| Research background and purpose:Bladder cancer is a malignant tumor in the bladder mucosa,the urothelial carcinoma?transitional cell carcinoma?is the main type.It is the most common malignant tumor of urinary system,its incidence and mortality rate increased year by year in the world,the male incidence rate is 3-4 times higher than in females.In China,the bladder cancer takes the eighth place in the incidence of male malignant tumors and takes the twelfth place of female malignant tumors.The peak incidence is 65 years.With the increase of age,the incidence of bladder cancer is gradually increased.The majority is superficial bladder cancer,also named as non-muscle invasive bladder cancer?NMIBC?,transurethral resection of bladder tumor?TURBT?is the main treatment for non-muscle invasive bladder cancer in clinical.The early surgery is the key to the treatment of bladder cancer.The main causes of death are high postoperative recurrence rate and higher malignant degree.Intravesical chemotherapy and immunotherapy are widely used in the treatment of superficial bladder cancer after the surgery.Postoperative intravesical chemotherapy can effectively reduce the recurrence rate,improve the prognosis of patients and improve the survival rate.However,there are still 2/3 of patients with recurrence.Bacillus Calmette-Guérin?BCG?bladder perfusion is the gold standard for superficial bladder cancer immunotherapy.It is considered as the most effective chemotherapy after failure perfusion,but there are still30-45%patients with non-response,20%patients can not tolerate its side effects.There is still a certain proportion of patients can not tolerate the side effects,such as bleeding cystitis,hematopoietic and immune suppression,the quality of life is seriously affected.To improve the efficacy and reduce the side effects of BCG is a hot research for curing bladder cancer in current.BCG combined with chemotherapy is available for clinical treatment.The method is superior to that of BCG single perfusion,and it can reduce the dosage of BCGArsenic trioxide?As2O3?is the main component of arsenic.As a traditional Chinese medicine,it can treat syphilis,tuberculosis and cancer.It has a long clinical history,in1990s,researchers found that the arsenic targeted therapy can treat the acute promyelocytic leukemia?APL?and it has achieved outstanding curative effect.Since then,the antitumor activity of arsenic trioxide aroused worldwide attention.In 1999,the State Food and Drug Administration?SFDA?of China approved the production of ATO injection for clinical treatment.In 2000,the U.S.Food and drug administration also approved ATO injection as the second-line drugs for the treatment of relapsed/refractory APL.The effect of arsenic trioxide for blood system diseases is significant.Through clinical research of application of ATO in the blood system tumors:the complete remission rate and long-term survival rate are higher,the recurrence rate and adverse reactions are less,and there are no cross resistance with other drugs.Recent years,ATO is gradually used in solid tumors,such as lung cancer,gastric cancer,breast cancer,prostate cancer,ovarian cancer,colon cancer and liver cancer.Many in vivo and in vitro experiments show that ATO can induce apoptosis of tumor cells selectively and without induce function in normal cells and has antiangiogenic effect of tumour.But there is an obvious dose and time dependence for the antitumor effect of ATO.The therapeutic window is narrow for ATO.The high dose of ATO has obvious toxicity to normal cells,so the effective route of administration and dosage is the key to determine the clinical efficacy.In order to ensure the anti-tumor efficacy and reduce the side effect,the low and effective drug concentration is the basic principle of clinical medication.Connexins?Cxs?have the same basic structure,there are 4 hydrophobic transmembrane segments,in which hydrophilic segments constitute 1 cytoplasmic loop and 6 conserved cysteine residues constitute 2 extracellular loop,the C-terminal and N-terminal of protein are in the cytoplasm.The carboxy terminal of Cxs is diversity and it is a recognized key part.It plays an extremely important role in protein interaction.The cell gap junction?GJs?which is a transmembrane channel structure is composed of two connexin six polymer,there are many unpaired hemichannels on the surface of the cell.They are similar to the cell gap junctions.The connexin hemichannels allow to exchange ion which molecular weight is under 1000D,metabolites and the second messengers(GSH,cAMP,cGMP,IL-6,IL-10,K+,Ca2+,NO and IP3),large molecules are not allowed?small RNAs is except?.Connexins play an important role in the information between cells and cell matrix,regulation of cell metabolism,proliferation and differentiation.There have been more than 20 kinds of cell gap junction proteins have been identified,there are GJs in nearly all the other cell types except for skeletal muscle,maturation erythrocyte and sperm.The most widely distribution in tissue and the most closely with tumor is Cx43.The down-regulation of Cx43 expression is related to tumor occurrence,development and metastasis in a variety of malignant tumor cells.The study of bladder carcinoma confirmed high expression of Cx43 protein was found in adjacent normal bladder tissue and low