[논문]H460 인체 폐암세포에서 길경 물 추출물에 의해 유도된 세포사멸에서 자가포식의 역할

홍수현; 최영현

doi:10.14374/hfs.2021.29.4.155

H460 인체 폐암세포에서 길경 물 추출물에 의해 유도된 세포사멸에서 자가포식의 역할
The Role of Autophagy in Apoptosis Induced by Water Extract of Platycodonis Radix in H460 Human Lung Cancer Cells 원문보기

大韓韓醫學方劑學會誌 = Herbal formula science, v.29 no.4, 2021년, pp.155 - 165

홍수현 (동의대학교 한의과대학 생화학교실) , 최영현 (동의대학교 한의과대학 생화학교실)

Abstract ▼ AI-Helper

Objectives : Recent studies have suggested that Platycodonis Radix has various pharmacological effects such as anti-cancer, antioxidant, anti-asthma, anti-diabetes, anti-obesity, hepatoprotective, and cardiovascular protection effects. The aim of this study was to investigate the role of water extract of Platycodonis Radix (WPR)-induced autophagy in H460 human lung cancer cells. Methods : H460 cells were treated with WPR and cell viability was calculated by an MTT assay. To evaluate changes in apoptosis- and autophagy-related genes, Western blotting was performed. Two kinds of autophagy inhibitors, 3-Methyladenine (3-MA) and bafilomycin A1, were pretreated to confirm the role of WPR-induced autophagy. Results : WPR reduced the viability of H460 cells in a treatment concentration-dependent manner, which was associated with induction of apoptosis. It was also confirmed that WPR induced autophagy based on the formation of specific intracellular vacuoles and changes in the expression of autophagy-related genes. Interestingly, pretreatment with 3-MA and bafilomycin A1 increased WPR-induced cytotoxicity and apoptosis. Conclusions : WPR induced autophagy at low concentrations and early stages of treatment, but promoted apoptosis at high concentrations and late stages. Moreover, WPR-induced autophagy had a cytoprotective role in H460 cells.

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표/그림 (5)

그림 Fig. 1. WPR stimulation attenuated cell growth and proliferation in human large cell lung carcinoma H460 cells. A) H460 cells were seeded at an initial density of 2.8 × 10⁵ cells per ml in 6 well plates. After 24 h, cells were treated with the indicated concentrations of WPR for 24 h. Cell viability was measured by an MTT assay. B) Cells were stimulated with WPR 4 ㎎/㎖ for the indicated time points. A and B) Each point represents the mean ± standard deviation (SD) of three independent experiments. (*p<0.05, compared with controls). C) Morphologic changes were photographed under an inverted-microscope. Magnification, X50
그림 Fig. 2. WPR treatment induced apoptosis which was in part dependent on caspase activation in H460 cells. A) H460 cells were seeded at an initial density of 2.8 × 10⁵ cells per ml in 6 well plates. After 24 h, cells were treated with the indicated concentrations of WPR for 24 h. The cells harvested and flow cytometer analysis was performed for analysis of DNA content using flow cytometry. The frequencies of cells in the sub-G1 phase were presented. Each point represents the mean ± SD of three independent experiments. (*p<0.05, compared with controls). B) H460 cells were treated by WPR 4 ㎎/㎖ for the indicated times. Representative fluorescence microscopy images of nuclei celled with DAPI were presented. Magnification, X200. C) Western blot analysis was performed for the expression of casapas-3, PARP, Bcl-2 and Bid using whole lysates of WPR-treated cells. β-actin was used to ensure the equal protein loading.
그림 Fig. 3. WPR induced autophagy in H460 cells. A) Creation of vacuoles were observed by an inverted-microscope in cells stimulated with WPR (3 ㎎/㎖) for the indicated times (magnification, X200). B) The expression level of autophagy-related proteins was investigated by immunoblot analysis. β-actin was used as a loading control. C) Immunofluorescence images representing the distribution of the autophagic marker LC3 in 3 ㎎/㎖ WPR-treated cells for 24 h were taken with a fluorescence microscope (magnification, X200). D) After WPR treatment (3 or 4 ㎎/㎖) for 24 h, autophagic cells were quantified in three random image fields by counting cells containing >5 LC3 punctate staining and expressed as the mean ± SD of three independent (^*p<0.05, compared with control group).
그림 Fig. 4. Attenuation of early phase autophagy inhibited autophagosome formation and enhanced the cytotoxicity and apoptosis in WPR-stimulated H460 cells. A) Immunofluorescence images showing alternative punctate patterns of LC3 localization in cells treated with 3 ㎎/㎖ WPR in the presence or absence of 1 mM 3-MA for 24 h were presented (magnification, X 200). B) Autophagic cells were quantified in three random image fields by counting cells containing >5 LC3 punctate staining and expressed as the mean ± SD of three independent (^*p<0.05, compared with WPR-only treated group). C) Changes in Beclin-1 expression in cells cultured under the same conditions were presented. β-actin was used as a loading control. D) Cells were pretreated with the 3-MA for 1 h and then WPR was treated for 24 h. The cell viability was measured by an MTT assay. E) Analysis of DNA content was performed using flow cytometry, and results represent the mean ± SD of three independent experiments (^*p<0.05, compared with WPR-only treated group).
그림 Fig. 5. Reduction of late phase autophagy inhibited autophagosome formation and enhanced WPR-induced cytotoxicity and apoptosis in H460 cells. A) Immunofluorescence images were shown using fluorescence microscopy suggesting changes in the punctate patterns of LC3 localization in H460 cells treated with 3 ㎎/㎖ WPR in the presence or absence of bafilomycin A1 for 24 h (magnification, X 200). B) Autophagic cells were quantified in three random image fields by counting cells containing >5 LC3 punctate staining and expressed as the mean ± SD of three independent (^*p<0.05, compared with WPR-only treated group). C) Changes in Beclin-1 and PARP expression in cells cultured under the same conditions were presented. β-actin was used as a loading control. D) Cells were treated with 3 or 4 ㎎/㎖ of WPR in the presence or absence of Bafilomycin A1 for 24 h. Cell viability was measured by an MTT assay. E) Analysis of DNA content was performed using flow cytometry, and results represent the mean ± SD of three independent experiments (^*p<0.05, compared with WPR-only treated group).

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