Levels In K562 Cells Research Articles

Effect of Csn-B Combined with Imatinib on Apoptosis of Chronic Myeloid Leukemia Cells through Nur77/Pim-1/Drp1 Pathway

To investigate the anti- chronic myelogenous leukemia (CML) activity of Nur77-specific agonist Csn-B combined with imatinib by promoting Nur77 expression, and explore the potential role of its signaling pathway. Firstly, CCK-8 and Transwell assay were used to detect the inhibitory effects of Csn-B, imatinib, and their combination on the proliferation and migration of K562 cells. Furthermore, the apoptosis rate of K562 cells treated with Csn-B, imatinib, and their combination was detected by flow cytometry. The expression levels of Nur77, Pim-1, Drp1, p-Drp1 S616, Bcl-2 and Bax in K562 cells were detected by Western blot. Finally, the expression levels of reactive oxygen species (ROS) in K562 cells treated with Csn-B, imatinib and their combination were detected by immunofluorescence assay. The level of Nur77 in CML patients decreased significantly compared with normal population in dataset of GSE43754 (P < 0.001). Csn-B combined with imatinib could significantly inhibit the proliferation and migration of K562 cells (both P < 0.001), and induce apoptosis (P < 0.001). Csn-B promoted Nur77 expression in K562 cells, and synergistically enhanced imatinib sensitivity when combined with imatinib. Csn-B combined with imatinib could significantly enhanced ROS levels in K562 cells and mitochondria compared with single-drug treatment (both P < 0.001). Csn-B combined with imatinib can enhance ROS expression and induce apoptosis of K562 cells through Nur77/Pim-1/Drp1 pathway.

STK10 mutations block erythropoiesis in acquired pure red cell aplasia via impairing ribosome biogenesis.

Acquired pure red cell aplasia (PRCA) is anemia associated with the absence of erythroblasts and is characterized by persistent and easy recurrence. However, the underlying mechanisms of acquired PRCA remain obscure, and the role of gene mutations in the pathogenesis of acquired PRCA is not fully characterized. In the present study, we detected thirty newly diagnosed patients with acquired PRCA using whole exome sequencing, and a potential role for STK10 in acquired PRCA was uncovered. The mRNA levels of STK10 in three patients with STK10 mutations were decreased. These three patients had a poor response to immunosuppressive therapy and two died in the follow-up period. Here we report that knockdown of STK10 inhibits erythroid differentiation and promotes apoptosis of K562 cells. We show that knockdown of STK10 resulted in inhibition of ribosome biogenesis and reduced ribosome levels in K562 cells. We also show that the p53 signaling pathway is activated by knockdown of STK10. Our results imply that ribosome biogenesis downregulation together with pathological p53 activation prevents normal erythropoiesis. Our study uncovers a new pathophysiological mechanism leading to acquired PRCA driven by STK10 mutations.

Plant-derived extracts P2Et and Anamu-SC affect NO and ROS levels in leukemic cells

Leukemic cells often show high nitric oxide (NO) and reactive oxygen species (ROS) levels. These can lead to resistance to apoptosis and therapy and increased proliferation. Plant-derived extracts decrease chemoresistance in cancer cells. In this study, we evaluated the effects of the plant-derived extracts P2Et (Caesalpinia spinosa) and Anamu-SC (Petiveria alliacea) and their combination with chemotherapeutic agents on NO and ROS levels in leukemic cell lines K562 and Reh. NO and ROS were determined using the DAF-FM DA and H2DCFDA probes. The mean fluorescence intensity for each variable was measured by flow cytometry. The extracts showed an antioxidant effect on both cell lines leading to a significant decrease in ROS levels without decreasing cell viability. Anamu-SC also increased NO levels in K562 cells when combined with idarubicin. Both extracts reduced the number of leukemic cells after 12 hours of treatment. Further studies are necessary to evaluate their effect on primary human leukemia cells. These findings suggest the potential of P2Et and Anamu-SC as adjuncts in leukemia treatment.

