J Huazhong Univ Sci Technol［Med Sci］ DOI 10.1007/s11596-014-1345-6 34(5):740-744,2014 J Huazhong Univ Sci Technol［Med Sci］ 34(5):2014
740
Quercetin Suppresses HeLa Cells by Blocking PI3K/Akt Pathway* Tao XIANG (项 涛), Yong FANG (方 勇)#, Shi-xuan WANG (王世宣) Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014
Summary: To explore the effect of quercetin on the proliferation and apoptosis of HeLa cells, HeLa cells were incubated with quercetin at different concentrations. Cell viability was evaluated by MTT assay, cell apoptosis was detected by Annexin-Ⅴ/PI double labeled cytometry and DNA ladder assay. Cell cycle was flow cytometrically determined and the morphological changes of the cells were observed under a fluorescence microscope after Hoechst 33258 staining and the apoptosis-related proteins in the HeLa cells were assessed by Western blotting. The results showed that quercetin significantly inhibited the growth of HeLa cells and induced obvious apoptosis in vitro in a time- and dose-dependent manner. Moreover, quercetin induced apoptosis of HeLa cells in cell cycle-dependent manner because quercetin could induce arrest of HeLa cells at G0/G1 phase. Quercetin treatment down-regulated the expression of the PI3K and p-Akt. In addition, quercetin could down-regulate expression of bcl-2, up-regulate Bax, but exerted no effect on the overall expression of Akt. We are led to conclude that quercetin induces apoptosis via PI3k/Akt pathways, and quercetin has potential to be used as an anti-tumor agent against human cervix cancer. Key words: quercetin; HeLa cells; PI3K; p-AKT; Bc1-2; Bax
Invasive cervical cancer is one of the most common forms of female malignancies, and according to an estimate, in 2008 alone, there were approximately 530 000 new cases of the cancer and 275 000 cervical-cancer-related deaths worldwide[1]. Nowadays, growing number of young patients suffer from cervical cancer and the pathologic types of the malignancies undergo changes. Clinically, the platinum-based combination chemotherapy has been playing a major role in treatment of cervical cancer. Nonetheless, the toxicity of the chemotherapy and resistance to chemotherapeutic agents substantially restrict the application of the platinum-based combination chemotherapy. Herbal medicines, as complementary and alternative medical therapies, have been used for the treatment of cancers and have been gaining attention for its low toxicity and effectiveness among the general population in the United States[2]. Quercetin (3, 30, 40, 5, 7-pentahydroxy flavones) is a strong anti-oxidant and one of the most ubiquitous bioflavonoids in the plant kingdom. Quercetin possesses anti-tumor effect on a variety of tumors and is a promising anti-tumor agent that works by inducing cell-cycle arrest and/or apoptosis, or inhibiting the reproduction and migration of tumor cells[3]. In this study, we examined the effect of quercetin on the viability of HeLa cells in vitro and explored if it suppresses HeLa cells by blocking PI3K/Akt pathway, with an attempt to understand the mechanism of its anti-tumor Tao XIANG, E-mail: [email protected] # Corresponding author, E-mail: [email protected] * This project was supported by the National Natural Science Foundation of China (No. 81071663).
