Growth Of K562 Cells Research Articles

A Lipid-Sensitive Spider Peptide Toxin Exhibits Selective Anti-Leukemia Efficacy through Multimodal Mechanisms.

Anti-cancer peptides (ACPs) represent a promising potential for cancer treatment, although their mechanisms need to be further elucidated to improve their application in cancer therapy. Lycosin-I, a linear amphipathic peptide isolated from the venom of Lycosa singorensis, shows significant anticancer potential. Herein, it is found that Lycosin-I, which can self-assemble into a nanosphere structure, has a multimodal mechanism of action involving lipid binding for the selective and effective treatment of leukemia. Mechanistically, Lycosin-I selectively binds to the K562 cell membrane, likely due to its preferential interaction with negatively charged phosphatidylserine, and rapidly triggers membrane lysis, particularly at high concentrations. In addition, Lycosin-I induces apoptosis, cell cycle arrest in the G1 phase and ferroptosis in K562 cells by suppressing the PI3K-AKT-mTOR signaling pathway and activating cell autophagy at low concentrations. Furthermore, intraperitoneal injection of Lycosin-I inhibits tumor growth of K562 cells in a nude mouse xenograft model without causing side effects. Collectively, the multimodal effect of Lycosin-I can provide new insights into the mechanism of ACPs, and Lycosin-I, which is characterized by high potency and specificity, can be a promising lead for the development of anti-leukemia drugs.

Review of Sacred Herbal Medicine Aegle marmelos: A Potent Metastasis Inhibitor

Abstract: Aegle marmelos (A. marmelos) appears to be a significantly used ayurvedic medicine. This is a brilliantly composed nutritious fruit with carbohydrates, proteins, vitamins like riboflavin, thiamine, niacin, fatty acids, and minerals. Scientific studies have proved that A. marmelos has phytochemicals: carotenoids, phenolic, alkaloids, pectins, tannins, coumarins, flavonoids and terpenoids. Recent research on the effects of A. marmelos proved its anticancer, antimicrobial, cardioprotective, antidiabetic, and hepatoprotective activities. Extracts of various parts of plants such as leaves, bark, stems, fruits, and pulp subjected to preclinical studies conducted on cell lines showed apoptosis induction, inhibition of cell cycle and inhibition of proliferation of cells. Anti-cancer activity of A. marmelos was studied on selected cell lines of lung cancer, breast cancer, colon cancer and leukaemia. A. marmelos reported significant inhibitory effect on cell proliferation in cell line studies on A549, Ehrlich Ascites Carcinoma, MNU, DMBA, MCF-7, k562 cells, t-lymphoid Jurkat cells, b lymphoid Raji cells, erythroleukemic Hel cells, melanoma Colo 38 cells which supports the anticancer potential of it. Imperatorin has shown an antiproliferative effect on several cancer cell lines. The hydroethanolic extract of A. marmelos leaves showed a decrease in cell viability on A549 Human lung cancer cell line which works by fold change over control of the β-Catenin-m RNA and Wnt-mRNA expression of A549 and tumour growth suppression in DMBA induced carcinogenesis in rats. In human promyelocytic leukaemia, HL-60 cells, imperatorin from A. marmelos has caused cytochrome c-dependent apoptosis. Drugs interfere with the proliferative and survival signals that suppress the growth of K562 cells by blocking BCR-ABL1. Proper awareness, development of formulation and consumption of it make this drug scale up commercially.

Abstract 4404: EZH2 modulates mRNA splicing in chronic myeloid leukemia through the repression of splicing factors and exerts part of its oncogenic function through the regulation of CELF2

