Human Chronic Myeloid Leukemia Cells Research Articles

Cepharanthine inhibits dengue virus production and cytokine secretion

Dengue virus (DENV) infection is a public health problem in tropical and subtropical regions. It can cause a spectrum of clinical manifestations ranging from mild dengue fever (DF) to severe dengue haemorrhagic fever (DHF) and potentially life-threatening disease including dengue shock syndrome (DSS). Severe DENV infection is caused by high viral load and cytokine storm in dengue-infected patients. Currently, there is no specific antiviral drug for DENV infection. An anti-DENV agent that demonstrates inhibitory effects on both DENV replication and cytokine secretion is urgently needed. In this study, cepharanthine (CEP), which is an anti-inflammatory, anti-HIV, and anti-tumor compound isolated from Stephania cepharantha Hayata, was tested for inhibition of DENV infection. We investigated the efficacy of CEP to inhibit DENV infection, replication, and cytokine production. The inhibitory effect of CEP treatment was studied in DENV-infected human chronic myeloid leukemia (K562) cells. The levels of DENV E protein and DENV production were determined by flow cytometry and FFU assay, respectively. CEP treatment significantly reduced viral E protein and viral production in all DENV-1, 2, 3, 4 serotypes. In addition, CEP treatment reduced the IL-6 proinflammatory cytokine production in DENV-infected A549 cells. Taken together, CEP has inhibitory effects on DENV infection specifically at the initial viral replication states and proinflammatory cytokine secretion, and is a promising candidate for further development as an anti-DENV treatment.

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.

PHYTOCHEMICAL CONSTITUENTS AND ANTILEUKEMIC EFFECTS OF JUNIPERUS OXYCEDRUS EXTRACT

Aim: Many genetic and environmental factors can be effective in the process of cancerization. Preventing the progression of leukemia may be possible by controlling the pathways involving mechanisms such as apoptosis and autophagy. When the literature is examined, there are studies showing the effects of various types of juniper on various cancer cell lines, including human chronic myeloid leukemia cells, but the signal pathways in which they act are not fully known. In this study, the anticancer effects of Juniperus oxycedrus extract on K-562 human chronic myeloid leukemia cells were investigated. Method: After the cells were treated with the Juniperus oxycedrus extract, cytotoxicity and gene expression analyzes were performed. Changes in the expression of Akt, the member of the PI3K/Akt/mTOR signaling pathway; caspase 3, which is one of the main effective genes in the pathways regulating apoptosis; and the apoptosis suppressor BCL-2 gene, which is an oncogene, were investigated. Results: According to the MTT test results, Juniperus oxycedrus extract showed over approximately 50% cell viability in K-562 cells at all doses. The most appropriate dose of Juniperus oxycedrus fruit extract in this research was determined as 50 µg/ mL considering cell viability. After the gene expression analysis, it was observed that Akt expression increased 1.092 times, BCL-2 expression decreased approximately 0.3 times, and caspase 3 expression increased 1.2 times. Conclusions: Constituents of Juniperus oxycedrus plant may have apoptotic effects on chronic myeloid leukemia cells.

Ketoconazole Reverses Imatinib Resistance in Human Chronic Myelogenous Leukemia K562 Cells.

Chronic myeloid leukemia (CML) is a hematologic disorder characterized by the oncogene BCR-ABL1, which encodes an oncoprotein with tyrosine kinase activity. Imatinib, a BCR-ABL1 tyrosine kinase inhibitor, performs exceptionally well with minimal toxicity in CML chemotherapy. According to clinical trials, however, 20–30% of CML patients develop resistance to imatinib. Although the best studied resistance mechanisms are BCR-ABL1-dependent, P-glycoprotein (P-gp, a drug efflux transporter) may also contribute significantly. This study aimed to establish an imatinib-resistant human CML cell line, evaluate the role of P-gp in drug resistance, and assess the capacity of ketoconazole to reverse resistance by inhibiting P-gp. The following parameters were determined in both cell lines: cell viability (as the IC50) after exposure to imatinib and imatinib + ketoconazole, P-gp expression (by Western blot and immunofluorescence), the intracellular accumulation of a P-gp substrate (doxorubicin) by flow cytometry, and the percentage of apoptosis (by the Annexin method). In the highly resistant CML cell line obtained, P-gp was overexpressed, and the level of intracellular doxorubicin was low, representing high P-gp activity. Imatinib plus a non-toxic concentration of ketoconazole (10 μM) overcame drug resistance, inhibited P-gp overexpression and its efflux function, increased the intracellular accumulation of doxorubicin, and favored greater apoptosis of CML cells. P-gp contributes substantially to imatinib resistance in CML cells. Ketoconazole reversed CML cell resistance to imatinib by targeting P-gp-related pathways. The repurposing of ketoconazole for CML treatment will likely help patients resistant to imatinib.

