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The anti-tumor effect of ginsenoside Rh4 in MCF-7 breast cancer cells in vitro and in vivo
Biochemical and Biophysical Research Communications xxx (2018) 1e6
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The anti-tumor effect of ginsenoside Rh4 in MCF-7 breast cancer cells in vitro and in vivo Zhiguang Duan a, b, 1, Bo Wei a, b, 1, Jianjun Deng a, b, Yu Mi a, b, Yangfang Dong a, b, Chenhui Zhu a, b, Rongzhan Fu a, b, Linlin Qu a, b, Daidi Fan a, b, * a
Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, 229 North Taibai Road, Xi'an, Shaanxi 710069, China Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, 229 North Taibai Road, Xi'an, Shaanxi 710069, China
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a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 March 2018 Accepted 22 March 2018 Available online xxx
Breast cancer is a tremendous threat to humans in many countries, and thus we need to find safe and effective drugs for treatment. Ginsenoside Rh4 has been reported to be present in processed ginseng. However, few studies have focused on its anti-tumor activity. In this study, we investigated the inhibitory effects of ginsenoside Rh4 on MCF-7 breast cancer cells and the pathways that promote apoptosis in vitro. To study the effect of ginsenoside Rh4 in vivo, xenograft models were randomly divided into 3 groups (the control group, 10 mg/kg/d Rh4, 20 mg/kg/d Rh4, n ¼ 10 per group), the ginsenoside Rh4 injection method was i.p. The results showed that ginsenoside Rh4 effectively inhibited proliferation, arrested the cell cycle in S phase and induced apoptosis in MCF-7 cells by flow cytometry. Morphological changes caused by ginsenoside Rh4-induced apoptosis were also observed by Hoechst 33342 staining. Westernblot analyses indicated that the apoptosis-inducing effects of ginsenoside Rh4 were associated with the external pathway by decreasing Bcl-2, increasing Bax, and activating caspase-8, -3 and PARP. Moreover, ginsenoside Rh4 significantly inhibited the growth of MCF-7 tumor cells in vivo. These results suggested that ginsenoside Rh4 could be a potentially effective anti-tumor drug for breast cancer. © 2018 Elsevier Inc. All rights reserved.
Keywords: Apoptosis Breast cancer Ginsenoside MCF-7 cells Xenograft model
1. Introduction The morbidity and mortality rates of breast cancer have remained at a high level, which poses a tremendous threat to women in many countries [1]. Chemotherapy has become the main treatment method. However, it has serious side effects, such as diarrhea and emesis, increasing the suffering of patients [2,3]. Therefore, it is imperative to develop a new method to treat or prevent breast cancer. Ginseng, a type of precious herbal medicine, has attracted increasing attention from Western countries due to its extensive pharmacological activity and relative safety [4e6]. It has been reported to possess anti-oxidant functions, improving
Abbreviations: Rh4, ginsenoside Rh4; PEG, >polyethylene glycol. * Corresponding author. Shaanxi Key Laboratory of Degradable Biomedical Materials, School of chemical engineering, Northwest University, 229 North Taibai Road, Xi'an, Shaanxi 710069, China. E-mail addresses: [email protected] (Z. Duan), 201520752@stumail. nwu.edu.cn (B. Wei), [email protected] (D. Fan). 1 These authors contributed equally to this work.
immunity, and anti-tumor effects [7e9]. The multiple pharmacological activities of ginseng are considered to be due to the presence of various ginsenosides [10]. Ginsenosides, which are the major component of ginseng, have been widely reported to have anti-tumor activity, especially for ginsenoside Rg3 and Rh2. Ginsenoside Rg3 has potential antiproliferation and apoptosis-inducing effects in human gastric cancer AGS cells and colon cancer HT29 cells [11,12]. Ginsenoside Rh2 can significantly inhibit cell growth and induce apoptosis via activation of caspase-8 and -9 in human Hepatoma SK-HEP-1 cells [13,14]. Ginsenoside Rh4 (Rh4) was isolated in processed ginseng [15]. Rh4 has been reported to inhibit human leukemia K562 cells [16]. However, there are no reports or studies about the anti-tumor effect of Rh4 on human breast cancer MCF-7 cells, and its mechanism remains unknown. In this paper, based on the above considerations, we have studied the anti-tumor effect of Rh4 on MCF-7 cells and its mechanism through cell experiments including MTT assay, cell cycle assay, apoptosis assay and detection of protein content in vitro.
