ABSTRACT
Radiation therapy is an essential modality in the treatment of colorectal cancers. Radiation exerts an antiangiogenic effect on tumors, inhibiting endothelial proliferation and survival in the tumor microvasculature. However, damage from low levels of irradiation can induce a paradoxical effect, stimulating survival in endothelial cells. We used human intestinal microvascular endothelial cells (HIMEC) to define effects of radiation on these gut-specific endothelial cells. Low-level irradiation (1-5 Gy) activates NF-kappaB and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is involved in cell cycle reentry and cell survival in HIMEC. A downstream target of PI3K/Akt is mammalian target of rapamycin (mTOR), which contributes to endothelial proliferation and angiogenesis. The aim of this study was to investigate the signaling molecules involved in the radiosensitizing effects of curcumin on HIMEC subjected to low levels of irradiation. We have demonstrated that exposure of HIMEC to low levels of irradiation induced Akt and mTOR phosphorylation, which was attenuated by curcumin, rapamycin, LY294002, and mTOR small interference RNA (siRNA). Activation of NF-kappaB by low levels of irradiation was inhibited by curcumin, SN-50, and mTOR siRNA. Curcumin also induced apoptosis by induction of caspase-3 cleavage in irradiated HIMEC. In conclusion, curcumin significantly inhibited NF-kappaB and attenuated the effect of irradiation-induced prosurvival signaling through the PI3K/Akt/mTOR and NF-kappaB pathways in these gut-specific endothelial cells. Curcumin may be a potential radiosensitizing agent for enhanced antiangiogenic effect in colorectal cancer radiation therapy.
Subject(s)
Curcumin/pharmacology , Endothelial Cells/drug effects , Intracellular Signaling Peptides and Proteins/metabolism , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Radiation-Sensitizing Agents/pharmacology , Abnormalities, Radiation-Induced/drug therapy , Animals , Caspase 3/genetics , Caspase 3/metabolism , Cell Death/drug effects , Cell Death/radiation effects , Cell Survival/drug effects , Cell Survival/radiation effects , Dose-Response Relationship, Drug , Endothelial Cells/radiation effects , Forkhead Box Protein O1 , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Regulation , Gene Silencing , Humans , Intestines/blood supply , Intestines/drug effects , Intestines/radiation effects , Intracellular Signaling Peptides and Proteins/genetics , Male , Microvessels/cytology , NF-kappa B/genetics , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , RNA, Small Interfering , Rats , Rats, Sprague-Dawley , TOR Serine-Threonine KinasesABSTRACT
Small membrane-bound extracellular organelles known as articular cartilage matrix vesicles (ACVs) participate in pathologic mineralization in osteoarthritic articular cartilage. ACVs are also present in normal cartilage, although they have no known functions other than mineralization. Recently, RNA was identified in extracellular vesicles derived from mast cells, suggesting that such vesicles might carry coding information from cell to cell. We found that ACVs from normal porcine and human articular cartilage and primary chondrocyte conditioned media contained 1 microg RNA/80 microg ACV protein. No DNA could be detected. RT-PCR of ACV RNA demonstrated the presence of full length mRNAs for factor XIIIA, type II transglutaminase, collagen II, aggrecan, ANKH and GAPDH. RNA in intact ACVs was resistant to RNase, despite the fact that ACV preparations contained measurable levels of active RNases. Significantly, radiolabeled RNA in ACVs could be transferred to unlabeled chondrocytes by co-incubation and produced changes in levels of chondrocyte enzymes and proteins. The demonstration that ACVs contain mRNAs suggests that they may function to shuttle genetic information between articular cells and indicate novel functions for these structures in articular cartilage.
