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Leukemia is a malignant tumor with high heterogeneity and a complex evolutionary process. It is difficult to resolve the heterogeneity and clonal evolution of leukemia cells by applying traditional bulk sequencing techniques, thus preventing a deep understanding of the mechanisms of leukemia development and the identification of potential therapeutic targets. However, with the development and application of single-cell sequencing technology, it is now possible to investigate the gene expression profile, mutations, and epigenetic features of leukemia at the single-cell level, thus providing a new perspective for leukemia research. In this article, we review the recent applications and advances of single-cell sequencing technology in leukemia research, discuss its potential for enhancing our understanding of the mechanisms of leukemia development, discovering therapeutic targets and personalized treatment, and provide reference guidelines for the significance of this technology in clinical research. [ABSTRACT FROM AUTHOR]

Magonia pubescens is a natural species from the Brazilian cerrado biome. Its fruits and seeds are used in the treatment of seborrheic dermatitis, a common inflammatory skin disease. In this work, the known compounds lapachol, stigmasterol, maniladiol and scopoletin were isolated from hexane and dichloromethane extracts of M. pubescens branches. The aqueous extract of this material was fractioned through a liquid-liquid partition and the obtained fractions were analyzed by UHPLC-MS/MS. The results obtained were compared with data from three databases, leading to the putative identification of 51 compounds from different classes, including flavonoids, saponins and triterpenes. The cytotoxicity of aqueous fractions was assayed against breast cancer (MDA-MB-231) and leukemia (THP-1 and K562) cells. The best activity was observed for fraction AE3 against MDA-MB-231 cells (IC50 30.72 µg.mL−1). [ABSTRACT FROM AUTHOR]

Naphthylisoquinoline (NIQ) alkaloids are rising as a promising class of secondary metabolites with pharmaceutical potential. NF-κB has already been recognized as a significant modulator of cancer proliferation and drug resistance. We have previously reported the mechanisms behind the cytotoxic effect of dioncophylline A, an NIQ monomer, in leukemia cells. In the current study, we have investigated the cytotoxic effect of jozimine A2, an NIQ dimer, on leukemia cells in comparison to a second, structurally unsymmetric dimer, michellamine B. To this end, molecular docking was applied to predict the binding affinity of the dimers towards NF-κB, which was then validated through microscale thermophoresis. Next, cytotoxicity assays were performed on CCRF-CEM cells and multidrug-resistant CEM/ADR5000 cells following treatment. Transcriptome analysis uncovered the molecular networks affected by jozimine A2 and identified the cell cycle as one of the major affected processes. Cell death modes were evaluated through flow cytometry, while angiogenesis was measured with the endothelial cell tube formation assay on human umbilical vein endothelial cells (HUVECs). The results indicated that jozimine A2 bound to NF-κB, inhibited its activity and prevented its translocation to the nucleus. In addition, jozimine A2 induced cell death through apoptosis and prevented angiogenesis. Our study describes the cytotoxic effect of jozimine A2 on leukemia cells and explains the interactions with the NF-κB signaling pathway and the anticancer activity. [ABSTRACT FROM AUTHOR]

Background: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) can accurately detect relative gene expression levels in biological samples. However, widely used reference genes exhibit unstable expression under certain conditions. Methods and results: Here, we compared the expression stability of eight reference genes (RPLP0, RPS18, RPL13, EEF1A1, β-actin, GAPDH, HPRT1, and TUBB) commonly used in liproxstatin-1 (Lip-1)-treated K562 cells using RNA-sequencing and RT-qPCR. The expression of EEF1A1, ACTB, GAPDH, HPRT1, and TUBB was considerably lower in cells treated with 20 μM Lip-1 than in the control, and GAPDH also showed significant downregulation in the 10 μM Lip-1 group. Meanwhile, when we used geNorm, NormFinder, and BestKeeper to compare expression stability, we found that GAPDH and HPRT1 were the most unstable reference genes among all those tested. Stability analysis yielded very similar results when geNorm or BestKeeper was used but not when NormFinder was used. Specifically, geNorm and BestKeeper identified RPL13 and RPLP0 as the most stable genes under 20 μM Lip-1 treatment, whereas RPL13, EEF1A1, and TUBB were the most stable under 10 μM Lip-1 treatment. TUBB and EEF1A1 were the most stable genes in both treatment groups according to the results obtained using NormFinder. An assumed most stable gene was incorporated into each software to validate the accuracy. The results suggest that NormFinder is not an appropriate algorithm for this study. Conclusions: Stable reference genes were recognized using geNorm and BestKeeper but not NormFinder. Overall, RPL13 and RPLP0 were the most stable reference genes under 20 μM Lip-1 treatment, whereas RPL13, EEF1A1, and TUBB were the most stable genes under 10 μM Lip-1 treatment. [ABSTRACT FROM AUTHOR]

In order to study the chemical constituents from Carpesium cernuum and their inhibitory effects on leukemia cells in vitro. The chemical constituents from ethyl acetate fraction of C. cernuum were isolated and purified by silica gel column chromatography, Sephadex LH-20 column chromatography and macroporous adsorption resin, and their structures were identified by means of various spectroscopic techniques such as 1H NMR, 13C NMR and MS. The inhibitory effects of compounds 1-10 on leukemia cells (K562, HEL) in vitro were determined by MTT assay. The results were as follows: (1) Eleven compounds were isolated and identified as 2, 9-epoxy-5, 9-dihydroxy-8-angeloyloxy-11-methoxymethyl-4(15)-germacraen-6, 12-olide (1), cardivin D (2), cernuumolide I (3), cernuumolide J (4), 8-hydroxy-9, 10-diisobutyryloxythymol (5), (2E, 6Z, 10E, 12R)-7-［(acetyloxy)methyl］-3, 11, 15-trimethylhexadeca-2, 6, 10, 14-tetraene-1, 12-diol (6), 9, 10-dihydroxyoctadecanoate (7), 1, 6-dihydroxy-8-hydroxymethyl-anthraquinone (8), emodin (9), 4-megastigmen-3, 9-dione (10), β-sitosterol (11). Among them, Compound 1 was identified as a new compound, compounds 5, 7-10 were isolated from Carpesium for the first time, compounds 2, 5-10 were isolated from C. cernuum for the first time. (2) The results of activity test showed that cardivin D (2), cernuumolide I (3) and cernuumolide J (4) had good inhibitory effects on leukemia cells in vitro. The IC50 of compounds 2-4 against K562 cells and HEL cells were (2.27 ± 0.46), (5.53 ± 0.41), (3.90 ± 0.80) μmol·L-1 and (1.84 ± 0.14), (2.36 ± 0.90), (2.31 ± 1.17) μmol·L-1, respectively. The study enriches the chemical constituents of C. cernuum, and provides a material basis for the development of anti-leukemia drugs.

In order to study the chemical constituents from Carpesium cernuum and their inhibitory effects on leukemia cells in vitro. The chemical constituents from ethyl acetate fraction of C. cernuum were isolated and purified by silica gel column chromatography, Sephadex LH-20 column chromatography and macroporous adsorption resin, and their structures were identified by means of various spectroscopic techniques such as 1H NMR, 13C NMR and MS. The inhibitory effects of compounds 1-10 on leukemia cells (K562, HEL) in vitro were determined by MTT assay. The results were as follows: (1) Eleven compounds were isolated and identified as 2, 9-epoxy-5, 9-dihydroxy-8-angeloyloxy-11-methoxymethyl-4 (15)-germacraen-6, 12-olide (1), cardivin D (2), cernuumolide I(3), cernuumolide J(4), 8-hydroxy-9, 10-diisobutyryloxythymol (5), (2E, 6Z, 10E, 12R)-7-[(acetyloxy)methyl]-3, 11, 15-trimethylhexadeca-2, 6, 10, 14-tetraene-1, 12-diol(6), 9, 10-dihydroxyoctadecanoate(7), 1, 6-dihydroxy-8-hydroxymethyl-anthraquinone (8), emodin (9), 4-megastigmen-3, 9-dione(10), β-sitosterol(11). Among them, Compound 1 was identified as a new compound, compounds 5, 7-10 were isolated from the Carpesium for the first time, compounds 2, 5-10 were isolated from C. cernuum for the first time. (2) The results of activity test showed that cardivin D(2), cernuumolide I(3)and cernuumolide J(4)had good inhibitory effects on leukemia cells in vitro. The IC50 of compounds 2-4 against K562 cells and HEL cells were(2.27 ± 0.46), (5.53 ± 0.41), (3.90 ± 0.80) μmol·L-1 and(1.84 ± 0.14), (2.36 ± 0.90), (2.31 ± 1.17) μmol·L-1, respectively. Thus, the study enriches the chemical constituents of C. cernuum, and provides a material basis for the development of anti-leukemia drugs. [ABSTRACT FROM AUTHOR]

