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Dihydrotanshinone I Induces Apoptosis Through Reactive Oxygen Species–Mediated Oxidative Stress in AGS Human Gastric Cancer Cells

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Dihydrotanshinone I Induces Apoptosis Through Reactive Oxygen Species–Mediated Oxidative Stress in AGS Human Gastric Cancer Cells

  1. 1. 27 OBJECTIVE: To investigate the anticancer actions of dihydrotanshinone I (DHTS) in AGS human gastric cancer cells. STUDY DESIGN: Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Intracellular reactive oxygen spe- cies (ROS) levels were determined using flow cytome­ try. Caspase activities were measured with fluorometric assay. RESULTS: Results from MTT assay showed that DHTS significantly inhibited AGS cell viability in dose- and time-dependent manners. Elevated intracellular ROS levels and increased apoptotic cells were observed in DHTS-treated AGS cells. In addition, activation of caspase-3 and caspase-8, rather than caspase-9, was no- ticed in DHTS-treated AGS cells. Furthermore, block- ing ROS generation with N-acetylcysteine markedly re- versed DHTS-induced cell apoptosis. CONCLUSION: All the findings strongly suggest that DHTS can initiate ROS generation and induce oxida­ tive stress and cell apoptosis in AGS human gastric cancer cells, which deserves to be further developed as an anticancer agent. (Anal Quant Cytopathol Histpathol 2019;41:27–32) Keywords:  alternative medicine; apoptosis; apo- ptosis/drug effects; cell line, tumor; cell prolifera­ tion/drug effects; complementary therapies; Dan­ shen; dihydrotanshinone I; gastric cancer; humans; mitogen-activated protein kinases/metabolism; reactive oxygen species; Salvia miltiorrhiza. Gastric cancer is one of the leading causes of cancer-related death in the world, with low prog­ noses and high mortality.1 Physical surgery is still the most effective way to treat gastric cancer.2 However, the survival rate remains low due to lim- ited therapy and delayed diagnosis approaches for gastric cancer. Chemotherapy is one optional way in treating gastric cancer, especially in those patients at the late stage with metastasis.3 Natural products and their derivatives, such as vitamins, curcumin, and green tea, show great potential ben­ efits to control the growth of cancers.4-6 Analytical and Quantitative Cytopathology and Histopathology® 0884-6812/19/4101-0027/$18.00/0 © Science Printers and Publishers, Inc. Analytical and Quantitative Cytopathology and Histopathology® Dihydrotanshinone I Induces Apoptosis Through Reactive Oxygen Species–Mediated Oxidative Stress in AGS Human Gastric Cancer Cells Xiongdong Zhong, M.Sc., Ni Gu, M.Sc., Lei Chen, B.Sc., and Yunlong Pan, Ph.D. From the Departments of General Surgery, The First Affiliated Hospital of Jinan University and The People’s Hospital of Zhuhai, Guang- zhou, Guangdong Province, P.R. China. Dr. Zhong is Associate Chief Physician, Department of General Surgery, The First Affiliated Hospital of Jinan University, and Depart­ ment of General Surgery, The People’s Hospital of Zhuhai. Dr. Gu is Resident Physician, Department of General Surgery, The People’s Hospital of Zhuhai. Mr. Chen is Chief Physician, Department of General Surgery, The People’s Hospital of Zhuhai. Dr. Pan is Professor, Department of General Surgery, The First Affiliated Hospital of Jinan University. Address correspondence to:  Yunlong Pan, Ph.D., Department of General Surgery, The First Affiliated Hospital of Jinan University, No. 613 Huangpu Road, Tianhe District, Guangzhou 510632, Guangdong Province, P.R. China (yunlongpan@21cn.com). Financial Disclosure:  The authors have no connection to any companies or products mentioned in this article.
