Publications by authors named "Aneesh Chandrasekharan"

13 Publications

  • Page 1 of 1

Correction to: Molecular hybridization combining tumor homing and penetrating peptide domains for cellular targeting.

Drug Deliv Transl Res 2021 Sep 5. Epub 2021 Sep 5.

Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13346-021-01051-zDOI Listing
September 2021

Molecular hybridization combining tumor homing and penetrating peptide domains for cellular targeting.

Drug Deliv Transl Res 2021 Jul 31. Epub 2021 Jul 31.

Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India.

A complete peptide-based drug delivery unit has been designed with a tumor homing domain chemically linked to a syndiotactic cell-penetrating domain. The designed peptides were synthesized, characterized, and tested in vitro for cellular uptake and cytotoxicity evaluation. The differential uptake, cellular internalization, negligible hemotoxicity, selective toxicity to MDA-MB-231 breast cancer cells, and the superior penetration in three-dimensional MDA-MB-231 tumorospheres confirm their utility as a promising delivery vector.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13346-021-01035-zDOI Listing
July 2021

Peptide-based delivery vectors with pre-defined geometrical locks.

RSC Med Chem 2020 Nov 13;11(11):1303-1313. Epub 2020 Aug 13.

Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati Guwahati-781039 Assam India

Design of peptide-based targeted delivery vectors with attributes of specificity and selective cellular targeting by fixing their topology and resulting electrostatic fingerprint is the objective of this study. We formulated our peptide design platform by utilizing the possibilities of side-chain induced geometric restrictions in a typical peptide molecule. Conceptually, we locked the conformation of the RGD/NGR motif of tumor homing peptides (THPs) by mutating glycine in these motifs with d-proline and tailed the peptides with a syndiotactic amphipathic segment for cellular penetration. The designed peptides were synthesized, characterized, and tested on various cell lines, including breast cancer (MDA-MB-231), cervical cancer (HeLa), osteosarcoma (U2-OS) and non-cancer mammary epithelial cells (MCF-10A), by flow cytometry and confocal microscopy. The results showed differential cellular uptake in different cell types, as a result of the distinct electrostatic fingerprint encoded in their design. The uptake of serum pre-treated peptides by cells reveals the retention of peptide activity even after the incubation with serum. In addition, peptide-methotrexate (MTX) conjugates compared to the methotrexate drug showed enhanced apoptotic cell death in MTX-resistant MDA-MB-231 cells, indicating the increase in MTX bioavailability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0md00229aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126890PMC
November 2020

Geometry encoded functional programming of tumor homing peptides for targeted drug delivery.

J Control Release 2021 05 13;333:16-27. Epub 2021 Mar 13.

Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India. Electronic address:

Poly-peptide molecules have shown promising applications in drug delivery and tumor targeting. A series of tumor homing peptides were designed by exhaustively sampling low energy geometrical basins of amino acids at specific sites of a peptide molecule to induce a conformational lock. This peptide library was pruned to a limited set of eight molecules, employing electrostatic interactions, docking, and molecular dynamics simulations. These designed and optimized peptides were synthesized and tested on various cell lines, including breast cancer (MDA-MB-231), cervical cancer (HeLa), osteosarcoma (U2-OS), and non-cancerous mammary epithelial cells (MCF-10A) using confocal microscopy and flow cytometry. Peptides show differential uptake in cancerous MDA-MB-231, HeLa, U2-OS, and non-cancerous MCF-10A cells. Confocal imaging verified their ability to penetrate even in 3D tumorospheres of MDA-MB-231 cells. Further, experiments of mitochondrial membrane potential depolarization and Caspase-3 activation confirmed that their cytotoxic effects are by apoptosis. Homing ability of the designed peptides in in vivo system and fluorescence imaging with clinical samples of human origin have further confirmed that the in vitro studies are qualitatively identical and quantitatively comparable in their ability to selectively recognize tumor cells. Overall, we present a roadmap for the functional programming of peptide-based homing and penetrating molecules that can perform selective tumor targeting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jconrel.2021.03.010DOI Listing
May 2021

Mitochondria targeted redox GFP reveals time and dose dependent onset and progression of mitochondrial oxidation with diverging cell death decisions during photodynamic therapy.

Photodiagnosis Photodyn Ther 2020 Sep 15;31:101921. Epub 2020 Jul 15.

Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thycaud P.O., Thiruvananthapuram, Kerala 695014, India. Electronic address:

Background: Photodynamic therapy (PDT) is a successful cancer treatment modality. In vitro, in vivo, and clinical studies with different photosensitizers reveal diverging cell fates, including apoptosis, necrosis, autophagy, and non-specific forms of cell death. The mode of action and efficacy of PDT is mediated through free radical generation and is highly dependent on diverse variables such as nature, dose, metabolism of photosensitizer, irradiation energy, and irradiation cycle.

Aim: Discovery of newer photosensitizers and optimization of PDT approaches to achieve a clinically relevant form of cell death called apoptosis requires better in vitro real-time methods. Oxidative damage and mitochondrial permeabilization are critical signaling events involved in photodamage and apoptosis. Hence, mitochondrial damage detection is an appropriate target signaling for mechanistic evaluation of PDT.

Methodology: We report mitochondria-targeted redox GFP expressing cells as a sensitive system to test and validate important variables of PDT using the photosensitizer 5-Aminolevulinic acid (5-ALA) as a model. An independent FRET-based caspase sensor cell was also used to study the impact of the photosensitizer dosage and irradiation duration on the mode of cell death.

Results: The study reveals that the cancer cells expressing mt-roGFP are extremely sensitive to monitor mitochondrial oxidation induced by PDT. The extent of mitochondrial redox changes induced by PDT can be determined using these sensor cells by real-time image-based approaches. These approaches provide sufficient temporal resolution that is required to fine-tune and optimize the PDT conditions. The degree of oxidation of the probe is highly dependent on the dosage of photosensitizer and duration of light irradiation, which determines the nature of cell death. A real-time caspase sensor probe further confirmed that the caspase-dependent and caspase-independent nature of cell death is in high correlation with the extent of mitochondrial oxidation. A condition that triggers rapid and extreme mito-oxidation seems to favor necrosis, while delayed and slowly progressing redox changes contribute to caspase-dependent apoptosis.

Conclusion: The study confirms that temporal analysis of mitochondrial oxidation is a reliable biomarker for fine-tuning PDT conditions to achieve the desired outcome. This can be achieved using stable cancer cell lines expressing mitochondria-targeted roGFP by ratiometric imaging.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pdpdt.2020.101921DOI Listing
September 2020

Water-Templated, Polysaccharide-rich Bioartificial 3D Microarchitectures as Extra-Cellular Matrix Bioautomatons.

ACS Appl Mater Interfaces 2020 May 22;12(18):20912-20921. Epub 2020 Apr 22.

Amity Institute of Biotechnology, Amity University Haryana, Amity Education Valley Gurugram, Manesar, Panchgaon, Haryana 122413, India.

This is the first report of exploiting the "quasi-spherical" shape of water molecules for recapitulating a true human extracellular matrix (ECM). Herein, water behaved as a quasi-spherical porogen, for engineering polysaccharide-rich and chemically defined 3D-microarchitecture, with semi-interpenetrating networks (S-IPNs). Furthermore, their viscoelastic behavior along with a heterogeneous, fibroporous morphology, facilitated instructive, self-remodeling of the bioartificial scaffolds, thence effectively permitting and promoting the growth of 3D tumor spheroids of divergent origins. The hybrid composites displayed reproducible, uniform tumor spheroids with a -depth of ∼65 ± 2 μm in case of human adenocarcinoma (DLD-1) and ∼54 ± 3 μm for human glioblastoma cells (U-251) (vs. nonuniform spheroids, on Agarose matrix). Thereafter, their capacity for anticancer drug screening was examined using limited cancer drugs. The conflicting drug screening results for Etoposide's reduced efficacy on glioblastoma cells cultured on our 3D matrix could be ascribed to decreased drug access and thus lower ingression. Nonetheless, adenocarcinoma's resistance to Camptothecin was paralleled. Moreover, their potential for real-time, high-content, phenotypic precision oncology was affirmed by the exceptional transparency of the synthesized composite. Since this 3D microarchitecture typifies ECM bioautomaton, this matrix can also be wielded for precision oncology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsami.0c01012DOI Listing
May 2020

A reporter cell line for real-time imaging of autophagy and apoptosis.

Toxicol Lett 2020 Jun 25;326:23-30. Epub 2020 Feb 25.

