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.
Here, we show that Withanolide D (C4β-C5β,C6β-epoxy-1-oxo-,20β, 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.
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.
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.
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.
The SARS-CoV-2 (COVID-19) pandemic is a worldwide public health emergency with widespread impact on health care delivery. Unforeseen challenges have been noted during administration of usual ...haematology care in these unusual COVID-19 times. Medical services have been overstretched and frontline health workers have borne the brunt of COVID-19 pandemic. Movement restrictions during lockdown prevented large sections of population from accessing health care, blood banks from holding blood drives, and disrupted delivery of diagnostic hematology services. The disruption in hematology care due to COVID-19 pandemic in India has been disproportionately higher compared to other subspecialities as hematology practice in India remains restricted to major cities. In this review we chronicle the challenges encountered in caring for hematology patients during the COVID-19 pandemic in India and put forth recommendations for minimizing their impact on provision of hematology care with special emphasis on hematology practice in lower and middle income countries (LMICs).
•Identification of 79 differentially regulated proteins in B-ALL patient CD19+ cells.•PCA revealed genetic anomaly based variations in malignant CD19+ B-cell proteome.•De-regulation of metabolic, ...cytoskeletal, stress response & signalling proteins.•Candidate proteins showing characteristic de-regulation in B-ALL B-cell proteomes.
With an objective of defining potential diagnostic and/or therapeutic markers and the mechanism of cellular transformation, we present a comparative proteomic study of B-lymphocytes from B-cell acute lymphoblastic leukemia (B-ALL) patients and normal controls, using two-dimensional gel electrophoresis and MALDI ToF/ToF tandem mass spectrometry. Our study led to the identification of 79 differentially regulated proteins in the malignant cells including proteins participating in proteostasis, cytoskeletal organization, redox homeostasis, and signal transduction pathways relevant to leukemogenesis. Principal component analysis displayed immunophenotype-/genotype-dependent variations in the malignant cell proteome. Our study adds new insights to the leukemogenic B-cell biology and prognostic stratifications.
Over 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-alpha2-6-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.
A 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.
Using 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.
A 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.
Summary
Regulatory T (Treg) cells act to suppress activation of the immune system and thereby maintain immunological homeostasis and tolerance to self‐antigens. The frequency and suppressing activity ...of Treg cells in general are high in different malignancies. We wanted to identify the role and regulation of CD4+ CD25+ FoxP3+ Treg cells in B‐cell acute lymphoblastic leukaemia (B‐ALL). We have included patients at diagnosis (n = 54), patients in clinical remission (n = 32) and normal healthy individuals (n = 35). These diagnosed patients demonstrated a lower number of CD4+ CD25+ cells co‐expressing a higher level of FoxP3, interleukin‐10, transforming growth factor‐β and CD152/CTLA‐4 than the normal population. Treg cells from patients showed a higher suppressive capability on CD4+ CD25− responder T (Tresp) cells than normal. The frequency and immunosuppressive potential of CD4+ CD25+ FoxP3+ Treg cells became high with the progression of malignancy in B‐ALL. Relative distribution of Tresp and Treg cells was only ~5 : 1 in B‐ALL but ~35 : 1 in normal healthy individuals, further confirming the elevated immunosuppression in patients. A co‐culture study at these definite ex vivo ratios, indicated that Treg cells from B‐ALL patients exhibited higher immunosuppression than Treg cells from normal healthy individuals. After chemotherapy using the MCP841 protocol, the frequency of CD4+ CD25+ cells was gradually enhanced with the reduction of FoxP3, interleukin‐10 positivity corresponded with disease presentation, indicating reduced immunosuppression. Taken together, our study indicated that the CD4+ CD25+ FoxP3+ Treg cells played an important role in immunosuppression, resulting in a positive disease‐correlation in these patients. To the best of our knowledge, this is the first detailed report on the frequency, regulation and functionality of Treg cells in B‐ALL.
Abstract Altered sialylation occurs in essentially all types of human and experimental cancers. Although, aberrant sialylation is believed to mainly due to altered sialyltransferase (ST) level, so ...far, expression pattern of different STs in acute lymphoblastic leukemia has never been investigated. Accordingly, the aim of our study was to monitor the changes in mRNA expression of ST6Gal I, ST3Gal V and ST8Sia I in patients by real-time PCR, which may provide prognostic information useful in defining appropriate therapeutic options. Our data demonstrated that ST6Gal I and ST3Gal V mRNA were up-regulated in lymphoblasts whereas its presence was negligible in non-malignant donors. In contrast, ST8SiaI was downregulated in patients. The extents of linkage-specific sialylation of glycoconjugates were found to be associated with disease establishment. Additionally, ST6Gal I and ST3Gal V were positively correlated with the high risk of the disease ( P = 0.0032 and 0.0016). This differential ST level can be used as biomarker with the molecular method of quantitative PCR and may be useful to discriminate normal and cancer patients.
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