Expression of thimet oligopeptidase (THOP) modulated by oxidative stress in human multidrug resistant (MDR) leukemia cells

Thimet oligopeptidase (THOP) is a cytosolic metallopeptidase known to regulate the fate of post-proteasomal peptides, protein turnover and peptide selection in the antigen presentation machinery (APM) system. Oxidative stress influences THOP expression and regulates its proteolytic activity, generating variable cytosolic peptide levels, possibly affecting the immune evasion of tumor cells. In the present work, we examined the association between THOP expression/activity and stress oxidative resistance in human leukemia cells using the K562 cell line, a chronic myeloid leukemia (CML), and the multidrug-resistant (MDR) Lucena 1 (K562-derived MDR cell line) as model. The Lucena 1 phenotype was validated under vincristine treatment and the relative THOP1 mRNA levels and protein expression compared to K562 cell line. Our data demonstrated increased THOP1 gene and protein levels in K562 cells in contrast to the oxidative-resistant Lucena 1, even after H2O2 treatment, suggesting an oxidative stress dependence in THOP regulation. Further, it was observed higher basal levels of reactive oxygen species (ROS) in K562 compared to Lucena 1 cell line using DHE fluorescent probe. Since THOP activity is dependent on its oligomeric state, we also compared its proteolytic activity under reducing agent treatment, which demonstrated that its function modulation with respect to changes in redox state. Finally, the mRNA expression and FACS analyses demonstrated a reduced expression of MHC I only in K562 cell line. In conclusion, our results highlight THOP redox modulation, which could influence antigen presentation in multidrug resistant leukemia cells.

Ormeloxifene, a nonsteroidal antifertility drug promotes megakaryocyte differentiation in leukemia cell line K562.

Ormeloxifene (ORM) (3,4-trans-2,2-dimethyl-3-phenyl-4-p-(β-pyrrolidinoethoxy) phenyl-7-methoxychroman), world's first nonsteroidal selective estrogen receptor modulatorapproved for contraception in India has been shown to have potential anticancer activities. Here, we show that ORMcan induce megakaryocyte and myeloid (granulocytic) but not erythroid differentiation in multipotent human myeloid leukemia cell line K562. We show that ORM at an IC50 of 7.5 µM can induce morphological changes similar to megakaryocytes in K562 cells. ORM led to increase in levels of megakaryocytic differentiation markers (CD41 and CD61) as well as key transcription factors GATA1 and AML1. We further show that ORM induces megakaryocytic differentiation in K562 cells through ERK activation and induction of autophagy in a fashion similar to other known inducers of megakaryocytic differentiation such as phorbol esters. In addition, as shown earlier, we yet again observed that ORM led to activation of caspases since their inhibition through pan-caspase inhibitor mitigated megakaryocytic differentiation as they led to significant decrease in CD41 and CD61. Because induction of megakaryocytic differentiation in K562 involves growth arrest and exit from cell cycle, we also observed an increase in levels of p21 and p27 with decrease in c-Myc protein levels in K562 cells treated with 7.5 µM ORM for 24 and 48 h,respectively. Taken together, these findings indicate that ORM can markedly induce megakaryocytic differentiation in K562 cells.

Neobaicalein, a flavonoid from the Scutellaria litwinowii Bornm. & Sint. ex Bornm. induced apoptosis in human leukemic cell lines.

Neobaicalein is one of the rich plant flavonoids isolated from the roots of Scutellaria spp. In this study, we evaluated and compared cytotoxic activity and the related apoptosis mechanisms of neobaicalein from Scutellaria litwinowii Bornm. & Sint. ex Bornm on apoptosis-proficient HL-60 cells and apoptosis-resistant K562 cells. Cell viability, cell apoptosis, caspase activity, and apoptosis-related protein expression were measured using MTS assay, propidium iodide (PI) staining and flow cytometry, caspase activity assay, and western blot analysis, respectively. Neobaicalein significantly reduced cell viability in a dose-dependent manner using the MTS assay (P<0.05). The IC50 values (µM) against HL-60 and K562 cells after 48 hr treatment were 40.5 and 84.8, respectively. Incubation of HL-60 and K562 cells with 25, 50, and 100 µM neobaicalein for 48 hr, significantly increased the number of apoptotic cells and showed cytotoxic effects compared with the control group. Treatment with neobaicalein significantly increased Fas (P<0.05) and the cleaved form of PARP (P<0.05), and decreased the Bcl-2 levels (P<0.05) in HL-60 cells, whereas neobaicalein significantly increased Bax (P<0.05) and the cleaved form of PARP (P<0.05), and the caspases of the extrinsic and intrinsic pathways including caspases-8 (P<0.0001), -9 (P<0.01), and effector caspase-3 (P<0.0001) levels in K562 cells compared with the control group. It seems neobaicalein might cause cytotoxicity and cell apoptosis through interaction with the different apoptosis-related proteins of apoptotic pathways in HL-60 and K562 cells. Neobaicalein may exert a beneficial protective effect in slowing the progression of hematological malignancies.