Abstract The polycomb protein EZH2 is a commonly deregulated or mutated transcriptional regulator in various cancers. In Chronic Myeloid Leukemia (CML) it has been reported to be upregulated and associated with transcriptional reprograming, rendering CML leukemic stem cells (LSCs) sensitive to EZH2 inhibitor treatment. Mechanistically this seems to be linked to the silencing of apoptotic target genes by EZH2-mediated histone H3 lysine 27 tri-methylation (H3K27me3). We hypothesized that the regulation of apoptotic genes is not the only pathway responsible for its oncogenic function, and that EZH2 might also act as a modulator of the mRNA splicing landscape. To test this, we treated the CML cell line K562 with the EZH2 inhibitors GSK126 and EPZ-6438 and performed RNA-seq followed by splicing analysis. Using two different bioinformatics tools, we detected differential splicing of several hundreds of splicing events. However, differentially spliced genes were not associated with increased EZH2 or H3K27me3 signals, suggesting an indirect mechanism of splicing regulation. Indeed, we found 11 splicing factors transcriptionally upregulated upon EZH2 inhibitor treatment. CELF2 was identified as top candidate to mediate part of the EZH2 inhibitor induced phenotype. Upon overexpression, we observed 1) reduced cell growth, viability, and colony formation of K562 cells, 2) a change in the splicing landscape, significantly overlapping with EZH2 inhibitor induced changes, 3) the downregulation of cMYC signaling, also found upon EZH2 inhibitor treatment. To test if CELF2 is indeed acting as EZH2-regulated tumor suppressor in CML, we extended our study to CML patient samples: we performed RNA-seq followed by splicing and gene expression analysis in seven CML patient-derived CD34+ samples and eight healthy donor-derived CD34+ samples, as well as three CML patient samples transduced with CELF2-containing expression constructs. While we found Celf2 mRNA downregulated in CML derived samples, overexpression led to decreased colony formation and cMYC signaling, validating our findings from K562 cells. We also performed an analysis of CML associated splicing changes and found hundreds of deregulated splicing events, of which a significant portion overlapped with CELF2 overexpression mediated changes, suggesting direct regulation by CELF2. Based on this data we propose a novel role of EZH2, where it exerts part of its oncogenic function in CML through the transcriptional repression of splicing factors, in particular CELF2, leading to splicing deregulation, increased cMYC signaling, and enhanced cell growth and viability. Finally, data from other publicly available EZH2 inhibitor treated cancer models suggests that splicing modulation by EZH2 might not be restricted to CML. Citation Format: Reinhard Brunmeir, Ying Li, Baohong Lin, Leong Qiao Zheng, Henry Yang, Wee Joo Chng. EZH2 modulates mRNA splicing in chronic myeloid leukemia through the repression of splicing factors and exerts part of its oncogenic function through the regulation of CELF2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4404.

Renin-Angiotensin System Inhibitors Suppress the Growth of Leukemia Cells.

The renin-angiotensin system (RAS) regulates blood pressure. The RAS is also related to cell growth, and its activation has been reported in various cancer cells. Therefore, we investigated the effects of RAS inhibitors on the in vitro growth of leukemia cell lines. THP-1, MV4-11, and TMD7 cells derived from acute myeloid leukemia, K-562 cells from chronic myeloid leukemia, and Jurkat and KOPT-K1 cells from T-lymphoblastic leukemia (T-ALL) with NOTCH1 mutations were used. We used four RAS inhibitors: the renin inhibitor aliskiren, angiotensin-converting enzyme 1 inhibitor captopril, angiotensin II type 1 receptor antagonist azilsartan, and angiotensin II type 2 receptor antagonist PD123319. Cells were cultured with the inhibitors and cell growth was assessed using a colorimetric assay. The expression of signaling proteins was assessed using immunoblotting. Treatment with aliskiren, azilsartan, or PD123319 suppressed the growth of all cell lines. Captopril treatment suppressed the growth of K-562, KOPT-K1, and MV4-11 cells. Flow cytometric analysis revealed that the growth suppression was due to the induction of apoptosis. Their suppressive effects on normal lymphocytes were milder than those on leukemia cells. Treatment with these inhibitors decreased MYC expression, induced caspase3 and PARP cleavage, and suppressed mTOR signaling. The treatment also suppressed NOTCH1 signaling in T-ALL cells. RAS inhibitors can be repurposed as molecular-targeted drugs for leukemia. However, the concentrations of the inhibitors were much higher than those in the plasma of patients with hypertension. Therefore, further investigation is required for their clinical use.

Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner.

Our findings unveiled that iNKT cells recognize and kill CD1d-negative target tumors via the anti-iNKT TCR mAb bound to CD32 at the tumor site, thereby bridging iNKT cells and CD1d-negative tumors. These findings shed light on the therapeutic potential of anti-iNKT TCR mAbs in NKT cell-based immunotherapy to treat CD1d-negative CD32+ cancers.

Effect of Extracellular Vesicles Derived From Peripheral Blood Mononuclear Cells on K562 Leukemia Cell Line

Recently, extracellular vesicles (EVs) are generating considerable interest in terms of their ability. EVs defined themselves as a route for intercellular communication between the origin cells and the recipient ones by transferring information. This paper investigates the influence of peripheral blood mononuclear cells derived EVs on proliferation and apoptosis of the chronic myelogenous leukemia cell line k562. Trypan blue staining was used to detect cell viability subsequently, metabolic activity was assessed by the MTT assay. Cell apoptosis and cell cycle progression were evaluated using flowcytometric assay after treatment of k562 cell line with MNC derived EVs. Our results showed that MNC-EVs have no inhibitory effect on k562 cell growth and proliferation. Our data did not reveal any significant variations in the case of enhancing k562 cell line growth following treatment with MNC derived EVs. It seems tumour-derived extracellular vesicles support tumour cells growth by communicating with each other through their extracellular vesicles.

