No abstract

Epidemiological data suggest that consumption of fruits and vegetables has been associated with a lower incidence of cancer. Cyanidin-3-glucoside (C3G), a compound found in blackberry and other food products, was shown to possess chemopreventive and chemotherapeutic activity in the present study.

This study was designed to identify the molecular mechanisms of phosphatidylinositol 3-kinase (PI3K)-induced actin filament remodeling and cell migration. Expression of active forms of PI3K, v-P3k or Myr-P3k, was sufficient to induce actin filament remodeling to lead to an increase in cell migration, as well as the activation of Akt in chicken embryo fibroblast (CEF) cells. Either the inhibition of PI3K activity using a PI3K-specific inhibitor, LY-294002, or the disruption of Akt activity restored the integrity of actin filaments in CEF cells and inhibited PI3K-induced cell migration. We also found that expression of an activated form of Akt (Myr-Akt) was sufficient to remodel actin filaments to lead to an increase in cell migration, which was unable to be inhibited by the presence of LY-294002. Furthermore, we found that p70S6K1 kinase was a downstream molecule that can mediate the effects of both PI3K and Akt on actin filaments and cell migration. Overexpression of an active form of p70S6K1 was sufficient to induce actin filament remodeling and cell migration in CEF cells, which requires Rac activity. These results demonstrate that activation of PI3K activity alone is sufficient to remodel actin filaments to increase cell migration through the activation of Akt and p70S6K1 in CEF cells.

One of the hallmarks of integrin signaling is an increase in cell migration and invasion, both of which are associated with actin filament rearrangements. Integrin-linked kinase (ILK) is a cytoplasmic effector of integrin receptors. ILK is known to be involved in multiple cellular functions. However, the signaling pathways involved in ILKmediated cellular structure and motility remain to be elucidated. Here, we have demonstrated that overexpression of ILK was sufficient to induce actin filament rearrangements, to form cell motility structures, and to increase cell migration and invasion in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. This corresponds with the activation of both Akt and p70 ribosomal protein S6 kinase (p70S6K1). Overexpression of dominantnegative mutants of Akt inhibited ILK-dependent activation of p70S6K1, indicating that Akt is upstream of p70S6K1 in response to ILK signaling. Overexpression of ILK was sufficient to induce Rac1 activation, which was abolish by a PI3K inhibitor, indicating that Rac1 activity is involved in ILK signaling in a PI3K dependent manner. Inhibition of Akt, Rac1, or p70S6K1 inhibited the effects of ILK on actin filaments and cell migration, suggesting a regulatory role of the PI3K/Akt/p70S6K1/Rac1 signaling pathway in response to ILK signaling. We have shown that overexpression of a dominant-negative ILK was sufficient to abolish fibronectin peptide (PHSRN)-induced rearrangements of actin filaments and cell migration and invasion. Taken together, our results identify a mechanism through which ILK can regulate both integrin-associated rearrangements of actin filaments and cell migration and invasion at the integrin receptor-proximal region.

Inflammation and oxidative stress are associated with cancer, atherosclerosis, and other chronic diseases. Dietary flavonoids have been reported to possess antiinflammatory and antioxidant properties, but their mechanisms of action and structure-activity relations have not been fully investigated. We hypothesized that differences in antioxidant activity between the structurally similar flavones, luteolin and chrysin (differing only in B-ring hydroxylation patterns), would differentially affect inflammation-associated Cox-2 expression and PGE2 formation. Pretreatment of RAW 264.7 macrophage-like cells with 25, 50, or 100 micromol/L concentrations of luteolin inhibited lipopolysaccharide (LPS)-induced Cox-2 protein expression (P < 0.0001). Chrysin pretreatment did not reduce LPS-induced Cox-2 protein expression at any level tested. Conversely, both luteolin and chrysin completely suppressed LPS-induced PGE2 formation (P < 0.001). Luteolin, but not chrysin, inhibited xanthine/xanthine oxidase-generated superoxide formation at 100 micromol/L in a cell-free system (P < 0.001). Although both luteolin and chrysin reduced LPS-induced hydroxyl radical formation relative to the positive control (P < 0.001), luteolin was superior to chrysin (P = 0.003). In summary, luteolin and chrysin suppressed PGE2 formation equally well, despite differential effects on Cox-2 protein expression and on superoxide and hydroxyl radical scavenging. These data indicate that flavones may display similar antiinflammatory activity via different mechanisms.