effect and to find a new anti-tumor alternative. 1 MATERIALS AND METHODS 1.1 Subjects Quercetin was purchased from Sigma (Aldrich, USA). It was stored under –20°C and diluted with Dulbecco’s modified Eagle medium (DMEM) into the desired concentrations before use. Also from Sigma Company were 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT), DNA ladder extraction kit, Rabbit anti-human antibodies against PI3k, T-Akt, p-Akt, Bcl-2 and Bax. DMEM and fetal bovine serum (FBS) were obtained from Gibco, USA. Hoechst 33258 was procured from Beyotime Institute of Biotechnology, China. Annexin Ⅴ-FITC/propidium iodide (PI) assay kit was from Jingmei Biotechnology Co. Ltd. (China). HRP-labeled sheep anti-rabbit secondary antibody was procured from Santa Cruz Biotechnology, USA. Bio-Rad Protein Assay kit and Enhanced chemiluminescence (ECL) kit came from Amersham, Sweden. 1.2 Cell Culture The HeLa cell line was obtained from the China Center for Type Culture Collection, Wuhan University, China and was grown in DMEM supplemented with 10% FBS, and 100 μg/mL of gentamicin, 100 IU/mL of penicillin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. The cells were passaged every 1 or 2 day(s), and the cells with over 98 % activity in the logarithmic growth phase were used in subsequent experiments. 1.3 MTT Assay The effect of quercetin on cell viability was determined by MTT assay. Briefly, HeLa cells (2×105
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J Huazhong Univ Sci Technol［Med Sci］ 34(5):2014
cells/mL, 200 µL/well) were seeded into 96-well plates and then incubated for 24 h at 37°C in the presence of 12.5–200 µmol/L quercetin. Culture medium of the identical volume containing DMSO served as control. Subsequently, 20 mL MTT solution (5 mg/mL) was added into each well for continuous incubation for another 4 h. Then the culture medium was removed, and the formazan product was dissolved with 150 μL DMSO by shaking for 10 min. The plates were then read on a Bio-Rad M450 system at a test wavelength of 492 nm. The survival rate of tumor cells incubated with quercetin at different concentrations was calculated as follows: Survival rate (%)=[1–Absorbance (A) of drug treated cells/A of control]×100%. All experimental data were based on at least three independent experiments. 1.4 Detection of Apoptosis with Hoechst 33258 Apotosis was morphologically detected by using Hoechest 33258 staining. About 5×105 HeLa cells on the cover-slips in the 6-well plates were treated with 25 µmol/L quercetin (concentration at IC50) for 24 h. Cells were washed twice with phosphate-buffered saline (PBS, pH 7.4) and fixed in 75% ethanol for 15 min. They were washed with PBS, incubated in 2.5 mg/mL Hoechst 33258 solution at room temperature for 20 min in the dark and washed with PBS 3 times. The cells were then observed and photographs were taken under an inverted fluorescent microscope (Olympus, Japan). The apoptosis rate was calculated based on five different but equal visual areas where the number of apoptotic cells and total cell number were counted. 1.5 Annexin Ⅴ-FITC/PI Assay Quercetin-induced apoptosis of HeLa cells was flow cytometrically measured by using annexin Ⅴ -FITC. Cells were incubated with quercetin at different concentrations (0, 25, 50 and 100 µmol/L) for 24 h. Cells were harvested, washed with cold PBS twice and then re-suspended in 1×binding buffer at a density of 1×106 cells/mL. Afterwards, 100 µL cell suspension was transferred into a FCM tube, and 5 µL annexin V-FITC and 10 µL PI were added to the cell suspension. The sample was fully mixed by gently vortexing. The stained samples were incubated for 15 min at room temperature in the dark. An additional 300 µL 1×binding buffer was added to each tube. Samples were detected within 1 h on a flow cytometer (BD FACS Calibur, USA). 1.6 DNA Ladder Assay HeLa cells were incubated with quercetin at different concentrations (25, 50 and 100 µmol/L), harvested by centrifugation, washed twice with cold PBS, with DNA extracted by following the instructions of DNA ladder extraction kit, and then dissolved in TE solution without
DNase. The sample was then electrophored at 20 V on agarose gel for 4 h, and then analyzed on an image analysis system (KODAK DNA gel image analysis 2.0 system). 1.7 Cell Cycle Analysis HeLa cells were treated by incubation with different concentrations (0, 50, 100 and 150 µmol/L) of quercetin for 24 h, and 1×106 cells were then harvested by lowspeed centrifugation and fixed in ice-cold 70% ethanol overnight at 4°C. Cell pellets were re-suspended in PBS with 0.9 mL 1% TritonX-100 and 100 µL 1 mg/mL RNaseA. The solution was incubated for 30 min at 37°C. Cells were stained with 300–500 µL PI solution in the dark at 4°C for 30 min. Samples were then flow cytometrically detected. 