The Influence of N-Acetyl-selenomethionine on Two RONS-Generating Cancer Cell Lines Compared to N-Acetyl-methionine.

N-acetyl-selenomethionine (NASeLM), a representative of the selenium compounds, failed to convince in clinical studies and cell cultures that it neither inhibits cancer growth nor has a chemoprotective effect. This study aims to find out whether NASeLM shows a growth-inhibiting property compared to the carrier substance N-Acetyl-L-methionine (NALM) on two different cancer cells, namely Jurkat cells and MTC-SK cells. METHODS: Jurkat and MTC-SK cells were cultured in the absence or presence of varying concentrations (0-500 µg/mL) of NASeLM and NALM solutions. After 0, 24, 48, and 72 h, mitochondrial activity, cancer cell membrane CP levels, cell growth, and caspase-3 activity were assessed in aliquots of Jurkat and MTC-SK cells. RESULTS: Both substances, NASeLM and NALM, were similarly able to inhibit cell growth and mitochondrial activity of Jurkat cells in a concentration-dependent and time-dependent manner up to 70%. Only the determination of caspase activity showed that only NASeLM was able to increase this to almost 40% compared to the control as well as the same lack of NALM. However, the experiments on MTC-SK cells showed a clear difference in favor of NASeLM compared to NALM. While NASeLM was able to reduce cell growth to up to 55%, the same amount of NALM was only at around 15%, which turned out to be highly significant (p < 0.001). The same could also be measured for the reduction in MTC-SK mitochondrial activity. Time dependence could also be recognized: the longer both substances, NASeLM and NALM, were incubated, the higher the effect on cell growth and mitochondrial activity, in favour of NASeLM. Only NASeLM was able to increase caspase-3 activity in MTC-SK cells: at 250 µg/mL NASeLM, caspase-3 activity increased significantly to 28% after 24 and 48 h compared to the control (14%) or the same NALM concentration (14%). After 72 h, this could still increase to 37%. A further increase in the NASeLM concentration did not result in higher caspase-3 activity. CONCLUSION: NASeLM could clearly increase caspase-3 activity in both cell types, Jurkat or MTC-SK cells, and thus induce cell death. NALM and NASeLM showed a reduction in cell growth and mitochondrial activity in both cell lines: While NALM and NASeLM showed almost identical measurements on Jurkat cells, NASeLM was much more effective on MTC-SK than the non-selenium-containing carrier, indicating that it has additional anti-chemoprotective effects.

Phytochemical study of Magonia pubescens A. St.-Hil. and cytotoxicity of branches aqueous extract on breast cancer and leukemia cells.

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).

Jozimine A2, a Dimeric Naphthylisoquinoline (NIQ) Alkaloid, Shows In Vitro Cytotoxic Effects against Leukemia Cells through NF-κB Inhibition.

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.

Indirubin, an Active Component of Indigo Naturalis, Exhibits Inhibitory Effects on Leukemia Cells via Targeting HSP90AA1 and PI3K/Akt Pathway.

