calpain and Urinary-Bladder-Neoplasms

To investigate the mechanism by which long non-coding RNA TUG1 affects bladder cancer cell migration and invasion.. The expressions of TUG1 and miR-29c-3p were examined by quantitative RT-PCR (qRT-PCR) in 10 bladder cancer tissues and 5 bladder cancer cell lines. Trans-well assay was used to detect the changes in migration and invasion abilities of bladder cancer T24 cells after TUG1 knockdown using RNA interference technique, and the alteration in the expression of CAPN7 was also detected. The expression of CAPN7 was examined in T24 cells overexpressing mir-29c-3p by Western blotting, and luciferase reporter assay was performed to confirm the targeting of miR-29c-3p to TUG1 and CAPN7. The effects of CAPN7 overexpression and sh-TUG1 on the migration and invasion of T24 cells were investigated.. The expression of TUG1 was up-regulated and mir-29c-3p was down-regulated significantly in bladder cancer tissue with a negative correlation between their expressions. TUG1 knockdown significantly inhibited the migration and invasion of T24 cells (. Mir-29c-3p targeting TUG1 affects the migration and invasion of bladder cancer cells by regulating the expression of CAPN7.

Topics: Calpain; Cell Line, Tumor; Cell Proliferation; Humans; MicroRNAs; RNA, Long Noncoding; Urinary Bladder Neoplasms

Calpain 3 (Capn3), also named p94, is a skeletal muscle tissue-specific protein known to be responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Recent experimental studies have hypothesized a pro-apoptotic role of Capn3 in some melanoma cell lines. So far the link between calpain3 and tumors comes from in vitro studies. The objective of this study was to describe Capn3 activation in naturally occurring urothelial tumors of the urinary bladder in cattle.. Here we describe, for the first time in veterinary and comparative oncology, the activation of Capn3 in twelve urothelial tumor cells of the urinary bladder of cattle. Capn3 protein was initially identified with nanoscale liquid chromatography coupled with tandem mass spectrometry (nano LC-MS/MS) in a co-immunoprecipitation experiment on E2F3, known to be a transcription factor playing a crucial role in bladder carcinogenesis in humans. Capn3 expression was then confirmed by reverse transcription polymerase chain reaction (RT-PCR). Finally, the Ca(2+)-dependent proteolytic activity of Capn3 was assayed following ion exchange chromatography. Morphologically, Capn3 expression was documented by immunohistochemical methods. In fact numerous tumor cells showed an intracytoplasmic immunoreactivity, which was more rarely evident also at nuclear level. In urothelial tumors, bovine papillomavirus type 2 (BPV-2) DNA was amplified by PCR and the expression of E5 protein, the major oncogenic protein of BVP-2, was detected by western blotting, immunohistochemistry, and immunofluorescence. E2F3 overexpression and pRb protein downregulation were shown by western blotting.. The role of capn3 protein in urothelial cancer of the urinary bladder remains to be elucidated: further studies would be required to determine the precise function of this protease in tumor development and progression. However, we suggest that activated Capn3 may be involved in molecular pathways leading to the overexpression of E2F3, which in turn could be responsible for urothelial tumor cell proliferation also in cattle, though other mechanisms are likely to exist. If further studies corroborate the important role of Capn3 in urothelial tumors of the urinary bladder, cattle with urinary tumors may prove useful as animal model for bladder carcinogenesis.

Topics: Animals; Bovine papillomavirus 1; Calcium; Calpain; Cattle; E2F3 Transcription Factor; Gene Expression Regulation, Neoplastic; Neoplasms, Glandular and Epithelial; Papillomavirus Infections; Reverse Transcriptase Polymerase Chain Reaction; Urinary Bladder Neoplasms; Urothelium