No abstract

The actin filament-associated protein and Src-binding partner, AFAP-110, is an adaptor protein that links signaling molecules to actin filaments. AFAP-110 binds actin filaments directly and multimerizes through a leucine zipper motif. Cellular signals downstream of Src 527F can regulate multimerization. Here, we determined recombinant AFAP-110 (rAFAP-110)-bound actin filaments cooperatively, through a lateral association. We demonstrate rAFAP-110 has the capability to cross-link actin filaments, and this ability is dependent on the integrity of the carboxy terminal actin binding domain. Deletion of the leucine zipper motif or PKC phosphorylation affected AFAP-110's conformation, which correlated with changes in multimerization and increased the capability of rAFAP-110 to cross-link actin filaments. AFAP-110 is both a substrate and binding partner of PKC. On PKC activation, stress filament organization is lost, motility structures form, and AFAP-110 colocalizes strongly with motility structures. Expression of a deletion mutant of AFAP-110 that is unable to bind PKC blocked the effect of PMA on actin filaments. We hypothesize that upon PKC activation, AFAP-110 can be cooperatively recruited to newly forming actin filaments, like those that exist in cell motility structures, and that PKC phosphorylation effects a conformational change that may enable AFAP-110 to promote actin filament cross-linking at the cell membrane.

The SH2 and SH3 binding partner AFAP-110 is a tyrosine phosphorylated substrate of Src. AFAP-110 has been hypothesized to link Src to actin ®laments, which may contribute to the eects of Src upon actin ®lament integrity. However, it has been unclear what eect activated Src (Src 527F ) has upon AFAP-110 structure or function and whether AFAP-110 plays a role in actin ®lament integrity. We report here that the carboxy terminal 127 amino acids of AFAP-110 are comprised of an a-helical region that contains a leucine zipper motif. This indicated the potential of AFAP-110 to selfassociate. Expression of the carboxy terminus as a fusion protein (GST-cterm) will permit anity absorption of cellular AFAP-110. The integrity of the a-helical leucine zipper motif in GST-cterm is required for anity absorption, but binding is not due to a classical leucine zipper interaction. Co-expression of Src 527F , unlike cSrc, will abrogate anity absorption of AFAP-110 with GST-cterm. These data indicate that Src 527F has aected a change in the carboxy terminal structure that renders AFAP-110 unavailable for anity absorption. Superose chromatography demonstrate that AFAP-110 will fractionate as a monomer or multimer, indicating AFAP-110 can be detected in a selfassociated form in cell lysates. Co-expression of Src 527F resulted in AFAP-110 fractionating with a molecular weight that predicts only a multimeric population. Deletional mutagenesis also indicate a biological role for the carboxy terminus in cellular localization and actin ®lament integrity. Deletion of the entire carboxy terminal a-helix (84 amino acids) will not permit AFAP-110 to eciently colocalize with actin ®laments or the cell membrane. Deletion of only the leucine zipper region of the carboxy terminal a-helix (44 amino acids) from AFAP-110 (AFAPA Dzip ) demonstrate that both AFAP Dlzip and actin ®laments are repositioned into rosette-like structures, similar to the eects of Src 527F , while co-expression of AFAP-110 with cSrc will not aect actin ®laments. These data indicate that AFAP-110 can play an important role in modulating actin ®lament integrity through carboxy terminal interactions that can be aected by Src .