Biserka Relic

NF-κB transcription factor induces drug resistance through MDR1 expression in cancer cells

The ubiquitous NF-κB transcription factor has been reported to inhibit apoptosis and to induce drug resistance in cancer cells. Drug resistance is the major reason for cancer therapy failure and neoplastic cells often develop multiple mechanisms of drug resistance during tumor progression. We observed that NF-κB or P-glycoprotein inhibition in the HCT15 colon cancer cells led to increased apoptotic cell death in response to daunomycin treatment. Interestingly, NF-κB inhibition through transfection of a plasmid coding for a mutated IκB-α inhibitor increased daunomycin cell uptake. Indeed, the inhibition of NF-κB reduced mdr1 mRNA and P-glycoprotein expression in HCT15 cells. We identified a consensus NF-κB binding site in the first intron of the human mdr1 gene and demonstrated that NF-κB complexes could bind with this intronic site. Moreover, NF-κB transactivates an mdr1 promoter luciferase construct. Our data thus demonstrate a role for NF-κB in the regulation of the mdr1 gene expression in cancer cells and in drug resistance.

The umbilical cord matrix is a better source of mesenchymal stem cells (MSC) than the umbilical cord blood

Many studies have drawn attention to the emerging role of MSC (mesenchymal stem cells) as a promising population supporting new clinical concepts in cellular therapy. However, the sources from which these cells can be isolated are still under discussion. Whereas BM (bone marrow) is presented as the main source of MSC, despite the invasive procedure related to this source, the possibility of isolating sufficient numbers of these cells from UCB (umbilical cord blood) remains controversial. Here, we present the results of experiments aimed at isolating MSC from UCB, BM and UCM (umbilical cord matrix) using different methods of isolation and various culture media that summarize the main procedures and criteria reported in the literature. Whereas isolation of MSC were successful from BM (10:10) and (UCM) (8:8), only one cord blood sample (1:15) gave rise to MSC using various culture media [DMEM (Dulbeccos modified Eagles medium) +5% platelet lysate, DMEM+10% FBS (fetal bovine serum), DMEM+10% human UCB serum, MSCGM®] and different isolation methods [plastic adherence of total MNC (mononuclear cells), CD3+/CD19+/CD14+/CD38+‐depleted MNC and CD133+‐ or LNGFR+‐enriched MNC]. MSC from UCM and BM were able to differentiate into adipocytes, osteocytes and hepatocytes. The expansion potential was highest for MSC from UCM. The two cell populations had CD90+/CD73+/CD105+ phenotype with the additional expression of SSEA4 and LNGFR for BM MSC. These results clearly exclude UCB from the list of MSC sources for clinical use and propose instead UCM as a rich, non‐invasive and abundant source of MSC.

Regulation of HER-2 oncogene expression by cyclooxygenase-2 and prostaglandin E2

The oncoprotein HER-2/neu is a prosurvival factor and its overexpression has been correlated with adverse prognosis in breast cancers. High levels of the cyclooxygenase-2 (COX-2), a proinflammatory and antiapoptotic enzyme, were detected in HER-2-positive tumors and this observation was linked to an HER-2-mediated induction of COX-2 gene transcription. Here, we report that COX-2 expression, and synthesis of its major enzymatic product, PGE2, leads in turn to an enhanced HER-2 expression. Moreover, COX-2 enzymatic inhibition dramatically reduced HER-2 protein levels, efficiently increased the cancer cells sensitility to chemotherapeutic treatment and acted in synergy with HER-2 inhibitor, trastuzumab. Therefore, we propose an original model where HER-2 and COX-2 transcriptionally regulate each other in a positive loop.

