Kaushik Bhattacharya

Apoptotic effects of mahanine on human leukemic cells are mediated through crosstalk between Apo-1/Fas signaling and the Bid protein and via mitochondrial pathways

Apo-1 (Fas/CD95), a cell surface receptor, triggers apoptosis after binding to its physiological ligand, Apo-1L (FasL/CD95L). This study reports that mahanine, purified from the leaves of Murraya koenigii, has a dose- and time-dependent anti-proliferative activity in acute lymphoid (MOLT-3) and chronic myeloid (K562) leukemic cell lines and in the primary cells of leukemic and myeloid patients, with minimal effect on normal immune cells including CD34(+) cells. Leukemic cells underwent phosphatidylserine externalization and DNA fragmentation, indicating mahanine-induced apoptosis. An increase in reactive oxygen species suggests that the mahanine-induced apoptosis was mediated by oxidative stress. A significant drop in the Bcl2/Bax ratio, the loss of mitochondrial transmembrane potential as well as cytochrome c release from the mitochondria to the cytosol suggested involvement of the mitochondrial pathway of apoptosis. Cytochrome c release was followed by the activation of caspase-9, caspase-3 and caspase-7, and cleavage of PARP in both MOLT-3 and K562 cells. In MOLT-3 cells, formation of the Fas-FasL-FADD-caspase-8 heterotetramer occurred, leading to the cleavage of Bid to its truncated form, which consequently resulted in formation of the mitochondrial transmembrane pore. The incubation of MOLT-3 cells with mahanine in the presence of caspase-8 inhibitor or FasL-neutralizing NOK-2 antibody resulted in the decrease of mahanine-induced cell death. Mahanine was also a potent inhibitor of K562 xenograft growth, which was evident in an athymic nude mice model. In summary, these results provide evidence for involvement of the death receptor-mediated extrinsic pathway of apoptosis in the mahanine-induced anticancer activity in MOLT-3 cells, but not in K562 cells, which are deficient in Fas/FasL.

In situ synthesized TiB-TiN reinforced Ti6Al4V alloy composite coatings: microstructure, tribological and in-vitro biocompatibility.

Wear resistant TiB-TiN reinforced Ti6Al4V alloy composite coatings were deposited on Ti substrate using laser based additive manufacturing technology. Ti6Al4V alloy powder premixed with 5wt% and 15wt% of boron nitride (BN) powder was used to synthesize TiB-TiN reinforcements in situ during laser deposition. Influences of laser power, scanning speed and concentration of BN on the microstructure, mechanical, in vitro tribological and biological properties of the coatings were investigated. Microstructural analysis of the composite coatings showed that the high temperature generated due to laser interaction with Ti6Al4V alloy and BN results in situ formation of TiB and TiN phases. With increasing BN concentration, from 5wt% to 15wt%, the Youngs modulus of the composite coatings, measured by nanoindentation, increased from 170±5GPa to 204±14GPa. In vitro tribological tests showed significant increase in the wear resistance with increasing BN concentration. Under identical test conditions TiB-TiN composite coatings with 15wt% BN exhibited an order of magnitude less wear rate than CoCrMo alloy-a common material for articulating surfaces of orthopedic implants. Average top surface hardness of the composite coatings increased from 543±21HV to 877±75HV with increase in the BN concentration. In vitro biocompatibility and flow cytometry study showed that these composite coatings were non-toxic, exhibit similar cell-materials interactions and biocompatibility as that of commercially pure titanium (CP-Ti) samples. In summary, excellent in vitro wear resistance, high stiffness and suitable biocompatibility make these composite coatings as a potential material for load-bearing articulating surfaces towards orthopaedic implants.

Oxidative inhibition of Hsp90 disrupts the super-chaperone complex and attenuates pancreatic adenocarcinoma in vitro and in vivo.

