[논문]비기환, 대칠기탕 및 목향빈랑환 열수 추출물에 의한 인간 간세포암종 HepG2 세포의 세포사멸 유도에 미치는 자가포식의 역할

박상은; 홍수현; 최영현

doi:10.14374/hfs.2022.30.2.67

[국내논문] 비기환, 대칠기탕 및 목향빈랑환 열수 추출물에 의한 인간 간세포암종 HepG2 세포의 세포사멸 유도에 미치는 자가포식의 역할
The Role of Autophagy on the Induction of Apoptosis by Water Extracts of Bigihwan, Daechilgitang and Mokwhyangbinranghwan in HepG2 Human Hepatocellular Carcinoma Cells 원문보기

大韓韓醫學方劑學會誌 = Herbal formula science, v.30 no.2, 2022년, pp.67 - 83

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

Abstract ▼ AI-Helper

Objectives : In this study, the anticancer activity of water extracts of three herbal medicine formulas, Bigihwan (BGH), Daechilgitang (DCGT) and Mokwhyangbinranghwan (MHBRH) listed in Donguibogam, was evaluated in HepG2 cells, a human hepatocellular carcinoma cell line. Methods : We investigated whether the cell viability of HepG2 cells was inhibited by the treatment of water extracts of three prescriptions, and whether their viability inhibitory effect was related to the induction of apoptosis. In addition, the role of autophagy on the induction of apoptosis by the treatment of these extracts was investigated. Results : The anticancer activity of the three water extracts on HepG2 cells was due to induction of apoptosis, not necrosis. Among them, BGH activated the caspase-dependent intrinsic apoptosis pathway associated with mitochondrial dysfunction. However, autophagy was induced more than 2-fold in DCGT-treated HepG2 cells, and the anticancer activity of DCGT was enhanced 1.5-fold in the presence of an autophagy inhibitor, but was attenuated in BGH and MHBRH-treated cells. Conclusion : The results of this study indicate that DCGT-induced autophagy was involved in the inhibition of apoptosis, whereas autophagy by BGH and MHBRH was related to induction of apoptosis.

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표/그림 (8)

표 Table 1. Composition of Bigihwan, Daechilgithang and Mokwhyangbinranghwan
표 Table 2. List of primary and secondary antibodies used for immunoblotting
그림 Fig. 1. Inhibition of cell viability and induction of apoptosis by water extracts of Bigihwan (BGH), Daechilgitang (DCGT) and Mokwhyangbinranghwan (MHBRH) in HepG2 cells. Cells were treated with different concentrations of BGH, DCGT and MHBRH for 24 h. (A) After treatment, cell viability was measured by the MTT assay. (B) The percentages of apoptotic cells by annexin V and PI staining were expressed as a percentage of the total number of annexin V-positive cells. Data are expressed as the mean ± SD (^*p <0.05 and ^***p <0.001 compared to untreated cells). (C) Morphological changes in cells treated with 1.5 mg/mml of each extract were captured using a phase-contrast microscope (Scale bar: 200 μm).
그림 Fig. 2. Effects of necrostatin-1 (NEC), a necrosis inhibitor, on cell survival inhibition and apoptosis induction by BGH, DCGT and MHBRH in HepG2 cells. Cells were pre-treated with 200 μM NEC for 1 h, and maintained in the presence or absence of 1.5 mg/ml BGH, DCGT and MHBRH for 24 h. (A) Cell viability was measured by the MTT assay. (B) The populations of apoptotic cells were determined by expressing the numbers of annexin V-positive cells. The data are presented as the mean ± SD of three independent experiments (^***p <0.001 compared to control; N.S., not significant compared to treated cells).
$Fig. 3. Effects of BGH, DCGT and MHBRH on the expression of Bax, Bcl-2, PARP and cytochrome c, the levels of MMP and activity of caspase-3 in HepG2 cells. Cells were stimulated with the indicated concentrations of BGH, DCGT and MHBRH for 24 h. (A) After treatment, Western blotting with total protein isolated from cells was performed using the indicated antibodies and ECL detection system. Actin was used as an internal control. (B) The cells were stained with JC-1 and then changes of MMP were determined by a flow cytometer. (C) Expression levels of cytochrome c using cytoplasmic and mitochondrial fractions were investigated by Western blot analysis. Actin and COX VI in each protein extract were presented as loading controls. (D) The activity of caspase-3 was examined using a colorimetric caspase-3 assay kit. (B and D) The data indicated the mean ±SD (*p <0.05, **p <0.01 and ***p <0.001 compared to untreated cells).$ 그림 Fig. 3. Effects of BGH, DCGT and MHBRH on the expression of Bax, Bcl-2, PARP and cytochrome c, the levels of MMP and activity of caspase-3 in HepG2 cells. Cells were stimulated with the indicated concentrations of BGH, DCGT and MHBRH for 24 h. (A) After treatment, Western blotting with total protein isolated from cells was performed using the indicated antibodies and ECL detection system. Actin was used as an internal control. (B) The cells were stained with JC-1 and then changes of MMP were determined by a flow cytometer. (C) Expression levels of cytochrome c using cytoplasmic and mitochondrial fractions were investigated by Western blot analysis. Actin and COX VI in each protein extract were presented as loading controls. (D) The activity of caspase-3 was examined using a colorimetric caspase-3 assay kit. (B and D) The data indicated the mean ±SD (^*p <0.05, ^**p <0.01 and ^***p <0.001 compared to untreated cells).
그림 Fig. 4. Effects of a pan-caspase inhibitor, z-VAD-fmk, on cell survival inhibition and apoptosis induction by BGH, DCGT and MHBRH in HepG2 cells. Cells were pre-treated with 50 μM z-VAD-fmk for 1 h, and maintained in the presence or absence of 1.5 mg/ml BGH, DCGT and MHBRH for 24 h. (A) Cell viability was measured by the MTT assay. (B) The populations of apoptotic cells were determined by expressing the numbers of annexin V-positive cells. The data are presented as the mean ± SD of three independent experiments (^***p <0.001 compared to control;^#p <0.05 and ^###p <0.001 compared to treated cells; N.S., not significant compared to treated cells).
그림 Fig. 5. Induction of autophagy by BGH, DCGT and MHBRH in HepG2 cells. Cells were treated with different concentrations of BGH, DCGT and MHBRH for 24 h. (A) After treatment, cells were stained with Cyto-ID, and analyzed by flow cytometry. The frequencies of autophagic cells were presented. Each bar indicated the mean ±SD (^*p <0.05 and ^***p <0.001 compared to untreated cells). (B) Western blotting with total protein isolated from cells was performed using the indicated antibodies and ECL detection system. Actin was used as an internal control.
그림 Fig. 6. Effects of autophagy inhibitors, Baf A1 and 3-MA, on cell survival inhibition and apoptosis induction by BGH, DCGT and MHBRH in HepG2 cells. Cells were pre-treated with 5 μM Baf A1 or 1 mM 3-MA for 1 h, and cultured in the presence or absence of 1.5 mg/ml BGH, DCGT and MHBRH for 24 h. (A) Cell viability was measured by the MTT assay. (B) The populations of apoptotic cells were determined by expressing the numbers of annexin V-positive cells. The data are presented as the mean ± SD of three independent experiments (^***p <0.001 compared to control;^#p <0.05, ^##p <0.01 and ^###p <0.001 compared to treated cells; N.S., not significant compared to treated cells).

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