Joyjyoti Das

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.

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.

Environment Activatable Nanoprodrug: Two-Step Surveillance in the Anticancer Drug Release

Remedial cancer therapy deals with a large number of theranostic applications. However, systems, so far known, are only capable of single surveillance for both diagnostic and therapeutic modes of action. A nanosystem, which can be localized to the cancer and deliver the chemotherapeutic agent on demand, will provide effective therapeutic activity. Herein, we designed a single component nanoprodrug ANPD-X (Activatable Nano Pro-Drug-X) which indentified the tumor sites by fluorescent color change (signal 1, blue to green fluorescence) using H2O2-mediated oxidation of boronate fluorophore. In the next step, precise spatiotemporal irradiation of light only on identified tumor sites resulted in the release of anticancer drug chlorambucil. The real time information on drug release was achieved by a second fluorescence color change (signal 2, green to blue fluorescence). Thus, nanoprodrug ANPD-X provided overall two-step surveillance in the anticancer drug delivery. Activation of the ANPD-X after addition of H2O2 and drug release upon photoirradiation was investigated in vitro by monitoring its fluorescence in the HeLa cell line.

Development of gelatin/carboxymethyl chitosan/nano-hydroxyapatite composite 3D macroporous scaffold for bone tissue engineering applications

The present study delineates a relatively simpler approach for fabrication of a macroporous three-dimensional scaffold for bone tissue engineering. The novelty of the work is to obtain a scaffold with macroporosity (interconnected networks) through a combined approach of high stirring induced foaming of the gelatin/carboxymethyl chitosan (CMC)/nano-hydroxyapatite (nHAp) matrix followed by freeze drying. The fabricated macroporous (SGC) scaffold had a greater pore size, higher porosity, higher water retention capacity, slow and sustained enzymatic degradation rate along with higher compressive strength compared to that of non-macroporous (NGC, prepared by conventional freeze drying methodology) scaffold. The biological studies revealed the increased percentage of viability, proliferation, and differentiation as well as higher mineralization of differentiated human Whartons jelly MSC microtissue (wjhMSC-MT) on SGC as compared to NGC scaffold. RT-PCR also showed enhanced expression level of collagen type I, osteocalcin and Runx2 when seeded on SGC. μCT and histological analysis further revealed a penetration of cellular spheroid to a greater depth in SGC scaffold than NGC scaffold. Furthermore, the effect of cryopreservation on microtissue survival on the three-dimensional construct revealed significant higher viability upon revival in macroporous SGC scaffolds. These results together suggest that high stirring based macroporous scaffolds could have a potential application in bone tissue engineering.

Cell penetrating peptides from agglutinin protein of Abrus precatorius facilitate the uptake of Imatinib mesylate.

Targeted drug delivery is of paramount importance for cancer patients. Cell penetrating peptides (CPPs) have emerged as potent vehicles for this purpose. Herein, we demonstrate CPP- like properties of two peptides: NH2-SGASDDEEIAR-COOH (SR11) and NH2-ICSSHYEPTVRIGGR-COOH (IR15), derived from the tryptic digest of Abrus precatorius agglutinin. Both IR15 and SR11 were found to be non-toxic at lower doses (up to 50 μg/ml). These two peptides entered into HeLa cells through lipid raft-mediated endocytosis within 15 min and penetrated the nuclear membrane in 60 min of incubation. Co-treatment of peptides (20 μg/ml) and Imatinib (5 μM) in HeLa cells increased uptake of the drug by ∼ 55% and lowered the IC50 value to one-third in comparison to the drug added exclusively. However, co-treatment of TAT peptide (standard CPP) did not alter the Imatinib uptake significantly. In summary, we have identified two novel CPPs from tryptic digest of Abrus agglutinin which increased the cellular uptake of Imatinib upon co-administration. Further studies may result in deciphering a novel mode of drug delivery.

A spiropyran–coumarin platform: an environment sensitive photoresponsive drug delivery system for efficient cancer therapy

In spite of inventing several anticancer agents the clinical payoff still remains unsatisfactory because of their severe host toxicity due to their nonspecific biodistribution in the body. To achieve high efficiency in anti-cancer drug delivery, thus, we designed and developed a single component photoresponsive drug delivery system, a fusion of two platforms spiropyran and coumarin, which synchronizes two controlling factors: first, the lower pH of cancer tissue, which acts as an internal control and leads to the ring opening of spiropyran resulting in a distinct colour change and fluorescence activation of coumarin; and second, the release of the anti-tumor drug by the externally controlled light. Highly fluorescent nature and promising biocompatibility make the SP-Cou-Cbl system suitable for cell imaging and in vitro studies.

