Tumor Biol. DOI 10.1007/s13277-014-2390-2
RESEARCH ARTICLE
Huaier aqueous extract inhibits stem-like characteristics of MCF7 breast cancer cells via inactivation of hedgehog pathway Xiaolong Wang & Ning Zhang & Qiang Huo & Mingjuan Sun & Lun Dong & Yan Zhang & Guangwei Xu & Qifeng Yang
Received: 25 May 2014 / Accepted: 23 July 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract The theory of targeting cancer stem-like cells (CSCs) provides novel strategy for cancer treatment. In the present study, we examined the inhibitory effect of Huaier aqueous extract on eradicating breast cancer stem cells and explored the underlying mechanisms. Our data demonstrated that various concentrations of Huaier extract significantly decreased the viabilities, numbers, and sizes of mammospheres. After incubation with Huaier extract for 24 h, the clonogenicity of MCF7 cell line was obviously impaired, along with less holoclones. In addition, Huaier extract reduced the number of cells expressing CD44+/CD24− and decreased the level of stem cell markers (OCT-4, NESTIN, and NANOG). The hedgehog (Hh), notch, and Wnt/βcatenin pathways were essential stem cell signaling pathways involved in regulating CSC renewal and maintenance. We reported that the inhibitory effect of Huaier extract was partly depended on the inactivation of Hh pathway. These findings provided experimental evidence that Huaier extract was a promising therapeutic drug for eliminating the breast cancer stem cells.
X. Wang : N. Zhang : Q. Huo : M. Sun : L. Dong : Q. Yang Department of Breast Surgery, Qilu Hospital, Shandong University, Wenhua Xi Road No. 107, Jinan 250012, Shandong, People’s Republic of China Y. Zhang Qilu Hospital, Shandong University, Wenhua Xi Road No. 107, Jinan 250012, Shandong, People’s Republic of China G. Xu : Q. Yang (*) Key Laboratory of Experimental Teratology, Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Wenhua Xi Road No. 44, Jinan 250012, Shandong, People’s Republic of China e-mail: [email protected]
Keywords Cancer stem cells . Huaier extract . Hedgehog . Breast cancer . MCF7
Introduction Globally, breast cancer has already become the most frequently diagnosed cancer among females, with an incidence of almost 1.4 million in 2008 [1]. Although there has been a small decrease in breast cancer-related deaths (from 14.4/ 100,000 in 2002 to 13.9/100,000 in 2008), breast cancer remains the leading cause of cancer-related mortality among women worldwide, which is responsible for almost 14 % of all cancer deaths [2, 3]. Standard therapies for cancer patients include surgery to resect primary localized tumor and radiochemotherapy to eradicate systemic spreading. Although great progresses have been made in the treatment of patients, metastasis and chemoresistance remain the two major causes of death in breast cancer patients [4]. It has been reported that the majority of deaths caused by cancer were related with the metastasis of chemoresistant cancer cells to vital organs, including the lung, bone, liver, and brain [5, 6]. Indeed, Siegel and colleagues reported that 5-year relative survival rate of breast cancer varied from 99 % for localized tumors to 84 % for regional diseases and 23 % for metastasis diseases [7]. Presently, increasing data have demonstrated that tumor metastasis, relapse, and therapeutic resistance of cancer patients may be due to the fact that tumor growth is supported by a small subpopulation of cancer stem-like cells (CSC) or tumor-initiating cells (TICs) [8–10]. CSCs are tumor cells with enhanced capacity for tumor generation. They are capable of dividing asymmetrically to produce one stem cell, which enables the capacity for self-renewal, and one progenitor cell, which allows them to produce phenotypically diverse cancer cells that constitute tumors. In addition to their serious
Tumor Biol.
suspension single cells were plated at a density of 10,000 cells/ml and grown in a serum-free DMEM/F12 medium (Gibco-BRL, Rockville, IN, USA), supplemented with 5 μg/ml bovine insulin, 20 ng/ml EGF, 20 ng/ml bFGF, and 1× B27 supplement.
