Activity In K562 Cells Research Articles

A pectic polysaccharide from Typhonii Rhizoma: Characterization and antiproliferative activity in K562 cells through regulating mitochondrial function and energy metabolism

The pectic polysaccharide WTRP-A0.2b (43 kDa) has been isolated from Typhonii rhizoma and analyzed in terms of its structural features, anti-tumor activities and mechanism of action. NMR, FT-IR, monosaccharide composition, and enzymology demonstrate that WTRP-A0.2b is composed of rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II) and homogalacturonan (HG) domains with mass ratios of 3.7:1:1.7, respectively. The RG-I domains contain a highly branched structure that is substituted primarily with β-D-1,4-galactan, α-L-1,5-arabinan, and AG-II. The HG domains contain un-esterified and methyl-esterified and/or acetyl-esterified oligogalacturonides with a degree of polymerization of 1–8. In vitro experiments demonstrate that WTRP-A0.2b inhibits proliferation of K562 cells by inducing mitochondrial damage and suppressing glycolysis. This activity promotes mitochondrial permeability, increases production of reactive oxygen species (ROS), boosts extracellular oxygen consumption and adenosine triphosphate (ATP) content, while it decreases uncoupling protein-2 (UCP2) expression and lactic acid content. Our results provide valuable insight for screening natural polysaccharide-based anti-tumor effects of polysaccharides from Typhonii rhizoma.

Capsazepine activates amiloride-insensitive ENaC-like channels in human leukemia cells

Sodium influx carried out by ion channels is one of the main regulators of water-salt and volume balance in cells of blood origin. Previously, we described amiloride-insensitive ENaC-like channels in human myeloid leukemia K562 cells; the intracellular regulatory mechanisms of the channels are associated with actin cytoskeleton dynamics. Recently, an extracellular mechanism of ENaC-like channels activation in K562 cells by the action of serine protease trypsin has been revealed. The other extracellular pathways that modulate ENaC (epithelial Na+ channel) activity and sodium permeability in transformed blood cells are not yet fully investigated. Here, we study the action of capsazepine (CPZ), as δ-ENaC activator, on single channel activity in K562 cells in whole-cell patch clamp experiments. Addition of CPZ (2 μM) to the extracellular solution caused an activation of sodium channels with typical features; unitary conductance was 15.1 ± 0.8 pS. Amiloride derivative benzamil (50 μM) did not inhibit their activity. Unitary currents and conductance of CPZ-activated channels were higher in Na+-containing extracellular solution than in Li+, that is one of the main fingerprints of δ-ENaC. The results of RT-PCR analysis and immunofluorescence staining also confirmed the expression of δ-hENaC (as well as α-, β-, γ-ENaC) at the mRNA and protein level. These findings allow us to speculate that CPZ activates amiloride-insensitive ENaC-like channels that contain δ-ENaC in К562 cells. Our data reveal a novel extracellular mechanism for ENaC-like activation in human leukemia cells.

In vitro and in silico perspectives to explain anticancer activity of a novel syringic acid analog ((4-(1H-1, 3-benzodiazol-2-yl)-2, 6-dimethoxy phenol)) through apoptosis activation and NFkB inhibition in K562 leukemia cells

Syringic acid (SA) is an active carcinogenesis inhibitor; however, the low bioavailability and unstable functional groups hinder its activity. Here, a chemically synthesized novel SA analog (SA10) is evaluated for its anticancer activity using in-vitro and in-silico studies. K562 cell line study revealed that SA10 had shown a higher rate of inhibition (IC50 = 50.40 μg/mL) than its parental compound, SA (IC50 = 96.92 μg/mL), at 50 μM concentration. The inhibition ratio was also been evaluated by checking the expression level of NFkB and Bcl-2 and showing that SA10 has two-fold increase in the inhibitory mechanism than SA. This result demonstrates that SA10 acts as an NFkB inhibitor and an apoptosis inducer. Further, molecular docking and simulation have been performed to get insights into the possible inhibitory mechanism of SA and SA10 on NFkB at the atomistic level. The molecular docking results exemplify that both SA and SA10 bind to the active site of NFkB, thereby interfering with the association between DNA and NFkB. SA10 exhibits a more robust binding affinity than SA and is firmly docked well into the interior of the NFkB, as confirmed by MM-PBSA calculations. In a nutshell, the Benzimidazole scaffold containing SA10 has shown more NFkB inhibitory activity in K562 cells than SA, which could be helpful as an ideal therapeutic NFkB inhibitor for treating cancers.

