XIAP Inhibitor Embelin Inhibits Bladder Cancer Survival And Invasion In Vitro

Background:Bladder cancer is the most common urological malignancy around the world, incidence rate of bladder cancer is in the first 11 malignant tumors, ranking 7th in the men and after the first 10 in women. An estimated 429,800 new cases of bladder cancer and 165,100 deaths occurred in 2012 worldwide. In China, the incidence of bladder cancer among men is No.7 systemic cancer, women came in after the first 10. According to the National Cancer Registry area in 2009,the total incidence rates of bladder cancer is 6.61/10 million, of which men 11.41/100,000 and women 3.51/ 100,000. In recent years, the incidence rate of bladder cancer is increased year by year in China.The characteristics of bladder cancer disease are as follows:1, bladder cancer can occur at any age, even children, but the main age of onset is in middle age, and its incidence increases with age.2, Approximately 70%-85% of patients are diagnosed as non muscle invasive bladder cancer (NMIBC), including Ta、T1 and Tis.3, about 10-30% of patients with superficial bladder cancer eventually develop into muscle invasive bladder cancer or metastatic bladder cancer.4,10%-15% of patients with muscle invasive bladder cancer (MIBC) metastasis have occurred at the time of diagnosis, even after radical cystectomy, up to 50% of the patients will be transferred. 5, histological type of bladder cancer is urothelial carcinoma, accounting for more than 90% of bladder cancer, which is moderately sensitive to chemotherapy.According to diagnosis and treatment guidelines, the characteristics of treatment of bladder cancer disease are as follows:superficial bladder cancer, transurethral resection of bladder tumor (TRUBT)+chemotherapy; T2-T4a, radical cystectomy and pelvic lymphadenectomy+urinary diversion, if necessary, neoadjuvant chemotherapy and (or) and adjuvant chemotherapy or (radiotherapy); T4b, palliative operation plus chemotherapy and (or) radiotherapy.and then we found that chemotherapy is always throughout the treatment of bladder cancer.Although we had improved the diagnosis and treatment of bladder cancer in recent years, new treatments of bladder cancer are immunotherapy, photodynamic therapy and molecular targeted therapy and etal. but the recurrence rate of superficial bladder cancer is still high. Although the therapy of muscle invasive bladder cancer (MIBC) are including radical cystectomy, radiotherapy and chemotherapy, patients finally associate with a strong propensity toward deadly metastases and with a low 5-year survival rate. Difficulties in bladder cancer treatment is easy to recur, and the key of improve the survival and prognosis is to reduce the recurrence rate and keep control of tumor metastasis and invasion. Therefore, we must pay attention to bladder cancer diagnosis, treatment, deepening exploration to find new measures and discover new mechanism, and it also has been concerned about the current field of oncology.With the development of molecular biology and genetic engineering technology, the researchers have made a lot of encouraging results that we found some new regulatory sites, as well as regulatory proteins which regulate the relationship between signaling pathways and further define the bladder cancer pathogenesis, regulatory mechanism for the development of a more cutting-edge diagnostic methods, more sophisticated methods of treatment as a new target.Apoptosis is a way to customize the death gene regulation, and to stabilize the multicellular living organisms in this way, which is significant imbalance in cancer cells and apoptosis mechanism closely linked.the former are often able to induce the latter occurs. Inhibitor of apoptosis proteins IAPs (inhibitor of apoptosis protein) have a significant impact on tumor emergence and developments highly conserved family of recently discovered endogenous inhibition of apoptosis,which can play an endogenous apoptosis inhibition. X-linked inhibitor of apoptosis protein (XIAP) is one of the most potent caspase inhibitors among apoptosis protein family.The antiapoptotic activity of XIAP is due to its ability of binding to and inhibiting the activation of initiator caspase-9, as well as the effector caspases (caspase-3 and caspase-7), which are vital for the execution of apoptosis, and XIAP is also through a variety of signaling pathways involved in inhibition of apoptosis in the apoptosis-inducing endogenous or exogenous cause. Therefore, in the caspase pathway, XIAP is the only country to exert inhibitory effect of IAPs in the initial stage and effector stage.XIAP has been confirmed in a variety of human tissues, significantly higher expression in a variety of malignancies,whicn often indicates a poor prognosis patients. XIAP is now considered as a potential target of IAPs family for cancer therapeutics. Yang etal have found three apoptosis inhibitory protein livin, survivin, XIAP expression in bladder cancer cells,while, at the same time when knockdowning the three inhibitor of apoptosis