Evaluation of the Effect of Curcumin and Imatinib on BCR-ABL Expression Gene in Chronic Human k562 Cells

Background and Aims: Detection of overexpression in tumor-inhibiting genes provides valuable information for leukemia diagnosis and prognosis. Chronic myeloid leukemia (CML) is a stem cell disorder determined by a well-defined genetic anomaly involving BCR-ABL translocation in the Philadelphia chromosome. Curcumin is a chemo-preventive agent for the primary cancer targets, such as the breast, prostate, lung, stomach, duodenum, colon cancers, and leukemias. Imatinib (Gleevec®, Glivec®) is a synthetic tyrosine kinase inhibitor that treats CML. This study aimed to investigate Curcumin and Imatinib's effect on K562, a human CML cell line that expresses p210 BCR-ABL.
 Materials and Methods: In this study, Curcumin nanomicelles and Imatinib's apoptotic effects on the K562 cells and the expression of BCR-ABL were studied. BCR-ABL gene expression was evaluated using real-time polymerase chain reaction.
 Results: The findings indicated a decrease in the desired gene expression, but the BCR-ABL gene expression of the samples treated with Curcumin nanomicelles did not differ significantly from Imatinib (group control). The amount fold change of the BCR-ABL gene for Imatinib and Curcumin nanomicelle was 0.497 and 0.540, respectively.
 Conclusions: The present study showed that treating cellular category k562 with Curcumin nanomicelle and Imatinib reduces the BCR-ABL gene expression. Also, data showed that the Curcumin nanomicelle and Imatinib induced the apoptotic process.

IL-2 and Zoledronic Acid Therapy Restores the In Vivo Anti-Leukemic Activity of Human Lymphocytes Pre-Exposed to Simulated Microgravity.

We have previously shown that the anti-tumor activity of human lymphocytes is diminished in vitro after 12-hours pre-exposure to simulated microgravity (SMG). Here we used an immunocompromised mouse model to determine if this loss of function would extend in vivo, and to also test the efficacy of IL-2 and zoledronic acid (ZOL) therapy as a potential countermeasure against SMG-induced immune dysfunction. We adoptively transferred human lymphocytes that were exposed to either SMG or 1G-control into NSG-Tg (Hu-IL15) mice 1-week after they were injected with a luciferase-tagged human chronic myeloid leukemia (K562) cell line. Tumor growth was monitored 2x weekly with bioluminescence imaging (BLI) for up to 6-weeks. Mice that received lymphocytes exposed to SMG showed greater tumor burden compared to those receiving lymphocytes exposed to 1G (week 6 BLI: 1.8e10 ± 8.07e9 versus 2.22e8 ± 1.39e8 photons/second; p < 0.0001). Peak BLI was also higher in the SMG group compared to 1G-control (2.34e10 ± 1.23e10 versus 3.75e8 ± 1.56e8 photons/second; p = 0.0062). Exposure to SMG did not affect the ability of human lymphocytes to engraft or evoke xeno-graft-versus-host disease in the mice. Additionally, we injected the mice with IL-2 and zoledronic acid (ZOL) to expand and activate the anti-tumor activity of NK cells and γ δ-T cells, respectively. This treatment was found to revive the loss of anti-leukemic function observed in vivo when lymphocytes were pre-exposed to SMG. Microgravity plays a contributory role in loss of tumor control in vivo. Immuno-stimulating agents like ZOL+IL-2 may offer an important countermeasure for immune dysregulation during prolonged spaceflight.

