DNA is described. DNA footprinting using an oligonucleotide as substrate showed the complexes’ preference for thymine base rich sites. It is worth notifying that the complexes interact with the Src homology SH3 domain of the Abl tyrosine kinase protein. Abl protein is involved in signal transduction and implicated in the development of chronic myelogenous leukemia. Nuclear magnetic resonance (NMR) studies of the interaction of complex 2 with the Abl-SH3 domain showed that the most affected residues were T79, G97, W99, and Y115.

PF‑114, a novel selective inhibitor of BCR‑ABL tyrosine kinase, is a potent inducer of apoptosis in chronic myelogenous leukemia cells.

A t(9;22) chromosomal translocation which forms the chimeric tyrosine kinase breakpoint cluster region (BCR)‑Abelson murine leukemia viral oncogene homolog1(ABL) is a key mechanism underlying the pathogenesis of chronic myelogenous leukemia(CML). Pharmacological inhibition of BCR‑ABL with imatinib (Gleevec) has been reported as an effective targeted therapy; however, mutations (including the kinase domain of ABL) suppress the efficacy of inhibitors. PF‑114, a derivative of the third generation BCR‑ABL inhibitor ponatinib, demonstrated a high inhibitory activity against wild-type and mutant BCR‑ABL forms, such as the clinically important T315I. Furthermore, PF‑114 exhibited preferential kinase selectivity, safety, notable pharmacokinetic properties and therapeutic efficacy in a murine model. Investigation into the mechanisms of CML cell death revealed an exceptional potency of PF‑114 (at low nanomolar concentrations) for the CML‑derived K562 cell line, whereas leukemia cell lines that lack the chimeric tyrosine kinase were markedly more refractory. The molecular ordering of events mechanistically associated with K562 cell death included the dephosphorylation of CrkL adaptor protein followed by inhibition of ERK1/2 and Akt, G1 arrest, a decrease of phosphorylated Bcl‑2‑associated death promoter, Bcl‑2‑like protein11, BH3 interacting‑domain death agonist, Bcl‑extra large and Bcl‑2 family apoptosis regulator, and reduced mitochondrial transmembrane potential. Increased AnnexinV reactivity, activation of caspases and poly(ADP‑ribose)polymerase cleavage were proposed to lead to internucleosomal DNA fragmentation. Thus, PF‑114 may be a potent inducer of apoptosis in CML cells. Nevertheless, activation of STAT3 phosphorylation in response to PF‑114 may permit cell rescue; thus, a combination of BCR‑ABL and STAT3 inhibitors should be considered for improved therapeutic outcome. Collectively, the targeted killing of BCR‑ABL‑positive cells, along with other beneficial properties, such as invivo characteristics, suggests PF‑114 as a potential candidate for analysis in clinical trials with CML patients.

Pt(II) complexes of the type trans-[PtCl2(DMSO) (hydrazide)] and cis-[PtCl2(hydrazide)2]: Solvolysis and cytotoxic activity

The present work describes the synthesis, solvolysis and cytotoxic activity of Pt (II) complexes of the type trans-[PtCl2 (DMSO) (L)] and cis-[PtCl2(L)2], where L = benzhydrazide (BH), 4-nitrobenzhydrazide (4NH) and 4-(trifluoromethyl)benzhydrazide (4 TF). The structures of these complexes were proposed using spectroscopic methods. All complexes undergo fast solvolysis generating four identical species in DMSO, being that the species cis- and trans-[PtCl2 (DMSO) (L)] are the most important. As to the stability in DMF, Pt (II) complexes of the type cis-[PtCl2(L)2] are very stable, while Pt (II) complexes of the type trans-[PtCl2 (DMSO) (L)] react at a slower rate than those verified in DMSO. The cytotoxic activity of the compounds was evaluated in a chronic myelogenous leukemia cell line. A relationship between solvolysis and cytotoxic activity was verified. Moreover, our results showed that some Pt (II) complexes were more cytotoxic than the free ligands, carboplatin and cisplatin.

Role of SIRT1 in hematologic malignancies.

Sirtuin 1 (SIRT1) is a protein deacetylase, which regulates various physiological activities by deacetylating different protein substrates. An increasing number of studies have revealed critical roles of SIRT1 in different aspects of cancers including metabolism, proliferation, genomic instability, and chemotherapy resistance. Depending on the protein targets in a certain oncogenic context, SIRT1 may play a unique role in each individual blood cancer subtype. Our previous work showed that activation of SIRT1 in primitive leukemia cells of acute myeloid leukemia (AML) and chronic myelogenous leukemia (CML) promotes disease maintenance. On the other hand, an SIRT1 agonist was shown to disrupt maintenance of myelodysplastic syndrome (MDS) stem cells and holds promise as a potential therapeutic approach. Herein, we present a concise summary of the different functions of SIRT1 in hematologic malignancies.

