| Transforming growth factor beta (TGFβ) is a protein that has special regulatory effects on the growth and differentiation of a variety of cells. Early studies demonstrated that the TGFβsignaling pathways were abnormal in pancreatic carcinoma, thereby favoring the tumor cells to escape the growth inhibition exerted by TGFβ. The present study was aimed to characterize the expression of TGFβ1 and its type-1 receptor (TGFβR1) in different pancreatic tissues and cell lines and its clinical significance. The molecular mechanisms concerning TGFβ1 signaling pathways in the oncogenesis and progression of pancreatic cancer were also discussed.Part OneExpression of TGFβ1 and TGFβR1 in Different Pancreatic TissuesPurpose: To study the expression of TGFβ1 and its type-1 receptor (TGFβR1) in different pancreatic tissues.Methods: Using immunohistochemistry, the expression of TGFβ1 and TGFβR1 was studied in 30 cases of pancreatic carcinoma and their adjacent tissues. Ten cases of chronic pancreatitis and 10 normal pancrease as controls were also studied. The relevance between TGFβ1 and TGFβR1 levels and the different characteristics of pancreatic carcinomas were analyzed by using the Stata7.0 software. Parameters analyzed included age (under sixty versus over sixty), gender (male versus female), tumor size (less than 3 cm versus more than 3 cm), histological types (good differentiation, median differentiation, and poor differentiation), and TNM stages , respectively.Results: The positive expression rates of TGFβ1 in normal pancreas, chronic pancreatitis and pancreatic carcinomas were 30%, 50% and 83.3%, respectively (P＜0.05). The positive expression rates of TGFβR1 in normal pancreas, chronic pancreatitis and pancreatic carcinomas were 90%, 80% and 40%, respectively (P＜0.05). The levels of TGFβ1 were positively correlated with TNM stages, but were independent of age, gender, tumor size, and histological types. The levels of TGFβR1 were independent of age, gender, tumor size, histological types and TNM stages though increasing trend of TGFβ1 was observed in group of histological typesPart TwoExpression of TGFβ1 and TGFβR1 in AsPC-1 and BxPC-3 Cell linesPurpose: To further study the expression of TGFβ1 and TGFβR1 in AsPC-1 and BxPC-3 pancreatic carcinoma cell lines.Methods: The levles of TGFβ1 and TGFβR1 mRNAs and proteins were studied in BxPC-3 (primary pancreatic cancer) and AsPC-1 (pancreatic cancer with metastasis potentials) pancreatic carcinoma cell lines using quantitative RT-PCR and Western blot analysis.Results: Compared with primary pancreatic cancer cell line BxPC-3, the AsPC-1cells had much higher levels of TGFβ1 and TGFβR1 mRNAs and their proteins, which might be related to the increased metastasis potential of this cell line.Part ThreeEffects of TGFβ1 on the growth of BxPC-3 Cells and the MechanismsPurpose: To study the effects of TGFβ1 on growth of BxPC-3 pancreatic carcinoma cells, and also to develop a hypothesis concerning its mechanisms of action.Methods: To evaluate the effects of TGFβ1 on the growth of BxPC-3 cells, BxPC-3 cells were cultured with 2.0ng/ml TGFβ1 from 1 to 7 days with normal cultured BxPC-3 cells as contral. In addition, BxPC-3 cells were cultured in the presence of 1.0, 2.0, 4.0, 6.0, 8.0ng/ml TGFβ1 for 4 days with normal cultured BxPC-3 cells as contral. The rate of cell growth was determined by MTT assay. Cell cycle distribution was determined by FCM analysis after PI staining. Cyclin D1 expression was measured by Western blot analysis.Results: Compared with controls, TGFβ1 revealed a growth-inhibitory effect on BxPC-3 cells which showed both time-dependent and dose-dependent pattern. The maximal inhibition rate cultured with 2.0ng/ml TGFβ1 was 12.5%, which was observed on the 4th day of examination. FCM analysis showed G1/S arrest of the cell cycle in TGFβ1-treated cells. In addition, the cyclin D1 expression was also reduced significantly.Part FourAnalysis of Cell Cycle-Associated Genes Regulated by TGFβ1 in BxPC-3 cellPurpose: To identify the cell cycle-associated genes that was expressed following treatment of BxPC-3 cells with TGFβ1.Methods: Changes in 24 cell cycle-associated genes were determined in BxPC-3 cells treated with 2.0ng/ml TGFβ1, by using a specially designed gene chip.Results: Three genes were found unchanged, 3 genes (namely, p27KIP1, DMTF1 and TIEG) were up-regulated, and 17 genes were down-regulated. One of the genes was deleted for characterization due to an experimental error. It was noted that some of these genes were associated with the G1/S cell cycle transition (e.g., GSPT1, ASK, CDK4, SKP2, cyclin C), some of them were transcription factors (e.g., E2F3 and E2F5), and TRAD is a serine/threonine kinase with Dbl- and pleckstrin homology domains. ConclusionsBased on the studies described above, the following conclusions could be drawn:1. The decreasing trend of TGFβ1 levels in pancreatic cancers and chronic pancreatitis, together with its positive correlation with the TNM grades, suggested that TGFβ1 may be a clinically useful parameter for diagnosis, scoring, and prognostic evaluation of different types of pancreatic cancer patients.2. TGFβR1 down-regulation appears to be a quite common phenomenon in pancreatic cancers, and this may be one of the mechanisms that favor the escape of tumor cells from TGFβR1's growth-inhibitory effect.3. Pancreatic cancer cells with metastasis potentials have much higher levels of TGFβ1 and TGFβR1 than primary pancreatic cancer cells. This finding suggested that the expression of TGFβ1 and TGFβR1 had closely related with malignancy of pancreatic cancers.4. Although smad4 is depleted in BxPC-3 cells, exogenous TGFβ1 could still produce a growth inhibition in BxPC-3 pancreatic carcinoma cells, possibly through G1/S cell cycle arrest and cyclin D1 down-regulation.5. Tissue microarray analysis revealed multiple alterations of the cell cycle-associated genes that are regulated by TGFβ1. This finding suggested the existence of cross-talking between the Smad-dependent and Smad-independent pathways of TGFβ1, which may mediate the growth-inhibitory effect of TGFβ1 in pancreatic carcinoma.
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