The differential role of the lipid raft-associated protein flotillin 2 for progression of myeloid leukemia

AbstractLipid raft-associated proteins play a vital role in membrane-mediated processes. The lipid microdomain-associated protein flotillin 2 (FLOT2), which has a scaffolding function, is involved in polarization, as well as in actin cytoskeletal organization of primitive and mature hematopoietic cells and has been associated with different malignancies. However, its involvement in myeloid leukemias is not well studied. Using murine transplantation models, we show here that the absence of FLOT2 from leukemia-initiating cells (LICs) altered the disease course of BCR-ABL1+ chronic myeloid leukemia (CML), but not of MLL–AF9-driven acute myeloid leukemia (AML). While FLOT2 was required for expression of the adhesion molecule CD44 on both CML- and AML-LIC, a defect in the cytoskeleton, cell polarity, and impaired homing ability of LIC was only observed in FLOT2-deficient BCR-ABL1+ compared with MLL-AF9+ cells. Downstream of CD44, BCR-ABL1 kinase-independent discrepancies were observed regarding expression, localization, and activity of cell division control protein 42 homolog (CDC42) between wild-type (WT) and FLOT2-deficient human CML and AML cells. Inhibition of CDC42 by ML141 impaired the homing of CML LIC and, thereby, CML progression. This suggested that alteration of both CD44 and CDC42 may be causative of impaired CML progression in the absence of FLOT2. In summary, our data suggest a FLOT2-CD44-CDC42 axis, which differentially regulates CML vs AML progression, with deficiency of FLOT2 impairing the development of CML.

Leelamine Modulates STAT5 Pathway Causing Both Autophagy and Apoptosis in Chronic Myelogenous Leukemia Cells.

Simple SummaryAutophagy is a cellular mechanism that is essential for removing misfolded proteins and damaged organelles. Moreover, the aberrant activation of signal transducer and activator of transcription 5 (STAT5), which can regulate cellular survival and homeostasis, has been often observed in different malignancies. In this study, we demonstrate that leelamine inhibits the STAT5 phosphorylation while inducing autophagy as well as apoptosis in chronic myeloid leukemia cells. Leelamine induces autophagy by stimulating the expression of Atg7, beclin-1, and the production of autophagosomes, which leads to substantial inhibition of STAT5 activation.Leelamine (LEE) has recently attracted significant attention for its growth inhibitory effects against melanoma, breast cancer, and prostate cancer cells; however, its impact on hematological malignancies remains unclear. Here, we first investigate the cytotoxic effects of LEE on several human chronic myeloid leukemia (CML) cells. We noted that LEE stimulated both apoptosis and autophagy in CML cells. In addition, the constitutive activation of signal transducer and activator of transcription 5 (STAT5) was suppressed substantially upon LEE treatment. Moreover, STAT5 knockdown with small interfering RNA (siRNA) increased LEE-induced apoptosis as well as autophagy and affected the levels of various oncogenic proteins. Thus, the targeted mitigation of STAT5 activation by LEE can contribute to its diverse anticancer effects by enhancing two distinct cell death pathways.

Aberrant Expressional Profiling of Small RNA by Cold Atmospheric Plasma Treatment in Human Chronic Myeloid Leukemia Cells.

Small RNAs (sRNAs), particularly microRNAs (miRNAs), are functional molecules that modulate mRNA transcripts and have been implicated in the etiology of various types of cancer. Cold atmospheric plasma (CAP) is a physical technology widely used in the field of cancer treatment after exhibiting extensive lethality on cancer cells. However, few studies have reported the exact role of miRNAs in CAP-induced anti-cancer effects. The aim of the present study was to determine whether miRNAs are involved in CAP-induced cytotoxicity by using high-throughput sequencing. Our research demonstrated that 28 miRNAs were significantly changed (17 upregulated and 11downregulated) following 24 h of treatment with a room-temperature argon plasma jet for 90 s compared with that of the untreated group in human chronic myeloid leukemia K562 cells. GO enrichment analysis revealed that these target genes were related to cell organelles, protein binding, and single-organism processes. Furthermore, KEGG pathway analysis demonstrated that the target genes of differentially expressed miRNAs were primarily involved in the cAMP signaling pathway, AMPK signaling pathway, and phosphatidylinositol signaling system. Taken together, our study demonstrated that CAP treatment could significantly alter the small RNA expression profile of chronic myeloid leukemia cells and provide a novel theoretical insight for elucidating the molecular mechanisms in CAP biomedicine application.