Cancer Research Program, Rajiv Gandhi Centre for Biotechnology (Research Centre, University of Kerala), Poojappura, Thycaud P.O., Thiruvananthapuram, Kerala, 695014, India. Electronic address:

Simultaneous detection of autophagy and apoptosis is important in drug discovery and signaling studies. Here we report, a real-time reporter cell line for the simultaneous detection of apoptosis and autophagy at single-cell level employing stable integration of two fluorescent protein reporters of apoptosis and autophagy. Cells stably expressing EGFP-LC3 fusion was developed initially as a marker for autophagy and subsequently stably expressed with inter-mitochondrial membrane protein SMAC with RFP fusion to detect mitochondrial permeabilization event of apoptosis. The cell lines faithfully reported the LC3 punctae formation and release of intermembrane proteins in response to diverse apoptotic and autophagic stimuli.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.toxlet.2020.02.011DOI Listing
June 2020

The ubiquitin ligase FBXW7 targets the centriolar assembly protein HsSAS-6 for degradation and thereby regulates centriole duplication.

J Biol Chem 2020 04 21;295(14):4428-4437. Epub 2020 Feb 21.

School of Biology, Indian Institute of Science Education and Research, Thiruvananthapuram, Vithura, Thiruvananthapuram 695551, Kerala, India

Formation of a single new centriole from a pre-existing centriole is strictly controlled to maintain correct centrosome number and spindle polarity in cells. However, the mechanisms that govern this process are incompletely understood. Here, using several human cell lines, immunofluorescence and structured illumination microscopy methods, and ubiquitination assays, we show that the E3 ubiquitin ligase F-box and WD repeat domain-containing 7 (FBXW7), a subunit of the SCF ubiquitin ligase, down-regulates spindle assembly 6 homolog (HsSAS-6), a key protein required for procentriole cartwheel assembly, and thereby regulates centriole duplication. We found that FBXW7 abrogation stabilizes HsSAS-6 and increases its recruitment to the mother centriole at multiple sites, leading to supernumerary centrioles. Ultrastructural analyses revealed that FBXW7 is broadly localized on the mother centriole and that its presence is reduced at the site where the HsSAS-6-containing procentriole is formed. This observation suggested that FBXW7 restricts procentriole assembly to a specific site to generate a single new centriole. In contrast, during HsSAS-6 overexpression, FBXW7 strongly associated with HsSAS-6 at the centriole. We also found that SCF interacts with HsSAS-6 and targets it for ubiquitin-mediated degradation. Further, we identified putative phosphodegron sites in HsSAS-6, whose substitutions rendered it insensitive to FBXW7-mediated degradation and control of centriole number. In summary, SCF targets HsSAS-6 for degradation and thereby controls centriole biogenesis by restraining HsSAS-6 recruitment to the mother centriole, a molecular mechanism that controls supernumerary centrioles/centrosomes and the maintenance of bipolar spindles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.AC119.012178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135989PMC
April 2020

Oxyresveratrol drives caspase-independent apoptosis-like cell death in MDA-MB-231 breast cancer cells through the induction of ROS.

Biochem Pharmacol 2020 03 20;173:113724. Epub 2019 Nov 20.

Amrita School of Biotechnology, Amrita Vishwa Vidyapeetham, Amritapuri, Clappana P.O., Kollam, Kerala 690525, India. Electronic address:

Earlier studies from our laboratory have demonstrated that Oxyresveratrol (OXY), a hydroxyl-substituted stilbene, exhibits potent inhibition of human melanoma cell proliferation. The present study defines a cytotoxic effect of OXY on the highly chemo-resistant, triple-negative human breast cancer cell line MDA-MB-231. OXY-mediated cell death resulted in accumulation of cells at the sub-G1 phase of the cell cycle, induced chromatin condensation, DNA fragmentation, phosphatidylserine externalization and PARP cleavage, indicative of apoptosis. Interestingly, morphology and cell viability studies with the pan-caspase inhibitor, QVD-OPH revealed that OXY-induced cell death was caspase-independent. Docking studies also showed that OXY can bind to the S1 site of caspase-3, and could also exert an inhibitory effect on this executioner caspase. The immunoblot analysis demonstrating the absence of caspase cleavage during cell death further confirmed these findings. OXY was also observed to induce the production of reactive oxygen species, which caused the depolarization of the mitochondrial membrane resulting in translocation of Apoptosis Inducing Factor (AIF) into the nucleus. Pretreatment of the cells with N-Acetyl Cysteine antioxidant prevented cell death resulting from OXY treatment. Thus, OXY initiates ROS-mediated, apoptosis-like cell death, involving mitochondrial membrane depolarization, translocation of AIF into the nucleus, and DNA fragmentation, resulting in caspase-independent cell death in MDA-MB-231 cells. The cytotoxicity manifested by OXY was also observed in 3D cell culture models and primary cells, thereby providing a basis for the utilization of OXY as a novel template for the future design of anticancer therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bcp.2019.113724DOI Listing
March 2020

Molecular profiling of anastatic cancer cells: potential role of the nuclear export pathway.

