Atrial fibrillation - Can HbA1c levels really predict the risk?

BACKGROUND: Diabetes is associated with higher mortality and worse post-operative outcomes in patients undergoing coronary artery bypass grafting and HbA1c levels have consistently been reported to be associated with adverse post-operative outcomes. However, the role of HbA1c still remains unclear with regards to the occurrence of atrial fibrillation. METHOD: Data for the patients undergoing off-pump coronary artery bypass grafting was analysed in a retrospective fashion. Patients were divided into-those with HbA1c < 6.5% and those with HbA1c ≥ 6.5% and the incidence of atrial fibrillation observed in these two groups. We also compared patient who developed atrial fibrillation in the post-operative period and compared them with those who did not. RESULTS: Of the 5259 patients included in the study HbA1c was <6.5 in 2808 (53.4%) patients and was ≥6.5 in 2451 (46.6%) patients; 623 (11.8%) patients in our study developed atrial fibrillation. Onset of atrial fibrillation in the post-operative period was seen most commonly 235 (38.3%) on between 24 and 48 h after the operation with more than half of them 338 (54.2%) occurring within the first 48 h. On multivariate analysis, HbA1c was not a risk factor for atrial fibrillation (odd's ratio 1.144, 95% confidence interval 0.967-1.354). Only increased age (odd's ratio 1.08; 95% confidence interval 1.069-1.091); EuroSCORE (odd's ratio 1.073; 95% confidence interval 1.048-1.099); history of recent MI (odd's ratio 0.768; 95% confidence interval 0.606-0.971) and peripheral vascular disease (odd's ratio 1.667; 95% confidence interval 1.091-2.517) were found to be independently associated with increased risk of atrial fibrillation in the post-operative period. CONCLUSIONS: After adjusting for confounders HbA1c levels do not independently predict risk of atrial fibrillation after off-pump coronary artery bypass grafting.

Autophagy-independent induction of LC3B through oxidative stress reveals its non-canonical role in anoikis of ovarian cancer cells.

Cancer cells display abnormal redox metabolism. Autophagy, anoikis and reactive oxygen species (ROS) play a regulatory role during metastasis. LC3 is a well-known essential molecule for autophagy. Therefore, we wanted to explore the molecular interplay between autophagy, anoikis, and ROS in relation to LC3B. We observed enhanced LC3B level along with increased expression of p62 and modulation of other autophagy-related molecules (Atg 3, 5, 7, 12, 16L1 and Beclin1) by inducing oxidative-stress in ovarian cancer cells using a ROS-producing pro-oxidant molecule. Surprisingly, enhanced LC3B was unable to induce autophagosome formation rather promoted anoikis. ROS-induced inhibition of autophagosome-formation is possibly due to the instability of autophagy initiator, ULK1 complex. Moreover, such upregulation of LC3B via ROS enhanced several apoptotic molecules. Silencing LC3B reduced these apoptotic molecules and increased when overexpressed, suggesting its role in apoptosis. Furthermore, LC3B-dependent apoptosis was decreased by inhibiting ROS, indicating a possible link between ROS, LC3B, and apoptosis. Additionally, ROS-induced enhanced LC3B promoted detachment-induced cell death (anoikis). This was further reflected by reduced cell adhesion molecules (integrin-ß3 and focal adhesion kinase) and mesenchymal markers (snail and slug). Our in vitro experimental data was further confirmed in primary tumors developed in syngeneic mice, which also showed ROS-mediated LC3B enhancement along with reduced autophagosomes, integrin-ß3 and focal adhesion kinase ultimately leading to the decreased tumor mass. Additionally, primary cells from high-grade serous carcinoma patient's ascites exhibited LC3B enhancement and autophagy inhibition through ROS which provided a clinical relevance of our study. Taken together, this is the first evidence for a non-canonical role of LC3B in promoting anoikis in contrast to autophagy and may, therefore, consider as a potential therapeutic target molecule in ovarian cancer. Taken together, autophagy-inhibition may be an alternative approach to induce apoptosis/anoikis in cancer.