expression in bladder urothelial carcinoma cell.Cx43 gene mutation is rare,the regulation of its expression and function process mainly at the transcriptional level and after that.It was found that ATO could regulate the expression of CX43 to produce biological effects.However,the current mechanism and the anti-tumor effect and mechanism of ATO combined with BCG are still blank.A detailed assessment of the effectiveness and safety is needed by in vitro and in vivo experimental models in order to develop the new drugs for bladder cancer.At present,the most commonly used bladder cancer in vitro model is human bladder cancer cell line,which not only can be used to study the molecular mechanism of the occurrence and development of bladder cancer,but also can be used to assess the effectiveness of drug.The advantages of in vitro model are controllable experimental conditions,easy operation,less time and low development costs;its disadvantage is that the cell line is from single tumor cells and grows in vitro,so it can not fully simulate bladder cancer organization and its micro growth environment such as matrix and blood vessels.The side effects of chemotherapy drugs are not predicted.So the efficacy and safety evaluation of chemotherapy drugs in clinic is not enough by the in vitro study,the in vivo experiment is also needed.The most commonly used animal model is mice or nude mice xenograft tumor model.The reasons are the small animal size,short breeding cycle,clear genetic background,the similar characteristics of physiological and biochemical metabolism with human.In order to better demonstrate the efficacy,safety and the dynamic characteristics of new chemotherapy drugs,animal model of bladder cancer is essential.In view of this,this study will use the human bladder cancer cell lines BIU-87,T24,5637to design the extreme relevant in vitro experimental system with clinic,followed by the establishment of bladder cancer xenograft nude mice model.So the efficacy and safety of arsenic trioxide chemotherapy for bladder cancer can be fully demonstrated from in vitro and in vivo system,in order to lay a scientific and stable foundation for the clinical application of arsenic trioxide for the treatment of bladder cancer.The main purposes of this experiment are:?1?To culture the human bladder cancer cell line BIU-87,T24,5637 and other bladder cancer cells in vitro,through a variety of experimental techniques and methods to establish bladder cancer model.?2?To culture human bladder cancer cell lines in vitro,by giving the different concentrations of ATO,to simulate the influence of different ATO dosages on the proliferation of tumor cells.To discuss the relationship between the effects of arsenic trioxide on connexin 43 and antitumor effects form cellular and molecular level.?3?The xenograft nude mice animal model was establishment by subcutaneous injection of human bladder cancer cell 5637,though intraperitoneal injection of different drugs,to analyze the effects of intervention methods for tumor cells,to analyze the effect and possible anti-tumor effect mechanism of ATO combined with BCG intravesical therapy for the treatment of bladder xenograft cancer preliminarily.Methods:1.The bladder cancer cell lines NBT-II,5637,BIU-87,T24 and PBS were cultured in DMEM culture medium with 10%fetal bovine serum.After the cells were cultured to the logarithmic growth phase,the cells were weight suspended by culture medium and adjust cell the concentration,they were injected into Balb/c female nude mice.According to the different cell injection,the mice were divided into 5 groups,NBT-II group,5637 group,BIU-87 group,T24 group and PBS group,subcutaneous tumor formation and growth in mice were observed and analyzed,to determine the type of modeling and to lay the foundation for in vivo modeling.2.5637 bladder cancer cell lines were used to build in vitro simulated experimental system for intravesical chemotherapy after the surgery.According to the different intervention methods,they were divided into 6 groups,?1?normal culture group?without any intervention?;?2?5mol/L group?5 mol/L As2O3 intervention?;?3?10 mol/L group?10 mol/L As2O3intervention?;?4?15 mol/L group?5 mol/L As2O3 intervention?;?5?20 mol/L group?10mol/L As2O3 intervention?;?6?25 mol/L group?10 mol/L As2O3 intervention?.After modeling for 24h,48h,cell proliferation was observed and protein expression levels were analyzed in each group.The survival rate of bladder cancer cell was detected by CCK-8and ATO influence on cell apoptosis was obsered by flow cytometry,the Cx43,Nrf2,JNK,JNK-1 expression levels were detected by Western blot method,PCR was used to detect the CX43mRNA level,the annexin-V/FITC kit was used for detecting apoptosis under flow cytometry.The molecular mechanism of the arsenic trioxide on gap junction protein 43 and half channel for bladder cancer were detected and analyzed,and the change of Cx43 expression was detected.3.The bladder cancer xenograft model was established by subcutaneous injection of human bladder cancer cell 5637.When the average tumor volume reached 100mm3150mm3,according to the tumor volume and body weight of mice,they were randomly divided into 4 groups,9 mice in each group.The difference between each group was less than 10%of the mean tumor.The drug was given by the following scheme:?1?The