Abstract 333: Interleukin 23 receptor is a critical regulator of the mitotic spindle and necessary for AML viability

Abstract Classically, the interleukin-23 receptor (IL-23R) is expressed on the surface of T cells where activation by its ligand, interleukin-23 (IL-23) leads to T cell activation through the JAK/STAT pathway. Here, we identify a novel localization and function for IL-23R in which intracellular IL-23R is necessary for AML viability by regulating mitotic spindle and centrosome formation. IL-23R was identified as a potential target through InterLinked Therapeutics’ ITX machine learning platform. IL-23R was identified through this unbiased in silico screen as a top hit and a potential regulator of the G2/M checkpoint and mitotic spindle. We confirmed that IL-23R protein was overexpressed >2-fold in 6 of 8 primary AML patient samples compared to mean IL-23R expression in normal hematopoietic cells. Consistent with prior studies, we confirmed cell surface localization of IL-23R in double negative T cells by flow cytometry and confocal microscopy. In contrast, only small amounts of IL-23R were detected on the surface of AML cells and primary AML patient samples. Rather, IL-23R was located intracellularly as demonstrated by flow cytometry, confocal microscopy, and immunoblotting of subcellular fractions. We confirmed intracellular localization of IL-23R in AML cells using 4 different antibodies targeted against 4 different epitopes of the receptor. We used BioID mass spectrometry to identify novel protein interactors with intracellular IL-23R and identified 61 interacting proteins. 36 of those 61 were nuclear or cytoplasmic in localization and the largest subset was associated with mitotic spindle regulation. By Proximity Ligation Assay, we confirmed the interactions between endogenous IL-23R with known mitotic spindle proteins NUMA, TMEM201, BAG6, and TACC1 in OCI-AML2 cells and primary AML samples. By confocal microscopy, we discovered that IL-23R co-localized with the mitotic spindle and centrosomes in AML cells. Genetic knockout of IL-23R increased spindle defects with multipolarity, lagging chromosomes, and spindle orientation errors. Interestingly, heterodimeric partner of IL-23R, IL12Rβ1 was also detected to be co-localized with mitotic spindles but the cytokine, IL-23, could not be detected. Knockdown of IL-23R also reduced cell growth in OCI-AML2, TEX, K562, NB4, and U937 cells. IL-23R knockdown also reduced the clonogenic growth and engraftment efficiency of TEX leukemia cells into murine bone marrow. In summary, intracellular IL-23R is a critical regulator of mitosis in AML and is necessary for AML viability. This study highlights a novel function and localization of IL-23R in AML and identifies a potential therapeutic target. Citation Format: Nathan Duong, Dilshad H. Khan, Geethu E. Thomas, Rose Hurren, Jong Bok Lee, Jonathan St-Germain, Lily Drimmer, Yongran Yan, Neil MacLean, Marcela Gronda, Brandon D. Brown, Brian Raught, Andrea Arruda, Hong Chang, Mark D. Minden, Li Zhang, Steven M. Kornblau, Vito Spadavecchio, Aaron D. Schimmer. Interleukin 23 receptor is a critical regulator of the mitotic spindle and necessary for AML viability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 333.

Bioisosteric replacement of 1H-1,2,3-triazole with 1H-tetrazole ring enhances anti-leukemic activity of (5-benzylthiazol-2-yl)benzamides