1.8 Western Blotting HeLa cells were treated with different concentrations of quercetin or left untreated to serve as control group. The samples were homogenized on ice in RIPA buffer containing protease inhibitors. Proteins (50 mg) were dissolved in 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane. After being blocked with 5 % non-fat milk, the membrane was incubated with primary antibodies (1:1500 dilution) against PI3k, T-Akt, p-Akt, Bcl-2 and Bax at 4°C overnight and then incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit IgG (1:2000 dilution) for 90 min. Protein expression was visualized by enhanced chemiluminescence reaction and densitometrically measured. Each experiment was performed in triplicate to obtain the mean value. 1.9 Statistical Analysis Experimental data were expressed as ±s. For intra-group comparison, the F test was used. All statistical analyses were performed by using the statistics software package SPSS 16.0. Statistical significance was set at a P
740
Quercetin Suppresses HeLa Cells by Blocking PI3K/Akt Pathway* Tao XIANG (项 涛), Yong FANG (方 勇)#, Shi-xuan WANG (王世宣) Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014
Summary: To explore the effect of quercetin on the proliferation and apoptosis of HeLa cells, HeLa cells were incubated with quercetin at different concentrations. Cell viability was evaluated by MTT assay, cell apoptosis was detected by Annexin-Ⅴ/PI double labeled cytometry and DNA ladder assay. Cell cycle was flow cytometrically determined and the morphological changes of the cells were observed under a fluorescence microscope after Hoechst 33258 staining and the apoptosis-related proteins in the HeLa cells were assessed by Western blotting. The results showed that quercetin significantly inhibited the growth of HeLa cells and induced obvious apoptosis in vitro in a time- and dose-dependent manner. Moreover, quercetin induced apoptosis of HeLa cells in cell cycle-dependent manner because quercetin could induce arrest of HeLa cells at G0/G1 phase. Quercetin treatment down-regulated the expression of the PI3K and p-Akt. In addition, quercetin could down-regulate expression of bcl-2, up-regulate Bax, but exerted no effect on the overall expression of Akt. We are led to conclude that quercetin induces apoptosis via PI3k/Akt pathways, and quercetin has potential to be used as an anti-tumor agent against human cervix cancer. Key words: quercetin; HeLa cells; PI3K; p-AKT; Bc1-2; Bax
Invasive cervical cancer is one of the most common forms of female malignancies, and according to an estimate, in 2008 alone, there were approximately 530 000 new cases of the cancer and 275 000 cervical-cancer-related deaths worldwide[1]. Nowadays, growing number of young patients suffer from cervical cancer and the pathologic types of the malignancies undergo changes. Clinically, the platinum-based combination chemotherapy has been playing a major role in treatment of cervical cancer. Nonetheless, the toxicity of the chemotherapy and resistance to chemotherapeutic agents substantially restrict the application of the platinum-based combination chemotherapy. Herbal medicines, as complementary and alternative medical therapies, have been used for the treatment of cancers and have been gaining attention for its low toxicity and effectiveness among the general population in the United States[2]. Quercetin (3, 30, 40, 5, 7-pentahydroxy flavones) is a strong anti-oxidant and one of the most ubiquitous bioflavonoids in the plant kingdom. Quercetin possesses anti-tumor effect on a variety of tumors and is a promising anti-tumor agent that works by inducing cell-cycle arrest and/or apoptosis, or inhibiting the reproduction and migration of tumor cells[3]. In this study, we examined the effect of quercetin on the viability of HeLa cells in vitro and explored if it suppresses HeLa cells by blocking PI3K/Akt pathway, with an attempt to understand the mechanism of its anti-tumor Tao XIANG, E-mail: [email protected] # Corresponding author, E-mail: [email protected] * This project was supported by the National Natural Science Foundation of China (No. 81071663).
effect and to find a new anti-tumor alternative. 1 MATERIALS AND METHODS 1.1 Subjects Quercetin was purchased from Sigma (Aldrich, USA). It was stored under –20°C and diluted with Dulbecco’s modified Eagle medium (DMEM) into the desired concentrations before use. Also from Sigma Company were 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT), DNA ladder extraction kit, Rabbit anti-human antibodies against PI3k, T-Akt, p-Akt, Bcl-2 and Bax. DMEM and fetal bovine serum (FBS) were obtained from Gibco, USA. Hoechst 33258 was procured from Beyotime Institute of Biotechnology, China. Annexin Ⅴ-FITC/propidium iodide (PI) assay kit was from Jingmei Biotechnology Co. Ltd. (China). HRP-labeled sheep anti-rabbit secondary antibody was procured from Santa Cruz Biotechnology, USA. Bio-Rad Protein Assay kit and Enhanced chemiluminescence (ECL) kit came from Amersham, Sweden. 1.2 Cell Culture The HeLa cell line was obtained from the China Center for Type Culture Collection, Wuhan University, China and was grown in DMEM supplemented with 10% FBS, and 100 μg/mL of gentamicin, 100 IU/mL of penicillin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. The cells were passaged every 1 or 2 day(s), and the cells with over 98 % activity in the logarithmic growth phase were used in subsequent experiments. 1.3 MTT Assay The effect of quercetin on cell viability was determined by MTT assay. Briefly, HeLa cells (2×105
