Differentially expressed genes on the growth of mouse Leydig cells treated with standardised Eurycoma longifolia extract

Khurshid Ahmed,Nor Amira; Lim,Shern Kwok; Pandian,Ganesh   N.; Sugiyama,Hiroshi; Lee,Chong Yew; Khoo,Boon Yin; Chan,Kit Lam

doi:10.3892/mmr.2020.11485

Differentially expressed genes on the growth of mouse Leydig cells treated with standardised Eurycoma longifolia extract

Authors:
- Nor Amira Khurshid Ahmed
- Shern Kwok Lim
- Ganesh N. Pandian
- Hiroshi Sugiyama
- Chong Yew Lee
- Boon Yin Khoo
- Kit Lam Chan
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Affiliations: Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Penang, Malaysia, World Premier International Research Initiative‑Institute for Integrated Cell‑Material Sciences (WPI‑iCeMS), Kyoto University, Kyoto 606-8501, Japan, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia
Published online on: September 2, 2020 https://doi.org/10.3892/mmr.2020.11485
Pages: 3645-3658
Copyright: © Khurshid Ahmed et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Eurycoma (E.) longifolia Jack (Tongkat Ali) is a widely applied medicine that has been reported to boost serum testosterone and increase muscle mass. However, its actual biological targets and effects on an in vitro level remain poorly understood. Therefore, the present study aimed to investigate the effects of a standardised E. longifolia extract (F2) on the growth and its associated gene expression profile in mouse Leydig cells. F2, even at lower doses, was found to induce a high level of testosterone by ELISA. The level was as high as the levels induced by eurycomanone and formestane in Leydig cells. However, Leydig cells treated with F2 demonstrated reduced viability, which was likely due to the diminished cell population at the G0/G1 phase and increased cell population arrested at the S phase in the cell cycle, as assessed by MTT assay and flow cytometry, respectively. Cell viability was revived when the treatment time‑point was prolonged to 96 h. Genome‑wide gene analysis by reverse transcription‑quantitative PCR of F2‑treated Leydig cells at 72 h, when the cell growth was not revived, and 96 h, when the cell growth had started to revive, revealed cyclin‑dependent kinase‑like 2 (CDKL2) to be a potential target in regulating the viability of F2‑treated Leydig cells. Functional analysis, as analysed using GeneMANIA Cytoscape program v.3.6.0 (https://genemania.org/), further suggested that CDKL2 could act in concert with Casitas B‑lineage lymphoma and sphingosine kinase 1 interactor‑A‑kinase anchoring protein domain‑containing genes to regulate the viability of F2‑treated Leydig cells. The findings of the present study provide new insights regarding the potential molecular targets associated with the biological effect of E. longifolia extract on cell growth, particularly on the cell cycle, which could aid in enhancing the bioefficacy and reducing the toxicity of this natural product in the future.

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