Expression And Significance Of Muscarinic Receptor Subtypes, VEGF And Endostatin In Human Prostate Tissues

Research background and objects:Prostate which is an important organ of male adult has many important functions, such as prostatic fluid secretion, ejaculation assisting, urination and so on. The physiological and functional disorder of the prostate and its pathological changes can cause many diseases, even affect the physical and mental health of men. Benign prostatic hyperplasia is,of the highest incidence disease among tumors of men in our country. Prostate cancer is one of the most common malignant tumors for men in western country. Although the incidence of prostate cancer is low in our country, it has the tendency to increase in recent years. Chronic prostatitis affects patients' life because of severe symptoms and unsatisfied treatment, so that it continuously harasses urologists all over the world. Prostatic diseases' disadvantages are serious enough to call our attention, but the mechanism of these diseases is still unknown and the treatment is not so satisfied.Detective and aggressive treatment of early prostate cancer resulted in a significant decline of overall mortality rates. However, the treatment of advanced tumor stages is still a crucial problem. Knowledge on molecular alterations taking place in the development and progression of prostate cancer increased rapidly in the past few years, thanks also to the use of novel techniques that enabled to perform broad gene expression profiling of tumors. This better understanding of the molecular biology of prostate cancer led to the establishment of novel strategies to delay/inhibit tumor progression.The growth and function of prostate is regulated by many nervous systems, including cholinergic and epinephrine nerves systems. In the past few years the construction and function of prostate have been deeply researched. The researches of autonomic nerve system's regulation for function of the prostate demonstrated that the regulation of nerve system was intimately related to the genesis and development of prostatic diseases. Great success has been obtained in the research of alpha receptor inhibitor used in the treatment of benign prostatic hyperplasia, and androgen-blocking has been successfully used in the endocrine treatment of prostate cancer. But cholinergic muscarinic receptor's functions in regulating the prostatic physiologic function, disease genesis and development is still unclear. And also there has been no such kind of drugs used in the treatment of prostatic diseases.In order to better understand the roles of cholinergic muscarinic receptors in prostate and to apply cholinergic drugs in the treatment of prostatic diseases, we should make out the distribution characteristics of muscarinic receptor in normal human prostatic tissue. Up to now there are only researches about muscarinic receptor on benign hyperplastic prostate tissue and prostatic tumor cell lines, but the report of distribution regularity in different zones of normal human prostate is only fewer. The only report is done by our research team about the distribution regularity of the three kinds of muscarinic receptor subtypes in normal human prostate in 2006The cholinergic receptor expression was prevalent in many tumors, in which mostly higher than in the normal tissue. The research about breast carcinoma and other tumors revealed that M₃ receptor was intimately related with the carcinogenesis and classification of tumor. The activity of cholinergic receptor could provoke the cell proliferate through different signal pathway. The previous research found out that M₁, M₂. M₃ receptors could stimulate cell growth and differentiation, but M₄ was not found having such funetions. Furthermore M₃ receptor is the main subtype to play the role of regulator, and be changed much in tumor generation. To understand the expression rule of M₃ receptor in healthy, hyperplastic, cancerous prostate tissue is very important for making clear the role of M₃ receptor and its molecular biologic mechanism in genesis and development of prostatic tumor.The proliferation of tumor cells and the formation of new blood vessels were important steps in the process of the genesis and development of tumor. In the research of breast cancer LMM3 cell line, it was found that the main subtype was M3 and its expression quantity was 40 folds of LM3 cell. The cholinergic receptor agonist CARB could provoke the growth and neovascular genesis of LMM3 cell, but this function could be suppressed by the selective M3 receptor antagonist. It was also demonstrated that CARB could promote the expression of VEGF-A, which could be inhibited by selective M3 receptor antagonist pf-HHSiD. All these findings told us that M3 receptor played an important role in LMM3 cell growth and neovascular genesis. But the researches about M 3 receptor in prostatic hyperplasia and cancer was much fewer, and the fewer finished study was done based on not strict normal control.Vascular endothelial growth factor(VEGF) is an important factor in neogenesis of tumor vessels. VEGF and its receptor are highly expressed in most malignant tumors, through para/autoendocrine reaction, promoted endothelial cell division, proliferation and induced vascular neogenesis, directly affecting tumor cell and provoking it to grow. In prostate, VEGF could be synthesized by normal duct endothelial cell, adenoma cell and infiltrative lymphocyte.The stronger the metastatic characteristic of human prostatic cancer cell was, the higher VEGF expression would be, and neovascular genesis would be more effectively promoted by