Effects of IL-6 and its soluble receptor on proteoglycan synthesis and NO release by human articular chondrocytes: comparison with IL-1. Modulation by dexamethasone☆

Contradictory results have been reported on the effects and role of IL-6 on proteoglycan (PG) synthesis. Having shown recently that in vitro IL-6 depends on the presence of soluble IL-6 receptor alpha (sIL-6Ralpha) to fully exert its effects on chondrocytes, we conducted the present study to analyse the effects of IL-6 on PG synthesis by human articular chondrocytes in the presence of sIL-6Ralpha. PG synthesis was quantified by specific ELISA using a monoclonal antibody (MAB) raised against the keratan sulphate region of PG as a capture antibody, and a MAB to the acid binding region as a detector. It proved specific for PG from primary (differentiated) chondrocytes. In the absence of sIL-6Ralpha, IL-6 had a slight inhibitory effect on PG synthesis by articular chondrocytes. sIL-6Ralpha alone also had slight but consistent inhibitory effects. When adding sIL-6Ralpha at concentrations of 50 ng/ml corresponding to levels found in synovial fluid, the effects of IL-6 increased consistently. However, even at optimal concentrations (30-100 ng/ml of IL-6sR per 100 ng/ml of IL-6), maximal inhibition (48%) did not equal the degree of inhibition achieved by IL-1 at 1 ng/ml (66%). Similar effects, although slightly weaker, were observed on osteoarthritic cells. Dexamethasone, over a wide range of concentrations, markedly enhanced proteoglycan synthesis and completely reversed the downregulatory effects of IL-1 and IL-6 + sIL-6Ralpha. The effects of IL-1 were partially inhibited by an anti-IL-6 antibody. Finally, unlike IL-1, IL-6 + sIL-6Ralpha only weakly stimulated nitric oxide (NO) synthesis. In conclusion, sIL-6Ralpha potentiates the inhibitory effect of IL-6 on PG synthesis by articular chondrocytes, but the overall effect of IL-6 + IL-6sR is moderate compared to the effects of IL-1.

TNF-alpha protects human primary articular chondrocytes from nitric oxide-induced apoptosis via nuclear factor-kappaB

TNF-α plays a key role in rheumatoid arthritis, but its effect on chondrocyte survival is still conflicting. In the present study, we tested how TNF-α influences chondrocyte survival in response to nitric oxide (NO)-related apoptotic signals, which are abundant during rheumatoid arthritis. Human primary articular chondrocytes or cartilage explants were pretreated with TNF-α for 24 hours and then treated with the proapoptotic NO donor sodium-nitro-prusside (SNP) for an additional 24 hours. TNF-α pretreatment markedly protected chondrocytes from SNP-induced cell death. Preincubation of chondrocytes with TNF-α inhibited both SNP-induced high-molecular weight DNA fragmentation and annexin V-FITC binding. Of interest, TNF-α induced persistent nuclear factor-κB (NF-κB)-DNA binding activity even in the presence of SNP, mirroring apoptosis protection effects. Both the TNF-α antiapoptotic effect and NF-κB-DNA binding activity were significantly inhibited by NF-κB inhibitors, Bay 11-7085, MG-132, and adenovirus-expressing mutated IκB-α. Phosphatidylinositol-3 kinase inhibitor LY 294002 also markedly inhibited the antiapoptotic effect of TNF-α. In primary chondrocytes, TNF-α induced expression of the antiapoptotic protein Cox-2, which persisted in the presence of SNP, and a specific Cox-2 inhibitor significantly blocked the TNF-α protective effect. We therefore conclude that TNF-α–mediated protection of chondrocytes from NO-induced apoptosis acts through NF-κB and requires Cox-2 activity.

15-deoxy-delta12,14-prostaglandin J2 inhibits Bay 11-7085-induced sustained extracellular signal-regulated kinase phosphorylation and apoptosis in human articular chondrocytes and synovial fibroblasts

