Cissus quadrangularis Linn. Stem Ethanolic Extract Liberates Reactive Oxygen Species and Induces Mitochondria Mediated Apoptosis in KB Cells

Background: Cissus quadrangularis Linn. (CQ) commonly known as Hadjod (Family: Vitaceae) is usually distributed in India and Sri Lanka and contains several bioactive compounds responsible for various metabolic and physiologic effects. Objective: In this study, the biological effects of CQ ethanolic extract were evaluated by in vitro and supported by in silico analysis on KB oral epidermoid cancer cell line. Materials and methods: Anti-cancer potential of ethanolic extract of CQ stem against KB oral epidermoid cancer cells was evaluated in terms of morphological analysis, nuclei staining, liberation of reactive oxygen species (ROS), cell cycle arrest, mitochondrial membrane potential (MMP) and p53 and Bcl-2 protein expression which reveal the induction of apoptosis along with supporting in silico analysis. Results: Ethanolic extract of CQ stem contains various bioactive compounds responsible for cancer cell morphological alterations, liberation of ROS, G1 phase cell cycle arrest and decreased MMP along with up-regulation of p53 and down-regulation of Bcl-2. By employing in silico approach, we have also postulated that the CQ extract active constituents sequester Bcl-2 with higher affinity as compared to p53, which may be the reason for induction of growth arrest and apoptosis in KB cells. Conclusion: Our data indicate that the CQ extract has a remarkable apoptotic effect that suggests that it could be a viable treatment option for specific types of cancers. Summary: Cissus quadrangularis stem ethanolic extract induces apoptosis and cell cycle arrest at G1 phaseIt liberates (ROS) and mitochondria mediated apoptosisIt upregulates p53 and down-regulates Bcl-2 protein expressionIn silico studies indicates that the active constituents of CQ binds Bcl-2 with higher affinity as compared to p53

Sirtuins and Autophagy in Age-Associated Neurodegenerative Diseases: Lessons from the C. elegans Model

Age-associated neurodegenerative diseases are known to have “impaired protein clearance” as one of the key features causing their onset and progression. Hence, homeostasis is the key to maintaining balance throughout the cellular system as an organism ages. Any imbalance in the protein clearance machinery is responsible for accumulation of unwanted proteins, leading to pathological consequences—manifesting in neurodegeneration and associated debilitating outcomes. Multiple processes are involved in regulating this phenomenon; however, failure to regulate the autophagic machinery is a critical process that hampers the protein clearing pathway, leading to neurodegeneration. Another important and widely known component that plays a role in modulating neurodegeneration is a class of proteins called sirtuins. These are class III histone deacetylases (HDACs) that are known to regulate various vital processes such as longevity, genomic stability, transcription and DNA repair. These enzymes are also known to modulate neurodegeneration in an autophagy-dependent manner. Considering its genetic relevance and ease of studying disease-related endpoints in neurodegeneration, the model system Caenorhabditis elegans has been successfully employed in deciphering various functional outcomes related to critical protein molecules, cell death pathways and their association with ageing. This review summarizes the vital role of sirtuins and autophagy in ageing and neurodegeneration, in particular highlighting the knowledge obtained using the C. elegans model system

Titanium dioxide nanoparticles as guardian against environmental carcinogen benzo[alpha]pyrene.

Polycyclic aromatic hydrocarbons (PAH), like Benzo[alpha]Pyrene (BaP) are known to cause a number of toxic manifestations including lung cancer. As Titanium dioxide Nanoparticles (TiO2 NPs) have recently been shown to adsorb a number of PAHs from soil and water, we investigated whether TiO2 NPs could provide protection against the BaP induced toxicity in biological system. A549 cells when co-exposed with BaP (25 µM, 50 µM and 75 µM) along with 0.1 µg/ml,0.5 µg/ml and 1 µg/ml of TiO2 NPs, showed significant reduction in the toxic effects of BaP, as measured by Micronucleus Assay, MTT Assay and ROS Assay. In order to explore the mechanism of protection by TiO2 NP against BaP, we performed in silico studies. BaP and other PAHs are known to enter the cell via aromatic hydrocarbon receptor (AHR). TiO2 NP showed a much higher docking score with AHR (12074) as compared to the docking score of BaP with AHR (4600). This indicates a preferential binding of TiO2 NP with the AHR, in case if both the TiO2 NP and BaP are present. Further, we have done the docking of BaP with the TiO2 NP bound AHR-complex (score 4710), and observed that BaP showed strong adsorption on TiO2 NP itself, and not at its original binding site (at AHR). TiO2 NPs thereby prevent the entry of BaP in to the cell via AHR and hence protect cells against the deleterious effects induced by BaP

Binding Sites of AHR-TiO₂ NP complex.

<p>(Tyr 145, Ser 151, Phe 148, Leu 369, Asn 673. Asn 366, Agr 384, Pro 385, Leu 413, Try 719, Gln383, Phe 406, Glu 488, Pro 665, Gln 667, Tyr 696, Gln 671, Asp 144, Ser 682, Gln 149).</p

BaP structure as genrated with help of CORINA online server.

<p>BaP structure as genrated with help of CORINA online server.</p

Percentage viability of the cells exposed to 75 µM BaP, to 0.1, 0.5 and 1.0 µg/ml of TiO₂ NPs and co-exposure to 0.1, 0.5 and 1.0 µg/ml of TiO₂ NPs along with 75 µM, for 6, 12 & 24 h, as measured by MTT assay.

<p>*p<0.05 indicates significance. a– as compared to control, b- as compared to BaP treated. MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide; BaP: Benzo[alpha]Pyrene, TiO₂ NPs: titanium dioxide nanoparticles.</p

Binding Sites of AHR-BaP complex.

<p>(Pro180, Ser181, Cys183, Gly187, Leu196, Val200, Asn204, Leu259, Pro260, Leu265, Ala269, Thr270, Leu272, Pro274).</p

Binding Sites of AHR - TiO₂ NP complex & BaP.

<p>(Gln 666, Try 719, Phe 700, Pro 669, Gln 698, Thr 408, Phe 406, Phe 675, Thr 696).</p

Role of AHR in BaP internalization.

<p>(A) Internalization of BaP in to cell through AHR, metabolic conversion to BPDE and interaction of BPDE with DNA. (B) Preferential binding of TiO₂NP with AHR. TiO₂ NP bound to AHR blocks the internalization of BaP, preventing its metabolic conversion to BPDE and finally avoiding DNA damage.</p

Percentage change in ROS generation following 2, 6, 12 and 24 h of exposure to various concentrations of BaP.

<p>*p<0.05 considered as significant. BaP: Benzo[alpha]Pyrene; ROS: Reactive Oxygen Species.</p