  2. 2. Dihydrotanshinone I (DHTS) is a natural bio­ logical tanshinone identified from the herbal med­ icine called Danshen (Salvia miltiorrhiza), which has been widely used for treating cardiac-cerebral vas­ cular diseases and hepatitis for a thousand years in China. DHTS has been demonstrated to have diverse biological activities, including antibacteri- al,7 platelet aggregation inhibition,8 antiangiogen­ esis,9 antiosteoclastogenesis,10 etc. Recently, DHTS was shown to inhibit various cancers including colon cancer,11,12 prostate cancer,13 liver cancer,14 breast cancer,15 and cervical cancer.16 The molec- ular mechanisms for the anticancer action of DHTS included induction of apoptosis,12,15,16 ac- tivation of autophagy,12 induction of cell cycle arrest,15 etc. However, to date, the effect of DHTS on gastric cancer has not been comprehensively elucidated. Reactive oxygen species (ROS) are produced by various redox metabolic reactions, which play important roles in numerous physiological and pathological processes, including arteriosclerosis and carcinogenesis.17 ROS, especially H2O2, are key elements for inflammatory cell recruitment.18 Fur­ thermore, ROS work as second messengers to in- duce diverse redox-sensitive signaling molecules, for example, the stress-activated p38 MAP kinases.19 The activation of p38 MAPK by ROS will eventual­ ly induce cell death, including apoptosis, necrosis, and autophagy.20 In the current study the anticancer action of DHTS was investigated in human gastric cancer cell line AGS and human gastric epithelial cell line GES-1. The underlying mechanisms for the pro- apoptotic effects of DHTS were also evaluated using numerous methods. Materials and Methods All experiments were approved by the Research Committee of The First Affiliated Hospital of Jinan University and carried out in accordance with the approved guidelines. Reagents Dihydrotanshinone I (DHTS, purity >98%) was obtained from TianJin Zhongxin Pharmaceutical Group Co. Ltd. (Tianjin, China). RPMI culture medium, N-acetylcysteine (NAC), 2’7’-dichlorodi­ hydrofluorescein diacetate (H2DCFDA), annexin V/PI (propidium iodide) apoptosis kit, and di- methylsulfoxide (DMSO) were purchased from Sigma (St. Louis, Missouri, USA). Fetal bovine serum and penicillin-streptomycin were purchased from Tianjin Hao Yang Biological Manufacture Co. Ltd. (Tianjin, China). Unless stated otherwise, other reagents were purchased from Sigma. DHTS was dissolved in DMSO and stocked at −20°C, which was diluted to the desired concentrations with fresh culture medium before usage. The final concentration of DMSO was lower than 0.5% in fresh medium, which showed no obvious effect on cell viability. Cell Culture Human gastric cancer cell line AGS and human gastric epithelial cell line GES-1 were obtained from the Cell Bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C under 5% CO2. Cell Viability Assay The effects of DHTS on cell viability of AGS cells and GES-1 cells were measured with 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Briefly, the cells were seed­ ed in 96-well plates (4×103 cells/well) and then incubated with DHTS (0, 5, 10, 20 μM) for 48 hours. The medium was then replaced with fresh culture medium containing 20 μL of 5 mg/mL MTT solution. After further incubation for 4 hours, the supernatants were replaced with 100 μL DMSO to dissolve the formed blue formazan crystal. Absorbances at a wavelength of 570 nm were collected using a multiple fluorescent plate reader (Bio-Rad Laboratories, USA). The data were shown as the percentage of cell viability compared with vehicle control group. Measurement of Intracellular Reactive Oxygen Species (ROS) Level The intracellular ROS levels were determined using a fluorogenic probe H2DCFDA. In brief, AGS cells were treated with different concentra­ tions of DHTS with or without NAC (500 μM) for 2 or 4 hours. Then, the cells were washed twice with phosphate buffered saline (PBS) and incu- bated with H2DCFDA (30 μM) at 37°C for 30 min­ utes. After incubation the cells were then washed twice and resuspended in fresh PBS. The fluores­ cence of 10,000 individual cells from each group were collected and analyzed using a FACStar flow 28 Analytical and Quantitative Cytopathology and Histopathology® Zhong et al
  3. 3. cytometer (Becton Dickinson, Franklin Lakes, New Jersey, USA). Oxidative Stress Detection Reduced glutathione (GSH)/oxidized disulfide (GSSG) ratio was measured to determine oxida­ tive stress in AGS cells with DHTS treatment. In this study the concentrations of total glutathione (T-GSH), GSH, and GSSG were measured using enzymatic methods. T-GSH was measured by testing 5,5-dithio-bis(2-nitrobenzoic) acid (DTNB)- GSSG reductase recycling. GSSG was determined using 5-thio-2-nitrobenzoic acid (TNB) generated from reduced GSH reaction with DTNB. The sig­ nal measured at 410 nm was used to assay the TNB formation. The reduced GSH level was calculated by subtracting GSSG from T-GSH. Measurement of Apoptotic Cells by Flow Cytometry The apoptotic cells in DHTS-treated AGS cells were determined