CREB/Sp1-mediated MCL1 expression and NFκB-mediated ABCB1 expression modulate the cytotoxicity of daunorubicin in chronic myeloid leukemia cells.

Although some studies have hinted at the therapeutic potential of daunorubicin (DNR) in chronic myeloid leukemia (CML), the mechanism by which DNR induces CML cell death is unclear. Therefore, this study aimed to investigate DNR-induced cell death signaling pathways in CML cell lines K562 and KU812. DNR-triggered apoptosis in K562 cells was characterized by inhibition of MCL1 expression, while restoration of MCL1 expression protected K562 cells from DNR-mediated cytotoxicity. In addition, DNR induced NOX4-dependent ROS production, leading to the activation of p38 MAPK and inactivation of Akt and ERK. Activated p38 MAPK stimulated protein phosphatase 2A-dependent dephosphorylation of CREB. Since Akt-mediated activation of ERK reduced ß-TrCP mRNA stability, the inactivation of Akt-ERK axis increased ß-TrCP expression, which in turn promoted proteasomal degradation of Sp1. Inhibition of CREB phosphorylation and Sp1 expression simultaneously reduced MCL1 transcription and protein expression. DNR-induced MCL1 suppression was not reliant on its ability to induce DNA damage. In addition, DNR induced the expression of drug exporter ABCB1 in K562 cells through the p38 MAPK/NFκB-mediated pathway, while imatinib or ABT-199 inhibited the DNR-induced effect. The combination of imatinib or ABT-199 with DNR showed synergistic cytotoxicity in K562 cells by increasing intracellular DNR retention. Cumulatively, our data indicate that DNR induces MCL1 downregulation in K562 cells by promoting p38 MAPK-mediated dephosphorylation of CREB and inhibiting the Akt-ERK axis-mediated Sp1 protein stabilization. Furthermore, experimental evidence indicates that DNR-induced death of KU812 cells occurs through a similar pathway.

Functional and structural properties of cardiotoxin isomers produced by blocking negatively charged groups.

In this study, we investigated the functional roles of Asp40, Asp57, and C-terminal Asn60 in Naja atra cardiotoxin 3 (CTX3) structure and function by modifying these three carboxyl groups with semicarbazide. The conjugation of the carboxyl groups with semicarbazide produced two conformational isomers whose gross and fine structures were different from those of CTX3. The blocking of the carboxyl groups increased the structural flexibility of CTX3 in response to trifluoroethanol-induced effect. Despite presenting modest to no effect on decreasing the induction of permeability in zwitterionic phospholipid vesicles, the carboxyl group-modified CTX3 showed a marked reduction in its permeabilizing effect on anionic phospholipid vesicles in comparison to that of the native protein. Compared with native CTX3, carboxyl group-modified CTX3 exhibited lower activity in inducing membrane leakage in U937 cells. The CD spectra of lipid-bound toxins and the color transition of polydiacetylene/lipid assay showed that the membrane interaction mode of CTX3 was distinctly changed by the modification in the carboxyl groups. Given that the selective modification of Asp40 does not cause the conformational isomerization of CTX3, our data indicate that the carboxyl groups in Asp57 and Asn60 are essential in maintaining the structural topology of CTX3. Furthermore, modification of carboxyl groups changes the interdependence between the infrastructure and the global conformation of CTX3 in modulating membrane permeabilizing activity.

NOXA-mediated degradation of MCL1 and BCL2L1 causes apoptosis of daunorubicin-treated human acute myeloid leukemia cells.

Daunorubicin (DNR) is used clinically to treat acute myeloid leukemia (AML), while the signaling pathways associated with its cytotoxicity are not fully elucidated. Thus, we investigated the DNR-induced death pathway in the human AML cell lines U937 and HL-60. DNR-induced apoptosis in U937 cells accompanied by downregulation of MCL1 and BCL2L1, upregulation of Phorbol-12-myristate-13-acetate-induced protein 1 (NOXA), and mitochondrial depolarization. DNR induced NOX4-mediated reactive reactive oxygen species (ROS) production, which in turn inactivated Akt and simultaneously activated p38 mitogen-activated protein kinase (MAPK). Activated p38 MAPK and inactivated Akt coordinately increased GSK3ß-mediated cAMP response element-binding protein (CREB) phosphorylation, which promoted NOXA transcription. NOXA upregulation critically increased the proteasomal degradation of MCL1 and BCL2L1. The same pathway was also responsible for the DNR-induced death of HL-60 cells. Restoration of MCL1 or BCL2L1 expression alleviated DNR-induced mitochondrial depolarization and cell death. Furthermore, ABT-199 (a BCL2 inhibitor) synergistically enhanced the cytotoxicity of DNR in AML cell lines. Notably, DNR-induced DNA damage was not related to NOXA-mediated degradation of MCL1 and BCL2L1. Collectively, these results indicate that the upregulation of NOXA expression through the NOX4-ROS-p38 MAPK-GSK3ß-CREB axis results in the degradation of MCL1 and BCL2L1 in DNR-treated U937 and HL-60 cells. This signaling pathway may provide insights into the mechanism underlying DNR-triggered apoptosis in AML cells.

