Bibhas Roy

Role of PI3K/Akt/mTOR and MEK/ERK pathway in Concanavalin A induced autophagy in HeLa cells

Concanavalin A (Con A), a mannose or glucose specific legume lectin, is well known for its anti-proliferative and cytotoxic effect on different types of cancer cells, through its binding to the membrane receptors leading to a major stimulus for the induction of distinct metabolic responses. Recently it has been also been proved that, Con A induces autophagy in hepatoma cells through internalization and mitochondria mediated pathway involving a mitochondrial interacting protein named Bcl2/E1B-19kDa protein-interacting protein 3 (BNIP3). Through this current endeavor, we propose a membrane associated pathway involved in Con A induced autophagy, taking Human cervical cancer (HeLa) cell as a cancer model. Here, we deciphered the role of membrane mediated phosphatidylinositol 3 kinase (PI3K)/Akt/mTOR (mammalian target of rapamycin) and MEK/Extracellular signal-regulated kinases (ERK) pathway in Con A induced autophagy in HeLa cells. Subsequently, we found that Con A treatment suppresses the PI3K/Akt/mTOR and up regulates the MEK/ERK pathway leading to the activation of autophagy. This study will further help us to understand the mechanism behind the autophagic pathway induced by Con A and simultaneously it will strengthen its effective use as a prospective cancer chemo-therapeutic.

Characterization and lectin microarray of an immunomodulatory heteroglucan from Pleurotus ostreatus mycelia

Glucans isolated from various mushroom and mycelia sources are interestingly being studied nowadays as a potent therapeutic agent. The present work was focused on the isolation, characterization and immunomodulatory study of a novel water soluble glucan from the pure mycelia of Pleurotus ostreatus. The extracted glucan was found to have a high molecular weight of ∼2.7 × 10(6)Da and mainly comprised of glucose, mannose and fucose in a ratio of 3:2:1 with both β and α linkages. Presence of terminal or interior glucose, mannose and fucose residues was also revealed using a high throughput miniaturized platform of lectin microarray. The heteroglucan folded into a triple helical conformation and exhibited enhanced immune cell activation and anti-tumor potential in tumor bearing mice model. Thus, potential biological functions incorporated in these glucan molecules acts in accord with its structural property and exploration of such structure-function relationship will unveil its diverse mechanism of action.

Cobalt doped proangiogenic hydroxyapatite for bone tissue engineering application

The present study delineates the synthesis and characterization of cobalt doped proangiogenic-osteogenic hydroxyapatite. Hydroxyapatite samples, doped with varying concentrations of bivalent cobalt (Co(2+)) were prepared by the ammoniacal precipitation method and the extent of doping was measured by ICP-OES. The crystalline structure of the doped hydroxyapatite samples was confirmed by XRD and FTIR studies. Analysis pertaining to the effect of doped hydroxyapatite on cell cycle progression and proliferation of MG-63 cells revealed that the doping of cobalt supported the cell viability and proliferation up to a threshold limit. Furthermore, such level of doping also induced differentiation of the bone cells, which was evident from the higher expression of differentiation markers (Runx2 and Osterix) and better nodule formation (SEM study). Western blot analysis in conjugation with ELISA study confirmed that the doped HAp samples significantly increased the expression of HIF-1α and VEGF in MG-63 cells. The analysis described here confirms the proangiogenic-osteogenic properties of the cobalt doped hydroxyapatite and indicates its potential application in bone tissue engineering.