using flow cytometry with Annexin V/PI apoptosis detection kit under the manufacturer’s instructions. In brief, AGS cells were treated with DHTS with or without NAC (500 μM) for 48 hours and washed 3 times with ice-cold PBS. Annexin V-FITC (5 μL) and PI (1 mg/ mL) were then added into the cells and further incubated for another 30 minutes. The stained cells were washed twice with PBS, and data from 20,000 cells of each group were collected and analyzed by a BD flow cytometer. Detection of Caspase Activity Caspase-3, caspase-8, and caspase-9 activities were measured with fluorometric assay kits following the manufacturer’s instructions (Abcam). Briefly, AGS cells were collected and lysed with lysis buf­ fer. Proteins (20 μg) were incubated with caspase- 3 substrate Ac-DEVD-AMC, caspase-8 substrate Ac-LEDH-AMC, or caspase-9 substrate Ac-IETD- AMC (50 μg) at 37°C for 2 hours, respectively. The mixtures were then transferred into a 96-well microplate with black bottom. Fluorescences at 400 nm and 505 nm for noncleaved substrate and cleaved substrate, respectively, were recorded in a fluorescent plate reader (Millipore, USA). Statistical Analysis The results were analyzed by the analysis of vari­ ance (ANOVA) followed by Student’s t test using GraphPad Prism version 5.0 (GraphPad Co., USA). All data were shown as means±standard devia­ tion (SD) from 3 independent experiments. In all comparisons, p<0.05 was considered statistically significant. Results DHTS Inhibited Cell Proliferation in AGS Cells MTT assay was used to access the effects of DHTS on cell proliferation in human gastric cancer cell AGS and human gastric epithelial cell GES-1. As shown in Figure 1A, treatment with DHTS (5, 10, and 20 μM) markedly inhibited cell proliferation in AGS cells in a dose-dependent manner. In the meantime, DHTS showed no obvious effects in GES-1 cell treatment with the same concentrations (Figure 1B). DHTS Induced Oxidative Stress in AGS Cells The intracellular ROS level and the GSH/GSSG ratio were measured to determine oxidative stress in AGS cells under DHTS treatment. The results showed that DHTS treatment (10 μM) significant- ly increased the intracellular ROS level after incu­ bation for 2 or 4 hours. NAC could remarkably reverse the induction of intracellular ROS levels in AGS with DHTS treatment (Figure 2A). In ad- dition, DHTS (10 μM) significantly inhibited the ratio of GSH/GSSG in AGS cells, while NAC could recover the inhibition of the ratio of GSH/GSSG in AGS cells (Figure 2B). DHTS Induced Cell Apoptosis in AGS Cells The apoptotic cells in DHTS-treated AGS cells were determined using flow cytometry with Annexin V/PI double staining. As shown in Figure 3, the apoptotic cells were significantly increased by DHTS (10 μM) treatment. In addition, the pro­ apoptotic effect of DHTS was inhibited by NAC (500 μM) pretreatment. DHTS Activated Caspase-3 and -8 Activities in AGS Cells As shown in Figure 4, treatment of AGS cells with DHTS (10 μM) resulted in remarkable elevations of caspase-3 and -8 activities, rather than caspase-9. The activations of caspase-3 and -8 by DHTS in AGS cells were further inhibited by NAC, a ROS inhibitor. Discussion Gastric cancer is one leading malignancy in the world, with an increased mortality rate. Although chemoresistance becomes a big concern, chemo­ Volume 41, Number 1/February 2019 29 Dihydrotanshinone I in Gastric Cancer Cells
  4. 4. therapy remains one of the effective options for treating gastric cancer. It is urgent to develop nov- el chemotherapeutic agents. Due to the diversity of molecular structures, natural products are con­ sidered as a valuable source of novel chemothera­ peutic agents. DHTS, a tanshinone from Danshen (Salvia miltiorrhiza), exhibits various biological ac- tivities.8,12 In the present study the results indicated that DHTS exerted promising antitumor effects by inducing cell apoptosis through generation of ROS and activation of p38 MAPK pathway in human gastric cancer cells. Apoptosis, known as programmed cell death, causes diverse cell changes including cell shrink­ age, cell membrane blebbing, nuclear and DNA fragmentation, and consequently cellular dysfunc­ tion.21 Apoptosis is controlled by a broad range of cell signals originating intracellularly or extra­ 30 Analytical and Quantitative Cytopathology and Histopathology® Zhong et al Figure 1  Effects of DHTS on cell viability of (A) AGS and (B) GES-1 cells. Data are expressed as means±SD. **p<0.01, ***p<0.001 when compared with vehicle control group. Figure 2 Effects of DHTS on ROS and GSH/GSSG levels in AGS cells. (A) AGS cells were treated with DHTS (10 μM) with or without NAC (500 μM) for 2 or 4 h. The intracellular ROS level was determined using a fluorogenic probe H2DCFDA. (B) AGS cells were treated with DHTS (10 μM) with or without NAC (500 μM) for 4 h. Reduced glutathione (GSH)/oxidized disulfide (GSSG) ratio was measured to determine oxidative stress in AGS cells. Data are expressed as means±SD. **p<0.01 when compared with vehicle control group; #p<0.05 when compared with DHTS treatment group.
