GAO Yu (高 宇), LIU Li-kun (劉麗坤), LI Yi-fang (李宜放),WANG Dong (王 棟), WANG Xi-xing (王晞星)

1. Shanxi Provincial Hospital of Traditional Chinese Medicine, Shanxi Provincial Institute of Traditional Chinese Medicine,Taiyuan 030012, China

2. Graduate School of Shanxi Provincial Institute of Traditional Chinese Medicine, Taiyuan 030012, China

ABSTRACT Objective:To investigate the molecular mechanism of Ruanjian Sanjie Capsules (軟堅(jiān)散結(jié)膠囊) through regulating NOTCH signaling pathway to promote human clear cell renal cell carcinoma 7 860 cell apoptosis.Methods:A laser confocal microscope was used to observe the nuclear displacement of apoptosis-related proteins, and the expression of Notch1, NICD, Bcl-2, and Bax was detected by Western blotting (WB).Results:Laser confocal microscopy showed that the immunofluorescence intensity of Bcl-2 protein gradually decreased with the increase of Ruanjian Sanjie Capsules (軟堅(jiān)散結(jié)膠囊)drug-containing serum concentration, and the fluorescence intensity of Bax protein gradually increased with the increase of dose concentration, and it entered the nucleus to varying degrees; WB detection showed that Bcl-2, Notch1, and NICD protein expression levels were inversely correlated with the drugcontaining serum dose concentration, that is, protein expression decreased with the increase of drugcontaining serum concentration, while Bax protein expression increased gradually with the increase of serum concentration. Afte 7 860 cells knock out Notch1, the expression of related proteins was consistent with the expression trend of the drug-containing serum group.Conclusion:Ruanjian Sanjie Capsule's (軟堅(jiān)散結(jié)膠囊) low, medium and high dose concentration of drug-containing serum can promote 7 860 cell apoptosis, and it is concentration-dependent. The molecular mechanism of apoptosis is directly related with Ruanjian Sanjie Capsule (軟堅(jiān)散結(jié)膠囊) blocking Notch signaling pathway activation.

KEYWORDS Renal clear cell carcinoma; Apoptosis; Notch; Ruanjian Sanjie Capsules (軟堅(jiān)散結(jié)膠囊)

The incidence of kidney cancer in China is showing a rapid growth trend. Among them, the incidence of renal clear cell carcinoma is the highest.About a quarter of patients have metastasized when they are first diagnosed, and the 5-year survival rate of such patients is less than 10%[1]. For the treatment of kidney cancer, surgery is the first choice, but the effect is not good, and radiotherapy and chemotherapy are not very sensitive. Molecular targeted drugs and immunotherapy have become the main treatment methods for kidney cancer in recent years. However, due to the narrow application range of such medicines, the side effects are relatively large, and drug resistance occurs frequently[2]. At the same time, it is expensive and difficult to promote,and there are fewer beneficiaries. Therefore, we urgently need to find medicines with strong antitumor effect, long-lasting curative effect, low toxicity,and can be widely used in patients with advanced renal cancer. Our previous research found that Ruanjian Sanjie Capsules (軟堅(jiān)散結(jié)膠囊) can induce 7 860 cell apoptosis and inhibit proliferation[4],but its target and molecular mechanism are not yet clear. In this study, in vitro experiments revealed the molecular mechanism of Ruanjian Sanjie Capsules(軟堅(jiān)散結(jié)膠囊) inducing apoptosis of renal cancer cells by regulating Notch signaling pathway.

MATERIAL

Preparation of Medicines and Drug-Containing Serum

Ruanjian Sanjie Capsules (軟堅(jiān)散結(jié)膠囊) is prepared and quality controlled by the Preparation Department of Shanxi Provincial Hospital of Traditional Chinese Medicine. The composition includes Radix Ranunculi Ternati, Pseudobulbus Cremastrae seu Pleiones, Rhizoma Curcumae,Spica Prunellae, Bulbus Fritillariae Thunbergii,Gecko, Endothelium Corneum Gigeriae Galli,Concha Ostreae, etc..

Drug-containing serum: 40 SD rats were randomly divided into 4 groups, Ruanjian Sanjie Capsule (軟堅(jiān)散結(jié)膠囊) high, medium and low dose administration group and control group. Rat dosage = clinical dosage × animal equivalent dose coefficient (according to body surface area) × serum dilution in the medium, and 630 mg/(kg·d) was the middle dose group. Gavage was given twice a day,once in the morning and once in the evening, for 7 consecutive days. The control group had a normal diet. Two hours after the last gavage, anesthesia was performed and blood was collected through the inferior vena cava. Standing for 2.5 hours, separate the serum naturally, centrifuge at 2 500 r/min for 25 minutes, inactivate in a water bath at 56 ℃for 30 minutes, filter and sterilize with a 0.22 μm microporous membrane to obtain sterile blank serum and Ruanjian Sanjie Capsule (軟堅(jiān)散結(jié)膠囊) drugcontaining serum. After loading into EP tube, put it in the refrigerator at -80 ℃ for later use.

