Chia-Hui Huang

Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressed c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression.

Amsacrine-induced apoptosis of human leukemia U937 cells is mediated by the inhibition of AKT- and ERK-induced stabilization of MCL1

Previous studies have attributed the anticancer activity of amsacrine to its inhibitory effect on topoisomerase II. However, 9-aminoacridine derivatives, which have the same structural scaffold as amsacrine, induce cancer cell apoptosis by altering the expression of BCL2 family proteins. Therefore, in the present study, we assessed whether BCL2 family proteins mediated the cytotoxic effects of amsacrine on human leukemia U937 cells. Amsacrine-induced apoptosis of U937 cells was characterized by caspase-9 and caspase-3 activation, increased intracellular Ca2+ concentration, mitochondrial depolarization, and MCL1 down-regulation. Amsacrine induced MCL1 down-regulation by decreasing its stability. Further, amsacrine-treated U937 cells showed AKT degradation and Ca2+-mediated ERK inactivation. Blockade of ERK-mediated phosphorylation of MCL1 inhibited the effect of Pin1 on the stabilization of MCL1, and AKT degradation promoted GSK3β-mediated degradation of MCL1. Restoration of ERK phosphorylation and AKT expression abrogated amsacrine-induced MCL1 down-regulation. Moreover, MCL1 over-expression inhibited amsacrine-induced depolarization of mitochondria membrane and increased the viability of amsacrine-treated cells. Taken together, our data indicate that amsacrine abolishes ERK- and Pin1-mediated stabilization of MCL1 and promotes GSK3β-mediated degradation of MCL1, leading to activate mitochondria-mediated apoptosis pathway in U937 cells.

Gallic acid-capped gold nanoparticles inhibit EGF-induced MMP-9 expression through suppression of p300 stabilization and NFκB/c-Jun activation in breast cancer MDA-MB-231 cells

Triple-negative breast cancers (TNBCs) are highly invasive and have a higher rate of distant metastasis. Matrix metalloproteinase-9 (MMP-9) plays a crucial role in EGF/EGFR-mediated malignant progression and metastasis of TNBCs. Various studies have revealed that treatment with gallic acid down-regulates MMP-9 expression in cancer cells, and that conjugation of phytochemical compounds with gold nanoparticles (AuNPs) increases the anti-tumor activity of the phytochemical compounds. Thus, the effect of gallic acid-capped AuNPs (GA-AuNPs) on MMP-9 expression in EGF-treated TNBC MDA-MB-231 cells was analyzed in the present study. The so-called green synthesis of AuNPs by means of gallic acid was performed at pH10, and the resulting GA-AuNPs had spherical shape with an average diameter of approximately 50nm. GA-AuNPs notably suppressed migration and invasion of EGF-treated cells, and inhibited EGF-induced MMP-9 up-regulation. GA-AuNPs abrogated EGF-induced Akt/p65 and ERK/c-Jun phosphorylation, leading to down-regulation of MMP-9 mRNA and protein expression in EGF-treated cells. Meanwhile, EGF-induced p300 stabilization was found to be involved in MMP-9 expression, whereas GA-AuNPs inhibited the EGF-promoted stability of the p300 protein. Although GA-AuNPs and gallic acid suppressed EGF-induced MMP-9 up-regulation via the same signaling pathway, the effective concentration of gallic acid was approximately 100-fold higher than that of GA-AuNPs for inhibition of MMP-9 expression in EGF-treated cells to a similar extent. Collectively, our data indicate that, in comparison with gallic acid, GA-AuNPs have a superior ability to inhibit EGF/EGFR-mediated MMP-9 expression in TNBC MDA-MB-231 cells. Our findings also point to a way to improve the anti-tumor activity of gallic acid.

Quinacrine induces the apoptosis of human leukemia U937 cells through FOXP3/miR-183/β-TrCP/SP1 axis-mediated BAX upregulation

&NA; Quinacrine, which is clinically used as an antimalarial drug, has anti‐cancer activity. However, mechanism underlying its cytotoxic effect remains to be completely elucidated. In the present study, we investigated the cytotoxic effect of quinacrine on human leukemia U937 cells. Quinacrine‐induced apoptosis of U937 cells was accompanied with ROS generation, mitochondrial depolarization, and BAX upregulation. Quinacrine‐treated U937 cells showed ROS‐mediated p38 MAPK activation and ERK inactivation, which in turn upregulated FOXP3 transcription. FOXP3‐mediated miR‐183 expression decreased &bgr;‐TrCP mRNA stability and suppressed &bgr;‐TrCP‐mediated SP1 degradation, thus increasing SP1 expression in U937 cells. Upregulated SP1 expression further increased BAX expression. BAX knock‐down attenuated quinacrine‐induced mitochondrial depolarization and increased the viability of quinacrine‐treated cells. Together, our data indicate that quinacrine‐induced apoptosis of U937 cells is mediated by mitochondrial alterations triggered by FOXP3/miR‐183/&bgr;‐TrCP/SP1 axis‐mediated BAX upregulation. HighlightsQuinacrine induces U937 cell apoptosis via upregulation of BAX.Quinacrine induces FOXP3 expression via p38 MAPK activation and ERK inactivation.FOXP3‐mediated miR‐183 expression reduces &bgr;‐TrCP mRNA stability.Suppression of &bgr;‐TrCP‐mediated SP1 degradation increases BAX expression.Quinacrine elicits FOXP3/miR‐183/&bgr;‐TrCP/SP1 axis‐mediated BAX upregulation.