VEGF which was synthesized and secreted by malignant cell. The VEGF receptor expressed on both the vessel endothelium of prostatic tumor and the tumor cell showed that VEGF not only stimulated vascular endothelial proliferation by paraendocrine way but also regulated tumor cell proliferation by autoendocrine way.Endostatin was XVIII collagen C-terminal amino segment which was isolated from culture solution of vascular endothelioma. It could specifically inhibit endothelial cell proliferation, metastasis and tumorous angiogenesis, and could induce cell apoptosis, so that it made the primary and metastatic site of tumor stay in dormant state and stop growing.To study VEGF, endostatin and M₃ receptor's relation in normal and tumor prostate is very important for further understanding the mechanism of tumorigenesis,development, and selecting the target of treatment in late stage tumor. At present the research on this aspect about human prostatic tumor has not been reported.For the above reason my research firstly studied the mRNA expression of muscarinic receptor subtypes in each zone of normal prostate and discussed the expression difference of each subtype among different zones. Then we detected the expression of those three kinds of receptors, among the normal, hyperplastic and cancerous prostate, using Western blot, which were highly expressed in the prostatic tissue and had intimate relationship with the tumor genesis. We gained the protein expression difference of each subtype in different zones of prostate tissue and analyzed the role of different subtype in normal human prostate,and in the genesis.developmeiit of diseases. At last using RT-PCR, immunohistochemistry, Westsern blot, we comparatively studied the expression of VEGF and endostatin in prostatic tissue at genetic and protein levels, together with the expression of M₃ receptor in normal, hyperplastic and cancerous prostate, and discussed their roles in genesis and development of prostatic tumor.These researches were important for understanding M receptor, VEGF and endostatin's roles in prostate's function educing and disease generation, also it is more important for developing new drugs for prostatic diseases .Material and method:Materials:The fresh normal prostates were obtained from healthy adult donators who were died of accident. The samples of hyperplastic, cancerous prostates were taken from the hospitalized patients in The Second Hospital of SDU from 2000 to 2006, and from prostatectomy or core biopsy. All the tissues were put in nitrogen liquid and kept in -80℃refrigerator for further using. Seven samples of normal prostate were used in detecting M receptor subtype in each zone of prostate. The specimens used in detecting M₃ receptor, VEGF, endostatin at genetic and protein levels included 36 normal prostates, 36 hyperplastic ones, and 8 cancerous ones.The samples detected with immunohistochemistry were paraffin-embedded ones. The normal prostatic tissue was from fresh corpses of healthy adults. The hyperplastic, cancerous samples were from hospitalized patients of The Second Hospital of SDU from 2000 to 2006, taken with prostatectomy or care biopsy. The samples were soaked in 10% formalin and paraffin-embedded. There were 36 normal prostate, 36 hyperplastic ones and 36 cancerous ones. All the operations were taken on super clean bench. During taking fresh samples, the operating knife blade should be changed for different site. Every sample of normal prostate was taken out 3 pieces, respectively used in RT-PCR,western blot and made into paraffin-embedded block which would be used in immunohistochemistry.Methods:I .Detect muscarinic receptor subtypes'genetic expression in each zone of normal human prostate with real-time fluorescent quantitative RT-PCRProcess the experiment material with RNase. Levigate the tissue at low temperature. Extract the total mRNA. Synthesize cDNA. Carry out PCR amplification reaction. Real Time PCR reaction condition: 95℃10s, 95℃5s, 60℃45s, 40 cycles; Every receptor's threshold cycle(Ct) value of each site which we got was transformed into its initial relative concentration withâ–³Ct way. The transformation formula:â–³Ct=Ct_sample-Ct_β-actin. The mean relative quantity = 2^-â–³Ct . Apply SAS9.0 software in statistical treatment. The statistical method was variance analysis to pair-matched data. And q test was used to compare every two groups. The difference was considered as statistically significant when P<0.05.II .Using RT-PCR detect M₃ receptor, VEGF, endostatin's expression in normal,hyperplastic, cancerous prostate at genetic level:The experiment material was processed with RNase. Levigate the tissue at low tempreture. Extract the total RNA.The RNA was then reverse transcribed into cDNA. Input gel electrophoresis picture to the American Kodak gel analystic system.Use 1 D Image Analysis Software to analyse the expression intensity.Calculate the relative coefficient with the formula below.The relative coefficient=cytokine expression intensity/β-actin expression intensityIII. Detect VEGF, endostatin, CD34's expression in normal, hyperplastic, and cancerous prostate with immunohistochemical methodFirstly we cut 4 serial sections with thickness 4 micrometres from the paraffin block. Fetal tissue with PBS instead of first Ab serves as negative control. The 3 slices of every group were respectively used in VEGF , endostatin and CD34's immunohistochemical detection. The other one was HE stained. Every sample was inspected by the pathologist. In the result evaluation the positive stained should be brown. M₃, VEGF, endostatin's staining result assessment: to classify them