Effect of fluidic transport on the reaction kinetics in lectin microarrays

Lectins are the proteins which can distinguish glycosylation patterns. They are frequently used as biomarkers for progressions of several diseases including cancer. As the lectin microarray based prognosis devices miniaturize the process of glycoprofiling, it is anticipated that their performance can be augmented by integration with microfluidic framework. This is analogous to microfluidics based DNA arrays. However, unlike small oligonucleotide microarrays, it remains uncertain whether the binding reaction-kinetic parameters can be considered invariant of imposed hydrodynamics, for relatively larger and structure sensitive molecules such as lectins. Here we show, using two standard lectins namely Concanavalin A and Abrus Agglutinin, that the steady state binding efficiency unexpectedly declines beyond a critical shear rate magnitude. This observation can be explained only if the associated reaction constants are presumed to be functions of hydrodynamic parameters. We methodically deduce the shear rate dependence of association and dissociation constants from the comparison of experimental and model-simulation trends. The aforementioned phenomena are perceived to be the consequences of strong hydrodynamic perturbations, culminating into molecular structural distortion. The exploration, therefore, reveals a unique coupling between reaction kinetics and hydrodynamics for biomacromolecules and provides a generic scheme towards futuristic microfluidics-coupled biomedical assays.

On-chip lectin microarray for glycoprofiling of different gastritis types and gastric cancer

An on-chip lectin microarray based glycomic approach is employed to identify glyco markers for different gastritis and gastric cancer. Changes in protein glycosylation have impact on biological function and carcinogenesis. These altered glycosylation patterns in serum proteins and membrane proteins of tumor cells can be unique markers of cancer progression and hence have been exploited to diagnose various stages of cancer through lectin microarray technology. In the present work, we aimed to study the alteration of glycan structure itself in different stages of gastritis and gastric cancer thoroughly. In order to perform the study from both serum and tissue glycoproteins in an efficient and high-throughput manner, we indigenously developed and employed lectin microarray integrated on a microfluidic lab-on-a-chip platform. We analyzed serum and gastric biopsy samples from 8 normal, 15 chronic Type-B gastritis, 10 chronic Type-C gastritis, and 6 gastric adenocarcinoma patients and found that the glycoprofile obtained from tissue samples was more distinctive than that of the sera samples. We were able to establish signature glycoprofile for the three disease groups, that were absent in healthy normal individuals. In addition, our findings elucidated certain novel signature glycan expression in chronic gastritis and gastric cancer. In silico analysis showed that glycoprofile of chronic gastritis and gastric adenocarcinoma formed close clusters, confirming the previously hypothesized linkage between them. This signature can be explored further as gastric cancer marker to develop novel analytical tools and obtain in-depth understanding of the disease prognosis.

Abrus Agglutinin, a type II ribosome inactivating protein inhibits Akt/PH domain to induce endoplasmic reticulum stress mediated autophagy-dependent cell death

Abrus agglutinin (AGG), a type II ribosome‐inactivating protein has been found to induce mitochondrial apoptosis. In the present study, we documented that AGG‐mediated Akt dephosphorylation led to ER stress resulting the induction of autophagy‐dependent cell death through the canonical pathway in cervical cancer cells. Inhibition of autophagic death with 3‐methyladenine (3‐MA) and siRNA of Beclin‐1 and ATG5 increased AGG‐induced apoptosis. Further, inhibiting apoptosis by Z‐DEVD‐FMK and N‐acetyl cysteine (NAC) increased autophagic cell death after AGG treatment, suggesting that AGG simultaneously induced autophagic and apoptotic death in HeLa cells. Additionally, it observed that AGG‐induced autophagic cell death in Bax knock down (Bax‐KD) and 5‐FU resistant HeLa cells, confirming as an alternate cell killing pathway to apoptosis. At the molecular level, AGG‐induced ER stress in PERK dependent pathway and inhibition of ER stress by salubrinal, eIF2α phosphatase inhibitor as well as siPERK reduced autophagic death in the presence of AGG. Further, our in silico and colocalization study showed that AGG interacted with pleckstrin homology (PH) domain of Akt to suppress its phosphorylation and consequent downstream mTOR dephosphorylation in HeLa cells. We showed that Akt overexpression could not augment GRP78 expression and reduced autophagic cell death by AGG as compared to pcDNA control, indicating Akt modulation was the upstream signal during AGGs ER stress mediated autophagic cell death. In conclusion, we established that AGG stimulated cell death by autophagy might be used as an alternative tumor suppressor mechanism in human cervical cancer.

