Cytopathological image analysis using deep-learning networks in microfluidic microscopy.

Cytopathologic testing is one of the most critical steps in the diagnosis of diseases, including cancer. However, the task is laborious and demands skill. Associated high cost and low throughput drew considerable interest in automating the testing process. Several neural network architectures were designed to provide human expertise to machines. In this paper, we explore and propose the feasibility of using deep-learning networks for cytopathologic analysis by performing the classification of three important unlabeled, unstained leukemia cell lines (K562, MOLT, and HL60). The cell images used in the classification are captured using a low-cost, high-throughput cell imaging technique: microfluidics-based imaging flow cytometry. We demonstrate that without any conventional fine segmentation followed by explicit feature extraction, the proposed deep-learning algorithms effectively classify the coarsely localized cell lines. We show that the designed deep belief network as well as the deeply pretrained convolutional neural network outperform the conventionally used decision systems and are important in the medical domain, where the availability of labeled data is limited for training. We hope that our work enables the development of a clinically significant high-throughput microfluidic microscopy-based tool for disease screening/triaging, especially in resource-limited settings.

The newly synthesized 2-(3-hydroxy-5-methoxyphenyl)-6,7-methylenedioxyquinolin-4-one triggers cell apoptosis through induction of oxidative stress and upregulation of the p38 MAPK signaling pathway in HL-60 human leukemia cells.

The aim of the present study was to discover the signaling pathways associated with 2-(3-hydroxy-5-methoxy-phenyl)-6,7-methylenedioxyquinolin-4-one (YYK1)-induced apoptosis in HL-60 human leukemia cells. YYK1 induced cytotoxic effects, cell morphological changes, decreased the cell number and increased reactive oxygen species (ROS) production and loss of mitochondrial membrane potential (ΔΨm) in HL-60 cells. YYK1-induced apoptosis was confirmed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Results from colorimetric assays and western blot analysis indicated that activities of caspase-7/-3, caspase-8 and caspase-9 were increased in YYK1-treated HL-60 cells. Western blot analysis showed that the protein levels of extrinsic apoptotic proteins (Fas/CD95, FasL and FADD), intrinsic related proteins (cytochrome c, Apaf-1, AIF and Endo G), the ratio of Bax/Bcl-2 and phosphorylated p38 MAPK were increased in HL-60 cells after YYK1 treatment. Cell apoptosis was significantly reduced after pre-treatment with N-acetylcysteine (NAC; a ROS scavenger) or diphenyleneiodonium chloride (DPI; a NADPH oxidase inhibitor). Blockage of p38 MAPK signaling by SB202190 abolished YYK1-induced Fas/CD95 upregulation and apoptosis in HL-60 cells. We conclude that YYK1 induces both of extrinsic and intrinsic apoptotic pathways via ROS-mediated activation of p38 MAPK signaling in HL-60 human leukemia cells in vitro.

Induction of caspase-3-dependent apoptosis in human leukemia HL-60 cells by δ-elemene.

  Î´-Elemene, an antitumor component, is a chemical compound isolated from Curcuma wenyujin, a Chinese traditional herb. We examined whether δ-elemene could inhibit cell growth and cell cycle progression and induce apoptosis in human leukemia HL-60 cells. The results demonstrated that δ-elemene induces significant apoptosis of HL-60 cells, as shown by MTT assay, annexin V (AnV) binding of externalized phosphatidylserine (PS), and the mitochondrial probe JC-1 using flow cytometry. HL-60 cells treated with δ-elemene showed high percentages in the early apoptotic and late apoptoctic/necrotic stages, as well as caspase-3 activation of HL-60 cells. By monitoring the changes in cell cycle profiles, we confirmed that δ-elemene could interfere with the cell cycle in the G2/M phase and induce apoptosis in HL-60 cells in a time-dependent manner. Caspase-3 plays a direct role in proteolytic cleavage of the cellular proteins responsible for progression to apoptosis. Therefore we examined apoptosis in HL-60 cells after exposure to δ-elemene and measured caspase-3 activities with or without Z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk, a broad-spectrum caspase inhibitor) pretreatment using flow cytometric analysis. The results showed that δ-elemene could induce caspase-3 activation as detected by the decrease in δ-elemene-induced caspase-3 activities after treatment with z-VAD-fmk. In the present study, δ-elemene activated typical caspase-dependent apoptosis in HL-60 cells, as demonstrated by an inhibitory effect of z-VAD-fmk on this cell death. During δ-elemene-induced apoptosis, cytochrome c and apoptosis-inducing factor were released into the cytosol and BAX was translocated from the cytosol to mitochondria. However, these were not prevented by z-VAD-fmk. In conclusion, our study demonstrated that δ-elemene could induce G2/M cell cycle transition and trigger apoptosis through a caspase-3-dependent pathway.