We aim to study the inhibitory effect of alkaline serine protease (ASPNJ) on lymphocytic leukemia Jurkat cells and its related mechanism through examining the expression of membrane proteins or membrane‐associated proteins. MTT assay and trypan blue staining were used to detect the inhibitory effect of ASPNJ on the proliferation and growth of Jurkat cells. Wright‐Giemsa staining was used to observe the effect of ASPNJ on the morphology of Jurkat cells. The effect of ASPNJ on Jurkat cell apoptosis was detected by flow cytometry. Two‐dimensional electrophoresis‐mass spectrometry (2‐DE‐MS) was used to detect and identify the differentially expressed proteins of Jurkat cells treated with ASPNJ (4 μg/mL, 3 h), of which three were selected and verified by Western blot. ASPNJ significantly inhibited the proliferation of leukemia cells (Raji, U937, and Jurkat), caused obvious morphological changes, and induced apoptosis of Jurkat cells. ASPNJ also increased the sensitivity of Jurkat cells to vincristine (VCR). Seven differentially expressed proteins were obtained through 2DE‐MS, of which Peroxiredoxin‐6 (PRDX6), Calcium‐binding protein (CHP1), and 40S ribosomal protein SA (RPSA) were validated. ASPNJ can cause significant toxic effects on Jurkat cells and enhance the effects of VCR. The mechanism of action of ASPNJ on Jurkat cells may be related to differentially expressed proteins such as PRDX6. This study provides a new experimental basis and direction for antileukemia research. [ABSTRACT FROM AUTHOR]

Naphthylisoquinoline (NIQ) alkaloids are rising as a promising class of secondary metabolites with pharmaceutical potential. NF-κB has already been recognized as a significant modulator of cancer proliferation and drug resistance. We have previously reported the mechanisms behind the cytotoxic effect of dioncophylline A, an NIQ monomer, in leukemia cells. In the current study, we have investigated the cytotoxic effect of jozimine A2, an NIQ dimer, on leukemia cells in comparison to a second, structurally unsymmetric dimer, michellamine B. To this end, molecular docking was applied to predict the binding affinity of the dimers towards NF-κB, which was then validated through microscale thermophoresis. Next, cytotoxicity assays were performed on CCRF-CEM cells and multidrug-resistant CEM/ADR5000 cells following treatment. Transcriptome analysis uncovered the molecular networks affected by jozimine A2 and identified the cell cycle as one of the major affected processes. Cell death modes were evaluated through flow cytometry, while angiogenesis was measured with the endothelial cell tube formation assay on human umbilical vein endothelial cells (HUVECs). The results indicated that jozimine A2 bound to NF-κB, inhibited its activity and prevented its translocation to the nucleus. In addition, jozimine A2 induced cell death through apoptosis and prevented angiogenesis. Our study describes the cytotoxic effect of jozimine A2 on leukemia cells and explains the interactions with the NF-κB signaling pathway and the anticancer activity.

The aim of this study was to explore the mechanism of antitumor effect of (E)-6-morpholino-9-(styrylsulfonyl)-9H-purine (6-Morpholino-SPD) and (E)-6-amino-9-(styrylsulfonyl)-9H-purine (6-Amino-SPD). The effects on apoptosis induction, mitochondrial potential, and accumulation of ROS in treated K562 cells were determined by flow cytometry. The RT-PCR method was used to measure the expression of Akt, CA IX, caspase 3, and cytochrome c genes, as well as selected miRNAs. Western blot analysis was used to determine the expression of Akt, cytochrome c, and caspase 3. The results demonstrate the potential of the tested derivatives as effective antitumor agents with apoptotic-inducing properties. In leukemic cells treated with 6-Amino-SPD, increased expression of caspase 3 and cytochrome c genes was observed, indicating involvement of the intrinsic mitochondrial pathway in the induction of apoptosis. Conversely, leukemic cells treated with 6-Morpholino-SPD showed reduced expression of these genes. The observed downregulation of miR-21 by 6-Morpholino-SPD may contribute to the induction of apoptosis and disruption of mitochondrial function. In addition, both derivatives exhibited increased expression of Akt and CA IX genes, suggesting activation of the Akt/HIF pathway. However, the exact mechanism and its relations to the observed overexpression of miR-210 need further investigation. The acceptable absorption and distribution properties predicted by ADMET analysis suggest favorable pharmacokinetic properties for these derivatives. [ABSTRACT FROM AUTHOR]

Pilocarpine is a selective M1/M3 agonist of muscarinic acetylcholine receptor subtypes. Muscarinic acetylcholine receptors are G protein-coupled receptors. These receptors are different drug targets. The aim of the present work was to investigate the effect of pilocarpine on the expression of M3 muscarinic acetylcholine receptor, the AChE activity, IL-8 release response, and proliferation in K562 cells, via muscarinic receptor activation. Human chronic myeloid leukemic cell cultures were incubated with drugs. Proliferation assays were performed by BrdU assay. Expression of M3 muscarinic acetylcholine receptor and apoptosis proteins such as bcl, bax, cyt C, and caspases was assessed with the semiquantitative Western blotting method. Pilocarpine inhibits chronic myeloid cell proliferation and M3 muscarinic acetylcholine receptor protein expression. Pilocarpine increases caspase-8 and -9 expression levels, upregulating the proapoptotic protein Bax and downregulating the expression levels of the antiapoptotic protein Bcl-2. The apoptotic activity of pilocarpine is associated with an increase in AChE activity. M3 muscarinic acetylcholine receptors can activate multiple signal transduction systems and mediate inhibitory effects on chronic myeloid K562 cell proliferation depending on the presence of 1% FBS conditions. This apoptotic effect of pilocarpine may be due to the concentration of pilocarpine and the increase in AChE level. Our results suggest that inhibition of cell proliferation by inducing apoptosis of pilocarpine in K562 cells may be one of the targets. M3 selective agonist may have therapeutic potential in chronic myeloid leukemia. [ABSTRACT FROM AUTHOR]

To explore the molecular mechanisms of action underlying the antileukemia activities of darinaparsin, an organic arsenical approved for the treatment of peripheral T–cell lymphoma in Japan, cytotoxicity of darinaparsin was evaluated in leukemia cell lines NB4, U-937, MOLT-4 and HL-60. Darinaparsin was a more potent cytotoxic than sodium arsenite, and induced apoptosis/necrosis in NB4 and HL-60 cells. In NB4 cells exhibiting the highest susceptibility to darinaparsin, apoptosis induction was accompanied by the activation of caspase-8/-9/-3, a substantial decrease in Bid expression, and was suppressed by Boc-D-FMK, a pancaspase inhibitor, suggesting that darinaparsin triggered a convergence of the extrinsic and intrinsic pathways of apoptosis via Bid truncation. A dramatic increase in the expression level of γH2AX, a DNA damage marker, occurred in parallel with G2/M arrest. Activation of p53 and the inhibition of cdc25C/cyclin B1/cdc2 were concomitantly observed in treated cells. Downregulation of c-Myc, along with inactivation of E2F1 associated with the activation of Rb, was observed, suggesting the critical roles of p53 and c-Myc in darinaparsin-mediated G2/M arrest. Trolox, an antioxidative reagent, suppressed the apoptosis induction but failed to correct G2/M arrest, suggesting that oxidative stress primarily contributed to apoptosis induction. Suppression of Notch1 signaling was also confirmed. Our findings provide novel insights into molecular mechanisms underlying the cytotoxicity of darinaparsin and strong rationale for its new clinical application for patients with different types of cancer. [ABSTRACT FROM AUTHOR]

Simple Summary: PARP inhibitors (PARPi) have been administered to treat BRCA1/2-mutated/deficient malignancies. Nevertheless, the resistance to PARPi is emerging in experimental and clinical interventions. Importantly, the resistance originated from diverse mechanisms, therefore requiring tremendous efforts to identify mechanistic aspects and develop combinational therapies to prevent the resistance and/or restore the efficiency of PARPi in cancer cells. Here, we review pre-existing and acquired resistance to PARPi and propose potential therapeutic solutions. The advanced development of synthetic lethality has opened the doors for specific anti-cancer medications of personalized medicine and efficient therapies against cancers. One of the most popular approaches being investigated is targeting DNA repair pathways as the implementation of the PARP inhibitor (PARPi) into individual or combinational therapeutic schemes. Such treatment has been effectively employed against homologous recombination-defective solid tumors as well as hematopoietic malignancies. However, the resistance to PARPi has been observed in both preclinical research and clinical treatment. Therefore, elucidating the mechanisms responsible for the resistance to PARPi is pivotal for the further success of this intervention. Apart from mechanisms of acquired resistance, the bone marrow microenvironment provides a pre-existing mechanism to induce the inefficiency of PARPi in leukemic cells. Here, we describe the pre-existing and acquired mechanisms of the resistance to PARPi-induced synthetic lethality. We also discuss the potential rationales for developing effective therapies to prevent/repress the PARPi resistance in cancer cells. [ABSTRACT FROM AUTHOR]

