[논문]산수유 추출물에 의한 LNCaP 전립선 세포의 증식 억제 및 양성 전립선 비대증 유발 인자의 발현에 미치는 영향

김민영; 지선영; 황보현; 이혜숙; 홍수현; 최영현

doi:10.15429/jkomor.2021.21.1.10

산수유 추출물에 의한 LNCaP 전립선 세포의 증식 억제 및 양성 전립선 비대증 유발 인자의 발현에 미치는 영향
Inhibition of Proliferation of LNCaP Prostate Cells by Corni Fructus Extract Is Associated with a Decrease in the Expression of Benign Prostatic Hyperplasia-Causing Factors 원문보기

한방비만학회지 = Journal of Korean Medicine for Obesity Research, v.21 no.1, 2021년, pp.10 - 21

김민영 (동의대학교 한의과대학 생화학교실) , 지선영 (동의대학교 한의과대학 생화학교실) , 황보현 (동의대학교 한의과대학 생화학교실) , 이혜숙 (동의대학교 한의과대학 생화학교실) , 홍수현 (동의대학교 한의과대학 생화학교실) , 최영현 (동의대학교 한의과대학 생화학교실)

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Objectives: Benign prostatic hyperplasia (BPH) is a progressive pathological condition characterized by excessive proliferation of the prostate. In this study, we evaluated the effect of Corni Fructus water extract (CF) on the promotion of prostate cell proliferation by dihydrotestosterone (DHT). Methods: The effect of CF on the proliferation of LNCaP prostate cells was evaluated, and DHT was treated to induce an in vitro BPH model. To study the mechanism of inhibition of cell proliferation and BPH by CF, changes in the expression of key factors related to cell cycle and BPH were investigated. We further investigated the effect on the production of reactive oxygen species (ROS) and nitric oxide (NO) to evaluate the antioxidant and anti-inflammatory efficacy of CF. Results: Inhibition of LNCaP cell proliferation by CF was associated with decreased expression of cyclin D1 and cyclin A and increased expression of cyclin-dependent kinase inhibitor p21. CF also suppressed expression of BPH inducing factors such as 5α-reductase type 2 and androgen receptor (AR) as well as prostate specific antigen (PSA). Furthermore, CF significantly blocked DHT-induced LNCaP cell proliferation and effectively attenuated DHT-induced expression of BPH mediators and cyclins. In addition, CF inhibited DHT-induced oxidative and inflammatory reactions by inhibiting production of ROS and NO. Conclusion: Our results demonstrated that CF probably acted as 5α-reductase type 2 inhibitor, preventing the 5α-reductase type 2-AR signaling pathway, thereby reducing the conversion of testosterone to DHT and the expression of PSA, which is at least correlated with the antioxidant and anti-inflammatory activities of CF.