Triazoloacridone C-1305 abrogates the restriction checkpoint in cells lacking functional p53 and promotes their accumulation in the G2/M phase of the cell cycle.

Triazoloacridone C-1305, a new topoisomerase II inhibitor, exhibits potent cytostatic activity toward various tumours under in vitro and in vivo conditions. Interestingly, mouse cells lacking poly(ADP-ribose) polymerase-1 are much more sensitive to C-1305 than their normal counterparts. In the present study we tested the hypothesis that the functional status of p53 in tumour cells might have an impact on the efficiency of C-1305 in experiments with both p53-deficient human HL-60 promyelocytic leukemia cells and human MCF-7 breast cancer cells harboring a functional p53 pathway. Exposure of both cancer cell lines to C-1305 reduced the number of viable cells in a time- and concentration-dependent manner. Remarkably, however, HL-60 cells were much more strongly affected than MCF-7 cells. Measurements of DNA concentrations in single cells revealed that C-1305 arrested the tested cancer cells at the G/M transition. Analysis of the cell cycle and apoptosis regulators revealed that C-1305 strongly elevated phosphorylation of CDK1 at the inhibitory sites (Thr14/Tyr15) in HL-60 cells. Furthermore, C-1305 increased phosphorylation of pRb protein and CDK2 at Thr160 in HL60 cells, but not in MCF-7 cells. These observations suggest that C-1305 abrogates the restriction checkpoint and promotes G1/S transition in cells lacking functional p53.

[Search of novel bioactive natural products from plant sources--novel structures and biological activities--].

Over 30 years, our laboratory has been involved in the search of bioactive natural products from plant sources of several plant families, Rutaceae, Guttiferae, Avicenniaceae, and so on. In this review, novel structures of acridone alkaloids, carbazole alkaloids, coumarins, depsidones, and so on isolated in our laboratory will be showed. In addition, some results of assay of biological activities of the isolated compounds also will be described.

Cyclin-dependent kinase 1 inhibitor RO-3306 enhances p53-mediated Bax activation and mitochondrial apoptosis in AML.

Cyclin-dependent kinase (CDK) 1 and the murine double minute 2 homolog (MDM2)-p53 interaction are potential therapeutic targets in cancer, and their inhibition has been reported to be more proapoptotic in malignant cells compared to normal cells. We investigated the effect of CDK1 inhibition on p53 signaling after simultaneous dual blockade using the CDK1 inhibitor RO-3306 and the MDM2 inhibitor Nutlin-3 in AML. Treatment of growing AML cells with RO-3306 induced G2/M-phase cell cycle arrest and apoptosis in a dose- and time-dependent manner. We found that RO-3306 acts cooperatively with Nutlin-3 to induce mitochondrial apoptosis in a cell cycle-independent fashion. RO-3306 downregulated expression of the antiapoptotic proteins Bcl-2 and survivin and blocked p53-mediated induction of p21 and MDM2. CDK1 siRNA experiments showed that reduced CDK1 expression affects p53-induced p21 transactivation. We suggest that RO-3306 actively enhances downstream p53 signaling to promote apoptosis and that a combination strategy aimed at both inhibiting CDK1 and activating p53 signaling is potentially effective in AML, where TP53 mutations are rare and downstream p53 signaling is intact.

[Effect of reactive oxygen species during the leukemogenic process associated with exposure to benzene].