ABT-263-induced MCL1 upregulation depends on autophagy-mediated 4EBP1 downregulation in human leukemia cells

The present study aimed to investigate the pathway related to MCL1 expression in ABT-263-treated human leukemia U937 cells. ABT-263 upregulated MCL1 protein expression but did not affect its mRNA level and protein stability. Notably, ABT-263 increased 4EBP1 mRNA decay and thus reduced 4EBP1 expression. Overexpression of 4EBP1 abrogated ABT-263-induced MCL1 upregulation. ABT-263-induced activation of IKKα/β-NFκB axis elicited autophagy of U937 cells, leading to reduced mRNA stability of 4EBP1. Inhibition of the IKKα/β-NFκB axis or autophagy mitigated the effect of ABT-263 on 4EBP1 and MCL1 expression. Amsacrine enhanced the cytotoxicity of ABT-263 in human leukemia U937, HL-60, and Jurkat cells because of its inhibitory effect on the IKKα/β-NFκB-mediated pathway. Our data indicate that ABT-263 alleviates the inhibitory effect of 4EBP1 on MCL1 protein synthesis through IKKα/β-NFκB-mediated induction of autophagy, and suggest a promising strategy to improve anti-leukemia therapy with ABT-263.

The Association Between p38 MAPK‐Mediated TNF‐α/TNFR2 up‐Regulation and 2‐(4‐Aminophenyl)‐7‐Methoxybenzothiazole‐Induced Apoptosis in Human Leukemia U937 Cells

The primary cause of treatment failures in acute myeloid leukemia is usually associated with defects in the apoptotic pathway. Several studies suggest that 2‐(4‐aminophenyl)‐7‐methoxybenzothiazole (7‐OMe‐APBT) may potentially induce apoptosis of cancer cells. Thus, the present study was conducted to explore the cytotoxic effect of 7‐OMe‐APBT on human leukemia U937 cells. The apoptosis of human leukemia U937 cells induced by 7‐OMe‐APBT was characterized by an increase in mitochondrial membrane depolarization, procaspase‐8 degradation, and tBid production. Down‐regulation of FADD blocked 7‐OMe‐APBT‐induced procaspase‐8 degradation and rescued the viability of 7‐OMe‐APBT‐treated cells, suggesting the involvement of a death receptor‐mediated pathway in 7‐OMe‐APBT‐induced cell death. Increased TNF‐α expression, TNFR2 expression, and p38 MAPK phosphorylation were noted in 7‐OMe‐APBT‐treated cells. Pretreatment with a p38 MAPK inhibitor abolished 7‐OMe‐APBT‐induced TNF‐α and TNFR2 up‐regulation. 7‐OMe‐APBT stimulated p38 MAPK/c‐Jun‐mediated transcriptional up‐regulation of TNFR2, while the increased TNF‐α mRNA stability led to TNF‐α up‐regulation in 7‐OMe‐APBT‐treated cells. Treatment with 7‐OMe‐APBT up‐regulated protein phosphatase 2A catalytic subunit α (PP2Acα) expression via the p38 MAPK/c‐Jun/ATF‐2 pathway, which, in turn, promoted tristetraprolin (TTP) degradation. Pretreatment with a protein phosphatase 2A inhibitor or TTP over‐expression abrogated TNF‐α up‐regulation in 7‐OMe‐APBT‐treated cells. Abolishment of TNF‐α up‐regulation or knock‐down of TNFR1/TNFR2 by siRNA restored the viability of 7‐OMe‐APBT‐treated cells. Taken together, our data indicate a connection between p38 MAPK‐mediated TNF‐α and TNFR2 up‐regulation and 7‐OMe‐APBT‐induced TNF‐α‐mediated death pathway activation in U937 cells. The same pathway also elucidates the mechanism underlying 7‐OMe‐APBT‐induced death of human leukemia HL‐60 cells. J. Cell. Physiol. 230: 130–141, 2016.