Cell Culture and Grouped Administration

RPMI 1 640 medium was used to adhere to the human renal clear cell carcinoma cell line 7 860 (provided by the Shanghai Cell Bank of the Chinese Academy of Sciences), and add 10% blank serum/10% Ruanjian Sanjie Capsules (軟堅(jiān)散結(jié)膠囊) drug-containing serum, 1% double antibody to the medium. After the cells were cultured in an incubator and expanded for 3 generations, cells in the logarithmic growth phase were taken for experiments. Grouping: Negative control group:7 860+10% blank serum; low-dose group:7 860+10% low-dose Ruanjian Sanjie Capsule (軟堅(jiān)散結(jié)膠囊) drug-containing serum; medium-dose group: 7 860+10% medium-dose Ruanjian Sanjie Capsule (軟堅(jiān)散結(jié)膠囊) drug-containing serum;high-dose group: 7 860+10% high-dose Ruanjian Sanjie Capsule (軟堅(jiān)散結(jié)膠囊) drug-containing serum; DAPT group: 7 860+10% medium-dose Ruanjian Sanjie Capsule (軟堅(jiān)散結(jié)膠囊) drugcontaining serum + DAPT (40 μmol/l); siNotch1 group: 7 860+10% medium dose Ruanjian Sanjie Capsule (軟堅(jiān)散結(jié)膠囊) drug-containing serum +si Notch1; scramble group: 7 860+10% medium dose Ruanjian Sanjie Capsule (軟堅(jiān)散結(jié)膠囊) drugcontaining serum + siRNA.

Main Reagents and Equipment

Antibodies: rabbit anti-human monoclonal antibody anti-Notch1 (Sigma), Anti-Notch1 intracellular domain antibody (Sigma), rabbit antihuman monoclonal antibody Bcl-2 (Santa Cruz),mouse anti-human monoclonal antibody Bax(Santa Cruz). Western blot detection of protein:cell lysate, BCA protein quantification kit, PAGE gel kit, pre-stained protein marker, buffer, Tris,glycine, methanol, PVDF membrane, ultra-sensitive luminescent solution, X-ray film (Beijing Solarbio Co., Ltd.). Cell transfection: Lipofectamine 2000(Invitrogen), siRNA (Shanghai Gima Biological Co.,Ltd.), DAPT (Notch signal pathway blocker) (Sigma).Main instruments: Amersham electrophoresis instrument (Bioscience, Sweden), laser confocal microscope (OLYMPUS FV1000).

METHODS

FITC Immunofluorescence Double Staining Method to Detect Nuclear Displacement of Apoptotic Proteins

The cell suspension is inoculated into a 24-well plate with a glass slide. After the cells adhere to the wall, the medicine medium is changed, washed after 48 hours, fixed with formaldehyde, and blocked with goat serum was for 1 hour. Add the primary antibody at 4 ℃ overnight, add the fluorescent secondary antibody, and incubate at 37 ℃ for 1 hour. Add an appropriate amount of DAPI to stain the nucleus,block the light for 10 minutes, and add a drop of antifluorescence quenching mounting solution to mount the slide. The displacement of the protein nucleus was detected with a laser confocal microscope and photos were taken.

Western Blot to Detect Protein Expression

After 48 hours of culture, the cells of each group were collected, lysed, and total protein was extracted.The total protein content was determined by the BCA method and a standard curve was drawn. The protein samples were subjected to gel electrophoresis,transferred to membranes for 2-4 hours, and blocked with 5% BSA solution at 4 ℃ overnight. Add the primary antibody and incubate on shaking plate at room temperature for 2 hours; after washing the membrane, add the secondary antibody and continue to incubate at room temperature for 2 hours. Finally,ECL develops color.

Cell Transfection

After gently mixing 1 640, siRNA and diluted lipofectamine 2 000 reagent, leave it at room temperature for 20 minutes to form a liposome mixture.Add this mixture to a 24-well plate 24 hours after inoculation of the cells, and incubate for 48 hours for successful transfection. And then the testing can be performed.

Statistical Analysis

The experimental data obtained were analyzed by SPSS17.0 statistical software, and the results of measurement data were represented by. The comparison between multiple groups was performed by single-factor analysis of variance, and the pairwise comparison between means was performed by test. Results with P＜0.05 was considered as a statistically significant difference.