2-Methoxyestradiol combined with ascorbic acid facilitates the apoptosis of chronic myeloid leukemia cells via the microRNA-223/Fms-like tyrosine kinase 3/phosphatidylinositol-3 kinase/protein kinase B axis

ABSTRACT Chronic myeloid leukemia (CML) is a malignant myeloproliferative tumor. 2-Methoxyestradiol (2-ME) is an endogenous estrogen metabolite that shows efficacy in human malignancies. Ascorbic acid (AA) possesses antioxidant activity. This study explored the mechanism of 2-ME combined with AA in the apoptosis of CML cells. Firstly, human CML cell lines were treated with 2-ME and AA. The cell viability, apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were detected. miR-223 expression in CML cells was detected. In addition, CML cells were transfected with miR-223 inhibitor. The binding relationship between miR-223 and FLT3 was verified. Subsequently, the FLT3 was overexpressed or silenced for the function rescue experiment to confirm the role of FLT3 in CML cell apoptosis. The expression levels of key factors of the PI3K/AKT pathway were detected. Finally, xenograft nude mouse models were established for in vivo verification. 2-ME + AA treatment inhibited CML cell viability and promoted apoptosis, elevated ROS content, and reduced MMP. 2-ME + AA treatment promoted miR-223 expression in CML cells. miR-223 targeted FLT3. Moreover, miR-223 inhibitor or FLT3 overexpression partially annulled the effect of 2-ME + AA on CML cells. 2-ME + AA inhibited the PI3K/AKT pathway via the miR-223/FLT3 axis. Furthermore, 2-ME + AA suppressed CML xenograft growth in mice. Collectively, 2-ME + AA promoted miR-223 expression and suppressed FLT3 and the PI3K/AKT pathway, thereby facilitating the apoptosis of CML cells and inhibiting CML xenograft growth in mice.

Design, synthesis, and antitumor activity of novel sorafenib derivatives

AbstractNew series of 18 compounds were synthesized using sorafenib derivatives as parent structure and p‐aminoacetophenone as raw materials. The structures of the newly synthesized compounds were confirmed on the basis of 1H, 13C NMR and HRMS (Supporting Information). Their antitumor activities against human lung adenocarcinoma cells A549 (A549), prostate cancer cells PC‐3 (PC‐3), human chronic myeloid leukemia cells (K562), and human liver cancer cells (HepG2) in vitro were evaluated, using Sorafenib as a positive control drug. Bioactivity assays indicated that these compounds showed good antitumor activities toward tested four cancer cell lines. In particular, compound 7n showed potent inhibitory activity with IC50 of 7.39 ± 1.51 μM toward K562. The mechanism and the apoptosis inducing effect of 7n against k562 cell line were studied. The results showed that compound 7n blocked K562 cells in G2/M phase to induce cell apoptosis with a concentration and time‐dependent manner.

Anticancer Mechanism of Parthenolide Analogue PTLA-1 on Adriamycin Resistant Human Chronic Myeloid Leukemia Cell Line K562/ADR

Objective: To investigate the anticancer effect and mechanism of parthenolide analogue (PTLA-1) on adriamycin resistant human chronic myeloid leukemia cell line K562/ADR cells. Methods K562/ADR cells were cultured in vitro. The inhibitory effects of PTLA-1 on cell proliferation were detected by MTT assay, and the effects of PTLA-1 on K562/ADR cell cycle, apoptosis, mitochondrial membrane potential (ΔΨm) and reactive oxygen species were detected by flow cytometry. The effects of PTLA-1 on K562/ADR cell cycle and apoptosis related proteins expressions were detected by Western blot. Results PTLA-1 had higher inhibitory activity on K562/ADR cells than parthenolide. It inhibited the expressions of Cyclin B1/D1 and CDK1/2/4/6, resulting in the arrest of K562/ADR cells in G0/G1 phase; PTLA-1 induced apoptosis of K562/ADR cells in mitochondrial pathway. PTLA-1 could induce mitochondrial membrane potential (ΔΨm) and down regulate the expressions of reactive oxygen species, Bcl-2 and p-Akt proteins, and up regulate the expressions of Bax, cyto c, caspase 9/3 proteins. The chromatin of K562/ADR cells contracted and apoptotic bodies appeared. Conclusion Parthenolide analogue PTLA-1 could inhibit cell proliferation by causing K562/ADR cells to block in G0/G1 phase. Secondly, it could induce K562/ADR cells to apoptosis through caspase dependent mitochondrial pathway, which was related to Akt signal pathway. It had certain anticancer therapeutic potential for chronic myeloid leukemia.

