Bak Compensated for Bax in p53-null Cells to Release Cytochrome c for the Initiation of Mitochondrial Signaling during Withanolide D-Induced Apoptosis

The goal of cancer chemotherapy to induce multi-directional apoptosis as targeting a single pathway is unable to decrease all the downstream effect arises from crosstalk. Present study reports that Withanolide D (WithaD), a steroidal lactone isolated from Withania somnifera, induced cellular apoptosis in which mitochondria and p53 were intricately involved. In MOLT-3 and HCT116p53+/+ cells, WithaD induced crosstalk between intrinsic and extrinsic signaling through Bid, whereas in K562 and HCT116p53−/− cells, only intrinsic pathway was activated where Bid remain unaltered. WithaD showed pronounced activation of p53 in cancer cells. Moreover, lowered apoptogenic effect of HCT116p53−/− over HCT116p53+/+ established a strong correlation between WithaD-mediated apoptosis and p53. WithaD induced Bax and Bak upregulation in HCT116p53+/+, whereas increase only Bak expression in HCT116p53−/− cells, which was coordinated with augmented p53 expression. p53 inhibition substantially reduced Bax level and failed to inhibit Bak upregulation in HCT116p53+/+ cells confirming p53-dependent Bax and p53-independent Bak activation. Additionally, in HCT116p53+/+ cells, combined loss of Bax and Bak (HCT116Bax−Bak−) reduced WithaD-induced apoptosis and completely blocked cytochrome c release whereas single loss of Bax or Bak (HCT116Bax−Bak+/HCT116Bax+Bak−) was only marginally effective after WithaD treatment. In HCT116p53−/− cells, though Bax translocation to mitochondria was abrogated, Bak oligomerization helped the cells to release cytochrome c even before the disruption of mitochondrial membrane potential. WithaD also showed in vitro growth-inhibitory activity against an array of p53 wild type and null cancer cells and K562 xenograft in vivo. Taken together, WithaD elicited apoptosis in malignant cells through Bax/Bak dependent pathway in p53-wild type cells, whereas Bak compensated against loss of Bax in p53-null cells

Over-expressed IgG2 antibodies against O-acetylated sialoglycoconjugates incapable of proper effector functioning in childhood acute lymphoblastic leukemia

Earlier studies have demonstrated an over-expression of 9-O-acetylated sialoglycoconjugates (9-OAcSGs) on lymphoblasts, concomitant with high titers of anti-9-OAcSGs in childhood acute lymphoblastic leukemia (ALL). The present study was aimed to evaluate whether this high induction of anti-9-OAcSGs at disease presentation contributes toward immune surveillance. Accordingly, anti- 9-OAcSGs were affinity purified from sera of ALL patients and normal individuals, and their specificity toward the glycotope having terminal 9-O-acetylated sialic acid-linked subterminal N-acetyl galactosamine (GalNAc) in a2–6 manner (9-OAcSAa2–6GalNAc) was established by hemagglutination assay, flow cytometry and confocal microscopy. Subclass distribution of anti-9-OAcSGs revealed a predominance of IgG2 in ALL. Analysis of glycosylation of anti-9-OAcSGs purified from sera of ALL patients (IgGALL) and normal individuals (IgGN) by digoxigenin glycan enzyme assay, fluorimetric estimation, gas–liquid chromatography and lectin-binding assays demonstrated that disease-specific antibodies differ in content and nature as compared with normal controls. Enhanced amount of 9-OAcSA-specific IgG2 induced in ALL was unable to trigger activation of FccR, the complement cascade and cell-mediated cytotoxicity, although its glycotope-binding ability remains unaffected. Interestingly, only IgG1N emerged as the potent mediator of cell-mediated cytotoxicity, complement fixation and activator of effector cells through FccR. In ALL, the observed subclass switching of anti-9-OAcSGs to IgG2, alteration in their glycosylation profile along with impairment of a few Fc-glycosylation-sensitive effector functions hints toward a disbalanced homeostasis, thereby evading the host defense. These findings justify further evaluation of the mechanism for functional unresponsiveness of antibodies and production of 9-OAcSA-specific chimeric antibodies with normal Fc domain for therapeutic application

Co-expression of 9-O-acetylated sialoglycoproteins and their binding proteins on lymphoblasts of childhood acute lymphoblastic leukaemia: an anti-apoptotic role

Enhanced levels of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2GPs) as disease-associated molecules was reported to act as signaling molecules for promoting survival of lymphoblasts in childhood acute lymphoblastic leukemia (ALL). Here, we searched for potential physiological ligands for Neu5,9Ac2GPs that could be involved in modulating the survival of lymphoblasts. Accordingly, we examined the presence of binding proteins for Neu5,9Ac2GPs on cell lines and primary cells of patients with B- and T-ALL, at presentation of the disease. Peripheral blood mononuclear cells from normal healthy donors and cells from myeloid leukemia patients were used for comparison. Neu5,9Ac2GPs-binding proteins (BPs) were specifically detected on the surface of both T- and B-ALL-lymphoblasts and ALL-cell lines along with the consistent presence of Neu5,9Ac2GPs. The Neu5,9Ac2GPs and BPs also co-localized on the cell surface and interacted specifically in vitro. Apoptosis of lymphoblasts, induced by serum starvation, was reversed in the presence of purified Neu5,9Ac2GPs due to possible engagement of BPs, and the anti-apoptotic role of this interaction was established. This is the first report of the presence of potential physiological ligands for disease-associated molecules like Neu5,9Ac2GPs, the interaction of which is able to trigger an anti-apoptotic signal conferring a survival advantage to leukemic cells in childhood ALL