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그림 Fig. 1. Effect of CF on the cell proliferation and apoptosis of LNCaP cells. Cells were treated with the indicated concentrations of CF for 48 h. (A) Cell viability was measured by the metabolic-dye-based MTT assay. Data are expressed as the mean±standard errors (n=3). The statistical analysis was conducted using analysis of variance between groups (*P<0.05 when compared to control). (B and C) After 48 h treatment with increasing concentrations of CF, cells were stained with PI. The cell DNA was analyzed by flow cytometry. (B) Representative histograms are presented. (C) The average values of the cell frequency corresponding to each phase of the cell cycle are shown. (D) Cell apoptosis was detected by flow cytometry following annexin-V and PI double staining. The frequencies of apoptosis positive cells were quantified by expressing the number of annexin V-positive cells. CF: Corni Fructus water extract, MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide, PI: propidium iodide.
그림 Fig. 2. Effect of CF on the expression of cell cycle regulators in LNCaP cells. Cells were treated with the indicated concentrations of CF for 48 h. (A) Total cell lysates were prepared. Western blotting was then performed using the indicated antibodies and an enhanced chemiluminescence detection system. β-actin was used as an internal control. (B) The expression of each protein was indicated as a fold change relative to the control. Quantitative analysis of mean pixel density was performed using the ImageJ^® software (U.S. National Institutes of Health, Bethesda, MD, USA). The results are presented as the mean±standard errors (n=3, *P<0.05 when compared t o control). CF: Corni F ructus w ater e xtract, CDK: cyclin-dependent kinase.
그림 Fig. 3. Effect of CF on the expression of BPH regulators in LNCaP cells. Cells were treated with the indicated concentrations of CF for 48 h. (A) Total cell lysates were prepared. Western blotting was then performed using the indicated antibodies and an enhanced chemiluminescence detection system. β-actin was used as an internal control. (B) The expression of each protein was indicated as a fold change relative to the control. Quantitative analysis of mean pixel density was performed using the ImageJ^® software (U.S. National Institutes of Health, Bethesda, MD, USA). The results are presented as the mean±standard errors (n=3, *P<0.05 when compared to control). CF: Corni Fructus water extract, BPH: benign prostatic hyperplasia, AR: androgen receptor, SRC1: steroid receptor coactivator 1, Arnt1: arylhydrocarbon receptor nuclear translocator 1, PSA: prostate specific antigen.
그림 Fig. 4. Effect of CF on cell proliferation and apoptosis of DHT-treated LNCaP cells. Cells were pre-treated with or without the indicated concentrations of CF for 1 h before 10 nM DHT treatment for 48 h. (A) Cell viability was measured by the metabolic-dye-based MTT assay. Data are expressed as the mean±standard errors (n=3, *P<0.05 when compared to control; ^#P<0.05 when compared to the control DHT-treated cells). (B and C) After pre-treatment with of CF in the presence or absence of DHT, cells were stained with PI. The cell DNA was analyzed by flow cytometry. (B) Representative histograms are presented. (C) The average values of the cell frequency corresponding to each phase of the cell cycle are shown. (D) Cell apoptosis was detected by flow cytometry following annexin-V and PI double staining. The frequencies of apoptosis positive cells were quantified by expressing the number of annexin V-positive cells. CF: Corni Fructus water extract, DHT: dihydrotestosterone, MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide, PI: propidium iodide.
그림 Fig. 5. Effect of CF on the expression of cell cycle and BPH regulators in LNCaP cells. Cells were pre-treated with or without 0.6 mg/ml CF for 1 h before 10 nM DHT treatment for 48 h. (A) Total cell lysates were prepared. Western blotting was then performed using the indicated antibodies and an enhanced chemiluminescence detection system. β-actin was used as an internal control. (B) The expression of each protein was indicated as a fold change relative to the control. Quantitative analysis of mean pixel density was performed using the ImageJ^® software (U.S. National Institutes of Health, Bethesda, MD, USA). The results are presented as the mean±standard errors (n=3, *P<0.05 when compared to control; ^#P<0.05 when compared to DHT-treated cells). CF: Corni Fructus water extract, BPH: benign prostatic hyperplasia, DHT: dihydrotestosterone, AR: androgen receptor, SRC1: steroid receptor coactivator 1, PSA: prostate specific antigen.
그림 Fig. 6. Effect of CF on the production of ROS and NO in DHT-stimulated LNCaP cells. Cells were pre-treated with or without 0.6 mg/ml CF for 1 h before 10 nM DHT treatment for 1 h (A) or 48 h (B-D). The cells were incubated at 37℃ in the dark for 20 min with a culture medium containing 10 μM DCF-DA to monitor ROS production. (A) The degree of ROS production was measured with a flow cytometer. Quantitative data of DCF-DA intensity of CF-treated cells with or without 10 nM DHT. (B) The nitrite concentration in culture media was evaluated by Greiss reaction. (C) Total cell lysates were prepared. Western blotting for eNOS was then performed using an enhanced chemiluminescence detection system. β-actin was used as an internal control. (D) The expression of eNOS protein was indicated as a fold change relative to the control. Quantitative analysis of mean pixel density was performed using the ImageJ^® software (U.S. National Institutes of Health, Bethesda, MD, USA). The results are presented as the mean±standard errors (n=3, *P<0.05 when compared to control; ^#P<0.05 when compared to DHT-treated cells). CF: Corni Fructus water extract, ROS: reactive oxygen species, NO: nitric oxide, DHT: dihydrotestosterone, DCF-DA: 2',7'-dichlorodihydrofluorescein diacetate, eNOS: endothelial nitric oxide synthetase.

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