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J Huazhong Univ Sci Technol［Med Sci］ 34(5):2014
cells/mL, 200 µL/well) were seeded into 96-well plates and then incubated for 24 h at 37°C in the presence of 12.5–200 µmol/L quercetin. Culture medium of the identical volume containing DMSO served as control. Subsequently, 20 mL MTT solution (5 mg/mL) was added into each well for continuous incubation for another 4 h. Then the culture medium was removed, and the formazan product was dissolved with 150 μL DMSO by shaking for 10 min. The plates were then read on a Bio-Rad M450 system at a test wavelength of 492 nm. The survival rate of tumor cells incubated with quercetin at different concentrations was calculated as follows: Survival rate (%)=[1–Absorbance (A) of drug treated cells/A of control]×100%. All experimental data were based on at least three independent experiments. 1.4 Detection of Apoptosis with Hoechst 33258 Apotosis was morphologically detected by using Hoechest 33258 staining. About 5×105 HeLa cells on the cover-slips in the 6-well plates were treated with 25 µmol/L quercetin (concentration at IC50) for 24 h. Cells were washed twice with phosphate-buffered saline (PBS, pH 7.4) and fixed in 75% ethanol for 15 min. They were washed with PBS, incubated in 2.5 mg/mL Hoechst 33258 solution at room temperature for 20 min in the dark and washed with PBS 3 times. The cells were then observed and photographs were taken under an inverted fluorescent microscope (Olympus, Japan). The apoptosis rate was calculated based on five different but equal visual areas where the number of apoptotic cells and total cell number were counted. 1.5 Annexin Ⅴ-FITC/PI Assay Quercetin-induced apoptosis of HeLa cells was flow cytometrically measured by using annexin Ⅴ -FITC. Cells were incubated with quercetin at different concentrations (0, 25, 50 and 100 µmol/L) for 24 h. Cells were harvested, washed with cold PBS twice and then re-suspended in 1×binding buffer at a density of 1×106 cells/mL. Afterwards, 100 µL cell suspension was transferred into a FCM tube, and 5 µL annexin V-FITC and 10 µL PI were added to the cell suspension. The sample was fully mixed by gently vortexing. The stained samples were incubated for 15 min at room temperature in the dark. An additional 300 µL 1×binding buffer was added to each tube. Samples were detected within 1 h on a flow cytometer (BD FACS Calibur, USA). 1.6 DNA Ladder Assay HeLa cells were incubated with quercetin at different concentrations (25, 50 and 100 µmol/L), harvested by centrifugation, washed twice with cold PBS, with DNA extracted by following the instructions of DNA ladder extraction kit, and then dissolved in TE solution without
DNase. The sample was then electrophored at 20 V on agarose gel for 4 h, and then analyzed on an image analysis system (KODAK DNA gel image analysis 2.0 system). 1.7 Cell Cycle Analysis HeLa cells were treated by incubation with different concentrations (0, 50, 100 and 150 µmol/L) of quercetin for 24 h, and 1×106 cells were then harvested by lowspeed centrifugation and fixed in ice-cold 70% ethanol overnight at 4°C. Cell pellets were re-suspended in PBS with 0.9 mL 1% TritonX-100 and 100 µL 1 mg/mL RNaseA. The solution was incubated for 30 min at 37°C. Cells were stained with 300–500 µL PI solution in the dark at 4°C for 30 min. Samples were then flow cytometrically detected. 1.8 Western Blotting HeLa cells were treated with different concentrations of quercetin or left untreated to serve as control group. The samples were homogenized on ice in RIPA buffer containing protease inhibitors. Proteins (50 mg) were dissolved in 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane. After being blocked with 5 % non-fat milk, the membrane was incubated with primary antibodies (1:1500 dilution) against PI3k, T-Akt, p-Akt, Bcl-2 and Bax at 4°C overnight and then incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit IgG (1:2000 dilution) for 90 min. Protein expression was visualized by enhanced chemiluminescence reaction and densitometrically measured. Each experiment was performed in triplicate to obtain the mean value. 1.9 Statistical Analysis Experimental data were expressed as ±s. For intra-group comparison, the F test was used. All statistical analyses were performed by using the statistics software package SPSS 16.0. Statistical significance was set at a P