Mir-424 and Mir-503 Regulates Cobll1 Expression during the CML Progression

Up- or down-regulation of many miRNAs plays an important role in chronic myeloid leukemia (CML), however underlying mechanisms remain to be elucidated. Here, we report miRNA-424 and miR-503 regulates Cobll1 expression during CML progression.In previous our study, Cobll1 is a novel oncoprotein to associate with drug resistance and CML progression and highly expressed in blast crisis (BC). Also, overexpression of Cobll1 for blast formation and independently of BCR-ABL1 could be achieved in several ways. It could be by altering unknown signaling pathway(s), and being independent from BCR-ABL1. We searched for miRNAs that can regulate the expression level of Cobll1 to examine potential regulators of Cobll1 .To find candidate miRNAs of Cobll1, we were predicted to miRNA (miR-15a, miR-16, miR-103, miR-107, miR-424 and miR-503) by the algorithm in the TargetScan software. To validation target miRNA of Cobll1, we analyzed the expression levels of these miRNAs in CP and BC samples of 2 CML patients. Interestingly, miR-424 and miR-503 expression were significantly reduced in BC, which indicates that expression of Cobll1 and miR-424 or miR-503 is negative correlation.We next performed that eighty one CML samples (41 CP and 40 BC) were analyzed to determine the levels of the Cobll1 protein/miR-424 and miR-503 by Western blotting/qRT-PCR. Amazingly, we found that miR-424 and miR-503 were significantly downregulated highly expressed Cobll1 BC samples (n=21) compared to Cobll1 less expressed BC samples (n=19) and CP samples (n=41). Interestingly, there were down-regulations in expression of miR-424 and miR-503 that had high co-relation with Cobll1 expression in CML progression.To assess the effects of miR-424 or miR-503 regulates Cobll1 expression, we make ectopically expressing plasmids of wild-type Cobll1 3'-UTR and mutant Cobll1 3'-UTR. Transfection of miR-424 or miR-503 reduced luciferase activity in K562 cells ectopically expressing wild-type Cobll1 3'-UTR, but not a mutant Cobll1 3'-UTR. In addition, overexpression of miR-424 or miR-503 significantly reduced the endogenous Cobll1 levels in K562 cells. Consistently, the knockdown of Cobll1 by the miRNAs sensitized K562 cells to nilotinib-induced apoptosis. Taken together, our findings suggest that miR-424 and miR-503 plays a critical role CML by targeting directly targeting the Cobll1. These results provide the clue of miR-424 and miR-503 as a potential therapeutic target for CML progression by regulating the Cobll1 expression. DisclosuresNo relevant conflicts of interest to declare.