OBJECTIVE: To study the effect of reactive oxygen species (ROS) during the leukemogenic process associated with exposure to benzene. METHODS: HL-60 was treated with 3 micromol/L benzoquinone (BQ). Generation of ROS in cells was measured by DCFH-DA method. For proliferation assays,cells were stained with alamar blue dye and counted. RESULTS: ROS production and the proliferation of cell were all increased in BQ-treated cells (13.10 +/- 0.15, 185% +/- 30.00%) as compared with control cells (11.32 +/- 0.09, 100% +/- 0.00%) (P < 0.05); The addition of catalase just before BQ addition reduced ROS generation to basal levels and decreased the growth of cell (P < 0.05). CONCLUSION: ROS may play an important role in the process of proliferation of HL-60 cells induced by BQ.

CHR-2797: an antiproliferative aminopeptidase inhibitor that leads to amino acid deprivation in human leukemic cells.

CHR-2797 is a novel metalloenzyme inhibitor that is converted into a pharmacologically active acid product (CHR-79888) inside cells. CHR-79888 is a potent inhibitor of a number of intracellular aminopeptidases, including leucine aminopeptidase. CHR-2797 exerts antiproliferative effects against a range of tumor cell lines in vitro and in vivo and shows selectivity for transformed over nontransformed cells. Its antiproliferative effects are at least 300 times more potent than the prototypical aminopeptidase inhibitor, bestatin. However, the mechanism by which inhibition of these enzymes leads to proliferative changes is not understood. Gene expression microarrays were used to profile changes in mRNA expression levels in the human promyelocytic leukemia cell line HL-60 treated with CHR-2797. This analysis showed that CHR-2797 treatment induced a transcriptional response indicative of amino acid depletion, the amino acid deprivation response, which involves up-regulation of amino acid synthetic genes, transporters, and tRNA synthetases. These changes were confirmed in other leukemic cell lines sensitive to the antiproliferative effects of CHR-2797. Furthermore, CHR-2797 treatment inhibited phosphorylation of mTOR substrates and reduced protein synthesis in HL-60 cells, both also indicative of amino acid depletion. Treatment with CHR-2797 led to an increase in the concentration of intracellular small peptides, the substrates of aminopeptidases. It is suggested that aminopeptidase inhibitors, such as CHR-2797 and bestatin, deplete sensitive tumor cells of amino acids by blocking protein recycling, and this generates an antiproliferative effect. CHR-2797 is orally bioavailable and currently undergoing phase II clinical investigation in the treatment of myeloid leukemia.

Interactions between Shiga toxins and human polymorphonuclear leukocytes.

Human intestinal infections by Shiga toxin (Stx)-producing Escherichia coli cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), which represents the main cause of acute renal failure in early childhood. In HUS, Stx released in the gut enter the bloodstream and are targeted to renal endothelium. The mechanism of toxin delivery is still a matter of debate, although the role of polymorphonuclear leukocytes (PMN) as a Stx carrier has been indicated. The aim of this paper was to better define the interactions between Stx and human PMN. Direct and indirect flow cytometric analysis and binding experiments with radiolabeled toxins demonstrated that Stx bind to the surface of human mature PMN but not to immature PMN from G-CSF-treated donors. The use of the human myeloid leukemia cell (HL-60) model for inducible cell differentiation confirmed that the toxin binding occurs only after granulocytic differentiation. Stx binding caused a delay of the spontaneous apoptosis of PMN, as shown by the delayed appearance of apoptotic nuclei and activation of caspase 3 and by the higher number of cells negative to the annexin V-binding assay after 48 h. Moreover, flow cytometric analysis of mixed Stx-positive and Stx-negative PMN populations showed that the toxins were transferred from positive to negative PMN. The delayed, spontaneous apoptosis and the passage of the toxic ligand from older PMN to new, mature cells entering the circulation from the bone marrow may explain the previously reported persistence of Stx in the blood of children with HUS.

Side-chain modified vitamin D analogs induce rapid accumulation of VDR in the cell nuclei proportionately to their differentiation-inducing potential.

1,25-Dihydroxyvitamin D(3) (1,25D) regulates gene transcription through a nuclear vitamin D receptor (VDR) which acts as a ligand-regulated transcription factor. Some structural vitamin D analogs (VDAs) are selective in their biological actions, because they retain cell-differentiating potential, while their calcemic activity is reduced. In this article we have shown that in untreated HL60 cells the expression level of VDR is low, in spite of constant presence of VDR mRNA. Furthermore we have shown that one of the most rapid effects of either 1,25D or VDAs is nuclear accumulation of VDR, which is proportional to the differentiation-inducing potential of given analog. We observed this effect not only in HL60 cells, but also in blast cells isolated from patients with acute myeloid leukemias. After longer incubation time of the cells with various VDAs, the expression levels of VDR have become unrelated to the final differentiation effect.