https://doi.org/10.1016/j.bbrc.2018.03.174 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: Z. Duan, et al., The anti-tumor effect of ginsenoside Rh4 in MCF-7 breast cancer cells in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.174
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Then, nude mice with tumors were injected with Rh4 to observe the anti-tumor effect of the drugs in vivo. The results showed that the inhibition and apoptosis rate of MCF-7 cells increased significantly after Rh4 treatment. Rh4 induced cell apoptosis via external apoptotic pathway activation due to changes in the levels of related proteins. In vivo, Rh4 significantly inhibited the growth of MCF-7 tumor cells. All of the studies will provide the basis for developing Rh4 as an urgentlyneeded therapeutic drug for human breast cancer. 2. Materials and methods 2.1. Chemicals and reagents Rh4 (purity99.0%) was purchased from Puruifa Technology Development Co., Ltd (Chengdu, China). MTT was obtained from Solarbio Co., Ltd (Beijing, China). The Hoechst 33342 fluorescent dye kit and RIPA cell lysates were obtained from Beyotime Institute of Biotechnology Co., Ltd (Shanghai, China). Polyethylene glycol (PEG) 400, which was of medical grade, was obtained from Hua Ri Pharmaceutical Co., Ltd (Hunan, China). Primary antibodies to procaspase-8, procaspase-9, procaspase-3, cleaved caspase-3, PARP, cleaved PARP, Bax, Bcl-2 and GAPDH were purchased from Abcam (CB, US). The secondary antibody to IgG was from Gene Tex (Texas, USA). All staining solutions and other chemicals were purchased from standard commercial suppliers. 2.2. Cell culture The human breast cancer MCF-7 cells were purchased from the American Type Culture Collection (VA, USA). All the cells were cultured with RPMI-1640 (HyClone) medium containing 10% FBS (HyClone) and 1% penicillinestreptomycin (HyClone) at 37 C under a humidified 5% CO2 atmosphere. 2.3. MTT assay Cell proliferation was measured using the MTT assay for MCF7 cells. Rh4 was dissolved in RPMI-1640 culture media containing 0.1% DMSO at final concentrations of 0, 50, 100, 150, 200, and 250 mg/mL. The MCF-7 cells were grown in 96-well plates at 9 103 cells per well, incubated at 37 C for 24 h and then treated with various concentrations of Rh4 for 48 h. The control cells were exposed to culture media containing 0.1% DMSO. Then, 50 mL of MTT solution (5 mg/mL) was added to each well. Cells were incubated for three additional hours. Finally, 150 mL of DMSO was added to dissolve the form azan crystals. The absorbance at 570 nm was measured by scanning with a microplate reader (Power Wave XS2, Bio-tek Instruments Inc., USA). 2.4. Cell cycle assay The MCF-7 cells were plated in 6-well plates and treated with various concentrations of Rh4 (0, 75 and 150 mg/mL) for 24 h. The cells were collected, washed with PBS, and fixed with 75% ethanol at 4 C overnight. Then, the cells were stained with 50 mg/mL of PI solution containing 1 mg/mL of RNase A for 15 min in the dark at 37 C. The cell cycle was detected using flow cytometry (Becton Dickson, CA). 2.5. Annexin V-FITC/PI apoptosis assay The MCF-7 cells were plated in the 6-well plates, and treated with the various concentration of Rh4 (0, 75 and 150 mg/mL) for 24 h. The cells were harvested, washed with PBS and centrifuged
(1000 rpm, 5 min). The supernatant was discarded, and the cells were re-suspended in 1 binding buffer. Then, the cells were stained with a combination of Annexin V-FITC/PI (AV/PI) solution and incubated for 15 min in the dark at 37 C. Finally, the cells were analyzed using flow cytometry (Becton Dickson, CA).
2.6. Hoechst 33342 fluorescent staining assay The cells were plated in 6-well plates and treated with various concentrations of Rh4 (0, 50, 100, 150, 200 and 250 mg/mL) for 24 h. The supernatant was discarded, and the cells were washed twice with PBS twice. Then, 100 mL of staining solution was added to each well. After incubation at 37 C for 20 min, the stained cells were observed using a fluorescence microscope (Nikon, Japan).
2.7. Western blotting The MCF-7 cells were grown in 6-well plates at 3e4 105 cells per well and treated with Rh4 (150 mg/mL). After a 24-h incubation, the whole-cell extracts were prepared using RIPA buffer containing 1 mM PMSF. Proteins in the whole-cell extracts were separated by 10% SDS-PAGE and then transferred to PVDF membranes. The membrane was blocked with 5% skim milk dissolved in TBST buffer and incubated with primary and secondary antibodies in turn. Bound antibodies were observed using ECL Western Blotting Detection Reagent.
2.8. Xenograft models and treatment All experiments were performed in compliance with the relevant laws and institutional guidelines and were conducted with the approval of the Animal Committee of Northwest University. This study was supported by the Shaanxi Key Laboratory of Degradable Biomedical Materials (C02170021) and Northwest University (170105034), and the research staff received training about humane endpoints by the Animal Committee. Thirty female BALB/c athymic nude mice, 4 weeks of age, were from SJA Lab Animal Co., Ltd (Hunan, China). They were fed in standard laboratory conditions (room temperature, 50%e60% humidity, 12 h light/dark cycle) and allowed free access to normal food and water for 7 days before the experiment, which lasted 35 days. The health and behavior of the mice were monitored every day. All the mice were eventually euthanized by asphyxia with inhalation of carbon dioxide in a closed environment and approximately 5 min elapsed before euthanasia. No mouse died before meeting the criteria for euthanasia. The MCF-7 cell xenograft model was established as previously reported [17]. Briefly, cells were suspended in serum-free culture medium at a density of 4 106 cells in 0.2 mL per mouse and injected subcutaneously at the left forelimb pit. After 10 days of injection, the tumor-bearing mice were randomly divided into 3 groups (the control group, 10 mg/kg/d Rh4, and 20 mg/kg/d Rh4, n ¼ 10 per group) according to the size of the tumor and body weight and were i.p. with vehicle (PEG400: water ¼ 50%:50%, v/v) and Rh4 (10 and 20 mg/kg/d) for 25 days, respectively. Tumor diameter and body weight were measured once 5 days. Tumor volume was estimated using the formula [0.5 a b2], where “a” is the long diameter and “b” is the short diameter. After 25 days of Rh4 treatment, all nude mice were killed and the growth of the tumors was measured. The tumors were removed from nude mice, immobilized with 4% paraformaldehyde and embedded in paraffin. Sections were then cut, and H&E staining was performed as previously described [18].
