The effect of resveratrol and its methylthio-derivatives on the Nrf2-ARE pathway in mouse epidermis and HaCaT keratinocytes

Resveratrol is the most extensively studied stilbene derivative. We previously showed that methylthiostilbenes were more effective inhibitors of CYP1A1 and 1B1 activity than resveratrol. In this study, we investigated whether resveratrol and its methylthio-substituted derivatives, i.e. 3-M-4′-MTS (S2), 3,5-DM-4′-MTS (S5) and 3,4,5-TM-4′-MTS (S7) could activate Nrf2 signaling in the mouse epidermis and in human keratinocytes. Western blot analysis showed translocation of Nrf2 from the cytosol to the nucleus in both models. All of the tested stilbenes increased GST activity, but resveratrol was the most effective inducer. Moreover, only resveratrol increased the protein level of GSTP in the mouse epidermis. GSTM was enhanced in HaCaT cells after the treatment with derivatives S2 and S5. The same effect was observed for GSTP in the case of compound S2. Resveratrol and its derivatives reduced the NQO2 protein level in HaCaT cells. Thus, it is possible that increased expression of GSTP or GSTM and GST activity was linked with NQO2 inhibition in these cells. The results of this study indicate that resveratrol and its methylthioderivatives activate Nrf2 not only in the mouse epidermis, but also in human keratinocytes. Upregulating GST isozymes might be particularly important for deactivating chemical carcinogens, such as PAH.     

The store-operated calcium entry pathways in human carcinoma A431 cells: functional properties and activation mechanisms

Activation of phospholipase C (PLC)-mediated signaling pathways in nonexcitable cells causes the release of Ca2+ from intracellular Ca2+ stores and activation of Ca2+ influx across the plasma membrane. Two types of Ca2+ channels, highly Ca2+-selective ICRAC and moderately Ca2+-selective ISOC, support store-operated Ca2+ entry process. In previous patch-clamp experiments with a human carcinoma A431 cell line we described store-operated Imin/ICRACL plasma membrane Ca2+ influx channels. In the present paper we use whole-cell and single-channel recordings to further characterize store-operated Ca2+ influx pathways in A431 cells. We discovered that (a) ICRAC and ISOC are present in A431 cells; (b) ICRAC currents are highly selective for divalent cations and fully activate within 150 s after initiation of Ca2+ store depletion; (c) ISOC currents are moderately selective for divalent cations (PBa/PCs = 14.5) and require at least 300 s for full activation; (d) ICRAC and ISOC currents are activated by PLC-coupled receptor agonists; (e) ISOC currents are supported by Imin/ICRACL channels that display 8.5-10 pS conductance for sodium; (f) ICRAC single channel conductance for sodium is estimated at 0.9 pS by the noise analysis; (g) Imin/ICRACL channels are activated in excised patches by an amino-terminal fragment of InsP3R1 (InsP3R1N); and (h) InsP3 binding to InsP3R1N is necessary for activation of Imin/ICRACL channels. Our findings provide novel information about store-operated Ca2+ influx pathways in A431 cells.     

Autocrine saturation of pro-urokinase receptors on human A431 cells

Single-chain pro-urokinase (pro-uPA) is present both in the medium and lysate of the A431 epidermoid carcinoma cell line. Most of the cell-associated pro-uPA is on the cell surface, as shown by indirect immunofluorescence and by surface iodination. Pro-uPA is not an integral membrane protein but is bound to a specific surface receptor that is completely saturated. A mild acid treatment uncovers the surface receptors by dissociating pro-uPA. Resaturation of uncovered receptors has been studied by reincubating cells in normal medium; within 40 min, 50% of the free sites are reoccupied. Excess uPA-specific antibodies prevent rebinding of ligand to the receptors. Thus, A431 cells first secrete uPA, which then binds to the surface receptor. We propose that the synthesis of uPA and uPA receptor by the same cell may provide a pathway for the activation of the metastatic potential of malignant cells.     

