| Background:RCC is the most common malignancy of the urinary system, in the past decade, its incidence has been increasing. It is estimated that in 2012,64,770 people worldwide were diagnosed with kidney cancer, and there were 13 570 patients died of kidney cancer. The incidence ratio of renal cell carcinoma in male and female patients is 2:1. The incidence of kidney cancer increased year by year, this phenomenon may be considered with relevant and early diagnosis. Of course, this is mainly due to the popularity of imaging examinations for early detection of cancer-related kidney. Since 1992, male patients with kidney cancer each year 2% rate increase; since 1998, female kidney cancer patients increased at 3% rate per year, a large part of these patients had metastasis when diagnosed, including 30% of patients with locally advanced cancer.It is well known that advanced kidney cancer is not sensitive to radiotherapy and chemotherapy, but in order to improve the quality of life of patients and make a treatment to bone metastasis, local recurrence and distant lymph node metastasis, palliative radiotherapy can achieve pain relief effect. In recent years, we carried out a clinical stereotactic radiotherapy (y knife, x-ray knife, three-dimensional conformal radiotherapy) for the treatment of recurrent lesions. Cytokines are thought to have efficacy in patients with advanced renal cell carcinoma, particularly with interferon-a and interleukin-2. The anti-tumor mechanisms of interferon-a:inhibition of tumor cell oncogene expression and DNA synthesis; direct inhibition of tumor cell proliferation; inducing terminal differentiation of tumor cells, promoting differentiation antigen expression; activating lymphokines to enhance NK cell-mediated ADCC activity and T cells, granulocytes and monocytes cytotoxic cells; inhibition of tumor angiogenesis and growth factor receptors to reduce tumor cell proliferation. Development of molecular biology make such renal cancer signaling pathway become a key target for the treatment of advanced kidney cancer, these molecular target plays an important role in the treatment of advanced kidney cancer. Common clinical molecular targeted drugs include anti-angiogenesis drugs, regulation of cell growth and angiogenesis signaling proteins (mTOR) inhibitors. The US Food and Drug Administration (FDA) has approved six kinds of molecular targeted drugs for the treatment of metastatic renal cell carcinoma (mRCC):sunitinib, pazopanib, sorafenib, bevacizumab, sirolimus, everolimus. However, the efficacy of these drugs targeting are very limited and expensive, so an economic and effective anti-kidney cancer drugs are need to be found.Chinese medicine is a traditional medicine of China, playing a very important role in the treatment of multiple diseases. According to historical records, as early as the Song Dynasty, Chinese doctors started to use traditional Chinese medicine to treat cancer, and getting a good effect, but the mechanisms is still unknown. In recent years, with the development of Chinese and Western medicine, many doctors combine Western medicine with Chinese medicine in the treatment of advanced tumors, the treatment mechanisms may include:reducing the pain of cancer patients, improving clinical symptoms of cancer patients, providing immunity of cancer patients, used as alternative way of adjuvant therapy, and many oncologists also combine chemotherapy with traditional Chinese medicine to treat patients with advanced cancer, improvng the quality of life of cancer patients, prolonging median survival of cancer patients.Currently, many Chinese medicine are used in anti-tumor treatment and many scholars began to study the molecular mechanisms of traditional Chinese medicine. Studies have shown that saffron organizations can reduce the expression role of VEGF and fibroblast growth factor in gastric adenocarcinoma BGC-823 cells, through reducing the effect of MMP-9 in degradation of extracellular matrix. Centipede extraction can inhibit tumor angiogenesis in liver cancer, and the mechanism may be that Chinese medicine can reduce the expression of tumor cells angiopoietin-2 and VEGF-related. Orange peel extraction can reduce S180 cell cycle and promote apoptosis of tumor cells, thereby inhibiting the growth of tumor cells. Common turmeric can inhibit the prolifetation of gastric cancer cell SGC-7091 by inhibiting the secretion of IGF-1 growth of gastric cancer cells. Thus, the role of traditional Chinese medicine on tumor treatment is more and more important, and its anti-tumor mechanism will also be studied.Now, with the deepening study of Chinese herbal medicine, many Chinese medicine wased found to has a lot of therapeutic effect in urinary tract tumors. Studies have shown that Taxol can inhibit the growth of human bladder cancer cells, mitomycin C can inhibit the growth of tumor cells in mouse testis. North bean root extract can inhibit bladder cancer T-24 and 786-0 cells growth, and its anti-tumor mechanism may be associated with tumor cell early apoptosis and tumor cell growth-related signaling pathway.Saikosaponin (Saikosaponin, SS) is the main active ingredient of Bupleurum, divided into nine kinds of a, b, c, d, of which Saikosaponin d (SSd) pharmacological has the strongest effection, the earlier study display that SSd may have:(1) a pharmacologically active antipyretic, analgesic and anti-inflammatory effects, and commonly used as non-steroidal anti-inflammatory drugs, and may also inhibit the COX activity, affect the metabolism of arachidonic acid; (2) SSd exhibit various antitumor effects, and can regulate cell growth and function of the immune system. In the late 1980s, the Japanese