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Manilkara zapota (L.) P. Royen Leaf Extract Derived Silver Nanoparticles Induce Apoptosis in Human Colorectal Carcinoma Cells Without Affecting Human Lymphocytes or Erythrocytes

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Abstract

Plant-derived synthesis of silver nanoparticles (AgNPs) has found wide biomedical applications including cancer cure. This report deals with biosynthesis of silver nanoparticles (MZLAgNPs) employing leaf extracts of Manilkara zapota (L.) under optimized conditions. Characterization of MZLAgNPs using UV-Vis spectroscopy, FTIR, XRD, and FESEM analyses revealed that the particles were predominantly spherical averaging 24 nm in size. Their cellular effects were assessed by MTT assay, fluorescence, and scanning electron microscopy of cells stained with propidium iodide, acridine orange/ethidium bromide, and annexin V-FITC to visualize signs of apoptosis. Evaluation of cell proliferation by clonogenic assay, wound healing ability by scratch assay and cell cycle distribution by flow-cytometry was also carried out. Apoptosis-related gene expressions were analyzed by RTq-PCR and western blot analysis. MZLAgNPs selectively inhibited growth of colorectal carcinoma HCT116, HeLa, and non-small lung carcinoma A549 cells, dose-dependently with IC50 concentrations of 8, 16, and 29 μg/mL respectively, following 72-h treatment, without affecting growth of normal human lymphocytes and erythrocytes. Apoptosis induction was observed by fluorescence and scanning electron microscopy. Overproduction of reactive oxygen species (ROS), reduction of mitochondrial membrane potential, upregulation of apoptotic-related genes - PUMA, cas-3, cas-8, cas-9, and BAX, expression of caspase 3, and occurrence of PARP cleavage were observed in MZLAgNPs/cisplatin treated cells. Taken together, our results clearly demonstrate the therapeutic potential of biogenic MZLAgNPs as an effective agent for killing colorectal carcinoma cells by apoptosis induction.

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Acknowledgements

The authors would like to thank the National Institute of Technology, Calicut, for use of SEM and EDX facility. Thanks are also due to our sister departments of Chemistry and Physics and Central Sophisticated Instruments Facility, University of Calicut, for allowing use of facilities to carry out FTIR, XRD, and SEM analysis and Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram for allowing use of their FACS facility. VSS acknowledges technical help by Jobish Joseph with the figures.

Funding

This study received financial support from Calicut University by way of Research fellowship to VSS.

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Authors and Affiliations

  1. Recombinant DNA Laboratory, Department of Biotechnology, University of Calicut, Malappuram, Kerala, 673635, India

    V. S. Shaniba, Ahlam Abdul Aziz & P. R. Manish Kumar

  2. School of Health sciences, University of Calicut, Malappuram, Kerala, 673635, India

    P. R. Jayasree

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  1. V. S. Shaniba
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Correspondence to P. R. Manish Kumar.

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The blood samples used for hemolysis assay and isolation of lymphocytes for lymphocyte culture and MTT assay was willingly self-donated by the first author (Shaniba V.S.) and the second author (Ahlam Abdul Aziz) of the manuscript submitted. It may be noted that according to the Indian Council for Medical Research, New Delhi, India, Chapter-II, page no. 11–12, the ethical approval for this research was not deemed to be necessary. According to this guideline, proposals which present less than minimal risks are exempted from the ethical review process.

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Shaniba, V.S., Aziz, A.A., Jayasree, P.R. et al. Manilkara zapota (L.) P. Royen Leaf Extract Derived Silver Nanoparticles Induce Apoptosis in Human Colorectal Carcinoma Cells Without Affecting Human Lymphocytes or Erythrocytes. Biol Trace Elem Res 192, 160–174 (2019). https://doi.org/10.1007/s12011-019-1653-6

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