Previous studies demonstrated that CD4+ T cells can uptake tumor antigenpulsed dendritic cell-derived exosomes (DEXO), which harbor tumor antigen peptide/pMHC I complex and costimulatory molecules and show potent effects on inducing antitumor immunity. However, in preliminary study, CD4+ T cells targeted by leukemia cell-derived exosomes (LEXs) did not show the expected effects in inducing effective anti-leukemia immunity, indicating that LEX is poorly immunogenetic largely due to an inadequate costimulatory capacity. Therefore, LEX-based anti-leukemia vaccines need to be optimized. In this study, we constructed a novel LEX-based vaccine by combining CD4+ T cells with costimulatory molecules gene-modified LEXs, which harbor upregulated CD80 and CD86, and the anti-leukemia immunity of CD80 and CD86 gene-modified LEX-targeted CD4+ T cells was investigated. We used lentiviral vectors encoding CD80 and CD86 to successfully transduced the L1210 leukemia cells, and the expression of CD80 and CD86 was remarkably upregulated in leukemia cells. The LEXs highly expressing CD80 and CD86 were obtained from the supernatants of gene-transduced leukemia cells. Our data have shown that LEX-CD8086 could promote CD4+ T cell proliferation and Th1 cytokine secretion more efficiently than control LEXs. Moreover, CD4+ TLEX-CD8086 expressed the acquired exosomal costimulatory molecules. With acquired costimulatory molecules, CD4+ TLEX-CD8086 can act as APCs and are capable of directly stimulating the leukemia cell antigen-specific CD8+ CTL response. This response was higher in potency compared to that noted by the other formulations. Furthermore, the animal study revealed that the CD4+ TLEXCD8086 significantly inhibited tumor growth and prolonged survival of tumorbearing mice than other formulations did in both protective and therapeutic models. In conclusion, this study revealed that CD4+ TLEX-CD8086 could effectively induce more potential anti-leukemia immunity than LEX-CD8086 alone, suggesting that the utilization of a costimulatory molecule gene-modified leukemia cell-derived exosome-targeted CD4+ T cell vaccine may have promising potential for leukemia immunotherapy. [ABSTRACT FROM AUTHOR]

Previous studies demonstrated that CD4+ T cells can uptake tumor antigen-pulsed dendritic cell-derived exosomes (DEXO), which harbor tumor antigen peptide/pMHC I complex and costimulatory molecules and show potent effects on inducing antitumor immunity. However, in preliminary study, CD4+ T cells targeted by leukemia cell-derived exosomes (LEXs) did not show the expected effects in inducing effective anti-leukemia immunity, indicating that LEX is poorly immunogenetic largely due to an inadequate costimulatory capacity. Therefore, LEX-based anti-leukemia vaccines need to be optimized. In this study, we constructed a novel LEX-based vaccine by combining CD4+ T cells with costimulatory molecules gene-modified LEXs, which harbor upregulated CD80 and CD86, and the anti-leukemia immunity of CD80 and CD86 gene-modified LEX-targeted CD4+ T cells was investigated. We used lentiviral vectors encoding CD80 and CD86 to successfully transduced the L1210 leukemia cells, and the expression of CD80 and CD86 was remarkably upregulated in leukemia cells. The LEXs highly expressing CD80 and CD86 were obtained from the supernatants of gene-transduced leukemia cells. Our data have shown that LEX-CD8086 could promote CD4+ T cell proliferation and Th1 cytokine secretion more efficiently than control LEXs. Moreover, CD4+ TLEX-CD8086 expressed the acquired exosomal costimulatory molecules. With acquired costimulatory molecules, CD4+ TLEX-CD8086 can act as APCs and are capable of directly stimulating the leukemia cell antigen-specific CD8+ CTL response. This response was higher in potency compared to that noted by the other formulations. Furthermore, the animal study revealed that the CD4+ TLEX-CD8086 significantly inhibited tumor growth and prolonged survival of tumor-bearing mice than other formulations did in both protective and therapeutic models. In conclusion, this study revealed that CD4+ TLEX-CD8086 could effectively induce more potential anti-leukemia immunity than LEX-CD8086 alone, suggesting that the utilization of a costimulatory molecule gene-modified leukemia cell-derived exosome-targeted CD4+ T cell vaccine may have promising potential for leukemia immunotherapy.

The aim of this study was to explore the mechanism of antitumor effect of (E)-6-morpholino-9-(styrylsulfonyl)-9H-purine (6-Morpholino-SPD) and (E)-6-amino-9-(styrylsulfonyl)-9H-purine (6-Amino-SPD). The effects on apoptosis induction, mitochondrial potential, and accumulation of ROS in treated K562 cells were determined by flow cytometry. The RT-PCR method was used to measure the expression of Akt, CA IX, caspase 3, and cytochrome c genes, as well as selected miRNAs. Western blot analysis was used to determine the expression of Akt, cytochrome c, and caspase 3. The results demonstrate the potential of the tested derivatives as effective antitumor agents with apoptotic-inducing properties. In leukemic cells treated with 6-Amino-SPD, increased expression of caspase 3 and cytochrome c genes was observed, indicating involvement of the intrinsic mitochondrial pathway in the induction of apoptosis. Conversely, leukemic cells treated with 6-Morpholino-SPD showed reduced expression of these genes. The observed downregulation of miR-21 by 6-Morpholino-SPD may contribute to the induction of apoptosis and disruption of mitochondrial function. In addition, both derivatives exhibited increased expression of Akt and CA IX genes, suggesting activation of the Akt/HIF pathway. However, the exact mechanism and its relations to the observed overexpression of miR-210 need further investigation. The acceptable absorption and distribution properties predicted by ADMET analysis suggest favorable pharmacokinetic properties for these derivatives.

Spin probes can be used to monitor biological membranes, including the penetration of different molecules into cells. The aim of the present studies was an investigation of the endocytosis process of two spin labels—2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) and 4-hydroxy-TEMPO (TEMPOL)—into yeast cells and a leukemia cell line (HL-60, ATCC CCL-240) by Electron Spin Resonance (ESR). The ESR method is helpful for the direct detection of free radicals. The cell incubation and endocytosis of spin probes were carried out at 310 K. In contrast, the ESR measurements of yeast cells and a leukemia cell line with spin probes were at 240 K. Spectral differentiation was observed; hence, the spin probes present in suspension and attached to the cell membrane could be distinguished. The ESR signal changes of spin probes depended on spin probe concentration, cell number, and type of cell (healthy/cancerous). Additionally, the effect of external factors (oxygen and vitamin C) on the ESR signal decay of spin markers in the cell solution was established. The experimental results prove that the spin probes (TEMPO and TEMPOL) could scavenge free radicals inside the cell. At the same time, the mechanism of spin probe interaction in suspension was determined based on the measurements at low temperatures. [ABSTRACT FROM AUTHOR]

Hydroxytyrosol (HT), the main representative of polyphenols of olive oil, has been described as one of the most powerful natural antioxidants, also showing anti-inflammatory, antimicrobial, cardioprotective and anticancer activity in different type of cancers, but has been little studied in hematological neoplasms. The objective of this work was to evaluate the anticancer potential of HT in acute human leukemia T cells (Jurkat and HL60) and the anti-inflammatory potential in murine macrophages (Raw264.7). For this, cytotoxicity tests were performed for HT, showing IC50 values, at 24 h, for Jurkat, HL60 and Raw264.7 cells, of 27.3 µg·mL−1, 109.8 µg·mL−1 and 45.7 µg·mL−1, respectively. At the same time, HT caused cell arrest in G0/G1 phase in both Jurkat and HL60 cells by increasing G0/G1 phase and significantly decreasing S phase. Apoptosis and cell cycle assays revealed an antiproliferative effect of HT, decreasing the percentage of dividing cells and increasing apoptosis. Furthermore, HT inhibited the PI3K signaling pathway and, consequently, the MAPK pathway was activated. Inflammation tests revealed that HT acts as an anti-inflammatory agent, reducing NO levels in Raw264.7 cells previously stimulated by lipopolysaccharide (LPS). These processes were confirmed by the changes in the expression of the main markers of inflammation and cancer. In conclusion, HT has an anticancer and anti-inflammatory effect in the cell lines studied, which were Raw264.7, Jurkat, and HL60, and could be used as a natural drug in the treatment of liquid cancers, leukemias, myelomas and lymphomas. [ABSTRACT FROM AUTHOR]

