| Purpose: To investigate the effect of dihydrotestosterone(DHT) on the gene transcription and expression of Smad3 and Smad4, two down stream signal proteins of TGF- β signal transmission, in androgen dependent prostate cancer cell line LNCaP, and whether this effect can be suppressed by the androgen receptor inhibitor Flutamide. And to indirectly determine whether there is a crosstalk between the androgen receptor(AR) signal passway and the transforming growth factor- β (TGF- β) signal passway. Methods: The androgen dependent prostate cancer cell line LNCaP was cultured in RPMI 1640 medium which contained 10% fetal bovine serum(FBS) for 48 hours. Then the cells were cultured for another 48 hours in serum free medium andtreated with different concentrations of DHT(D) and Flutamide(F) in five groups: (1) control group: D-,F-; (2)low DHT group: D 2nM, F-; (3)moderate DHT group: D 10nM, F-; (4)high DHT group: D 50nM, F-; (5)DHT with Flutamide group: D 10nM, F100nM. Cells were harvested after the medium was drawn out. Cells in different groups were counted with cytometer under microscope. The PSA concentrations were measured by radioimmunoassay. Quantitative reverse transcription PCR(RT-PCR) was used to detect the mRNA of PSA, Smad3 and Smad4. The expression of Smad3 and Smad4 protein were detected by Western-blot analysis. Each data in control group was used as a basic calibrator(giving the value=1), and the values used in final analysis of relative data in treated groups were normalized on the basic calibrator(value for analysis=detected data/relative data of control). Three duplicates were carried out in each case, and thedata in results was the mean of those in all the duplicates ( x ± SE) . The statistical analysis was performed using the software SPSS for Windows 11.0. Results: Data in control group, the low DHT group, the moderate DHT group, the high DHT group and the DHT with Flutamide group for the cell density were 1.00± 0.00, 1.32 ± 0.33, 4.53 ± 0.86, 3.12 + 0.66, and 1.31+0.23, respectively, for the PSA concentration were 1.00± 0.00, 1.51+0.12, 5.28 + 0.44, 2.92+0.27, and 1.12 + 0.23 respectively, for the PSA mRNA were 1.00 ± 0.00, 1.44 + 0.11, 4.81+0.37, 3.10 + 0.19, and 1.08 + 0.15,respectively, for the Smad3 mRNA were 1.00+0.00, 1.22 + 0.11, 2.02 + 0.15, 2.04 +0.13, and 1.32 + 0.17, respectively, for Smad4 mRNA were 1.00±0.00, 0.80 + 0.11, 0.72 + 0.07, 0.90 + 0.10, and 0.90 + 0.11, respectively, for Smad3 protein were 1.00+ 0.00, 1.38 + 0.12, 2.40 + 0.21, 2.10±0.14, and 1.58±0.14 respectively, for Smad4 protein were 1.00 + 0.00, 0.46 ±0.09, 0.42 ±0.10, 0.42 ±0.06, and 0.62 ±0.08 respectively. DHT at different concentrations in the medium significantly enhanced the proliferation of LNCaP cell when compared with the control (p<0.05). And Flutamidedecreased this effect of DHT. Different concentrations of DHT up-regulated the transcription and expression of PSA mRNA compared with the control (p<0.05). This up-regulation of PSA mRNA transcription reduced by DHT was depressed significantly by Flutamide (p<0.05). At higher concentration, DHT enhanced the transcription of Smad3 mRNA (p<0.05). Different concentration of DHT increased the expression of Smad3 protein (p<0.05). Flutamide inhibited the up-regulation of both Smad3 mRNA transcription and expression significantly (p<0.05). DHT at lOnM suppressed the transcription of Smad4 significantly (p<0.05). There was a considerable suppression of Smad4 expression at the presence of DHT in different concentration (p<0.05). And the degree of this suppression was more significant than those of DHT on Smad4 mRNA transcription. Flutamide inhibited the suppression of DHT on both Smad4 mRNA transcription and expression. Conclusions: DHT can enhance the proliferation of LNCaP cell, the transcription and expression of Smad3, while it decreases the transcription and expression of Smad4 in LNCaP cell line. There is a possible crosstalk between the AR signal passway and TGF- P signal passway in the level of Smads.Purpose: Prostate cancer is the most common type malignant tumor in adult men in the Western developed countries. With the improving life condition, the rising cognition and the improving detection methods on this disease, the incidence of prostate cancer in our country has been becoming higher in recent years. Being a key growth regulatory factor, transforming growth factor- P (TGF- P) regulates the proliferation, differentiation and apoptosis in many cells. It also plays a critical role in the genesis and development of cancers. The Smad superfamily is the downstream signaling proteins of TGF- P superfamily signaling transduction. They conduct the TGF- P signal from the cell membrane to the nucleus where they regulate gene transcription and expression. It is confirmed that the disorder of Smads expression is contributed to the genesis and development