Mahanine drives pancreatic adenocarcinoma cells into endoplasmic reticular stress-mediated apoptosis through modulating sialylation process and Ca2+-signaling.

Endoplasmic reticulum (ER) stress results from protein unfolding/misfolding during cellular maturation, which requires a coordinated action of several chaperones and enzymes and Ca2+ signalling. ER-stress possibly has a positive effect on survival of pancreatic cancer cell. Therefore, detailed insights into this complex signaling network are urgently needed. Here, we systematically analyzed the impact of ER stress-mediated unfolded protein response (UPR) and Ca2+-signaling cross-talk for the survival of pancreatic adenocarcinoma (PDAC) cells. We observed enhanced ER activity and initiation of UPR signaling induced by a carbazole alkaloid (mahanine). This event triggers a time-dependent increase of intracellular Ca2+ leakage from ER and subsequently Ca2+ signaling induced by enhanced reactive oxygen species (ROS) produced by this pro-oxidant agent. In addition, we observed an altered glycosylation, in particular with regard to reduced linkage-specific sialic acids possibly due to decreased sialyltransferase activity. Changes in sialylation entailed enhanced expression of the ganglioside GD3 in the treated cells. GD3, an inducer of apoptosis, inhibited pancreatic xenograft tumor. Taken together, our study describes a molecular scenario how PDAC cells are driven into apoptosis by mahanine by UPR-driven ER stress-associated and ROS-mediated calcium signaling and possibly defective sialylation.

Benefits of Using High-Volume-Low-Pressure Tracheal Tube in Children Undergoing Congenital Cardiac Surgery: Evidence From a Prospective Randomized Study.

BACKGROUND: In the past 2 decades, usage of high-volume-low-pressure microcuffed tracheal tubes in smaller children has increased. However, there is paucity of evidence of its usage in smaller children undergoing congenital cardiac surgery. The aim of this study was to assess if microcuff endotracheal tubes in neonates and younger children undergoing congenital cardiac surgery is associated with better outcomes than uncuffed tubes. METHODS: We carried out this single-center, prospective, randomized study between June and November 2016. Eighty patients were randomized into those receiving microcuff tracheal tubes and conventional uncuffed tubes. Primary outcome was stridor postextubation. Secondary outcomes measured included number of tube changes, volume of anesthetic gases required, and cost incurred. RESULTS: The 2 groups were comparable in terms of baseline characteristics and duration of intubation. Incidence of stridor was significantly higher in conventional uncuffed tubes (12 [30%] vs 4 [10%]; P = .04) and so was the number of tube changes required (17/40 [42.5%] vs 2/40 [5%]; P ≤ .001). Tube change was associated with more than 3-fold risk of stridor (odds ratio = 3.92; 95% confidence interval = 1.23-12.43). Isoflurane (29.14 ± 7.01 mL vs19.2 ± 4.81 mL; P < .0001) and oxygen flow requirement ( P < .0001) and the resultant cost (7.46 ± 1.4 vs 5.77 ± 1.2 US$; P < .0001) were all significantly higher in the conventional uncuffed group. CONCLUSION: Microcuff pediatric tracheal tube is associated with significantly lower incidence of stridor, tube changes, and anesthetic gas requirement. This leads to significant cost reduction that offsets the higher costs associated with usage of a microcuff tracheal tube.

A Glycomic Approach Towards Identification of Signature Molecules in CD34+ Haematopoietic Stem Cells from Umbilical Cord Blood.

Umbilical cord blood (UCB) is a powerful storehouse for normal CD34+ haematopoietic stem cells (HSCs), often used for allogeneic bone marrow (BM) transplantation in malignant and non-malignant diseases. The glycomic especially the sialoglycomic aspect of these HSCs has been unravelled in this study. Cell surface expression of the glycans with the related enzymatic activities has been compared with the BM of childhood acute lymphoblastic leukaemia, a common BM-associated malignancy. An enhanced cell surface expression of α2,3-linked sialic acid, P- and E-selectins, and intercellular adhesion molecule along with reduced expression of L-selectin distinguishes CD34+ HSCs of UCB from leukaemic samples. More importantly, high expression of O-acetylated sialoglycoproteins, a hallmark of lymphoblasts, is drastically reduced in the CD34+ HSCs of UCB and is substantiated by the low activity of sialylate-O-acetyltransferase and high sialidase activity. In contrast, a significant variation is evident in the expression of sialic acid, α2,6-linked sialic acids, and the sialyltransferase activity. Taken together, these studies indicate a few signature molecules, forming a unique glycomic template, which may be a potential indicator, reassuring the normal profile of these stem cells, to be used for future transplantation.