Please cite this article in press as: Z. Duan, et al., The anti-tumor effect of ginsenoside Rh4 in MCF-7 breast cancer cells in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.174
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2.9. Statistical analysis The results were expressed as means and standard deviations, and statistical significance was performed using Student's t-test with SPSS software. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Effect of Rh4 on proliferation of and cell cycle MCF-7 cells in vitro As shown in Fig. 1A, after 48 h of Rh4 treatment, the proliferation of MCF-7 cells was obviously inhibited and the inhibition rate was dose-dependent within the concentration ranges tested. Among these, when the concentration of Rh4 reached 150 mg/mL, it led to a significant increase in cell growth inhibition, which was 62.96 ± 2.31%. The proportion of cells in each cycle was calculated and plotted as a bar graph (Fig. 1B). The graph shows that the Rh4 treatment significantly increased the percentage of cells in S phase, and decreased the percentage of cells in G1 phase in a dose-dependent manner. Specifically, the percentage of cells in S phase increased from 25.15 ± 0.79% of the control to 40.54 ± 0.84% of the cells treated with Rh4 (150 mg/mL).
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As shown in Fig. 2B, MCF-7 cells were subjected to morphological changes in apoptosis after treatment with Rh4. From the unstained cells, with an increase in the concentration of Rh4 in the culture medium, the number of cells observed in the field of view decreased significantly. Moreover, some cells appeared shrunken, rounded, and removed from the bottom of petri dishes, especially when the concentration reached 250 mg/mL, whose almost all of the cells had undergone morphological changes. From the cells stained by Hoechst 33342 staining solution, the addition of Rh4 allowed some cells to manifest brighter blue fluorescence than that of the control. The parts of the yellow arrows show apoptotic bodies. When the concentration of Rh4 reached 150 mg/mL, apoptotic bodies appeared. These apoptotic bodies differed in shape and the cytoplasm began to condense, which indicates that most cells have been disrupted. 3.3. Expression of apoptosis-related proteins in MCF-7 cells treated with Rh4 After Rh4 treatment, the protein expression levels of procaspase-3, -8, Bcl-2 and PARP were markedly down-regulated, and the protein expression levels of cleaved caspase-3, Bax and cleaved PARP were significantly up-regulated. The changes in the expression of the above proteins were dose-dependent. However, the protein expression level of procaspase-9 was maintained (Fig. 3AeC).
3.2. Rh4 induces cell apoptosis of MCF-7 cells 3.4. Effect of Rh4 on the growth of MCF-7 cells in nude mice The Rh4-treated cells were stained with AV/PI and detected by flow cytometry. After treatment with Rh4 for 24 h, the distribution of cells in the early and late apoptosis areas increased. Among these, the proportion of early apoptotic cells was significantly dosedependent (Fig. 2A). Rh4 at 150 mg/mL efficiently induced MCF7 cell apoptosis after treatment, with an early apoptosis rate of 17.10 ± 0.47%.
As shown in Fig. 4A, compared with the control group, mice injected with Rh4 gained weight quickly, especially in the high dose group. Furthermore, the tumor growth rate of the Rh4-treated group was far slower than that of the control group, which can be observed from the size of the xenograft (Fig. 4B and C). Of these, the average tumor volume of the control increased by 636.77% after
Fig. 1. The cell inhibition rate and periodic distribution of MC.
Please cite this article in press as: Z. Duan, et al., The anti-tumor effect of ginsenoside Rh4 in MCF-7 breast cancer cells in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.174
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Fig. 2. The effect of Rh4 on cell apoptosis in MCF-7 cells. (A).
Fig. 3. The expression of apoptosis-associated proteins in MCF-7.
Please cite this article in press as: Z. Duan, et al., The anti-tumor effect of ginsenoside Rh4 in MCF-7 breast cancer cells in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.174
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Fig. 4. Effect of Rh4 on the growth of MCF-7 cells in mice. (A).
