Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer related death. It is lethal in nearly all patients, due to an almost complete chemoresistance. Most if not all drugs that pass preclinical tests successfully, fail miserably in the patient. This raises the question whether traditional 2D cell culture is the correct tool for drug screening. The objective of this study is to develop a simple, high-throughput 3D model of human PDAC cell lines, and to explore mechanisms underlying the transition from 2D to 3D that might be responsible for chemoresistance. Methods: Several established human PDAC and a KPC mouse cell lines were tested, whereby Panc-1 was studied in more detail. 3D spheroid formation was facilitated with methylcellulose. Spheroids were studied morphologically, electron microscopically and by qRT-PCR for selected matrix genes, related factors and miRNA. Metabolic studies were performed, and a panel of novel drugs was tested against gemcitabine. Results: Comparing 3D to 2D cell culture, matrix proteins were significantly increased as were lumican, SNED1, DARP32, and miR-146a. Cell metabolism in 3D was shifted towards glycolysis. All drugs tested were less effective in 3D, except for allicin, MT100 and AX, which demonstrated effect. Conclusions: We developed a high-throughput 3D cell culture drug screening system for pancreatic cancer, which displays a strongly increased chemoresistance. Features associated to the 3D cell model are increased expression of matrix proteins and miRNA as well as stromal markers such as PPP1R1B and SNED1. This is supporting the concept of cell adhesion mediated drug resistance.

OBJECTIVE: Although Smad3 has been considered as a downstream mediator of transforming growth factor-β (TGF-β) signaling in diabetes complications, the role of Smad7 in diabetes remains largely unclear. The current study tests the hypothesis that Smad7 may play a protective role and has therapeutic potential for diabetic kidney disease. RESEARCH DESIGN AND METHODS: Protective role of Smad7 in diabetic kidney disease was examined in streptozotocin-induced diabetic mice that have Smad7 gene knockout (KO) and in diabetic rats given Smad7 gene transfer using an ultrasound-microbubble-mediated technique. RESULTS: We found that mice deficient for Smad7 developed more severe diabetic kidney injury than wild-type mice as evidenced by a significant increase in microalbuminuria, renal fibrosis (collagen I, IV, and fibronectin), and renal inflammation (interleukin-1β [IL-1β], tumor necrosis factor-α [TNF-α], monocyte chemoattractant protein-1 [MCP-1], intracellular adhesion molecule-1 [ICAM-1], and macrophages). Further studies revealed that enhanced renal fibrosis and inflammation in Smad7 KO mice with diabetes were associated with increased activation of both TGF-β/Smad2/3 and nuclear factor-κB (NF-κB) signaling pathways. To develop a therapeutic potential for diabetic kidney disease, Smad7 gene was transferred into the kidney in diabetic rats by an ultrasound-microbubble-mediated technique. Although overexpression of renal Smad7 had no effect on levels of blood glucose, it significantly attenuated the development of microalbuminuria, TGF-β/Smad3-mediated renal fibrosis such as collagen I and IV and fibronectin accumulation and NF-κB/p65-driven renal inflammation including IL-1β, TNF-α, MCP-1, and ICAM-1 expression and macrophage infiltration in diabetic rats. CONCLUSIONS: Smad7 plays a protective role in diabetic renal injury. Overexpression of Smad7 may represent a novel therapy for the diabetic kidney complication.

We have shown previously that the heavy metal‐responsive transcriptional activator MTF‐1 regulates the basal and heavy metal‐induced expression of metallothioneins. To investigate the physiological function of MTF‐1, we generated null mutant mice by targeted gene disruption. Embryos lacking MTF‐1 die in utero at approximately day 14 of gestation. They show impaired development of hepatocytes and, at later stages, liver decay and generalized edema. MTF‐1−/− embryos fail to transcribe metallothionein I and II genes, and also show diminished transcripts of the gene which encodes the heavy‐chain subunit of the γ‐glutamylcysteine synthetase, a key enzyme for glutathione biosynthesis. Metallothionein and glutathione are involved in heavy metal homeostasis and detoxification processes, such as scavenging reactive oxygen intermediates. Accordingly, primary mouse embryo fibroblasts lacking MTF‐1 show increased susceptibility to the cytotoxic effects of cadmium or hydrogen peroxide. Thus, MTF‐1 may help to control metal homeostasis and probably cellular redox state, especially during liver development. We also note that the MTF‐1 null mutant phenotype bears some similarity to those of two other regulators of cellular stress response, namely c‐Jun and NF‐κB (p65/RelA).

The inhibitory Smad7, a direct target gene for transforming growth factor-β (TGF-β), mediates TGF-β1–induced apoptosis in several cell types. Herein, we report that apoptosis of human prostate cancer PC-3U cells induced by TGF-β1 or Smad7 overexpression is caused by a specific activation of the p38 mitogen-activated protein kinase pathway in a TGF-β–activated kinase 1 (TAK1)- and mitogen-activated protein kinase kinase 3 (MKK3)-dependent manner. Expression of dominant negative p38, dominant negative MKK3, or incubation with the p38 selective inhibitor [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], prevented TGF-β1–induced apoptosis. The expression of Smad7 was required for TGF-β–induced activation of MKK3 and p38 kinases, and endogenous Smad7 was found to interact with phosphorylated p38 in a ligand-dependent manner. Ectopic expression of wild-type TAK1 promoted TGF-β1–induced phosphorylation of p38 and apoptosis, whereas dominant negative TAK1 reduced TGF-β1–induced phosphorylation of p38 and apoptosis. Endogenous Smad7 was found to interact with TAK1, and TAK1, MKK3, and p38 were coimmunoprecipitated with Smad7 in transiently transfected COS1 cells. Moreover, ectopically expressed Smad7 enhanced the coimmunoprecipitation of HA-MKK3 and Flag-p38, supporting the notion that Smad7 may act as a scaffolding protein and facilitate TAK1- and MKK3-mediated activation of p38.

Background. The present study tested the hypothesis that disruption of Smad7 function may accelerate renal fibrosis and inflammation. Methods. This was investigated in a unilateral ureteral obstruction (UUO) model induced in wild-type (WT) and Smad7ΔE1 mice in which functional Smad7 is disrupted by deleting exon I in the Smad7 gene. Renal fibrosis and inflammation after UUO were examined by histology, real-time PCR, western blot analyses and immunohistochemistry. Results. Seven days after UUO, severe tubulointerstitial fibrosis developed in WT mice as evidenced by a marked increase in α-SMA, collagen I and III extracellular matrix. This was associated with a significant upregulation of renal TGF-β1 and CTGF and activation of Smad2/3. Interestingly, compared to WT UUO mice, Smad7ΔE1 mice with UUO exhibited a further increase in TGF-β/Smad2/3-dependent renal fibrosis. Moreover, compared to WT UUO mice, deletion of the Smad7 gene also sustained NF-κB activation and thus enhanced further renal inflammation such as macrophage infiltration and upregulation of TNF-α, MCP-1, OPN and ICAM-1. Conclusion. Smad7 is a critical negative regulator of TGF-β/Smad2/3 and NF-κB signalling and plays a negative regulating role in both renal fibrosis and inflammation after UUO. Results from this study further support the notion that Smad7 may be a therapeutic agent for kidney diseases.