side effects, most current available therapeutic agents can only kill the bulk of cancer cells within a tumor, while CSCenriched cells may be left to regenerate more malignant tumors [11, 12]. Therefore, it is highly desirable to develop a non-toxic, natural treatment that can target CSCs. Traditional Chinese medicine (TCM) has been used in China for thousands of years and still holds an important position in primary health care in most parts of China. Over the past several years, as a rich source for identification of novel drugs for cancer therapy, natural products from TCM have attracted great interests around the world. Recent studies have demonstrated that several dietary compounds, such as sulforaphane [13], mulberry leaf extract [14], and epigallocathechin gallate [15] are potent to eliminate CSCs through several regulatory mechanisms. These findings led us to investigate the anti-CSC activity of Trametes robiniophila Murr. (Huaier extract). Huaier extract is a TCM. The cancer preventive effects of Huaier extract have been widely supported by results from cell and animal models. We have demonstrated that Huaier extract inhibited breast cancer growth via inducing apoptosis [16], anti-angiogenic activities [17], and suppressing ER signaling pathway [18] by using a series of breast cancer cell lines. However, the effects of Huaier extract on cancer stem cells have been largely unknown. In this study, we explored if Huaier extract could inhibit the stemness of MCF7 breast cancer cell line, which is the most widely used cell model for breast CSC study [19–22].
The effect of Huaier extract on the viabilities of MCF7 spherederived cells was examined by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, single-cell suspensions from mammospheres were maintained in the sphere cell culture and seeded in 96-well plates at a density of 3,000 cells per well in the presence or absence of Huaier extract. After indicated time of treatment, 20 μl of MTT solution (5 mg/ml) was added to each well and incubated for 4–6 h at 37 °C. The MTT formazan crystal was then centrifuged at 1,000×g for 10 min followed by dissolving in DMSO, and the absorbance was measured by the Microplate Reader (Bio-Rad, Hercules, CA, USA).
Materials and methods
Clonogenic assay
Cell cultures and reagents Human breast cancer cell line MCF7 cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were routinely grown in Dulbecco’s modified of Eagle’s medium (DMEM) (Gibco-BRL, Rockville, IN, USA) supplemented with 10 % fetal bovine serum (Haoyang Biological Manufacturer CO., Ltd., Tianjin, China), 100 U/ml penicillin and 100 μg/ml streptomycin in 5 % CO2 at 37 °C. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and bovine insulin were obtained from SigmaAldrich (St. Louis, MO, USA). B27 supplement (50×) was purchased from Gibco. Electuary ointment of Huaier extract was kindly provided by Gaitianli Medicine Co., Ltd. (Jiangsu, China). And, the stock solutions were generated as previously described [16, 17].
To determine the inhibitory effect of Huaier extract on the clonogenic characteristic of single cancer cell, clonogenic assay was performed as previously described with some modifications [24]. Briefly, 106 cells were first plated in 25-cm2 cell culture flask. After incubation at 37 °C overnight, the cells were treated with indicated concentrations of Huaier extract or vehicle as negative control. Twenty-four hours later, the cells were replated at a density of 1,000 cells/60 mm plate in triplicate and incubated for 18 days. The media were refreshed every 5 days. Colonies were classified as holoclone, meroclone, and paraclone according to their morphologies. Their represent pictures were taken by an Olympus digital camera (Olympus, Tokyo, Japan). Then, colonies that formed were stained with 0.01 % crystal violet and counted. The stained colonies were photographed using an Olympus Live View Digital SLR camera.
Sphere cell culture
Flow cytometry analysis
Sphere cell culture was manufactured according to the published protocol with some modifications [23]. Briefly,
Cell surface markers were detected by flow cytometry analysis following previous protocols [25]. Briefly, cells were
MTT assay
Sphere formation assay Adherent MCF7 cells were gently trypsinized, washed, and then diluted in the sphere cell culture. Media were replenished every 3–4 days. The numbers and sizes of mammospheres were determined using Olympus digital camera (Olympus, Tokyo, Japan). Mammosphere volume was calculated using the following formula: (length×width2)/2.
