| Background Pancreatic cancer is a kind of highly malignant neoplasm. Themortality of this tumor almost equals to its incidence. Pancreatic cancer is also geneticallymutated, involving the alterations of dozens of oncogens and tumor suppressor genes aswell as certain growth factors. TGF-β1 (Transforming Growth Factor-β1) is one of theimportant growth factors that play a pivotal role in the tumorigenesis of pancreatic cancer.According to the Smad4 status, the signaling pathway can be divided into two branches,Smad4-dependent and independent ways. The Smad4-independent way of TGF-β1 may beof more importance than its contradictive in the progression of pancreatic cancer. ObjectiveThe current research was aimed to study the biologic effects of the Smad4-independentpathway of TGF-β1 on the pancreatic cancer cell line and to elucidate the differentiallyexpressed genes in the signaling pathway as well as their roles in the progression ofpancreatic cancer. Methods 1. Gene transfection, cell morpholic observation,flowcytometry and wound healing assay were used to detect the effects of TGF-β1 on thepancreatic cancer cell line BxPC3 haboring homozygous loss of SMAD4; 2. By thetechnique of suppression subtractive hybridization (SSH), we constructed a substractedcDNA library of differentially expressed genes resulting from TGF-β1 overexpression onBxPC3; Amplification of the library was carried out with the E. coli strain JM109 andreverse northern blot was used to confirm the targeting genes; 3. Immunohistochemisty wasapplied to screen the distribution of TGF-β1, PKCαand P-gp between cancerous andnormal tissues of pancreas; Also correlation among these proteins was evaluated; MTTassay was used to observe the sensitivity of BxPC3 to different kinds of anti-cancer drugs(5-Fu, Gemzar, Oxaliplatin, Cisplatin, CPT-11 and Epirubicin) and influence ofstably-transfection with full-length cDNA of TGF-β1 as well as TGF-β1 in vitro stimulation (5ng/ml and 10ng/ml) on the cytotoxicity of drugs; P-gp and PKC-αproteinexpression was examined by Western blot; p38 and ERK1/2 as well as their activatedphosphorated forms were evaluated; We performed small interference RNA targetingⅡreceptor of TGF-β1 and specific inhibitor of PKC-α, Go6976 to block the signalingpathway of TGF-β1 and observed their effects on the drug-resistance of BxPC3. Results 1.Transfection of TGF-β1 changed the outlook of BxPC3 into spindle-like shape. Thegrowth rate of BxPC3 transfected with TGF-β1 slowed down since the fourth day,compared with control groups. Flow cytometry showed that S phage cells percent were(27.53%±0.0242), (26.32%±0.0136) and (17.01%±0.0265) in BxPC3, vector group andTGF-β1 group, respectively. Less cells entered into S phage after TGF-β1 function(p＜0.0001), but no difference between BxPC3 and vector group (p=0.4811). 2. TGF-β1related subtractive library with high subtractive efficiency was set up successfully. Theamplified library contained 300 positive clones. Reverse northern blot showed that 32clones were actually differentially expressed. After sequencing and blastn searching, wefound 10 genes up-regulated and 12 down-regulated, 13 of which habour known functionand 9 unknown. 3. P-gp and TGF-β1 were expressed in 88.1% and 80.9% of pancreaticcancer, respectively. The total positivity of PKC-αbetween cancer and normal pancreatictissue was similar, but it differed in the cellular distribution. Cancer cells mainly expressedmembranous PKC-a, while more normal ductal cells were cytoplasmic positivite. TGF-β1was positively correlated with membranous PKC-αor P-gp. Also the latter two proteinswere significantly related. Cisplatin had the most powerful effect on killing the tumor cells,Oxaliplatin, 5-Fu and CPT-11 had the moderate and Gemzar and Epirubicin had the leasteffect. Cells transfected with plasmid containing full-length cDNA of TGF-β1 was alsoless sensitive to cisplatin than the origin one. After TGF-β1 stimulation for 24 hours,BxPC3 showed more resistance to cisplatin and Western blot revealed upregulation of P-gpand PKC-αwas involved. Also, p38 or ERK signaling pathway were activated duingTGF-β1 functioning. Both RNAi targeting TβRⅡand PKC-αinhibitor, Go6976,reversed the drug resistance of BxPC3 in some extence. Conclusions 1. TheSmad4-independent pathway of TGF-β1 can not only induce the EMT phenotype inpancreatic cancer BxPC3 but inhibit its growth rate; 2. Differentially expressed genes revealed that this signaling pathway concerned multiple biologic aspects of pancreaticcancer, for instance, desmoplasia, immunoregulation, drug resistance and some basic cellevents; 3. The Smad4-independent pathway of TGF-β1 might be involved in themulti-drug-resistance of pancreatic cancer. The use of TbetaR-Ⅱor PKC-αinhibitor maybe of clinical and practical importance in treating pancreatic cancer.
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