25 days of administration, while the Rh4 low and high dose groups increased by 418.18% and 272.54%, respectively (Fig. 4B). The state of MCF-7 cells can be observed in Fig. 4D, and compared with the control group, there were large areas of necrosis in tumors of the 20 mg/kg Rh4-treated group. 4. Discussion Chemotherapy methods currently used for breast cancer produce many side effects, including pain, and seriously affect the health of patients. The purpose of this article is to explore the feasibility and safety of Rh4, which may be a new anti-breast cancer drug. To test the cytotoxic effects of Rh4 in MCF-7 cells, the study was started with an analysis of the inhibition rate of Rh4 on MCF-7 breast cancer cells, and the viability of Rh4-treated cells was measured using the MTT assay. From the results, Rh4 exerted significant anti-proliferative activity against MCF-7 cells, which indicated that Rh4 inhibited MCF-7 cell growth in vitro, and it is powerful evidence that Rh4 could be a new anti-breast cancer drug. Next, to investigate the effect of Rh4 on the cell cycle, the Rh4treated cells were stained with PI and examined by flow cytometry. From the results, the number of cells in S phase obviously increased in an Rh4 dose-dependent manner, which indicated that Rh4 induced S phase arrest of the cell cycle in MCF-7 cells. S phase is the DNA synthesis phase, and a large number of cells were blocked during this period, showing that Rh4 can produce an effect in the early stage of cell division [19]. There are many reasons for the decrease in the cell proliferation rate, and apoptosis is an essential physiological mechanism of cell death [20]. The effect of Rh4 on MCF-7 cell apoptosis was studied with a flow cytometry assay, which can be used to quantify the ratio of early and late cell apoptosis. From the results, after the treatment
of MCF-7 cells with Rh4, the number of apoptotic cells increased significantly, and the changes in the proportion of early apoptotic cells showed a concentration-dependent behavior. These results showed that the main pathway of Rh4 inhibition may be apoptosis. When cells in the high content group were observed without staining, many of them had been removed from the bottom of the plate, and the morphology of the remaining cells showed apoptotic characteristics. Fluorescent staining was used to further confirm that Rh4 induces MCF-7 cell apoptosis. When cells were observed with Hoechst 33342, many apoptotic bodies were apparent, also showing that Rh4 has an obvious effect on apoptosis of MCF7 cells [21]. To further clarify the apoptotic mechanism induced by Rh4 in MCF-7 cells, the expression levels of apoptosis-related proteins, especially cysteinyl aspartate-specific proteinase, in cells were analyzed using Western blotting [22]. The Bcl-2 gene, a type of oncogene, can inhibit cell apoptosis, and the Bax gene is the major apoptosis gene in the human body [23,24]. Changes in the levels of these two proteins are consistent with the state of apoptosis in Rh4-treated cells. As a central link and executor in the process of apoptosis, the caspase family has received extensive attention in recent years [25]. Among all of the caspase members, caspase-8 and -9 are the executors of the external and internal apoptotic pathways, respectively [26]. Based on the results, Rh4-induced MCF-7 cell death occurred via the external apoptotic pathway activation for caspase cleavage, as caspase-8 induction but not caspase-9 was observed. Caspase-3, an essential apoptotic effector, can cause some cellular changes associated with apoptosis, such as cytoskeletal breakdown and nuclear death. Caspase-3 can also lead to the cleavage of PARP during apoptosis [27,28]. In addition, an increase in the activity of Ca2þ/Mg2þ-dependent endonucleases negatively regulated by PARP results in cleavage of the DNA between
Please cite this article in press as: Z. Duan, et al., The anti-tumor effect of ginsenoside Rh4 in MCF-7 breast cancer cells in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.174
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nucleosomes [29]. The changes in caspase-3 and PARP expression indicated that Rh4 may improve the activity of Ca2þ/Mg2þdependent endonucleases to promote apoptosis in MCF-7 cells. Rh4 has exerted potent anti-tumor activity in vitro, but whether it can exert effect in vivo was not known. The MCF-7 cell xenograft model was established and administered vehicle or Rh4. Rh4 administration can helped the mice gain body weight quickly and made them more active, which shows that Rh4 is helpful for cancer treatment first. The size of the tumor is indispensable for reflecting the efficacy of Rh4, and Rh4 significantly reduced tumor size. Meanwhile, the subcutaneous tumors were examined by H&E staining, and the results indicated that Rh4 suppressed tumor growth by altering the proliferation and morphology of MCF-7 tumor cells in vivo. In conclusion, Rh4 showed a high inhibitory effect on MCF7 cells in vitro and in vivo. Rh4 inhibited proliferation, arrested the cell cycle in S phase and induced apoptosis in MCF-7 cells in vitro, and the possible mechanisms of the effect could be attributed to the external pathway activation. In addition, Rh4 significantly suppressed the growth of MCF-7 cells in vivo. Therefore, Rh4 could be a potentially effective anti-tumor drug for breast cancer.
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
Conflicts of interest No potential conflicts of interest were reported by the authors. Acknowledgements This study was financially supported by the National Natural Science Foundation of China (21476182, 21776227, 21776228); Shaanxi Key Laboratory of Degradable Biomedical Materials Programe (2014SZS07-K04, 2014SZS07-P05, 14JS104); Shaanxi R&D Center of Biomaterials and Fermentation Engineering Programe (2015HBGC-04).
