This procedure highlights the availability of autologous culture expanded bone marrow mesenchymal cell transplantation for the repair of articular cartilage defects in humans.

To inhibit arthritis upstream of inflammatory cytokine release and matrix metalloproteinase (MMP) action, we designed de novo a small-molecule inhibitor of c-Fos/activator protein-1 (AP-1) using three-dimensional (3D) pharmacophore modeling. This model was based on the 3D structure of the basic region-leucine zipper domain of AP-1-DNA complex. Administration of this inhibitor prevented type II collagen-induced arthritis from day 21, before the onset of arthritis, or from day 27, resolved arthritis after its onset. Suppression of disease was accomplished by reducing the amounts of inflammatory cytokines and MMPs in vivo in sera and joints and in vitro in synovial cell and chondrocyte cultures. The primary action of this molecule was the inhibition of matrix-degrading MMPs and inflammatory cytokines including interleukin 1beta; this molecule also synergized with anti-tumor necrosis factor alpha to inhibit arthritis. Thus, selective inhibition of c-Fos/AP-1 resolves arthritis in a preclinical model of the disease.

Although adherent junctions have been extensively studied, the role of tight junctions in cancer cell invasion is not sufficiently explored. We investigated whether claudin-1, a component of tight junctions, regulated invasion activity in oral squamous cell carcinoma (OSC) cells. The expression of claudin-1, activity of matrix metalloproteinase (MMP)-2, and cleavage of laminin-5 ;2 chains were assessed by Western blot analysis, immunohistochemistry, and zymography in OSC cell lines (OSC-4 and NOS-2, highly invasive; OSC-7, weakly invasive) and their xenografts in severe combined immunodeficient (SCID) mice. The influence of claudin-1 small interfering RNA (siRNA) on the invasion activity of the cell lines was also investigated. Compared with OSC-7, both OSC-4 and NOS-2 more strongly expressed claudin-1 and possessed high activities of MMP-2 and MMP-9. Tumors formed in the tongues of SCID mice xenografted with OSC-4, NOS-2, and OSC-7 immunohistochemically revealed strong, moderate, and weak expression of laminin-5 ;2 chains, respectively, and laminin-5 ;2 chains were secreted in the conditioned medium of the cancer cells in parallel with the in vivo results. Claudin-1 siRNA largely suppressed the invasion of OSC-4 and decreased the activation of MMP-2, the expression of membrane-type MMP-1 (MT1-MMP), and the cleavage of laminin-5 ;2. In addition, not only antibodies against MT1-MMP and epidermal growth factor receptor (EGFR) but also MMP-2 and EGFR inhibitors strongly suppressed the invasion activity of OSC-4. These results suggest that claudin-1 upregulates cancer cell invasion activity through activation of MT1-MMP and MMP-2, which results in enhanced cleavage of laminin-5 ;2 chains. (Cancer Res 2006; 66(10): 5251-7)