Previously, we discovered that N-(5-benzyl-1,3-thiazol-2-yl)-4-(5-methyl-1H-1,2,3-triazol-1-yl)benzamide possessed a remarkable cytotoxic effect on 28 cancer cell lines with IC50 < 50 μM, including 9 cancer cell lines, where IC50 was in the range of 2.02–4.70 μM. In the present study, we designed a novel N-(5-benzylthiazol-2-yl)amide compound 3d that was synthesized using the original bioisosteric replacement of 1H-1,2,3-triazole ring by the 1H-tetrazole ring. A significantly enhanced anticancer activity in vitro with an excellent anti-leukemic potency towards chronic myeloid leukemia cells of the K-562 line was demonstrated. Two compounds – 3d and 3l - were highly cytotoxic at nanomolar concentrations towards various tumor cells of the following lines: K-562, NCI-H460, HCT-15, KM12, SW-620, LOX IMVI, M14, UACC-62, CAKI-1, and T47D. As a highlight, the compound N-(5-(4-fluorobenzyl)thiazol-2-yl)-4-(1H-tetrazol-1-yl)benzamide 3d inhibited the growth of leukemia K-562 cells and melanoma UACC-62 cells with IС50 of 56.4 and 56.9 nM (SRB test), respectively. The viability of leukemia K-562 and pseudo-normal HaCaT, NIH-3T3, and J774.2 cells was measured by the MTT assay. Together with SAR analysis, it allowed the selection of a lead compound 3d, which demonstrated the highest selectivity (SI = 101.0) towards treated leukemic cells. The compound 3d caused DNA damage (single-strand breaks detected by the alkaline comet assay) in the leukemic K-562 cells. The morphological study of the K-562 cells treated with compound 3d revealed changes consistent with apoptosis. Thus, the bioisosteric replacement in (5-benzylthiazol-2-yl)amide scaffold proved to be a perspective approach in the design of novel heterocyclic compounds with enhanced anticancer potential.

The role of CSE1L silencing in the regulation of proliferation and apoptosis via the AMPK/mTOR signaling pathway in chronic myeloid leukemia

ABSTRACTObjectivesChromosome segregation 1-like (CSE1L) is abundant and strongly expressed in solid tumors. However, the expression and role of CSE1L in chronic myeloid leukemia(CML) remain largely unknown.Materials and methodsThe relative expression levels of CSE1L in bone marrow granulocytes from patients with primary CML and non-hematologic controls were measured by flow cytometry. Cell counting kit-8 analysis, DNA Content Quantitation Assay, and Annexin V-PE/7-AAD staining were applied to assess the effects of CSE1L knockdown on cell proliferation, cell cycle progression, and apoptosis.ResultsElevated expression of CSE1L was detected in bone marrow granulocytes of patients with primary CML. In the CML cell line K562 cells, CSE1L knockdown impaired cell proliferation blocked the cell cycle shift from G0/G1 phase to the S phase, and promoted apoptosis. Knockdown of CSE1L reduced Bcl-2 protein expression and increased Bax protein expression. Meanwhile, knockdown of CSE1L enhanced the expression of phospho-AMPK protein and decreased the expression of phospho-mTOR protein. The expression of total AMPK and mTOR proteins was not affected. In addition, CSE1L expression levels were decreased in imatinib-treated K562 cells.ConclusionsCSE1L plays a pivotal role in K562 cell survival and growth. These functions may be partially dependent on the AMPK/mTOR signaling pathway to achieve. In addition, CSE1L may have had a future impact on the treatment of CML patients.

Synthesis and biological assessment of indole derivatives containing penta-heterocycles scaffold as novel anticancer agents towards A549 and K562 cells

Herein, a new series of 2-chloro-N-(5-(2-oxoindolin-3-yl)-4H-pyrazol-3-yl) acetamide derivatives containing 1,3,4-thiadiazole (10a–i) and 4H-1,2,4-triazol-4-amine (11a–r) moiety was designed, synthesised as novel anticancer agents. The antiproliferative activity values indicated that compound 10 b stood as the most potent derivative with IC50 values of 12.0 nM and 10 nM against A549 and K562 cells, respectively. Mechanism investigation and docking studies of 10 b indicated that it possessed good apoptosis characteristic and dose-dependent growth arrest of A549 and K562 cells, blocked cell cycle into G2/M phase. Interestingly, 10 b suppressed the growth of A549 and K562 cells via modulation of EGFR and p53-MDM2 mediated pathway.