Ultrasound single-beam acoustic tweezer system has attracted increasing attention in the field of biomechanics. Cell biomechanics play a pivotal role in leukemia cell functions. To better understand and compare the cell mechanics of the leukemia cells, herein, we fabricated an acoustic tweezer system in-house connected with a 50-MHz high-frequency cylinder ultrasound transducer. Selected leukemia cells (Jurkat, K562, and MV-411 cells) were cultured, trapped, and manipulated by high-frequency ultrasound single beam, which was transmitted from the ultrasound transducer without contacting any cells. The relative deformability of each leukemia cell was measured, characterized, and compared, and the leukemia cell (Jurkat cell) gaining the highest deformability was highlighted. Our results demonstrate that the high-frequency ultrasound single beam can be utilized to manipulate and characterize leukemia cells, which can be applied to study potential mechanisms in the immune system and cell biomechanics in other cell types. [ABSTRACT FROM AUTHOR]

Although previous studies have shown anti-cancer activity of betulinic acid (BA), a pentacyclic triterpenoid, against various cancer lines, the underlying molecular mechanisms are not well elucidated. In this study, we evaluated the mechanisms involved in the anti-cancer efficacy of BA in U937 human myeloid leukemia cells. BA exerted a significant cytotoxic effect on U937 cells through blocking cell cycle arrest at the G2/M phase and inducing apoptosis, and that the intracellular reactive oxygen species (ROS) levels increased after treatment with BA. The down-regulation of cyclin A and cyclin B1, and up-regulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1 revealed the G2/M phase arrest mechanism of BA. In addition, BA induced the cytosolic release of cytochrome c by reducing the mitochondrial membrane potential with an increasing Bax/Bcl-2 expression ratio. BA also increased the activity of caspase-9 and -3, and subsequent degradation of the poly (ADP-ribose) polymerase. However, quenching of ROS by N-acetyl-cysteine, an ROS scavenger, markedly abolished BA-induced G2/M arrest and apoptosis, indicating that the generation of ROS plays a key role in inhibiting the proliferation of U937 cells by BA treatment. Taken together, our results provide a mechanistic rationale that BA exhibits anti-cancer properties in U937 leukemia cells through ROS-dependent induction of cell cycle arrest at G2/M phase and apoptosis.

Many quinazoline derivatives with pharmacological properties, such as anticancer activity, have been synthesized. Fourteen quinazoline derivatives bearing a substituted sulfonamide moiety (4a–n) were previously synthesized and fully characterized. These compounds exerted antiproliferative activity against cell lines derived from solid tumors. Herein, the antileukemic activities of these compounds (4a–n) against two different leukemia cell lines (Jurkat acute T cell and THP-1 acute monocytic) were investigated. Our investigation included examining their activity in vivo in a zebrafish embryo model. Remarkably, compounds 4a and 4d were the most potent in suppressing cell proliferation, with an IC50 value range of 4–6.5 µM. Flow cytometry analysis indicated that both compounds halted cell progression at the G2/M phase and induced apoptosis in a dose-dependent manner. RT-PCR and Western blot analyses also showed that both compounds effectively induced apoptosis by upregulating the expression of proapoptotic factors while downregulating that of antiapoptotic factors. In vivo animal toxicity assays performed in zebrafish embryos indicated that compound 4d was more toxic than compound 4a, with compound 4d inducing multiple levels of teratogenic phenotypes in zebrafish embryos at a sublethal concentration. Moreover, both compounds perturbed the hematopoiesis process in developing zebrafish embryos. Collectively, our data suggest that compounds 4a and 4d have the potential to be used as antileukemic agents. [ABSTRACT FROM AUTHOR]

An interaction between acute myeloid leukemia (AML) cells and endothelial cells in the bone marrow seems to play a critical role in chemosensitivity on leukemia treatment. The endothelial niche reportedly enhances the paracrine action of the soluble secretory proteins responsible for chemoresistance in a vascular endothelial growth factor A (VEGF-A)/VEGF receptor 2 (VEGFR-2) signaling pathway–dependent manner. To further investigate the contribution of VEGF-A/VEGFR-2 signaling to the chemoresistance of AML cells, a biochemical assay system in which the AML cells were cocultured with human endothelial EA.hy926 cells in a monolayer was developed. By coculture with EA.hy926 cells, this study revealed that the AML cells resisted apoptosis induced by the anticancer drug cytarabine. SU4312, a VEGFR-2 inhibitor, attenuated VEGFR-2 phosphorylation and VEGF-A/VEGFR-2 signaling–dependent endothelial cell migration; thus, this inhibitor was observed to block VEGF-A/VEGFR-2 signaling. Interestingly, this inhibitor did not reverse the chemoresistance. When VEGFR-2 was knocked out in EA.hy926 cells using the CRISPR–Cas9 system, the cytarabine-induced apoptosis of AML cells did not significantly change compared with that of wild-type cells. Thus, coculture-induced chemoresistance appears to be independent of VEGF-A/VEGFR-2 signaling. When the transwell, a coculturing device, separated the AML cells from the EA.hy926 cells in a monolayer, the coculture-induced chemoresistance was inhibited. Given that the migration of VEGF-A/VEGFR-2 signaling–dependent endothelial cells is necessary for the endothelial niche formation in the bone marrow, VEGF-A/VEGFR-2 signaling contributes to chemoresistance by mediating the niche formation process, but not to the chemoresistance of AML cells in the niche. • Coculture with endothelial cells inhibits the drug-induced apoptosis of AML cells. • The chemoresistance is not reversed by the inhibition or knockout of VEGFR-2. • The VEGFR-2 pathway is dispensable for endothelial cells-induced AML chemoresistance. [ABSTRACT FROM AUTHOR]

The in vitro antibacterial effect of a synthetic new peptide NJP9-A originated from natural marine invertebrates and its cytotoxicity, selectivity and one possible mechanism of action on human leukemia cells were investigated. NJP9-A showed significant bacteriostasis against Staphylococcus aureus (including methicillin-resistant S. aureus), and had a very low hemolysis efficiency on the human red blood cells. NJP9-A selectively bound to and inhibited the proliferation of leukemia cells, but had almost no inhibitory effect on the proliferation of HEK293 and HUVEC cells. The underlying mechanism of NJP9-A's anti-leukemia cell effect may involve changing the expression of PIK3R1 (PI3K p85 regulatory subunit 1), and the corresponding PI3K signal transduction pathway. NJP9-A inhibits Gram-positive bacteria and is cytotoxic to leukemia cells. The underlying mechanism of NJP9-A's anti-leukemia cell effect is still unclear, but it may be related to the alteration of PIK3R1 expression. [ABSTRACT FROM AUTHOR]

To explore the molecular mechanisms of action underlying the antileukemia activities of darinaparsin, an organic arsenical approved for the treatment of peripheral T–cell lymphoma in Japan, cytotoxicity of darinaparsin was evaluated in leukemia cell lines NB4, U-937, MOLT-4 and HL-60. Darinaparsin was a more potent cytotoxic than sodium arsenite, and induced apoptosis/necrosis in NB4 and HL-60 cells. In NB4 cells exhibiting the highest susceptibility to darinaparsin, apoptosis induction was accompanied by the activation of caspase-8/-9/-3, a substantial decrease in Bid expression, and was suppressed by Boc-D-FMK, a pancaspase inhibitor, suggesting that darinaparsin triggered a convergence of the extrinsic and intrinsic pathways of apoptosis via Bid truncation. A dramatic increase in the expression level of γH2AX, a DNA damage marker, occurred in parallel with G2/M arrest. Activation of p53 and the inhibition of cdc25C/cyclin B1/cdc2 were concomitantly observed in treated cells. Downregulation of c-Myc, along with inactivation of E2F1 associated with the activation of Rb, was observed, suggesting the critical roles of p53 and c-Myc in darinaparsin-mediated G2/M arrest. Trolox, an antioxidative reagent, suppressed the apoptosis induction but failed to correct G2/M arrest, suggesting that oxidative stress primarily contributed to apoptosis induction. Suppression of Notch1 signaling was also confirmed. Our findings provide novel insights into molecular mechanisms underlying the cytotoxicity of darinaparsin and strong rationale for its new clinical application for patients with different types of cancer.