Modulation of TET2 Activity with Ascorbic Acid in Myeloid Neoplasia: Diagnostic and Therapeutic Implications

TET2 mutations (TET2MT) are ubiquitous in myeloid neoplasia, but are most frequent in MDS and MDS/MPN. TET2 is an iron-catalyzed dioxygenase critical in active DNA demethylation that progressively oxidizes 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5CaC). As 5hmC residues are not recognized by DNMT3A, their methylation pattern is not copied onto the daughter DNA strand, leading to net DNA demethylation: TET2 can also therefore be considered a demethylase.Ascorbic acid (AA) is required as a prosthetic group of TET-mediated cytosine demethylation. AA may increase the residual function/throughput of intact TET2 in cases with heterozygous TET2MT and restore hydroxymethylation, thus reversing the expected epigenetic phenotype. We hypothesized that in heterozygous TET2MT neoplasms, AA alone or in combination may constitute a rational targeted therapy to restore TET2 function.We first assessed the effects of AA on TET2 in cell-free experiments. We combined purified wild-type TET2 (TET2WT) with a synthetic poly mC substrate and 0-4 mM AA, and measured levels of 5mC, 5hmC, 5fC, and 5CaC via 2D-UPLC-MS/MS profiling. AA increased the ratio of 5hmC, 5CaC, and 5fC/5mC in a dose-dependent fashion (peak effect 4.5 to 9). To model heterozygous TET2MT seen in myeloid neoplasia, we combined TET2WT with a catalytically-dead TET2S1898F in a 1:1 ratio. AA again increased the ratio of 5hmC, 5fC, and 5CaC to 5mC (peak effect 3 to 6, p<.0001); AA had no effect on TET2S1898F alone.To determine if AA TET2 upregulation could be reproduced in human cells, K562 and MOLM-13 AML lines were treated for 24 hours with 0-1000 uM of AA before subjecting them to 2D-UPLC-MS/MS analysis. AA increased 5hmC levels in K562 cells by 1.8-fold (500 uM, p=.04) and 1.7-fold (1000 uM, p=.15), and increased 5fC levels by 2.1-fold (500 uM, p <0.0001) and 3.1-fold (1000 uM, p=.035). Similarly, AA increased 5hmC levels in MOLM13 cells by 1.8-fold (500 uM, p=.01) and 2-fold (1000 uM, p<.0001), and increased 5fC levels by 3.6-fold (500 uM, p<.0001; 1000 uM, p=.01).We then studied the effect of TET2 loss on leukemogenesis in dual Tet2+/- ; Gulo -/- knockout mice unable to produce endogenous AA, making them ideal to determine the effect of exogenous AA on in vivo TET2 function. Bone marrow samples isolated from Tet2+/- ; Gulo -/- mice had a 1.7-fold reduction in 5hmC (p<.0001), a 2-fold reduction in 5fC (p=.0009), and a 3.4-fold reduction in 5CaC (p<.0001). Treatment of Tet2+/- ; Gulo -/- mice with 3.3 g/L ascorbic acid (in the drinking water for 6 months) partially rescued these DNA oxidation products in their bone marrow cells, with significantly increased levels of 5fC (p = .02) and 5CaC (p =.04) compared to untreated Tet2+/- ; Gulo -/- mice. Compared to TET2WT mice, Tet2+/- ; Gulo -/- mice had only a 1.2-fold reduction in 5hmC (p = 0.2), a 1.15-fold reduction in 5fC (p = .004), and a 1.7-fold reduction in 5CaC (p =.3).Similar to the TET2 knockout mice results, 8/8 of TET2MT patient samples profiled had reduced levels of at least one TET2-dependent DNA oxidation product. Intriguingly, 24-hour treatment with 250 uM AA treatment had no significant effect on any TET2 MT bone marrow samples; in TET2 WT samples, 250 uM AA increased 5fC levels by 2-fold (p =.014) and 5CaC levels by 3-fold (p =.0025), though it nonsignificantly reduced 5hmC levels by 80% (p =.069).In our study, AA significantly increased the levels of TET2-dependent DNA oxidation products in purified TET2, Tet2+/- ; Gulo -/- knockout mice, TET2WT human cell lines, and TET2WT patient samples. Therapeutic AA may therefore play a role in treating myeloid neoplasia with IDH1 or IDH2 mutations, which have structurally-intact but functionally hypoactive TET2 from loss of the IDH-catalyzed TET2 substrate alpha-ketoglutarate. Given that AA was unable to restore TET2 activity in samples from TET2MT patients, AA may not be beneficial in TET2MT myeloid neoplasia.Our study also tells a cautionary tale about using 5hmC levels alone to estimate TET2 activity. 5hmC levels were normal in 3/8 (37.5%) of patients with pathogenic TET2MT; simultaneous measurement of multiple TET2-dependent products is therefore important to accurately assess in-vivo TET2 activity and determine who might benefit from therapeutic AA intervention. DisclosuresGreenberg:Acetylon Pharmaceuticals Inc.: Patents & Royalties: Treatment of Protein Degradation Disorders; Oisin Biotechnologies: Consultancy. Makishima:Yasuda Medical Foundation: Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees; Apellis Pharmaceuticals: Consultancy; Ra Pharma: Consultancy.

