Research On The Immunol Mechanism And Treatment Of Murine BTCC Of BCG And BCG Extracts

Bladder transitional cell carcinoma (BTCC) is the most common malignant tumor of urinary system with a trend of rising morbidity. It has the characteristic of mμltiple and recurrence, 70%-80% of which is superficial bladder cancer. It is reported that the recurrence rate of cancer is 50%-70% after TURBt or partial cystectomy and the malignant degree and infiltrate ability are rising. So it is important to do the bladder instillation of drugs to prevent the recurrence of tumor.By now there are two kinds of drugs for bladder instillation. One is chemical medicine such as MMC (mitomycin), eAMI (epirubicin), TESPA (thiotepa) and so on. The other is immune intensive agent such as IL-2, INF-γand so on. Chemical medicine has few side effects, but because it can activate the antidrug genes, which highly express the P-gp and make tumor cells insensitive to the medicine, the recurrence rate of cancer is high. For example, the five-year recurrence rate of cancer is 42%-49% for MMC and 44%-51% for TESPA.Bacillus Chalmette-Guerin (BCG) is the earliest biologic agent in tumor treatment, which is much effective in the prevention of recurrence and advance of cancer. The recurrence rate of superficial bladder cancer post operation for BCG is 18.9%. Besides, BCG has the treatment effect, the efficiency rate for residual small tumor is 50%-60%,the complete efficiency rate for carcinoma in situ is 70%-75%,the total efficiency rate of release over 5 years is 70%.It can treat the upper urinary tract transitional cell carcinoma when it is instillated to this area.The instillation of BCG is the most effective method in the prevention of recurrence of superficial bladder cancer, but it can bring some side effects. Cystitis symptoms including frequency of micturition, precipitant urination, urodynia and dysuria occurred in most patients and hematuria occur occasionally. Sometimes granμloma of urethra and bladder may occur. High fever, discomfort and temporary influ-like symptoms are usually seen. Serious side effects occur in 5% of patients, 10% of those are related to BCG sepsis.7cases were reported to dead of BCG sepsis due to bladder instillation. In clinic, some patients can not finish the entire treatment course because of side effects. So it is an urgent problem to resolve that some substance must be found to replace BCG, which has the biologic activity of BCG and no side effects.Research on the immune mechanism and treatment of murine BTCC of BCG, BCG extract (BCGE2) and Ag85BObjective: Transitional cell carcinoma of the bladder is the most common cancer in urinary system, and the high recurrence rate is a serious problem for urinologist. Therefore, it is very necessary to find an effective medicine to suppress the growth and cut down the recurrence of BTCC. It is generally believed that BCG is the most effective medicine for BTCC by now, but it has many adverse effects. Ag85B is one of the most important composition of BCG and the most principal secreting protein of Mycobacterium, inducing violent Th1 immunological reaction, playing a significant role in synthesizing Mycobacterium cell wall and having the peculiarity of binding Fibronectin. Ag58B can induce the reaction of Th1 immunological cell ,which secret IL-2,TNF-αand IFN-γetc. The change of these three kinds of cytokines is essential to study the therapeutical mechanism in BTCC treatment. The final course of BCG in treatment of BTCC is to kill the tumor cells or induce apoptosis. Making use of light microscope and transmission electron microscope to observe the tumor cell necrosis and Apoptosis and of FCM to detect Cell Cycle and apoptosis can give us a preliminary approach in treatment of BTCC with BCG, BCGE2 and Ag85B in order to provide a good medicine for treatment of BTCC and to improve therapeutic efficacy of this disease.Material and Methods1 Establishment of BTCC transplantable model of T739 mouse1.1 BTT739 cell line was provided by the Department of pathology, the First Shanghai People'Hospital. Conventional culture to log phase growth.T739 mice were provided by Laboratory Animal Center, Tumor Research Institute, Chinese Academy of Medical Sciences, and raised in Laboratory Animal Center, Hebei Medical University.1.2 We choose 48 samples of T739 mouse aging from 4 to 6 weeks old, weighting 18~22 grams. The mice were randomly divided into 4 groups, half of them being female in each group. Each group is randomly divided into 2 small groups, half of them being female in each small group. Each mice is injected hypodermically BTT739 cell line (5.0×10~6/l) 0.1ml to left armpit.2 Medicines intervene the growth of bladder transitional cell. Taking their blood samples and dissecting the tumor issue. The mice of each group were injected hypodermically with the same volume of Normal saline, BCG, BCGE2 and Ag85B near the carcinoma on the first, the fifth, the ninth, the thirteenth, the seventeenth day. The normal saline group was as the control group, other three groups were as the treatment groups. The mice of one small group in each big group were killed. Collect the serum after centrifuging their blood and weight the tumor after the dissection. Tumor Inhibition Rate (TIR) is calculated according to mean tumor weight and its formula: TIR= (1-mean tumor weight of treatment group/ mean tumor weight of control group)×100%.Each sample was separated into three pieces for the examination of light microscope, electron microscope and FCM. The mice of another small group in each big group were used to observe the period of their lives after the transplantation. After the observation we can calculate Survival Prolong Rate (SPR), its formula being: SPR= (mean days of survival in treatment group/mean days of survival in control group-1)×100%.3 Double antibody sandwich ABC-ELISA detected the level of IL-2, TNF-αand IFN-γof