Leukemia is a malignant tumor with high heterogeneity and a complex evolutionary process. It is difficult to resolve the heterogeneity and clonal evolution of leukemia cells by applying traditional bulk sequencing techniques, thus preventing a deep understanding of the mechanisms of leukemia development and the identification of potential therapeutic targets. However, with the development and application of single-cell sequencing technology, it is now possible to investigate the gene expression profile, mutations, and epigenetic features of leukemia at the single-cell level, thus providing a new perspective for leukemia research. In this article, we review the recent applications and advances of single-cell sequencing technology in leukemia research, discuss its potential for enhancing our understanding of the mechanisms of leukemia development, discovering therapeutic targets and personalized treatment, and provide reference guidelines for the significance of this technology in clinical research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Xie, An, Yang, Wang, Guo, Luo, Huang and Sun.)

Efforts to overcome multidrug resistance in cancer have led to the development of several novel strategies including photodynamic therapy (PDT). PDT is based on the use of photosensitizers (PSs) photoactivation, which causes the formation of reactive oxygen species that can induce cell death. In the last decade, the development of new PSs has been significantly accelerated. Recently, acridine-3,6-dialkyldithiourea hydrochlorides (AcrDTUs) have been investigated as a new group of PSs and we have shown that PDT/AcrDTUs caused cell death of mouse leukemic cells L1210. In this study, we investigated the efficacy of PDT/AcrDTUs for the treatment of L1210/VCR cells as a model of chemoresistant cells (overexpressing P-glycoprotein, P-gp). The photoactivation (365 nm, 1.05 J/cm2) increased the cytotoxicity of AcrDTUs 10-15 times. Inhibition of P-gp (verapamil) has been shown to have no significant effect on the accumulation of propyl-AcrDTU (the most potent derivative) in L1210/VCR cells. The intracellular distribution of this acridine derivative has been studied. Prior to irradiation of the resistant cells, propyl-AcrDTU was sequestered mainly in the cytosol, partly in the mitochondria, and, unlike in the sensitive cells, the AcrDTU was not found in the lysosomes. PDT with 1 µM propyl-AcrDTU induced cell shrinkage and "ladder DNA" formation, and although a drastic decrease of the intracellular ATP level was observed at the same time, there was no increase in extracellular LDH activity. AIF in the nucleus can induce DNA fragmentation and we have actually observed a mitochondrio-nuclear translocation of AIF. We concluded that AcrDTUs are photocytotoxic against L1210/VCR cells and that mitochondria play an important role in cell death induced by PDT. [ABSTRACT FROM AUTHOR]

Myricitrin, a naturally occurring flavonoid in Madhuca longifolia, possesses several medicinal properties. Even though our earlier work revealed its role against the proliferation of acute myelogenous leukemia cells (HL-60), its molecular mechanisms have not yet been revealed. The current study aims to explore the molecular mechanisms of myricitrin (isolated from an ethnomedicinal drug Madhuca longifolia) to induce apoptosis in HL-60 cells. Treatment with IC-50 dose of myricitrin (353 µM) caused cellular shrinkage and cell wall damage in HL-60 cells compared to untreated control cells. Myricitrin treatment reduced the mitochondrial membrane potential (22.95%), increased DNA fragmentation (90.4%), inhibited the cell survival proteins (RAS, B-RAF, & BCL-2) and also induced pro-apoptotic proteins (p38, pro-caspase-3, pro-caspase-9 and caspase-3) in the HL-60 cells. The present study provides scientific evidence for the apoptosis caused by myricitrin in HL-60 leukemia cells. Hence, the phytochemical myricitrin could be considered as a potential candidate to develop an anticancer drug after checking its efficacy through suitable pre-clinical and clinical studies. [ABSTRACT FROM AUTHOR]

Spin probes can be used to monitor biological membranes, including the penetration of different molecules into cells. The aim of the present studies was an investigation of the endocytosis process of two spin labels—2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) and 4-hydroxy-TEMPO (TEMPOL)—into yeast cells and a leukemia cell line (HL-60, ATCC CCL-240) by Electron Spin Resonance (ESR). The ESR method is helpful for the direct detection of free radicals. The cell incubation and endocytosis of spin probes were carried out at 310 K. In contrast, the ESR measurements of yeast cells and a leukemia cell line with spin probes were at 240 K. Spectral differentiation was observed; hence, the spin probes present in suspension and attached to the cell membrane could be distinguished. The ESR signal changes of spin probes depended on spin probe concentration, cell number, and type of cell (healthy/cancerous). Additionally, the effect of external factors (oxygen and vitamin C) on the ESR signal decay of spin markers in the cell solution was established. The experimental results prove that the spin probes (TEMPO and TEMPOL) could scavenge free radicals inside the cell. At the same time, the mechanism of spin probe interaction in suspension was determined based on the measurements at low temperatures.

Hydroxytyrosol (HT), the main representative of polyphenols of olive oil, has been described as one of the most powerful natural antioxidants, also showing anti-inflammatory, antimicrobial, cardioprotective and anticancer activity in different type of cancers, but has been little studied in hematological neoplasms. The objective of this work was to evaluate the anticancer potential of HT in acute human leukemia T cells (Jurkat and HL60) and the anti-inflammatory potential in murine macrophages (Raw264.7). For this, cytotoxicity tests were performed for HT, showing IC50 values, at 24 h, for Jurkat, HL60 and Raw264.7 cells, of 27.3 µg·mL−1, 109.8 µg·mL−1 and 45.7 µg·mL−1, respectively. At the same time, HT caused cell arrest in G0/G1 phase in both Jurkat and HL60 cells by increasing G0/G1 phase and significantly decreasing S phase. Apoptosis and cell cycle assays revealed an antiproliferative effect of HT, decreasing the percentage of dividing cells and increasing apoptosis. Furthermore, HT inhibited the PI3K signaling pathway and, consequently, the MAPK pathway was activated. Inflammation tests revealed that HT acts as an anti-inflammatory agent, reducing NO levels in Raw264.7 cells previously stimulated by lipopolysaccharide (LPS). These processes were confirmed by the changes in the expression of the main markers of inflammation and cancer. In conclusion, HT has an anticancer and anti-inflammatory effect in the cell lines studied, which were Raw264.7, Jurkat, and HL60, and could be used as a natural drug in the treatment of liquid cancers, leukemias, myelomas and lymphomas.

Although previous studies have shown anti-cancer activity of betulinic acid (BA), a pentacyclic triterpenoid, against various cancer lines, the underlying molecular mechanisms are not well elucidated. In this study, we evaluated the mechanisms involved in the anti-cancer efficacy of BA in U937 human myeloid leukemia cells. BA exerted a significant cytotoxic effect on U937 cells through blocking cell cycle arrest at the G2/M phase and inducing apoptosis, and that the intracellular reactive oxygen species (ROS) levels increased after treatment with BA. The down-regulation of cyclin A and cyclin B1, and up-regulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1 revealed the G2/M phase arrest mechanism of BA. In addition, BA induced the cytosolic release of cytochrome c by reducing the mitochondrial membrane potential with an increasing Bax/Bcl-2 expression ratio. BA also increased the activity of caspase-9 and -3, and subsequent degradation of the poly (ADP-ribose) polymerase. However, quenching of ROS by N-acetyl-cysteine, an ROS scavenger, markedly abolished BA-induced G2/M arrest and apoptosis, indicating that the generation of ROS plays a key role in inhibiting the proliferation of U937 cells by BA treatment. Taken together, our results provide a mechanistic rationale that BA exhibits anti-cancer properties in U937 leukemia cells through ROS-dependent induction of cell cycle arrest at G2/M phase and apoptosis. [ABSTRACT FROM AUTHOR]

Many quinazoline derivatives with pharmacological properties, such as anticancer activity, have been synthesized. Fourteen quinazoline derivatives bearing a substituted sulfonamide moiety (4a–n) were previously synthesized and fully characterized. These compounds exerted antiproliferative activity against cell lines derived from solid tumors. Herein, the antileukemic activities of these compounds (4a–n) against two different leukemia cell lines (Jurkat acute T cell and THP-1 acute monocytic) were investigated. Our investigation included examining their activity in vivo in a zebrafish embryo model. Remarkably, compounds 4a and 4d were the most potent in suppressing cell proliferation, with an IC50 value range of 4–6.5 µM. Flow cytometry analysis indicated that both compounds halted cell progression at the G2/M phase and induced apoptosis in a dose-dependent manner. RT-PCR and Western blot analyses also showed that both compounds effectively induced apoptosis by upregulating the expression of proapoptotic factors while downregulating that of antiapoptotic factors. In vivo animal toxicity assays performed in zebrafish embryos indicated that compound 4d was more toxic than compound 4a, with compound 4d inducing multiple levels of teratogenic phenotypes in zebrafish embryos at a sublethal concentration. Moreover, both compounds perturbed the hematopoiesis process in developing zebrafish embryos. Collectively, our data suggest that compounds 4a and 4d have the potential to be used as antileukemic agents.