Targeting Stat3 with G-quartet oligodeoxynucleotides in human cancer cells

Stat3 is an oncogene that is activated in many human cancer cells. Genetic approaches that disrupt Stat3 activity result in inhibition of cancer cell growth and enhanced cell apoptosis supporting the development of novel drugs targeting Stat3 for cancer therapy. G-quartet oligodeoxynucleotides (ODNs) were demonstrated to be potent inhibitors of Stat3 DNA binding activity in vitro with the G-quartet ODN, T40214, having an IC(50) of 7 microM. Computer-simulated docking studies indicated that G-quartet ODNs mainly interacted with the SH2 domain of Stat3 and were capable of inserting between the SH2 domains of Stat3 dimers bound to DNA. We demonstrated that the G-rich ODN T40214, which forms a G-quartet structure at intracellular but not extracellular K+ ion concentrations, is delivered efficiently into the cytoplasm and nucleus of cancer cells where it inhibited IL-6-stimulated Stat3 activation and suppressed Stat3-mediated upregulation of bcl-x and mcl-1 gene expression. Thus, G-quartet represents a new class of drug for targeting of Stat3 within cancer cells.     

Dependence of EGF receptor and STAT factor activation on redox of A431 cells

Reactive oxygen species (ROS) were established to play an important role in cellular signaling as second messengers by integrating different pathways. Recently, we showed that EGF initiated a rapid tyrosine phosphorylation of both EGF-receptor and STAT factors with simultaneous increase in the intracellular ROS level. Now, we have investigated the effect of intracellular red-ox state on EGF- and H2O2-induced activation of EGF receptor, STAT1 and STAT3. We demonstrated that the pretreatment of A431 cells with antioxidant N-acetyl-L-cysteine (NAC) partly reduced the level of EGF-induced phosphorylation of proteins under investigation. Besides, H2O2-induced activation of EGF receptor, and STAT factors was fully prevented by NAC pretreatment. The inhibition of ROS generation by DPI declined EGF-dependent activation of EGF receptor and STAT factors to basal level. Our results demonstrate the essential role of cellular red-ox status in the modulation of EGF-mediated activation of receptor and STAT factors. We have postulated that EGF-induced ROS generation is a very important initial event promoting physiological activation of EGF receptor and subsequent STAT factor activation.     

Cutting edge: TCR stimulation by antibody and bacterial superantigen induces Stat3 activation in human T cells.

Recent data show that TCR/CD3 stimulation induces activation of Stat5 in murine T cells. Here, we show that CD3 ligation by mAb and Staphylococcal enterotoxin (SE) induce a rapid, gradually accumulating, long-lasting tyrosine, and serine phosphorylation of Stat3 (but not Stat5) in allogen-specific human CD4+ T cell lines. In contrast, IL-2 induces a rapid and transient tyrosine and serine phosphorylation of Stat3. Compared with IL-2, CD3 ligation induces a delayed Stat3 binding to oligonucleotide probes from the ICAM-1 and IL-2R alpha promoter. CD3-mediated activation of Stat3 is almost completely inhibited by a Src kinase inhibitor (PP1), whereas IL-2-induced Stat3 activation is unaffected. In conclusion, we show that CD3 ligation by mAb and SE triggers a rapid, PP1-sensitive tyrosine and serine phosphorylation of Stat3 in human CD4+ T cells. Moreover, we provide evidence that TCR/CD3 and IL-2 induce Stat3 activation via distinct signaling pathways.     

STAT1 and STAT3 activation by oxidative stress in A431 cells involves Src-dependent EGF receptor transactivation

Different cellular signal transduction cascades are affected by environmental stressors (UV-radiation, gamma-irradiation, hyperosmotic conditions, oxidants). In this study, we examined oxidative stress-evoked signal transduction pathways leading to activation of STATs in A431 carcinoma cells. Oxidative stress, initiated by addition of H2O2 (1-2 mM) to A431 cells, activates STAT3 and, to a lesser extent, STAT1 in dose- and time-dependent manner. Maximum phosphorylation levels were observed after a 2 minutes stimulation at 1-2 mM H2O2. Phosphorylation was blocked by AG1478, a pharmacological inhibitor of the epidermal growth factor receptor tyrosine kinase, implicating intrinsic EGF receptor tyrosine kinase in this process. Consistent with this observation, H2O2-stimulated EGFR tyrosine phosphorylation was abolished by specific Src kinase family inhibitor CGP77675, implicating Src in H2O2-induced EGFR activation. An essential role for Src and JAK2 in STATs activation was suggested by three findings. 1. Src kinase family inhibitor CGP77675 blocked STAT3 and STAT1 activation by H2O2 in a concentration-dependent manner. 2. In Src-/-fibroblasts, activation of both STAT3 and STAT1 by H2O2 was significantly attenuated. 3. Inhibiting JAK2 activity with the specific inhibitor AG490 reduced the level of H2O2-induced STAT3 phosphorylation, but not STAT1 in A431 cells. These data show essential roles for Src and JAK2 inactivation of STAT3. In contrast, H2O2-mediated activation of STAT1 requires only Src kinase activity. Herein, we postulate also that H2O2-induced STAT activation in carcinoma cells involves Src-dependent EGFR transactivation.     