GSK3ß suppression inhibits MCL1 protein synthesis in human acute myeloid leukemia cells.

Previous studies have shown that glycogen synthase kinase 3ß (GSK3ß) suppression is a potential strategy for human acute myeloid leukemia (AML) therapy. However, the cytotoxic mechanism associated with GSK3ß suppression remains unresolved. Thus, the underlying mechanism of N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418)-elicited GSK3ß suppression in the induction of AML U937 and HL-60 cell death was investigated in this study. Our study revealed that AR-A014418-induced MCL1 downregulation remarkably elicited apoptosis of U937 cells. Furthermore, the AR-A014418 treatment increased p38 MAPK phosphorylation and decreased the phosphorylated Akt and ERK levels. Activation of p38 MAPK subsequently evoked autophagic degradation of 4EBP1, while Akt inactivation suppressed mTOR-mediated 4EBP1 phosphorylation. Furthermore, AR-A014418-elicited ERK inactivation inhibited Mnk1-mediated eIF4E phosphorylation, which inhibited MCL1 mRNA translation in U937 cells. In contrast to GSK3α, GSK3ß downregulation recapitulated the effect of AR-A014418 in U937 cells. Transfection of constitutively active GSK3ß or cotransfection of constitutively activated MEK1 and Akt suppressed AR-A014418-induced MCL1 downregulation. Moreover, AR-A014418 sensitized U937 cells to ABT-263 (BCL2/BCL2L1 inhibitor) cytotoxicity owing to MCL1 suppression. Collectively, these results indicate that AR-A014418-induced GSK3ß suppression inhibits ERK-Mnk1-eIF4E axis-modulated de novo MCL1 protein synthesis and thereby results in U937 cell apoptosis. Our findings also indicate a similar pathway underlying AR-A014418-induced death in human AML HL-60 cells.

SIRT3, PP2A and TTP protein stability in the presence of TNF-α on vincristine-induced apoptosis of leukaemia cells.

The contribution of vincristine (VCR)-induced microtubule destabilization to evoke apoptosis in cancer cells remains to be resolved. Thus, we investigated the cytotoxic mechanism of VCR on U937 and HL-60 human leukaemia cell lines. We discovered that VCR treatment resulted in the up-regulation of TNF-α expression and activation of the death receptor pathway, which evoked apoptosis of U937 cells. Moreover, VCR induced microtubule destabilization and mitotic arrest. VCR treatment down-regulated SIRT3, and such down-regulation caused mitochondrial ROS to initiate phosphorylation of p38 MAPK. p38 MAPK suppressed MID1-modulated degradation of the protein phosphatase 2A (PP2A) catalytic subunit. The SIRT3-ROS-p38 MAPK-PP2A axis inhibited tristetraprolin (TTP)-controlled TNF-α mRNA degradation, consequently, up-regulating TNF-α expression. Restoration of SIRT3 and TTP expression, or inhibition of the ROS-p38 MAPK axis increased the survival of VCR-treated cells and repressed TNF-α up-regulation. In contrast to suppression of the ROS-p38 MAPK axis, overexpression of SIRT3 modestly inhibited the effect of VCR on microtubule destabilization and mitotic arrest in U937 cells. Apoptosis of HL-60 cells, similarly, went through the same pathway. Collectively, our data indicate that the SIRT3-ROS-p38 MAPK-PP2A-TTP axis modulates TNF-α expression, which triggers apoptosis of VCR-treated U937 and HL-60 cells. We also demonstrate that the apoptotic signalling is not affected by VCR-elicited microtubule destabilization.

Autophagic HuR mRNA degradation induces survivin and MCL1 downregulation in YM155-treated human leukemia cells.