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.

Genetic Predisposition to the Mortality in Septic Shock Patients: From GWAS to the Identification of a Regulatory Variant Modulating the Activity of a CISH Enhancer.

The high mortality rate in septic shock patients is likely due to environmental and genetic factors, which influence the host response to infection. Two genome-wide association studies (GWAS) on 832 septic shock patients were performed. We used integrative bioinformatic approaches to annotate and prioritize the sepsis-associated single nucleotide polymorphisms (SNPs). An association of 139 SNPs with death based on a false discovery rate of 5% was detected. The most significant SNPs were within the CISH gene involved in cytokine regulation. Among the 139 SNPs associated with death and the 1311 SNPs in strong linkage disequilibrium with them, we investigated 1439 SNPs within non-coding regions to identify regulatory variants. The highest integrative weighted score (IW-score) was obtained for rs143356980, indicating that this SNP is a robust regulatory candidate. The rs143356980 region is located in a non-coding region close to the CISH gene. A CRISPR-Cas9-mediated deletion of this region and specific luciferase assays in K562 cells showed that rs143356980 modulates the enhancer activity in K562 cells. These analyses allowed us to identify several genes associated with death in patients with septic shock. They suggest that genetic variations in key genes, such as CISH, perturb relevant pathways, increasing the risk of death in sepsis patients.

Pro-apoptotic activity of novel synthetic isoxazole derivatives exhibiting inhibitory activity against tumor cell growth in vitro.

In order to develop potential anticancer agents stimulating apoptosis, novel 3,4-isoxazolediamide and 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine derivatives have been synthetized. The original structures of geldanamycin and radicicol, which are known natural heat shock protein (HSP) inhibitors, were deeply modified because both of them exhibit several drawbacks, such as poor solubility, hepatotoxicity, intrinsic chemical instability or deprivation of the in vivo activity. This novel class of synthetic compounds containing the isoxazole nucleus exhibited potent and selective inhibition of HSP90 in previous studies. Biological assays (focusing on in vitro antiproliferative effects and pro-apoptotic activity) in human erythroleukemic K562 cells (as a model system referring to tumor cells grown in suspension), glioblastoma U251-MG and glioblastoma temozolomide (TMZ)-resistant T98G cell lines (two model systems referring to tumor cells grown attached to the flask), were performed. Almost all isoxazole derivatives demonstrated significant antiproliferative and pro-apoptotic activities, showing induction of both early and late apoptosis of K562 cells. Different effects were observed on the glioma U251-MG and T98G cells, depending on the structure of the analogues. Antiproliferative and pro-apoptotic activities in K562 cells were associated with the activation of the erythroid differentiation program. The present study demonstrated that 3,4-isoxazolediamide and 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine derivatives should be considered for in vivo studies focusing on the development of anticancer drugs acting, at least partially, via activation of apoptosis.

Functional Evaluation and Mechanism Study of Kel As an Oncogenic Driver in Acute Erythroleukemia