Silibinin is the main biologically active component of silymarin extract and consists of a mixture 1:1 of two diastereoisomeric flavonolignans, namely silybin A (1a) and silybin B (1b), which we call here silybins. Despite the high interest in the activity of this flavonolignan, there are still few studies that give due attention to the role of its stereochemistry and, there is still today a strong need to investigate in this area. In this regard, here we report a study concerning the radical scavenger ability and the antiproliferative activity on different cell lines, both of silybins and phosphodiester-linked silybin dimers. An efficient synthetic strategy to obtain silybin dimers in an optical pure form (6aa, 6ab and 6bb) starting from a suitable building block of silybin A and silybin B, obtained by us from natural extract silibinin, was proposed. New dimers show strong antioxidant properties, determined through hydroxyl radical (HO●) scavenging ability, comparable to the value reported for known potent antioxidants such as quercetin. A preliminary screening was performed by treating cells with 10 and 50 μM concentrations for 48 h to identify the most sensitive cell lines. The results show that silibinin compounds were active on Jurkat, A375, WM266, and HeLa, but at the tested concentrations, they did not interfere with the growth of PANC, MCF-7, HDF or U87. In particular, both monomers (1a and 1b) and dimers (6aa, 6ab and 6bb) present selective anti-proliferative activity towards leukemia cells in the mid-micromolar range and are poorly active on normal cells. They exhibit different mechanisms of action in fact all the cells treated with the 1a and 1b go completely into apoptosis, whereas only part of the cells treated with 6aa and 6ab were found to be in apoptosis.

Fentanyl is a common sedative/analgesic used for intrathecal chemotherapy injection in children with acute leukemia. Given the contradictory findings that fentanyl has both inhibitory and stimulatory activities in cancer cells, we investigated the biological effects of fentanyl alone and its combination with standard of care in acute myeloid leukemia (AML) cells at all stages of development. We showed that fentanyl at clinically relevant concentration inhibited growth and colony formation of AML differentiated cells and committed progenitors without affecting their survival. Compared to AML cells without FLT3 mutation, cells harboring FLT3‐ITD mutation are likely to be more sensitive to fentanyl. However, fentanyl did not affect the most primitive AML stem cells. Fentanyl significantly augmented the efficacy of cytarabine but not midostaurin in AML differentiated cells and committed progenitors. We further demonstrated that fentanyl inhibited AML cells via suppressing Ras/Raf/MEK/ERK and STAT5 pathway, and this was not dependent on opioid receptor system. Our findings demonstrate the anti‐leukemia activity of fentanyl and synergistic effects between fentanyl and cytarabine in AML, via opioid receptor‐independent suppression of Ras and STAT5 pathways. Our work is the first to suggest the beneficial effects of fentanyl in children with leukemia. [ABSTRACT FROM AUTHOR]

A new Zn(II)-containing coordination complex [ZnBrL] (1) based on the Schiff base ligand(E)-1-(((3-(piperidin-1-yl)propylidene)amino)methyl)naphthalen-2-ol (HL) has been successfully prepared using a slow volatilization method. A mechanical grinding method was applied for preparing the crystalline complex 1 in the nano-region, which was studied by both of the scanning electron microscopy and transmission electron microscopy. In addition, its inhibitory effect on ARH-77 human leukemia cells were evaluated. First, the Cell Counting Kit-8 detection kit was used to detect the influence of nano 1 on the cell viability. Next, the cell colony formation assay was conducted for the determination of the cell proliferation. The trans-well assay was performed to explore the migration and invasion of the cells after nano 1 treatment. [ABSTRACT FROM AUTHOR]

Synthesis of molecular hybrids, obtained by combination of two or more pharmacophoric groups of different bioactive substances in order to produce more efficient drugs, is now a frequently used approach in medicinal chemistry. Following this strategy, we synthetized a library of 3-methylidene-1-tosyl-2,3-dihydro-1,8-naphthyridin-4(1H)-ones, combining a 1,8-naphthyridin-4-one motif with an exo-methylidene bond conjugated with a carbonyl group, pharmacophoric units that are present in many natural, biologically active compounds with anticancer potential. We reasoned that such bifunctional conjugates may have enhanced cytotoxic activity. The title compounds were synthesized in a four step reaction sequence. β-Ketophosphonate, obtained from methyl N-tosylnicotinate and diethyl methylphosphonate, was reacted with various aldehydes giving 3-diethoxyphosphoryl-2,3-dihydro-1,8-naphthyridin-4(1H)-ones as keto-enol tautomers. Later, these compounds were transformed into 3-methylidene-1-tosyl-2,3-dihydro-1,8-naphthyridin-4(1H)-ones applying the Horner-Wadsworth-Emmons methodology. Then, the cytotoxicity of the new compounds was assessed on two cancer cell lines, promyelocytic leukemia HL-60 and breast cancer adenocarcinoma MCF-7, and for comparison, on human umbilical vein endothelial cells HUVEC. The most active and selective analog, 2-ethyl-3-methylidene-1-tosyl-2,3-dihydro-1,8-naphthyridin-4(1H)-one 4 a was chosen for more detailed studies on HL-60 cell line, to determine molecular mechanisms of its anticancer activity. It was shown that 4 a strongly inhibited proliferation and induced apoptosis which could be attributed to its ability to cause DNA damage., (© 2023 Wiley‐VCH GmbH.)

The studies concerned the expression of sulfurtransferases and cystathionine beta-synthase in six human leukemia cell lines: B cell acute lymphoblastic leukemia-B-ALL (REH cells), T cell acute lymphoblastic leukemia-T-ALL (DND-41 and MOLT-4 cells), acute myeloid leukemia—AML (MV4-11 and MOLM-14 cells), and chronic myeloid leukemia—CML (K562 cells). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were performed to determine the expression of thiosulfate sulfurtransferase, 3-mercaptopyruvate sulfurtransferase, gamma-cystathionase, and cystathionine beta-synthase on the mRNA and protein level. Interestingly, we found significant differences in the mRNA and protein levels of sulfurtransferases and cystathionine beta-synthase in the studied leukemia cells. The obtained results may contribute to elucidating the significance of the differences between the studied cells in the field of sulfur compound metabolism and finding new promising ways to inhibit the proliferation of various types of leukemic cells by modulating the activity of sulfurtransferases, cystathionine beta-synthase, and, consequently, the change of intracellular level of sulfane sulfur as well as H2S and reactive oxygen species production.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a highly selective and promising anticancer agent due to its specific apoptosis-inducing effect on tumor cells, rather than most normal cells. TRAIL is currently under investigation for use in the treatment of leukemia. However, the resistance of leukemic cells to TRAIL-induced apoptosis may limit its efficacy. The mechanisms of leukemic cell resistance to antitumor immunity remains a topical issue. In this work, we have found an increase in the resistance to TRAIL-induced cell death in human leukemia THP-1 cells, which was caused by differentiation into a macrophage-like phenotype in high-density culture in vitro. Stressful conditions, manifested by the inhibition of cell growth and the activation of cell death in high-density culture of THP-1 cells, induced the appearance of cells adhered to culture dishes. The THP-1ad cell line was derived by selection of these adhered cells. The genetic study, using STR and aCGH assays, has shown that THP-1ad cells were derived from THP-1 cells due to mutagenesis. The THP-1ad cells possessed high proliferative potential and a macrophage-like immunophenotype. The adhesion of THP-1ad cells to the extracellular matrix was mediated by αVβ5 integrin. The cytokine production, as well as the rise of intracellular ROS and NO activities by LPS in THP-1ad cell culture, were characteristic of macrophage-like cells. The THP-1ad cells were found to appear to increase in resistance to TRAIL-induced cell death in comparison with THP-1 cells. The mechanism of the increase in TRAIL-resistance can be related to a decrease in the expression of death receptors DR4 and DR5 on the THP-1ad cells. Thus, the macrophage-like phenotype formation with the maintenance of a high proliferative potential of leukemic cells, caused by stress conditions in high-density cell cultures in vitro, can induce an increase in resistance to TRAIL-induced cell death due to the loss of DR4 and DR5 receptors. The possible realization of these events in vivo may be the reason for tumor progression.