Investigation of the role of RCAN1 Gene in apoptosis

Our lab is interested in understanding the molecular basis of chemotherapeutic evoked apoptosis and resistance in leukemic lymphoblasts. The long‐term goal of our research is to improve the therapeutic effectiveness of chemotherapeutic agents. Antileukemic agents and signaling pathway inhibitors induce apoptosis by regulating gene expression; a drawback of this treatment is the development of resistance to these agents. The RCAN1 (Regulator of Calcineurin 1) gene is located on chromosome 21 and is one of many genes implicated in apoptosis. Upregulation of RCAN1 correlates with sensitivity to antileukemic agents and signaling pathway inhibitors. The protein product of this gene, RCAN1, binds to the protein phosphatase calcineurin, inhibiting its activity, and altering cellular responses to survival and death triggers. We are testing the hypothesis that if the protein function of the RCAN1 is knocked out, cells will be protected from apoptosis. We are screening a series of antileukemic agents and signaling pathway inhibitors, utilizing paired wild‐type and RCAN1 knockout HAP1 cell lines, to determine which agents might mediate an apoptotic response via RCAN1 involvement. HAP1 is a human near‐haploid cell line derived from the male chronic myelogenous leukemia (CML) cell line KBM‐7, allowing for efficient CRISPR‐mediated genome editing. Viability of cells exposed to various agents was evaluated by MTT assays and transcript levels were monitored via reverse‐transcription‐PCR analysis of RNA extracted from treated cells. MTT assays show that RCAN1 knockout offered the greatest protection from apoptosis caused by the AKT inhibitor MK‐2206, when compared to wildtype cells. Upregulation of BIM, a proapoptotic protein, by MK‐2206 was reduced in RCAN1 knockout HAP1 cells compared to the wild‐type HAP1 cells. Our results suggest that RCAN1 may influence the AKT pathway to mediate apoptosis.Support or Funding InformationThis research is supported by GM081099 awarded to Rheem Medh and NIH GM008395 for student support of Alejandra Fausto.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Andrographolide and its potent derivative exhibit anticancer effects against imatinib-resistant chronic myeloid leukemia cells by downregulating the Bcr-Abl oncoprotein

Chronic myelogenous leukemia (CML) is clinically treated with imatinib, which inhibits the kinase activity of the Bcr-Abl oncoprotein. However, imatinib resistance remains a common clinical issue. Andrographolide, the major compound of the medicinal plant Andrographis paniculata, was reported to exhibit anticancer activity. In this study, we explored the therapeutic potential of andrographolide and its derivative, NCTU-322, against both imatinib-sensitive and imatinib-resistant human CML cell lines. Both andrographolide and NCTU-322 downregulated the Bcr-Abl oncoprotein in imatinib-resistant CML cells through an Hsp90-dependent mechanism similar to that observed in imatinib-sensitive CML cells. In addition, NCTU-322 had stronger effects than andrographolide on downregulation of Bcr-Abl oncoprotein, induction of Hsp90 cleavage and cytotoxicity of CML cells. Notably, andrographolide and NCTU-322 could induce differentiation, mitotic arrest and apoptosis of both imatinib-sensitive and imatinib-resistant CML cells. Finally, the anticancer activity of NCTU-322 against imatinib-resistant CML cells was demonstrated in vivo. In summary, our data demonstrated that andrographolide and NCTU-322 inhibit Bcr-abl function via a mechanism different from that of imatinib, and they induced multiple anticancer effects in both imatinib-sensitive and resistant CML cells. Our findings demonstrate that andrographolide and NCTU-322 are potential therapeutic agents again CML.

Discovery of the Oncogenic Parp1, a Target of bcr-abl and a Potential Therapeutic, in mir-181a/PPFIA1 Signaling Pathway

miR-181a is downregulated in leukemia and affects its progression, drug resistance, and prognosis. However, the exact mechanism of its targets in leukemia, particularly in chronic myelogenous leukemia (CML), has not previously been established. Here, we use a multi-omics approach to demonstrate that protein tyrosine phosphatase, receptor type, f polypeptide, leukocyte common antigen (LAR) interacting protein (liprin), alpha 1 (PPFIA1) is a direct target for miR-181a in CML. Phospho-array assay shows that multiple phosphorylated proteins, particularly KIT signaling molecules, were downregulated in PPFIA1 inhibition. Additionally, PPFIA1 bound PARP1, a common molecule downstream of both PPFIA1 and BCR/ABL, to upregulate KIT protein through activation of nuclear factor kappa B (NF-κB)-P65 expression. Targeted inhibition of PPFIA1 and PARP1 downregulated c-KIT level, inhibited CML cell growth, and prolonged mouse survival. Overall, we report a critical regulatory miR-181a/PPFIA1/PARP1/NF-κB-P65/KIT axis in CML, and our preclinical study supports that targeted PPFIA1 and PARP1 may serve as a potential CML therapy.

A modified DAW-22 compound F-B1 inhibits Bcr/Abl and induces apoptosis in chronic myelogenous leukemia cells.