RESULTS

Observation of the Nuclear Displacement of Bcl-2 and Bax Protein in Each Group by Laser Confocal Microscope

In renal cancer cells, Bcl-2 and Bax proteins were stable in the cytoplasm. When tumor cells underwent apoptosis, Bax protein was activated.Its intracellular expression was enhanced and the protein entered into the nucleus, while Bcl-2 expression in the cell is reduced. Observation under a laser confocal microscope found that FITClabeled Bcl-2 and Bax proteins showed green fluorescence, and DAPI-labeled cell nuclei showed blue fluorescence. The overlapping part showed bluewhite fluorescence. The experimental results showed that the immunofluorescence intensity of Bcl-2 protein gradually weakened after 7 860 cells were treated with Ruanjian Sanjie Capsule (軟堅(jiān)散結(jié)膠囊)low, medium and high drug serum for 48 hours, and the high dose group was the weakest. Bax protein fluorescence intensity gradually increased with the increase of drug serum concentration, and the protein can be seen to enter the nucleus. As shown in Figure 1.

Figure 1. Confocal Laser Double Staining Method to Detect the Expression of Bcl-2 and Bax Protein after the Action of Different Concentrations of Ruanjian Sanjie Capsule (軟堅(jiān)散結(jié)膠囊) Drug Serum

WB Detection of Notch1, Nicd, Bcl-2 and Bax Protein Expression in 7 860 Cells after Action of Ruanjian Sanjie Capsule (軟堅(jiān)散結(jié)膠囊)-Containing Serum

After Ruanjian Sanjie capsule low, medium and high drug-containing serum were treated on 7 860 cells for 48 hours, Notch1, NICD, Bcl-2 and Bax proteins were detected by WB. The results showed that the serum groups containing Ruanjian Sanjie capsules (軟堅(jiān)散結(jié)膠囊) at each dose could significantly reduce the expression levels of Notch1,NICD, and Bcl-2 protein, and the Bax protein expression levels increased with the increase of serum concentration, which were statistically different from the negative control group (P＜0.05).As shown in Figure 2.

Figure 2. Comparison of the Relative Expression Levels of Notch1, NICD, Bcl-2, and Bax Protein after the Action of Ruanjian Sanjie Capsules(軟堅(jiān)散結(jié)膠囊) Containing Serum

WB Detection of Blocking Notch Signaling Pathway on the Expression of Bcl-2 and Bax Apoptosis Proteins in 7860 Cells

The 7860 cells transfected with Notch1 siRNA or DAPT were added with a medium-dose concentration of drug-containing serum medium and incubated for 48 hours. The expression levels of Notch1 and NICD protein decrease in the cell group after gene knockout by WB detection compared with the single-use serum group (P＜0.05), indicating successful gene knockout;and after Notch signal blockade, the Bcl-2 protein expression level was significantly decreased than that of the cell group without knockout genes (P＜0.05). On the contrary, Bax protein was significantly increased(P＜0.05). It is suggested that based on the action of Ruanjian Sanjie Capsules (軟堅(jiān)散結(jié)膠囊) containing serum, blocking Notch signaling pathway can more obviously induce 7 860 cell apoptosis. As shown in Figure 3.

Figure 3. Relative Expression Levels of Notch1,NICD, Bcl-2 and Bax Proteins after Treatment of Cells with siNotch1 and DAPT

DISCUSSION

Ruanjian Sanjie Capsule (軟堅(jiān)散結(jié)膠囊) is an effective medicine for treating kidney cancer.Kidney cancer can be attributed to the diseases of"hematuria" and "kidney accumulation" in traditional Chinese medicine. Kidney cancer occurs because a variety of internal and external pathogenic factors acting on the body, causing the body's Qi (氣), blood and body fluid to function abnormally. Blood stasis in the meridian and water dampness stagnation leads to phlegm and blood stasis. Long-term accumulation will transform into heat and toxin, leanding to"carcinoma toxin generating in the kidney". At the same time, phlegm, blood stasis and toxin are glued to each other, resulting in abnormal functions of the internal organs, Qi (氣) and blood, and further promoting the development of the disease[3].Therefore, the phlegm-stasis-toxin interaction is the basic pathogenesis for the occurrence and development of kidney cancer throughout the disease. The treatment should be promoting blood circulation and removing blood stasis, resolving phlegm and softening hardness, clearing away heat and removing toxin. Our hospital's self-prepared Ruanjian Sanjie Capsule (軟堅(jiān)散結(jié)膠囊) is guided by the method of activating blood circulation,softening hardness and removing toxin. The clinical treatment of kidney cancer is very effective. We will further explore its anti-cancer mechanism from the molecular level.

