Suchandra Chowdhury

Arsenic induced apoptosis in malignant melanoma cells is enhanced by menadione through ROS generation, p38 signaling and p53 activation

IntroductionResistance to apoptosis is a prominent feature of melanoma. Pharmacological concentration of arsenic in combination with a widely known oxidant, menadione was explored in this study to synergistically sensitize malignant melanoma cells to apoptosis. The molecular mechanism of apoptosis and the signaling-pathways involved were thoroughly investigated.Materials methods and resultsMenadione synergized NaAsO2 to significantly increase ROS generation and facilitate the major apoptotic signaling events: alteration of mitochondrial membrane potential, cytochrome c release and anti-apoptotic protein Bcl-2 down-regulation and subsequent activation of caspase-9 and caspase-3 followed by poly-ADP-ribose polymerase-1 cleavage. Antioxidant N-acetyl-l-cysteine antagonized these events. Investigation of the signaling-pathway revealed significant suppression of AP-1 activity but not NF-κB upon NaAsO2 and menadione application. An increase in p38 phosphorylation and p53 protein expression did also dictate the apoptotic response. Suppression of p38 activation with SB203580 and inhibition of p53 expression by siRNA attenuated apoptosis. Transfection of p53, in p53 null HCT cells augmented the apoptotic events. Moreover, the treatment also led to tumor size reduction in BALB/c mice developed by intra-dermal B16 mouse melanoma cell injection; however, it had no detectable pro-proliferative or pro-apoptotic effect on non-tumor keratinocytes, normal fibroblasts or PBMC.ConclusionThis study thus provides an insight into innovative mechanisms of melanoma sensitization, a proper cure against which is still elusive. Taken together, our data also provides the first evidence of arsenic activity accentuation by menadione through modulation of specific signaling-pathways.

Role of C-reactive protein in complement-mediated hemolysis in Malaria

Human C-reactive protein (CRP) is a clinically important classical acute phase protein. Although CRP has been reported to bind with many nucleated cells, the direct binding of CRP to erythrocytes in diseases remains largely unexplored. The main focus of the present study was to investigate the binding of disease-specific CRP to erythrocytes of same patients. Distinct molecular variant of disease-specific CRP was affinity purified from sera of malaria patients (CRPMal). This CRP showed strong binding with malaria erythrocytes (RBCMal) as confirmed by flow cytometric analysis (FACS), enzyme-linked immunosorbent assays (ELISA), and radio binding assays. Calcium and phosphoryl choline (PC) were found to be essential for this interaction. A 2.3-fold increased binding of induced CRP to RBCMal as compared to normal erythrocytes (RBCN) confirmed disease-specificity. Preincubation of RBCMal with unconjugated CRP showed 3–5 fold inhibition. The association constant of CRP and RBCMal was 4.7 × 106 cpm/μg with the corresponding number of receptors/cell being 4.3 × 105. The effector function of CRPMal has been demonstrated by its potency to activate the complement pathway. An optimal dose of 10 μg/ml of CRP induced three-fold higher hemolysis of patient erythrocytes as compared to RBCN. These studies provide direct evidence for an important phagocytic functional interaction of this acute-phase protein by triggering the CRP-complement pathway after the binding of CRPMal with RBCMal. Hemolysis as triggered by this pathway may be one of the causative factors of anemia, a common clinical manifestation of this disease.

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 (Neu5,9Ac2-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 Neu5,9Ac2-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 Vmax 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.

Flow-cytometric monitoring of disease-associated expression of 9-O-acetylated sialoglycoproteins in combination with known CD antigens, as an index for MRD in children with acute lymphoblastic leukaemia: a two-year longitudinal follow-up study