[17]
[18]
[19]
[20]
[21]
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The anti-tumor effect of ginsenoside Rh4 in MCF-7 breast cancer cells in vitro and in vivo Zhiguang Duan a, b, 1, Bo Wei a, b, 1, Jianjun Deng a, b, Yu Mi a, b, Yangfang Dong a, b, Chenhui Zhu a, b, Rongzhan Fu a, b, Linlin Qu a, b, Daidi Fan a, b, * a
Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, 229 North Taibai Road, Xi'an, Shaanxi 710069, China Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, 229 North Taibai Road, Xi'an, Shaanxi 710069, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 March 2018 Accepted 22 March 2018 Available online xxx
Breast cancer is a tremendous threat to humans in many countries, and thus we need to find safe and effective drugs for treatment. Ginsenoside Rh4 has been reported to be present in processed ginseng. However, few studies have focused on its anti-tumor activity. In this study, we investigated the inhibitory effects of ginsenoside Rh4 on MCF-7 breast cancer cells and the pathways that promote apoptosis in vitro. To study the effect of ginsenoside Rh4 in vivo, xenograft models were randomly divided into 3 groups (the control group, 10 mg/kg/d Rh4, 20 mg/kg/d Rh4, n ¼ 10 per group), the ginsenoside Rh4 injection method was i.p. The results showed that ginsenoside Rh4 effectively inhibited proliferation, arrested the cell cycle in S phase and induced apoptosis in MCF-7 cells by flow cytometry. Morphological changes caused by ginsenoside Rh4-induced apoptosis were also observed by Hoechst 33342 staining. Westernblot analyses indicated that the apoptosis-inducing effects of ginsenoside Rh4 were associated with the external pathway by decreasing Bcl-2, increasing Bax, and activating caspase-8, -3 and PARP. Moreover, ginsenoside Rh4 significantly inhibited the growth of MCF-7 tumor cells in vivo. These results suggested that ginsenoside Rh4 could be a potentially effective anti-tumor drug for breast cancer. © 2018 Elsevier Inc. All rights reserved.
Keywords: Apoptosis Breast cancer Ginsenoside MCF-7 cells Xenograft model
1. Introduction The morbidity and mortality rates of breast cancer have remained at a high level, which poses a tremendous threat to women in many countries [1]. Chemotherapy has become the main treatment method. However, it has serious side effects, such as diarrhea and emesis, increasing the suffering of patients [2,3]. Therefore, it is imperative to develop a new method to treat or prevent breast cancer. Ginseng, a type of precious herbal medicine, has attracted increasing attention from Western countries due to its extensive pharmacological activity and relative safety [4e6]. It has been reported to possess anti-oxidant functions, improving
Abbreviations: Rh4, ginsenoside Rh4; PEG, >polyethylene glycol. * Corresponding author. Shaanxi Key Laboratory of Degradable Biomedical Materials, School of chemical engineering, Northwest University, 229 North Taibai Road, Xi'an, Shaanxi 710069, China. E-mail addresses: [email protected] (Z. Duan), 201520752@stumail. nwu.edu.cn (B. Wei), [email protected] (D. Fan). 1 These authors contributed equally to this work.
immunity, and anti-tumor effects [7e9]. The multiple pharmacological activities of ginseng are considered to be due to the presence of various ginsenosides [10]. Ginsenosides, which are the major component of ginseng, have been widely reported to have anti-tumor activity, especially for ginsenoside Rg3 and Rh2. Ginsenoside Rg3 has potential antiproliferation and apoptosis-inducing effects in human gastric cancer AGS cells and colon cancer HT29 cells [11,12]. Ginsenoside Rh2 can significantly inhibit cell growth and induce apoptosis via activation of caspase-8 and -9 in human Hepatoma SK-HEP-1 cells [13,14]. Ginsenoside Rh4 (Rh4) was isolated in processed ginseng [15]. Rh4 has been reported to inhibit human leukemia K562 cells [16]. However, there are no reports or studies about the anti-tumor effect of Rh4 on human breast cancer MCF-7 cells, and its mechanism remains unknown. In this paper, based on the above considerations, we have studied the anti-tumor effect of Rh4 on MCF-7 cells and its mechanism through cell experiments including MTT assay, cell cycle assay, apoptosis assay and detection of protein content in vitro.