Nuclear organization of PML bodies in leukaemic and multiple myeloma cells.

The nuclear arrangement of promyelocytic leukaemia nuclear bodies (PML NBs) was studied in vitro after the cell treatment by clinically used agents such as all-trans retinoic acid (RA) in human leukaemia and cytostatics or gamma radiation in multiple myeloma cells. In addition, the influence of phorbol ester (PMA) on PML NBs formation was analyzed. A reduced number of PML bodies, which led to relocation of PML NBs closer to the nuclear interior, mostly accompanied RA- and PMA-induced differentiation. Centrally located PML NBs were associated with transcriptional protein RNAP II and SC35 regions, which support importance of PML NBs in RNA processing that mostly proceeds within the nuclear interior. Conversely, the quantity of PML NBs was increased after cytostatic treatment, which caused re-distribution of PML NBs closer to the nuclear envelope. Here we showed correlations between the number of PML NBs and average Centre-to-PML distances. Moreover, a number of cells in S phase, especially during differentiation, influenced number of PML NBs. Studying the proteins involved in PML compartment, such as c-MYC, cell-type specific association of c-MYC and the PML NBs was observed in selected leukaemic cells undergoing differentiation, which was accompanied by c-MYC down-regulation.

Multistep regulation of telomerase during differentiation of HL60 cells.

Using three different differentiation agents (1alpha, 25 dihydroxyvitamin D3, all-trans-retinoic acid, and Am80), down-regulation of telomerase activity was found to be a common response during the monocytic or granulocytic differentiation of human acute myeloblastic leukemia cell line 60 (HL60) cells. Rapid down-regulation of telomerase transcription occurred during early differentiation of HL60 cells prior to G(1) arrest. Akt kinase activity was suppressed after 6 h of differentiation along with inhibition of telomerase activity, and the extent of the suppression that occurred while maintaining telomerase protein expression suggested the post-translational regulation of telomerase activity. Recombinant Akt dose-dependently increased telomerase activity, and telomerase was inhibited at the transcriptional and post-translational levels by LY294002, suggesting that PI-3K/Akt is one of the key signaling proteins involved in telomerase regulation. Each of the three differentiation agents caused a significant increase of signaling proteins (including Akt) at 3 days after the initiation of differentiation. Changes of acetyl-histone H4, which regulates transcription of the telomerase gene, were observed before the activation of Akt. This finding suggests that epigenetic control of telomerase transcription occurs before activation of Akt during the late stage of differentiation. These results indicate that telomerase activity is regulated by at least two mechanisms during granulocytic and monocytic differentiation, with one mechanism being transcriptional and the other being post-translational.

Synthesis and antiproliferative activities of isoindigo and azaisoindigo derivatives.

In the course of structure-activity relationship studies, diversely substituted 1-(beta- d-acetylatedglucopyranosyl)isoindigo derivatives were prepared from indolines. New 7'-azaisoindigo analogues were also synthesized by coupling a glycosylated isatine and a 7-azaindolin-2-one derivative. Compounds containing a 7'-azaisoindigo framework have never been described before. To get an insight into the substitution pattern required for the best biological potencies, their antiproliferative activities were evaluated toward a human buccal carcinoma cell line (KB) and two human myeloid leukaemia cell lines (K562, HL60).

Caspase-8 preferentially senses the apoptosis-inducing action of NG-18, a Gambogic acid derivative, in human leukemia HL-60 cells.