Tumor Biol.
harvested and resuspended with Pharmingen Stain Buffer to a final concentration of 2×107 cells/ml. Then, fluorochromeconjugated mouse anti-human CD44 or CD24 in combination with their respective isotype controls was added and incubated for 20 min on ice, protected from light. After being washed twice, the cells were resuspended in 0.5 ml Stain Buffer and analyzed by a FACScan flow cytometer (Becton Dickinson, Franklin lakes, NJ, USA).
differences among multiple groups, followed by Fisher’s post hoc test for significance. P
RESEARCH ARTICLE
Huaier aqueous extract inhibits stem-like characteristics of MCF7 breast cancer cells via inactivation of hedgehog pathway Xiaolong Wang & Ning Zhang & Qiang Huo & Mingjuan Sun & Lun Dong & Yan Zhang & Guangwei Xu & Qifeng Yang
Received: 25 May 2014 / Accepted: 23 July 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract The theory of targeting cancer stem-like cells (CSCs) provides novel strategy for cancer treatment. In the present study, we examined the inhibitory effect of Huaier aqueous extract on eradicating breast cancer stem cells and explored the underlying mechanisms. Our data demonstrated that various concentrations of Huaier extract significantly decreased the viabilities, numbers, and sizes of mammospheres. After incubation with Huaier extract for 24 h, the clonogenicity of MCF7 cell line was obviously impaired, along with less holoclones. In addition, Huaier extract reduced the number of cells expressing CD44+/CD24− and decreased the level of stem cell markers (OCT-4, NESTIN, and NANOG). The hedgehog (Hh), notch, and Wnt/βcatenin pathways were essential stem cell signaling pathways involved in regulating CSC renewal and maintenance. We reported that the inhibitory effect of Huaier extract was partly depended on the inactivation of Hh pathway. These findings provided experimental evidence that Huaier extract was a promising therapeutic drug for eliminating the breast cancer stem cells.
X. Wang : N. Zhang : Q. Huo : M. Sun : L. Dong : Q. Yang Department of Breast Surgery, Qilu Hospital, Shandong University, Wenhua Xi Road No. 107, Jinan 250012, Shandong, People’s Republic of China Y. Zhang Qilu Hospital, Shandong University, Wenhua Xi Road No. 107, Jinan 250012, Shandong, People’s Republic of China G. Xu : Q. Yang (*) Key Laboratory of Experimental Teratology, Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Wenhua Xi Road No. 44, Jinan 250012, Shandong, People’s Republic of China e-mail: [email protected]
Keywords Cancer stem cells . Huaier extract . Hedgehog . Breast cancer . MCF7
Introduction Globally, breast cancer has already become the most frequently diagnosed cancer among females, with an incidence of almost 1.4 million in 2008 [1]. Although there has been a small decrease in breast cancer-related deaths (from 14.4/ 100,000 in 2002 to 13.9/100,000 in 2008), breast cancer remains the leading cause of cancer-related mortality among women worldwide, which is responsible for almost 14 % of all cancer deaths [2, 3]. Standard therapies for cancer patients include surgery to resect primary localized tumor and radiochemotherapy to eradicate systemic spreading. Although great progresses have been made in the treatment of patients, metastasis and chemoresistance remain the two major causes of death in breast cancer patients [4]. It has been reported that the majority of deaths caused by cancer were related with the metastasis of chemoresistant cancer cells to vital organs, including the lung, bone, liver, and brain [5, 6]. Indeed, Siegel and colleagues reported that 5-year relative survival rate of breast cancer varied from 99 % for localized tumors to 84 % for regional diseases and 23 % for metastasis diseases [7]. Presently, increasing data have demonstrated that tumor metastasis, relapse, and therapeutic resistance of cancer patients may be due to the fact that tumor growth is supported by a small subpopulation of cancer stem-like cells (CSC) or tumor-initiating cells (TICs) [8–10]. CSCs are tumor cells with enhanced capacity for tumor generation. They are capable of dividing asymmetrically to produce one stem cell, which enables the capacity for self-renewal, and one progenitor cell, which allows them to produce phenotypically diverse cancer cells that constitute tumors. In addition to their serious
Tumor Biol.
suspension single cells were plated at a density of 10,000 cells/ml and grown in a serum-free DMEM/F12 medium (Gibco-BRL, Rockville, IN, USA), supplemented with 5 μg/ml bovine insulin, 20 ng/ml EGF, 20 ng/ml bFGF, and 1× B27 supplement.