BACKGROUND: Pinellia ternata can be cultivated artificially and is easy to purify. Its extract can inhibit the cell proliferation of chronic myeloid leukemia, acute myelogenous leukemia M3, and T-cell acute lymphoblastic leukemia, but the underlying mechanism remains unclear. OBJECTIVE: To explore the mechanism of action of pinellia ternata extract on apoptosis of three kinds of leukemia cells. METHODS: Chronic myeloid leukemia cell lines (K562), acute myeloid leukemia–M3 cell line (HL-60), acute T lymphocyte leukemia cell line (C8166) were incubated in five kinds of concentration in pinellia ternata extracts in vitro for 24, 48, and 72 hours, respectively. Cell counting kit-8 assay was used to assess the influence of three kinds of leukemia cell proliferation to screen the optimal inhibitory effect of moderate (300 mg/L) and high (500 mg/L) concentration. Flow cytometry was used to determine the incidence of early apoptosis of three kinds of leukemia cells. The mRNA and protein expression levels of Bax, Bcl-2 and Caspase-3 were detected by RT-PCR and western blot assay. RESULTS AND CONCLUSION: Cell counting kit-8 assay results showed that five different concentrations of pinellia extracts could inhibit the proliferation of three kinds of leukemia cells. The inhibition rates of medium concentration (300 mg/L) and high concentration (500 mg/L) were significantly higher than those of low concentration (100 mg/L) and control group (P < 0.01). Flow cytometry (Annexin PE/7AAD and Annexin V/PI) double staining showed that moderate and high concentrations of pinellia extract could induce early apoptosis of three kinds of leukemia cells. RT-PCR results revealed moderate and high concentrations of pinellia extract could down-regulate the mRNA expression level of Bcl-2, and up-regulate the mRNA expression levels of Bax, Bcl-2, Caspase-3 (P < 0.05, P < 0.01). Western blot assay results showed that the Bcl-2 protein bands of K562 and C8166 cells were significantly weakened after 72 hours of incubation with moderate and high concentrations of pinellia tubulin extract, while no significant changes were observed in HL-60 cells. Bax and caspase-3 protein bands were significantly enhanced. In summary, pinellia ternata extract can effectively inhibit the proliferation of myeloid leukemia and lymphocytic leukemia cells and promote their apoptosis via regulating Bax/Bcl-2 and Caspase-3 protein expression. [ABSTRACT FROM AUTHOR]

The chemical investigation of the leaves and stem bark of Acacia polyacantha (Fabaceae) led to the isolation of a new oleanane-type triterpenoid saponin named polyacanthoside A 1 together with fifteen known compounds. Their structures were established from spectral , mainly HRESIMS, 1D NMR and 2D NMR and by comparison with literature data. The cytotoxicity of compound 1 and the analogues 8 as well as doxorubicin was determined in a panel of 9 cancer cell lines including sensitive and drug resistant phenotypes. Unlike the analogue 8, compound 1 as well as doxorubicin displayed cytotoxic effects in all the 9 tested cancer cell lines with IC50 values ranged from 8.90 μM (towards CCRF-CEM leukemia cells) to 35.21 μM (towards HepG2 hepatocarcinoma cells) for compound 1 and from 0.02 μM (towards CCRF-CEM leukemia cells) to 66.83 μM (against CEM/ADR5000 leukemia cells) for doxorubicin. [ABSTRACT FROM AUTHOR]

Several kinds of solvents have been applied to Nepenthes extractions exhibiting antioxidant and anticancer effects. However, they were rarely investigated for Nepenthes ethyl acetate extract (EANT), especially leukemia cells. The purpose of the present study was to evaluate the antioxidant properties and explore the antiproliferation impact and mechanism of EANT in leukemia cells. Five standard assays demonstrated that EANT exhibits antioxidant capability. In the cell line model, EANT dose-responsively inhibited cell viabilities of three leukemia cell lines (HL-60, K-562, and MOLT-4) based on 24 h MTS assays, which were reverted by pretreating oxidative stress and apoptosis inhibitors (N-acetylcysteine and Z-VAD-FMK). Due to similar sensitivities among the three cell lines, leukemia HL-60 cells were chosen for exploring antiproliferation mechanisms. EANT caused subG1 and G1 cumulations, triggered annexin V-detected apoptosis, activated apoptotic caspase 3/7 activity, and induced poly ADP-ribose polymerase expression. Moreover, reactive oxygen species, mitochondrial superoxide, and mitochondrial membrane depolarization were generated by EANT, which was reverted by N-acetylcysteine. The antioxidant response to oxidative stress showed that EANT upregulated mRNA expressions for nuclear factor erythroid 2-like 2 (NFE2L2), catalase (CAT), thioredoxin (TXN), heme oxygenase 1 (HMOX1), and NAD(P)H quinone dehydrogenase 1 (NQO1) genes. Moreover, these oxidative stresses led to DNA damage (γH2AX and 8-hydroxy-2-deoxyguanosine) and were alleviated by N-acetylcysteine. Taken together, EANT demonstrated oxidative stress-dependent anti-leukemia ability to HL-60 cells associated with apoptosis and DNA damage.

Ethanol has been shown to exhibit therapeutic properties as an ablative agent alone and in combination with thermal ablation. Ethanol may also increase sensitivity of cancer cells to certain physical and chemical antitumoral agents. The aim of our study was to assess the potential influence of nontoxic concentrations of ethanol on hyperthermia therapy, an antitumoral modality that is continuously growing and that can be combined with classical chemotherapy and radiotherapy to improve their efficiency. Human leukemia cells were included as a model in the study. The results indicated that ethanol augments the cytotoxicity of hyperthermia against U937 and HL60 cells. The therapeutic benefit of the hyperthermia/ethanol combination was associated with an increase in the percentage of apoptotic cells and activation of caspases-3, -8 and -9. Apoptosis triggered either by hyperthermia or hyperthermia/ethanol was almost completely abolished by a caspase-8 specific inhibitor, indicating that this caspase plays a main role in both conditions. The role of caspase-9 in hyperthermia treated cells acquired significance whether ethanol was present during hyperthermia since the alcohol enhanced Bid cleavage, translocation of Bax from cytosol to mitochondria, release of mitochondrial apoptogenic factors, and decreased of the levels of the anti-apoptotic factor myeloid cell leukemia-1 (Mcl-1). The enhancement effect of ethanol on hyperthermia-activated cell death was associated with a reduction in the expression of HSP70, a protein known to interfere in the activation of apoptosis at different stages. Collectively, our findings suggest that ethanol could be useful as an adjuvant in hyperthermia therapy for cancer.

Cephalotaxine (CET) is a natural alkaloid with potent antileukemia effects. However, its underlying molecular mechanism has not been well understood. In this study, we verified that CET significantly inhibited the viability of various leukemia cells, including HL-60, NB4, Jurkat, K562, Raji and MOLT-4. RNA-sequencing and bioinformatics analysis revealed that CET causes mitochondrial function change. Mechanism research indicated that CET activated the mitochondrial apoptosis pathway by reducing the mitochondrial membrane potential, downregulating anti-apoptotic Bcl-2 protein and upregulating pro-apoptotic Bak protein. In addition, the autophagy signaling pathway was highly enriched by RNA-seq analysis. Then, we found that CET blocked the fluorescence colocation of MitoTracker Green and LysoTracker Red and upregulated the level of LC3-II and p62, which indicated that autophagy flow was impaired. Further results demonstrated that CET could impair lysosomal acidification and block autophagy flow. Finally, inhibiting autophagy flow could aggravate apoptosis of HL-60 cells induced by CET. In summary, this study demonstrated that CET exerted antileukemia effects through activation of the mitochondria-dependent pathway and by impairing autophagy flow. Our research provides new insights into the molecular mechanisms of CET in the treatment of leukemia.

