Roles of paxillin phosphorylation in IL-3 withdrawal-induced Ba/F3 cell apoptosis.

BACKGROUND: Ba/F3, a mouse pro-B cell line, is dependent on IL-3 for its survival and proliferation. IL-3 withdrawal causes cells to round, stop in G1 phase, then undergo apoptosis. Additionally, IL-3 is known to induce tyrosine phosphorylation of paxillin, a scaffold and signaling protein. We previously determined that overexpression of paxillin prohibited Ba/F3 cell apoptosis induced by IL-3 withdrawal. OBJECTIVE: Address whether phosphorylation is essential for the anti-apoptotic effect of overexpressed paxillin. METHODS: Mutations were introduced into paxillin cDNA at five phosphorylation sites-Y31F, Y40F, Y118F, Y181F, S273A, or S273D. After overexpression of paxillin mutants in Ba/F3 cells, the apoptotic proportions of cell populations were measured by an annexin V conjugation assay while cells were undergoing IL-3 withdrawal. RESULTS: The anti-apoptotic effect of paxillin overexpression was abolished by site-directed mutagenesis replacing Y31, Y40, Y118, and Y181 with phenylalanine, and S273 with aspartic acid. In contrast, the mutation replacing S273 with alanine had no effect on the anti-apoptotic effect. CONCLUSION: The above results suggest that paxillin-mediated phosphorylation at Y31, Y40, Y118, and Y181 is essential for the anti-apoptotic effect of paxillin overexpression in Ba/F3 cells and contributes to the cell survival signaling pathway triggered by IL-3. Conversely, phosphorylation at S273 is involved in the negative regulation of the anti-apoptotic action of overexpressed paxillin.

Redox regulation of the tumor suppressor PTEN by the thioredoxin system and cumene hydroperoxide.

Intracellular redox status influences the oxidation and enzyme activity of the tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN). Cumene hydroperoxide (CuHP), an organic hydroperoxide, is a known tumor promoter. However, molecular targets and action mechanism of CuHP in tumor promotion have not been well characterized. In this study, we investigated the effect of CuHP on the redox state of PTEN in HeLa cells. In addition, the intracellular reducing system of oxidized PTEN was analyzed using a biochemical approach and the effect of CuHP on this reducing system was also analyzed. While PTEN oxidized by hydrogen peroxide is progressively converted to its reduced form, PTEN was irreversibly oxidized by exposure to CuHP in HeLa cells. A combination of protein fractionation and mass analysis showed that the reducing system of PTEN was comprised of NADPH, thioredoxin reductase (TrxR), and thioredoxin (Trx). Although CuHP-mediated PTEN oxidation was not reversible in cells, CuHP-oxidized PTEN was reactivated by the exogenous Trx system, indicating that the cellular Trx redox system for PTEN is inactivated by CuHP. We present evidence that PTEN oxidation and the concomitant inhibition of thioredoxin by CuHP results in irreversible oxidation of PTEN in HeLa cells. In addition, ablation of peroxiredoxin (Prdx) enhanced CuHP-induced PTEN oxidation in cells. These results provide a new line of evidence that PTEN might be a crucial determinant of cell fate in response to cellular oxidative stress induced by organic hydroperoxides.

Redox Regulation of the Tumor Suppressor PTEN by Hydrogen Peroxide and Tert-Butyl Hydroperoxide.

Organic peroxides and hydroperoxides are skin tumor promoters. Free radical derivatives from these compounds are presumed to be the prominent mediators of tumor promotion. However, the molecular targets of these species are unknown. Phosphatase and tensin homologs deleted on chromosome 10 (PTEN) are tumor suppressors that play important roles in cell growth, proliferation, and cell survival by negative regulation of phosphoinositol-3-kinase/protein kinase B signaling. PTEN is reversibly oxidized in various cells by exogenous and endogenous hydrogen peroxide. Oxidized PTEN is converted back to the reduced form by cellular reducing agents, predominantly by the thioredoxin (Trx) system. Here, the role of tert-butyl hydroperoxide (t-BHP) in redox regulation of PTEN was analyzed by using cell-based and in vitro assays. Exposure to t-BHP led to oxidation of recombinant PTEN. In contrast to H2O2, PTEN oxidation by t-BHP was irreversible in HeLa cells. However, oxidized PTEN was reduced by exogenous Trx system. Taken together, these results indicate that t-BHP induces PTEN oxidation and inhibits Trx system, which results in irreversible PTEN oxidation in HeLa cells. Collectively, these results suggest a novel mechanism of t-BHP in the promotion of tumorigenesis.

