Determination of correlations in multivariate count data with informative observation times.

We consider there are various types of recurrent events and the total number of occurrences are collected at the random observation times. It has concerned that the observation process may not be independent to the multivariate event processes, hence the total counts and observation times may be correlated and the dependence may exist among different types of the event processes as well. Many methods have developed nonparametric models to accommodate such unknown structures; however, it is difficult to assess and directly quantify their correlation relationships. A multivariate frailty model is proposed to this study, in which the event and observation processes are linked by frailty variables whose joint distribution can be implicitly specified through the multivariate normal distribution with some unknown covariance matrix. The Bayesian inference method is conducted to obtain the estimates of the regression coefficients and correlation parameters. We use a form of trigonometric functions to represent the covariance matrix, so that it meets the positive-definiteness condition efficiently during the estimation schemes. The simulation studies demonstrate the utility of the proposed models. We apply the model to a skin cancer prevention study, and aim to determine the covariate and association effects. We found treatment is significant in determining the duration of examination times; prior-counts, age and gender are significant variables on the occurrence rates of tumor counts. Using the covariance matrix to access the underlying dependent structure, the mutual correlations among them are all positive, and the basal cell counts are more related to the examination times.

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.

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.

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.

Inhibition of Sp1-mediated survivin and MCL1 expression cooperates with SLC35F2 and myeloperoxidase to modulate YM155 cytotoxicity to human leukemia cells.

Although YM155 is reported to suppress survivin (also known as BIRC5) expression in cancer cells, its cytotoxic mechanism in human acute myeloid leukemia (AML) cells has not been clearly resolved. In this study, we analyzed the mechanistic pathways that modulate the sensitivity of human AML U937 and HL-60 cells to YM155. YM155 induced apoptosis in AML cells, which was characterized by p38 MAPK phosphorylation and downregulation of survivin and MCL1 expression. Phosphorylated p38 MAPK causes autophagy-mediated Sp1 degradation, thereby inhibiting the transcription of survivin and MCL1. The reduction of survivin and MCL1 levels further facilitated Sp1 protein degradation through autophagy. The restoration of Sp1, survivin, or MCL1 expression protected U937 and HL-60 cells from YM155-mediated cytotoxicity. U937 and HL-60 cells were continuously exposed to hydroquinone (HQ) to generate U937/HQ and HL-60/HQ cells, which showed increased SLC35F2 expression. The increase in SLC35F2 expression led to an increase in the sensitivity of U937/HQ cells to YM155-mediated cytotoxicity, whereas no such effect was observed in HL-60/HQ cells. Of note, myeloperoxidase (MPO) activity in HL-60 and HL-60/HQ cells enhanced YM155 cytotoxicity in these cells, and the enforced expression of MPO also increased the sensitivity of U937 cells to YM155. Taken together, we conclude that p38 MAPK-modulated autophagy inhibits Sp1-mediated survivin and MCL1 expression, which, in turn, leads to the death of U937 and HL-60 cells following YM155 treatment. In addition, our data indicate that SLC35F2 increases the sensitivity of U937 cells to YM155-mediated cytotoxicity, whereas MPO enhances YM155 cytotoxicity in U937 and HL-60 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.

Modification of carboxyl groups converts α-lactalbumin into an active molten globule state with membrane-perturbing activity and cytotoxicity.

We investigated whether the modification of the negatively charged carboxyl groups with semicarbazide could confer membrane-disrupting and cytotoxic properties to bovine α-lactalbumin (LA). MALDI-TOF analysis revealed that eighteen of the twenty-one carboxyl groups in LA were coupled with semicarbazide molecules. Measurement of circular dichroism spectra and Trp fluorescence quenching studies showed that semicarbazide-modified LA (SEM-LA) had a molten globule-like conformation that retained the α-helix secondary structure but lost the tertiary structure of LA. Compared to LA, SEM-LA had a higher structural flexibility in response to trifluoroethanol- and temperature-induced structural transitions. In sharp contrast to LA, SEM-LA exhibited membrane-damaging activity and cytotoxicity. Furthermore, SEM-LA-induced membrane permeability promoted the uptake of daunorubicin and thereby its cytotoxicity. The microenvironment surrounding the Trp residues of SEM-LA was enriched in positive charges, as revealed by iodide quenching studies. The binding of SEM-LA with lipid vesicles altered the positively charged cluster around Trp residues. Although LA and SEM-LA displayed similar lipid-binding affinities, the membrane interaction modes of SEM-LA and LA differed. Collectively, these results suggest that blocking of negatively charged residues enables the formation of a molten-globule conformation of LA with structural flexibility and increased positive charge, thereby generating functional LA with membrane-disrupting activity and cytotoxicity.