BACKGROUND: This research intended to predict the active ingredients and key target genes of Indigo Naturalis in treating human chronic myeloid leukemia (CML) using network pharmacology and conduct the invitro verification. METHODS: The active components of Indigo Naturalis and the corresponding targets and leukemia-associated genes were gathered through public databases. The core targets and pathways of Indigo Naturalis were predicted through protein-protein interaction (PPI) network, gene ontology (GO) function, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Next, after intersecting with leukemia-related genes, the direct core target gene of Indigo Naturalis active components was identified. Subsequently, HL-60 cells were stimulated with indirubin (IND) and then examined for cell proliferation using CCK-8 assay and cell cycle, cell apoptosis, and mitochondrial membrane potential using flow cytometry. The content of apoptosis-associated proteins (Cleaved Caspase 9, Cleaved Caspase 7, Cleaved Caspase 3, and Cleaved parp) were detected using Western blot, HSP90AA1 protein, and PI3K/Akt signaling (PI3K, p-PI3K, Akt, and p-Akt) within HL-60 cells. RESULTS: A total of 9 active components of Indigo Naturalis were screened. The top 10 core target genes (TNF, PTGS2, RELA, MAPK14, IFNG, PPARG, NOS2, IKBKB, HSP90AA1, and NOS3) of Indigo Naturalis active components within the PPI network were identified. According to the KEGG enrichment analysis, these targets were associated with leukemia-related pathways (such as acute myeloid leukemia and CML). After intersecting with leukemia-related genes, it was found that IND participated in the most pairs of target information and was at the core of the target network; HSP90AA1 was the direct core gene of IND. Furthermore, the in-vitro cell experiments verified that IND could inhibit the proliferation, elicit G2/M-phase cell cycle arrest, enhance the apoptosis of HL-60 cells, reduce mitochondrial membrane potential, and promote apoptosis-related protein levels. Under IND treatment, HSP90AA1 overexpression notably promoted cell proliferation and inhibited apoptosis. Additionally, IND exerted tumor suppressor effects on leukemia cells by inhibiting HSP90AA1 expression. CONCLUSION: IND, an active component of Indigo Naturalis, could inhibit CML progression, which may be achieved via inhibiting HSP90AA1 and PI3K/Akt signaling expression levels.

Selection of reference genes in liproxstatin-1-treated K562 Leukemia cells via RT-qPCR and RNA sequencing.

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, ß-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.

Synthesis And Anticancer Activity Of New Hybrid 3-Methylidene-2,3-Dihydro-1,8-Naphthyridinones.

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. ß-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.

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

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

CADM1 impairs the effect of miR-1246 on promoting cell cycle progression in chemo-resistant leukemia cells.

The interruption of normal cell cycle execution acts as an important part to the development of leukemia. It was reported that microRNAs (miRNAs) were closely related to tumorigenesis and progression, and their aberrant expression had been demonstrated to play a crucial role in numerous types of cancer. Our previous study showed that miR-1246 was preferentially overexpressed in chemo-resistant leukemia cell lines, and participated in process of cell cycle progression and multidrug resistant regulation. However, the underlying mechanism remains unclear. In present study, bioinformatics prediction and dual luciferase reporter assay indicated that CADM1 was a direct target of miR-1246. Evidently decreased expression of CADM1 was observed in relapsed primary leukemia patients and chemo-resistant cell lines. Our results furtherly proved that inhibition of miR-1246 could significantly enhance drug sensitivity to Adriamycin (ADM), induce cell cycle arrest at G0/G1 phase, promote cell apoptosis, and relieve its suppression on CADM1 in K562/ADM and HL-60/RS cells. Interference with CADM1 could reduce the increased drug sensitivity induced by miR-1246 inhibition, and notably restore drug resistance by promoting cell cycle progression and cell survival via regulating CDKs/Cyclins complexes in chemo-resistant leukemia cells. Above all, our results demonstrated that CADM1 attenuated the role of miR-1246 in promoting cell cycle progression and cell survival, thus influencing multidrug resistance within chemo-resistant leukemia cells via CDKs/Cyclins. Higher expression of miR-1246 and lower expression of CADM1 might be risk factors for leukemia.

[Effect of Sunitinib on Pyroptosis of Drug-Resistant Leukemia K562/ADR Cells and Its Pathway].