PHF6 Maintains Acute Myeloid Leukemia Via Regulating NF-Κappab Signaling Pathway

Acute myeloid leukemia (AML) is a major hematopoietic malignancy characterized by the uncontrolled expansion of the immature myeloid cells in the hematopoietic system. Current chemotherapy regimens remain ineffective as assessed by low remission and 5-year-survival rate. Many efforts have been dedicated to decipher the molecular events underlying AML transformation. PHF6 is a conserved epigenetic regulator and has been found to be frequently altered in hematologic malignancies. Previous studies demonstrated that PHF6 plays a suppressive role in T-ALL leukemogenesis. However, the functional role(s) of PHF6 in AML initiation and progression remains unknown. Here in this study, we analyzed the mRNA expression of PHF6 in blood cells from healthy donors and leukemia cells from AML patients, and found that the PHF6 expression was significantly increased in AML cells when compared with that of normal PMNs (www.Bloodspot.com) (p < 0.05). Significantly, the average survival time of AML patients with high PHF6 expression was much shorter than that of AML patients with low PHF6 expression (p = 0.022). Overexpressing of PHF6 (PHF6 OE) in Kasumi-1 or K562 leukemia cell lines showed increased growth of Kasumi-1 and K562 cells, decreased apoptosis of Kasumi-1 cells. Additionally, we knocked down (KD) PHF6 expression in AML cells and CD34+ cord blood cells, and found that PHF6 KD significantly decreased the growth of Kasumi-1 and promoted cell apoptosis, while had little effects on apoptosis of CD34+ cord blood cells. These results suggested that PHF6 is required for the growth of myeloid leukemia cells but is dispensable for normal blood cells. To further evaluate the functional role of Phf6 in leukemogenesis in vivo, we generated Phf6 KO+RUNX1-ETO9a (RE9a) and Phf6 KO+MLL-AF9 (MA9) AML mice. We found the survival time of Phf6 KO+RE9a mice was significantly longer than that of WT Phf6+RE9a mice. The Phf6 KO+RE9a mice showed much milder disease phenotypes than WT Phf6+RE9a mice, including lower counts of GFP+ leukemia cells in the PB and BM, higher body weights, lower spleen and liver weights, and lower degree of extramedullary infiltration in spleen, liver, lung and brain. Extreme limiting dilution transplantation assays demonstrated a marked decrease in leukemia stem cell (LSC) activity in Phf6 KO+RE9a cells when compared with Phf6 WT+RE9a cells. Consistent with RE9a-induced AML mouse model, PHF6 deficiency impaired the self-renewal of LSCs and delayed the progression of MA9-induced AML, supporting a general oncogenic role of Phf6 in sustaining myeloid leukemia. To investigate the underlying molecular mechanisms of Phf6 in regulating AML development, we performed RNAseq to analyze the transcriptome programing changes associated with Phf6 deficiency in isogenic Phf6 WT+RE9a or Phf6 KO+RE9aAML cells. Gene set enrichment analysis (GSEA) showed up-regulation of apoptosis and down-regulation of NF-κB-regulated gene expressions in Phf6 KO+RE9a cells, suggesting that Phf6 depletion might promote AML cell apoptosis by blocking the NF-κB signaling. Immunocytochemistry studies showed that PHF6 depletion partially inhibited the nuclear translocation of p50. Co-immunoprecipitation experiments revealed that PHF6 directly bound to p50 in myeloid leukemia cells, suggesting that PHF6 might activate NF-κB signaling pathways by binding with p50 and maintaining the nuclear translocation of p50. To confirm this, we treated PHF6 OE AML cells with BAY11-7082 (a selective NF-κB inhibitor), and found BAY11-7082 significantly inhibited the growth of PHF6 OE Kasumi-1 cells in vitro. In summary, we revealed that PHF6 promoted RE9a- or MA9-induced AML progression in mice by activating NF-κB signaling pathway, and promoting P50 nuclear translocation. Our study thus defined a unique role for Phf6 in regulating NF-κB signaling pathway in AMLs, and potentially can be used as a therapeutic target for treating myeloid leukemia patients.

Intracellular IL-23 Receptor (IL-23R) Is Necessary for AML Viability and Regulates Mitotic Spindle and Centrosome Formation