The Bcr/Abl kinase is an oncogenic fusion protein that plays a central role in the pathogenesis of chronic myeloid leukemia (CML). Some small-molecule kinase inhibitors such as imatinib were developed in the treatment of CML; however, resistant to imatinib is an emerging problem of CML therapy. Hence, additional approaches or compounds targeting leukemogenic cells are required. F-B1 is a new compound obtained by modifying DAW-22, a natural sesquiterpenoid coumarin, which was isolated from traditional Chinese medicine Ferula ferulaeoides (Steud.) Korov. F-B1 was found to inhibit the growth of myelogenous leukemia cell lines, that is, K562 cells bearing wild-type Bcr/Abl and imatinib-resistant K562G cells. F-B1 potently down-regulated the mRNA and protein levels of Bcr/Abl, followed by suppression of the downstream molecules such as Akt, externally regulated kinases, and nuclear factor κB. In addition, F-B1 also induced cell apoptosis by impairing the balance between proapoptotic protein Bax and antiapoptotic proteins Bcl-2 and Bcl-XL and increased the activity of mitochondrial-dependent apoptosis in nude mouse xenografts. Experimental validation results together demonstrated that F-B1 can inhibit Bcr/Abl fusion proteins in K562 and K562G cells, implying that F-B1 might be a promising drug to treat CML, especially the imatinib-resistant CML.

Hydroxychavicol sensitizes imatinib-resistant chronic myelogenous leukemia cells to TRAIL-induced apoptosis by ROS-mediated IAP downregulation.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of cytokine superfamily, induces apoptosis in a number of tumor cells through the activation of extrinsic apoptotic pathway but shows little or no cytotoxicity toward normal cells. However some tumor cells are inherently resistant to TRAIL-mediated apoptosis, which needs to be addressed to establish TRAIL as a potential chemotherapeutic drug. In this study, our aim was to manipulate TRAIL-apoptosis pathway by hydroxychavicol (HCH), a polyphenol from Piper betel leaf, for the induction of apoptosis in TRAIL resistant chronic myeloid leukemia cell. When imatinib-resistant K562 cells were treated with HCH, it made these K562 cells sensitive to TRAIL. It was observed that HCH downregulated antiapoptotic proteins XIAP and FLIP, whereas the expression of TRAIL receptors, DR4 and DR5, remains unchanged. Moreover, we observed that reactive oxygen species or ROS played a crucial role in the downregulation of FLIP and XIAP because ROS scavenger significantly reversed the decrease of XIAP, and FLIP. Ubiquitin-proteasome pathway was observed to play a crucial role in the downregulation of XIAP and FLIP, as proteasomal inhibitor MG132 significantly reversed the downregulation of XIAP and FLIP. In conclusion, this study demonstrates the combinatorial treatment of TRAIL and HCH as promising alternative therapeutic approach to treat the imatinib-resistant leukemia, which are also resistant to TRAIL.

Research Progress of C-Myc in Chronic Myelogenous Leukemia --Review

As a potential target for cancer treatment, the C-Myc high expresses abnormally in a variety of solid tumors and hematological malignancies in humans. In hematologic malignancies, the increasing expression of C-Myc is associated with poor prognosis of patients diffuse large B-cell lymphoma. However, some studies have shown that high expression of C-Myc might be one of the mechanisms of disease progression and abrupt change, therefore, it can promote the transformation of chronic myeloid leukemia. Besides, the high expression of C-Myc may lead to the decrease of the sensitivity of chronic myelogenous leukemia cells to imatinib. Therefore, C-Myc has been concerned in the study of imatinib drug resistance. This review will focuses on the latest research progress on the mechanism of C-Myc in chronic myeloid leukemia in recent years.

Effects of carbachol on apoptosis in human chronic myelogenous leukemic K562 cell line

Objectives: Muscarinic receptors mediate diverse actions ofacetylcholine in the central nervous system and in non-nervoustissues innervated by the parasympathetic nervous system.Our study aims to evaluate the potential association of theM3 muscarinic receptor with K562 cell proliferation and death.Materials and Methods: Cell proliferation was evaluatedby bromodeoxyuridine (BrDU) incorporation. To show early,late apoptosis and cell death, cells were labelled with AnnexinV, propidium iodide (PI) and analyzed by flow cytometry. Nuclearextracellular signal-regulated kinase (ERK/pERK) expressionwas measured by western blot analysis.Results: Treatment with carbachol (CCh) for 48h decreased cellnumber. Exposing K562 cells to CCh for 24h decreased the number ofearly apoptotic cells but did not change the number of late apoptotic andnecrotic cells. CCh treatment for 48h increased the number of necroticcells, but decreased the number of early and late apoptotic cells. Inresponse to CCh, nuclear ERK expression was increased and this effectwas reversed by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide(4DAMP). Nuclear pERK expression was decreased in CCh treatedcells, 4DAMP did not reverse the effect.Conclusion: Our data suggest that cholinergic agonist CChaffects cell proliferation in K562 cells not only through muscarinicreceptors but also through other cholinergic receptors.