Background Acute erythroleukemia (AEL) is defined as an distinct subtype (<5%) of AML in FAB classification, characterized by predominant erythropoiesis. It is associated with a poor prognosis, with a median overall survival (OS) of 3-14 months from diagnosis. No recurrent cytogenetic abnormality is specific of AEL. Through "Oncomine" Database, we found that KEL was significantly up-regulated in AEL patients, compared with other types of AML and normal patients. It has been reported that KEL promoter exhibited a strong transcriptional erythroid activity in K562 cells, but its role in the development of AEL remains unclear. Circular RNAs (circRNAs) have been implicated in various biological processes. Here we found one circRNA transcripted from KEL gene (circ-KEL), which also had a high expression in AEL, may play a role of coordinate regulation with KEL. The present study aimed to investigate the potential modulation of KEL in the development and progression of AEL. Method In our study, we collected blood samples of 11 AEL patient, 20 other types of AML patients and 20 normal patients. qPCR and Western blot analysis were performed to examine the expression of KEL and circ-KEL. We set up the erythroid differentiation models in vitro using two AEL cells lines (K562 and HEL) , induced with 50 μM hemin from day1 to day 7. The oncogenic functions of KEL were further measured in AEL cell lines by performing RNA interfering (RNAi), immunofluorescence (IF) staining, CCK8 assay and flow cytometry. The expression levels of γ-globin and FUT1 as well as the erythroid surface markers TER119 and GPA were examined to evaluate their function in erythroid differentiation. The proliferative ability of AEL cells was determined by CCK-8 assay. AEL animal models were established using NCG mice transplanted with K562 (NC and KEL over-expression). The tumor formation rate, body weight and survival curve were investigated to evaluate the oncogenic functions of KEL in-vivo. Protein antibody microarray was used to analyze the activation of related pathways after KEL over-expression in AEL cells. Moreover, we explored the molecular mechanisms of KEL and verified the interactions among GATA1 and H3K27ac as well as the PKC/RSK2/CREB pathway by performing ChIP, RIP, dual-luciferase reporter assay and Western blotting. Result KEL was specifically higher expressed in AEL clinical samples/cells lines, consistent with the results of the database. The over-expression of KEL could up-regulate the expression of GATA1, thus promoting the erythroid differentiation of AEL cells. Moreover, protein antibody microarray results showed that the up-regulation of KEL in AEL cells leads to the activation of multiple proliferation and PDL1 related pathways. After subsequent verification, it was verified that PKC/RSK2/CREB pathway was significantly activated. Meanwhile, the expression of PDL1 was highly correlated with KEL. We found that the higher expression of KEL in AEL cells could up-regulate PDL1 expression through activating the phosphorylation of relative transcript factors on PDL1 DNA promoter. In-vivo experiment showed that KEL over-expression may accelerate the leukemia progression. In addition, we discovered that JQ1 could be used as a inhibitor of KEL, which could effectively decrease the expression of GATA1 and PDL1. A number of circRNAs have been reported to act as microRNA (miRNA) sponges, which regulate target gene expression by interacting with miRNA. Our results revealed that circ-KEL could serve as a sponge for miR-671 and up-regulate miR-617 functional target KEL, thus promote the erythroid differentiation. Conclusion Our study reveals the oncogenic role and a novel regulatory mechanism of KEL in AEL progression. For the first time, we demonstrated that KEL was specifically highly expressed in AEL cells, leading to the dysregulation of erythroid differentiation through up-regulating GATA1, and on the other hand, promoting cell proliferation via PKC/RSK2/CREB pathway. Circ-KEL which also had a high expression in AEL, may play a role of coordinate regulation with KEL. These findings may provide a promising strategy for future diagnosis, biomarker discoveries and treatment for AEL patients. Figure Disclosures No relevant conflicts of interest to declare.

Antitumor effect of depsidones from lichens on tumor cell lines and experimental murine melanoma

Lichens have exhibited numerous biological activities, including growth inhibition of tumor cells. This study evaluated the antiproliferative activity of hypostictic and salazinic acids against tumor cell lines (B16-F10, PC-03, MCF7, HT-29, HEP-G2, K562 and 786-0) by the SRB assay in vitro and antitumor activity in experimental murine melanoma in vivo. Activation of caspase-3 was quantified by flow cytometry. The murine experimental melanoma model B16-F10 was used in BALB/c mice for evaluation of antitumor activity. Hypostictic acid showed significant antiproliferative activity in K562 cells (GI50 2.20μM), B16-F10 (GI50 13.78μM) and 786-0 (GI50 14.24μM), whereas salazinic acid was more active against K562 cells (GI50 64.36μM), HT-29 (GI50 67.91μM) and B16-F10 (GI5078.64μM). Quantification of capase-3 revealed that the test compounds did not increase the expression of that enzyme. In the in vivo antitumor evaluation in B16-F10 melanoma, the isolated compounds inhibited tumor growth in relation to weight and volume. Hypostictic acid (16.7mg/kg) inhibited 72% and salazinic acid 88% of tumor volume (p<0.05). The results indicated that, both in the in vitro and in vivo models, the compounds evaluated showed antiproliferative and antitumor activities.