Inhibition of USP1, a new partner of Bcr-Abl, results in decrease of Bcr-Abl level in K562 cells.

To analyze interaction of ubiquitin specific peptidase 1 (USP1) with Bcr-Abl and to assess the relation between USP1 functional activity and Bcr-Abl expression in K562 chronic myeloid leukemia cells. The interaction between USP1and Bcr-Abl in K562cells was analyzed by co-immunoprecipitation, Western blot analysis, and confocal microscopy. A direct interaction between Bcr-Abl oncoprotein and USP1protein in K562cells was established by co-immunoprecipitation. Immunofluorescence analysis and confocal microscopy revealed that Bcr-Abl/USP1protein complex is formed in the cell nucleus. The inhibition of USP1protein activity by ML323reduced the level of Bcr-Abl oncoprotein in K562cells. USP1protein has been identified as a new protein partner of Bcr-Abl oncoprotein in chronic myeloid leukemia. The relationship between the functional activity of USP1protein and the level of Bcr-Abl oncoprotein has been demonstrated, suggesting that the targeted inhibition of USP1 activity could be a challenging approach for reducing Bcr-Abl expression.

WT1 regulates cyclinA1 expression in K562cells.

The restricted expression of Wilms tumor1 (WT1) and cyclinA1 (CCNA1) in normal tissues, as opposed to their abnormal expression in leukemia demonstrates the applicability of WT1 and CCNA1 as cancer antigens for immunotherapy, and as markers for prognosis and relapse. In this study, the WT1 and CCNA1 mRNA levels were found to be elevated in bone marrow samples from pediatric acute promyelocytic leukemia (APL or AML‑M3) patients, and to be quite varied in pediatric acute lymphocytic leukemia (ALL) patients, compared to non‑leukemic bone marrow controls. Consistent with the observed upregulation of both WT1 and CCNA1 in APL, WT1 overexpression elevated the CCNA1 mRNA levels in K562 leukemia cells. Treatment with curcumin decreased the WT1 levels in K562cells, and also decreased CCNA1 protein expression. The examination of the CCNA1 promoter identified potential canonical WT1 binding sites within the 3‑kb region upstream of the transcription start site. Chromatin immunoprecipitation and luciferase reporter assays confirmed WT1 binding and the activation of the CCNA1 promoter. Furthermore, the GC‑rich core CCNA1 promoter region provided additional non‑canonical WT1 activation sites, as revealed by promoter assays. The importance of the GC‑rich core region of the CCNA1 promoter was confirmed by treating the K562cells with mithramycinA, which blocks the binding of zinc finger transcription factors to GC‑rich sequences. MithramycinA subsequently suppressed both CCNA1 promoter activity and protein expression in the K562cells. Taken together, the data from the WT1 overexpression, and curcumin and mithramycinA treatment experiments, as well as those from chromatin binding assays, along with inferences from patient RNA analyses, establish a plausible link between WT1 and CCNA1, and support the functional significance of an elevated WT1 expression in leukemia, which may also affect CCNA1 expression.

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.

Analysis of the Effects of δ-Tocopherol on RAW264.7 and K562 Cells Based on 1H NMR Metabonomics.