Trident cold atmospheric plasma blocks three cancer survival pathways to overcome therapy resistance

Therapy resistance is responsible for most cancer-related death and is mediated by the unique ability of cancer cells to leverage metabolic conditions, signaling molecules, redox status, and other pathways for their survival. Interestingly, many cancer survival pathways are susceptible to disturbances in cellular reactive oxygen species (ROS) and may therefore be disrupted by exogenous ROS. Here, we explore whether trident cold atmospheric plasma (Tri-CAP), a gas discharge with exceptionally low-level ROS, could inhibit multiple cancer survival pathways together in a murine cell line model of therapy-resistant chronic myeloid leukemia (CML). We show that Tri-CAP simultaneously disrupts three cancer survival pathways of redox deregulation, glycolysis, and proliferative AKT/mTOR/HIF-1α signaling in this cancer model. Significantly, Tri-CAP blockade induces a very high rate of apoptotic death in CML cell lines and in primary CD34+ hematopoietic stem and progenitor cells from CML patients, both harboring the therapy-resistant T315I mutation. In contrast, nonmalignant controls are minimally affected by Tri-CAP, suggesting it selectively targets resistant cancer cells. We further demonstrate that Tri-CAP elicits similar lethality in human melanoma, breast cancer, and CML cells with disparate, resistant mechanisms and that it both reduces tumor formation in two mouse models and improves survival of tumor-bearing mice. For use in patients, administration of Tri-CAP may be extracorporeal for hematopoietic stem cell transplantation therapy, transdermal, or through its activated solution for infusion therapy. Collectively, our results suggest that Tri-CAP represents a potent strategy for disrupting cancer survival pathways and overcoming therapy resistance in a variety of malignancies.

8-Hydroxydaidzein Downregulates JAK/STAT, MMP, Oxidative Phosphorylation, and PI3K/AKT Pathways in K562 Cells.

A metabolite isolated from fermented soybean, 8-hydroxydaidzein (8-OHD, 7,8,4′-trihydroxyisoflavone, NSC-678112), is widely used in ethnopharmacological research due to its anti-proliferative and anti-inflammatory effects. We reported previously that 8-OHD provoked reactive oxygen species (ROS) overproduction, and induced autophagy, apoptosis, breakpoint cluster region-Abelson murine leukemia viral oncogene (BCR-ABL) degradation, and differentiation in K562 human chronic myeloid leukemia (CML) cells. However, how 8-OHD regulates metabolism, the extracellular matrix during invasion and metastasis, and survival signaling pathways in CML remains largely unexplored. High-throughput technologies have been widely used to discover the therapeutic targets and pathways of drugs. Bioinformatics analysis of 8-OHD-downregulated differentially expressed genes (DEGs) revealed that Janus kinase/signal transducer and activator of transcription (JAK/STAT), matrix metalloproteinases (MMPs), c-Myc, phosphoinositide 3-kinase (PI3K)/AKT, and oxidative phosphorylation (OXPHOS) metabolic pathways were significantly altered by 8-OHD treatment. Western blot analyses validated that 8-OHD significantly downregulated cytosolic JAK2 and the expression and phosphorylation of STAT3 dose- and time-dependently in K562 cells. Zymography and transwell assays also confirmed that K562-secreted MMP9 and invasion activities were dose-dependently inhibited by 8-OHD after 24 h of treatment. RT-qPCR analyses verified that 8-OHD repressed metastasis and OXPHOS-related genes. In combination with DisGeNET, it was found that 8-OHD’s downregulation of PI3K/AKT is crucial for controlling CML development. A STRING protein–protein interaction analysis further revealed that AKT and MYC are hub proteins for cancer progression. Western blotting revealed that AKT phosphorylation and nuclear MYC expression were significantly inhibited by 8-OHD. Collectively, this systematic investigation revealed that 8-OHD exerts anti-CML effects by downregulating JAK/STAT, PI3K/AKT, MMP, and OXPHOS pathways, and MYC expression. These results could shed new light on the development of 8-OHD for CML therapy.

