Endometrial cancer, also known as uterine body cancer, refers to a group of epithelial malignant tumors that originated from the endometrium, most of which originated from the endometrial gland and it is called endometrial adenocarcinoma. Endometrial cancer is one of the three most common malignant tumors in the female reproductive tract, accounting for 8% of the total malignant tumors in the female body and 20% to 30% of the malignant tumors in the female reproductive tract [
Treatment with traditional Chinese medicine (TCM) can improve the effect on radiotherapy and chemotherapy in clinic and also reduce the toxic and side effects of radiotherapy and chemotherapy on digestive tract and hematopoietic system. For patients with advanced endometrial cancer or those who cannot operate, radiotherapy and chemotherapy can also be treated with TCM. TCM holds that the main pathogenic factors of endometrial cancer are related to Yin and Yang deficiency, fatigue, faint praise, dampness, and heat. Bu shen Yang xue (BSYX) prescription was derived from the works of traditional Chinese medicine named Jing yue’s complete work and was made up of many herbs: Herba epimedium (Yin yang huo), dodder (Tu si zi), eucommia ulmoides (Du zhong), dogwood (Shan zhu yu), ripe radix rehmanniae (Shu di huang), salvia miltiorrhiza (Dang shen), wolfberry (Gou qi), angelica sinensis (Dang gui), paeoniae (Shao yao), and Ligusticum wallichii (chuan xiong), mixed with licorice (Gan cao) and herbs. BSYX has the characteristics of nourishing liver and kidney, invigorating qi and activating blood to collect Yin, replenishing without stagnation, nourishing without satiety and satiety, and making kidney gas abundant and decayed to heaven. BSYX has been used in treating gynecologic disease in clinic, including immune premature ovarian failure, infertility, and acted on the development of the endometrium [
Endometrial carcinoma is a hormone-dependent malignancy. When estrogen decreases after menopause, facilitates follicle-stimulating hormone (FSH) will increase. FSH is the core hormone in mammalian reproductive process. High FSH has large impact on women’s reproductive and perimenopausal physiological pathology, and the signaling pathway downstream of FSH receptor (FSHR) has become a new research field. During human proliferation and secretion, FSHR protein is expressed in endometrial epithelial cells and interstitial cells, and the expression of secretory phase is increased. FSH may regulate endometrial function [
There is evidence that the phosphoinositide-3-kinase (PI3K) pathway is the most commonly activated pathway in cancer, such as human cancer cell lines [
BSYX prescription contains eleven medicinal materials that include Herba epimedium, dodder, eucommia ulmoides, dogwood, ripe radix rehmanniae, salvia miltiorrhiza, wolfberry, angelica sinensis, paeoniae, and Ligusticum wallichii, mixed with licorice. These dried slices were acquired from Beijing Tongrentang Drugstore in China. The raw herbs were first boiled by 8-fold of water (v/w) for 2 h at atmosphere pressure. The decoction was then collected; an additional 6-fold of water was further added and boiled for 1.5h. The second decoction was separated and mixed with the first, which was then concentrated by heating at low-atmosphere pressure condition. Finally an extract of 1 mg/mL was prepared in a large scare for detailed experimental investigations.
Human endometrial carcinoma Ishikawa cells, an estrogen-responsive cell line derived from a well-differentiated endometrioid carcinoma, were obtained from the National Infrastructure of Cell Resource, China. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM high glucose) (Gibco BRL. Grand Island, NY, USA), with 10% fetal bovine serum (Gibco BRL. Grand Island, NY, USA), 100 U/ml penicillin, and 100 U/mL of streptomycin in a humidified atmosphere of 5% CO2 at 37°C.