δ-Tocopherol (δ-TOH) is a form of vitamin E with higher bioactivity. In this study, we studied the bioactivity of δ-TOH using the IC50 of δ-TOH on RAW264.7 (80 μM) and K562 (110 μM) cells. We compared the differential metabolites from the cell lines with and without δ-TOH treatment by 1H NMR metabonomics analysis. It was found that δ-TOH affected the protein biosynthesis, betaine metabolism, and urea cycle in various ways in both cell lines. Metabolic levels of the cell lines were changed after treatment with δ-TOH as differential metabolites were produced. The betaine level in RAW264.7 cells was reduced significantly, while the l-lactic acid level in K562 cells was significantly enhanced. The metabolic changes might contribute to the switch of the respiration pattern from aerobic respiration to anaerobic respiration in K562 cells. These results are helpful in further understanding the subtoxicity of δ-TOH.

Xiao-Ai-Ping inhibits proliferation and induces apoptosis of chronic myeloid leukemia cells through downregulating β-catenin signaling pathway

// Tongtong Wang 2, * , Jing Du 3, * , Bingyu Chen 1 , Wei Zhang 4 , Zhen Wang 1 , Ke Hao 1 , Xin Wang 2 , Yi Zhou 2 , Qiaojuan Zhu 2 , Xiangmin Tong 2 and Ying Wang 1, 2 1 Department of Blood Transfusion, Zhejiang Provincial People&rsquo;s Hospital, People&rsquo;s Hospital of Hangzhou Medical College, Hangzhou, Zhejiang 310014, China 2 Clinical Research Institute, Zhejiang Provincial People&rsquo;s Hospital, People&rsquo;s Hospital of Hangzhou Medical College, Hangzhou, Zhejiang 310014, China 3 Department of Laboratory Medicine, Zhejiang Provincial People&rsquo;s Hospital, People&rsquo;s Hospital of Hangzhou Medical College, Hangzhou, Zhejiang 310014, China 4 Department of Hepatopancreatobiliary Surgery, Zhejiang Provincial People&rsquo;s Hospital, People&rsquo;s Hospital of Hangzhou Medical College, Hangzhou, Zhejiang 310014, China * These authors contributed equally to this work Correspondence to: Ying Wang, email: nancywangying@163.com Xiangmin Tong, email: tongxiangmin11@163.com Keywords: Xiao-Ai-Ping; chronic myeloid leukemia; proliferation; apoptosis; &beta;-catenin Received: August 08, 2017&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Accepted: November 16, 2017&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Published: January 04, 2018 ABSTRACT The active ingredients of Xiao-Ai-Ping (XAP) injection are Marsdeniae tenacissimae extraction, which is a traditional Chinese herb has been used to treat multiple diseases for a long time. However, the possible roles and mechanisms of XAP in chronic myeloid leukemia (CML) still remain unknown. In this study, we examined the effects of XAP on proliferation and apoptosis of K562 cells (CML cell line) and peripheral blood mononuclear cell (PBMCs) from CML patients. Here, we found that XAP could inhibit the proliferation of K562 cells and induce the apoptosis of K562 cells via mitochondrial pathway. In addition, XAP inhibited the migration of K562 cells through downregulating the expression levels of chemokine SDF-1 and its receptor CXCR-4. In addition, XAP down-regulated &beta;-catenin and CyclinD1 level, and up-regulated GSK3&beta; level in K562 cells. Interestingly, over-expression of &beta;-catenin restored the XAP-induced apoptosis of K562 cells. Furthermore, XAP significantly inhibited the proliferation and induced the apoptosis of PBMCs from CML patients, compared with healthy people. In conclusion, XAP could inhibit the proliferation and migration of CML cells, and induce the apoptosis of CML cells through down-regulation of &beta;-catenin and SDF1/CXCR-4 signaling.

Abstract 5481: Role and regulation of Peroxiredoxin antioxidant proteins in K562 leukemia cells