protein gene, found that the proliferation and invasion of bladder cancer T24 cells decreased at the same time, and caspase-3, caspase-7, caspase-9 activity was significantly increased in bladder cancer cells, which infer these three genes can become more important targeted gene therapy of bladder cancer.Embelin is a Chinese herb extracts,and initially exhibits various medicinal effects including anti-infective, anti-inflammatory and anti-cell proliferation and so on. Embelin is identified as a novel small molecule inhibitors of XIAP in 2004 because Nikolovska-Coleska by computer screening discoveryed that Embelin could combine with binding sites of second mitochondrial activator (Smac) of Baculovirus 1AP repeat 3 (BIR3) domains of X-linked inhibitor of apoptosis protein (XIAP).Some domestic and foreign researchers have applied Embelin on tumor cells in breast cancer, pancreatic cancer, stomach cancer and leukemia in vivo and in vitro and confirmed Embelin can inhibit tumor cell proliferation by inhibiting XIAP and promoting tumor cell apoptosis. Hu Rong etal confirmed Embelin can reverse the K562/D to DNR resistance by promoting apoptosis.so Embelin is expected to become a promising anti-cancer drug, However, therapeutic effect of Embelin to human bladder cancer is not yet determined.It is an important topic in oncology research at present that looking for efficiency, low toxicity of natural medicine as an anti-invasion and metastasis research and development of drugs and exploring the mechanism of tumor invasion and metastasis. Therefore, we carried out study of XIAP inhibitor Embelin inhibiting bladder cancer survival and invasion in vitro, and to explore possible mechanisms to provide experimental data for the the prevention and treatment of bladder cancer.Objective:1.Detect the expression and the distributtion level of XIAP protein in bladder cancer cells. To explore the significance of high expression of XIAP in tumor occurrence and development by IHC, as well as the inhibitory effect of Embelin on the expression of XIAP in tumor cells after adding different doses of Embelin byWestern Blot.2. By using different concentrations of Embelin to process bladder cancer T24 and 5637 to explore inhibitory effect of Embelin on the proliferation in tumor cells at different times by CCK8,and to explore its mechanism of inhibition.3. By testing cancer cell T24 cells treated with different doses of Embelin by Transwell, to explore inhibition rate of migration and invasion of cancer cell T24 cells, explore its mechanism of inhibition effect of migration and invasion on cancer cell T24 cells.4. By using flow cytometry to detect cancer cells T24 cells and 5637 which have treated with different concentrations of Embelin, to explore the apoptosis influence of bladder cancer T24 cells and 5637. By Western blot hybridization to detecte XIAP, PI3K, Akt and p-Akt protein of bladder cancer T24 cells treated by Embelin,to explore its mechanism of apoptosis.Methods:1. Cell culture and drug intervention:the human bladder cancer cell line T24 and 5637 were maintained in RPMI 1640 medium supplemented with10%fetal bovine serum (FBS). All media contained 100 units of penicillin/mL and 100 μig of streptomycin/mL. All cell lines were maintained in a humidified incubator at 5% CO2 and 37 ℃. The cells that entered the logarithmic growth period were selected for experiment. We selected different concentration of the Embelin group, meanwhile setting DMSO blank control group. The experiment time was as followed:12h,24h, 48h. All trials were repeated three times.2. Measurement of the survival rates of cells with CCK-8 assay:a total of 5×103 /mL cells were seeded into 96-well plates and cultured overnight with 200 μL each well, and added to the culture medium containing agents of different concentrations(five different concentrations:5,10,20,25,35 μmol/L) or control PBS with 100μLeach well, each concentration for parallel 4 wells after adherence. After culturing at a temperature of 37 C for 12h,24h,48h, the medium was replaced with 100μLfresh medium and 10μLCCK-8 solution. The cells were incubated for additional 4 h and the absorbance was measured at 450 nm by an Enspire microplate reader (PerkinElmer, USA). The cell viability and IC50 values were calculated.According to the following formula to calculate the cell survival rate:survival rate of tumor cells (%)= experimental group A value/control group A value ×100%.3. Determination of cell apoptosis by flow cytometry:after culturing for 12h,24h, 48h apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit. Cells were detached by trypsinization and washed three times in PBS, centrifuged at 1,000rpm for 5min and resuspended in 195 μLAnnexin V-FITC binding buffer. 