We examined the influence of ROS on the phosphorylation and complex formation of Bcl-2 family proteins in Mn-superoxide dismutase (SOD) antisense-transfected squamous cell carcinoma cells, OSC-4 cells. The increase of intracellular ROS level induced by cis-diamminedichloroplatinum (CDDP) and γ γ γ γ-ray treatment was greater in antisense-transfected cells than in control vector-transfected cells, and apoptosis was more extensively induced in the former. Antisense-transfected cells expressed high levels of Bax and Bak, but low levels of Bcl-2 and Bcl-X L when treated with CDDP, peplomycin, 5-fluorouracil or γ γ γ γ-rays. After treatment with these agents, the phosphorylation of protein kinase A, Bcl-2 (Thr56) and Bad (Ser155) was increased, especially in antioxidant (N-acetylcysteine and pyrrolidine dithiocarbamate)-pretreated control cells, but the phosphorylation levels were very low in the antisense-transfected cells. Bcl-2 ubiquitination was increased, but ubiquitination of Bad and Bax was decreased in the antisense-transfected cells, although their ubiquitination was increased by the antioxidants. These results reveal that ROS induce apoptosis by regulating the phosphorylation and ubiquitination of Bcl-2 family proteins, resulting in increased proapoptotic protein levels and decreased antiapoptotic protein expression. here are multiple signal pathways to induce apoptosis, including those originating from mitochondria and Fas. 1-4)The signal originating from Fas and Fas-associated proteins passes to caspase 3 through caspase 8 and other caspases. 5-7)The mitochondrial signal is also transduced to caspase 3 via cytochrome c, apoptosis protease-activating factor-1, ATP, and caspase 9. 8) These two pathways exhibit cross-talk with a proapoptotic Bcl-2 family protein, Bid. 9) Bcl-2 family proteins including Bid control the release of cytochrome c from mitochondria regulating VDAC. 10)Bcl-2 family proteins are divided into two groups, proapoptotic and antiapoptotic, according to their chemical structure, that is, BH3 and BH4.11, 12) Proapoptotic and antiapoptotic proteins form heterodimers and inhibit each other's activity. [13][14][15] The dimerization is influenced by phosphorylation of the amino acid residues of the proapoptotic members, Bax, Bak, and Bik.14-17) The expression level of each Bcl-2 family protein is controlled by transcription, heterodimerization, and ubiquitination.13-26) Phosphorylation of antiapoptotic Bcl-2 family proteins inhibits the binding of these proteins and polyubiquitin. [27][28][29][30] Apoptosis is therefore deeply associated with the phosphorylation of Bcl-2 family proteins.Recently, it has been established that some growth factors induce survival signal-activating kinases such as PKA and PKB, the MAP family, ERK1/2, and AP-1. [31][32][33][34] The activation of these kinases finally results in an increase in antiapoptotic Bcl-2 expression. 29,35,36) Suppression of these kinase activities is, therefore, required for the induction of apoptosis.ROS possess both cell-impairing and cel...

Cytokine production was investigated in oral keratinocytes and tissue-infiltrated mononuclear cells (TIMC) obtained from patients with oral lichen planus (OLP). The numbers of cells producing interleukin (IL)-1 beta, IL-4, IL-6, granulocyte colony-stimulating factor, and tumor necrosis factor-alpha per 10(4) cells in keratinocytes from patients with OLP were determined by enzyme-linked immunospot assay. These levels were two to threefold greater than those in keratinocytes from chronically inflamed gingiva and 10 to 20-fold of those from the intact gingiva. The concentrations of these cytokines in the culture supernatants of keratinocytes were correlated with the number of cytokine-producing cells. Compared with TIMC in the gingiva and peripheral blood mononuclear cells, TIMC in OLP were more cytokine-productive, with larger numbers of cytokine-producing cells that expressed more cytokine messengers. More IL-6, IL-2, and IL-10 were generated from TIMC in OLP, whereas less granulocyte colony-stimulating factor was generated. After pretreatment with IL-2, TIMC from OLP patients generated more IL-6 than did peripheral blood mononuclear cells, and IL-4-pretreated TIMC from the patients released larger amounts of IL-2, IL-6, and IL-10. These results indicate that keratinocytes play a critical role in OLP through production of large amounts of cytokines, that TIMC are stimulated in situ and differentiated to produce cytokines characteristic of OLP, and that the inflammatory condition of OLP is determined by the local cytokine network.

The most abundant green tea polyphenol, epigallocatechin-3-gallate (EGCG), was found to induce differential effects between tumor cells and normal cells. Nevertheless, how normal epithelial cells respond to the polyphenol at concentrations for which tumor cells undergo apoptosis is undefined. The current study tested exponentially growing and aged primary human epidermal keratinocytes in response to EGCG or a mixture of the four major green tea polyphenols. EGCG elicited cell differentiation with associated induction of p57/KIP2 within 24 h in growing keratinocytes, measured by the expression of keratin 1, filaggrin, and transglutaminase activity. Aged keratinocytes, which exhibited low basal cellular activities after culturing in growth medium for up to 25 days, renewed DNA synthesis and activated succinate dehydrogenase up to 37-fold upon exposure to either EGCG or the polyphenols. These results suggest that tea polyphenols may be used for treatment of wounds or certain skin conditions characterized by altered cellular activities or metabolism.Green tea polyphenols (referred to as GTPPs, to include the four major polyphenols: epicatechin, epigallocatechin, epcatechin-3-gallate, and epigallocatechin-3-gallate (EGCG) have been identified to possess chemopreventive and apoptotic activity against certain cancers, whereas normal epidermal keratinocytes follow a survival pathway that has not been fully elucidated (Chen et al

