Bio-Medical Materials and Engineering 24 (2014) 1019–1025 DOI 10.3233/BME-130899 IOS Press
1019
Licochalcone A inhibiting proliferation of bladder cancer T24 cells by inducing reactive oxygen species production Jiangtao Jianga, Xuan Yuana, Hong Zhaoa, Xinyan Yana , Sun Xilingb,∗ and Qiusheng Zhenga,* a
School of Pharmacy, Shihezi University, Shihezi, 832002, Xinjiang, China School of integrated traditional Chinese and Western Medicine, Binzhou Medical College, Yantai 264000, Shandong, China
b
Abstract. The aim of this study was to determine the relationship between proliferation inhibition and the production of reactive oxygen species (ROS) induced by Licochalcone A (LCA). Cell viability was evaluated using sulforhodamine B (SRB) assay. Intracellular ROS level was assessed using the 2, 7-dichlorofluorescein diacetate (H2DCFDA) probe and dihydroethidium (DHE) probe assay. The results indicate that LCA inhibits human bladder cancer T24 proliferation in a concentration-dependent manner, with an IC50 value of approximately 55 µM. The LCA-induced ROS production is inhibited by the co-treatment of LCA and free radical scavenger N-acetyl-cysteine (NAC), on the contrary, the proliferation rate and ROS production increase when treated by the combination of LCA and L-buthionine-(S,R)-sulfoximine (BSO). The ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) decreases in a concentration-dependent manner. The results suggest that LCA inhibits proliferation by increasing intracellular ROS levels resulted in an oxidative stress status in T24 cells. Keywords: Licochalcone A, T24 cells, ROS, Cell proliferation, GSH, GSSG
1. Introduction Bladder cancer is a common international public health problem, and it represents the 4th most common malignancy among men and the 8th most frequent cause of male cancer deaths [1, 2]. In recent years, bladder cancer has been usually cured with surgery, chemotherapy, or combinations of chemotherapy and radiotherapy, but all these method have associated limitations [3]. Patients diagnosed with localized transitional cell carcinoma have a 5-year survival rate above 90%. However, patients with regional and distant metastatic disease have a 5-year survival rate below 50% and 10%, respectively [1]. Therefore, the current therapy for bladder cancer is not satisfactory and it is crucial to find a novel treatment to combat the disease with less side effects and recurrence rate.
∗
Address for Correspondence: Binzhou Medical College, Yantai 264000, Shandong, China. Phone: +86 05356886837; fax: +86 005356886837; E-mail addresses: [email protected]; Shihezi University, beierlu, shihezi, China. Phone: +86 09932057003; fax: +86 099320570; E-mail addresses: [email protected]. 0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
1020
J. Jiang et al. / LCA inhibiting proliferation of bladder cancer T24 cells by inducing ROS production
Different from normal cells, tumor cells possess independent survival advantages with unlimited proliferation, which make tumors malignant. They constitutively generate large but tolerable amounts of reactive oxygen species (ROS) to constantly activate redox-sensitive transcription factors and responsive genes that are involved in the survival, proliferation and apoptosis of cancer cells. With such a reliance on ROS, it is anticipated that if the redox state of cancer cells can be regulated, the redox-sensitive responsive genes can then be manipulated and consequently cancer cell growth will be controlled. Recently, natural products have become one of the main sources in modern drug discovery for its active components with anticancer potential and less side effects, especially for anticancer drugs. Some of natural products are approved by the United States Food and Drug Administration [4]. Licorice is one of the most commonly prescribed herbs in China for therapeutic effects on various diseases ranging from microbial infection to cancer [5-7], and is therefore expected to be an effective medicine without serious side effects. Licochalcone A (LCA) is a flavonoid extracted from licorice root (Glycyrrhiza glabra) and has antiparasitic, antibacterial and anti-tumor properties [8-9]. LCA reportedly inhibits cell proliferation in gastric [7], colon [9], ovarian [10], prostatic [11], and skin cancer cells [12]. These studies strongly support the use of LCA in chemoprevention of human cancer. The aim of this study was to determine the relationship between proliferation inhibition and the production of ROS induced by LCA. 2. Materials and Methods 2.1. Materials LCA (purity ≥ 98%) was purchased from Zhongxin Pharmaceutical Group Co., Ltd. (Tianjin, China). RPMI (Roswell Park Memorial Institute) Medium 1640 was obtained from GIBCO (Invitrogen Corporation). Unless indicated otherwise, other reagents were purchased from Sigma. 2.2. Cell culture and treatment Human bladder cancer T24 cells were purchased from Cell Bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in RPMI 1640 medium supplemented with 10% FBS (v/v), 100 U/ml penicillin and 100 μg/ml streptomycin in humidified atmosphere with 5% CO2 at 37°C. Cells were allowed to attach for 24 h before treatment. LCA was dissolved in DMSO and diluted with fresh medium to achieve the desired concentration. The final concentration of DMSO did not exceed 0.2% in the fresh medium, and DMSO at this concentration has no significant effect on the cell viability. 2.3. Cell viability assay T24 cells were trypsinized and seeded into 96-well plates at 1.0×105 cells/ml. Thereafter, cells were exposed to LCA (0, 10, 20, 40, 60 and 80 μM) for 24 h followed by extra incubation in fresh medium for another 24 h. The effect of LCA-induced cytotoxicity was evaluated using sulforhodamine B (SRB) assay [13]. Optical density (OD) was detected at a wavelength of 490 nm.