Hemin induces autophagy in a leukemic erythroblast cell line through the LRP1 receptor.

Hemin is an erythropoietic inductor capable of inducing autophagy in erythroid-like cell lines. Low-density lipoprotein receptor-related protein 1 (LRP1) is a transmembrane receptor involved in a wide range of cellular processes, such as proliferation, differentiation, and metabolism. Our aim was to evaluate whether LRP1 is responsible for hemin activity in K562 cells, with the results demonstrating a three-fold increase in LRP1 gene expression levels (P-values <0.001) when assessed by quantitative real-time RT-PCR (qRT-PCR). Moreover, a 70% higher protein amount was observed compared with control condition (P-values <0.01) by Western blot (WB). Time kinetic assays demonstrated a peak in light chain 3 (LC3) II (LC3II) levels after 8 h of hemin stimulation and the localization of LRP1 in the autophagosome structures. Silencing LRP1 by siRNA decreased drastically the hemin-induced autophagy activity by almost 80% compared with control cells (P-values <0.01). Confocal localization and biochemical analysis indicated a significant redistribution of LRP1 from early endosomes and recycling compartments to late endosomes and autophagolysosomes, where the receptor is degraded. We conclude that LRP1 is responsible for hemin-induced autophagy activity in the erythroblastic cell line and that hemin–LRP1 complex activation promotes a self-regulation of the receptor. Our results suggest that hemin, via the LRP1 receptor, favors erythroid maturation by inducing an autophagic response, making it a possible therapeutic candidate to help in the treatment of hematological disorders.

PO-039 Examination of the anti-tumour and neuropathic side effect responses of first generation and second generation proteasome inhibitors

IntroductionThe overall goal in cancer treatment is to damage cancer cell macromolecules that causes the cancer cells to die. But in the treatment process, the defense mechanisms developed by the cancer cells affect the response to the treatment. As we have shown in our studies, cancer cells after anti-cancer stress have about 10–20 times more proteasome activation than healthy cells. Therefore, proteasome inhibition plays an important role in the prevention of resistance and recurrence in cancer treatment.Bortezomib, the first generation proteasome inhibitor, is used intensively, in hematologic cancer types, and significantly increases patient survival. However, it causes significant side effects such as neuropathy. New proteasome inhibitors are being produced to reduce side effects in the market. These inhibitors include clinical studies as well as preclinical studies. However, the lack of side-effect studies causes the early termination of clinical trials.Material and methodsIn this study, we compared the anti-tumour effects and neural toxicities of first generation and second-generation proteasome inhibitors in co-culture model. In this direction, human neural progenitor cells and K562 leukaemia cells were used. Co-cultures were incubated with 100 nM proteasome inhibitors for 24 hour and effects on the cancer cells and neural cells were analysed separately. Apoptotic cell death was evaluated with Annexin V/PI double staining by flow cytometry in K562 cells, and also PARP, Caspase 9 protein levels have been analysed in both cells. Protein oxidation related parameters such as protein carbonyls, ubiquitinated proteins, and HSP levels were investigated for understanding the stress response in both cells. Additionally, cytoskeleton proteins β-actin and β- tubulin changes were assessed in neural cells with confocal microscopy.Results and discussionsOur data showed that new proteasome inhibitors are less toxic in neural cells when compared to bortezomib. Protein carbonyls and ubiquitinated proteins were highest in bortezomib treated cells. On the other hand, especially carfilzomib has a lower anti-tumour activity in K562 cells when compared to bortezomib.ConclusionThis study may bring a highlight for the clinical usage and side effects of new proteasome inhibitors.

Unintended target effect of anti-BCL-2 DNAi.