5μLAnnexin V-FITC was added and mixed. Then, the cells were stained in the dark for 10min at room temperature. After that, cells were centrifuged at 1,000rpm for 5min and resuspended in 190μLof Annexin V-FITC binding buffer. Last, lOμLpropidium iodide staining solution was added and mixed. The cells were kept on ice in the dark and immediately subjected to flow cytometry analysis. The data were analyzed using the Cell Quest software. The experiment was repeated three times.4. Measurement Migration and invasion of bladder cancer T24 Cell by transwell.transwell insert with a pore size of 8 1m from Corning, Inc. (Corning, NY) was used to determine tumor cell migration capacity. After 24 h, the cells were starved in medium without fetal calf serum (FCS) for 24 h, and then the cells were resuspended in the FCS-free medium and placed in the top chambers in triplicate. The cells remaining on the upper membrane were removed with cotton wool, whereas the cells that had migrated to the bottom of the membrane were then fixed with 95% ethanol and stained with 0.1% crystal violet. Five visual fields of each insert were randomly chosen and photographed under a light microscope at 200 x magnification. All experiments were performed in triplicate.5. Immunohistochemical Staining:all specimens were fixed in 10% buffered formalin for 24 h and embedded in paraffin. Sections (4-μm) were placed on silane-coated slides. After deparaffinization and rehydration, slides were placed into 3% hydrogen peroxidase for 15 min, and were autoclaved at 121℃ in citrate buffer (10 mM, pH 6.0) for 10 min for antigen activation. After cooling at room temperature (RT) for 20 min, specimens were incubated with each blocking buffer for 15 min at RT, and were then incubated with an anti-XIAP antibody (BD Biosciences) at 4℃ overnight. Immunohistochemical staining was performed using a standard avidin-biotin complex method with a streptavidin-biotin-peroxidase kit (Nichirei, Tokyo, Japan).3,3’-diaminobenzidine was used as a chromogen. Counterstaining was performed with hematoxylin. Negative controls were treated without antibody. Two independent pathologists blinded to the clinicopathologic variables, evaluated the immunostaining. The immunostaining score was evaluated based on the whole slide. The results were recorded by assessing the percentage of positive cells and the intensity of the staining (1, mild; 2, moderate; 3, intense) on the tumoral and the nontumoral areas for each slide.6. Protein extraction and Western blot:bladder cancer cells were seeded onto six-well plates the day before transfections were performed. Forty-eight hours after transfection, cells were lysed with RIPA buffer containing protease inhibitors (Beyotime biotechnology, Haimen, China). The concentrations of the proteins were determined using a BCA Protein Assay Kit (Beyotime biotechnology, Haimen, China). Samples were thawed in 5 x SDS-PAGE sample loading buffer, vortexed and then denatured at 100℃ for 5 min and placed on ice for 5 min. Cell lysates (approximately 30 μg of protein) were loaded on an 8% SDS-PAGE gel and subsequently transferred to μpolyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) by Wet Electrophoretic Transfer (Bio-Rad Laboratories). The membranes were blocked for 1 h at room temperature, and incubated overnight at 4℃ with primary antibody in Tris buffered saline with 0.05% tween (TBST) containing 5% non-fat milk. After washing three times with TBST, the membrane was incubated at room temperature for 2h with horseradish peroxidase-conjugated secondary antibody diluted with TBST, and then visualized using commercial ECL kit (Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. The protein bands were quantified using ImageJ 1.33 software (NIH), and the data were normalized to β-actin.7.Statistical analysis:a 11 statistical analyses were performed using SPSS 15.0 for Windows. Results were expressed as mean±SE. Chi-squared analysis was done to evaluate the significance of differences between the experiental groups. For a single comparison of two groups, Student’s t test was used. A P-value of less than 0.05 was considered to be statistically significant.Results:1.①Inhibitory effect of different doses of Embelin on the expression of XIAP in bladder cancer cells:Bladder cancer cells T24 were treated with a dose of 0, 5μmol/L,20μmol/L,35 μmol/L of Embelin effect,the results showed that the signal of the expression of XIAP hybridization becomes shallow banding with the dose of Embelin increased, which suggeste that the expression of XIAP in cancer cells decreased gradually,and indicate that with increase of dose of Embelin, expression inhibition effect of XIAP in tumor cells enhanced.