Nanofabrication of methylglyoxal with chitosan biopolymer: a potential tool for enhancement of its anticancer effect

The normal metabolite methylglyoxal (MG) specifically kills cancer cells by inhibiting glycolysis and mitochondrial respiration without much adverse effect upon normal cells. Though the anticancer property of MG is well documented, its gradual enzymatic degradation in vivo has prompted interest in developing a nanoparticulate drug delivery system to protect it and also to enhance its efficacy. Materials and methods: MG-conjugated chitosan nanoparticles (Nano-MG) were prepared by conjugating the carbonyl group of MG with the amino group of chitosan polymer (Schiff’s base formation). Nano-MG were characterized in detail using the dynamic light scattering method, zeta potential measurement, Fourier transform infrared spectroscopy, and transmission electron microscopic analysis. Amount of MG anchored to Nano-MG, stability of Nano-MG, and in vitro release of MG from Nano-MG were estimated spectrophotometrically. Ehrlich ascites carcinoma (EAC) cells, human breast cancer cell line HBL-100, and lung epithelial adenocarcinoma cell line A549 were used as test systems to compare Nano-MG with bare MG in vitro. Cytotoxicity to EAC cells was evaluated by the trypan blue dye exclusion test, and cell viability of HBL-100 and A549 cells were studied using 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis of HBL-100 cells was assessed by flow cytometry and confocal microscopy. In vivo studies were performed on both EAC cells inoculated and also in sarcoma-180-induced solid tumor-bearing Swiss albino mice to assess the anticancer activity of Nano-MG in comparison to bare MG with varying doses, times, and administrative routes. Results: Fourier transform infrared spectroscopy revealed the presence of imine groups in Nano-MG due to conjugation of the amino group of chitosan and carbonyl group of MG with diameters of nanoparticles ranging from 50–100 nm. The zeta potential of Nano-MG was +21 mV and they contained approximately 100 μg of MG in 1 mL of solution. In vitro studies with Nano-MG showed higher cytotoxicity and enhanced rate of apoptosis in the HBL-100 cell line in comparison with bare MG, but no detrimental effect on normal mouse myoblast cell line C2C12 at the concerned doses. Studies with EAC cells also showed increased cell death of nearly 1.5 times. Nano-MG had similar cytotoxic effects on A549 cells. In vivo studies further demonstrated the efficacy of Nano-MG over bare MG and found them to be about 400 times more potent in EAC-bearing mice and nearly 80 times more effective in sarcoma-180-bearing mice. Administration of ascorbic acid and creatine during in vivo treatments augmented the anticancer effect of Nano-MG. Conclusion: The results clearly indicate that Nano-MG may constitute a promising tool in anticancer therapeutics in the near future

Landslide susceptibility modeling in a complex mountainous region of Sikkim Himalaya using new hybrid data mining approach

Landslide is recognized as one of the greatest threats in the complex mountainous regions of Sikkim Himalaya. Therefore, landslide susceptibility modeling (LSMs) has become an ideal tool for managing landslide disasters. Keeping this fact in view, researchers always try to develop optimal models for better performance in LSMs. Thus, the present research study proposed a novel ensemble approach of Alternating Decision Tree (ADTree) and Quantum-Particle Swamp Optimization (QPSO) algorithm and stand-alone of ADTree, QPSO and Random Forest for LSMs in the Rangpo River Basin, India. A total of 342 historical landslide datasets with 14 appropriate landslide causative factors were used for optimal LSMs. The models robustness was appraised via receiver operating characteristics and others statistical indices. Results indicated that QPSO-ADTree model outperformed other models. Overall, the proposed novel ensemble model can be applied as a promising approach for precise LSMs in several complex mountainous regions of the globe

Probable mechanism of WithaD induced apoptosis.

<p>WithaD treatment induces p53 activation and mitochondrial apoptosis in p53wt cells in Bax and Bak dependent manner. However, in p53-deficient cells, lack of Bax function is complemented with Bak in WithaD-induced mitochondrial apoptosis.</p

WithaD inhibit in vitro and in vivo K562 xenograft growth.

<p>(A) WithaD could prolong the survival time of nude mice injected with K562. The tumor load of treated mice (10 mg/kg body weight) was visibly lower than untreated control mice. (B) WithaD significantly reduced tumor volume. Each point represents mean of three tumors. (C) Status of PI-positive cells in untreated and WithaD-treated tumor cells. PI-positive cells (▪) were determined with respect to PI unstained cells (□). (D) Tissue section of spleen, lungs and liver of nude mice (control and WithaD treated) determined by H&E staining and observed both in 20× and 40×.</p

Bax and Bak were critical mediator of WithaD-mediated apoptosis.

<p>(A) Expression of p53, Bax, Bak and Cytochrome c were evaluated in the lysate of HCT116 cells proficient for Bax and Bak (Bax+/Bak+), deficient for either Bax (Bax−/Bak+) or Bak (Bax+/Bak−) or deficient for both Bax and Bak (Bax−/Bak−) after WithaD (0–2 µM) treatment for 15 hr by Western blot analysis. Each sub-lines were treated with WithaD (0–4 µM) for (B) 24 and (C) 48 hr and viabilities were determined by MTT assay. (D) Apoptosis induction was assessed by flow cytometric detection of each sub-lines by 7-AAD staining. ‘*’ indicates the difference was statistically significant (<i>P</i><0.005) between HCT116Bax+/Bak+ and HCT116Bax−/Bak− cells.</p