Blocking of negative charged carboxyl groups converts Naja atra neurotoxin to cardiotoxin-like protein.

Naja atra cobrotoxin and cardiotoxin 3 (CTX3) exhibit neurotoxicity and cytotoxicity, respectively. In the present study, we aimed to investigate whether the carboxyl groups of cobrotoxin play a role in structural constraints, thereby preventing cobrotoxin from exhibiting cytotoxic activity. Six of the seven carboxyl groups in cobrotoxin were conjugated with semicarbazide. Measurement of circular dichroism spectra and Trp fluorescence quenching showed that the gross conformation of semicarbazide-modified cobrotoxin (SEM-cobrotoxin) and cobrotoxin differed. In sharp contrast to cobrotoxin, SEM-cobrotoxin demonstrated membrane-damaging activity and cytotoxicity, which are feature more characteristic of CTX3. Furthermore, both SEM-cobrotoxin and CTX3 induced cell death through AMPK activation. Analyses of the interaction between polydiacetylene/lipid vesicles and fluorescence-labeled lipids revealed that SEM-cobrotoxin and cobrotoxin adopted different membrane-bound states. The structural characteristics of SEM-cobrotoxin were similar to those of CTX3, including trifluoroethanol (TFE)-induced structural transformation and membrane binding-induced conformational change. Conversely, cobrotoxin was insensitive to the TFE-induced effect. Collectively, the data of this study indicate that blocking negatively charged residues confers cobrotoxin with membrane-damaging activity and cytotoxicity. The findings also suggest that the structural constraints imposed by carboxyl groups control the functional properties of snake venom α-neurotoxins during the divergent evolution of snake venom neurotoxins and cardiotoxins.

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.

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.

Inhibition of EGFR pathway promotes the cytotoxicity of ABT-263 in human leukemia K562 cells by blocking MCL1 upregulation.

ABT-263 induces MCL1 upregulation in cancer cells, which confers resistance to the drug. An increased understanding of the mechanism underlying ABT-263-induced MCL1 expression may provide a strategy to improve its tumor-suppression activity. The present study revealed that ABT-263 reduced the turnover of MCL1 mRNA, thereby upregulating MCL1 expression in human K562 leukemia cells. Furthermore, ABT-263-induced EGFR activation promoted AGO2 phosphorylation at Y393 and reduced miR-125b maturation. Treatment with EGFR inhibitors mitigated MCL1 upregulation induced by ABT-263. Additionally, lithium chloride (LiCl) alleviated ABT-263-induced MCL1 upregulation through EGFR-AGO2 axis-modulated miR-125b suppression. Ectopic expression of dominant negative AGO2(Y393F) or transfection with miR-125b abolished ABT-263-induced upregulation of MCL1 mRNA and protein levels. Co-treatment with either EGFR inhibitors or LiCl collaboratively enhanced ABT-263 cytotoxicity, while MCL1 overexpression eliminated this synergistic effect. Collectively, our data reveal that ABT-263 increases EGFR-mediated AGO2 phosphorylation, which in turn suppresses miR-125b-mediated MCL1 mRNA degradation in K562 cells. The suppression of this signaling pathway results in the synergistic cytotoxic effect of EGFR inhibitors or LiCl and ABT-263.

Status of Asp29 and Asp40 in the Interaction of Naja atra Cardiotoxins with Lipid Bilayers.