scholar Takuo’s earliest studies reported the anti-tumor effection of SSd, then, many scholars pay close attention in Chaihu and SSd, especially in its anti-canner effection, including liver cancer, thyroid carcinoma, cervical cancer, lung cancer and other tumor. Currently, few studies have been reported about SSd in urologic oncology. In MIN YAO’s study found, SSd can inhibit DU145 tumor cell growth by inhibiting apoptotic cells, and reduce cell cycle in the G0/G1 phase, but now no research has showed the therapeutic effect of SSd in renal cell. In the present study, we were the first time to investigate the anti-proliferative and apoptotic effect of SSd on renal cell carcinoma and the possible mechanics.Methods:1. Cell viability was assessed using a tetrazolium-based assay (MTT assay). One thousand cells in 50 μl of media per well were plated in 96-well plates. Cells treated with or without different doses of SSd were incubated for various times, and then incubated with 0.5 mg/ml of MTT at 37℃ for 1 h. Subsequently, the supernatant was dropped and dissolved with DMSO. Colorimetric analysis using a 96-well microplate reader was performed at the wavelength of 490 nm. The experiments were performed in triplicate.2. Colony formation assay. RCC cells (769-P and 786-0) were respectively seeded in 24-well plates (100 cells/well) and cultured with different doses of SSd (10, 20 and 30 μM) for 14 days before staining. The colonies were stained with crystal violet and counted.3. Cell cycle detection assay. After cells reached 60-80% confluence, they were treated with different doses of SSd (0 and 10 μM). After 48 h, cells were washed twice with PBS and fixed with 70% ethanol for 1h at 4℃, and then washed with PBS and resuspended with PI solution (0.05 mg/ml) containing RNase, and incubated at room temperature in the dark for 30 min. DNA content was then analyzed using the flow cytometer.4. Quantitative detection of apoptosis. Cells (769-P and 786-0) were exposed to different doses of SSd (10 and 20 μM) for 48 h. The cells were collected and subjected to Annexin V and propidium iodide (PI) staining using an Annexin V-FITC Apoptosis Detection kit, following the protocol provided by the manufacturer. Apoptotic cells were then analyzed by flow cytometry.5. Western blot analysis. Cells were lysed in RIPA buffer (50 mM Tris-HCl/pH 7.4,1% NP-40,150 mM NaCl,1 mM EDTA,1 mM PMSF,1 mM Na3VO4,1 mM NaF,1 mM okadaic acid, and 1 mg/ml aprotinin, leupeptin, and pepstatin) with Protease Inhibitor Cocktail (Roche Inc.). Individual samples (20 μg protein) were prepared for electrophoresis run on 10-15% SDS-PAGE gel and then transferred onto PVDF membranes (Millipore). After blocking the membranes with 5% BSA in PBS for 1h at room temperature, the membranes were incubated with appropriate dilutions of specific primary antibodies overnight at 4℃. After washing, the blots were incubated with anti-rabbit, anti-mouse or anti-goat IgG HRPs for 1 h. The blots were developed in ECL mixture (Thermo Fisher Scientific Inc.).6. Statistical analyses. All statistical analyses were performed using SPSS 22.0. Quantitative data are presented as mean ± SE and the differences among various treatment groups were compared by one-way ANOVA, followed by Dunnett’s t-test for separate comparisons. When the comparison involved only 2 groups, Student’s t-test was used. P<0.05 was considered to indicate statistically significant differences.Result:1. MTT results suggest that SSd treatment can inhibit the proliferation of RCC, which is time-dependent and dose-dependent. Different concentrations of SSd (0,10,15,20μM) treatment of renal cancer cells (769-P and 786-0) 48 hours, cell survival rates were 100%,66.7%,35%,11% and 100%,68%,34%,19%. If a fixed concentration of 15μM after SSd, respectively, after treatment 0,24,48,72 hours at 769-P cells and 786-0 cells, cell survival rates were 100%,52%,30%,10%, and 99%,57%,29%,10%. And compared with control group, each group has significant differences. This means that SSd has effect on RCC, the higher the concentration, the stronger the effect, longer the treatment, the better the effect.2. Cloning experiments suggest that the number of colony cells in 769-P and 786-0 cells with different concentrations SSd (0,10,20,30μM) for 14 days, were 46,50,19,2 1 and 106,42,7,1, and compared with control group, each group has significant differences.3. Cell cycle experiments suggest that without SSd cell cycle rates of 769-P and 786-0 cells:G1 phase 48%, S phase 42%, G2 phase 10% and G1 phase 41%, S phase 54%, G2 phase 4%. After 20μM treatment of SSd 24-48h at 769-P and 786-0 cells, cell cycle rates:G1 phase 72%, S phase 24%, G2 phase 4% and G1 phase 72%, S phase 21%, G2 phase 7%. From that we can draw that SSd can induce cell cycle arrest in G0/G1 phase.4. Apoptosis experiments suggest SSd treated cells Annexin V-positive cells compared with control group, showed a significant increase in the trend, and there are significant differences, Annexin V-positive cells rates were 9.6% and 16.6% in the 769-P cells, Annexin V-positive cells rates were 21.2% and 49.8% in the 786-0 cells. We use the western blotting assay to study apoptosis related protein, and found that the expression of p53 protein increased.5. SSd inhibit RCC (769-P and 786-0) growth through EGFR/p38 MAPK signaling pathway. P-EGFR, P-MEK, P-p38 and its downstream of c-Fos protein significantly reduced after SSd treatment. Then separately with EGFR inhibitors AG1478 [28] and p38 inhibitor SB203580 treated RCC, we can find the amount of p-EGFR and p-p38 was significantly reduced, and the effect of reducing was similar with SSd. |
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