We have previously shown that nuclear factor-κB inhibition by adenovirus expressing mutated IκB-α or by proteasome inhibitor increases human articular chondrocytes sensibility to apoptosis. Moreover, the nuclear factor-κB inhibitor BAY11-7085, a potent anti-inflammatory drug in rat adjuvant arthritis, is itself a proapoptotic agent for chondrocytes. In this work, we show that BAY 11-7085 but not the proteasome inhibitor MG-132 induced a rapid and sustained phosphorylation of extracellular signal-regulated kinases (ERK1/2) in human articular chondrocytes. The level of ERK1/2 phosphorylation correlated with BAY 11-7085 concentration and chondrocyte apoptosis. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and its precursor prostaglandin (PG) D2 but not PGE2 and PGF2α rescued chondrocytes from BAY 11-7085-induced apoptosis. 15d-PGJ2 markedly inhibited BAY 11-7085-induced phosphorylation of ERK1/2. BAY 11-7085 also induced ERK1/2 phosphorylation and apoptosis in human synovial fibroblasts, and these reactions were down-regulated by 15d-PGJ2. Further analysis in synovial fibroblasts showed that only molecules that suppressed BAY 11-7085-induced phosphorylation of ERK1/2 (i.e. 15d-PGJ2, PGD2, and to a lesser extent, MEK1/2 inhibitor UO126, but not prostaglandins E2 and F2α or peroxisome proliferator-activated receptor-γ agonist ciglitazone) were able protect cells from apoptosis. These results suggested that the antiapoptotic effect of 15d-PGJ2 on chondrocytes and synovial fibroblasts might involve inhibition of ERK1/2 phosphorylation.

Genistein induces adipogenesis but inhibits leptin induction in human synovial fibroblasts

It was shown recently that synovial fibroblast transformation into adipocytes reduced the expression of interleukin-6 (IL-6) and IL-8. However, the synovial fibroblast adipogenesis was inhibited in inflammatory conditions induced by the tumor necrosis factor-α (TNF-α). Furthermore, adipogenesis is often accompanied by leptin production, a proinflammatory adipokine in rheumatic diseases. In this study, we tested the phytohormone genistein for adipogenic and anti-inflammatory properties on human synovial fibroblasts. Results showed that genistein was able to transform synovial fibroblasts into adipocytes that expressed perilipin-A and produced adiponectin, but not leptin. Furthermore, genistein enhanced glucocorticoid-mediated synovial fibroblast adipogenesis and, in parallel, downregulated glucocorticoid-induced leptin and leptin receptor. Endogenous and TNF-α-induced expressions of IL-6, IL-8, p38, p65 and C/EBP-β were also downregulated by genistein, showing its anti-inflammatory properties. Peroxisome proliferator- activated receptor-γ (PPAR-γ) agonist, rosiglitazone, had a synergic effect on genistein-induced adipogenesis, whereas the non-active tyrosine kinase inhibitor, daidzein, had a significantly inferior adipogenic activity than genistein. The Janus kinase-2 tyrosine kinase inhibitor, AG 490, mimicked the anti-leptin effect of genistein. These results showed that genistein-induced adipogenesis involves PPAR-γ induction and tyrosine kinase inhibition. In conclusion, genistein, alone or coupled with glucocorticoids, have both adipogenic and anti-inflammatory effects on synovial fibroblasts.

Acute-phase serum amyloid a in osteoarthritis: regulatory mechanism and proinflammatory properties.

Objective To determine if serum amyloid A (A-SAA) could be detected in human osteoarthritic (OA) joints and further clarify if high A-SAA level in joints result from a local production or from a diffusion process from abnormally elevated plasma concentration. Regulatory mechanism of A-SAA expression and its pro-inflammatory properties were also investigated. Methods A-SAA levels in serum and synovial fluid of OA (n = 29) and rheumatoid arthritis (RA) (n = 27) patients were measured and compared to matched-healthy volunteers (HV) (n = 35). In vitro cell cultures were performed on primary joint cells provided from osteoarthritis patients. Regulatory mechanisms were studied using Western-blotting, ELISA and lentiviral transfections. Results A-SAA was statistically increased in OA plasma patients compared to HV. Moreover, A-SAA level in OA plasma and synovial fluid increased with the Kellgren & Lauwrence grade. For all OA and RA patients, A-SAA plasma level was higher and highly correlated with its corresponding level in the synovial fluid, therefore supporting that A-SAA was mainly due to the passive diffusion process from blood into the joint cavity. However, A-SAA expression was also observed in vitro under corticosteroid treatment and/or under IL-1beta stimuli. A-SAA expression was down-regulated by PPAR-γ agonists (genistein and rosiglitazone) and up-regulated by TGF-β1 through Alk1 (Smad1/5) pathway. RhSAA induced proinflammatory cytokines (IL-6, IL-8, GRO-α and MCP-1) and metalloproteinases (MMP-1, MMP-3 and MMP-13) expression in FLS and chondrocytes, which expression was downregulated by TAK242, a specific TLR4 inhibitor. Conclusion Systemic or local A-SAA expression inside OA joint cavity may play a key role in inflammatory process seen in osteoarthritis, which could be counteracted by TLR4 inhibition.