The aim of this study was to investigate the mechanism of YM155 cytotoxicity in human chronic myeloid leukemia (CML) cells. YM155-induced apoptosis of human CML K562 cells was characterized by ROS-mediated p38 MAPK activation, mitochondrial depolarization, and survivin and MCL1 downregulation. Moreover, YM155-induced autophagy caused degradation of HuR mRNA and downregulation of HuR protein expression, which resulted in destabilized survivin and MCL1 mRNA. Interestingly, survivin and MCL1 suppression contributed to autophagy-mediated HuR mRNA destabilization in YM155-treated cells. Pretreatment with inhibitors of p38 MAPK or autophagy alleviated YM155-induced autophagy and apoptosis in K562 cells, as well as YM155-induced downregulation of HuR, survivin, and MCL1. Ectopic overexpression of HuR, survivin, or MCL1 attenuated the cytotoxic effect of YM155 on K562 cells. Conversely, YM155 sensitized K562 cells to ABT-199 (a BCL2 inhibitor), and circumvented K562 cell resistance to ABT-199 because of its inhibitory effect on survivin and MCL1 expression. Overall, our data indicate that YM155-induced apoptosis is mediated by inducing autophagic HuR mRNA degradation, and reveal the pathway responsible for YM155-induced downregulation of survivin and MCL1 in K562 cells. Our findings also indicate a similar pathway underlying YM155-induced death in human CML MEG-01 cells.

Arsenic trioxide-induced p38 MAPK and Akt mediated MCL1 downregulation causes apoptosis of BCR-ABL1-positive leukemia cells.

In this study, we investigated the mechanisms underlying arsenic trioxide (ATO)-induced death of human BCR-ABL1-positive K562 and MEG-01 cells. ATO-induced apoptotic death in K562 cells was characterized by ROS-mediated mitochondrial depolarization, MCL1 downregulation, p38 MAPK activation, and Akt inactivation. ATO-induced BCR-ABL1 downregulation caused Akt inactivation but not p38 MAPK activation. Akt inactivation increased GSK3ß-mediated MCL1 degradation, while p38 MAPK-mediated NFκB activation coordinated with HDAC1 suppressed MCL1 transcription. Inhibition of p38 MAPK activation or overexpression of constitutively active Akt increased MCL1 expression and promoted the survival of ATO-treated cells. Overexpression of MCL1 alleviated mitochondrial depolarization and cell death induced by ATO. The same pathway was found to be involved in ATO-induced death in MEG-01 cells. Remarkably, YM155 synergistically enhanced the cytotoxicity of ATO on K562 and MEG-01 cells through suppression of MCL1 and survivin. Collectively, our data indicate that ATO-induced p38 MAPK- and Akt-mediated MCL1 downregulation triggers apoptosis in K562 and MEG-01 cells, and that p38 MAPK activation is independent of ATO-induced BCR-ABL1 suppression.

Semiparametric copula-based analysis for treatment effects in the presence of treatment switching.

In controlled trials, "treatment switching" occurs when patients in one treatment group switch to alternative treatments during the trial, and poses challenges to treatment effect evaluation owing to crossover of the treatments groups. In this work, we assume that treatment switching can occur after some disease progression event and view the progression and death events as two semicompeting risks. The proposed model consists of a copula model for the joint distribution of time-to-progression (TTP) and overall survival (OS) up to the earlier of the two events, as well as a conditional hazard model for OS subsequent to progression. The copula model facilitates assessing the marginal distributions of TTP and OS separately from the association between the two events, and, in particular, the treatment effect on OS in the absence of treatment switching. The proposed conditional hazard model for death subsequent to progression allows us to assess the treatment switching (crossover) effect on OS given occurrence of progression and covariates. Semiparametric proportional hazards models are employed in the marginal models for TTP and OS. A nonparametric maximum likelihood procedure is developed for model inference, which is verified through asymptotic theory and simulation studies. The proposed analysis is applied to a lung cancer dataset to illustrate its real utility.

Compound C induces autophagy and apoptosis in parental and hydroquinone-selected malignant leukemia cells through the ROS/p38 MAPK/AMPK/TET2/FOXP3 axis.

Hydroquinone (HQ), a major metabolic product of benzene, causes acute myeloid leukemia (AML) elicited by benzene exposure. Past studies found that continuous exposure of human AML U937 cells to HQ selectively produces malignant U937/HQ cells in which FOXP3 upregulation modulates malignant progression. Other studies revealed that AMPK promotes TET2 activity on DNA demethylation and that TET2 activity is crucial for upregulating FOXP3 expression. This study was conducted to elucidate whether compound C, an AMPK inhibitor, blocked the AMPK-TET2-FOXP3 axis in AML and in HQ-selected malignant cells. We found higher levels of AMPKα, TET2, and FOXP3 expression in U937/HQ cells compared to U937 cells. Treatment of parental Original Article and HQ-selected malignant U937 cells with compound C induced ROS-mediated p38 MAPK activation, leading to a suppression of AMPKα, TET2, and FOXP3 expression. Moreover, compound C induced apoptosis and mTOR-independent autophagy. The suppression of the autophagic flux inhibited the apoptosis of compound C-treated U937 and U937/HQ cells, whereas co-treatment with rapamycin, a mTOR inhibitor, sensitized the two cell lines to compound C cytotoxicity. Overexpression of AMPKα1 or pretreatment with autophagic inhibitors abrogated compound C-induced autophagy and suppression of TET2 and FOXP3 expression. Restoration of AMPKα1 or FOXP3 expression increased cell survival after treatment with compound C. In conclusion, our results show that compound C suppresses AMPK/TET2 axis-mediated FOXP3 expression and induces autophagy-dependent apoptosis in parental and HQ-selected malignant U937 cells, suggesting that the AMPK/TET2/FOXP3 axis is a promising target for improving AML therapy and attenuating benzene exposure-induced AML progression.