Gambogic acid (GA) is the major active ingredient of gamboge secreted from a Chinese traditional medicine Garcinia hanburryi possessing potent anti-tumor activity. N-(2-ethoxyethyl)gambogamide (NG-18), a derivative of GA, also efficiently inhibits proliferation of cultured human tumor cells. The inhibition effect of NG-18 is associated with its ability to induce apoptosis. In the present study, NG-18 markedly induced leukemia HL-60 cells apoptosis, and the extrinsic and intrinsic apoptosis pathways were activated almost at the same time. NG-18-induced tumor cell apoptosis was associated with up-regulation of pro-apoptotic Bcl-2 family member Bax, and downregulation of anti-apoptotic protein Bcl-2. The NG-18-induced apoptosis was blocked completely by a pan-caspase inhibitor Z-VAD-FMK, indicating that caspases were functionally and actively involved in this process. The specific inhibition of caspase-8 activity using Z-IETD-FMK significantly blocked NG-18-induced apoptosis. In contrast, inhibition of other initiator caspases, caspase-2 or -9, using Z-VDVAD-FMK or Z-LEHD-FMK respectively had no effect on NG-18-induced apoptosis. Altogether, our data demonstrated that NG-18-induced apoptosis was dependent on caspases and caspase-8 acted as a key executor in the event.

Cyclosporin A potentiates the cytotoxic effects of methyl methanesulphonate in HL-60 and K562 cells.

Methyl methanesulphonate (MMS) is a DNA damaging agent, which induces oxidative stress, ATP depletion, and consequently, cell death, in HL-60 and K562 cells. The cell death induced by MMS predominantly exhibited the morphological and biochemical hallmarks of necrosis. A minor population of dying cells exhibited apoptotic hallmarks, especially in K562 cell cultures. Cyclosporin A (CsA) was used to modulate the MMS-induced cell death. Our results indicated that CsA did not prevent cells from dying, but changed the mode of death from necrotic to apoptotic. Surprisingly, CsA enhanced oxidative stress and increased the overall number of dead cells. Based on these results, we conclude that the modulatory effect of CsA on MMS-induced cell death might arise from an interference by CsA with mitochondrial metabolism, rather than from inhibition of the MMS efflux mediated by P-glycoprotein.

Biotransformation of sinapic acid catalyzed by Momordica charantia peroxidase.

Biotransformation of sinapic acid (1) with H2O2/Momordica charantia peroxidase, which exists in the widely used food M. charantia, at pH 5.0, 43 degrees C, in the presence of acetone resulted in six compounds, including four new compounds. Compound 2 was the first isolation of a new unique sinapic acid tetrameric derivate, which is formed by peroxidase catalysis in vitro. Besides 2, three other new sinapic acid dimers, 3-5 have also been isolated. Their structures were established on the basis of spectroscopic data. Compound 5 showed a stronger antioxidative activity than the parent sinapic acid (1). Compounds 4 and 5 significantly inhibited the growth of HL-60 cell at the concentration of 10-5 microl/L.

Bortezomib activity and in vitro interactions with anthracyclines and cytarabine in acute myeloid leukemia cells are independent of multidrug resistance mechanisms and p53 status.

PURPOSE: The proteasome inhibitor bortezomib may be effective in combination with cytarabine and anthracyclines in the treatment of acute myeloid leukemia (AML) by virtue of targeting aberrantly activated NF-kappaB in AML stem cells. We tested whether bortezomib cytotoxicity is affected by multidrug resistance (MDR) proteins expressed in AML cells. We also tested whether bortezomib interactions with cytarabine and anthracyclines are affected by p53, because proteasome inhibition stabilizes p53 and may thus cause cell cycle arrest. EXPERIMENTAL DESIGN: Bortezomib sensitivity of cell lines overexpressing P-glycoprotein, multidrug resistance protein-1, breast cancer resistance protein and lung resistance protein was studied in the presence and absence of established modulators of these transport proteins. Drug interactions during simultaneous and sequential exposure to bortezomib and anthracyclines or cytarabine in diverse ratios were evaluated by isobologram and combination index analyses in AML cell lines with wild type and inactive p53 and were correlated with cell cycle perturbations induced by bortezomib. RESULTS: Of the MDR mechanisms studied, only P-glycoprotein conferred resistance to bortezomib, and resistance was only twofold. Interactions between bortezomib and anthracylines and cytarabine changed from antagonistic to additive or synergistic with increasing drug activity levels and were not affected by p53 status. CONCLUSIONS: MDR proteins and p53 do not affect bortezomib cytotoxicity or in vitro interactions with anthracyclines or cytarabine, but these interactions are concentration-dependent, and this concentration-dependency should be considered in the design of combination regimens.