Primary human articular chondrocytes, dedifferentiated chondrocytes, and synoviocytes exhibit differential responsiveness to interleukin-4: Correlation with the expression pattern of the common receptor gamma chain

Interleukin (IL)‐4, which exhibits potent anti‐inflammatory activities, is of potential therapeutic value in destructive arthropathies. To further define the response of human joint cells to IL‐4, we analyzed the ability of this cytokine to modulate the effects of IL‐1β and growth factors. Freshly isolated chondrocytes, dedifferentiated chondrocytes, and synoviocytes were treated with IL‐4 before determination of nitric oxide (NO) and collagenase production in response to IL‐1β, or before proliferation assays in presence of IL‐1β, platelet‐derived growth factor (PDGF), or transforming growth factor (TGF)‐β. IL‐4 downregulated IL‐1β induced NO production in dedifferentiated chondrocytes and inhibited IL‐1β induced collagenase release, as well as IL‐1β and growth factor induced proliferation in dedifferentiated chondrocytes and synoviocytes. In contrast, IL‐4 had no effect in freshly isolated primary chondrocytes and in cartilage explants. The lack of response to IL‐4 in primary chondrocytes was associated with impaired signal transduction, as indicated by markedly decreased IL‐4 dependent tyrosine phosphorylation of signal transducer and activator of transcription (STAT)‐6. It also correlated with differences in the expression pattern of IL‐4 receptor (IL‐4R) subunits during chondrocyte dedifferentiation. Indeed, whereas the IL‐4Rα and IL‐13Rα′ subunits were expressed in all cell types, expression of the common receptor gamma chain was restricted to freshly isolated chondrocytes. In conclusion, IL‐4 downregulated IL‐1β‐induced catabolic events and cell proliferation in dedifferentiated chondrocytes and synoviocytes, but had no effects in freshly isolated chondrocytes. The difference in IL‐4 responsiveness between primary and dedifferentiated chondrocytes correlated with changes in proximal signaling events and in the expression pattern of IL‐4R subunits during cell dedifferentiation.

Insights on Molecular Mechanisms of Chondrocytes Death in Osteoarthritis

Osteoarthritis (OA) is a joint pathology characterized by progressive cartilage degradation. Medical care is mainly based on alleviating pain symptoms. Compelling studies report the presence of empty lacunae and hypocellularity in cartilage with aging and OA progression, suggesting that chondrocyte cell death occurs and participates to OA development. However, the relative contribution of apoptosis per se in OA pathogenesis appears complex to evaluate. Indeed, depending on technical approaches, OA stages, cartilage layers, animal models, as well as in vivo or in vitro experiments, the percentage of apoptosis and cell death types can vary. Apoptosis, chondroptosis, necrosis, and autophagic cell death are described in this review. The question of cell death causality in OA progression is also addressed, as well as the molecular pathways leading to cell death in response to the following inducers: Fas, Interleukin-1β (IL-1β), Tumor Necrosis factor-α (TNF-α), leptin, nitric oxide (NO) donors, and mechanical stresses. Furthermore, the protective role of autophagy in chondrocytes is highlighted, as well as its decline during OA progression, enhancing chondrocyte cell death; the transition being mainly controlled by HIF-1α/HIF-2α imbalance. Finally, we have considered whether interfering in chondrocyte apoptosis or promoting autophagy could constitute therapeutic strategies to impede OA progression.