Reactive oxygen species (ROS) constitute a group of highly reactive molecules that have evolved as regulators of important signaling pathways. It is now well accepted that moderate levels of ROS are required for several cellular functions, including gene expression. The production of ROS is elevated in tumor cells as a consequence of increased metabolic rate, gene mutation and relative hypoxia, and excess ROS are quenched by increased antioxidant enzymatic and nonenzymatic pathways in the same cells. Moderate increases of ROS contribute to several pathologic conditions, among which are tumor promotion and progression, as they are involved in different signaling pathways and induce DNA mutation. However, ROS are also able to trigger programmed cell death (PCD). Our review will emphasize the molecular mechanisms useful for the development of therapeutic strategies that are based on modulating ROS levels to treat cancer. Specifically, we will report on the growing data that highlight the role of ROS generated by different metabolic pathways as Trojan horses to eliminate cancer cells.

The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol‐dependent growth, estradiol treatment of human mammary cancer MCF‐7 cells triggers rapid and transient activation of the mitogen‐activated (MAP) kinases, erk‐1 and erk‐2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras‐GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP‐kinase pathway in MCF‐7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild‐type 67 kDa estradiol receptor cDNA that Cos cells become estradiol‐responsive in terms of erk‐2 activity. This finding, together with the inhibition by the pure anti‐estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF‐7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c‐src show that the estradiol receptor activates c‐src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c‐src is an initial and integral part of the signaling events mediated by the estradiol receptor.

Treatment of human prostate carcinoma-derived LNCaP cells with androgen or oestradiol triggers simultaneous association of androgen receptor and oestradiol receptor b with Src, activates the Src/Raf-1/ Erk-2 pathway and stimulates cell proliferation. Surprisingly, either androgen or oestradiol action on each of these steps is inhibited by both anti-androgens and anti-oestrogens. Similar ®ndings for oestradiol receptor a were observed in MCF-7 or T47D cells stimulated by either oestradiol or androgens. Microinjection of LNCaP, MCF-7 and T47D cells with SrcK ± abolishes steroid-stimulated S-phase entry. Data from transfected Cos cells con®rm and extend the ®ndings from these cells. Hormone-stimulated Src interaction with the androgen receptor and oestradiol receptor a or b is detected using glutathione S-transferase fusion constructs. Src SH2 interacts with phosphotyrosine 537 of oestradiol receptor a and the Src SH3 domain with a proline-rich stretch of the androgen receptor. The role of this phosphotyrosine is stressed by its requirement for association of oestradiol receptor a with Src and consequent activation of Src in intact Cos cells.

The molecular mechanisms by which ovarian hormones stimulate growth of breast tumors are unclear. It has been reported previously that estrogens activate the signal-transducing Src/p21 ras /Erk pathway in human breast cancer cells via an interaction of estrogen receptor (ER) with c-Src. We now show that progestins stimulate human breast cancer T47D cell proliferation and induce a similar rapid and transient activation of the pathway which, surprisingly, is blocked not only by anti-progestins but also by anti-estrogens. In Cos-7 cells transfected with the B isoform of progesterone receptor (PR B ), progestin activation of the MAP kinase pathway depends on co-transfection of ER. A transcriptionally inactive PR B mutant also activates the signaling pathway, demonstrating that this activity is independent of transcriptional effects. PR B does not interact with c-Src but associates via the N-terminal 168 amino acids with ER. This association is required for the signaling pathway activation by progestins. We propose that ER transmits to the Src/p21 ras /Erk pathway signals received from the agonist-activated PR B . These findings reveal a hitherto unrecognized cross-talk between ovarian hormones which could be crucial for their growth-promoting effects on cancer cells.