Green tea polyphenols (GTPPs) are considered beneficial to human health, especially as chemopreventive agents. Recently, cytotoxic reactive oxygen species (ROS) were identified in tumor and certain normal cell cultures incubated with high concentrations of the most abundant GTPP, (Ϫ)-epigallocatechin-3-gallate (EGCG). If EGCG also provokes the production of ROS in normal epithelial cells, it may preclude the topical use of EGCG at higher doses. The current study examined the oxidative status of normal epithelial, normal salivary gland, and oral carcinoma cells treated with EGCG, using ROS measurement and catalase and superoxide dismutase activity assays. The results demonstrated that high concentrations of EGCG induced oxidative stress only in tumor cells. In contrast, EGCG reduced ROS in normal cells to background levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-bromodeoxyuridine incorporation data were also compared between the two oral carcinoma cell lines treated by EGCG, which suggest that a difference in the levels of endogenous catalase activity may play an important role in reducing oxidative stress provoked by EGCG in tumor cells. It is concluded that pathways activated by GTPPs or EGCG in normal epithelial versus tumor cells create different oxidative environments, favoring either normal cell survival or tumor cell destruction. This finding may lead to applications of naturally occurring polyphenols to enhance the effectiveness of chemo/radiation therapy to promote cancer cell death while protecting normal cells.

The transcriptional factor hypoxia-inducible factor-1 (HIF-1) plays an important role in solid tumor cell growth and survival. Overexpression of HIF-1α α α α has been demonstrated in many human tumors and predicts a poor response to chemoradiotherapy. We examined the HIF-1α α α α-induced survival pathways in human oral squamous cell carcinoma cell (OSCC) lines. The results showed that forced expression of HIF-1α α α α suppressed hypoxia-induced apoptosis of OSCC lines by inhibiting cytochrome c release from mitochondria. Overexpression of HIF-1α α α α inhibited the generation of reactive oxygen species (ROS), elevation of intracellular Ca 2+ concentration, reduction of mitochondrial membrane potential, and cytosolic accumulation of cytochrome c, which resulted in the inactivation of caspase-9 and caspase-3. In addition, antiapoptotic Bcl-2 and Bcl-X L levels were increased and pro-apoptotic Bax and Bak levels were decreased in the HIF-1α α α α-overexpressing OSCC line. T he transcriptional factor hypoxia-inducible factor-1 (HIF-1) is one of the key regulators of oxygen homeostasis. HIF-1 is a heterodimer composed of HIF-1α and HIF-1β subunits, both belonging to the basic-helix-loop-helix PER-ARNT-SIM family of transcriptional factors. HIF-1β, an aryl hydrocarbon receptor nuclear translocator, is constitutively present in normoxic cells, whereas expression levels of HIF-1α are dependent on intracellular oxygen concentration. Under normoxic conditions, HIF-1α protein is rapidly degraded through the ubiquitin-proteasome pathway.(1,2) Degradation of HIF-1α is mediated by the ubiquitin E3 ligase complex, in which the von Hippel Lindau protein (pVHL) binds to the oxygen-dependent degradation domain in the HIF-1α subunits. (3,4) However, under hypoxic conditions, HIF-1α degradation is suppressed, and stabilized HIF-1α activates the transcription of a repertoire of target genes such as glucose transporters, glycolytic enzymes and angiogenic factors.(5-8) Solid tumors often have an insufficient blood supply because tumor cells grow faster than endothelial cells, so the newly formed vascular supply is frequently disorganized, which results in hypoxic regions in tumor tissues. (9)(10)(11) HIF-1α is therefore a resistive molecule against cancer therapy.The transcriptional factor HIF-1α is an important mediator in solid tumor development in vivo because of its promotion of angiogenesis and anaerobic metabolism and inhibition of apoptosis induction in tumor cells. (12)(13)(14) In fact, overexpression of HIF-1α protein coupled with a poor prognosis has been observed in human cancers. (15)(16)(17)(18)(19)(20) In addition, HIF-1α inhibits the induction of apoptosis in tumor cells. (21,22) Apoptosis is mediated by two distinct pathways: the mitochondrial apoptotic pathway and the death receptor (Fas-FasL)-mediated pathway. Decrease of the mitochondrial membrane potential (∆ψ m ) and release of cytochrome c from mitochondria are important apoptotic processes and members of the Bcl-2 family, including Bax, Bcl-2 and Bcl-X L , are c...