The IL-23 receptor (IL-23R) is a cell surface receptor classically expressed on T cells. In response to inflammatory stimuli, such as microbial infection, dendritic cells and macrophages secrete the cytokine IL-23, which stimulates IL-23R leading to increased T cell activation and proliferation. Here, we identify a novel function for IL-23R in which intracellular IL-23R is necessary for AML viability by regulating mitotic spindle and centrosome formation. An unbiased in silico screen was performed using the Interlinked Therapeutics ITX machine learning platform to identify novel targets and mechanisms in AML. This analysis identified IL-23R as a top hit and a potential regulator of the G2/M checkpoint and mitotic spindle. We confirmed IL-23R protein was over-expressed >2-fold in 6 of 8 primary AML patient samples compared to mean IL-23R expression (n=3) in normal hematopoietic cells. IL-23R is classically a cell surface receptor, and we confirmed cell surface localization in DNT (Double Negative T) cells by flow cytometry and confocal microscopy. In contrast, only small amounts of IL-23R were present on the cell surface of AML cells and primary AML patient samples. Rather, IL-23R was located intracellularly in the cytoplasm and nucleus as demonstrated by flow cytometry, confocal microscopy, and immunoblotting of subcellular fractions. To validate that we were indeed detecting IL-23R, we confirmed intracellular localization in AML cells using 4 different antibodies directed against 4 different epitopes of the receptor. To understand the function of intracellular IL-23R, we used BioID mass spectrometry to identify proteins that interact with IL-23R. Compared to the BioID controls, we identified 61 proteins that preferentially interacted with IL-23R. Of the 61 proteins, 36 are known cytoplasmic or nuclear proteins. Pathway analysis of interacting proteins identified mitotic spindle formation as the top pathway. Using a Proximity Ligation Assay and confocal microscopy, we validated the interaction of endogenous IL-23R with the known mitotic spindle proteins, NUMA, TMEM201, TACC1, and BAG6 in OCI-AML2 cells. In addition, by confocal microscopy, we demonstrated that IL-23R co-localizes with the mitotic spindle and centrosomes in AML. Knockdown and knockout of IL-23R in AML cells led to dysregulation of the mitotic spindle with multipolarity, lagging chromosomes, and spindle orientation errors. We then evaluated the effects of IL-23R knockdown on AML growth and viability. Knockdown or knockout of IL-23R reduced AML growth in OCI-AML2, TEX, K562, NB4, and U937 cells. In addition, knockdown of IL-23R led to a significant reduction in engraftment efficiency of TEX cells into murine bone marrow. To evaluate the effects of IL-23R depletion on normal hematopoiesis, we evaluated constitutive homozygote IL-23R knockout mice. IL23R-/- mice were bred in normal ratios and grew similar to wild type mice. IL-23R was associated with the mitotic spindle and centrosomes in hematopoietic cells of wild type mice and no IL-23R was detected in the knockout mice. Complete blood counts did not differ compared to wild type mice. The abundance of HSC (hematopoietic stem cell) and LSK (Lin−Sca-1+c-Kit+) hematopoietic stem cells were also similar between IL-23R -/- and wild type mice. In summary, we discovered that IL-23R is necessary for AML growth and viability by regulating the formation of the mitotic spindle and centrosome. Thus, we identified a new function and localization for IL-23R and highlight a new therapeutic target for this disease.

ACSL1 promotes imatinib-induced chronic myeloid leukemia cell senescence by regulating SIRT1/p53/p21 pathway

Although tyrosine kinase inhibitors (TKIs) improve the prognosis of chronic myeloid leukemia (CML) patients, resistance to TKIs and residual leukemia stem cells (LSCs) inevitably become the bottleneck of cure. Therefore, we need to explore novel treatment strategies based on conventional treatment strategies. Our previous study found that CML cell senescence may be one of the main factors to achieve clinical cure of CML. Studies have shown that lipid metabolism plays a key role in cellular senescence. Here, we found that long-chain acyl-CoA synthetase 1 (ACSL1) was significantly up-regulated in senescent CML cells. Furthermore, we demonstrated that overexpression of ACSL1 induces senescence and inhibits cell growth in K562 cells by altering cell cycle progression, and enhances the proliferation-inhibiting effect of imatinib. Overexpression of ACSL1 enhances imatinib-induced tumorigenic decline in K562 cells in vivo. Knockdown of ACSL1 reverses imatinib-induced senescence in K562 cells. Mechanistically, overexpression of ACSL1 induced senescence in K562 cells via the SIRT1/p53/p21 axis. Collectively, our study showed that ACSL1 promotes imatinib-induced K562 cells senescence and tumor growth by regulating SIRT1/p53/p21 pathway. The ACSL1/SIRT1/p53 signal axis is a novel mechanism of cell senescence in CML and a new potential target for eradication of CML LSCs.

Naturally Occurring Bicoumarin Compound Daphnoretin Inhibits Growth and Induces Megakaryocytic Differentiation in Human Chronic Myeloid Leukemia Cells.