https://doi.org/10.1016/j.bbrc.2018.03.174 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: Z. Duan, et al., The anti-tumor effect of ginsenoside Rh4 in MCF-7 breast cancer cells in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.174
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Then, nude mice with tumors were injected with Rh4 to observe the anti-tumor effect of the drugs in vivo. The results showed that the inhibition and apoptosis rate of MCF-7 cells increased significantly after Rh4 treatment. Rh4 induced cell apoptosis via external apoptotic pathway activation due to changes in the levels of related proteins. In vivo, Rh4 significantly inhibited the growth of MCF-7 tumor cells. All of the studies will provide the basis for developing Rh4 as an urgentlyneeded therapeutic drug for human breast cancer. 2. Materials and methods 2.1. Chemicals and reagents Rh4 (purity99.0%) was purchased from Puruifa Technology Development Co., Ltd (Chengdu, China). MTT was obtained from Solarbio Co., Ltd (Beijing, China). The Hoechst 33342 fluorescent dye kit and RIPA cell lysates were obtained from Beyotime Institute of Biotechnology Co., Ltd (Shanghai, China). Polyethylene glycol (PEG) 400, which was of medical grade, was obtained from Hua Ri Pharmaceutical Co., Ltd (Hunan, China). Primary antibodies to procaspase-8, procaspase-9, procaspase-3, cleaved caspase-3, PARP, cleaved PARP, Bax, Bcl-2 and GAPDH were purchased from Abcam (CB, US). The secondary antibody to IgG was from Gene Tex (Texas, USA). All staining solutions and other chemicals were purchased from standard commercial suppliers. 2.2. Cell culture The human breast cancer MCF-7 cells were purchased from the American Type Culture Collection (VA, USA). All the cells were cultured with RPMI-1640 (HyClone) medium containing 10% FBS (HyClone) and 1% penicillinestreptomycin (HyClone) at 37 C under a humidified 5% CO2 atmosphere. 2.3. MTT assay Cell proliferation was measured using the MTT assay for MCF7 cells. Rh4 was dissolved in RPMI-1640 culture media containing 0.1% DMSO at final concentrations of 0, 50, 100, 150, 200, and 250 mg/mL. The MCF-7 cells were grown in 96-well plates at 9 103 cells per well, incubated at 37 C for 24 h and then treated with various concentrations of Rh4 for 48 h. The control cells were exposed to culture media containing 0.1% DMSO. Then, 50 mL of MTT solution (5 mg/mL) was added to each well. Cells were incubated for three additional hours. Finally, 150 mL of DMSO was added to dissolve the form azan crystals. The absorbance at 570 nm was measured by scanning with a microplate reader (Power Wave XS2, Bio-tek Instruments Inc., USA). 2.4. Cell cycle assay The MCF-7 cells were plated in 6-well plates and treated with various concentrations of Rh4 (0, 75 and 150 mg/mL) for 24 h. The cells were collected, washed with PBS, and fixed with 75% ethanol at 4 C overnight. Then, the cells were stained with 50 mg/mL of PI solution containing 1 mg/mL of RNase A for 15 min in the dark at 37 C. The cell cycle was detected using flow cytometry (Becton Dickson, CA). 2.5. Annexin V-FITC/PI apoptosis assay The MCF-7 cells were plated in the 6-well plates, and treated with the various concentration of Rh4 (0, 75 and 150 mg/mL) for 24 h. The cells were harvested, washed with PBS and centrifuged
(1000 rpm, 5 min). The supernatant was discarded, and the cells were re-suspended in 1 binding buffer. Then, the cells were stained with a combination of Annexin V-FITC/PI (AV/PI) solution and incubated for 15 min in the dark at 37 C. Finally, the cells were analyzed using flow cytometry (Becton Dickson, CA).
2.6. Hoechst 33342 fluorescent staining assay The cells were plated in 6-well plates and treated with various concentrations of Rh4 (0, 50, 100, 150, 200 and 250 mg/mL) for 24 h. The supernatant was discarded, and the cells were washed twice with PBS twice. Then, 100 mL of staining solution was added to each well. After incubation at 37 C for 20 min, the stained cells were observed using a fluorescence microscope (Nikon, Japan).
2.7. Western blotting The MCF-7 cells were grown in 6-well plates at 3e4 105 cells per well and treated with Rh4 (150 mg/mL). After a 24-h incubation, the whole-cell extracts were prepared using RIPA buffer containing 1 mM PMSF. Proteins in the whole-cell extracts were separated by 10% SDS-PAGE and then transferred to PVDF membranes. The membrane was blocked with 5% skim milk dissolved in TBST buffer and incubated with primary and secondary antibodies in turn. Bound antibodies were observed using ECL Western Blotting Detection Reagent.
2.8. Xenograft models and treatment All experiments were performed in compliance with the relevant laws and institutional guidelines and were conducted with the approval of the Animal Committee of Northwest University. This study was supported by the Shaanxi Key Laboratory of Degradable Biomedical Materials (C02170021) and Northwest University (170105034), and the research staff received training about humane endpoints by the Animal Committee. Thirty female BALB/c athymic nude mice, 4 weeks of age, were from SJA Lab Animal Co., Ltd (Hunan, China). They were fed in standard laboratory conditions (room temperature, 50%e60% humidity, 12 h light/dark cycle) and allowed free access to normal food and water for 7 days before the experiment, which lasted 35 days. The health and behavior of the mice were monitored every day. All the mice were eventually euthanized by asphyxia with inhalation of carbon dioxide in a closed environment and approximately 5 min elapsed before euthanasia. No mouse died before meeting the criteria for euthanasia. The MCF-7 cell xenograft model was established as previously reported [17]. Briefly, cells were suspended in serum-free culture medium at a density of 4 106 cells in 0.2 mL per mouse and injected subcutaneously at the left forelimb pit. After 10 days of injection, the tumor-bearing mice were randomly divided into 3 groups (the control group, 10 mg/kg/d Rh4, and 20 mg/kg/d Rh4, n ¼ 10 per group) according to the size of the tumor and body weight and were i.p. with vehicle (PEG400: water ¼ 50%:50%, v/v) and Rh4 (10 and 20 mg/kg/d) for 25 days, respectively. Tumor diameter and body weight were measured once 5 days. Tumor volume was estimated using the formula [0.5 a b2], where “a” is the long diameter and “b” is the short diameter. After 25 days of Rh4 treatment, all nude mice were killed and the growth of the tumors was measured. The tumors were removed from nude mice, immobilized with 4% paraformaldehyde and embedded in paraffin. Sections were then cut, and H&E staining was performed as previously described [18].
