Inhibitory Mechanism of Combined Hydroxychavicol With Epigallocatechin-3-Gallate Against Glioma Cancer Cell Lines: A Transcriptomic Analysis

Amirah Abdul Rahman; Wan Zurinah Wan Ngah; Rahman Jamal; Suzana Makpol; Roslan Harun; Norfilza Mokhtar

doi:10.3389/fphar.2022.844199

Inhibitory Mechanism of Combined Hydroxychavicol With Epigallocatechin-3-Gallate Against Glioma Cancer Cell Lines: A Transcriptomic Analysis

Front Pharmacol. 2022 Mar 22:13:844199. doi: 10.3389/fphar.2022.844199. eCollection 2022.

Authors

Amirah Abdul Rahman^{1

2}, Wan Zurinah Wan Ngah^{2

3}, Rahman Jamal², Suzana Makpol³, Roslan Harun⁴, Norfilza Mokhtar⁵

Affiliations

¹ Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Kampus Sungai Buloh, Universiti Teknologi MARA, Cawangan Selangor, Sungai Buloh, Malaysia.
² UKM Medical Centre, UKM Medical Molecular Biology Institute (UMBI), Kuala Lumpur, Malaysia.
³ Department of Biochemistry, Faculty of Medicine, UKM Medical Centre, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.
⁴ KPJ Ampang Specialist Hospital, Ampang, Malaysia.
⁵ Department of Physiology, Faculty of Medicine, UKM Medical Centre, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.

Abstract

Emerging reports have shown therapeutic potential of hydroxychavicol (HC) and epigallocatechin-3-gallate (EGCG) against cancer cells, however high concentrations are required to achieve the anticancer activity. We reported the synergy of low combination doses of EGCG+HC in glioma cell lines 1321N1, SW1783, and LN18 by assessing the effects of EGCG+HC through functional assays. Using high throughput RNA sequencing, the molecular mechanisms of EGCG+HC against glioma cell lines were revealed. EGCG/HC alone inhibited the proliferation of glioma cell lines, with IC50 values ranging from 82 to 302 µg/ml and 75 to 119 µg/ml, respectively. Sub-effective concentrations of combined EGCG+HC enhanced the suppression of glioma cell growth, with SW1783 showing strong synergism with a combination index (CI) of 0.55 and LN18 showing a CI of 0.51. A moderate synergistic interaction of EGCG+HC was detected in 1321N1 cells, with a CI value of 0.88. Exposure of 1321N1, SW1783, and LN18 cells to EGCG+HC for 24 h induces cell death, with caspase-3 activation rates of 52%, 57%, and 9.4%, respectively. However, the dose for SW1783 is cytotoxic to normal cells, thus this dose was excluded from other tests. EGCG+HC induced cell cycle arrest at S phase and reduced 1321N1 and LN18 cell migration and invasion. Combined EGCG+HC amplified its anticancer effect by downregulating the axon guidance process and metabolic pathways, while simultaneously interfering with endoplasmic reticulum unfolded protein response pathway. Furthermore, EGCG+HC exerted its apoptotic effect through the alteration of mitochondrial genes such as MT-CO3 and MT-RNR2 in 1321N1 and LN18 cells respectively. EGCG+HC dynamically altered DYNLL1 alternative splicing expression in 1321N1 and DLD splicing expression in LN18 cell lines. Our work indicated the pleiotropic effects of EGCG+HC treatment, as well as particular target genes that might be investigated for future glioma cancer therapeutic development.

Keywords: apoptosis; epigallocatechin-3-gallate; gene expression; glioma; hydroxychavicol; synergism; transcriptomic.