Cell Oncol (Dordr) 2019 Oct 30;42(5):645-661. Epub 2019 May 30.

Cancer Research Division 1, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India.

Purpose: Anastasis is newly discovered process by which cells recover from late-stage apoptosis upon removal of a death stimulus. Recent reports suggest that cells may recover, even after the initiation of mitochondrial outer-membrane permeabilization (MOMP) and caspase activation. Here, we specifically studied the reversibility of late-stage apoptosis in cervical (HeLa) and breast (MDA-MB-231) cancer cells in relation to the extent of MOMP (limited or widespread). In addition, we explored the molecular factors involved in the anastatic process.

Methods: The extent of MOMP was assessed using time lapse confocal microscopic imaging, considering mitochondrial cytochrome c-GFP release as a marker for MOMP. Anastatic cells were generated by specifically recovering late-stage apoptotic (annexin V/PI positive) cervical and breast cancer cells. Molecular signaling events involved in death reversal were assessed using LC-MS/MS and qRT-PCR. Targeted chemical inhibition and shRNA-based gene silencing studies were employed to explore the role of the nuclear export pathway in anastasis and increased oncogenicity.

Results: Time-lapse imaging of drug-treated Cyt-c-GFP expressing cancer cells revealed cell recovery despite widespread MOMP. A few recovered anastatic cells were noted and these were found to proliferate through a selection-type of survival. They showed increased drug-resistance, migration and invasive potential compared to non-anastatic cancer cells. Network analysis using 49 proteins uniquely expressed in anastatic cells indicated upregulation of nuclear export/import, redox and Ras signaling pathways in both HeLa and MDA-MB-231 anastatic cells, indicating common molecular mechanisms in different cell types. Inhibition of XPO1 significantly reduced the recovery of apoptotic cells and abrogated acquired oncogenic transformation in the anastatic cancer cells.

Conclusions: Our study indicates that cancer cells can revert from apoptosis even after the induction of widespread MOMP. We noted a significant role of the nuclear-export pathway in the anastatic process of cancer cells. Inhibition of anastasis through the nuclear export pathway may be a potential therapeutic strategy for targeting drug-resistance, metastasis and recurrence problems during cancer treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13402-019-00451-1DOI Listing
October 2019

A high-throughput real-time in vitro assay using mitochondrial targeted roGFP for screening of drugs targeting mitochondria.

Redox Biol 2019 01 24;20:379-389. Epub 2018 Oct 24.

Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thycaud P.O., Thiruvananthapuram, Kerala 695014, India.

Most toxic compounds including cancer drugs target mitochondria culminating in its permeabilization. Cancer drug-screening and toxicological testing of compounds require cost-effective and sensitive high-throughput methods to detect mitochondrial damage. Real-time methods for detection of mitochondrial damage are less toxic, allow kinetic measurements with good spatial resolution and are preferred over end-stage assays. Cancer cell lines stably expressing genetically encoded mitochondrial-targeted redox-GFP2 (mt-roGFP) were developed and validated for its suitability as a mitochondrial damage sensor. Diverse imaging platforms and flow-cytometry were utilized for ratiometric analysis of redox changes with known toxic and cancer drugs. Key events of cell death and mitochondrial damage were studied at single-cell level coupled with mt-roGFP. Cells stably expressing mt-roGFP and H2B-mCherry were developed for high-throughput screening (HTS) application. Most cancer drugs while inducing mitochondrial permeabilization trigger mitochondrial-oxidation that can be detected at single-cell level with mt-roGFP. The image-based assay using mt-roGFP outperformed other quantitative methods of apoptosis in ease of screening. Incorporation of H2B-mCherry ensures accurate and complete automated segmentation with excellent Z value. The results substantiate that most cancer drugs and known plant-derived antioxidants trigger cell-death through mitochondrial redox alterations with pronounced ratio change in the mt-roGFP probe. Real-time analysis of mitochondrial oxidation and mitochondrial permeabilization reveal a biphasic ratio change in dying cells, with an initial redox surge before mitochondrial permeabilization followed by a drastic increase in ratio after complete mitochondrial permeabilization. Overall, the results prove that mitochondrial oxidation is a reliable indicator of mitochondrial damage, which can be readily determined in live cells using mt-roGFP employing diverse imaging techniques. The assay described is highly sensitive, easy to adapt to HTS platforms and is a valuable resource for identifying cytotoxic agents that target mitochondria and also for dissecting cell signaling events relevant to redox biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2018.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222140PMC
January 2019