J. Jiang et al. / LCA inhibiting proliferation of bladder cancer T24 cells by inducing ROS production
1021
2.4. Trypan blue exclusion test The lethality of LCA on T24 cells was determined by the trypan blue exclusion test [14]. After 24 h incubation with T24 at 0, 10, 20, 40, 60 and 80 μM, T24 cells were removed from culture medium and cells that excluded trypan blue were counted in a Neubauer chamber. 2.5. Detection of intracellular ROS levels Hydrogen peroxide (H2O2) and superoxide anions (·O2−) in T24 cells were assessed using the 2, 7-dichlorofluorescein (H2DCFDA) and dihydroethidium (DHE) probe [15, 16] respectively. In brief, cells were incubated with the indicated concentration of LCA with or without ROS scavengers (NAC) or GSH inhibitor (BSO). The treated cells were then washed in PBS and incubated with 30 μM of H2DCFDA or 30μM DHE at 37°C for 30 min. Fluorescence was detected using a fluorescent plate reader (Thermo Varioskan Flash 3001) and data expressed as the median fluorescence intensity (MFI). Then the stained cells were observed under a computer-assisted microscope (ZEISS, MIC00266). 2.6. Assessment of intracellular ratio of reduced to oxidized glutathione (GSH/GSSG) The intracellular GSH and GSSG levels were measured following the enzymatic method by assay kits purchased from the Nanking Jiancheng bio-engineering research institute (Nanking, China). In brief, cells were incubated with the indicated concentration of LCA with or without NAC or BSO, and the levels of total glutathione (GSH + GSSG) and GSSG were measured using a fluorescent plate reader (Thermo Varioskan Flash 3001) at 405 nm according to the manufacturer’s instructions. The level of reduced GSH was obtained by subtracting GSSG from total glutathione. 2.7. Statistical analysis Data obtained from different experiments were presented as mean ± standard (SD) from at least three independent experiments and evaluated by analysis of variance (ANOVA) followed by Student Newman-Keuls test. Values of P
1019
Licochalcone A inhibiting proliferation of bladder cancer T24 cells by inducing reactive oxygen species production Jiangtao Jianga, Xuan Yuana, Hong Zhaoa, Xinyan Yana , Sun Xilingb,∗ and Qiusheng Zhenga,* a
School of Pharmacy, Shihezi University, Shihezi, 832002, Xinjiang, China School of integrated traditional Chinese and Western Medicine, Binzhou Medical College, Yantai 264000, Shandong, China
b
Abstract. The aim of this study was to determine the relationship between proliferation inhibition and the production of reactive oxygen species (ROS) induced by Licochalcone A (LCA). Cell viability was evaluated using sulforhodamine B (SRB) assay. Intracellular ROS level was assessed using the 2, 7-dichlorofluorescein diacetate (H2DCFDA) probe and dihydroethidium (DHE) probe assay. The results indicate that LCA inhibits human bladder cancer T24 proliferation in a concentration-dependent manner, with an IC50 value of approximately 55 µM. The LCA-induced ROS production is inhibited by the co-treatment of LCA and free radical scavenger N-acetyl-cysteine (NAC), on the contrary, the proliferation rate and ROS production increase when treated by the combination of LCA and L-buthionine-(S,R)-sulfoximine (BSO). The ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) decreases in a concentration-dependent manner. The results suggest that LCA inhibits proliferation by increasing intracellular ROS levels resulted in an oxidative stress status in T24 cells. Keywords: Licochalcone A, T24 cells, ROS, Cell proliferation, GSH, GSSG
1. Introduction Bladder cancer is a common international public health problem, and it represents the 4th most common malignancy among men and the 8th most frequent cause of male cancer deaths [1, 2]. In recent years, bladder cancer has been usually cured with surgery, chemotherapy, or combinations of chemotherapy and radiotherapy, but all these method have associated limitations [3]. Patients diagnosed with localized transitional cell carcinoma have a 5-year survival rate above 90%. However, patients with regional and distant metastatic disease have a 5-year survival rate below 50% and 10%, respectively [1]. Therefore, the current therapy for bladder cancer is not satisfactory and it is crucial to find a novel treatment to combat the disease with less side effects and recurrence rate.