Ruanjian Sanjie Capsules (軟堅(jiān)散結(jié)膠囊) can induce apoptosis of renal cancer cells. Decreased apoptosis and (or) excessive proliferation are the biological basis of tumorigenesis, so "imbalance between apoptosis and proliferation" is the core mechanism for the development of renal cancer.Therefore, in the treatment of kidney cancer,we must focus on "apoptosis and proliferation imbalance". On the one hand, we must actively promote cell apoptosis. On the other hand, we must block the cell growth cycle and inhibit tumor cell proliferation. Kidney cancer cell apoptosis is regulated by a variety of genes and protein molecules. In 7 860 cells, Bcl-2, an anti-apoptotic factor, is highly expressed, while the expression of pro-apoptotic factor Bax is inhibited. In this study,under a laser confocal microscope, the drugcontaining serum of Ruanjian Sanjie Capsules (軟堅(jiān)散結(jié)膠囊) can promote the pro-apoptotic protein Bax in 7860 cells to enter the nucleus, gradually increasing the fluorescence color in the nucleus, and weakening the color of Bcl-2 protein. WB detection showed that the serum containing the drug at each dose concentration can significantly down-regulate Bcl-2 and up-regulate the expression level of Bax,and it has an obvious concentration-dependent.Ruanjian Sanjie Capsules (軟堅(jiān)散結(jié)膠囊) can induce apoptosis of renal cancer cells by downregulating the expression of Bcl-2 and up-regulating the expression of Bax in 7 860 cells.

Ruanjian Sanjie Capsule (軟堅(jiān)散結(jié)膠囊) can induce renal cancer cell apoptosis by blocking Notch signaling pathway. The Notch signaling pathway is involved in multiple stages in the course of cell life activities, including cell differentiation, proliferation,apoptosis and death. The Notch signaling pathway has four transmembrane receptors (Notch1-4),and the structure includes three parts of the extracellular domain (NEC), transmembrane region (TM) and intracellular region (NICD/ICN);five ligands (Jagged1, Jagged2, DLL1, DLL3 and DLL4) are expressed on the surface of adjacent cells and bind specifically to the four receptor molecules. A large number of studies have found that the disorder of the Notch signaling pathway is involved in the occurrence and development of a variety of tumors[4-6]. Notch1 can be both an oncogene and a tumor suppressor gene. Notch1 exhibits carcinogenic effects in gastric cancer,synovial sarcoma, chronic lymphocytic leukemia and colorectal cancer[7-10], and has antitumor effects on liver cancer, lung squamous cell carcinoma and squamous cell skin cancer[11]. Notch signaling is closely related to the occurrence and development of renal cancer. Studies have shown that there is overexpression of Notch1 protein in renal cancer cell lines and tumor specimens of renal cancer patients.Activation of overexpressed Notch1 can promote the proliferation of renal cancer cells, and silencing Notch1 can inhibit tumor cells growth, retard the cell growth cycle[12]. This study shows that Ruanjian Sanjie Capsules (軟堅(jiān)散結(jié)膠囊) can down-regulate the expression levels of Notch1 and NICD proteins in human renal clear cell carcinoma cells at various dose concentrations of Ruanjian Sanjie Capsules (軟堅(jiān)散結(jié)膠囊), and the down-regulation effect of highdose concentration is more obvious than that of the low and medium dose concentration. After 7 860 cells were co-treated with medicated serum and silenced Notch1 gene, the expression of Notch1 and NICD was significantly down-regulated compared with the drug-containing serum group alone, indicating that gene knockout was successful. At the same time, after co-treatment with medicated serum and silenced Notch1 gene, Bcl- 2 and Bax protein downregulated and up-regulated protein expression more significantly than the drug-containing serum group alone. This proves that Ruanjian Sanjie Capsule(軟堅(jiān)散結(jié)膠囊) can effectively induce renal cancer cell apoptosis by inhibiting Notch signaling pathway activity.

In summary, Ruanjian Sanjie Capsules (軟堅(jiān)散結(jié)膠囊) drug-containing serum can promote the apoptosis of human renal clear cell carcinoma cells, and its mechanism of action is related to blocking the Notch signaling pathway. Therefore, the effective inhibition of the Notch signaling pathway has great significance in the treatment of renal clear cell carcinoma. However, the occurrence and development of tumors is a complicated process.From a microscopic point of view, there are still many effective biological targets worthy of our attention.Ruanjian Sanjie Capsules (軟堅(jiān)散結(jié)膠囊) are compound prescriptions with complex ingredients,and their effective anti-cancer monomers are still unclear. We still need further in-depth exploration,and the results will provide experimental evidence for the clinical treatment of kidney cancer with Ruanjian Sanjie Capsules (軟堅(jiān)散結(jié)膠囊).