Preoperative Glycosylated Hemoglobin: A Risk Factor for Patients Undergoing Coronary Artery Bypass.

BACKGROUND: The prevalence of diabetes in the population of patients presenting with coronary artery disease continues to rise. The aim of this study was to assess whether high Glycosylated hemoglobin (HbA1c) was associated with adverse outcomes in patients undergoing elective coronary artery bypass grafting. METHODS: A retrospective observational study on prospectively collected data in 4,678 patients undergoing elective, isolated coronary artery bypass graft procedures in a single institution over a 4-year period was conducted. Patients were grouped into those with adequate preoperative control of hyperglycemia (HbA1c <6.5%) and those with suboptimal control (HbA1c ≥6.5%). Multivariable analysis using HbA1c as a binary independent variable was undertaken in the whole group. A subgroup analysis in diabetic patients and in nondiabetic patients was performed. The effect of HbA1c on outcomes at higher levels (HbA1c ≥8.0% and HbA1c ≥9.0%) was also assessed. RESULTS: A total of 4,678 patients (mean age, 58.8; male, 4,254) were included in the study. HbA1c was less than 6.5% in 2,476 (52.93%) patients and 6.5% or higher in 2,202 (47.07%) patients. On multivariate analysis, there was no difference in mortality rates between the groups (odds ratio, 1.36; 95% confidence interval [CI], 0.95 to 1.953; p = 0.08). Overall, an HbA1c of 6.5% or higher was an independent risk factor for respiratory complications (odds ratio, 1.05; 95% CI, 1.008 to 4.631; p = 0.01) and sternal dehiscence (odds ratio, 2.161; 95% CI, 1.008 to 4.63; p = 0.04). An association between HbA1c levels and adverse outcomes was not seen in nondiabetic patients. No additional adverse postoperative complications were seen with increasing HbA1c levels (HbA1c ≥8.0% and HbA1c ≥9.0%). CONCLUSIONS: An HbA1c level of 6.5% or higher in patients presenting for coronary artery bypass grafting was associated with a significant increase in the incidence of deep sternal wound infection and respiratory complications.

Disialoganglioside GD3-synthase over expression inhibits survival and angiogenesis of pancreatic cancer cells through cell cycle arrest at S-phase and disruption of integrin-ß1-mediated anchorage.

Gangliosides play important roles in the development, differentiation and proliferation of mammalian cells. They bind to other cell membrane components through their terminal sialic acids. Different gangliosides influence cellular functions based on the positions and linkages of sialic acids. Expression of gangliosides mainly depends on the status of sialic acid-modulatory enzymes, such as different types of sialyltransferases and sialidases. One such sialyltransferase, disialoganglioside GD3 synthase, is specifically responsible for the production of GD3. Pancreatic ductal adenocarcinoma, making up more than 90% of pancreatic cancers, is a fatal malignancy with poor prognosis. Despite higher sialylation status, the disialoganglioside GD3 level is very low in this cancer. However, the exact status and function of this disialoganglioside is still unknown. Here, we intended to study the intracellular mechanism of disialoganglioside GD3-induced apoptosis and its correlation with the adhesion and angiogenic pathways in pancreatic cancer. We demonstrated that disialoganglioside GD3 synthase-transfected cells showed enhanced apoptosis and it caused the arrest of these cells in the S-phase of the cell cycle. Integrins, a family of transmembrane proteins play important role in cell-cell recognition, invasion, adhesion and migration. disialoganglioside GD3 co-localised with integrin-ß1 and thereby inhibited it's downstream signalling in transfected cells. Transfected cells exhibited inhibition of cell adhesion with extracellular matrix proteins. Enhanced GD3 expression down regulated angiogenesis-regulatory proteins and inhibited epidermal growth factor/vascular endothelial growth factor-driven angiogenic cell growth in these cells. Taken together, our study provides support for the GD3-induced cell cycle arrest, disruption of integrin-ß1-mediated anchorage, inhibition of angiogenesis and thereby induced apoptosis in pancreatic cancer cells.