∗
Address for Correspondence: Binzhou Medical College, Yantai 264000, Shandong, China. Phone: +86 05356886837; fax: +86 005356886837; E-mail addresses: [email protected]; Shihezi University, beierlu, shihezi, China. Phone: +86 09932057003; fax: +86 099320570; E-mail addresses: [email protected]. 0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
1020
J. Jiang et al. / LCA inhibiting proliferation of bladder cancer T24 cells by inducing ROS production
Different from normal cells, tumor cells possess independent survival advantages with unlimited proliferation, which make tumors malignant. They constitutively generate large but tolerable amounts of reactive oxygen species (ROS) to constantly activate redox-sensitive transcription factors and responsive genes that are involved in the survival, proliferation and apoptosis of cancer cells. With such a reliance on ROS, it is anticipated that if the redox state of cancer cells can be regulated, the redox-sensitive responsive genes can then be manipulated and consequently cancer cell growth will be controlled. Recently, natural products have become one of the main sources in modern drug discovery for its active components with anticancer potential and less side effects, especially for anticancer drugs. Some of natural products are approved by the United States Food and Drug Administration [4]. Licorice is one of the most commonly prescribed herbs in China for therapeutic effects on various diseases ranging from microbial infection to cancer [5-7], and is therefore expected to be an effective medicine without serious side effects. Licochalcone A (LCA) is a flavonoid extracted from licorice root (Glycyrrhiza glabra) and has antiparasitic, antibacterial and anti-tumor properties [8-9]. LCA reportedly inhibits cell proliferation in gastric [7], colon [9], ovarian [10], prostatic [11], and skin cancer cells [12]. These studies strongly support the use of LCA in chemoprevention of human cancer. The aim of this study was to determine the relationship between proliferation inhibition and the production of ROS induced by LCA. 2. Materials and Methods 2.1. Materials LCA (purity ≥ 98%) was purchased from Zhongxin Pharmaceutical Group Co., Ltd. (Tianjin, China). RPMI (Roswell Park Memorial Institute) Medium 1640 was obtained from GIBCO (Invitrogen Corporation). Unless indicated otherwise, other reagents were purchased from Sigma. 2.2. Cell culture and treatment Human bladder cancer T24 cells were purchased from Cell Bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in RPMI 1640 medium supplemented with 10% FBS (v/v), 100 U/ml penicillin and 100 μg/ml streptomycin in humidified atmosphere with 5% CO2 at 37°C. Cells were allowed to attach for 24 h before treatment. LCA was dissolved in DMSO and diluted with fresh medium to achieve the desired concentration. The final concentration of DMSO did not exceed 0.2% in the fresh medium, and DMSO at this concentration has no significant effect on the cell viability. 2.3. Cell viability assay T24 cells were trypsinized and seeded into 96-well plates at 1.0×105 cells/ml. Thereafter, cells were exposed to LCA (0, 10, 20, 40, 60 and 80 μM) for 24 h followed by extra incubation in fresh medium for another 24 h. The effect of LCA-induced cytotoxicity was evaluated using sulforhodamine B (SRB) assay [13]. Optical density (OD) was detected at a wavelength of 490 nm.
J. Jiang et al. / LCA inhibiting proliferation of bladder cancer T24 cells by inducing ROS production
1021
2.4. Trypan blue exclusion test The lethality of LCA on T24 cells was determined by the trypan blue exclusion test [14]. After 24 h incubation with T24 at 0, 10, 20, 40, 60 and 80 μM, T24 cells were removed from culture medium and cells that excluded trypan blue were counted in a Neubauer chamber. 2.5. Detection of intracellular ROS levels Hydrogen peroxide (H2O2) and superoxide anions (·O2−) in T24 cells were assessed using the 2, 7-dichlorofluorescein (H2DCFDA) and dihydroethidium (DHE) probe [15, 16] respectively. In brief, cells were incubated with the indicated concentration of LCA with or without ROS scavengers (NAC) or GSH inhibitor (BSO). The treated cells were then washed in PBS and incubated with 30 μM of H2DCFDA or 30μM DHE at 37°C for 30 min. Fluorescence was detected using a fluorescent plate reader (Thermo Varioskan Flash 3001) and data expressed as the median fluorescence intensity (MFI). Then the stained cells were observed under a computer-assisted microscope (ZEISS, MIC00266). 2.6. Assessment of intracellular ratio of reduced to oxidized glutathione (GSH/GSSG) The intracellular GSH and GSSG levels were measured following the enzymatic method by assay kits purchased from the Nanking Jiancheng bio-engineering research institute (Nanking, China). In brief, cells were incubated with the indicated concentration of LCA with or without NAC or BSO, and the levels of total glutathione (GSH + GSSG) and GSSG were measured using a fluorescent plate reader (Thermo Varioskan Flash 3001) at 405 nm according to the manufacturer’s instructions. The level of reduced GSH was obtained by subtracting GSSG from total glutathione. 2.7. Statistical analysis Data obtained from different experiments were presented as mean ± standard (SD) from at least three independent experiments and evaluated by analysis of variance (ANOVA) followed by Student Newman-Keuls test. Values of P
