Birendra Behera

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.

Antitumor effect of soybean lectin mediated through reactive oxygen species-dependent pathway

AIMS The present study evaluated the potential role of soybean lectins (SBL) anticancer effect in vitro in different cancer cell lines and the therapeutic effectiveness in vivo in Daltons lymphoma (DL) bearing mice model. MAIN METHODS The effect of SBL on cell growth and viability was measured using MTT assay in different cancer cells in vitro. Apoptosis, autophagic cell death, DNA-damaging potential and reactive oxygen species (ROS) were analyzed in HeLa cells. The in vivo efficacy of SBL was demonstrated in Daltons lymphoma (DL) bearing mice. KEY FINDINGS SBL demonstrated clear, strong antiproliferative activity without affecting normal cells; however, heat denaturation of SBL diminished the antiproliferative efficacy of molecule as demonstrated by MTT assay. A sharp 74.51 ± 3.5% and 82.95 ± 5.8% inhibition of tumor cell proliferation in DL mice occurred when SBL was administered at a dosage of 1 and 2mg/kg body weight (i.p.), respectively, for ten days with the induction of autophagic and apoptotic cell death. An in vitro investigation revealed that SBL-mediated autophagy, apoptosis and DNA damage in HeLa cells were inflicted through the generation of ROS in a dose-dependent manner. Interestingly, pre-treating HeLa cells with N-acetylcysteine (NAC), a typical ROS scavenger, led to a noticeable reduction in SBL-induced autophagy, apoptosis and DNA-damaging activities, suggesting that SBLs antitumor potential was governed by ROS activation. SIGNIFICANCE In this study, we evaluated the apoptotic, autophagic death, and DNA-damaging effects of SBL in cancer cells, which may have the potential to be used as a phyto-derived protein for cancer therapy.

In vitro and in vivo antitumor effects of Peanut agglutinin through induction of apoptotic and autophagic cell death

In this study we unravel the mechanism underlying the antitumorigenic effects of Peanut agglutinin (PNA) isolated from Arachis hypogea in Daltons lymphoma (DL) bearing mice and elucidated the mechanism in vitro in HeLa cells. In vivo PNA administration at 1 and 2 mg/kg body weight reduced DL proliferation with increase in autophagic and apoptotic characteristics. In vitro data showed that PNA at 0.1-100 μg/ml dose exhibit selective antiproliferative activity on various cancer cell lines without displaying cytotoxic effect on normal cells. However, heat denatured PNA failed to show any antiproliferative activity. Moreover, PNA was found to induce autophagic and apoptotic cell death in HeLa cells. Exponential increase in reactive oxygen species (ROS) was proved to be the master signal for promoting PNA induced cell death in HeLa cells. Interestingly, when HeLa cells were pre-exposed with N-acetylcysteine (NAC) and followed to PNA treatment, there was sharp decline in autophagy, apoptosis and a concomitant abrogation of antiproliferative potential. PNA at lower doses was also seen to inflict senescence. Hence, this common culinary item derived molecule whose discovery dates back to late 1970s was for the first time evaluated mechanistically in vivo and in vitro as a novel naturally occurring therapeutic agent against cancer.

Abrus agglutinin suppresses human hepatocellular carcinoma in vitro and in vivo by inducing caspase-mediated cell death

Aim:Abrus agglutinin (AGG) from the seeds of Indian medicinal plant Abrus precatorius belongs to the class II ribosome inactivating protein family. In this study we investigated the anticancer effects of AGG against human hepatocellular carcinoma in vitro and in vivo.Methods:Cell proliferation, DNA fragmentation, Annexin V binding, immunocytofluorescence, Western blotting, caspase activity assays and luciferase assays were performed to evaluate AGG in human liver cancer cells HepG2. Immunohistochemical staining and TUNEL expression were studied in tumor samples of HepG2-xenografted nude mice.Results:AGG induced apoptosis in HepG2 cells in a dose- and time-dependent manner. AGG-treated HepG2 cells demonstrated an increase in caspase 3/7, 8 and 9 activities and a sharp decrease in the Bcl-2/Bax ratio, indicating activation of a caspase cascade. Co-treatment of HepG2 cells with AGG and a caspase inhibitor or treatment of AGG in Bax knockout HepG2 cells decreased the caspase 3/7 activity in comparison to HepG2 cells exposed only to AGG. Moreover, AGG decreased the expression of Hsp90 and suppressed Akt phosphorylation and NF-κB expression in HepG2 cells. Finally, AGG treatment significantly reduced tumor growth in nude mice bearing HepG2 xenografts, increased TUNEL expression and decreased CD-31 and Ki-67 expression compared to levels observed in the untreated control mice bearing HepG2 cells.Conclusion:AGG inhibits the growth and progression of HepG2 cells by inducing caspase-mediated cell death. The agglutinin could be an alternative natural remedy for the treatment of human hepatocellular carcinomas.

Chemical analysis of an immunostimulating (1→4)-, (1→6)-branched glucan from an edible mushroom, Calocybe indica.