according to the extent of positive cell coloration and positive cell percentage: i according to the coloration:no color score 0;buff 1;buffy 2;sable 3. ii according to the positive cell percentage: no positive cell score 0; positive cell<25% 1; positive cell 25%-50% 2; positive cell>50% 3;Then add the two score : 0-negative(-);1-2-weakly positive(+); 3-4-positive(++);5 -6-strongly positive(+++).The result assessment was carried out with double blind method. Every slice was inspected and counted by two pathologists respectively. We considered one single endothelium or one cluster of endothelium, which was CD34 positive stained as one microvessel. We counted the microvessel number of 3 different visual field where the vessel density was the highest in 200X view field. The mean value was treated as tumor's microvessel density.iv. Detect M₁, M₂, M₃, VEGF and endostatin's protein expression in normal, hyperplastic, cancerous prostate with western-blot methodFresh tissue were taken and weighted.Then the protein(we extracted membrane protein for M receptor, extracted the cytoplasmic protein for VEGF and endostatin) was extracted and denatured.Different samples were added in Bio-Rad vertical electrophoresis apparatus which had been perfused with weight percentage 10% separating gel and weight percentage 7.5% stacking gel, and ran for 90 min at stable voltage 120 volt. Then buffer transferred the protein in the gel onto PVDF membrane with transferring membrane, at stable voltage 100 volt for 60 min. The membrane was taken out and blocked by 7% skimmed milk for 60 min. Then it was incubated with first Ab overnight at 4℃, washed thoroughly and with the second Ab for 2 hours. Stained the membrane with DAB and scanned the picture. The picture was inputted into the American Kodak Image Analystc System. Using 1 D Image Analysis Software analyzed the expression density. The relative coefficient was calculated with the formula below:The Relative Coefficient=Cytokine expression density/β-actin expression densityResults:I .The fluorescent quantitation RT-PCR detection demonstrated that five muscarinic receptor subtypes were all expressed to different extent at genetic level in normal human prostate.There was difference among these receptors' expression in different zones. It was mainly M₂, M₃, and M₁ receptors expressed.In prefibromuscular zone, M₂ was obviously higher than other receptors(P<0.05),it is about three times of M₃'s expression; The difference among M₃, M₁, M₅, M₄'s expression was not statistically significant(P>0.05).In transitional zone, M₂'s expression was the highest (P<0.0001) in all the 5 receptors ,it is about 3 times of M₃'s.M₃'s was obviously higher than M4's(P<0.05).In central zone, M₂'s expression was the highest, but it was only statistically significant when it was compared with M4(P<0.05);the differences among M₂, M₁, M₃ and M₅ were not statistically significant(P>0.05).In peripheral zone there was no significant difference among those receptor subtypes (P>0.1).II .Western blot showed that there was protein expression difference among M₁, M2, M3 in normal, hyperplastic and cancerous prostatic tissue. In normal human prostate M₁ receptor expressed the highest(P<0.01). The differences of M3 receptor's expression in normal and pathological prostate tissue were statistically significance (P<0.05),while M₂ and M₁'s wasn't(P>0.05).The highest expression receptor in cancerous prostate tissue was M3 receptor(P<0.01).III. RT-PCR demonstrated that there was gene expression difference among M3 receptor, VEGF, endostatin in normal, hyperplastic and cancerous prostate tissue.M3 and VEGF's expression was higher in cancerous prostate than in normal prostate with significant difference (P<0.01).But endostatin's expression difference among the three different kinds of tissue wasn't statistically significant(P>0.05).It has positive correlation between M₃ and VEGF's expression (P<0.001) .IV .The immunohistochemistry revealed that there were protein expression differences of VEGF and endostatin in normal, hyperplastic and cancerous prostatic tissue. VEGF and endostatin's expression were higher in prostate cancer tissue than in BPH tissue, while their expression were lowest in normal prostate tissue with the significant differences (P<0.01). In prostate cancer the more the Gleason score was,the more higher VEGF and endostatin were expressed (P<0.05) .Furthermore the expression in the late clinical stage wre higher than in the early stage (P<0.05) .V .The Western blot indicated that VEGF and endostatin expressed highest in prostate cancer tissue ,moderate in BPH tissue, lowest in normal prostatic tissue with the significant differences (P<0.05 ) . It has positive correlation between VEGF and endostatin's expression (P<0.001) .Conclusions:I .There are genetic expressions of all the five muscarinic receptor subtypes in each zone of prostate, mainly are M₂, M₃, M₁.II .In normal human prostate, the genetic expression of M₂, M₃, M₁ is similar to their protein expression,M3 receptor's protein expression is higher in cancerous than in normal prostatic tissue, which means it concerned with tumorigenesis of prostate .III.M₃, VEGF and endostatin's expression in prostatic cancer is related with the tumor's clinical stage and pathological Gleason grading.IV. It has positive correlation between M₃, VEGF and endostatin's expression in human prostate tissue.M₂, M₃, VEGF and endostatin's expression is high in prostatic cancer tissue.V.M₃, VEGF, ES, and CD34 are related with genesis and development of prostatic tumor. They can be used as new targets of diagnosis and treatment for prostastic tumor.