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Effect of embelin on inhibition of cell growth and induction of apoptosis in Acanthamoeba castellanii

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Abstract

Acanthamoeba castellanii is the causative agent of fatal encephalitis and blinding keratitis. Current therapies remain a challenge, hence there is a need to search for new therapeutics. Here, we tested embelin (EMB) and silver nanoparticles doped with embelin (EMB–AgNPs) against A. castellanii. Using amoebicidal assays, the results revealed that both compounds inhibited the viability of Acanthamoeba, having an IC50 of 27.16 ± 0.63 and 13.63 ± 1.08 μM, respectively, while causing minimal cytotoxicity against HaCaT cells in vitro. The findings suggest that both samples induced apoptosis through the mitochondria-mediated pathway. Differentially expressed genes analysis showed that 652 genes were uniquely expressed in treated versus untreated cells, out of which 191 were significantly regulated in the negative control vs. conjugate. Combining the analysis, seven genes (ARIH1, RAP1, H3, SDR16C5, GST, SRX1, and PFN) were highlighted as the most significant (Log2 (FC) value ± 4) for the molecular mode of action in vitro. The KEGG analysis linked most of the genes to apoptosis, the oxidative stress signaling pathway, cytochrome P450, Rap1, and the oxytocin signaling pathways. In summary, this study provides a thorough framework for develo** therapeutic agents against microbial infections using EMB and EMB–AgNPs.

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Abbreviations

A. castellanii :

Acanthamoeba castellanii

AgNO3 :

Silver nitrate

AK:

Acanthamoeba Keratitis

ATCC:

American Type Culture Collection

BBB:

Blood–brain barrier

BF:

Biological process

CC:

Cellular component

CNS:

Central nervous system

CPR:

NADPH-cytochrome c reductase

DCF:

Dichlorofluorescein

DCFDA:

2′,7′-Dichlorodihydro-fluorescein diacetate

DEGs:

Differentially expressed genes

DEU:

Differential exon usage

DMSO:

Dimethyl sulfoxide

EMB:

Embelin

EMB–AgNPs:

Silver nanoparticles coated with embelin

FBS:

Fetal bovine serum

FE-SEM:

Field emission-scanning electron microscopy

FPKM:

Fragments per kilobases per million

GAE:

Granulomatous amoebic encephalitis

GO:

Gene ontology

H2O2 :

Hydrogen peroxide

HaCaT:

Human keratinocyte cell

HCS:

HiSeq Control Software

KEGG:

Kyoto Encyclopedia of Genes and Genomes

LDH:

Lactate dehydrogenase

MF:

Molecular function

NGS:

Next-generation sequencing

NPs:

Nanoparticles

PBS:

Phosphate-buffered saline

PCD:

Programmed cell death

PCR:

Polymerase chain reaction

Pen-strep:

Penicillin Streptomycin

PYG:

Proteose peptone, yeast extract and glucose

RCC1:

Regulator of chromosome condensation

ROS:

Reactive oxygen species

RPMI-1640:

Roswell Park Memorial Institute 1640 medium

SDS:

Sodium dodecyl sulfate

TEM:

Transmission electron microscope

UV–Vis:

Ultraviolet–visible

ΔΨm:

Mitochondrial membrane potential

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Acknowledgements

This research work was supported by Sunway University, Malaysia, Monash University, Malaysia, and the H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi.

Funding

This research work was supported by Sunway University, Malaysia (Individual Research Grant, GRTIN-IRG-10-2022) and Sindh Higher Education Commission (SHEC), Pakistan vide letter No. NO.DD/SHEC/1-14/2014, Project code SHEC/SRSP/Med-3/15/2021-21. Ruqaiyyah Siddiqui and Naveed Ahmed Khan were supported by the Air Force Office of Scientific Research (AFOSR), USA.

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Authors and Affiliations

  1. Department of Biological Sciences, School of Medical and Life Sciences, Sunway University, Subang Jaya, 47500, Selangor, Malaysia

    Usman Ahmed, Seng-Kai Ong & Ayaz Anwar

  2. H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan

    Khalid Mohammed Khan

  3. Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia

    Khalid Mohammed Khan

  4. Microbiota Research Center, Istinye University, 34010, Istanbul, Turkey

    Ruqaiyyah Siddiqui & Naveed Ahmed Khan

  5. Neuropharmacology Research Laboratory, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, 47500, Bandar Sunway, Malaysia

    Mohd Farooq Shaikh

  6. Department of Medical Laboratories Technology, College of Applied Medical Sciences, Taibah University, 42353, Madinah, Saudi Arabia

    Bader Saleem Alawfi

  7. School of Dentistry and Medical Sciences, Charles Sturt University, Orange, New South Wales, Australia

    Mohd Farooq Shaikh

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  1. Usman Ahmed
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Contributions

AA and NAK conceptualized the study. AA, RS, BSA, and NAK planned the study. UA and AA conducted the experiments and wrote the first draft of the manuscript. S-KO wrote the parts of NGS, KMK, RS, BSA, and MFS corrected the manuscript. NAK and AA supervised the entire study. All authors approved the final manuscript.

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Correspondence to Naveed Ahmed Khan or Ayaz Anwar.

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Ahmed, U., Ong, SK., Khan, K.M. et al. Effect of embelin on inhibition of cell growth and induction of apoptosis in Acanthamoeba castellanii. Arch Microbiol 205, 360 (2023). https://doi.org/10.1007/s00203-023-03698-3

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