Specific Targeting of Antiapoptotic Bcl-2 Proteins as a Radiosensitizing Approach in Solid Tumors.

Avoidance of therapy-induced apoptosis is a hallmark of acquired resistance towards radiotherapy. Thus, breaking resistance still challenges modern cancer therapy. The Bcl-2 protein family is known for its regulatory role in apoptosis signaling, making Bcl-2, Mcl-1 and Bcl-xL promising targets. This study evaluates the effects of highly specific inhibitors for Bcl-xL (WEHI-539), Bcl-2 (ABT-199) and Mcl-1 (S63845) as radiosensitizers. Covering a broad spectrum of solid tumors, Non-Small-Cell Lung Cancer (NSCLC), Head and Neck Squamous Cell Carcinoma (HNSCC) and synovial sarcoma cell lines were exposed to fractionated radiation as standard therapy with or without Bcl-2 protein inhibition. Protein expression was detected by Western blot and cell death was assessed by flow cytometry measuring apoptosis. In contrast to NSCLC, a high level of Bcl-xL and its upregulation during radiotherapy indicated radioresistance in HNSCC and synovial sarcoma. Radioresistant cell lines across all entities benefited synergistically from combined therapy with Bcl-xL inhibition and fractionated radiation. In NSCLC cell lines, Mcl-1 inhibition significantly augmented radiotherapy independent of the expression level. Our data suggest that among antiapoptotic Bcl-2 proteins, targeting Bcl-xL may break resistance to radiation in HNSCC, synovial sarcoma and NSCLC in vitro. In NSCLC, Mcl-1 might be a promising target that needs further investigation.

Knockdown of Atg7 Induces Nuclear-LC3 Dependent Apoptosis and Augments Chemotherapy in Colorectal Cancer Cells.

Autophagy is a catabolic process that enables cells to degrade obsolete content and refuel energy depots. In colorectal cancer (CRC) autophagy has been shown to promote tumorigenesis through energy delivery in the condition of uncontrolled proliferation. With this study, we aimed at evaluating whether autophagy sustains CRC cell viability and if it impacts therapy resistance. Initially, a colorectal cancer tissue micro array, containing mucosa (n = 10), adenoma (n = 18) and adenocarcinoma (n = 49) spots, was stained for expression of essential autophagy proteins LC3b, Atg7, p62 and Beclin-1. Subsequently, central autophagy proteins were downregulated in CRC cells using siRNA technology. Viability assays, flow cytometry and immunoblotting were performed and three-dimensional cell culture was utilized to study autophagy in a tissue mimicking environment. In our study we found an upregulation of Atg7 in CRC. Furthermore, we identified Atg7 as crucial factor within the autophagy network for CRC cell viability. Its disruption induced cell death via triggering apoptosis and in combination with conventional chemotherapy it exerted synergistic effects in inducing CRC cell death. Cell death was strictly dependent on nuclear LC3b, since simultaneous knockdown of Atg7 and LC3b completely restored viability. This study unravels a novel cell death preventing function of Atg7 in interaction with LC3b, thereby unmasking a promising therapeutic target in CRC.

Nuclear Translocation of RELB Is Increased in Diseased Human Liver and Promotes Ductular Reaction and Biliary Fibrosis in Mice.

BACKGROUND & AIMS: Cholangiocyte proliferation and ductular reaction contribute to the onset and progression of liver diseases. Little is known about the role of the transcription factor nuclear factor-κB (NF-κB) in this process. We investigated the activities of the RELB proto-oncogene NF-κB subunit in human cholangiocytes and in mouse models of liver disease characterized by a ductular reaction. METHODS: We obtained liver tissue samples from patients with primary sclerosing cholangitis, primary biliary cholangitis, hepatitis B or C virus infection, autoimmune hepatitis, alcoholic liver disease, or without these diseases (controls) from a tissue bank in Germany. Tissues were analyzed by immunohistochemistry for levels of RELB and lymphotoxin ß (LTB). We studied mice with liver parenchymal cell (LPC)-specific disruption of the cylindromatosis (CYLD) lysine 63 deubiquitinase gene (Cyld), with or without disruption of Relb (CyldΔLPC mice and Cyld/RelbΔLPC mice) and compared them with C57BL/6 mice (controls). Mice were fed 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or standard chow diets to induce biliary injury or were given injections of CCl4 to induce non-cholestatic liver fibrosis. Liver tissues were analyzed by histology, immunohistochemistry, immunoblots, in situ hybridization, and quantitative real-time polymerase chain reaction. Cholangiocytes were isolated from normal human liver, incubated with LTB receptor agonist, and transfected with small interfering RNAs to knock down RELB. RESULTS: In liver tissues from patients with primary sclerosing cholangitis, primary biliary cholangitis, chronic infection with hepatitis B or C virus, autoimmune hepatitis, or alcoholic liver disease, we detected increased nuclear translocation of RELB and increased levels of LTB in cholangiocytes that formed reactive bile ducts compared with control liver tissues. Human cholangiocytes, but not those with RELB knockdown, proliferated with exposure to LTB. The phenotype of CyldΔLPC mice, which included ductular reaction, oval cell activation, and biliary fibrosis, was completely lost from Cyld/RelbΔLPC mice. Compared with livers from control mice, livers from CyldΔLPC mice (but not Cyld/RelbΔLPC mice) had increased levels of mRNAs encoding cytokines (LTB; CD40; and tumor necrosis factor superfamily [TNFSF] members TNFSF11 [RANKL], TNFSF13B [BAFF], and TNFSF14 [LIGHT]) produced by reactive cholangiocytes. However, these strains of mice developed similar levels of liver fibrosis in response to CCl4 exposure. CyldΔLPC mice and Cyld/RelbΔLPC mice had improved liver function on the DDC diet compared with control mice fed the DDC diet. CONCLUSION: Reactive bile ducts in patients with chronic liver diseases have increased levels of LTB and nuclear translocation of RELB. RELB is required for the ductular reaction and development of biliary fibrosis in CyldΔLPC mice. Deletion of RELB and CYLD from LPCs protects mice from DDC-induced cholestatic liver fibrosis.

A systematic review and meta-analysis of the risk for development of varicose veins in women with a history of pregnancy.

OBJECTIVE: The association between pregnancy and the development of varicose veins is uncertain. We aimed to determine whether a history of pregnancy is associated with the development of varicose veins. METHODS: We performed a systematic literature search using the databases of PubMed, Embase, Robert Koch-Institut, and Cochrane Central and the references of included papers. Eligible studies were all epidemiologic observational studies in which the outcome "varicose veins" and pregnancy history were assessed. The quality of each study was evaluated on the basis of the Dutch Cochrane review checklist and by the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) statement. For our meta-analysis, a random effects model was applied to pool odds ratios and 95% confidence intervals across studies. RESULTS: We found nine eligible studies enrolling 17,109 women. Pregnancy was associated with a significant risk increase in developing varicose veins. The results of our meta-analysis suggest that the odds for women with a history of pregnancy in developing varicose veins significantly increases by 82% (odds ratio, 1.82; 95% CI, 1.43-2.33) compared with women with no history of pregnancy. As expected for epidemiologic observational studies, the heterogeneity was considerably high (I(2) = 81%). CONCLUSIONS: Our meta-analysis strongly supports the hypothesis that there is a significant and strong association between a history of pregnancy and varicose veins. However, qualitative and quantitative differences among studies were evident and were also reflected in a considerably high heterogeneity.