Coupling G2/M arrest to the Wnt/ß-catenin pathway restrains pancreatic adenocarcinoma.

ß-catenin plays a pivotal role in organogenesis and oncogenesis. Alterations in ß-catenin expression are common in pancreatic cancer, which is an extremely aggressive malignancy with a notably poor prognosis. In this report, we analyzed the apoptotic activity of withanolide-D (witha-D), a steroidal lactone that was purified from an Indian medicinal plant, Withania somnifera, and its underlying mechanism of action. Witha-D induced apoptosis in pancreatic ductal adenocarcinoma cells by prompting cell-cycle arrest at the G2/M phase. This lactone abrogated ß-catenin signaling in these cells regardless of disease grade, mutational status, and gemcitabine sensitivity. Witha-D also upregulated E-cadherin in most cells, thereby supporting the inversion of the epithelial-mesenchymal transition. Furthermore, the Akt/Gsk3ß kinase cascade was identified as a critical mediator of G2/M regulation and ß-catenin signaling. Witha-D deactivated Akt, which failed to promote Gsk3ß deactivation phosphorylation. Consequently, activated Gsk3ß facilitated ß-catenin destruction in pancreatic carcinoma cells. The knockdown of Chk1 and Chk2 further activated Akt and reversed the molecular signal. Taken together, the results of the current study represent the first evidence of ß-catenin signal crosstalk during the G2/M phase by functionally inactivating Akt via witha-D treatment in pancreatic cancer cells. In conclusion, this finding suggests the potential identification of a new lead molecule in the treatment of pancreatic adenocarcinoma.

Identification and analysis of o-acetylated sialoglycoproteins.

5-N-acetylneuraminic acid, commonly known as sialic acid (Sia), constitutes a family of N- and O-substituted 9-carbon monosaccharides. Frequent modification of O-acetylations at positions C-7, C-8, or C-9 of Sias generates a family of O-acetylated sialic acid (O-AcSia) and plays crucial roles in many cellular events like cell-cell adhesion, proliferation, migration, etc. Therefore, identification and analysis of O-acetylated sialoglycoproteins (O-AcSGPs) are important. In this chapter, we describe several approaches for successful identification of O-AcSGPs. We broadly divide them into two categories, i.e., invasive and noninvasive methods. Several O-AcSias-binding probes are used for this purpose. Detailed methodologies for step-by-step identification using these probes have been discussed. We have also included a few invasive analytical methods for identification and quantitation of O-AcSias. Several indirect methods are also elaborated for such purpose, in which O-acetyl group from sialic acids is initially removed followed by detection of Sias by several approaches. For molecular identification, we have described methods for affinity purification of O-AcSGPs using an O-AcSias-binding lectin as an affinity matrix followed by sequencing using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF) mass spectroscopy (MS). In spite of special attention, loss of O-acetyl groups due to its sensitivity towards alkaline pH and high temperature along with migration of labile O-acetyl groups from C7-C8-C9 during sample preparation is difficult to avoid. Therefore there is always a risk for underestimation of O-AcSias.

Regulation of O-acetylation of sialic acids by sialate-O-acetyltransferase and sialate-O-acetylesterase activities in childhood acute lymphoblastic leukemia.