Abstract Peroxiredoxins are a family of antioxidant proteins that have found to play a key role in many diseases, including cancer. A wide number of studies have found that Peroxiredoxin levels are elevated in several cancers, and reducing these levels can aid in inhibiting cell growth of cancer cells, suggesting that Peroxiredoxins confer resistance to reactive oxygen species (ROS)-induced cellular toxicity in cancer cells. Specific research has demonstrated that Peroxiredoxin levels are elevated in leukemia cells, and that targeting and inhibiting Peroxiredoxins in these cells can induce differentiation. Studies have also shown the use of doxorubicin as an effective chemotherapy treatment to significantly decrease cell proliferation and viability in leukemia. The purpose of this study is to examine Peroxiredoxin levels in K562 cells, a human chronic myelogenous leukemia cell line, and determine if high levels of Peroxiredoxins have the ability to protect cells from chemotherapy-induced death and possibly aid in chemoresistance. We first demonstrated that serum-deprived K562 cells exhibited significant growth inhibition over a 72 hour time period, but had no significant effect on viability. We also showed that treating K562 cells with 50 nM doxorubicin significantly decreased proliferation and viability. Corresponding changes in Peroxiredoxin protein levels are currently being analyzed to determine the effect of both growth-inhibition and chemotherapy-induced cytotoxicity on the Peroxiredoxin family in K562 cells, and siRNA experiments are being conducted to examine the protective role of Peroxiredoxins in these cells. Based on previous studies on MCF-7 cells conducted in our lab, we predict that Peroxiredoxin levels will be modified under both conditions, and that suppression of Prdxs by siRNA will increase chemotherapy susceptibility. Citation Format: Elizabeth Szabo, Shelley A. Phelan. Role and regulation of Peroxiredoxin antioxidant proteins in K562 leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5481. doi:10.1158/1538-7445.AM2017-5481

An enhancer haplotype may influence BCL11A expression levels and the response to hydroxyurea in β-thalassemia patients.

To identify the BCL11A intron-2 enhancer linkage disequilibrium (LD) block, harboring two previously identified SNPs, associating with the hydroxyurea response in β-thalassemia patients and the functional significance of this region. Several neighboring SNPs were genotyped in our cohort. The associating LD block was identified, and its function studied in K562 erythroid cells via CRISPR/Cas9 genome editing. A haplotype harboring three tag SNPs correlated significantly with the HU-response and BCL11A transcript levels in the patients' reticulocytes. Two deletions encompassing this LD block significantly reduced BCL11A transcript levels in K562 cells. Our data suggest an essential role for this LD block in BCL11A expression levels and the response to hydroxyurea in β-thalassemia patients.

Notch1-promoted TRPA1 expression in erythroleukemic cells suppresses erythroid but enhances megakaryocyte differentiation

The Notch1 pathway plays important roles in modulating erythroid and megakaryocyte differentiation. To screen the Notch1-related genes that regulate differentiation fate of K562 and HEL cells, the expression of transient receptor potential ankyrin 1 (TRPA1) was induced by Notch1 receptor intracellular domain (N1IC), the activated form of Notch1 receptor. N1IC and v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1) bound to TRPA1 promoter region to regulate transcription in K562 cells. Transactivation of TRPA1 promoter by N1IC depended on the methylation status of TRPA1 promoter. N1IC and Ets-1 suppressed the DNA methyltransferase 3B (DNMT3B) level in K562 cells. Inhibition of TRPA1 expression after Notch1 knockdown could be attenuated by nanaomycin A, an inhibitor of DNMT3B, in K562 and HEL cells. Functionally, hemin-induced erythroid differentiation could be suppressed by TRPA1, and the reduction of erythroid differentiation of both cells by N1IC and Ets-1 occurred via TRPA1. However, PMA-induced megakaryocyte differentiation could be enhanced by TRPA1, and the surface markers of megakaryocytes could be elevated by nanaomycin A. Megakaryocyte differentiation could be reduced by Notch1 or Ets-1 knockdown and relieved by TRPA1 overexpression. The results suggest that Notch1 and TRPA1 might be critical modulators that control the fate of erythroid and megakaryocyte differentiation.

Antitumor effects and mechanisms of Ganoderma extracts and spores oil.