An immunostimulating water-soluble glucan was isolated from hot aqueous extract of fruit bodies of an edible mushroom Calocybe indica. Structural investigation of the glucan was carried out using acid hydrolysis, methylation analysis, and NMR studies ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC). On the basis of above-mentioned experiments, the structure of the repeating unit of the polysaccharide was established as [see figure in text]. This glucan stimulated the splenocytes and thymocytes.

Glucan of a somatic hybrid mushroom, pfls1h: structural characterization and study of immunological activities

A water-soluble glucan (PS-I) was isolated from the aqueous extract of the fruit bodies of a hybrid mushroom, pfls1h of Pleurotus florida and Lentinus squarrosulus (Mont.) Singer. Structural characterization of PS-I was carried out using total hydrolysis, methylation analysis, periodate oxidation, and NMR experiments ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HSQC, and HMBC). Methylation analysis revealed that PS-I was composed of (1→3, 6), (1→3), (1→6)-linked and terminal β-d-glucopyranosyl residues in a relative proportion of approximately 1:1:1:1. The repeating unit of the glucan consists of a backbone chain of two (1→6)-β-d-glucopyranosyl residues, one of which is branched at O-3 position with (1→3)-β-d-glucopyranosyl and terminated with a β-d-glucopyranosyl residue. Study of immunological activity revealed that PS-I stimulates the splenocytes, thymocytes and macrophages.

Structural characterization and study of immunoenhancing properties of a glucan isolated from a hybrid mushroom of Pleurotus florida and Lentinula edodes

A water soluble glucan isolated from hot aqueous extract of fruit bodies of an edible hybrid mushroom Pfle1r of Pleurotus florida and Lentinula edodes showed macrophages, splenocytes, and thymocytes activation. The glucan consists of terminal, (1→3,6)-linked, and (1→6)-linked β-D-glucopyranosyl moieties in a molar ratio of nearly 1:1:3. On the basis of acid hydrolysis, methylation, periodate oxidation study, and NMR studies ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HSQC, and HMBC), the structure of the repeating unit of the glucan was established as: [structure: see text].

Prediction and validation of apoptosis through cytochrome P450 activation by benzo(a)pyrene

Polycyclic aromatic hydrocarbons (PAHs) processed by cytochrome P450 (CYP450) during metabolism is well reported to induce carcinogenesis. The present study has developed a new approach to examine apoptotic activity of a known PAH called benzo[a]pyrene (B[a]P), using protein-ligand and protein-protein interaction through in silico approach, followed by in vitro validation. In silico study showed that the conformational changes and energies involved in the binding of B[a]P to CYP1B1 was crucial with its target proteins. The data showed that activated B[a]P had high affinity to bind with aryl hydrocarbon receptor (AhR) with binding energy of -601.97kcal/mol. Interestingly, B[a]P-CYP1B1 complex showed strong binding affinity for caspase-8, -9, -3 with binding energy of -625.5, -479.3 and -514.2kcal/mol respectively. Moreover, the docking of specific caspase inhibitors in the complex showed weak interaction with low binding energy value as compared to B[a]P-CYP1B1 caspase complexes. To validate our in silico work, we showed B[a]P treated HaCaT cells triggered apoptosis with increase in caspase 8, caspase 9 and caspase 3/7 level. Further, in vitro work confirmed that B[a]P induced apoptosis was significantly suppressed in Ac-DEVD-CMK pre-treated cells. In addition, knockdown of CYP1B1 suppressed B[a]P induced apoptosis in HaCaT cells confirming a pivotal role of CYP1B1 in B[a]P induced apoptosis. Interestingly, through in silico modeling, we screened clotrimazole as a potent CYP1B1 inhibitor which completely inhibited B[a]P mediated activation. This hypothesis was validated by MTT assay, caspase activation measurement and showed remarkable inhibition of B[a]P induced cell death; thereby, highlighting a potent therapeutic role for industrial pollution associated diseases.

Structural studies of an immunoenhancing glucan of an ectomycorrhizal fungus Ramaria botrytis

A water-soluble glucan was isolated from the alkaline extract of an ectomycorrhizal fungus, Ramaria botrytis. On the basis of sugar analysis, methylation analysis, Smith degradation, partial hydrolysis, and 1D/2D NMR studies, the structure of the repeating unit of the glucan was established as: [structure: see text]. This glucan showed immunostimulating activity by NO production on RAW 264.7, a murine macrophage cell line. Splenocyte and thymocyte proliferation were measured using, respectively, single cell suspensions of spleen and thymus obtained from normal mice.

An immunostimulating water insoluble β-glucan of an edible hybrid mushroom: isolation and characterization.

An immunostimulating water-insoluble β-glucan isolated from hot alkaline extract of the fruiting bodies of an edible somatic hybrid mushroom of Pleurotus florida and Calocybe indica var. APK2 showed significant macrophage, splenocyte, and thymocyte activations. On the basis of total hydrolysis, methylation analysis, and NMR experiments ((1)H, (13)C, DQF-COSY, TOCSY, NOESY, DEPT-135, and HSQC), the repeating unit of the polysaccharide is established.