60 SPF four-week-old female nude mice (BALB/C) were provided by zhejiang Chinese Medical University Laboratory Animal Research Center. Mice were randomly divided into control group, model group, BSYX group (2.5, 5 and 10 g/kg), and Cisplatin group, and each group had 10 mice. Besides the control group, the models of subcutaneous tumor xenograft of nude mice with human endometrial carcinoma were established by right scapular subcutaneous injection of 1 × 107 Ishikawa tumor cell suspension. Tumor formation was about three weeks. Then BSYX group was treated with intragastric injection administration of BSYX,and the control group and model group were injected with normal saline for 14 days. Tumor volumes were gauged with vernier calipers regularly and the growth curves for tumors were plotted as mean ± SD of tumors from 8 mice. Tumor volume was evaluated according to the following formula: 1/2×L2×W (L = tumor length and W = tumor width). Antitumor rate = (C-T) /C×100% (C=the average volume of the tumor in the model group, T=the average volume of the transplanted tumor in the treatment group).
The mice were housed in a specific pathogen-free environment suitable for survival and were observed and measured on a regular basis. Later the mice were sacrificed and the tumors excised to compare the size of the tumor. All mouse experiments were approved by the animal ethics committee of Zhejiang University.
The tumor tissues were immersion-fixed in 10% neutral buffered formaldehyde solution and embedded in paraffin for a light microscopic study. The sections of formalin-fixed, paraffin-embedded tissue samples were deparaffinized in xylene and rehydrated in graded ethanols. The specimens were routinely processed and stained with HE for histopathological classification.
The ICR female mice weighing 18–22 g were randomly divided into following groups (n=10): a control group (normal saline); three BSYX groups (2.5g/kg, 5g/kg, and 10 g/kg). The control saline or BSYX groups were administered via intragastric injection every 2 days for five times. At day 14, all the animals were sacrificed and the drug serum and the blank serum were separated from heart blood. The blood was centrifuged at 4000 rpm for 10 min at 4°C to obtain serum. The serum was labeled and stored at −70°C.
Ishikawa cells were cultured and then divided into six groups: normal control group, FSH (50 mIU/ml), LY294002 (15
The CCK8 assay was performed using a CCK8 kit following the manufacturer’s protocol (Dojindo, Kumamoto, Japan). Briefly, cells were plated into 96-well plates (2 × 103 cells per well) in 100
Cell apoptosis/necrosis was evaluated using Annexin V-FITC/ PI staining. Cells were plated into 6-well plates. After treatment for 24 h, cells were collected and washed twice with cold phosphate-buffered saline (PBS). The cells were then resuspended in 500
Cells were seeded in 6-well plates at the density of 5 × 105cells and cultured until confluence. The cell monolayer was scratched with a sterile pipette yellow tip to produce a straight line, and the debris was washed out with culture medium. Medium and substances for treatment were renovated at 24 h or 48 h. The open gap was photographed with a camera at phase contrast microscope at the indicated times. The rate of healing was estimated by ImageJ software based on the area (in which the length is predetermined and the wideness varies) free of cells. Each determination represents the average of 3 individual experiments.
The total proteins from cell or tumor issue were harvested and lysed in RIPA buffer containing 1 mM phenyl methane sul-fonyl fluoride (Beyotime Biotechnology, Shanghai, China) and mixed with loading buffer. Protein concentration was determined by BCA kit (Beyotime Biotechnology, Shanghai, China). Proteins (10
The experimental data were analyzed using SPSS 19.0 software. Comparisons among multiple groups were made with one-way analysis of variance followed by Fisher’s least significant difference test, and all data were the mean ± standard deviation of at least three separate experiments;
After treatment for 14 days, we collected the tumor and calculated tumor volumes and the anti-tumor rates. As shown in Figures
Effect of BSYX on Tumor volume and histomorphology in mice tumor issue. After treating for 14 days, tumors from mice were collected (a), growth curve of tumors was obtained from tumor measure every four days (b), and the tumor volume and antitumor rate (c) were calculated. The tumor issue was observed with stained with H&E (magnification of all figures ×200) (d).