Green synthesis and characterization of silver and iron nanoparticles using Nerium oleander extracts and their antibacterial and anticancer activities

Medicinal plants can be used as reducing agents in the preparation of metal nanoparticles by green synthesis because of the chemotherapeutic and anti-infectious properties of natural compounds. Therefore, this paper reports the green synthesis of silver and iron nanoparticles from leaf and flower extracts of Nerium oleander and their capacity as anticancer and antimicrobial agents. Nanoparticle manufacturing and structural characterization of silver and iron nanoparticles are reported. The formation of nanoparticles is characterized by scanning electron microscopy with energy dispersive X-ray spectroscopy, UV-Vis and Fourier transform infrared (FTIR) spectroscopy. Nanoparticles formation was also investigated the surface charge, particle size, and distribution using zeta sizer analysis by DLS. Green synthesis of silver and iron nanoparticles using N. oleander showed different levels of selective cytotoxicity against K562 (human chronic myeloid leukemia cells) in low concentrations and were not cytotoxic to the HUVEC (human umbilical vein endothelial cells) in the same concentrations. Silver nanoparticles showed antibacterial activity against multidrug pathogens, while iron nanoparticles failed to show such activity. Results of the present research demonstrate the potential use of green synthesized nanoparticles in various biomedicine and pharmaceuticals fields in the future.

Preliminary study on the effect of B7H3 interaction with fibronectin on apoptosis of human chronic myeloid leukemia cells

目的探讨B7H3和纤维连接蛋白（FN）相互作用对人慢性髓性白血病细胞系K562细胞凋亡的影响。方法采用流式细胞术检测K562细胞中B7H3分子的表达，构建B7H3过表达细胞。采用免疫共沉淀技术检测B7H3与FN的相互作用。添加外源FN后，通过细胞实验检测细胞黏附和细胞凋亡的变化。Western blot法检测凋亡相关蛋白及PI3K/AKT信号通路的变化。结果① K562细胞低表达B7H3分子，慢病毒转染后得到稳定表达B7H3的细胞系K562 OE-B7H3及其对照细胞系K562 NC-B7H3细胞。②B7H3与FN之间存在相互作用（P＝0.036）。③B7H3与FN的相互作用促进细胞黏附（P<0.05），抑制细胞凋亡（P<0.05）。④B7H3与FN相互作用激活PI3K/AKT信号通路（P<0.05）。结论B7H3与FN相互作用促进了细胞黏附，可能通过激活PI3K/AKT信号通路抑制K562细胞的凋亡。

Salidroside inhibits chronic myeloid leukemia cell proliferation and induces apoptosis by regulating the miR-140-5p/wnt5a/β-catenin axis.

Salidroside, an active ingredient of Rhodiola rosea, exhibits antitumor effects in various types of cancer. However, the role of salidroside in chronic myeloid leukemia (CML) has not been elucidated. In the presents study, cell viability was assessed by CCK-8 assay, while apoptosis was detected by flow cytometry. Reverse transcription-quantitative PCR analysis was used to examine the expression levels of miR-140-5p in human CML cell lines. The expression levels of apoptosis and cell cycle-associated proteins and of the wnt5a/β-catenin signaling pathway were determined by western blot analysis. Bioinformatic analysis and luciferase reporter assays were employed to investigate the association between miR-140-5p and wnt5a. The results revealed that exposure of CML cells to salidroside (80 µM) inhibited cell proliferation and promoted apoptosis. In addition, salidroside treatment led to the upregulation of miR-140-5p expression. Furthermore, the inhibition of wnt5a/β-catenin signaling pathway and the pro-apoptotic effects induced by salidroside were attenuated by miR-140-5p silencing. Notably, wnt5a was revealed to be a direct target of miR-140-5p. The present findings indicated that salidroside exerted anti-CML effects through regulating miR-140-5p by suppressing the wnt5a/β-catenin signaling pathway. The present study provided evidence of the therapeutic role of salidroside in CML.

CRISPR-Cas9 Technology as a Tool to Target Gene Drivers in Cancer: Proof of Concept and New Opportunities to Treat Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy produced by a unique oncogenic event involving the constitutively active tyrosine-kinase (TK) BCR/ABL1. TK inhibitors (TKI) changed its prognosis and natural history. Unfortunately, ABL1 remains unaffected by TKIs. Leukemic stem cells (LSCs) remain, and resistant mutations arise during treatment. To address this problem, we have designed a therapeutic CRISPR-Cas9 deletion system targeting BCR/ABL1. The system was efficiently electroporated to cell lines, LSCs from a CML murine model, and LSCs from CML patients at diagnosis, generating a specific ABL1 null mutation at high efficiency and allowing the edited leukemic cells to be detected and tracked. The CRISPR-Cas9 deletion system triggered cell proliferation arrest and apoptosis in murine and human CML cell lines. Patient and murine-derived xenografts with CRISPR-edited LSCs in NOD SCID gamma niches revealed that normal multipotency and repopulation ability of CRISPR edited LSCs were fully restored. Normal hematopoiesis was restored, avoiding myeloid bias. To the best of our knowledge, we show for the first time how a CRISPR-Cas9 deletion system efficiently interrupts BCR/ABL1 oncogene in primary LSCs to bestow a therapeutic benefit. This study is a proof of concept for genome editing in all those diseases, like CML, sustained by a single oncogenic event, opening up new therapeutic opportunities.