Induced migration of tumor cells is generally considered to be one critical step in cancer progression to the invasive and metastatic stage. The implicit caveat of studies that show this is that other, unknown, signaling pathways and biophysical events are actually the operative rate-limiting steps, and not motility per se. Thus, to examine the hypothesis that motility is a single, but overall rate-limiting function required for invasion, disparate motility processes need be blocked with concordant effects on tumor invasion. Recently, we and others have described two signaling pathways that are critical to growth factor-induced motility but not mitogenesis. The key molecular switches are phospholipase C-gamma (PLCgamma) and calpain for cytoskeletal reorganization and rear detachment, respectively. We examined this hypothesis in a highly invasive tumor, bladder carcinoma. Three different human tumor cell lines, 253J-B-V, UMUC and T-24, were tested for invasiveness in vitro by transmigration of a Matrigel barrier. Inhibiting PLCgamma with the pharmacologic agent U73122 or the molecular dominant-negative PLCz construct reduced both invasiveness and motility. The same was noted when calpain was blocked using calpain inhibitor I (ALLN). These results demonstrate that one interventional target for limiting invasion is not necessarily an individual motility pathway but rather cell migration per se.

Topics: Calpain; Cell Movement; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Estrenes; Humans; Isoenzymes; Neoplasm Invasiveness; Phospholipase C gamma; Pyrrolidinones; Signal Transduction; Tumor Cells, Cultured; Type C Phospholipases; Urinary Bladder Neoplasms

Interleukin 1 alpha (IL-1 alpha) is synthesized as a 33 kDa form and proteolytically processed into a 17 kDa form. Although IL-1 alpha has no signal peptide, it is released from cells. To investigate the relationship between the processing and release of IL-1 alpha, human bladder carcinoma cells (HTB9 5637) which express IL-1 alpha constitutively, were treated with calcium ionophore (A23187). A23187 induced the processing of 33 kDa IL-1 alpha and selectively released processed 17 kDa IL-1 alpha, without any change in the release of 33 kDa IL-1 alpha. When extracellular calcium was chelated by EGTA, or when intracellular calpain was inhibited by the cell-permeable cysteine-protease inhibitor, E64d, the processing of 33 kDa IL-1 alpha was significantly blocked, the release of 33 kDa IL-1 alpha being unchanged. These results indicate that the release of IL-1 alpha was accompanied by the processing of 33 kDa IL-1 alpha.

Topics: Calcimycin; Calcium; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Egtazic Acid; Gene Expression; Humans; Interleukin-1; L-Lactate Dehydrogenase; Leucine; Methionine; Molecular Weight; Protein Processing, Post-Translational; Sulfur Radioisotopes; Tumor Cells, Cultured; Urinary Bladder Neoplasms

We describe here the involvement of calcium-activated neutral protease (CANP or calpain, EC 3.4.22.17) in calcium-dependent proteolytic processing of the precursor of human interleukin 1 alpha (IL-1 alpha) into mature IL-1 alpha. Calcium ionophore ionomycin enhanced proteolytic processing of pre-IL-1 alpha and the release of mature IL-1 alpha either from lipopolysaccharide (LPS)-activated human adherent mononuclear cells or from a human bladder carcinoma cell line (HTB9 5637) that constitutively produces human IL-1 alpha and -beta. The proteolytic processing of pre-IL-1 alpha was completely inhibited by EGTA. Similar calcium-dependent proteolytic processing of pre-IL-1 alpha was also observed with lysates of either LPS-activated human adherent mononuclear cells or HTB9 5637 cells. Since the optimal pH for processing was between 7 and 8, and E-64 (a cysteine protease inhibitor) and leupeptin (a serine and cysteine protease inhibitor) both inhibited this processing by cell lysates, we hypothesized that a calcium-activated neutral protease, CANP, might be responsible for this processing. This hypothesis was supported by data showing that the specific CANP inhibitor peptide inhibited this proteolysis in cell lysates in a dose-dependent fashion (IC50 = 0.05 microM) and that treatment of pre-IL-1 alpha with purified CANP yielded the 17-kDa mature form of IL-1 alpha, which has an amino terminus identical with that reported for mature human IL-1 alpha. Taken together, these findings indicate that calcium-dependent proteolytic processing of pre-IL-1 alpha is selectively mediated by CANP.

Topics: Calcium; Calpain; Cell Line; Egtazic Acid; Humans; Interleukin-1; Ionomycin; Kinetics; Lipopolysaccharides; Methionine; Monocytes; Protein Processing, Post-Translational; Tumor Cells, Cultured; Urinary Bladder Neoplasms