Enhancement of chemosensitivity toward peplomycin by calpastatin-stabilized NF-κB p65 in esophageal carcinoma cells: possible involvement of Fas/Fas-L synergism

Abstract

Chemosensitivity to anticancer drugs was compared between two human esophageal carcinoma cell lines, T.Tn and YES-6 cells. T.Tn cells were more resistant than YES-6 cells to peplomycin (PEP) but not to the other anticancer drugs such as camptothecin, mitomycin C and cytosine arabinoside. Western blot analysis showed higher expression levels of m-calpain and activated mu-calpain in T.Tn cells than in YES-6 cells. On the other hand, YES-6 cells showed a high expression level of calpastatin, which is a calpain-specific endogenous inhibitor. To investigate whether calpain activity was involved in the chemosensitivity, T.Tn cells were transfected with calpastatin cDNA in an inducible expression vector. The induction of calpastatin was accompanied by increased chemosensitivity to PEP. The increases in calpastatin levels were followed by serial increases in the expression levels of NF-kappaB p65 and Fas. Since purified m- or mu-calpain degraded NF-kappaB p65 in vitro, it is possible that calpastatin suppressed calpain-mediated degradation of NF-kappaB p65. Fas ligand (Fas-L) protein levels increased after treatment of the parental T.Tn and calpastatin-transfected cells with PEP, suggesting the synergism between calpastatin-induced Fas and PEP-induced Fas-L. These results suggest that calpain/calpastatin expression levels are effective markers for predicting the sensitivity of human esophageal carcinoma cells to PEP.

Cleavage of IκBα by calpain induces myocardial NF-κB activation, TNF-α expression, and cardiac dysfunction in septic mice

“…The temporal expression profile of IB␣ and NF-B proteins after LPS challenge suggests that IB␣ degradation preceded NF-B activation. In addition, NF-B p65 has also been reported as a potential substrate of calpain (17). Because the total NF-B p65 protein level was similar in control and septic mice in our study, we propose that the degradation of IB␣ by calpain induces NF-B p65 phosphorylation in septic mice.…”

“…Because IB␣, a substrate of calpain, is a key regulator of NF-B activation (17,30), we next evaluated the levels of IB␣ protein by Western blot analysis. In septic mice, myocardial IB␣ protein was significantly degraded at 1 h after LPS treatment (4 mg/kg; Fig.…”

“…One common type is IB␣, which binds NF-B in the cytoplasm and prevents its translocation to the nucleus by masking its nuclear localization signal (4). In vitro studies (17,30) have demonstrated that both IB␣ and NF-B p65, a subunit of NF-B, are substrates of calpain. In addition, studies (13,35) in nonseptic models have shown that inhibition of IB␣ degradation by calpain inhibitors prevented the translocation of NF-B from the cytosol to the nucleus and inhibited the expression of many NF-B-dependent genes, including TNF-␣.…”

Stimulation of p53 Transactivation Ability by Nicastrin in Mouse Fibroblasts

“…ras-NIH3T3 cells were transfected with expression plasmids and cultured for two days. Cell extracts were prepared by treatment with 0.5% Nonidet P-40, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 50 μM leupeptin, 50 μM antipain, 50 μM pepstatin A, and 50 μM ALLN for 10 minutes at 4 • C [30]. The cell lysate was centrifuged at 13 000 g for 10 minutes and the supernatant (cytoplasmic fraction) was used for detection of NCSTN, presenilin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).…”

Comparative proteomic analysis of the esophageal squamous carcinoma cell line EC109 and its multi-drug resistant subline EC109/CDDP

“…It has been reported that factors including growth-factor receptor, angiogenetic factors, tumor suppressor genes, cell cycle regulators, and DNA repair enzymes are potential EC chemo-sensitivity predictive markers (5)(6)(7)(8)(9)(10)(11). The development of techniques within genomics and proteomics enables extensive characterization of malignant tumors, which may help in understanding treatment resistance and/or treatment sensitivity on the global scale.…”