of some tumors, such as pancreatic cancer, colonic cancer, and endometrial cancer. This research aimed to investigate whether there is a difference in the expression of Smad2/3 and Smad4 between prostate cancer and normal prostate tissues, and whether there is a correlation between the grade and clinical stage of prostate cancer and the expression of Smad2/3 and Smad4. And to understand the relationship between the expression of Smad2/3 and Smad4. Methods: 60 specimens of prostate cancer were collected from patients who were diagnosed by pathology and underwent prostatectomy (18 underwent TURP) from 1996 to 2003. The average age ofpatients was 68 years (range 56-83). According to the Gleason score, 22 of the 60 were well-differentiated (scored 2~5), 16 were moderately differentiated (scored 6~8), and 22 were poorly differentiated (scored 9—10). Regarding the Whitmore-Jewettf clinical stage, 11 were at stage A, 24 were at stage B, 18 were at stage C, and 7 were at stage D. The normal prostate specimens were obtained from 18 new died cadavers with the mean age of 32(21 to 48) and pathologically identified having no prostatic disease. All of the specimens were fixed in buffered formalin (pH 7.4) and embedded with paraffin for the later cutting. SABC analyses: 4 u m sections were then cut from the paraffin block selected for each case and fixed on the slides and deparaffinized by routine techniques. The slides were treated with sodium citrate heat-induced epitope retrieval buffer for 15 minutes at 97°C. The non-specific antigens were covered with the goat serum. And then each slide was labeled with a 1:100 dilution of the specific antibodies to Smad2/3 or Smad4. The anti-Smad2/3 and anti-Smad4 antibodies were detected by adding biotinylated secondary antibodies, StreptAvidin-biotin complex, and 3,39-diaminobenzidine. The reaction was observed under a light microscope and would be stopped immediately with water. Sections were counterstained with hematoxylin. Two doctors independently evaluated the immunohistochemical labeling of the specimens. The labeling in each case was scored according to Wilentz's standards, as "Diffusely positive" labeling was defined as strong and uniform expression of Smad2/3 or Smad4 in the cytoplasm of cells, with focal expression of Smad2/3 or Smad4 in nuclei. "Focally positive" carcinomas contained two distinct populations of cells, those that labeled with the antibody to Smad2/3 or Smad4 and those that did not. Cases were regarded as "negative" only when no expression of Smad2/3 or Smad4 was seen in the cytoplasmic or nuclear compartments of cells. The Cross tabulations were analyzed with x or Fisher exact tests when appropriate. All of these tests were two-tailed. Therelationship between two factors was analyzed with nonparametric tests. Tests were performed using SPSS 11.0 for Windows. Results: The positive labeling of Smad2/3 and Smad4 was seen in the cytoplasmic and/or nuclear of the prostatic glandular cells. Some of the nuclear had been strongly labeled by the antibodies. Immunohistochemical labeling was rarely seen in the stroma cells. 31(51.7%) of the 60 prostate cancer tissues were positive with Smad2/3 immunohistochemical labeling (diffusely positive in 10, and focally positive in 21). 33(55.0%) of the 60 prostate cancer tissues were positive with Smad4 immunohistochemical labeling (diffusely positive in 9, and focally positive in 24). 18 cases were positive both with Smad2/3 and Smad4 immunohistochemical labeling. Of the 18 controls 13(72.2%) were positive with Smad2/3 immunohistochemical labeling (diffusely positive in 6, and focally positive in 7), while 15(83.3%) were positive with Smad4 immunohistochemical labeling (diffusely positive in 9, and focally positive in 6). 12 cases were positive both with Smad2/3 and Smad4 immunohistochemical labeling. The expression of Smad2/3 and Smad4 was decreased in prostate cancers compared with the normal controls. But only the difference of Smad4 expression between these two groups was statistically significant (p<0.05) . No correlation was seen between the expression of Smad2/3 and Smad4 in both prostate cancers and normal controls (p>0.05) . There was also no correlation between the expression of Smad2/3 and Smad4 and the grade and clinical stage of prostate cancer (p>0.05, respectively). Conclusions: The expression of Smad4 significantly decreased in prostate cancer compared with normal prostate tissue. The difference of Smad2/3 expression between these two groups was not significant. There was no correlation between the expression of Smad2/3 and Smad4 in both prostate cancers and normal controls. There was also no correlation between the expression of Smad2/3 and Smad4 and the grade and clinical stage of prostate cancer. The decreased expression of Smad4...
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