Enhanced expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) and 9-O-acetylated disialoganglioside (9-OAcGD3) was observed on lymphoblasts of childhood acute lymphoblastic leukemia (ALL). Sialate-O-acetyltransferase (SOAT) and sialate-O-acetylesterase (SIAE) are the two main enzymes responsible for the quantity of the O-acetyl ester groups on sialic acids (Sias). We have earlier shown an enhanced level of SOAT activity, capable of transferring acetyl groups to Sias of glycoconjugates in the microsomes of lymphoblasts of these children. We further observed a decreased SIAE activity in both lysosomal and cytosolic fractions of ALL cell lines and primary cells from bone marrow of patients compared with peripheral blood mononuclear cells from healthy donors, which preferentially hydrolyze O-acetyl groups at C-9 of Sia. The level of O-acetylated Sias in the cytosolic and the lysosomal fractions showed a good correlation with SIAE activity in the corresponding fractions. The apparent K(M) values for SIAE in the lysosomal and the cytosolic fractions from lymphoblasts of ALL patients are 0.38 and 0.39 mM, respectively. These studies demonstrate that both SIAE and SOAT activities seem to be responsible for the enhanced level of Neu5,9Ac(2) in lymphoblasts, which is a hallmark in ALL. This was subsequently confirmed by using an enzyme-linked immunosorbent assay that also demonstrated a steady decline in SOAT activities even in cell lysates of lymphoblasts during successful chemotherapy, like radioactive methods have shown earlier.

Mobilization of lymphoblasts from bone marrow to peripheral blood in childhood acute lymphoblastic leukaemia: role of 9-O-acetylated sialoglycoproteins.

Childhood acute lymphoblastic leukaemia is characterized by aberrant proliferation and accumulation of malignant lymphoblasts in bone marrow (BM), followed by their migration into circulation. An enhanced cell-surface expression of ALL-associated 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) was demonstrated. Present investigation reports a positive correlation between the increased density of Neu5,9Ac(2)-GPs on lymphoblasts and their mobilization from BM involving enhanced Neu5,9Ac(2) on CD45 demonstrating modulation of FAK and ERK molecules. In contrast, a small population of cells, identified as haematopoietic precursors, with comparatively lesser Neu5,9Ac(2)-GPs showed increased binding towards BM stroma. Thus, Neu5,9Ac(2)-GPs is a developmentally regulated oncofoetal antigen, whose up-regulation is imperative in the interaction between lymphoblasts and BM stroma, governing their mobilization into circulation.

Withanolide D induces apoptosis in leukemia by targeting the activation of neutral sphingomyelinase-ceramide cascade mediated by synergistic activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase.

BACKGROUND: Ceramide is an important second messenger that has diverse cellular and biological effect. It is a specific and potent inducer of apoptosis and suppressor of cell growth. In leukemia, chemoresistance generally developed due to deregulated ceramide metabolism. In combinatorial treatment strategies of leukemia, few components have the capability to increases ceramide production. Manipulation in ceramide production by physiological and pharmacological modulators therefore will give additive effect in leukemia chemotherapy. RESULTS: Here, we show that Withanolide D (C4ß-C5ß,C6ß-epoxy-1-oxo-,20ß, dihydroxy-20S,22R-witha-2,24-dienolide; WithaD), a pure herbal compound isolated from Withania somnifera could effectively induces apoptosis in a dose and time dependant manner both in myeloid (K562) and lymphoid (MOLT-4) cells being nontoxic to normal lymphocytes and control proliferative cells. WithaD potentially augment ceramide production in these cells. Downstream of ceramide, WithaD acted on MKK group of proteins and significantly increased JNK and p38MAPK phosphorylation. Pharmacological inhibition of p38MAPK and JNK proves their cooperative action on WithaD-induced cell death. Dissecting the cause of ceramide production, we found activation of neutral sphingomyelinase and showed neutral-sphingomyelinase 2 (N-SMase 2) is a critical mediator of WithaD-induced apoptosis. Knockdown of N-SMase 2 by siRNA and inhibitor of N-SMase (GW4869) significantly reduced WithaD-induced ceramide generation and phosphorylation of MKK4 and MKK3/6, whereas phosphorylation of MKK7 was moderately regulated in leukemic cells. Also, both by silencing of N-SMase 2 and/or blocking by GW4869 protects these cells from WithaD-mediated death and suppressed apoptosis, whereas Fumonisin B1, an inhibitor of ceramide synthase, did not have any effect. Additionally, WithaD effectively induced apoptosis in freshly isolated lymphoblasts from patients and the potent cell killing activity was through JNK and p38MAPK activation. CONCLUSION: Our results demonstrate that WithaD enhance the ceramide accumulation by activating N-SMase 2, modulate phosphorylation of the JNK and p38MAPK and induced apoptosis in both myeloid and lymphoid cells along with primary cells derived from leukemia patients. Taken together, this pure herbal compound (WithaD) may consider as a potential alternative tool with additive effects in conjunction with traditional chemotherapeutic treatment, thereby accelerate the process of conventional drug development.