Chrysin inhibits tumor promoter-induced MMP-9 expression by blocking AP-1 via suppression of ERK and JNK pathways in gastric cancer cells.

Cell invasion is a crucial mechanism of cancer metastasis and malignancy. Matrix metalloproteinase-9 (MMP-9) is an important proteolytic enzyme involved in the cancer cell invasion process. High expression levels of MMP-9 in gastric cancer positively correlate with tumor aggressiveness and have a significant negative correlation with patients' survival times. Recently, mechanisms suppressing MMP-9 by phytochemicals have become increasingly investigated. Chrysin, a naturally occurring chemical in plants, has been reported to suppress tumor metastasis. However, the effects of chrysin on MMP-9 expression in gastric cancer have not been well studied. In the present study, we tested the effects of chrysin on MMP-9 expression in gastric cancer cells, and determined its underlying mechanism. We examined the effects of chrysin on MMP-9 expression and activity via RT-PCR, zymography, promoter study, and western blotting in human gastric cancer AGS cells. Chrysin inhibited phorbol-12-myristate 13-acetate (PMA)-induced MMP-9 expression in a dose-dependent manner. Using AP-1 decoy oligodeoxynucleotides, we confirmed that AP-1 was the crucial transcriptional factor for MMP-9 expression. Chrysin blocked AP-1 via suppression of the phosphorylation of c-Jun and c-Fos through blocking the JNK1/2 and ERK1/2 pathways. Furthermore, AGS cells pretreated with PMA showed markedly enhanced invasiveness, which was partially abrogated by chrysin and MMP-9 antibody. Our results suggest that chrysin may exert at least part of its anticancer effect by controlling MMP-9 expression through suppression of AP-1 activity via a block of the JNK1/2 and ERK1/2 signaling pathways in gastric cancer AGS cells.

Assay of the redox state of the tumor suppressor PTEN by mobility shift.

PTEN is reversibly oxidized in various cells by exogenous hydrogen peroxide as well as by endogenous hydrogen peroxide generated when cells are stimulated with growth factors, cytokines and hormones. A gel mobility shift assay showed that oxidized PTEN migrated more rapidly than reduced PTEN on a non-reducing SDS-PAGE gel. Oxidized PTEN was reduced when treated with dithiothreitol. Supplementation of N-ethylmaleimide in the cell lysis buffer was critical for the apparent bands of oxidized and reduced PTEN. Formation of oxidized PTEN was abolished when the active site Cys(124) or nearby Cys(71) was replaced with Ser suggesting that Cys(124) and Cys(71) are involved in the formation of an intramolecular disulfide bond. These results show that the mobility shift assay is a convenient method to analyze the redox state of PTEN in cells.

Chrysin inhibits cell invasion by inhibition of Recepteur d'origine Nantais via suppressing early growth response-1 and NF-κB transcription factor activities in gastric cancer cells.

Cell invasion is one of crucial reasons for cancer metastasis and malignancy. Recepteur d'origine Nantais (RON) has been reported to play an important role in the cancer cell invasion process. High accumulation and activation of RON has been implicated in gastric adenocarcinoma AGS cells. Chrysin is a naturally occurring phytochemical, a type of flavonoid, which has been reported to suppress tumor metastasis. However, the effects of chrysin on RON expression in gastric cancer are not well studied. In the present study, we examined whether chrysin affects RON expression in gastric cancer, and if so, its underlying mechanism. We examined the effect of chrysin on RON expression and activity, via RT-PCR, promoter study, and western blotting in human gastric cancer AGS cells. Chrysin significantly inhibited endogenous and inducible RON expression in a dose-dependent manner. After demonstrating that Egr-1 and NF-κB are the critically required transcription factors for RON expression, we discovered that chrysin suppressed Egr-1 and NF-κB transcription factor activities. Additionally, the phorbol-12-myristate-13-acetate- (PMA) induced cell invasion was partially abrogated by chrysin and an RON antibody. Our results suggest that chrysin has anticancer effects at least by suppressing RON expression through blocking Egr-1 and NF-κB in gastric cancer AGS cells.

Piperine inhibits IL-1ß-induced IL-6 expression by suppressing p38 MAPK and STAT3 activation in gastric cancer cells.

Piperine, a kind of natural alkaloid found in peppers, has been reported to exhibit anti-oxidative and anti-tumor activities, both in vitro and in vivo. