Effects of triggers of senescence and senolysis in murine pancreatic cancer cells.

BACKGROUND: The combination of senescence triggers with senolytic drugs is considered a promising new approach to cancer therapy. Here, we studied the efficacy of the genotoxic agent etoposide (Eto) and irradiation in inducing senescence of Panc02 pancreatic cancer cells, and the capability of the Bcl-2 inhibitor navitoclax (ABT-263; Nav) to trigger senolysis. METHODS: Panc02 cells were treated with Eto or irradiated with 5-20 Gy before exposure to Nav. Cell survival, proliferation, and senescence were assessed by trypan blue staining, quantification of DNA synthesis, and staining of senescence-associated ß-galactosidase (SA-ß-Gal)-positive cells, respectively. Levels of mRNA were determined by real-time polymerase chain reaction, and protein expression was analyzed by immunoblotting. Panc02 cells were also grown as pancreatic tumors in mice, which were subsequently treated with Eto and Nav. RESULTS: Eto and irradiation had an antiproliferative effect on Panc02 cells that was significantly or tendentially enhanced by Nav. In vivo, Eto and Nav together, but not Eto alone, significantly reduced the proportion of proliferating cells. The expression of the senescence marker γH2AX and tumor infiltration with T-cells were not affected by the treatment. In vitro, almost all Eto-exposed cells and a significant proportion of cells irradiated with 20 Gy were SA-ß-Gal-positive. Application of Nav reduced the percentage of SA-ß-Gal-positive cells after irradiation but not after pretreatment with Eto. In response to triggers of senescence, cultured Panc02 cells showed increased protein levels of γH2AX and the autophagy marker LC3B-II, and higher mRNA levels of Cdkn1a, Mdm2, and PAI-1, while the effects of Nav were variable. CONCLUSIONS: In vitro and in vivo, the combination of senescence triggers with Nav inhibited tumor cell growth more effectively than the triggers alone. Our data also provide some evidence for senolytic effects of Nav in vitro.

Uncoupling protein 2 deficiency of non-cancerous tissues inhibits the progression of pancreatic cancer in mice.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a disease of the elderly mostly because its development from preneoplastic lesions depends on the accumulation of gene mutations and epigenetic alterations over time. How aging of non-cancerous tissues of the host affects tumor progression, however, remains largely unknown. METHODS: We took advantage of a model of accelerated aging, uncoupling protein 2-deficient (Ucp2 knockout, Ucp2 KO) mice, to investigate the growth of orthotopically transplanted Ucp2 wild-type (WT) PDAC cells (cell lines Panc02 and 6606PDA) in vivo and to study strain-dependent differences of the PDAC microenvironment. RESULTS: Measurements of tumor weights and quantification of proliferating cells indicated a significant growth advantage of Panc02 and 6606PDA cells in WT mice compared to Ucp2 KO mice. In tumors in the knockout strain, higher levels of interferon-γ mRNA despite similar numbers of tumor-infiltrating T cells were observed. 6606PDA cells triggered a stronger stromal reaction in Ucp2 KO mice than in WT animals. Accordingly, pancreatic stellate cells from Ucp2 KO mice proliferated at a higher rate than cells of the WT strain when they were incubated with conditioned media from PDAC cells. CONCLUSIONS: Ucp2 modulates PDAC microenvironment in a way that favors tumor progression and implicates an altered stromal response as one of the underlying mechanisms.

YAP activates pancreatic stellate cells and enhances pancreatic fibrosis.

BACKGROUND: Pancreatic stellate cells (PSCs) foster the progression of pancreatic adenocarcinoma and chronic pancreatitis (CP) by producing a dense fibrotic stroma. However, the incomplete knowledge of PSCs biology hampers the exploration of antifibrotic therapies. Here, we explored the role of the Hippo pathway in the context of PSCs activation and experimental CP. METHODS: CP model was created in rats with the tail vein injection of dibutyltin dichloride (DBTC). The expression of Yes-associated protein (YAP) in CP tissue was assessed. Primary and immortalized rats PSCs were treated with the YAP-inhibitor verteporfin. Furthermore, YAP siRNA was employed. Subsequently, DNA synthesis, cell survival, levels of α-smooth muscle actin (α-SMA) protein, presence of lipid droplets and PSCs gene expression were evaluated. Upstream regulators of YAP signaling were studied by reporter gene assays. RESULTS: In DBTC-induced CP, pronounced expression of YAP in areas of tubular structures and periductal fibrosis was observed. Verteporfin diminished DNA replication in PSCs in a dose-dependent fashion. Knockdown of YAP reduced cell proliferation. Primary cultures of PSCs were characterized by a decrease of lipid droplets and increased synthesis of α-SMA protein. Both processes were not affected by verteporfin. At the non-cytotoxic concentration of 100 nmol/L, verteporfin significantly reduced mRNA levels of transforming growth factor-ß1 (Tgf-ß1) and Ccn family member 1 (Ccn1). YAP signaling was activated by TGF-ß1, but repressed by interferon-γ. CONCLUSIONS: Activated YAP enhanced PSCs proliferation. The antifibrotic potential of Hippo pathway inhibitors warrants further investigation.

Cellular detection of the chemokine receptor CXCR4 in bovine mammary glands and its distribution and regulation on bovine leukocytes.

Mastitis has a high incidence in dairy cows. Experimental infection with Escherichia coli increased the number of leukocytes in milk and the gene expression of the chemokine receptor CXCR4 in mammary gland tissues. A link between CXCR4 expression and lipopolysaccharide sensing was demonstrated in other species using in vitro models. The receptor that binds the chemokine stomal cell-derived factor 1 might be associated with the inflammatory response in bovine mammary glands. However, studies in cows are rare, and data on the localization of CXCR4 in bovine mammary glands and its distribution in bovine leukocytes are lacking. Fatty acids (FA) affect the inflammatory response. In human peripheral blood monocytes, exposure to conjugated linoleic acids (CLA) decreases the expression of CXCR4, leading to a decreased inflammatory response in these cells. In this study, we analyzed the expression of CXCR4 in the mammary glands of dairy cows by immunohistochemistry (n = 5) and laser capture microdissection followed by qualitative PCR (n = 3). We characterized the surface expression of CXCR4 on bovine leukocytes, including monocyte subpopulations, first by flow cytometry (n = 5) and then confirmed these results by Western blotting (n = 3). Rumen fistulated dairy cows (n = 4; 126 ± 4 d in milk) were fitted with abomasal infusion tubes, arranged in a 4 × 4 Latin square design, and supplemented for 6 wk twice daily with rising doses of FA followed by a 3-wk washout period. Then, CXCR4 expression on leukocytes was analyzed. The cows received a corn-based diet and were supplemented with coconut oil delivering medium-chain FA (38 g/d), linseed-safflower oil mix delivering n-3 FA (EFA, 39 g of linseed oil and 2 g of safflower oil per day), Lutalin (cis-9,trans-11 and trans-10,cis-12 CLA, 5 g/d; BASF), and EFA + CLA. In the bovine mammary gland, the epithelial cells of the lactiferous duct, but not alveolar epithelial cells, showed clear CXCR4 protein and mRNA signals. Among the leukocyte subsets, monocytes displayed the highest percentage of CXCR4-positive cells (87%), whereas circulating neutrophils showed almost no CXCR4 surface expression (3%) but stored the receptor intracellularly. The percentage of CXCR4-positive leukocytes was not affected by the different FA supplements, but FA supplementation reduced the receptor abundance per cell (40% on average). In conclusion, CXCR4 was clearly detected in the lactiferous duct cells of the mammary gland but not in the alveolar epithelial cells. Compared with other leukocytes, bovine monocytes showed the highest signal intensity of CXCR4 on their surface, whereas granulocytes stored CXCR4 intracellularly. Supplementation with all the FA reduced the surface expression of CXCR4 per leukocyte and could therefore potentially affect the inflammatory status associated with the surface expression of CXCR4. The importance of our observations should be verified in cows with mastitis in the future.

Dietary Fatty Acids Affect Red Blood Cell Membrane Composition and Red Blood Cell ATP Release in Dairy Cows.

Diets of dairy cows are often based on maize silage (MS), delivering lower amounts of n-3 fatty acids (FA) compared to grass silage-based diets. The fatty acid composition of the cell membrane can affect the cell function. We evaluated the effects of an MS-based diet on bovine red blood cell (RBC) membrane FA composition and dietary effects on controlled ATP release of RBC. In trial 1, German Holstein cows were fed an MS-based total mixed ration for 24 weeks. The FA composition of RBC membranes from repeatedly taken blood samples was analysed in addition to the abundance of the RBC membrane protein flotillin-1, which is involved in, for example, cell signalling. In trial 2, four rumen fistulated MS-fed cows were abomasally infused in a 4 × 4 Latin square model with three successively increasing lipid dosages (coconut oil, linseed-safflower oil mix (EFA; rich in n-3 FA), Lutalin®, providing conjugated linoleic acids (CLA) or the combination of the supplements, EFA + CLA) for six weeks, followed by a three-week washout period. In trial 2, we analysed RBC ATP release, flotillin-1, and the membrane protein abundance of pannexin-1, which is involved in ATP release as the last part of a signalling cascade. In trial 1, the total amount of n-3 FA in RBC membranes decreased and the flotillin-1 abundance increased over time. In trial 2, the RBC n-3 FA amount was higher after the six-week infusion period of EFA or EFA + CLA. Furthermore, depending on the dosage of FA, the ATP release from RBC increased. The abundance of flotillin-1 and pannexin-1 was not affected in trial 2. It is concluded that changes of the membrane FA composition influence the RBC function, leading to altered ATP release from intact bovine RBC.