Epimedokoreanin C, A Prenylated Flavonoid Isolated from Epimedium Koreanum, Induces Non-Apoptotic Cell Death with the Characteristics of Methuosis in Lung Cancer Cells

Abstract

Background: Methuosis is a novel type of non-apoptotic cell death characterized by accumulation of cytoplasmic vacuoles. Identification of molecules that induce methuosis may provide alternative therapeutics for cancers that are refractory to apoptosis. Epimedokoreanin C (EKC) is a prenylated flavonoid isolated from a Chinese herb Epimedium koreanum. Methods: In this study, MTT assay, real-time cell analysis monitoring of cell growth and wound-healing assay were used for cell viability and migration evaluation. Flow cytometry was performed to assess apoptosis. Immunoblot analysis, immunofluorescence, pull-down assays and live cell imaging with fluorescent tracers were utilized to evaluate the regulation of signaling pathways.Results: EKC reduced cell viability accompanied by extreme vacuolation in lung cancer NCI-H292 cells. The EKC-induced cell death was clarified as non-apoptosis based on the absence of apoptotic changes. The vacuoles stimulated by EKC were supposed to be derived from macropinocytosis based on the engulfment of extracellular fluid tracer, Lucifer Yellow. The vacuoles acquired some characteristics of late endosomes supported that EKC-induced cell death could be described as methuosis. Rac1 and Arf6 were found to be regulated inversely after EKC treatment. Blocking Rac1 activation with the specific Rac1 inhibitor EHT 1864 prevented the accumulation of vacuoles induced by EKC markedly, suggested that the regulation of Rac1 and Arf6 was at least partial mechanism involved in EKC induced methuosis. EKC synergized the effects of doxorubicin and etoposide, demonstrating the effectiveness of using EKC to synergize conventional chemotherapy. Conclusions: Collectively, EKC was demonstrated as a methuosis-like cell death inducer in NCI-H292 cells. It has the potential to be used as an attractive prototype for developing drugs that could kill apoptosis-resistant cancer cells.

A Mulberry Diels-Alder-Type Adduct, Kuwanon M, Triggers Apoptosis and Paraptosis of Lung Cancer Cells through Inducing Endoplasmic Reticulum Stress

“…Pictures were acquired using a phase-contrast microscope (Olympus, IX71, Tokyo, Japan). The wound healing rate was calculated as: (area of original wound-area of wound at different time points)/area of original wound × 100% [ 41 ].…”

“…Cells were seeded and cultured in plate wells with cover-glass at the bottom, and, after treatment with KWM, cells were washed and incubated with Mito-Tracker or ER-Tracker, according to the protocol of the manufacturers. Images were collected by confocal microscope (LSM 700, Zeiss, Oberkochen, Germany) [ 41 ].…”

The Role and Therapeutic Potential of Macropinocytosis in Cancer

“…Rac1 and Arf6 regulation was partially involved in this methuosis. Furthermore, the effects of doxorubicin and etoposide were enhanced by EKC, suggesting that EKC may be a beneficial complement to conventional chemotherapy ( Liu et al, 2021 ). Isobavachalcone (IBC), a naturally occurring chalcone, is extracted from traditional Chinese medicines such as Psoralea corylifolia L. ( Kuete and Sandjo, 2012 ).…”