Daphnoretin extracted from the stem and roots of Wikstroemia indica (L.) C.A. Mey has been shown to possess antiviral and antitumor activities. Herein, we hypothesized that daphnoretin might induce megakaryocytic differentiation, thereby inhibiting the proliferation of cells and serving as a differentiation therapy agent for chronic myeloid leukemia (CML). Daphnoretin-treated K562 and HEL cells were examined for growth inhibition, cell morphology, and megakaryocyte-specific markers. Potential mechanisms of megakaryocytic differentiation of daphnoretin-treated K562 cells were evaluated. The results showed that daphnoretin inhibited the growth of K562 and HEL cells in a dose- and time-dependent manner. Flow cytometry analyses revealed that daphnoretin treatment slightly increased the proportion of sub-G1 and polyploid cells compared to that of dimethyl sulfoxide (DMSO)-treated control cells. Morphological examination showed that daphnoretin-treated K562 and HEL cells exhibited enlarged contours and multinucleation as megakaryocytic characteristics compared to DMSO-treated control cells. Daphnoretin treatment also dramatically enhanced the expression of megakaryocytic markers CD61 and CD41. Under optimal megakaryocytic differentiation conditions, daphnoretin increased the phosphorylation of STAT3 but not STAT5. In summary, daphnoretin inhibited cell growth and induced megakaryocytic differentiation in K562 and HEL cells. The efficacy of daphnoretin in vivo and in patients with CML may need further investigations for validation.

Targeted silencing of CDX2 gene with siRNA enhanced vincristine-induced inhibition of proliferation and induction of apoptosis of leukemia K562 cells

Purpose: To determine the effect of targeted silencing of CDX2 with siRNA on vincristine-induced inhibition of proliferation and apoptosis induction in leukemia K562 cells.&#x0D; Methods: K562 cells were divided into untreated group, vincristine group, blank group, and CDX2-siRNA group. The expression of CDX2 gene after CDX2-siRNA transfection was determined by reverse-transcription-polymerase chain reaction (RT-PCR) and immunoblotting, while MTT and flow cytometric procedures were used to evaluate the effect of vincristine on proliferation and apoptotic changes in K562 cells.&#x0D; Results: Protein expression of CDX2 did not change significantly in non-treated cells, vincristine group and blank group, while the expression of CDX2 protein in cells of CDX2-siRNA group was decreased significantly (p &lt; 0.05) while MTT assay results showed that the absorbance of CDX2-siRNA cells was significantly lower than those of the other three groups at 12, 24 and 48 h after CDX2 gene silencing. Flow cytometry showed markedly higher percentage apoptosis of CDX2-siRNA group than in other groups at 12, 24 and 48 h after CDX2 gene silencing. There was no difference in apoptosis level between vincristine group and blank group, but apoptosis was higher in these groups than in untreated group (p &lt; 0.05).&#x0D; Conclusion: Silencing CDX2 gene via targeting with siRNA enhances vincristine-induced suppression of growth and apoptotic changes in leukemia K562 cells, thereby enhancing the anti-tumor effect of vincristine.

Decreased expression of HBA1 and HBB genes in acute myeloid leukemia patients and their inhibitory effects on growth of K562 cells

ABSTRACT Background Besides the traditional roles of HBA1 and HBB, recent findings suggest that hemoglobin genes may have roles in other contexts. Objective In the present study, we aim to investigate a possible tumor-suppressor role of HBA1 and HBB in acute myeloid leukemia. Methods Quantitative real-time PCR (RT-qPCR) was performed to detect the expression levels of HBA1 and HBB in acute myeloid leukemia patients and AML cell lines. The transfected cells were analyzed for Cell Counting Kit-8 (CCK-8), apoptosis, and cell cycle assay. Results HBA1 and HBB genes were significantly decreased in acute myeloid leukemia patients and AML cell lines. Furthermore, in vitro approaches showed that overexpression of HBA1 and HBB inhibited proliferation, induced cell apoptosis, and blocked cell cycle process at the G2/M phase in K562 cells. Conclusion Our data indicated that HBA1 and HBB genes may be potential tumor-suppressor genes in acute myeloid leukemia.

Vandetanib drives growth arrest and promotes sensitivity to imatinib in chronic myeloid leukemia by targeting ephrin type‐B receptor 4

The oncogenic role of ephrin type‐B receptor 4 (EPHB4) has been reported in many types of tumors, including chronic myeloid leukemia (CML). Here, we found that CML patients have a higher EPHB4 expression level than healthy subjects. EPHB4 knockdown inhibited growth of K562 cells (a human immortalized myelogenous leukemia cell line). In addition, transient transfection of EPHB4 siRNA led to sensitization to imatinib. These growth defects could be fully rescued by EPHB4 transfection. To identify an EPHB4‐specific inhibitor with the potential of rapid translation into the clinic, a pool of clinical compounds was screened and vandetanib was found to be most sensitive to K562 cells, which express a high level of EPHB4. Vandetanib mainly acts on the intracellular tyrosine kinase domain and interacts stably with a hydrophobic pocket. Furthermore, vandetanib downregulated EPHB4 protein via the ubiquitin‐proteasome pathway and inhibited PI3K/AKT and MAPK/ERK signaling pathways in K562 cells. Vandetanib alone significantly inhibited tumor growth in a K562 xenograft model. Furthermore, the combination of vandetanib and imatinib exhibited enhanced and synergistic growth inhibition against imatinib‐resistant K562 cells in vitro and in vivo. These findings suggest that vandetanib drives growth arrest and overcomes the resistance to imatinib in CML via targeting EPHB4.