It is widely accepted that snake venom cardiotoxins (CTXs) target the plasma membranes of cells. In the present study, we investigated the role of Asp residues in the interaction of Naja atra cardiotoxin 1 (CTX1) and cardiotoxin 3 (CTX3) with phospholipid bilayers using chemical modification. CTX1 contains three Asp residues at positions 29, 40, and 57; CTX3 contains two Asp residues at positions 40 and 57. Compared to Asp29 and Asp40, Asp57 was sparingly modified with semi-carbazide, as revealed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass and mass/mass analyses. Thus, semi-carbazide-modified CTX1 (SEM-CTX1) mainly contained modified Asp29 and Asp40, while SEM-CTX3 contained modified Asp40. Compared to that of native toxins, trifluoroethanol easily induced structural transition of SEM-CTX1 and SEM-CTX3, suggesting that the structural flexibility of CTXs was constrained by Asp40. Modification of Asp29 and Asp40 markedly promoted the ability of CTX1 to induce permeability of cell membranes and lipid vesicles; CTX3 and SEM-CTX3 showed similar membrane-damaging activity. Modification of Asp residues did not affect the membrane-binding capability of CTXs. Circular dichroism spectra of SEM-CTX3 and CTX3 were similar, while the gross conformation of SEM-CTX1 was distinct from that of CTX1. The interaction of CTX1 with membrane was distinctly changed by Asp modification. Collectively, our data suggest that Asp29 of CTX1 suppresses the optimization of membrane-bound conformation to a fully active state and that the function of Asp40 in the structural constraints of CTX1 and CTX3 is not important for the manifestation of membrane-perturbing activity.

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.

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.

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.

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.

Naja atra Cardiotoxin 3 Elicits Autophagy and Apoptosis in U937 Human Leukemia Cells through the Ca2+/PP2A/AMPK Axis.

Cardiotoxins (CTXs) are suggested to exert their cytotoxicity through cell membrane damage. Other studies show that penetration of CTXs into cells elicits mitochondrial fragmentation or lysosome disruption, leading to cell death. Considering the role of AMPK-activated protein kinase (AMPK) in mitochondrial biogenesis and lysosomal biogenesis, we aimed to investigate whether the AMPK-mediated pathway modulated Naja atra (Taiwan cobra) CTX3 cytotoxicity in U937 human leukemia cells. Our results showed that CTX3 induced autophagy and apoptosis in U937 cells, whereas autophagic inhibitors suppressed CTX3-induced apoptosis. CTX3 treatment elicited Ca2+-dependent degradation of the protein phosphatase 2A (PP2A) catalytic subunit (PP2Acα) and phosphorylation of AMPKα. Overexpression of PP2Acα mitigated the CTX3-induced AMPKα phosphorylation. CTX3-induced autophagy was via AMPK-mediated suppression of the Akt/mTOR pathway. Removal of Ca2+ or suppression of AMPKα phosphorylation inhibited the CTX3-induced cell death. CTX3 was unable to induce autophagy and apoptosis in U937 cells expressing constitutively active Akt. Met-modified CTX3 retained its membrane-perturbing activity, however, it did not induce AMPK activation and death of U937 cells. These results conclusively indicate that CTX3 induces autophagy and apoptosis in U937 cells via the Ca2+/PP2A/AMPK axis, and suggest that the membrane-perturbing activity of CTX3 is not crucial for the cell death signaling pathway induction.

Naja atra cardiotoxins enhance the protease activity of chymotrypsin.

Snake venom cardiotoxins (CTXs) present diverse pharmacological functions. Previous studies have reported that CTXs affect the activity of some serine proteases, namely, chymotrypsin, subtilisin, trypsin, and acetylcholinesterase. To elucidate the mode of action of CTXs, the interaction of CTXs with chymotrypsin was thus investigated. It was found that Naja atra CTX isotoxins concentration-dependently enhanced chymotrypsin activity. The capability of CTX1 and CTX5 in increasing chymotrypsin activity was higher than that of CTX2, CTX3, and CTX4. Removal of the molecular beacon-bound CTXs by chymotrypsin, circular dichroism measurement, and acrylamide quenching of Trp fluorescence indicated that CTXs bound to chymotrypsin. Chemical modification of Lys, Arg, or Met residues of CTX1 attenuated its capability to enhance chymotrypsin activity without impairing their bond with chymotrypsin. Catalytically inactive chymotrypsin retained the binding affinity for native and modified CTX1. CTX1 and chemically modified CTX1 differently altered the global conformation of chymotrypsin and inactivated chymotrypsin. Moreover, CTX1 did not reduce the interaction of 2-(p-toluidino)-naphthalene-6-sulfonate (TNS) with chymotrypsin and inactivated chymotrypsin. Together with previous results revealing that TNS can bind at the hydrophobic region of active site in chymotrypsin, our data suggest that CTXs can enhance chymotrypsin activity by binding to the region outside the enzyme's active site.

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.