Subject(s)
Cartilage, Articular/metabolism , Chondrocytes/metabolism , RNA Transport , RNA, Messenger/metabolism , Aggrecans/genetics , Animals , Factor XIIIa/genetics , GTP-Binding Proteins/genetics , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/genetics , Humans , Phosphate Transport Proteins/genetics , Protein Glutamine gamma Glutamyltransferase 2 , RNA Stability , RNA, Messenger/chemistry , RNA, Messenger/genetics , Ribonucleases/chemistry , Ribonucleases/metabolism , Swine , Transglutaminases/genetics , Transport Vesicles/metabolismABSTRACT
OBJECTIVES: Tendon abnormalities, such as increased stiffness, thickness, and excess calcification, occur commonly in patients with diabetes mellitus and cause considerable disability. These changes are frequently attributed to increased cross-linking of extracellular matrix components by advanced glycation end-products (AGEs). However, cellular effects of AGEs, such as increased activity of the cross-linking transglutaminase (Tgase) enzymes, could also contribute to altered tissue biomechanics and calcification in diabetic tendons. We determined the effect of AGE-modified protein on tenocyte Tgase activity. RESEARCH DESIGN AND METHODS: Primary porcine tenocytes were exposed to N- carboxymethyl-lysine (CML)-modified type I collagen in high or normal glucose media. Protein and mRNA levels of the Tgase enzymes and Tgase activity levels were measured, as were markers of apoptosis. We also determined the effect of antioxidants on CML-collagen mediated Tgase activity. RESULTS: Carboxymethyl-lysine-collagen increased Tgase activity in tenocytes 2.3- to 5.6-fold over unmodified collagen controls in both normal and high glucose media, without altering enzyme protein levels. Anti-oxidant treatment reduced the effect of CML-collagen on Tgase activity. Deoxyribonucleic acid laddering and annexin V protein levels were not altered by CML-collagen exposure. CONCLUSIONS: Carboxymethyl-lysine-collagen increased Tgase activity in tenocytes, likely posttranslationally. Increased levels of Tgase-mediated cross-links may contribute to the excess calcification and biomechanical pathology seen in diabetic tendons.
Subject(s)
Glycation End Products, Advanced/metabolism , Tendons/enzymology , Transglutaminases/metabolism , Animals , Antioxidants/pharmacology , Apoptosis/drug effects , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Gene Expression Regulation, Enzymologic/drug effects , Lysine/analogs & derivatives , Lysine/pharmacology , RNA, Messenger/metabolism , Stifle/metabolism , Swine , Tendons/drug effects , Tetrazolium Salts/metabolism , Thiazoles/metabolism , Transglutaminases/geneticsABSTRACT
OBJECTIVE: Calcium pyrophosphate dihydrate (CPPD) crystals are commonly found in osteoarthritic joints and correlate with a poor prognosis. Intraarticular corticosteroids, such as dexamethasone (Dxm), are commonly used therapies for osteoarthritis with or without CPPD deposition. Dxm has variable effects in mineralization models. We investigated the effects of Dxm on CPPD crystal formation in a well established tissue culture model. METHODS: Porcine articular chondrocytes were incubated with ATP to generate CPPD crystals. Chondrocytes incubated with or without ATP were exposed to 1-100 nM Dxm in the presence of 45Ca. Mineralization was measured by 45Ca uptake in the cell layer. We also investigated the effect of Dxm on mineralization-regulating enzymes such as alkaline phosphatase, nucleoside triphosphate pyrophosphohydrolase (NTPPPH), and transglutaminase. RESULTS: Dxm significantly increased ATP-induced mineralization by articular chondrocytes. While alkaline phosphatase and NTPPPH activities were unchanged by Dxm, transglutaminase activity increased in a dose-responsive manner. Levels of Factor XIIIA mRNA and protein were increased by Dxm, while type II Tgase protein was unchanged. Transglutaminase inhibitors suppressed Dxminduced increases in CPPD crystal formation. CONCLUSION: These findings suggest a potential for Dxm to contribute to pathologic mineralization in cartilage and reinforce a central role for the transglutaminase enzymes in CPPD crystal formation.