Albendazole-Induced SIRT3 Upregulation Protects Human Leukemia K562 Cells from the Cytotoxicity of MCL1 Suppression.

Previous studies have shown that MCL1 stabilization confers cancer cells resistance to microtubule targeting agents (MTAs) and functionally extends the lifespan of MTA-triggered mitotically arrested cells. Albendazole (ABZ), a benzimidazole anthelmintic, shows microtubule-destabilizing activity and has been repositioned for cancer therapies. To clarify the role of MCL1 in ABZ-induced apoptosis, we investigated the cytotoxicity of ABZ on human leukemia K562 cells. Treatment with ABZ for 24 h did not appreciably induce apoptosis or mitochondrial depolarization in K562 cells, though it caused the mitotic arrest of K562 cells. ABZ-evoked p38 MAPK activation concurrently suppressed Sp1-mediated MCL1 expression and increased SIRT3 mRNA stability and protein expression. ABZ and A-1210477 (an MCL1 inhibitor) enhanced the cytotoxicity of ABT-263 (a BCL2/BCL2L1 inhibitor) to their effect on MCL1 suppression. Unlike ABZ, A-1210477 did not affect SIRT3 expression and reduced the survival of K562 cells. Overexpression of SIRT3 attenuated the A-1210477 cytotoxicity on K562 cells. ABZ treatment elicited marked apoptosis and ΔΨm loss in ABT-263-resistant K562 (K562/R) cells, but did not alter SIRT3 expression. Ectopic expression of SIRT3 alleviated the cytotoxicity of ABZ on K562/R cells. Collectively, our data demonstrate that ABZ-induced SIRT3 upregulation delays the apoptosis-inducing effect of MCL1 suppression on apoptosis induction in K562 cells.

Mixture regression models for the gap time distributions and illness-death processes.

The aim of this study is to provide an analysis of gap event times under the illness-death model, where some subjects experience "illness" before "death" and others experience only "death." Which event is more likely to occur first and how the duration of the "illness" influences the "death" event are of interest. Because the occurrence of the second event is subject to dependent censoring, it can lead to bias in the estimation of model parameters. In this work, we generalize the semiparametric mixture models for competing risks data to accommodate the subsequent event and use a copula function to model the dependent structure between the successive events. Under the proposed method, the survival function of the censoring time does not need to be estimated when developing the inference procedure. We incorporate the cause-specific hazard functions with the counting process approach and derive a consistent estimation using the nonparametric maximum likelihood method. Simulations are conducted to demonstrate the performance of the proposed analysis, and its application in a clinical study on chronic myeloid leukemia is reported to illustrate its utility.

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

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 GSK3ß-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.

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.

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.

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 GSK3ß-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 GSK3ß-mediated degradation of MCL1, leading to activate mitochondria-mediated apoptosis pathway in U937 cells.

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

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 ß-TrCP mRNA stability and suppressed ß-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/ß-TrCP/SP1 axis-mediated BAX upregulation.

Regression analysis for bivariate gap time with missing first gap time data.

We consider ordered bivariate gap time while data on the first gap time are unobservable. This study is motivated by the HIV infection and AIDS study, where the initial HIV contracting time is unavailable, but the diagnosis times for HIV and AIDS are available. We are interested in studying the risk factors for the gap time between initial HIV contraction and HIV diagnosis, and gap time between HIV and AIDS diagnoses. Besides, the association between the two gap times is also of interest. Accordingly, in the data analysis we are faced with two-fold complexity, namely data on the first gap time is completely missing, and the second gap time is subject to induced informative censoring due to dependence between the two gap times. We propose a modeling framework for regression analysis of bivariate gap time under the complexity of the data. The estimating equations for the covariate effects on, as well as the association between, the two gap times are derived through maximum likelihood and suitable counting processes. Large sample properties of the resulting estimators are developed by martingale theory. Simulations are performed to examine the performance of the proposed analysis procedure. An application of data from the HIV and AIDS study mentioned above is reported for illustration.

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.

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 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.