BackgroundOver expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs, abbreviated as OAcSGP) has been demonstrated as a disease-associated antigen on the lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). Achatinin-H, a lectin, has selective affinity towards terminal 9-O-acetylated sialic acids-α2-6-Nacetylated galactosamine. Exploring this affinity, enhanced expression of OAcSGP was observed, at the onset of disease, followed by its decrease with chemotherapy and reappearance with relapse. In spite of treatment, patients retain the diseased cells referred to as minimal residual disease (MRD) responsible for relapse. Our aim was to select a suitable template by using the differential expression of OAcSGP along with other known CD antigens to monitor MRD in peripheral blood (PB) and bone marrow (BM) of Indian patients with B- or T-ALL during treatment and correlate it with the disease status.MethodsA two-year longitudinal follow-up study was done with 109 patients from the onset of the disease till the end of chemotherapy, treated under MCP841protocol. Paired samples of PB (n = 1667) and BM (n = 999) were monitored by flow cytometry. Three templates selected for this investigation were OAcSGP+CD10+CD19+ or OAcSGP+CD34+CD19+ for B-ALL and OAcSGP+CD7+CD3+ for T-ALL.ResultsUsing each template the level of MRD detection reached 0.01% for a patient in clinical remission (CR). 81.65% of the patients were in CR during these two years while the remaining relapsed. Failure in early clearance of lymphoblasts, as indicated by higher MRD, implied an elevated risk of relapse. Soaring MRD during the chemotherapeutic regimen predicted clinical relapse, at least a month before medical manifestation. Irrespective of B- or T-lineage ALL, the MRD in PB and BM correlated well.ConclusionA range of MRD values can be predicted for the patients in CR, irrespective of their lineage, being 0.03 ± 0.01% (PB) and 0.05 ± 0.015% (BM). These patients may not be stated as normal with respect to the presence of MRD. Hence, MRD study beyond two-years follow-up is necessary to investigate further reduction in MRD, thereby ensuring their disease-free survival. Therefore, we suggest use of these templates for MRD detection, during and post-chemotherapy for proper patient management strategies, thereby helping in personalizing the treatment.

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.

9- O -Acetyl GD3 in Lymphoid and Erythroid Cells

Sialic acids are electronegatively charged sugars that contribute to the enormous structural diversity of complex carbohydrates, which are major constituents of mostly proteins and lipids of cell membranes and secreted macromolecules. They are usually positioned at the outer end of these molecules and thus are well suited for interacting with other cells, pathogens, or molecules in the cell environment. Sialic acids are 9-carbon-containing monosaccharides, and the structural diversity of glycan chains is further increased by the various modifications of sialic acids [1]. Amongst 50 known derivatives of sialic acids, 7-, 8-, and 9-O-acetylated derivatives (O-AcSA) are important constituents of the cell membrane and are known to influence many physiological and pathological processes [1, 2], including cell–cell adhesion, signaling, differentiation, and metastasis [3–6]. However, as O-acetyl esters from positions C-7 and C-8 spontaneously migrate to C-9, even under physiologic conditions, O-acetylation at C-9 is considered the most common biologically occurring modification [7]. The appearance of O-acetylated sialic acids on glycoproteins or glycolipids is cell-type specific and developmentally regulated, their synthesis and turnover being a highly orchestrated phenomenon. O-acetylation can have a significant role in cell physiology and can alter the functional effects of important molecular determinants in various disease conditions. In this chapter, we deal with the O-acetylation of glycosphingolipids (GSLs), specifically GD3 in both erythroid and lymphoid cells.

9-O-acetylated sialic acids differentiating normal haematopoietic precursors from leukemic stem cells with high aldehyde dehydrogenase activity in children with acute lymphoblastic leukaemia.

Childhood acute lymphoblastic leukaemia (ALL) originates from mutations in haematopoietic progenitor cells (HPCs). For high-risk patients, treated with intensified post-remission chemotherapy, haematopoietic stem cell (HSC) transplantation is considered. Autologous HSC transplantation needs improvisation till date. Previous studies established enhanced disease-associated expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs) on lymphoblasts of these patients at diagnosis, followed by its decrease with clinical remission and reappearance with relapse. Based on this differential expression of Neu5,9Ac2-GPs, identification of a normal HPC population was targeted from patients at diagnosis. This study identifies two distinct haematopoietic progenitor populations from bone marrow of diagnostic ALL patients, exploring the differential expression of Neu5,9Ac2-GPs with stem cell (CD34, CD90, CD117, CD133), haematopoietic (CD45), lineage-commitment (CD38) antigens and cytosolic aldehyde dehydrogenase (ALDH). Normal haematopoietic progenitor cells (ALDH+SSCloCD45hiNeu5,9Ac2-GPsloCD34+CD38−CD90+CD117+CD133+) differentiated into morphologically different, lineage-specific colonies, being crucial for autologous HSC transplantation while leukemic stem cells (ALDH+SSCloCD45loNeu5,9Ac2-GPshiCD34+CD38+CD90−CD117−CD133−) lacking this ability can be potential targets for minimal residual disease detection and drug-targeted immunotherapy.