IntroductionPrevious research suggested that a novel compound PNT2258 inhibits B-cell lymphoma 2 (BCL-2) transcription by DNA interference (DNAi) and demonstrated its activity in preclinical xenograft models and in a pilot Phase II clinical trial in non-Hodgkin’s lymphoma (NHL). While the drug downregulates BCL-2 at the promoter, mRNA, and protein levels, there is a significant homology (13–16 bases) between PNT100 and a number of promoters of genes involved in cell cycle regulation and survival. In this study, we identify cyclin-dependent kinase-4 (CDK4) as an unintended target gene of PNT2258 and examine its relevance to NHL.MethodsWe performed a Basic Local Alignment Search Tool (BLAST) homology search using PNT100 DNAi sequences. Also, we conducted CDK4 promoter assay in K562 cells and studied the protein expression of CDK4 in Wayne State University (WSU)-follicular small cleaved cell lymphoma (FSCCL), WSU-diffuse large cell lymphoma, and WSU-Waldenström’s macroglobulinemia (WM) lymphoma cells.ResultsBLAST homology search showed that PNT100 completely binds to BCL-2 gene as expected. However, there was 100% homology in a stretch of 14 bases (8–21) between PNT100 and CDK4. PNT2258 strongly inhibited CDK4 promoter activity in K562 cells. Moreover, CDK4 protein expression was significantly downregulated by PNT2258 in WSU-FSCCL and WSU-WM cell lines.DiscussionDNAi may work not only through knocking down the intended gene but also by knocking down other genes. PNT2258 affects CDK4 expression and promoter activity. Results of the present study suggest a broader mechanism of action for DNAi targeting both intended (BCL-2) and unintended (CDK4) genes.

Abstract 3450: Identification of a novel lncRNA involved in Bcr-Abl mediated cellular transformation

Abstract Aberrant expression of long noncoding RNAs (lncRNAs) is associated with various human cancers. However, little information is available about functional involvement of lncRNAs in tumorigenesis of chronic myeloid leukemia (CML) induced by Bcr-Abl oncogene. In this study, we performed a comprehensive analysis of lncRNAs in human CML cells using an lncRNA cDNA microarray combined with drug treatment. We identified an lncRNA termed lncRNA-up38 that was critically involved in Bcr-Abl-mediated cellular transformation. We found that expression of lncRNA-up38 was greatly upregulated by inhibiting Bcr-Abl kinase activity in K562 cells and leukemic cells derived from CML patients. Furthermore, our experiments demonstrated that lncRNA-up38 acted as a key regulator of 4EBP1 functioning during Bcr-Abl-mediated cellular transformation. Moreover, silencing lncRNA-up38 promoted leukemic cells survival and enhanced Bcr-Abl-mediated tumorigenesis, whereas forced expression of lncRNA-up38 sensitized leukemic cells to undergo apoptosis and inhibited Bcr-Abl-induced tumorigenesis. Additionally, our experiments have begun to address the mechanism of how lncRNA-up38 is regulated in the leukemic cells and showed that Bcr-Abl repressed lncRNA-up38 expression through c-Myc-dependent signaling. Taken together, these results reveal that Bcr-Abl-mediated cellular transformation is associated with downregulation of tumor-suppressor lncRNA-up38 and suggest a potential strategy for the treatment of Bcr-Abl-positive leukemia. Note: This abstract was not presented at the meeting. Citation Format: Jilong Chen, Xuefei Wang. Identification of a novel lncRNA involved in Bcr-Abl mediated cellular transformation [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 3450. doi:10.1158/1538-7445.AM2017-3450

The transcription factor GATA1 regulates NBEAL2 expression through a long-distance enhancer.

Gray platelet syndrome is named after the gray appearance of platelets due to the absence of α-granules. It is caused by recessive mutations in NBEAL2, resulting in macrothrombocytopenia and myelofibrosis. Though using the term gray platelets for GATA1 deficiency has been debated, a reduced number of α-granules has been described for macrothrombocytopenia due to GATA1 mutations. We compared platelet size and number of α-granules for two NBEAL2 and two GATA1-deficient patients and found reduced numbers of α-granules for all, with the defect being more pronounced for NBEAL2 deficiency. We further hypothesized that the granule defect for GATA1 is due to a defective control of NBEAL2 expression. Remarkably, platelets from two patients, and Gata1-deficient mice, expressed almost no NBEAL2. The differentiation of GATA1 patient-derived CD34+ stem cells to megakaryocytes showed defective proplatelet and α-granule formation with strongly reduced NBEAL2 protein and ribonucleic acid expression. Chromatin immunoprecipitation sequencing revealed 5 GATA binding sites in a regulatory region 31 kb upstream of NBEAL2 covered by a H3K4Me1 mark indicative of an enhancer locus. Luciferase reporter constructs containing this region confirmed its enhancer activity in K562 cells, and mutagenesis of the GATA1 binding sites resulted in significantly reduced enhancer activity. Moreover, DNA binding studies showed that GATA1 and GATA2 physically interact with this enhancer region. GATA1 depletion using small interfering ribonucleic acid in K562 cells also resulted in reduced NBEAL2 expression. In conclusion, we herein show a long-distance regulatory region with GATA1 binding sites as being a strong enhancer for NBEAL2 expression.