N-Benzyladriamycin-14-valerate (AD 198) cytotoxicty circumvents Bcr-Abl anti-apoptotic signaling in human leukemia cells and also potentiates imatinib cytotoxicity.

Bcr-Abl activity in chronic myelogenous leukemia (CML) results in dysregulated cell proliferation and resistance against multiple cytotoxic agents due to the constitutive activation of proliferative signaling pathways. Currently, the most effective treatment of CML is the inhibition of Bcr-Abl activity by imatinib mesylate (Gleevec). Imatinib efficacy is limited by development of resistance through either expression of Bcr-Abl variants that bind imatinib less avidly, increased expression of Bcr-Abl, or expression of multidrug transport proteins. N-Benzyladriamycin-14-valerate (AD 198) is a novel antitumor PKC activating agent that triggers rapid apoptosis through PKC-delta activation and mitochondrial depolarization in a manner that is unaffected by Bcl-2 expression. We demonstrate that Bcr-Abl expression does not confer resistance to AD 198. Further, AD 198 rapidly induces Erk1/2 and STAT5 phosphorylation prior to cytochrome c release from mitochondria, indicating that proliferative pathways are active even as drug-treated cells undergo apoptosis. At sub-cytotoxic doses, AD 198 and its cellular metabolite, N-benzyladriamycin (AD 288) sensitize CML cells to imatinib through a supra-additive reduction in the level of Bcr-Abl protein expression. These results suggest that AD 198 is an effective treatment for CML both in combination with imatinib and alone against imatinib-resistant CML cells.

MZ3 induces apoptosis in human leukemia cells.

PURPOSE: 4-(4-Bromophenyl)-2,3-dihydro-N,3-bis(3,4,5-trimethoxyphenyl)-2-oxoidmi-dazole-1-carboxamide (MZ3) is one of the synthesized combretastatin-A-4 analogues and has been reported that it displayed a promising specific activity against leukemia cell lines. Our purpose was to investigate the mechanism of MZ3's cytotoxicity. METHODS: Cytotoxicity was measured by MTT method, apoptosis was measured by flow cytometry. DNA fragmentation was tested by agarose gel electrophoresis. Mitochondrial membrane potential (DeltaPsim) was detected by JC1 staining and flow cytometry, while intracellular reactive oxygen species (ROS) was detected by 5-(and-6)-carboxy-2'-7'-dichlorofluorescin diacetate staining and flow cytometry. Protein expression was analyzed by western blotting. In vivo activity of MZ3 was assayed through severe combined immunodeficiency (SCID) mice model of human leukemia engrafts. RESULTS: MZ3 exhibited high anti-cancer activity in six leukemia cell lines, including two drug-resistant cell lines. MZ3 induced DNA fragmentation, and caused an elevation of ROS and a loss of DeltaPsim in HL60 cells. MZ3 also induced the activation of caspase-3, influenced the expression of Bcl-2 family members, MAPKs and other proteins relative to mitochondria-induced apoptosis. In addition, N-acetylcysteine cannot inhibit HL60 cell apoptosis caused by MZ3. Furthermore, a prolonged survival time was observed after treatment with MZ3 in SCID mice model of human leukemia engrafts. CONCLUSIONS: MZ3 is a potent compound against leukemia cell lines both in vitro and in vivo, and the mitochondrial pathway mediated by Bcl-2 protein family and MAPKs might be involved in signaling MZ3-induced apoptosis.

KN-62 analogues as potent differentiating agents of HL-60 cells.

KN-62, an inhibitor of the calmodulin-dependent protein kinases (CaMKs), enhances the terminal differentiation of retinoic acid sensitive human myeloid leukemia cell lines. In an effort to identify additional CaMK inhibitors that exhibit more potent activity in triggering leukemia cell differentiation, we synthesized 45 analogues of KN-62 and determined their ability to induce HL-60 cell differentiation. Sixteen of these novel analogues exhibited significant differentiation-inducing activity, and one analogue, AS-004, was five times more potent than KN-62 in inhibiting proliferation and inducing differentiation of HL-60 cells. Such KN-62 analogues and/or related compounds may prove useful in treating promyelocytic leukemia.