A Real-Time Image-Based Approach to Distinguish and Discriminate Apoptosis from Necrosis.

Curr Protoc Toxicol 2018 02 21;75:2.27.1-2.27.16. Epub 2018 Feb 21.

Cancer Research Program-1, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India.

Recent cell biology studies reveal that a cell can die through multiple pathways via distinct signaling mechanisms. Among these, apoptosis and necrosis are two distinct cell death pathways, and their detection and discrimination is vital in the drug discovery process and in understanding diverse biological processes. Although sensitive assays for apoptosis and necrosis are available, it is extremely difficult to adapt any of these methods to discriminate apoptosis-inducing stimuli from necrosis-inducing stimuli because of the acquisition of secondary necrosis by apoptotic cells when they are not phagocytosed. Essentially, any assay for discriminating apoptosis and necrosis needs to be carried out in real-time kinetic mode. Caspase 3 or 7 activation is observed in the majority of apoptotic cell death. Similarly, the absence of caspase 3/7 activation and cell membrane leakage are the two prominent indicators for necrotic cell death or necroptosis. The programmed form of necrosis, called pyroptosis, is also accompanied by membrane leakage and most often associated with activation of specific caspases such as caspase 1, 4, or 11, but not through caspase 3/7 activation. Here, a robust and sensitive real-time method is described to distinguish and discriminate apoptosis from necrosis. The assay utilizes stable integration of a genetically encoded fluorescence resonance energy transfer (FRET) probe for caspase 3/7 activation and the mitochondrion-targeted DsRed to identify necrotic cells. Caspase activation is determined by cleavage of the FRET probe; loss of soluble FRET probe with retention of mitochondrial red fluorescence indicates necrosis. This unit describes an important protocol for the generation of sensor cells expressing both probes, followed by detailed analysis of apoptosis and necrosis by microscopy imaging, confocal imaging, high-throughput imaging, and flow cytometry. © 2018 by John Wiley & Sons, Inc.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cptx.39DOI Listing
February 2018

Strategies for imaging mitophagy in high-resolution and high-throughput.

Eur J Cell Biol 2018 Jan 27;97(1):1-14. Epub 2017 Oct 27.

Cancer Research Program-1, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India. Electronic address:

The selective autophagic removal of mitochondria called mitophagy is an essential physiological signaling for clearing damaged mitochondria and thus maintains the functional integrity of mitochondria and cells. Defective mitophagy is implicated in several diseases, placing mitophagy as a target for drug development. The identification of key regulators of mitophagy as well as chemical modulators of mitophagy requires sensitive and reliable quantitative approaches. Since mitophagy is a rapidly progressing event and sub-microscopic in nature, live cell image-based detection tools with high spatial and temporal resolution is preferred over end-stage assays. We describe two approaches for measuring mitophagy in mammalian cells using stable cells expressing EGFP-LC3 - Mito-DsRed to mark early phase of mitophagy and Mitochondria-EGFP - LAMP1-RFP stable cells for late events of mitophagy. Both the assays showed good spatial and temporal resolution in wide-field, confocal and super-resolution microscopy with high-throughput adaptable capability. A limited compound screening allowed us to identify a few new mitophagy inducers. Compared to the current mitophagy tools, mito-Keima or mito-QC, the assay described here determines the direct delivery of mitochondrial components to the lysosome in real time mode with accurate quantification if monoclonal cells expressing a homogenous level of both probes are established. Since the assay described here employs real-time imaging approach in a high-throughput mode, the platform can be used both for siRNA screening or compound screening to identify key regulators of mitophagy at decisive stages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejcb.2017.10.003DOI Listing
January 2018
-->