②Immunohistochemical analysis of XIAP expression profiles in bladder cancer cells:Results showed that XIAP was significantly upregulated in bladder cancer cases, compared with the adjacent non-tumourous bladder tissues(P< 0.001). XIAP was predominantly stained in the cytoplasm, but it was stained diffusely and weakly in the nucleus. When we divided the bladder cancer group into invasive and noninvasive subgroups, IHC scores showed that XIAP was obviously upregulated in the invasive subgroup compared with the noninvasive subgroup (P<0.01).2. ①Inhibitory effect of Embelin on the proliferation of bladder cancer T24 cells:the inhibition rate of T24 cells which were not processed with Embelin after 24h, 48h,72h is substantially 0; when T24 cells were processed with a dose 5.0μmol/L of Embelin at different time, The average inhibition rate in 24h,48h and 72h were 11.896%,,12.664% and 15.000% respectively; when T24 cells were processed with a dose 10.0μmol/L of Embelin at different time, The average inhibition rate in 24h,48h and 72h were respectively 24.401%,30.906%,38.698%; when T24 cells were processed with a dose 20.0 μmol/L of Embelin at different time, The average inhibition rate in 24h,48h and 72h were respectively 34.968%,43.941%, 57.121%;when T24 cells were processed with a dose 25.0μmol/L of Embelin at different time, The average inhibition rate in 24h,48h and 72h were respectively 50.278%,55.322%,64.078%; when T24 cells were processed with a dose 30.0 μmol/ L of Embelin at different time, The average inhibition rate in 24h,48h and 72h were respectively 58.333%,79.333%,87.753%(P<0.05).②Inhibitory effect of Embelin on the proliferation of bladder cancer 5637 cells: the inhibition rate of 5637 cells which were not processed with Embelin after 24h, 48h,72h is substantially 0; when 5637 cells were processed with a dose 5.0μmol/L of Embelin at different time, The average inhibition rate in 24h,48h and 72h were 8.667%,12.104%,43.333% respectively; when 5637 cells were processed with a dose 10.0 μmol/L of Embelin at different time, The average inhibition rate in 24h,48h and 72h were respectively 31.075%,50.239%,56.000%; when 5637 cells were processed with a dose 20.0μmol/L of Embelin at different time, The average inhibition rate in 24h,48h and 72h were respectively 48.301%,72.333%,83.093%; when 5637 cells were processed with a dose 25.0 μmol/L of Embelin at different time, The average inhibition rate in 24h,48h and 72h were respectively 562.632%, 85.322%,94.078%; when 5637 cells were processed with a dose 30.0 μmol/L of Embelin at different time, The average inhibition rate in 24h,48h and 72h were respectively 65.333%,80.565%,96.832%(P<0.05).3.①Inhibitory effect of Embelin on the cell migration of bladder cancer T24 cells:the test results by Transwell show that the migration of T24 cells treated without Embelin is more obvious and the cells are more deeply stained in the photo section Aizen; after T24 cells treated with a dose of 0,5umol/L,20μmol/L,35 umol/L of Embelin,with the Embelin dose increases, photos blue dye gradually weaken, and the number and scope of the migration of T24 cells gradually reduce,the average number of cell migration were 333.333,285.38,214.035,95.906 (P<0.05).②Inhibitory effect of Embelin on the cell invasion of bladder cancer T24 cells: T24 cells without Embelin processing show stronger invasiveness and photos blue dye stained thick; These results show that Embelin plays a critical role in the inhibition on bladder cancer T24 cells migration and the average number of cell invasion were246.784,192.982,130.994,65.497 (P<0.05).4. ①Embelin induces apoptosis in bladder cancer cells:The results showed that Embelin could induces apoptosis in bladder cancer T24 cells and 5637 cells with the increase of 0,5μmol/L,20μmol/L,35 μmol/L of Embelin, the number of the upper right and tower quadrant gradually increased, which indicated that the number of apoptotic cells increased gradually, and apoptosis rate of T24 cells and 5637 cells were 3.849%,5.64%,12.067%,15.074% and 4.621%,7.347%,11.152%,14.815% respectively(p<0.01).②By Western blot hybridization, detection of XIAP,PI3K, Akt, and p-Akt protein results showed that with the increase of Embelin dose(0,5μmol/L,20μ/ L,35 μmol/L), the expression levels of hybridization becomes shallow banding with the dose of Embelin increased, which suggeste that the expression of XIAP,PI3K and p-Akt in cancer cells decreased significantly, the expression levels of Akt proteins stay stable,which further confirmed that Embelin inhibits cell growth by inducing apoptosis by inhibiting the expression of XIAP protein via PI3K/Akt pathway.Conclusion:1. XIAP was significantly upregulated in bladder cancer cases, and is mainly distributed in the cytoplasm of cancer cells. XIAP overexpression may have an important influence on the clinical stages of bladder cancer. Embelin can effectively inhibit the expression of XIAP protein, and in a certain range, with the increase of the dose, the inhibition effect is enhanced.2. Embelin could inhibit proliferation in human bladder cancer cells in a dose-time-dependent manner, indicating that Embelin may be a potential anticancer drug for bladder cancer patients.3. Compared with the control group, Embelin could significantly inhibited bladder cancer T24 cell migration and invasion ability, which has significant differences.Because of efftct of control the spread of tumor cells, Embelin has a high clinical value and good prospects for development to improve the condition of the patient.4. Embelin can induces apoptosis in human bladder cancer T24 and 5637 cells, and the mechanism is that Embelin can inhibits cell growth by inducing apoptosis by inhibiting the expression of XIAP protein via PI3K/Akt pathway.