The suppressive effect of arsenic trioxide on TET2-FOXP3-Lyn-Akt axis-modulated MCL1 expression induces apoptosis in human leukemia cells

ABSTRACT Arsenic trioxide (ATO) has been reported to inhibit the activity of Ten‐eleven translocation methylcytosine dioxygenase (TET). TET modulates FOXP3 expression, while dysregulation of FOXP3 expression promotes the malignant progression of leukemia cells. We examined the role of TET‐FOXP3 axis in the cytotoxic effects of ATO on the human acute myeloid leukemia cell line, U937. ATO‐induced apoptosis in U937 cells was characterized by activation of caspase‐3/‐9, mitochondrial depolarization, and MCL1 downregulation. In addition, ATO‐treated U937 cells showed ROS‐mediated inhibition of TET2 transcription, leading to downregulation of FOXP3 expression and in turn, suppression of FOXP3‐mediated activation of Lyn and Akt. Overexpression of FOXP3 or Lyn minimized the suppressive effect of ATO on Akt activation and MCL1 expression. Promoter luciferase activity and chromatin immunoprecipitation assays revealed the crucial role of Akt‐mediated CREB phosphorylation in MCL1 transcription. Further, ATO‐induced Akt inactivation promoted GSK3&bgr;‐mediated degradation of MCL1. Transfection of constitutively active Akt expression abrogated ATO‐induced MCL1 downregulation. MCL1 overexpression lessened the ATO‐induced depolarization of mitochondrial membrane and increased the viability of ATO‐treated cells. Thus, our data suggest that ATO induces mitochondria‐mediated apoptosis in U937 cells through its suppressive effect on TET2‐FOXP3‐Lyn‐Akt axis‐modulated MCL1 transcription and protein stabilization. Our findings also indicate that the same pathway underlies ATO‐induced death in human leukemia HL‐60 cells. HighlightsArsenic acid (ATO) induces U937 cell apoptosis through downregulation of MCL1.ATO downregulates FOXP3 expression via the inhibition of TET2 transcription.ATO‐induced FOXP3 downregulation suppresses Lyn‐mediated Akt activation.Inactivation of Akt reduces MCL1 transcription and promotes MCL1 degradation.ATO suppresses TET2‐FOXP3‐Lyn‐Akt axis‐modulated MCL1 expression.

A Turn-on Fluorescence Sensor for Heparin Detection Based on a Release of Taiwan Cobra Cardiotoxin from a DNA Aptamer or Adenosine-Based Molecular Beacon

This study presents two sensitive fluorescent assays for sensing heparin on the basis of the electrostatic interaction between heparin and Naja naja atra cardiotoxin 3 (CTX3). Owing to CTX3-induced folded structure of an adenosine-based molecular beacon (MB) or a DNA aptamer against CTX3, a reduction in the fluorescent signal of the aptamer or MB 5′-end labeled with carboxyfluorescein (FAM) and 3′-end labeled with 4-([4-(dimethylamino)phenyl]azo)-benzoic acid (DABCYL) was observed upon the addition of CTX3. The presence of heparin and formation of the CTX3–heparin complex caused CTX3 detachment from the MB or aptamer, and restoration of FAM fluorescence of the 5′-FAM-and-3′-DABCYL-labeled MB and aptamer was subsequently noted. Moreover, the detection of heparin with these CTX3-aptamer and CTX3-MB sensors showed high sensitivity and selectivity toward heparin over chondroitin sulfate and hyaluronic acid regardless of the presence of plasma. The limit of detection for heparin in plasma was determined to be 16 ng/mL and 15 ng/mL, respectively, at a signal-to-noise ratio of 3. This study validates the practical utility of the CTX3-aptamer and CTX3-MB systems for determining the concentration of heparin in a biological matrix.

Membrane-damaging activities of mannosylated ovalbumin are involved in its antibacterial action

Mannosylated ovalbumin (Man-OVA) prepared by modification of carboxyl groups with p-aminophenyl α-d-mannopyranoside shows an increase of net positive charge, which may enhance its binding to bacterial membrane. Thus, we aimed to investigate whether Man-OVA exerts antibacterial activity on Escherichia coli and Staphylococcus aureus via membrane-perturbing effect. Man-OVA inhibited the growth of E. coli and S. aureus, whereas ovalbumin (OVA) did not show any antibacterial activity. Moreover, Man-OVA induced an increase in the membrane permeability of E. coli and S. aureus, which was positively correlated to its bactericidal action. Morphological examination using scanning electron microscopy revealed that Man-OVA disrupted the bacterial membrane integrity. Destabilization of the lipopolysaccharide (LPS) layer and inhibition of lipoteichoic acid (LTA) biosynthesis in the cell wall increased the bactericidal effect of Man-OVA. In contrast to OVA, Man-OVA also induced leakage of bacterial membrane-mimicking liposomes. Color transformation of phospholipid/polydiacetylene membrane assay revealed that the membrane-interaction mode of Man-OVA was distinct from that of OVA. LPS and LTA suppressed the membrane-damaging activity of Man-OVA, whereas an increase in the Man-OVA concentration attenuated the inhibitory action of LPS and LTA. Taken together, our data indicate that the bactericidal activity of Man-OVA depends strongly on its ability to induce membrane permeability.