The Akt pathway has been frequently reported in cancer, so we examined Akt phosphorylation status. The results showed an evident increase of p-Akt/Akt in model group (
Effect of BSYX on levels of AKT, p-Akt, FSHR, Gankyrin, HIF-a, and cyclin D1 proteins in mice tumor. AKT, p-AKT, FSHR, Gankyrin, HIF-a, and cyclin D1 proteins levels were measured with Western blotting and normalized to GAPDH (a). Relative band intensities were used in order to quantify AKT, p-Akt, FSHR, Gankyrin, HIF-
Human endometrial carcinoma Ishikawa cells were stimulated for 24 h with serum of BSYX in a gradient concentration, FSH and LY294002, respectively. Cell ability was detected by CCK8 assay. As shown in Figure
Effect of BSYX on cell ability and apoptosis rate in human endometrial carcinoma Ishikawa cells. Cells were treated with FSH, LY284002, blank serum and drug serum containing BSYX for 24 h, then cell ability was detected by CCK8 assay and was calculated (a). Cells were double stained with annexin V-FITC and PI and analyzed by flow cytometry. And percentage of apoptotic cells was shown by histogram ((b) and (c)). Data are expressed as the mean ±SEM n = 3 in each group.
Effect of BSYX on levels of Bax and Bcl-2 proteins in human endometrial carcinoma Ishikawa cells. Bax and Bcl-2 proteins levels were measured with Western blotting and normalized to GAPDH (a). Relative band intensities were used in order to quantify Bax and Bcl-2 proteins (b) expression levels. Data are expressed as the mean ± SEM n = 3 in each group.
Ishikawa cells were stimulated for 24 h or 48 h with serum of BSYX in a gradient concentration. As shown in Figure
Effect of BSYX on migration ability in human endometrial carcinoma Ishikawa cells After BSYX treatment, the cell migration ability was analyzed by the wound healing assays (a) and the migration rate was calculated (b). Data are expressed as the mean ± SEM n = 3 in each group.
After Ishikawa cells were stimulated for 24 h with serum of BSYX in a gradient concentration, AKT, p-AKT, FSHR, Gankyrin, HIF-a, and cyclin D1 proteins were detected by western blot analysis. As shown in Figure
Effect of BSYX on levels of AKT, p-Akt, FSHR, Gankyrin, HIF-a, and cyclin D1 proteins in human endometrial carcinoma Ishikawa cells. AKT, p-Akt, FSHR, Gankyrin, HIF-
The incidence of endometrial cancer is increasing year by year and it is the second leading cause of deaths due to gynaecological cancer worldwide. Recent evidence showed that TCM has great potential in treating endometrial cancer including Qingrejiedu Lianhua soup [
The endometrium is now considered to be two kinds of pathogenesis: one is a kind of estrogen dependent; namely, endometrial hyperplasia or even cancer may occur under the long-term action of estrogen without progesterone antagonism. This type accounts for the majority of endometrial cancer. The other is nonestrogen-dependent and has no definite relationship with estrogen [
LY294002, a highly selective PI3K inhibitor, was used to block PI3K-dependent Akt phosphorylation. When exposed Ishikawa cells to FSH and LY294002, LY294002 reduced the tumor cell viability, increased cell apoptosis rate and proapoptosis Bax protein expression, decreased the anti-apoptosis Bcl-2 protein expression and the tumor cell migration ability, downregulated the levels of p-AKT/Akt, FSHR, Gankyrin, and cyclinD1 proteins expression, and upregulated the levels of HIF-
To sum up, 5 and 10 g/kg BSYX reduced tumor volume, changed pathological feature in mice tumor issue, also markedly reduced the human endometrial carcinoma Ishikawa cell growth ability and migration ability, and accelerated the apoptosis. The formulation of BSYX had treating effect on endometrial cancer and was related with FSH/PI3K/AKT/Gankyrin/HIF-
The data used to support the findings of this study are included within the article.
The authors declare that they have no conflicts of interest regarding the publication of this article.
Yue-qun Chen carried out the conception and design of the research, participated in the acquisition of data, and drafted the manuscript. Hua-li Fei carried out the analysis and interpretation of data. Hong-li Zhu conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.