  5. 5. cellularly, including caspases and the proteins of the bcl-2 family. Mitochondria plays a crucial role in mediating apoptosis through various apoptotic proteins, for example cytochrome c, whose major functions are the control of cellular metabolism and apoptosis and induction of the caspase cas­ cade as well.22 The caspases are a group of enzymes called cysteine proteases, and caspases working as the major executors of apoptotic processes. In our current study we demonstrated that DHTS induced remarkable elevations of caspase-3 and -8 activities, rather than caspase-9 (Figure 4). The activations of caspase-3 and -8 by DHTS in AGS cells were partially inhibited by NAC, a ROS in- hibitor, suggesting that the proapoptotic effects of DHTS are ROS dependent. Recently, various reports demonstrated that ROS served as central modulators in the apoptosis.23,24 In addition, ROS were also shown to induce apo­ ptosis through regulating phosphorylation and ac- tivation of the MAPK pathways, resulting in de- creased antiapoptotic protein levels and increased proapoptotic protein expression, and followed by cell death.23,24 Our present results showed that ROS generation was involved in DHTS-induced apoptosis in gastric cancer. Gastric cancer is a common malignant tumor affecting approximately 760,000 people worldwide each year. Although surgical removal is the most effective treatment, chemotherapy and radiation therapy are still used as palliative treatment for gastric cancer. DHTS firstly identified from Salvia miltiorrhiza exhibited potent anticancer activities through diverse mechanisms containing inhibi­ tion of cancer cell growth, induction of cell-cycle arrest, and reduction of tumor progression, etc.11-16 In the present study dihydrotanshinone I induced apoptosis through ROS-mediated oxidative stress in AGS human gastric cancer cells. In the future, it is warranted to verify the anticancer action of Volume 41, Number 1/February 2019 31 Dihydrotanshinone I in Gastric Cancer Cells Figure 3  Apoptosis induced by DHTS in AGS cells. AGS cells were treated with DHTS with or without NAC (500 μM) for 48 h. Annexin V-FITC (5 μL) and propidium iodide (1 mg/mL) were then added into the cells and further incubated for another 30 min. Data of 20,000 cells from each group were collected and analyzed by a BD flow cytometer. Data are expressed as means±SD. **p<0.01 when compared with vehicle control group; #p<0.05 when compared with DHTS treatment group. Figure 4  DHTS induced caspase activity in AGS cells. Caspase-3, -8, and -9 activity were monitored after DHTS treatment in AGS cells. Data are expressed as means±SD. **p<0.01 when compared with vehicle control group; #p<0.05 when compared with DHTS treatment group.
  6. 6. dihydrotanshinone I in a gastric cancer mouse mod­ el before applying to gastric cancer patients. Limitation of application of herbal medicines as drugs for cancer patients is probably due to the following reasons: (1) various secret components are added into highly complex personalized pre­ scriptions in some traditional medicines, (2) lack of sound proof of efficacy in treating specific cancer patients, and (3) long-term and short-term safety considerations, such as specific usage, minimal ef- fective dosage, and tolerable maximal dosage, etc. It is also important to note that there is still a long way to go before active ingredients from herbal medicines are accepted as treatment candidates in standard care for cancer patients. Taken together, the obtained results demon- strated that DHTS was able to inhibit cell prolif­ eration and induce ROS-mediated cell apoptosis. 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