Molecular Mechanisms Associated with Particulate and Soluble Heteroglycan Mediated Immune Response

Immune responses are outcomes of complex molecular machinery which occur inside the cells. Unravelling the cellular mechanisms induced by immune stimulating molecules such as glycans and determining their structure‐function relationship are therefore important factors to be assessed. With this viewpoint, the present study identifies the functional receptor binding unit of a well characterized heteroglycan and also delineates the cellular and molecular processes that are induced upon heteroglycan binding to specific cell surface receptors in immune cells. The heteroglycan was acid hydrolysed and it was revealed that 10–30 kDa fractions served as the functional receptor binding unit of the molecule. Increasing the size of 10–30 kDa heteroglycan showed prominent immune activity. The whole soluble heteroglycan was also conjugated with hyperbranched dendrimers so as to generate a particulate form of the molecule. Dectin‐1 and TLR2 were identified as the major receptors in macrophages that bind to particulate as well as soluble form of the heteroglycan and subsequently caused downstream signaling molecules such as NF‐κβ and MAPK to get activated. High levels of 1L‐1β and IL‐10 mRNA were observed in particulate heteroglycan treated macrophages, signifying that increasing the size and availability of the heteroglycan to its specific receptors is pertinent to its biological functioning. Upregulated expression of PKC and iNOS were also noted in particulate heteroglycan treated RAW 264.7 cells than the soluble forms. Taken together, our results indicate that biological functions of immunomodulatory heteroglycan are dependent on their size and molecular weight. J. Cell. Biochem. 117: 1580–1593, 2016.

PAMAM dendrimer grafted cellulose paper scaffolds as a novel in vitro 3D liver model for drug screening applications

Development of cheap and portable 3D liver models is a need of an hour for drug screening applications in the healthcare and diagnostic sector. An appropriate 3D liver model would support adhesion, proliferation, viability and functionality of the liver cells. With this perspective, the present study delineates the fabrication and characterization of PAMAM conjugated cellulose filter paper-based 3D liver model. Due to the absence of any cell adhesion motif, paper cannot directly be used for cell culture applications, thus, it was functionalized with PAMAM dendrimer using glutaraldehyde. PAMAM derivatized paper (PF) supported significantly higher adhesion, proliferation, and viability of the HepG2 cells in comparison to non-functionalized paper. Moreover, HepG2 cells maintained their functional aspects i.e., expression of mature hepatocyte markers albumin, CK19, CYP3 A4, MDR1 and SULT2 A1 on PF. Further, we showed the application of the PF-based 3D liver model for drug toxicity evaluations against hepatotoxins. The results showed a significantly higher sensitivity of the HepG2 cells in paper-based 3D culture as compared to 2D monolayer culture, which is similar to that of physiological drug-induced responses. In conclusion, PAMAM functionalized filter paper could serve as an efficient biomaterial platform for the development of microscale technologies, liver-associated diagnostics, and drug screening applications.

Hemodynamic shear stress induces protective autophagy in HeLa cells through lipid raft-mediated mechanotransduction

During metastatic dissemination, cancer cells experience shear stresses in narrow confinements of in vivo vasculature. Such stresses are currently known to influence a gamut of cellular processes. While a host of cells emanating from a primary tumor perish in circulation due to shear, some cells manage to migrate to distant niches and form secondary tumors. Current research focuses on how cancer cells avert such mechanical stresses and adapt themselves in order to survive. This study deals with the autophagic response of cervical cancer cells HeLa and its subline HeLa 229, exposed to physiological shear stresses in vitro, and evaluates its role as a pro-survival mechanism. It also delineates the probable mechanotransduction pathway that is involved in eliciting the stress-adaptive response in cervical cancer cells. Our results show that shear stress of physiological regime elicits protective autophagy in cervical cancer cells as an immediate response and inhibiting the same, leads to early onset of apoptosis. An effort to study the underlying mechanotransduction revealed that autophagy induction by shear stress requires intact lipid rafts which serve as signalling platforms to trigger phosphorylation of p38 mitogen activated protein kinases, leading to autophagy. This study thus gives novel insights into the mechanobiology of cervical cancer and hints at promising therapeutic targets in metastasis, the major cause of cancer mortality.

AMPK-mediated crosstalk of heteroglycan-induced reactive species and autophagic cascade in RAW 264.7 cells

Natural polysaccharides are currently being employed for macrophage activation due to their minimal cytotoxicity and side effects. Reactive species like nitric oxide (NO) and reactive oxygen species (ROS), induced by a novel immunostimulatory heteroglycan were found to regulate the autophagic machinery in RAW 264.7 cells via a feedback mechanism, through AMPK. Heteroglycan-induced autophagy enhanced NO generation while itself being negatively regulated by NO. Concomitantly, intracellular ROS triggered the autophagic machinery which in turn subdued ROS levels indicating a homeostatic mechanism for regulated immune function. This study may help in better understanding of regulated immunostimulation by natural polysaccharides.