Pancreatic cancer is almost always fatal, in part because of its delayed diagnosis, poor prognosis, rapid progression and chemoresistance. Oncogenic proteins are stabilized by the Hsp90, making it a potential therapeutic target. We investigated the oxidative stress‐mediated dysfunction of Hsp90 and the hindrance of its chaperonic activity by a carbazole alkaloid, mahanine, as a strategic therapeutic in pancreatic cancer. Mahanine exhibited antiproliferative activity against several pancreatic cancer cell lines through apoptosis. It induced early accumulation of reactive oxygen species (ROS) leading to thiol oxidation, aggregation and dysfunction of Hsp90 in MIAPaCa‐2. N‐acetyl‐L‐cysteine prevented mahanine‐induced ROS accumulation, aggregation of Hsp90, degradation of client proteins and cell death. Mahanine disrupted Hsp90‐Cdc37 complex in MIAPaCa‐2 as a consequence of ROS generation. Client proteins were restored by MG132, suggesting a possible role of ubiquitinylated protein degradation pathway. Surface plasmon resonance study demonstrated that the rate of interaction of mahanine with recombinant Hsp90 is in the range of seconds. Molecular dynamics simulation showed its weak interactions with Hsp90. However, no disruption of the Hsp90‐Cdc37 complex was observed at an early time point, thus ruling out that mahanine directly disrupts the complex. It did not impede the ATP binding pocket of Hsp90. Mahanine also reduced in vitro migration and tube formation in cancer cells. Further, it inhibited orthotopic pancreatic tumor growth in nude mice. Taken together, these results provide evidence for mahanine‐induced ROS‐mediated destabilization of Hsp90 chaperone activity resulting in Hsp90‐Cdc37 disruption leading to apoptosis, suggesting its potential as a specific target in pancreatic cancer.

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.

Sialoglycoproteins adsorbed by Pseudomonas aeruginosa facilitate their survival by impeding neutrophil extracellular trap through siglec-9

PA is an opportunistic pathogen that is commonly associated with severe infection in immunocompromised hosts. Siglec‐9 binds with Sias by cis interaction on the neutrophil surface, thereby reducing immunological activity. However, neutrophils bind with pathogens through trans interactions of siglec‐9 with Sias. Neutrophils kill invading pathogens by NETs, along with extracellular phagocytosis. Here, we report the mode of the adsorption of Sias by PA from host serum, the interaction of PA+Sias with human neutrophils, and the resulting neutrophil immunological activity. The α2–3‐linked sialoglycoproteins adsorbed by PA exhibited potent binding with the soluble siglec‐9‐Fc chimeras, CHO‐siglec‐9 and siglec‐9 on neutrophils. The binding between PA+Sias and neutrophils was blocked by the synthetic sialoglycan Neu5Acα2–3Galβ1–4GlcNAc, confirming the linkage‐specific, Sias–siglec‐9 interaction. The PA+Sias and siglec‐9 interaction on neutrophils reduced the level of ROS and the release of elastase, resulting in a reduction of NETs formation, demonstrating the role of the sialoglycoproteins adsorbed by PA in the weakening of neutrophil activity. The resistance of PA+Sias to NETs was made evident by the increased survival of PA+Sias. Moreover, the decrease in PA−Sias survival demonstrated the involvement of NETs formation in the absence of the Sias–siglec‐9 interaction. N‐actylcysteine or sivelestat‐pretreated neutrophils enhanced the survival of PA−Sias. DNAse‐pretreated neutrophils did not exhibit any NETs formation, resulting in the enhanced escape of PA−Sias. Taken together, one of the survival mechanisms of PA+Sias is the diminution of innate immunity via its adsorption of sialoglycoproteins by its engagement of the inhibitory molecule siglec‐9. This is possibly a general mechanism for pathogens that cannot synthesize Sias to subvert immunity.