The effect of FcγRIIA and FcγRIIB on coronary artery lesion formation and intravenous immunoglobulin treatment responses in children with Kawasaki disease.

Previous research has found patients with the FcγRIIIB NA1 variant having increased risk of intravenous immunoglobulin (IVIG) resistance in Kawasaki disease (KD). Our previous studies revealed that elevated FcγRIIA expression correlated with the susceptibility of KD patients. We conducted this research to determine whether and how Fcγ receptors affect the susceptibility, IVIG treatment response, and coronary artery lesions (CAL) of KD patients. The activating FcγRIIA and inhibitory FcγRIIB methylation levels of seven patients with KD and four control subjects were examined using HumanMethylation27 BeadChip. We enrolled a total of 44 KD patients and 10 control subjects with fevers. We performed real-time RT-PCR to determine the FcγRIIA and FcγRIIB expression levels, as well as a luciferase assay of FcγRIIA. We found a considerable increase in methylation of both FcγRIIA and FcγRIIB in KD patients undergoing IVIG treatment. Promoter methylation of FcγRIIA inhibited reporter activity in K562 cells using luciferase assay. The FcγRIIB mRNA expression levels were not found to increase susceptibility, CAL formation, or IVIG resistance. FcγRIIA mRNA expression levels were significantly higher in IVIG-resistant patients than in those that responded to IVIG during the pre-treatment period. Furthermore, the FcγRIIA/IIB mRNA expression ratio was considerably higher in KD patients with CAL than in those without CAL. FcγRIIA and FcγRIIB both demonstrated increased methylation levels in KD patients that underwent IVIG treatment. FcγRIIA expression influenced the IVIG treatment response of KD patients. The FcγRIIA/IIB mRNA expression ratio was greater in KD patients with CAL formation.

Abstract 46: Functional analysis of long noncoding RNAs in Bcr-Abl-mediated tumorigenesis

Abstract Human genome is pervasively transcribed and the majority does not have apparent protein coding potential, which is defined as noncoding RNA (ncRNA). Long noncoding RNAs (lncRNAs) are greater than 200 nucleotides in length, transcribed by RNA polymerase II or III, and poorly conserved. Aberrant expression of numerous functional lncRNAs is associated with various human cancers. Chronic myeloid leukemia (CML) is a hematologic malignancy associated with a translocation between chromosome 9 and 22 that results in the formation of bcr-abl hybrid gene which occurs in over 90% of CML cases. However, the role of lncRNAs in Bcr-Abl-mediated chronic myeloid leukemia (CML) is not fully understood. In this study, we performed a comprehensive analysis of lncRNAs in human CML cells using a microarray of cDNAs encoding lncRNAs. The microarray analysis revealed 338 upregulated lncRNAs and 108 downregulated lncRNAs after silencing Bcr-Abl in human CML cells. To identify functional lncRNAs that are important in Bcr-Abl-mediated transformation, we selected the most significantly affected lncRNAs for further studies. These lncRNAs were examined by ORF finder from NCBI and protein-coding potential was determined. We identified several lncRNAs that acted as a key regulator of Bcr-Abl-mediated cellular transformation. Expression of these lncRNAs was further examined in response to disruption of Bcr-Abl expression or by inhibiting Bcr-Abl kinase activity in K562 cells and leukemic cells derived from CML patients. Experiments including ectopic overexpression or knockdown of these lncRNAs were performed to address the role and mechanism of how these lncRNAs function in the leukemic cells. Together, these results reveal that several lncRNAs are critically involved in Bcr-Abl-mediated cellular transformation and suggest a potential strategy for the treatment of Bcr-Abl-positive leukemia. Note: This abstract was not presented at the conference. Citation Format: Guijie Guo, Xuefei Wang, Jilong Chen. Functional analysis of long noncoding RNAs in Bcr-Abl-mediated tumorigenesis. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 46.