arsenic trioxide group:As2O3 10mg/kg;?2?Gap26 group,As2O3 10mg/kg was given after blocked Gap26;?3?The blank control group,without adding drugs and blockers,given only 0.9%NaCl;?4?The United Group,As2O3 10mg/kg?BCG?and BCG 100g.They were given intervention for 10 days,before and after treatment the mice were observed and recorded,the changes of volume and tumor weight in each group were observed and compared.At the same time,Cx43expression levels were detected in each group.The intrinsic molecular mechanism of As2O3 and BCG on gap junction protein 43 and antitumor effect were detected and analyzed from the molecular level.4.The statistical analysis.Software SPSS 13.0 was used for data analysis.The effect on Cx43 and total protein p-Cx43 of different blockers in different treatment time were analyzed by factorial analysis.The separate effects of time and blocker were analyzed by single factor analysis of variance,Welch correction was used when variance inequality occurred.If it met the homogeneity of variance they were compared by LSD method,otherwise,they were compared by DunnettT3 method.The experimental data were expressed by mean square deviation?x±s?.The mean in each groups were compared by single one-way ANOVA.The data were compared by LSD method between groups,when variance inequality occurred,Dunnet T3 method was used.The grade data was analyzed by non parametric test?2 Independent Samples Test?.The significant level was a=0.05.Results:1.The condition of nude mice was good in 5637 group,BIU-87 group,NBT-II group and T24 group during the experiment.There was no significant decrease in body weight,no early death in mice and no obvious adverse reactions,the mice had the normal diet.After the injection of the same concentration of cell suspension,according to the standard of tumor volume of more than 0.1cm3,NBT-II group tumor was the highest,followed by the 5637 group.There was obvious difference in the nude mice bladder cancer tumor rate?P<0.05?;The body weight of nude mice decreased significantly over the time in bladder cancer xenograft model?P<0.05?;In NBT-II group and 5637 group,there were no significant differences in the tumor volume,tumor weight and the growth rate?P>0.05?.2.The CCK-8 method showed that As2O3 produceed significant effect at the concentration of 10umol/L As2O3 for inhibiting the proliferation of bladder cancer cells,and there was dependent relationship between the inhibitory effect and As2O3 dose.LD50 was about 20umol/L;When compared with the control group,Western Blot analysis showed that Cx43,Nrf2,JNK and JNK-1 protein expression were significantly up-regulated after As2O3 treatment for 12h,it increased with the dose addition,there was statistical differences between the different treatments of As2O3?P<0.05?.3.Before the administration,there was no significant difference in the tumor volume of each nude mice group?P>0.05?.After administration,tumor volume and tumor weight decreased significantly in As2O3 group and combined group,when compared with the data before administered,there was statistical significance?P<0.05?;After administration,in combined group,the volume and tumor weight were significant lower than that of the other groups?P<0.05?.In As2O3 group,the volume and tumor weight were significant lower than that of Gap26 group and control group,there was statistical significance between groups?P<0.05?;In combined group and As2O3 group,the tumor tissue necrosis was more,the residual nuclear division was reduced,in some regions,all tumor cell disappeared and necrosed.The brownish yellow granules were more within the cells,stained cells were positive.In Gap group,the tumor cell necrosis was not obvious,most of the tumor cells grew vigorously,yellow granules were rare,stained cells were weakly positive.In bland control group,tumor cell grew vigorously,mitosis was common,yellow granules were extremely rare,There was significant difference in CX43 levles between the groups?P<0.05?.Conclusion:There are significant differences in tumor formation rate of human bladder cancer T-24,BIU-87,5637 cells.In the process of bladder cancer model establishment,the human derived 5637 cells with higher tumor formation rate and better stability should be selected.In vitro system,low concentrations of arsenic trioxide can effectively inhibit the growth and increase the CX43 expression,and there are obvious time and dose dependent in the inhibitory effect of arsenic trioxide.After using the half channel specific inhibitor,the anti-tumor effects of arsenic trioxide significantly decreases,it suggests that the anti-tumor effects of arsenic trioxide related to hemichannel opening.Arsenic trioxide combined with BCG can significantly up-regulate the connexin expression,inhibit the growth of nude mouse xenografts bladder cancer cell,its effect is obviously better than that of simple arsenic trioxide.In summary,this study demonstrates the effectiveness and safety of arsenic trioxide for bladder cancer after chemotherapy from the clinical point of view by two kinds of experiments in vivo and in vitro system.It has been fully demonstrated the feasibility of arsenic trioxide in clinical treatment for bladder cancer.It has important practical application value in the field of translational medicine. |
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