Hydroxycitric Acid Inhibits Chronic Myelogenous Leukemia Growth through Activation of AMPK and mTOR Pathway.

Metabolic regulation of cancer cell growth via AMP-activated protein kinase (AMPK) activation is a widely studied strategy for cancer treatment, including leukemias. Recent notions that naturally occurring compounds might have AMPK activity led to the search for nutraceuticals with potential AMPK-stimulating activity. We found that hydroxycitric acid (HCA), a natural, safe bioactive from the plant Garcinia gummi-gutta (cambogia), has potent AMPK activity in chronic myelogenous leukemia (CML) cell line K562. HCA is a known competitive inhibitor of ATP citrate lyase (ACLY) and is widely used as a weight loss inducer. We found that HCA was able to inhibit the growth of K562 cells in in vitro and in vivo xenograft models. At the mechanistic level, we identified a direct interaction between AMPK and ACLY that seems to be sensitive to HCA treatment. Additionally, HCA treatment resulted in the co-activation of AMPK and the mammalian target of rapamycin (mTOR) pathways. Moreover, we found an enhanced unfolded protein response as observed by activation of the eIF2α/ATF4 pathway that could explain the induction of cell cycle arrest at the G2/M phase and DNA fragmentation upon HCA treatment in K562 cells. Overall, these findings suggest HCA as a nutraceutical approach for the treatment of CMLs.

Effect of D-mannose on Philadelphia chromosome-positive leukemia cells.

Although Abelson (ABL) tyrosine kinase inhibitors (TKIs) have demonstrated potency against chronic myeloid leukemia (CML), resistance to ABL TKIs can develop in CML patients after discontinuation of therapy. Glucose metabolism may be altered in CML cells because glucose is a key metabolite used by tumor cells. We investigated whether D-mannose treatment induced metabolic changes in CML cells and reduced CML growth in the presence of ABL TKIs. We investigated whether D-mannose treatment induced metabolic changes in CML cells and reduced CML growth in the presence of ABL TKIs. Treatment with D-mannose for 72h inhibited the growth of K562 cells. Combined treatment using ABL TKIs and D-mannose induced a significantly higher level of cytotoxicity in Philadelphia chromosome (Ph)-positive leukemia cells than in control cells. In the mouse model, severe toxicity was observed as evidenced by body weight loss in the ponatinib and D-mannose combination treatment groups. Our results indicate that metabolic reprogramming may be a useful strategy against Ph-positive leukemia cells. However, caution should be exercised during clinical applications.

Mesalazine induces apoptosis via mitochondrial pathway in K562 cell line.

Inflammation is an initial response of the body to infection and relationship between inflammation and cancer has been established. Nuclear factor kappa B (NF-κB) is a central factor in inflammation and its activity contributes to tumor progression and apoptosis prevention consequently leading to cancer promotion. As a result, NF-κB inhibitors can cause apoptosis. In this study, the effect of mesalazine as a NF-κB inhibitor on growth and apoptosis of K562 cells has been investigated. The K562 cells were first cultured in RPMI-1640 medium containing 10.00% fetal bovine serum. After that, they were treated for 72 hr with different concentrations of mesalazine (20.00, 40.00, 60.00 and 80.00 μM mL-1). The MTT assay was used to evaluate cell viability. Hoechst staining and RT-PCR of apoptosis related genes (Bcl-2 and Bax) were carried out to illustrate apoptosis induction and immunocytochemistry was performed to investigate changes in c-Myc protein level. According to the results of MTT assay, all of applied mesalazine concentrations decreased K562 cells viability. Hoechst staining showed that the fragmented nuclei increased indicating apoptosis induction. Immuno-cytochemical ‎ results showed that mesalazine decreased c-Myc in treated cells. The RT-PCR results also showed an increase in Bax and a decrease in Bcl-2 expressions in mesalazine-treated cells. As the results suggest, mesalazine reduces cell viability by inducing apoptosis in K562 cell line; therefore, it can be used as a candidate for the leukemia treatment.