Please cite this article in press as: Z. Duan, et al., The anti-tumor effect of ginsenoside Rh4 in MCF-7 breast cancer cells in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.174
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2.9. Statistical analysis The results were expressed as means and standard deviations, and statistical significance was performed using Student's t-test with SPSS software. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Effect of Rh4 on proliferation of and cell cycle MCF-7 cells in vitro As shown in Fig. 1A, after 48 h of Rh4 treatment, the proliferation of MCF-7 cells was obviously inhibited and the inhibition rate was dose-dependent within the concentration ranges tested. Among these, when the concentration of Rh4 reached 150 mg/mL, it led to a significant increase in cell growth inhibition, which was 62.96 ± 2.31%. The proportion of cells in each cycle was calculated and plotted as a bar graph (Fig. 1B). The graph shows that the Rh4 treatment significantly increased the percentage of cells in S phase, and decreased the percentage of cells in G1 phase in a dose-dependent manner. Specifically, the percentage of cells in S phase increased from 25.15 ± 0.79% of the control to 40.54 ± 0.84% of the cells treated with Rh4 (150 mg/mL).
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As shown in Fig. 2B, MCF-7 cells were subjected to morphological changes in apoptosis after treatment with Rh4. From the unstained cells, with an increase in the concentration of Rh4 in the culture medium, the number of cells observed in the field of view decreased significantly. Moreover, some cells appeared shrunken, rounded, and removed from the bottom of petri dishes, especially when the concentration reached 250 mg/mL, whose almost all of the cells had undergone morphological changes. From the cells stained by Hoechst 33342 staining solution, the addition of Rh4 allowed some cells to manifest brighter blue fluorescence than that of the control. The parts of the yellow arrows show apoptotic bodies. When the concentration of Rh4 reached 150 mg/mL, apoptotic bodies appeared. These apoptotic bodies differed in shape and the cytoplasm began to condense, which indicates that most cells have been disrupted. 3.3. Expression of apoptosis-related proteins in MCF-7 cells treated with Rh4 After Rh4 treatment, the protein expression levels of procaspase-3, -8, Bcl-2 and PARP were markedly down-regulated, and the protein expression levels of cleaved caspase-3, Bax and cleaved PARP were significantly up-regulated. The changes in the expression of the above proteins were dose-dependent. However, the protein expression level of procaspase-9 was maintained (Fig. 3AeC).
3.2. Rh4 induces cell apoptosis of MCF-7 cells 3.4. Effect of Rh4 on the growth of MCF-7 cells in nude mice The Rh4-treated cells were stained with AV/PI and detected by flow cytometry. After treatment with Rh4 for 24 h, the distribution of cells in the early and late apoptosis areas increased. Among these, the proportion of early apoptotic cells was significantly dosedependent (Fig. 2A). Rh4 at 150 mg/mL efficiently induced MCF7 cell apoptosis after treatment, with an early apoptosis rate of 17.10 ± 0.47%.
As shown in Fig. 4A, compared with the control group, mice injected with Rh4 gained weight quickly, especially in the high dose group. Furthermore, the tumor growth rate of the Rh4-treated group was far slower than that of the control group, which can be observed from the size of the xenograft (Fig. 4B and C). Of these, the average tumor volume of the control increased by 636.77% after
Fig. 1. The cell inhibition rate and periodic distribution of MC.
Please cite this article in press as: Z. Duan, et al., The anti-tumor effect of ginsenoside Rh4 in MCF-7 breast cancer cells in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.174
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Fig. 2. The effect of Rh4 on cell apoptosis in MCF-7 cells. (A).
Fig. 3. The expression of apoptosis-associated proteins in MCF-7.
Please cite this article in press as: Z. Duan, et al., The anti-tumor effect of ginsenoside Rh4 in MCF-7 breast cancer cells in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.174
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Fig. 4. Effect of Rh4 on the growth of MCF-7 cells in mice. (A).