Sialic acids acquired by Pseudomonas aeruginosa are involved in reduced complement deposition and siglec mediated host-cell recognition.

The opportunism of Pseudomonas aeruginosa (PA) in immunocompromised hosts prompted us to explore the potential role of sialic acids (Sia) in this phenomenon. Culture of PA in the presence of exogenous Sia resulted in linkage-specific incorporation of Sia which was associated with decreased complement deposition on the bacteria. Sia acquired by PA mediated enhanced binding of bacteria to recombinant-CHO cells expressing human siglec-7 or siglec-9, as well as to human NK-cells and monocytes naturally expressing these siglecs. Therefore, Sia may be acquired by PA in the host and contribute to bacterial pathogenicity and host-cell interactions via reduction of complement deposition and siglec-dependent recognition.

Down regulation of membrane-bound Neu3 constitutes a new potential marker for childhood acute lymphoblastic leukemia and induces apoptosis suppression of neoplastic cells.

Membrane-linked sialidase Neu3 is a key enzyme for the extralysosomal catabolism of gangliosides. In this respect, it regulates pivotal cell surface events, including trans-membrane signaling, and plays an essential role in carcinogenesis. In this report, we demonstrated that acute lymphoblastic leukemia (ALL), lymphoblasts (primary cells from patients and cell lines) are characterized by a marked down-regulation of Neu3 in terms of both gene expression (-30 to 40%) and enzymatic activity toward ganglioside GD1a (-25.6 to 30.6%), when compared with cells from healthy controls. Induced overexpression of Neu3 in the ALL-cell line, MOLT-4, led to a significant increase of ceramide (+66%) and to a parallel decrease of lactosylceramide (-55%). These events strongly guided lymphoblasts to apoptosis, as we assessed by the decrease in Bcl2/Bax ratio, the accumulation of Neu3 transfected cells in the sub G0-G1 phase of the cell cycle, the enhanced annexin-V positivity, the higher cleavage of procaspase-3. Therefore, the reduced expression of Neu3 in ALL could help lymphoblasts to survive, maintaining the cellular content of ceramide below a critical level. Interestingly, we found that Neu3 activity varied in relation to disease progression, increasing in clinical remission after chemotherapy, and decreasing again in patients that relapsed. In addition, a negative correlation was observed between Neu3 expression and the percentage of the ALL marker 9-OAcGD3 positive cells. Consequently, Neu3 could represent a new potent biomarker in childhood ALL, to assess the efficacy of therapeutic protocols and to rapidly identify an eventual relapse.

RETRACTED: Sialic acids, important constituents and selective recognition factors of Pseudomonas aeruginosa, an opportunistic pathogen.

This article has been retracted.

High level of sialate-O-acetyltransferase activity in lymphoblasts of childhood acute lymphoblastic leukaemia (ALL): enzyme characterization and correlation with disease status.

Previous studies had established an over-expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). Here, we report the discovery and characterization of sialate-O-acetyltransferase enzyme in ALL-cell lines and lymphoblasts from bone marrow of children diagnosed with B- and T-ALL. We observed a positive correlation between the enhanced sialate-O-acetyltransferase activity and the enhanced expression of Neu5,9Ac(2)-GPs in these lymphoblasts. Sialate-O-acetyltransferase activity in cell lysates or microsomal fractions of lymphoblasts of patients was always higher than that in healthy donors reaching up to 22-fold in microsomes. Additionally, the V (max) of this enzymatic reaction with AcCoA was over threefold higher in microsomal fractions of lymphoblasts. The enzyme bound to the microsomal fractions showed high activity with CMP-N-acetylneuraminic acid, ganglioside GD3 and endogenous sialic acid as substrates. N-acetyl-7-O-acetylneuraminic acid was the main reaction product, as detected by radio-thin-layer chromatography and fluorimetrically coupled radio-high-performance liquid chromatography. CMP and coenzyme A inhibited the microsomal enzyme. Sialate-O-acetyltransferase activity increased at the diagnosis of leukaemia, decreased with clinical remission and sharply increased again in relapsed patients as determined by radiometric-assay. A newly-developed non-radioactive ELISA can quickly detect sialate-O-acetyltransferase, and thus, may become a suitable tool for ALL-monitoring in larger scale. This is the first report on sialate-O-acetyltransferase in ALL being one of the few descriptions of an enzyme of this type in human.