mouse serum in each group.3.1 Detected the level of IL-2 of serum in each group. Establish a standard curve according to the operating instruction of the kit. Each well in microwell plate except standard wells was injected with 100μl sample and the reaction conditions were 120 min at 37℃.Then wash the plate and inject 50μl the first antibody fluid. After 60 min at 37℃wash again and pour 100μl enzyme labeled antibody. After washing the plate 60 min later, 100μl substrate fluid was added .The conditions were 10 min at 37℃in dark place. After 50μl stop buffer was injected, absorbance was measured at 492nm in a spectrophotometer. The amount of IL-2 concentration in mouse serum can be found according to the absorbance 3.1)3.2 Detected the level of TNF-αand IFN-γof mouse serum in each group. (The same as 3.1)4 FCM4.1 FCM for proliferation index of mouse tumor cell. The DNA of BTCC was stained by EB. The amount of DNA in BTCC was detected by FCM, after it was stained by EB. The proportion data of G0/1, S and G2M was analyzed by DNA cell cycle software. The PI of BTCC was calculated according to the following formula: PI(%)=(S+G2M)/(G0/1+S+G2M) ×100%。4.2 FCM for the proportion data of BTCC apoptosis. After stained and filtered, each sample become standard monoplast suspension and ready for being tested.5.1 Necrosis and Apoptosis of tumor cells was observed by light microscope and transmission electron microscope.Results1.1 All mice of four small groups was killed 18 days after injected tumor cells; the tumors were resected and weighted .The mean tumor weight (MTW) of Normal saline group,BCG group,BCGE2 group and Ag85B group was(6.67±1.54)g(,4.24±1.19)g(,3.90±1.58)g and (2.80±1.05)g.The tumor inhibition rate of treatment groups was 36.43%,41.52% and 58.02% respectively. The MTW of control group is much higher than those of the treatment groups and the difference is statistically significant. In the treatment groups the WTW of Ag85B group was the lighter than others with significantly difference, the difference between the rest groups is not statistically significant.1.2 The survival days of each group were 18.58±1.83 days, 24.42±5.51 days, 25.12±4.64 days and 30.25±5.43 days .The survival days of control group was much shorter than those of the treatment groups. The difference is statistically significant, the survival days of Ag85B group was the longest in the treatment groups with significantly difference, the difference between the rest groups is not statistically significant. The SPR of each treatment group was 31.43%, 35.20% and 62.81%. 2.1 Double antibody sandwich ABC-ELISA detected the level of IL-2 of serum in each group. The level of IL-2 of Ag85B was much higher than that of other two groups statistically, but BCG group and BCGE2 group is the same statistically.2.2 ELISA detected the level of TNF-αand IFN-γof mice serum. The level of TNF-αand IFN-γof Ag85B group was much higher than those of other two groups statistically, but BCG group and BCGE2 group is the same statistically.3.1 FCM for proliferation index of mouse BTCC. The mean PI of Normal saline group, BCG group, BCGE2 group and Ag85B group was ( 36.95±6.30 ) %, ( 29.65±3.48 ) %, (28.73±2.38)% and (21.40±2.00)%. The mean PI of Normal saline group was much higher than those of the treatment groups statistically. The PI of Ag85B group was the lowest statistically .BCG group and BCGE2 group was the same statistically.3.2 FCM for the proportion data of BTCC apoptosis. The mean proportion data of apoptosis of each group was (21.67±1.22)%, (24.78±1.75)%, (25.28±2.53)% and (26.18±3.39)%.The mean proportion data of apoptosis of the control group was much lower than that of other three groups and was different statistically. The proportion data of apoptosis of Ag85B group was the highest group with statistical difference, BCG group and BCGE2 group was the same statistically. 4.1 light microscope and transmission electron microscope examination. Tumor necrosis of the treatment groups was much more than the control group. Necrosis occurred occasionally in the control group; BCG group and BCGE2 group had a small- scale of necrosis; Ag85B group had a large-scale of necrosis. The apoptosis condition was the same in all groups.Conclusions1 BCG, BCGE2 and Ag85B can suppress the tumorous growth, prolong the survival period.2 BCG, BCGE2 and Ag85B can raise serum concentration of IL-2, TNF-αand IFN-γof the cancer-bearing mice. The effect of Ag85B is the best. The concentration change of IL-2, TNF-αand IFN-γmay be one of the important mechanism of these three kind of medicines in treating BTCC.3 Cells proliferation of the treatment groups was suppressed significantly and was much lower than that of the control group. Ag85B is the best also, its reason may be that other two substances have lots of antigens that interfere with each other. The apoptosis data in treatment groups was the same with the control group statistically, but there was an increase trend in the treatment groups and Ag85B group was a little higher than other groups. It may be necrosis rather than apoptosis that is the main factor in the BTCC treatment of the three substances. In addition, the results of light microscopic and transmission electron microscopic examination had also proved that is the case.4 BCG , BCGE2 and Ag85B have different molecular biological mechanism in the treatment of bladder transitional cell cancer. BCG contains variety of antigens, which play different roles, so we can explain why there are all kinds of adverse effects in the BTCC treatment. If the extract of BCG is not purified enough, its content may interfere with each other to affect its overall therapy. Ag85B is highly purified, using the antigen may reduce the therapeutical adverse effects, stimulate the immune system more effectively in the BTCC treatment. It can suppress the tumorous growth safely and efficiently lowering the recurrence rate of BTCC.