Spatiotemporal dynamics of doxorubicin elution from embolic beads within a microfluidic network.

Anticancer treatment using embolic drug-eluting beads (DEBs) has shown multifarious advantages compared to systemic chemotherapy. However, there is a growing need for a better understanding of the physical parameters governing drug-elution from embolic devices under physiologically relevant fluidic conditions. In the present study, we investigated the spatiotemporal dynamics of doxorubicin hydrochloride elution from drug-loaded hydrogel embolic beads within a microfluidic device consisting of a network of interconnected microchannels which replicates the architectural properties of microvascular systems. Drug-elution has been investigated experimentally at a single-bead level, using in-house developed microscopy- and spectrofluorimetry-based methods. Results demonstrated that the kinetics of drug-elution and the amount of eluted drug strongly depended on the location of the embolic event within the embolised channel (e.g. fractional amount of eluted drug after 3h was equal to ~0.2 and ~0.6 for completely-confined and partially-confined bead, respectively). Drug-elution from partially-confined bead showed a counterintuitive dependence on the local Reynolds number (and thus on the mean fluid velocity), as a result of dynamic changes in bead compressibility causing the displacement of the bead from the primary embolic site. Conversely, the kinetics of drug-elution from fully-confined bead was less affected by the local Reynolds number and bead displayed faster elution from the surface area exposed to the systemic flow, which was associated with the formation of fluid eddies nearby the bead post embolisation.

Assessing the immunomodulatory role of heteroglycan in a tumor spheroid and macrophage co-culture model system

The therapeutic benefits of glycans have garnered much attention over the last few decades with most studies being reported in 2D cultures or in animal models. The present work is therefore aimed to assess the effects of an immunomodulatory heteroglycan in a 3D milieu. Briefly, HT29 tumor spheroids were incubated with THP-1 macrophages at 1:1 ratio in a culture medium supplemented with immune stimulants such as heteroglycans or LPS. Spheroidal distortion, migration of tumor cells from the periphery of the spheroids and 46% of higher macrophage invasiveness was noted in heteroglycan-treated co-cultures with respect to control cultures. Histological sections of the treated co-cultures revealed the presence of high apoptotic tumor cells in the spheroidal periphery. CD11c and CD68 staining further suggested the predominance of macrophages in the vicinity of the apoptotic tumor cells. Such an in vitro created tissue system may thereby confirm the effectiveness of heteroglycan in activating the immune cells to exhibit tumor cytotoxic properties.

Groundnut oil based emulsion gels for passive and iontophoretic delivery of therapeutics

Abstract There is a persistent demand for an efficient drug delivery system, suitable for the delivery of both hydrophilic and lipophilic drugs. This study explores groundnut oil-based emulsion gels for the above-mentioned application. On the basis of stability, two representative gels OG5-80 (low oil content) and OG7-45 (high oil content) were studied further. Analysis of microarchitecture by ESEM and confocal microscopy, in conjugation with fluorescence recovery after photobleaching, confirmed the conversion of the dispersion phase from oil-continuous (OG7-45) to bicontinuous (OG5-80) with increasing water proportion. The gels were viscoelastic with unique stress relaxation properties. Passive and active (iontophoretic) release kinetics of the drugs showed differential release patterns. Mathematical modeling elucidated composition-dependent temporal variation in the drug release and stress relaxation patterns. In vitro cell viability study, cell cycle analysis, and immunocytochemistry divulged compatibility of the gels to human skin cells (keratinocytes). Drug-loaded gels were found active against B. subtilis and E. coli. Hence, groundnut oil-based emulsion gels can be an efficient and stable multimodal carrier system for the passive and the active delivery of both hydrophilic and lipophilic drugs.