We investigated the effect of the modulation of Na/H exchanger 1 (NHE1) on apoptosis, differentiation, and chemoresistance in acute myeloid leukemia (AML) cells to evaluate the possibility of NHE1 modulation as a novel therapeutic strategy for AML. The pHi of leukemia cell lines except KG1a was higher than that of normal bone marrow mononuclear cells (BM MNCs). Notably, in K562, cytarabine (AraC)-resistant OCI-AML2, and primary leukemia cells, pHi was significantly higher than that of normal BM MNCs. Western blotting and real-time quantitative PCR confirmed that the increased NHE1 expression was responsible for the higher pHi. Specifically, compared to CD34+CD38+ leukemia cells, the mean fluorescence intensity of NHE1 was significantly higher in CD34+CD38− leukemic stem cells. The out of range in pHi by treatment with an NHE inhibitor, the amiloride analogue 5-(N,N-hexamethylene) amiloride (HMA), or an NHE activator, phorbol 12-myristate 13-acetate (PMA), resulted in dose- and time-dependent inhibition of leukemia cell proliferation. PMA induced CD14+ differentiation of leukemia cells, whereas HMA induced cell cycle arrest at the G1 phase. HMA could induce apoptosis of leukemia cells even in AraC-resistant cells and showed an additive effect on apoptosis in AraC-sensitive cells. Our result revealed that AML cells prefer more alkalic intracellular moiety than normal BM MNCs following increased NHE1 expression and that NHE1 modulation can induce apoptosis and differentiation of AML cells. These findings imply that NHE1 is a potential target in cytotoxic or differentiation-induction treatment for AML. • Na/H exchanger 1(NHE1) modulation inhibited proliferation of leukemia cells. • PMA, an NHE1 activator, induced CD14+ differentiation of leukemia cells. • HMA, an NHE1 inhibitor, induced cell cycle arrest at G1 phase in leukemia cells. • NHE1 overexpression contributes to cytarabine resistance in OCI-AML2 cells. [ABSTRACT FROM AUTHOR]

Leukemia is a commonly seen disease caused by abnormal differentiation of hematopoietic stem cells and blasting in bone marrow. Despite drugs are used to treat the disease clinically, the influence of these drugs on leukemia cells' biomechanical properties, which are closely related to complications like leukostasis or infiltration, is still unclear. Due to non-adherent and viscoelastic nature of leukemia cells, accurate measurement of their elastic modulus is still a challenging issue. In this study, we adopted rate-jump method together with optical tweezers indentation to accurately measure elastic modulus of leukemia cells K562 after phorbol 12-myristate 13-acetate (PMA), all-trans retinoic acid (ATRA), Cytoxan (CTX), and Dexamethasone (DEX) treatment, respectively. We found that compared to control sample, K562 cells treated by PMA showed nearly a threefold increase in elastic modulus. Transwell experiment results suggested that the K562 cells treated with PMA have the lowest migration capability. Besides, it was shown that the cytoskeleton protein gene α-tubulin and vimentin have a significant increase in expression after PMA treatment by qPCR. The results indicate that PMA has a significant influence on protein expression, stiffness, and migration ability of the leukemia cell K562, and may also play an important role in the leukostasis in leukemia. • Quantitative measurement of K562 cells' elastic moduli using optical tweezer indentation with rate-jump model. • The elastic modulus of K562 cells had a three-fold increase after treated by PMA. • The K562 cells' migration capability decrease with an increasing elastic modulus. [ABSTRACT FROM AUTHOR]

Long‐noncoding RNAs (lncRNAs) is involved in the development of diverse diseases, including leukemia, while the role lncRNA HOX transcript antisense RNA (HOTAIR) played in leukemia remains unclear and in need of further investigation. Therefore, this study was conducted to explore the effects of lncRNA HOTAIR on the immunologic rejection of leukemia cells through the Wnt/β‐catenin in mice. Mice were administrated with HOTAIR mimics as well as small interfering RNA HOTAIR to explore the regulatory role of HOTAIR. The numbers of white blood cell (WBC) and platelet (PLT) and the content of hemoglobin in peripheral blood (PB) were determined. The cytokine level in PB was measured. T‐lymphocyte proliferation activity, Ig production by B cells, natural killer (NK) cell activity, and the proportion of cluster of differentiation 4 (CD4)/CD8 T cell subsets were detected. Expression of HOTAIR, β‐catenin, cyclinD1, GSK‐3β, and c‐Myc in bone marrow was determined. It was revealed that the WBC number increased, while the PLT number along with the hemoglobin content in PB decreased with the upregulated HOTAIR. Additionally, elevated HOTAIR led to decreased levels of transforming growth factor‐β, interferon‐γ, interleukin‐10, and tumor necrosis factor‐α in PB, proliferation activity in T‐lymphocyte, and inhibited Ig production, NK cell activity, and the ratio of CD4/CD8 T cell subsets in B‐lymphocyte. Furthermore, Wnt/β‐catenin was activated by overexpressing HOTAIR. Enhanced survival and proliferation were shown with increased expression of cyclinD1, GSK‐3β, and c‐Myc in the bone marrow of mice induced by overexpressing HOTAIR. These results indicate that restored HOTAIR reduces the immunologic rejection of leukemia cells in mice by activating Wnt/β‐catenin pathway. [ABSTRACT FROM AUTHOR]

Abstract Aim Studies have reported that micro (miR)-486-5p plays a crucial part in the progression of leukemia, however, to the best of our knowledge, few studies have been conducted on its mechanism in leukemia. In this study, the mechanism of miR-486-5p in leukemia cells was pointed out and its possible target genes were analyzed for the purpose of providing new therapeutic strategies for treating leukemia patients. Methods MiRNA expression of Leukemia cells (K562, Kasumi-1, and THP-1) and primary leukocytes was detected by Real-time Quantitative polymerase chain reaction(qPCR). The activity of the cells was assessed using the Cell Counting Kit-8 (CCK-8). Apoptotic cells were analyzed by a flow cytometer (FCM). Caspase-3 activation in leukemia cells was determined by Western blot. Targetscan 7.2 was used to predict the potential targets of miR-486-5p and further confirmed by dual-luciferase reporter assay. Result miR-486-5p was significantly down-regulated in leukemia cells. The over-expression of miR-486-5p notably increased the apoptosis and caspase-3 activity in leukemia cells. There was a predicted interaction site for miR-486-5p in the FOXO1 3′-UTR. Furthermore, this study showed that FOXO1 was significantly up-regulated in leukemia cells, the growth of which was depressed by the up-regulation of miR-486-5p. Conclusion miR-486-5p may inhibit the proliferation of leukemia cells and induce apoptosis through targeting FOXO1. Highlights • The relationship between miR-486-5p and human leukemia cells. • The related targets of miR-486-5p in human leukemia cells. • The action of FOXO1 on leukemia cells. [ABSTRACT FROM AUTHOR]

Highlights • Brightfield optical microscope images acquired by 20× and 40× objectives processed. • No cell staining and no fluorescent labeling. • Machine learning methods with color and size based object detection methods incorporated. • Neural Network, Random Forest and Support Vector Machine methods compared. • Detection and quantification of leukemia cells, micron size immunomagnetic beads and bead clusters performed on the images. Abstract Quantification of tumor cells is crucial for early detection and monitoring the progress of cancer. Several methods have been developed for detecting tumor cells. However, automated quantification of cells in the presence of immunomagnetic beads has not been studied. In this study, we developed computer vision based algorithms to quantify the leukemia cells captured and separated by micron size immunomagnetic beads. Color, size based object identification and machine learning based methods were implemented to quantify targets in the images recorded by a bright field microscope. Images acquired by a 40× or a 20× objective were analyzed, the immunomagnetic beads were detected with an error rate of 0.0171 and 0.0384 respectively. Our results reveal that the proposed method attains 91.6% precision for the 40× objective and 79.7% for the 20× objective. This algorithm has the potential to be the signal readout mechanism of a biochip for cell detection. [ABSTRACT FROM AUTHOR]

Leukemia, a highly malignant form of cancer, relies on early diagnosis for effective treatment and patient survival. Currently, leukemia is diagnosed by manually identifying blood cells through recognizing morphological features using staining techniques. In this work, we achieve rapid, accurate, label-free identification and categorization of four important leukemia cell lines to a subclass level using Raman spectroscopy and an identification model. The flat gold film-covered glass substrates ensure cell intact, good morphology and high-quality Raman spectra. Although the shapes of unstained erythrocytes, K562, U937 and Raji B cells are similar, their spectra are different. The sensitivity, the specificity and the accuracy of the unsupervised identification model (PCA-KMCA) of these four cell lines were 93.75%, 96.67%, and 97.50%, respectively. The accuracy of this model is higher than that of the supervised identification model (LDA-KNN) with an accuracy of 90.62%. Further, the unsupervised identification (PCA-KMCA) of Raji B and Jurkat cells could be greatly achieved, with sensitivity of 96.25%, specificity of 97.50% and accuracy of 96.88%. This method has great potential in the early diagnosis of leukemia, which can help doctors determine treatment plans faster and improve the prognosis of patients. • An unstained method is crucial for the rapid and accurate early diagnosis of diseases. • Different leukemia cell lines were differentiated at the protein level. • Unsupervised classification used Raman spectra to differentiate leukemia cell lines. • Raman spectra differentiated ALL subclasses via unsupervised classification. [ABSTRACT FROM AUTHOR]