OBJECTIVE: To investigate the inducing effect of sunitinib on the death of drug-resistant leukemia K562/ADR cells and the related signaling pathway. METHODS: K562/ADR cells were treated with different concentrations of sunitinib, and the cells were collected at 24, 48, 72, and 96 hours, respectively. MTS assay was used to detect the effect of sunitinib on the proliferation of K562/ADR cells, and the appropriate sunitinib intervention time and concentration were determined. QPCR and Western blot were used to detect the mRNA and protein expression levels of apoptosis-related genes in K562/ADR cells treated with sunitinib. Four different cell death inhibitors Nec-1, VX-765, CQ and Fer-1 were used to detect the death mode of K562/ADR cells treated with sunitinib. QPCR and Western blot were used to detect the mRNA and protein expression levels of pyroptosis-related genes in K562/ADR cells treated with sunitinib. RESULTS: Sunitinib significantly inhibited the proliferation of K562/ADR cells in a time - and concentration-dependent manner(R48 H=0.9579, r4 µg/ml=0.9740). The IC50 of sunitinib was (3.96±0.14) µg/ml at 48 hours. The mRNA and protein expression levels of apoptosis-related genes Bax, BCL-2 , Caspase-3 and Caspase-9 in K562/ADR cells treated with sunitinib did not change significantly. After treatment with four different cell death inhibitors, only the pyroptosis inhibitor VX-765 could significantly reverse the inhibitory effect of sunitinib on the proliferation of K562/ADR cells (P<0.01). The mRNA and protein expression levels of pyroptosis-related genes Caspase-1, Caspase-4, Caspase-5, NLRP3, GSDMD and IL-1ß in K562/ADR cells treated with sunitinib were significantly increased (P<0.01). CONCLUSION: Sunitinib can induce pyroptosis in drug-resistant leukemia K562/ADR cells. Further study of the signaling pathways related to pyroptosis may provide experimental basis for the treatment of drug-resistant leukemia.

The Mechanism of Anti-Tumor Activity of 6-Morpholino- and 6-Amino-9-Sulfonylpurine Derivatives on Human Leukemia Cells.

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.

Cytotoxicity of alkaline serine protease (ASPNJ) on Jurkat cells and its correlation with changes in the expression of membrane-associated proteins.

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.

Recognition of Nucleophosmin Mutant Gene Expression of Leukemia Cells Using Raman Spectroscopy.

As a label-free, nondestructive, and in situ detection method, Raman spectroscopy analysis of single cells has potential application value in biomedical fields such as cancer diagnosis. In this study, the Raman spectral characteristics of nucleophosmin (NPM1)-mutant acute myeloid leukemia (AML) cells and nonmutated AML cells were investigated, and the reasons for the differences in spectral peaks were explained in combination with transcriptomic analysis. Raman spectra of two AML cell lines without NPM1 mutation (THP-1 and HL-60) and the OCI-AML3 cell line carrying the NPM1 mutant gene were cultured and collected experimentally. It was found that the average Raman spectra of NPM1 mutant and nonmutated cells had intensity differences in multiple peaks corresponding to chondroitin sulfate (CS), nucleic acid, protein, and other molecules. The differentially expressed genes were identified by quantitative analysis of the gene expression matrix of the two types of cells, and their roles in the regulation of CS proteoglycan and protein synthesis were analyzed. The results showed that the differences between the two types of cells expressed by the single-cell Raman spectral information were consistent with the differences in transcriptional profiles. This research could advance the application of Raman spectroscopy in cancer cell typing.

Methyl-Thiol-Bridged Oxadiazole and Triazole Heterocycles as Inhibitors of NF-κB in Chronic Myelogenous Leukemia Cells.

Nuclear factor kappa beta (NF-κB) is a transcriptional factor that plays a crucial role in regulating cancer cell proliferation. Therefore, the inhibition of NF-κB activity by small molecules may be beneficial in cancer therapy. In this report, methyl-thiol-bridged oxadiazole and triazole heterocycles were synthesized via click chemistry and it was observed that the lead structure, 2-(((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5-(4-methoxybenzyl)-1,3,4-oxadiazole (4c), reduced the viability of MCF-7 cells with an IC50 value of 7.4 µM. Compound 4c also caused concentration-dependent loss of cell viability in chronic myelogenous leukemia (CML) cells. Furthermore, compound 4c inhibited the activation of NF-κB in human CML cells as observed by nuclear translocation and DNA binding assays. Functionally, compound 4c produced PARP cleavage and also suppressed expression of Bcl-2/xl, MMP-9, COX-2, survivin, as well as VEGF, resulting in apoptosis of CML cells. Moreover, ChIP assay showed that compound 4c decreased the binding of COX-2 to the p65 gene promoter. Detailed in silico analysis also indicated that compound 4c targeted NF-κB in CML cells. In conclusion, a novel structure bearing both triazole and oxadiazole moieties has been identified that can target NF-κB in CML cells and may constitute a potential novel drug candidate.