25 days of administration, while the Rh4 low and high dose groups increased by 418.18% and 272.54%, respectively (Fig. 4B). The state of MCF-7 cells can be observed in Fig. 4D, and compared with the control group, there were large areas of necrosis in tumors of the 20 mg/kg Rh4-treated group. 4. Discussion Chemotherapy methods currently used for breast cancer produce many side effects, including pain, and seriously affect the health of patients. The purpose of this article is to explore the feasibility and safety of Rh4, which may be a new anti-breast cancer drug. To test the cytotoxic effects of Rh4 in MCF-7 cells, the study was started with an analysis of the inhibition rate of Rh4 on MCF-7 breast cancer cells, and the viability of Rh4-treated cells was measured using the MTT assay. From the results, Rh4 exerted significant anti-proliferative activity against MCF-7 cells, which indicated that Rh4 inhibited MCF-7 cell growth in vitro, and it is powerful evidence that Rh4 could be a new anti-breast cancer drug. Next, to investigate the effect of Rh4 on the cell cycle, the Rh4treated cells were stained with PI and examined by flow cytometry. From the results, the number of cells in S phase obviously increased in an Rh4 dose-dependent manner, which indicated that Rh4 induced S phase arrest of the cell cycle in MCF-7 cells. S phase is the DNA synthesis phase, and a large number of cells were blocked during this period, showing that Rh4 can produce an effect in the early stage of cell division [19]. There are many reasons for the decrease in the cell proliferation rate, and apoptosis is an essential physiological mechanism of cell death [20]. The effect of Rh4 on MCF-7 cell apoptosis was studied with a flow cytometry assay, which can be used to quantify the ratio of early and late cell apoptosis. From the results, after the treatment
of MCF-7 cells with Rh4, the number of apoptotic cells increased significantly, and the changes in the proportion of early apoptotic cells showed a concentration-dependent behavior. These results showed that the main pathway of Rh4 inhibition may be apoptosis. When cells in the high content group were observed without staining, many of them had been removed from the bottom of the plate, and the morphology of the remaining cells showed apoptotic characteristics. Fluorescent staining was used to further confirm that Rh4 induces MCF-7 cell apoptosis. When cells were observed with Hoechst 33342, many apoptotic bodies were apparent, also showing that Rh4 has an obvious effect on apoptosis of MCF7 cells [21]. To further clarify the apoptotic mechanism induced by Rh4 in MCF-7 cells, the expression levels of apoptosis-related proteins, especially cysteinyl aspartate-specific proteinase, in cells were analyzed using Western blotting [22]. The Bcl-2 gene, a type of oncogene, can inhibit cell apoptosis, and the Bax gene is the major apoptosis gene in the human body [23,24]. Changes in the levels of these two proteins are consistent with the state of apoptosis in Rh4-treated cells. As a central link and executor in the process of apoptosis, the caspase family has received extensive attention in recent years [25]. Among all of the caspase members, caspase-8 and -9 are the executors of the external and internal apoptotic pathways, respectively [26]. Based on the results, Rh4-induced MCF-7 cell death occurred via the external apoptotic pathway activation for caspase cleavage, as caspase-8 induction but not caspase-9 was observed. Caspase-3, an essential apoptotic effector, can cause some cellular changes associated with apoptosis, such as cytoskeletal breakdown and nuclear death. Caspase-3 can also lead to the cleavage of PARP during apoptosis [27,28]. In addition, an increase in the activity of Ca2þ/Mg2þ-dependent endonucleases negatively regulated by PARP results in cleavage of the DNA between
Please cite this article in press as: Z. Duan, et al., The anti-tumor effect of ginsenoside Rh4 in MCF-7 breast cancer cells in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.174
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nucleosomes [29]. The changes in caspase-3 and PARP expression indicated that Rh4 may improve the activity of Ca2þ/Mg2þdependent endonucleases to promote apoptosis in MCF-7 cells. Rh4 has exerted potent anti-tumor activity in vitro, but whether it can exert effect in vivo was not known. The MCF-7 cell xenograft model was established and administered vehicle or Rh4. Rh4 administration can helped the mice gain body weight quickly and made them more active, which shows that Rh4 is helpful for cancer treatment first. The size of the tumor is indispensable for reflecting the efficacy of Rh4, and Rh4 significantly reduced tumor size. Meanwhile, the subcutaneous tumors were examined by H&E staining, and the results indicated that Rh4 suppressed tumor growth by altering the proliferation and morphology of MCF-7 tumor cells in vivo. In conclusion, Rh4 showed a high inhibitory effect on MCF7 cells in vitro and in vivo. Rh4 inhibited proliferation, arrested the cell cycle in S phase and induced apoptosis in MCF-7 cells in vitro, and the possible mechanisms of the effect could be attributed to the external pathway activation. In addition, Rh4 significantly suppressed the growth of MCF-7 cells in vivo. Therefore, Rh4 could be a potentially effective anti-tumor drug for breast cancer.
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Conflicts of interest No potential conflicts of interest were reported by the authors. Acknowledgements This study was financially supported by the National Natural Science Foundation of China (21476182, 21776227, 21776228); Shaanxi Key Laboratory of Degradable Biomedical Materials Programe (2014SZS07-K04, 2014SZS07-P05, 14JS104); Shaanxi R&D Center of Biomaterials and Fermentation Engineering Programe (2015HBGC-04).
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Please cite this article in press as: Z. Duan, et al., The anti-tumor effect of ginsenoside Rh4 in MCF-7 breast cancer cells in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.174