The p85-associated phosphatidylinositol (PI) 3-kinase/Akt pathway mediates the oestradiol-induced S-phase entry and cyclin D1 promoter activity in MCF-7 cells. Experiments with Src, p85alpha and Akt dominant-negative forms indicate that in oestradiol-treated cells these signalling effectors target the cyclin D1 promoter. Oestradiol acutely increases PI3-kinase and Akt activities in MCF-7 cells. In NIH 3T3 cells expressing ERalpha, a dominant-negative p85 suppresses hormone stimulation of Akt. The Src inhibitor, PP1, prevents hormone stimulation of Akt and PI3-kinase activities in MCF-7 cells. In turn, stimulation of Src activity is abolished in ERalpha-expressing NIH 3T3 fibroblasts by co-transfection of the dominant-negative p85alpha and in MCF-7 cells by the PI3-kinase inhibitor, LY294002. These findings indicate a novel reciprocal cross-talk between PI3-kinase and Src. Hormone stimulation of MCF-7 cells rapidly triggers association of ERalpha with Src and p85. In vitro these proteins are assembled in a ternary complex with a stronger association than that of the binary complexes composed by the same partners. The ternary complex probably favours hormone activation of Src- and PI3-kinase-dependent pathways, which converge on cell cycle progression.

No abstract

Background: Gliadins, a family of wheat proteins, are central to the pathogenesis of celiac disease (CD). In addition to 'immunogenic' effects, gliadin directly affects cultured cells and intestine preparations, and produces damage in vivo, via a separate 'toxic' peptide, such as A-gliadin p31-43 (P31-43). Aims: Understanding the molecular mechanisms underlying direct non T-cell mediated effects of gliadin peptides, and assessing their potential role in promoting CD.Method: Gliadin effects were tested on a number of cell lines and on cultured mucosa samples by evaluating cytoskeleton rearrangements, endocytosis, proliferation and apoptosis. Standard biochemical methods were used to assess prolonged epidermal growth factor receptor (EGFR) activation. Results: Crude gliadin peptic-tryptic peptides (PTG], or P31-43 alone, fully reproduce the effects of epidermal growth factor (EGF] on actin cytosketon, cell cycle and cell proliferation of various cell lines. Inhibitor studies demonstrate the role of EGFR in the early response to gliadin exposure, pointing to activation of the EGFR pathway. Peptide P31-43 is not similar to any EGFR ligand, but can delay inactivation of the EGFR interfering with its endocytosis. Gliadin-induced delay of EGFR endocytosis in cultured intestinal biopsies, together with S-phase entry of epithelial intestinal cells, confirm a role for EGFR activation in CD. Conclusion: The ability of gliadin peptides to delay EGFR inactivation through interference with the endocytic pathway suggests a model where gliadin fragments amplify the effects of trace amounts of EGF, and possibly of other growth factors, by prolonging receptor activation. The results, using cultures of coeliac intestinal biopsies, highlight the role of the EGF pathway in establishing and maintaining the typical atrophic and proliferative alterations of the small intestine in CD.

Under conditions of short-term hormone deprivation, epidermal growth factor (EGF) induces DNA synthesis, cytoskeletal changes, and Src activation in MCF-7 and LNCaP cells. These effects are drastically inhibited by pure estradiol or androgen antagonists, implicating a role of the steroid receptors in these findings. Interestingly, EGF triggers rapid association of Src with androgen receptor (AR) and estradiol receptor A (ERA) in MCF-7 cells or ERB in LNCaP cells. Here, we show that, through EGF receptor (EGFR) and erb-B2, EGF induces tyrosine phosphorylation of ER preassociated with AR, thereby triggering the assembly of ER/AR with Src and EGFR. Remarkably, experiments in Cos cells show that this complex stimulates EGF-triggered EGFR tyrosine phosphorylation. In turn, estradiol and androgen antagonists, through the Src-associated receptors, prevent Src activation by EGF and heavily reduce EGFR tyrosine phosphorylation and the subsequent multiple effects, including DNA synthesis and cytoskeletal changes in MCF-7 cells. In addition, knockdown of ERa or AR gene by small interfering RNA (siRNA) almost abolishes EGFR tyrosine phosphorylation and DNA synthesis in EGF-treated MCF-7 cells. The present findings reveal that steroid receptors have a key role in EGF signaling. EGFR tyrosine phosphorylation, depending on Src, is a part of this mechanism. Understanding of EGF-triggered growth and invasiveness of mammary and prostate cancer cells expressing steroid receptors is enhanced by this report, which reveals novel aspects of steroid receptor action. (Cancer Res 2005; 65(22): 10585-93)