13-Oxyingenol dodecanoate, a cytotoxic ingenol derivative, induces mitochondrial apoptosis and caspase-dependent Akt decrease in K562 cells.

13-Oxyingenol dodecanoate (13OD) is an ingenol derivative prepared from Chinese traditional medicine Euphorbia kansui without any report about its bioactivity. The present study demonstrated for the first time that 13OD displayed potent cytotoxicity against chronic myeloid leukemia K562 cells in vitro. 13OD inhibited proliferation, induced G2/M phase arrest, and exhibited potent apoptotic activity in K562 cells. In K562 cells, 13OD disrupted the mitochondrial membrane potential and induced high level of ROS, which played an indispensable role in 13OD-induced apoptosis. Further investigations on the molecular mechanisms revealed that total Akt protein level was decreased in a caspase-dependent way after treatment with 13OD; in addition, ERK was activated by 13OD, and this activation played a protective role in 13OD stimulation. Altogether, these results revealed that the cytotoxic ingenol derivative 13OD induced apoptosis with novel mechanisms for the proapoptotic function in cancer cells, and suggested that 13OD may serve as a lead template for rational drug design and for future anticancer agent development.

Antioxidant Activities and the In Vitro Effects on K562 Cell Viability of Flavonoids Obtained from Onion Skin by Different Extraction Methods

In order to effectively extract specific flavonoids with high biological activity from onionskin, NaNO2- Al(NO3)3-NaOH coloration method, spectrophotometry and MTT method were adopted to study the yield rate and content of flavonoids obtained from alcohol water and alkali water extraction processes, the antioxidant activities and the in vitro effects on K562 cell viability. The results indicated that yield rates of flavonoids obtained by alcohol water and alkali water extraction methods were 3.76% and 2.37%, and solid flavonoids contents were 42.20% and 76.21% respectively; under the same concentration of total flavonoids, alkali water extract has weaker effect on removing superoxide anions and DPPH • radicals, whereas they have similar effect on removing nitrite and hydroxyl free radicals; in the test concentration range (40 ∼ 200 µg / mL), they have similar effect on inhibiting the proliferation activity of K562 cells; the maximum inhibition rate of flavonoids obtained by alkali water extraction method is 77.71%, while maximum inhibition rate of flavonoid obtained by alcohol water extraction method is 70.64%. Both the flavonoids obtained from onionskins by alcohol water extraction method and alkali water extraction method have good biological activity. The alkali water extraction method is simple and without any alcohol consumption, and the product purity is high. Although the yield rate and certain biological activity of flavonoids obtained by alkali water extraction method are lower than those obtained by alcohol water extraction method, it still has certain significance in industrial production.

Analysis of the Biological Function of ELDF15 Using an Antisense Recombinant Expression Vector

ELDF15, homologous with AT2 receptor-interaction protein 1 (ATIP1), may play an important role in cell differentiation, proliferation, and carcinogenesis. We aimed to understand the biological function of ELDF15 via construction and transfection of a recombinant expression vector containing antisense ELDF15. Recombinant expression vectors were successfully constructed and transfected into K562 cells. A stable transfectant, known as pXJ41-asELDF15, stably produced antisense ELDF15. Compared with K562 and K562-zeo cells, K562- pXJ41-asELDF15 cells showed inhibition of cell proliferation. RT-PCR analysis showed that the expression and protein level of ELDF15 decreased significantly in K562 cells transfected with pXJ41-asELDF15. Expression of hemoglobin increased in K562 cells transfected with pXJ41-asELDF15 by benzidine staining. increases NBT reduction activity in K562 cells transfected with pXJ41-asELDF15.Colony forming efficiency in two-layer soft agar was clearly inhibited as assessed by electron microscopy. These results suggest that ELDF15 plays a potential role in cell differentiation, proliferation and carcinogenesis.