Withaferin A induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells of lymphoid and myeloid origin through mitochondrial death cascade.

Withaferin A (WA) is present abundantly in Withania somnifera, a well-known Indian medicinal plant. Here we demonstrate how WA exhibits a strong growth-inhibitory effect on several human leukemic cell lines and on primary cells from patients with lymphoblastic and myeloid leukemia in a dose-dependent manner, showing no toxicity on normal human lymphocytes and primitive hematopoietic progenitor cells. WA-mediated decrease in cell viability was observed through apoptosis as demonstrated by externalization of phosphatidylserine, a time-dependent increase in Bax/Bcl-2 ratio; loss of mitochondrial transmembrane potential, cytochrome c release, caspases 9 and 3 activation; and accumulation of cells in sub-G0 region based on DNA fragmentation. A search for the downstream pathway further reveals that WA-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2 and HSP27 in leukemic cells. The RNA interference of p38MAPK protected these cells from WA-induced apoptosis. The RNAi knockdown of p38MAPK inhibited active phosphorylation of p38MAPK, Bax expression, activation of caspase 3 and increase in Annexin V positivity. Altogether, these findings suggest that p38MAPK in leukemic cells promotes WA-induced apoptosis. WA caused increased levels of Bax in response to MAPK signaling, which resulted in the initiation of mitochondrial death cascade, and therefore it holds promise as a new, alternative, inexpensive chemotherapeutic agent for the treatment of patients with leukemia of both lymphoid and myeloid origin.

O-acetylation of GD3 prevents its apoptotic effect and promotes survival of lymphoblasts in childhood acute lymphoblastic leukaemia.

We have previously demonstrated induction of O-acetylated sialoglycoproteins on lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). These molecules promote survival of lymphoblasts by preventing apoptosis. Although O-acetylated sialoglycoproteins are over expressed, the status of O-acetylation of gangliosides and their role in lymphoblasts survival remains to be explored in ALL patients. Here, we have observed enhanced levels of 9-O-acetylated GD3 (9-O-AcGD3) in the lymphoblasts of patients and leukaemic cell line versus disialoganglioside GD3 in comparison to the normal cells. Localization of GD3 and 9-O-AcGD3 on mitochondria of patient's lymphoblasts has been demonstrated by immuno-electron microscopy. The exogenous administration of GD3-induced apoptosis in lymphoblasts as evident from the nuclear fragmentation and sub G0/G1 apoptotic peak. In contrast, 9-O-AcGD3 failed to induce such apoptosis. We further explored the mitochondria-dependent pathway triggered during GD3-induced apoptosis in lymphoblasts. GD3 caused a time-dependent depolarization of mitochondrial membrane potential, release of cytochrome c and 7.4- and 8-fold increased in caspase 9 and caspase 3 activity respectively. However, under identical conditions, an equimolar concentration of 9-O-AcGD3 failed to induce similar effects. Interestingly, 9-O-AcGD3 protected the lymphoblasts from GD3-induced apoptosis when administered in equimolar concentrations simultaneously. In situ de-O-acetylation of 9-O-AcGD3 with sodium salicylate restores the GD3-responsiveness to apoptotic signals. Although both GD3 and 9-O-acetyl GD3 localize to mitochondria, these two structurally related molecules may play different roles in ALL-disease biology. Taken together, our results suggest that O-acetylation of GD3, like that of O-acetylated sialoglycoproteins, might be a general strategy adopted by leukaemic blasts towards survival in ALL.