Novel Zn(II)-complex with hybrid chalcone-thiosemicarbazone ligand: Synthesis, characterization, and inhibitory effect on HTLV-1-infected MT-2 leukemia cells.

Chalcone and thiosemicarbazone have attracted attention due to their easy synthetic procedure and high success in the development of antiviral and antitumor, however, there are few biological data on the evaluation of chalcone-thiosemicarbazone hybrids and their complexation with metal ions. In this sense, the present work reports the synthesis and characterization of the hybrid (Z)-2-((E)-3-(4-chlorophenyl)-1-phenylallylidene)hydrazine-1-carbothioamide (CTCl) and its Zn(II)-complex (CTCl-Zn). The compounds were cell-based evaluated in terms of cytotoxicity against human T-cell lymphotropic virus type 1 (HTLV-1) infected leukemia cells (MT-2) and the experimental data were correlated with molecular docking calculations. The ligand and Zn(II)-complex were easily synthesized with a good yield - 57% and 79%, respectively. The dynamic of E/Z isomers with respect to the imine bond configuration of CTCl was evidenced by 1H NMR experiments in DMSO­d6, while the X-ray diffraction of CTCl-Zn showed that Zn(II) ion is tetracoordinated to two ligands in a bidentate mode and the metal ion lies on an intermediate geometry between the see-saw and trigonal pyramid. The ligand and complex exhibited low toxicity and the Zn(II)-complex is more cytotoxic than the ligand, with the corresponding IC50 value of 30.01 and 47.06 µM. Both compounds had a pro-apoptotic effect without the release of reactive oxygen species (ROS) and they can interact with DNA via minor grooves driven by van der Waals forces.

Cytotoxic Effects of Darinaparsin, a Novel Organic Arsenical, against Human Leukemia Cells.

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.

Potential antiproliferative and apoptotic effects of pilocarpine combined with TNF alpha in chronic myeloid leukemia cells.

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.

Pre-Existing and Acquired Resistance to PARP Inhibitor-Induced Synthetic Lethality.

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.

A novel costimulatory molecule gene-modified leukemia cell-derived exosome-targeted CD4+ T cell vaccine efficiently enhances anti-leukemia immunity.

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.

Differential Engraftment of Parental A20 PD-L1 WT and PD-L1 KO Leukemia Cells in Semiallogeneic Recipients in the Context of PD-L1/PD-1 Interaction and NK Cell-Mediated Hybrid Resistance.

The contribution of natural killer (NK) cells to tumor rejection in the context of programmed death-ligand 1/programmed death 1 (PD-L1/PD-1) blockade is a matter of intense debate. To elucidate the role of PD-L1 expression on tumor cells and the functional consequences of engaging PD-1 receptor on cytotoxic cells, PD-L1 expression was genetically inactivated and WT or PD-L1-deficient parental tumor cells were adoptively transferred intravenously into F1 recipients. The engraftment of PD-L1-deficient A20 tumor cells in the spleen and liver of F1 recipients was impaired compared with A20 PD-L1 WT tumor counterparts. To elucidate the mechanism responsible for this differential tumor engraftment and determine the relevance of the role of the PD-L1/PD-1 pathway in the interplay of tumor cells/NK cells, a short-term competitive tumor implantation assay in the peritoneal cavity of semiallogeneic F1 recipients was designed. The results presented herein showed that NK cells killed target tumor cells with similar efficiency regardless of PD-L1 expression, whereas PD-L1 expression on A20 tumor cells conferred significant tumor protection against rejection by CD8 T cells confirming the role of the co-inhibitory receptor PD-1 in the modulation of their cytotoxic activity. In summary, PD-L1 expression on A20 leukemia tumor cells modulates CD8 T-cell-mediated responses to tumor-specific antigens but does not contribute to inhibit NK cell-mediated hybrid resistance, which correlates with the inability to detect PD-1 expression on NK cells neither under steady-state conditions nor under inflammatory conditions.

Involvement of the PI3K/AKT Intracellular Signaling Pathway in the AntiCancer Activity of Hydroxytyrosol, a Polyphenol from Olea europaea, in Hematological Cells and Implication of HSP60 Levels in Its Anti-Inflammatory Activity.

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.