Abstract 3543: A long noncoding RNA plays a critical role in Bcr-Abl-mediated cellular transformation

Abstract It is well documented that malignant transformation of myeloid and lymphoid cells by Bcr-Abl involves the dysregulation or mutation of a variety of protein-coding genes that are normally involved in regulating the proliferation and survival of hematopoietic cells. However, the functional relevance of long noncoding RNAs (lncRNAs) and their in vivo regulation during Bcr-Abl-mediated tumorigenesis remain unknown, although recent studies have shown that aberrant expression of lncRNAs is associated with various human cancers. In this study, we performed a comprehensive analysis of lncRNAs in human CML cells using an lncRNA cDNA microarray, and identified an lncRNA termed lncRNA-BGL3 that acted as a key regulator of Bcr-Abl-mediated cellular transformation. Notably, we observed that lncRNA-BGL3 was highly induced in response to disruption of Bcr-Abl expression or by inhibiting Bcr-Abl kinase activity in K562 cells and leukemic cells derived from CML patients. Ectopic expression of lncRNA-BGL3 sensitized leukemic cells to undergo apoptosis and inhibited Bcr-Abl-induced tumorigenesis. Furthermore, transgenic mice expressing lncRNA-BGL3 were generated. We found that transgenic expression of lncRNA-BGL3 alone in mice was sufficient to impair primary bone marrow transformation by Bcr-Abl. Interestingly, we identified that lncRNA-BGL3 was a target of miR-17, miR-93, miR-20a, miR-20b, miR-106a and miR-106b, microRNAs that repress mRNA of PTEN. Further experiments demonstrated that lncRNA-BGL3 functioned as a competitive endogenous RNA (ceRNA) for binding these microRNAs to cross-regulate PTEN expression. Additionally, our experiments have begun to address the mechanism of how lncRNA-BGL3 is regulated in the leukemic cells and showed that Bcr-Abl repressed lncRNA-BGL3 expression through c-Myc-dependent DNA methylation. Taken together, these results reveal that Bcr-Abl-mediated cellular transformation critically requires silence of tumor suppressor lncRNA-BGL3 and suggest a potential strategy for the treatment of Bcr-Abl-positive leukemia. Citation Format: Guijie Guo, Qingzheng Kang, Qinghuang Chen, Ouyang Jing, Jilong Chen. A long noncoding RNA plays a critical role in Bcr-Abl-mediated cellular transformation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3543. doi:10.1158/1538-7445.AM2014-3543

A long noncoding RNA critically regulates Bcr-Abl-mediated cellular transformation by acting as a competitive endogenous RNA.

Aberrant expression of long noncoding RNAs (lncRNAs) is associated with various human cancers. However, the role of lncRNAs in Bcr-Abl-mediated chronic myeloid leukemia (CML) is unknown. In this study, we performed a comprehensive analysis of lncRNAs in human CML cells using an lncRNA cDNA microarray and identified an lncRNA termed lncRNA-BGL3 that acted as a key regulator of Bcr-Abl-mediated cellular transformation. Notably, we observed that lncRNA-BGL3 was highly induced in response to disruption of Bcr-Abl expression or by inhibiting Bcr-Abl kinase activity in K562 cells and leukemic cells derived from CML patients. Ectopic expression of lncRNA-BGL3 sensitized leukemic cells to undergo apoptosis and inhibited Bcr-Abl-induced tumorigenesis. Furthermore, transgenic (TG) mice expressing lncRNA-BGL3 were generated. We found that TG expression of lncRNA-BGL3 alone in mice was sufficient to impair primary bone marrow transformation by Bcr-Abl. Interestingly, we identified that lncRNA-BGL3 was a target of miR-17, miR-93, miR-20a, miR-20b, miR-106a and miR-106b, microRNAs that repress mRNA of phosphatase and tensin homolog (PTEN). Further experiments demonstrated that lncRNA-BGL3 functioned as a competitive endogenous RNA for binding these microRNAs to cross-regulate PTEN expression. Additionally, our experiments have begun to address the mechanism of how lncRNA-BGL3 is regulated in the leukemic cells and showed that Bcr-Abl repressed lncRNA-BGL3 expression through c-Myc-dependent DNA methylation. Taken together, these results reveal that Bcr-Abl-mediated cellular transformation critically requires silence of tumor-suppressor lncRNA-BGL3 and suggest a potential strategy for the treatment of Bcr-Abl-positive leukemia.