Delayed Expansion of Murine BCR-ABL1+ Leukemia By Genetic and Pharmacological Inhibition of NOX2

Reactive oxygen species (ROS), produced from reduction of oxygen by the myeloid NADPH oxidase (NOX2), are pivotal effector molecules in the innate immune defense against microbes, but imbalance between the generation and detoxification of ROS may be injurious to cell components. The malignant cells of patients with BCR-ABL1+ chronic myeloid leukemia (CML) show elevated ROS production, which has been implicated in the genetic instability BCR-ABL1+ cells. However, further studies are required to define the role of ROS in leukemogenesis. We have utilized genetically modified murine hematopoietic cells to determine the influence of NOX2-derived ROS for the in vivo expansion of transplanted BCR-ABL1+ cells. Irradiated C57BL/6J mice received transplants of wild-type (WT) or Nox2 knockout (Nox2 -KO) hematopoietic cells that were transduced to express GFP-labeled human BCR-ABL1 by use of the p210 construct cloned into a MSCV vector. In vitro experiments using the NOX2 inducer D-peptide confirmed that Nox2 -KO BCR-ABL1+ cells were devoid of ROS production via NOX2. Peripheral blood was drawn biweekly from mice receiving WT and Nox2 -KO transplants for fluorimetric quantification of BCR-ABL1+ cells. We observed prolonged survival for mice receiving Nox2 -KO vs. WT BCR-ABL1 transplants (n=43, p=0.017, logrank test) along with a trend towards reduced expansion of leukemic cells in vivo . To determine effects of pharmacological inhibition of NOX2 on leukemic expansion, the NOX2 inhibitor histamine dihydrochloride (HDC) was administered to mice carrying WT or Nox2 -KO BCR-ABL1+ transplants. In vitro experiments confirmed that HDC reduced NOX2-dependent induction of ROS formation in WT BCR-ABL1+ cells. Treatment of age-matched mice with HDC (1.5 mg three times per week i.p.) delayed the expansion of WT BCR-ABL1+ cells at four weeks after transplantation (n=18, p<0.05, t-test) but did not reduce the burden of leukemia in mice receiving Nox2 -KO BCR-ABL1+ transplants. Initial experiments using primary human CML cells revealed differential expression of redox-related genes in CD34+CD38- leukemic stem cells vs. cord blood stem cells. Our results implicate NOX2 as a conceivable therapeutic target in BCR-ABL1+ leukemia. DisclosuresGrauers Wiktorin:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer. Thoren:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer. Hellstrand:Immune Pharmaceuticals: Patents & Royalties: Filed or pending patents protecting the use of NOX2 inhibitors in cancer. Martner:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer.

RNA Binding Protein RBM47 Inhibits the K562 Cell Proliferation by Regulating HMGA2 mRNA Expression

To investigate the regulatory effects of RBM47 on HMGA2 and the function of RBM47 in human chronic myeloid leukemia cell K562. K562 cells were transduction by the overexpressed and knockdown RBM47 lentiviral vector. CCK-8 assay was used to detect the effect of RBM47 on the proliferation of K562 cells. Flow cytometry assay was used to detect the effect of RBM47 on the cell cycle progression of K562 cells. RNA immunoprecipitation assay was used to detect the association between RBM47 and HMGA2 mRNA. RT-qPCR was used to detect the effects of RBM47 on the stability of HMGA2 mRNA. Western blot was used to evaluate the effect of RBM47 on HMGA2 protein expression. The overexpressed RBM47 could inhibit the proliferation and cell cycle progression of K562 cells. However, the inhibitation of RBM47 could improve the proliferation and cell cycle progression of K562 cells. RBM47 combined with HMGA2 mRNA could promote the degradation of HMGA2 mRNA. Thus, the overexpressed RBM47 could decrease the expression of HMGA2 protein in K562 cells. RNA binding protein RBM47 can inhibit the proliferation of K562 cells by regulating HMGA2 expression.