side effects, most current available therapeutic agents can only kill the bulk of cancer cells within a tumor, while CSCenriched cells may be left to regenerate more malignant tumors [11, 12]. Therefore, it is highly desirable to develop a non-toxic, natural treatment that can target CSCs. Traditional Chinese medicine (TCM) has been used in China for thousands of years and still holds an important position in primary health care in most parts of China. Over the past several years, as a rich source for identification of novel drugs for cancer therapy, natural products from TCM have attracted great interests around the world. Recent studies have demonstrated that several dietary compounds, such as sulforaphane [13], mulberry leaf extract [14], and epigallocathechin gallate [15] are potent to eliminate CSCs through several regulatory mechanisms. These findings led us to investigate the anti-CSC activity of Trametes robiniophila Murr. (Huaier extract). Huaier extract is a TCM. The cancer preventive effects of Huaier extract have been widely supported by results from cell and animal models. We have demonstrated that Huaier extract inhibited breast cancer growth via inducing apoptosis [16], anti-angiogenic activities [17], and suppressing ER signaling pathway [18] by using a series of breast cancer cell lines. However, the effects of Huaier extract on cancer stem cells have been largely unknown. In this study, we explored if Huaier extract could inhibit the stemness of MCF7 breast cancer cell line, which is the most widely used cell model for breast CSC study [19–22].
The effect of Huaier extract on the viabilities of MCF7 spherederived cells was examined by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, single-cell suspensions from mammospheres were maintained in the sphere cell culture and seeded in 96-well plates at a density of 3,000 cells per well in the presence or absence of Huaier extract. After indicated time of treatment, 20 μl of MTT solution (5 mg/ml) was added to each well and incubated for 4–6 h at 37 °C. The MTT formazan crystal was then centrifuged at 1,000×g for 10 min followed by dissolving in DMSO, and the absorbance was measured by the Microplate Reader (Bio-Rad, Hercules, CA, USA).
Materials and methods
Clonogenic assay
Cell cultures and reagents Human breast cancer cell line MCF7 cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were routinely grown in Dulbecco’s modified of Eagle’s medium (DMEM) (Gibco-BRL, Rockville, IN, USA) supplemented with 10 % fetal bovine serum (Haoyang Biological Manufacturer CO., Ltd., Tianjin, China), 100 U/ml penicillin and 100 μg/ml streptomycin in 5 % CO2 at 37 °C. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and bovine insulin were obtained from SigmaAldrich (St. Louis, MO, USA). B27 supplement (50×) was purchased from Gibco. Electuary ointment of Huaier extract was kindly provided by Gaitianli Medicine Co., Ltd. (Jiangsu, China). And, the stock solutions were generated as previously described [16, 17].
To determine the inhibitory effect of Huaier extract on the clonogenic characteristic of single cancer cell, clonogenic assay was performed as previously described with some modifications [24]. Briefly, 106 cells were first plated in 25-cm2 cell culture flask. After incubation at 37 °C overnight, the cells were treated with indicated concentrations of Huaier extract or vehicle as negative control. Twenty-four hours later, the cells were replated at a density of 1,000 cells/60 mm plate in triplicate and incubated for 18 days. The media were refreshed every 5 days. Colonies were classified as holoclone, meroclone, and paraclone according to their morphologies. Their represent pictures were taken by an Olympus digital camera (Olympus, Tokyo, Japan). Then, colonies that formed were stained with 0.01 % crystal violet and counted. The stained colonies were photographed using an Olympus Live View Digital SLR camera.
Sphere cell culture
Flow cytometry analysis
Sphere cell culture was manufactured according to the published protocol with some modifications [23]. Briefly,
Cell surface markers were detected by flow cytometry analysis following previous protocols [25]. Briefly, cells were
MTT assay
Sphere formation assay Adherent MCF7 cells were gently trypsinized, washed, and then diluted in the sphere cell culture. Media were replenished every 3–4 days. The numbers and sizes of mammospheres were determined using Olympus digital camera (Olympus, Tokyo, Japan). Mammosphere volume was calculated using the following formula: (length×width2)/2.
Tumor Biol.
harvested and resuspended with Pharmingen Stain Buffer to a final concentration of 2×107 cells/ml. Then, fluorochromeconjugated mouse anti-human CD44 or CD24 in combination with their respective isotype controls was added and incubated for 20 min on ice, protected from light. After being washed twice, the cells were resuspended in 0.5 ml Stain Buffer and analyzed by a FACScan flow cytometer (Becton Dickinson, Franklin lakes, NJ, USA).
differences among multiple groups, followed by Fisher’s post hoc test for significance. P