TLR3 activation evokes IL-6 secretion, autocrine regulation of Stat3 signaling and TLR2 expression in human bronchial epithelial cells

Human bronchial epithelial cells exposed to synthetic double-stranded RNA (poly I:C) exhibited increased IL-6 and RANTES secretion and TLR2 expression that was inhibited following TLR3 silencing. Increased NF-κB and Stat3 phosphorylation were detected after poly I:C exposure and pretreatment with neutralizing antibody targeting IL-6 receptor α (IL-6Rα -nAb) or blocking Jak2 and Stat3 activity inhibited Stat3 phosphorylation. TLR2 up-regulation by poly I:C was also reduced by IL-6Rα-nAb and inhibitors of Jak2, Stat3 and NF-κB phosphorylation, whereas RANTES secretion was unaffected, but abolished following NF-κB inhibition. Treatment with exogenous IL-6 failed to increase TLR2. These findings demonstrate that TLR3 activation differentially regulates TLR expression through autocrine signaling involving IL-6 secretion, IL-6Rα activation and subsequent phosphorylation of Stat3. The results also indicate that NF-κB and Stat3 are required for TLR3-dependent up-regulation of TLR2 and that its delayed expression was due to a requirement for IL-6-dependent Stat3 activation.     

Endocytosis occurs independently of annexin VI in human A431 cells

Annexin VI is one of a family of calcium-dependent phospholipid-binding proteins. Although the function of this protein is not known, various physiological roles have been proposed, including a role in the budding of clathrin-coated pits (Lin et al., 1992. Cell. 70:283-291.). In this study we have investigated a possible endocytotic role for annexin VI in intact cells, using the human squamous carcinoma cell line A431, and report that these cells do not express endogenous annexin VI, as judged by Western and Northern blotting and PCR/Southern blotting. To examine whether endocytosis might in some way be either facilitated or inhibited by the presence of annexin VI, a series of A431 clones were isolated in which annexin VI expression was achieved by stable transfection. These cells expressed annexin VI at similar levels to other human cell types. Using assays for endocytosis and recycling of the transferrin receptor, we report that each of these cellular processes occurs with identical kinetics in both transfected and wild- type A431 cells. In addition, purified annexin VI failed to support the scission of coated pits in permeabilized A431 cells. We conclude that annexin VI is not an essential component of the endocytic pathway, and that in A431 cells, annexin VI fails to exert any influence on internalization and recycling of the transferrin receptor.     

Anti-apoptotic effects of curcumin on photosensitized human epidermal carcinoma A431 cells

Photodynamic treatment (PDT) can elicit a diverse range of cellular responses, including apoptotic cell death. Previously, we showed that PDT stimulates caspase-3 activation and subsequent cleavage and activation of p21-activated kinase 2 (PAK2) in human epidermal carcinoma A431 cells. Curcumin, the yellow pigment of Curcuma longa, is known to have anti-oxidant and anti-inflammatory properties. In the present study, using Rose Bengal (RB) as the photosensitizer, we investigated the effect of curcumin on PDT-induced apoptotic events in human epidermal carcinoma A431 cells. We report that curcumin prevented PDT-induced JNK activation, mitochondrial release of cytochrome c, caspase-3 activation, and cleavage of PAK2. Using the cell permeable dye DCF-DA as an indicator of reactive oxygen species (ROS) generation, we found that both curcumin and ROS scavengers (i.e., l-histidine, a-tocopherol, mannitol) abolished PDT-stimulated intracellular oxidative stress. Moreover, all these PDT-induced apoptotic changes in cells could be blocked by singlet oxygen scavengers (i.e., l-histidine, a-tocopherol), but were not affected by the hydroxyl radical scavenger mannitol. In addition, we found that SP600125, a JNK-specific inhibitor, reduced PDT-induced JNK activation as well as caspase-3 activation, indicating that JNK activity is required for PDT-induced caspase activation. Collectively, these results demonstrate that singlet oxygen triggers JNK activation, cytochrome c release, caspase activation and subsequent apoptotic biochemical changes during PDT and show that curcumin is a potent inhibitor for this process.