Mahanine, A DNA Minor Groove Binding Agent Exerts Cellular Cytotoxicity with Involvement of C-7-OH and −NH Functional Groups

Mahanine, a carbazole alkaloid is a potent anticancer molecule. To recognize the structure-activity correlation, mahanine was chemically modified. Antiproliferative activity of these derivatives was determined in 19 cancer cell lines from 7 different origins. Mahanine showed enhanced apoptosis compared to dehydroxy-mahanine-treated cells, indicating significant contribution of the C-7-OH group. O-Methylated-mahanine and N-methylated dehydroxy-mahanine-treated cells exhibited apoptosis only at higher concentrations, suggesting additional contribution of 9-NH group. Using biophysical techniques, we demonstrated that mahanine interacts with DNA through strong association with phosphate backbone compared to other derivatives but is unable to induce any conformational change in DNA, hence suggesting the possibility of being a minor groove binder. This was corroborated by molecular modeling and isothermal titration calorimetry studies. Taken together, the results of the current study represent the first evidence of involvement of C-7-OH and 9-NH group of mahanine for its cytotoxicity and its minor groove binding ability with DNA.

Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition

Toxicity reduction of cisplatin is necessary for improved treatment of cancer. Here we have demonstrated the synergistic growth-inhibitory effect of cisplatin on cervical cancer cells in-combination with a nontoxic herbal carbazole alkaloid, mahanine. Mahanine enhanced cisplatin-induced apoptosis and reduced its effective concentration ∼5-8 folds. Mahanine inhibited JAK1 and Src and subsequently promoted proteasome-mediated degradation of STAT3. This event was further enhanced in-combination with cisplatin and subsequently inhibited cancer cell migration. Collectively, our results revealed that mahanine may be a prospective agent to reduce the concentration of cisplatin in adjunct for the treatment of cancer and thereby decreasing its toxicity.

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.

Mahanine synergistically enhances cytotoxicity of 5-fluorouracil through ROS-mediated activation of PTEN and p53/p73 in colon carcinoma

Abstract5-Fluorouracil (5-FU) alone or in combination with other drugs is the main basis of chemotherapeutic treatment in colorectal cancer although patients with microsatellite instability generally show resistance to 5-FU treatment. The present investigation is focussed on the mechanistic insight of a pure herbal carbazole alkaloid, mahanine, as a single or in combination with 5-FU in colon cancer. We demonstrated that mahanine-induced apoptosis involved reactive oxygen species (ROS)-mediated nuclear accumulation of PTEN and its interaction with p53/p73. Mahanine and 5-FU in combination exerted synergistic inhibitory effect on cell viability. This combination also enhanced ROS production, increased tumour suppressor proteins and suppressed chemo-migration. Taken together, our results revealed that mahanine can be a potential chemotherapeutic agent with efficacy to reduce the concentration of toxic 5-FU in colon cancer.

Methylglyoxal induced activation of murine peritoneal macrophages and surface markers of T lymphocytes in Sarcoma-180 bearing mice: Involvement of MAP kinase, NF-κβ signal transduction pathway

Methylglyoxal profoundly stimulates hosts immune response against tumor cell by producing reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs) [Bhattacharyya, N., Pal, A., Patra, S., Haldar, A.K., Roy, S., Ray, M., 2008. Activation of macrophages and lymphocytes by methylglyoxal against tumor cells in the host. Int. Immunophar. 8 (11), 1503-1512]. Present study indicated that methylglyoxal stimulates iNOS activation by p38 MAPK-NF-kappa beta dependent pathway and ROS production by ERK and JNK activation in sarcoma-180 tumor bearing mice. Proinflammatory cytokines, for macrophage activation, IL-6 and IL-1 beta were also increased. Production of TLR 4 and TLR 9, which acts through the same signaling pathway, were also upregulated. Hence, concluded that methylglyoxal augmented the IL-6 and IL-1 beta, expression of TLR 4 and TLR 9 and produced MAPKs, important regulators of ROIs and RNIs. Methylglyoxal treatment also increased M-CSF, an upregulator of macrophage production. CD8 and CD4 molecules, associated with T(C) and T(H) cells respectively, were also increased. Overall methylglyoxal treatment is important for enhancement of macrophages and lymphocyte activation or immunomodulation against sarcoma-180 tumor.