Ganoderma lucidum is a popular herbal medicine used in China to promote health. Modern studies have disclosed that the active ingredients of Ganoderma can exhibit several effects, including antitumor effects and immunomodulation. The present study evaluated the antitumor effects of self-prepared Ganoderma extracts and spores oil, and investigated the possible underlying mechanisms by observing the effects of the extracts and oil on topoisomerases and the cell cycle. The results showed that Ganoderma extracts and spores oil presented dose-dependent inhibitory effects on tumor cells. The half maximal inhibitory concentration (IC50) values of Ganoderma extracts on HL60, K562 and SGC-7901 cells for 24 h were 0.44, 0.39 and 0.90 mg/ml, respectively; for Ganoderma spores oil, the IC50 values were 1.13, 2.27 and 6.29 mg/ml, respectively. In the in vivo study, the inhibitory rates of Ganoderma extracts (4 g/kg/d, intragastrically) on S180 and H22 cells were 39.1 and 44.6%, respectively, and for Ganoderma spores oil (1.2 g/kg/d, intragastrically) the inhibitory rates were 30.9 and 44.9%, respectively. Ganoderma extracts and spores oil inhibited the activities of topoisomerase I and II. Ganoderma spores oil was shown block the cell cycle at the transition between the G1 and S phases and induce a marked decrease in cyclin D1 levels in K562 cells, with no significant change in cyclin E level. These results suggest that the Ganoderma extracts and spores oil possessed antitumor effects in the in vitro and in vivo studies. The antitumor mechanisms of the extracts and spores oil were associated with inhibitory effects on topoisomerase I and II activities, and for Ganoderma spores oil, the antitumor effects may also be associated with decreased cyclin D1 levels, thus inducing G1 arrest in the cell cycle.

Chemical Proteomics Reveals Ferrochelatase as a Common Off-target of Kinase Inhibitors.

Many protein kinases are valid drug targets in oncology because they are key components of signal transduction pathways. The number of clinical kinase inhibitors is on the rise, but these molecules often exhibit polypharmacology, potentially eliciting desired and toxic effects. Therefore, a comprehensive assessment of a compound's target space is desirable for a better understanding of its biological effects. The enzyme ferrochelatase (FECH) catalyzes the conversion of protoporphyrin IX into heme and was recently found to be an off-target of the BRAF inhibitor Vemurafenib, likely explaining the phototoxicity associated with this drug in melanoma patients. This raises the question of whether FECH binding is a more general feature of kinase inhibitors. To address this, we applied a chemical proteomics approach using kinobeads to evaluate 226 clinical kinase inhibitors for their ability to bind FECH. Surprisingly, low or submicromolar FECH binding was detected for 29 of all compounds tested and isothermal dose response measurements confirmed target engagement in cells. We also show that Vemurafenib, Linsitinib, Neratinib, and MK-2461 reduce heme levels in K562 cells, verifying that drug binding leads to a loss of FECH activity. Further biochemical and docking experiments identified the protoporphyrin pocket in FECH as one major drug binding site. Since the genetic loss of FECH activity leads to photosensitivity in humans, our data strongly suggest that FECH inhibition by kinase inhibitors is the molecular mechanism triggering photosensitivity in patients. We therefore suggest that a FECH assay should generally be part of the preclinical molecular toxicology package for the development of kinase inhibitors.

Effect of Rapamycin on Expression of Survivin and Caspase-3 and Its Influence on K562 Cell Ultrastructure

To investigate the effect of rapamycin on the expression of survivin and caspase-3 at mRNA level in K562 cells and the influence of rapamycin on K562 cell ultrastructure. The effects of rapamycin at various concentration on K562 cell proliferation were analyzed by CCK8; the morphological characteristics of K562 cells was observed by transmission electron microscopy; the expression of survivin and caspase-3 at mRNA level in K562 cells treated with rapamycin was detected by RT-PCR. The proliferation of K562 cells was significantly inhibited by rapamycin. The apoptosis level of K562 cells increased with increase of rapamycin concentration, the expression of survivin at mRNA level decreased with increase of rapamycin concentration (P < 0.05). The expression of caspase-3 at mRNA level increased with increase of rapamycin concentration. Rapamycin can prornote K562 cell apoptosis through up-regulating caspase-3 level and reduceing survivin level.

Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line.

First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro transfection results from BCPV-siRNA, a newly developed biodegradable transfection agent, BCPV, is being probed for transfection performance in an animal model.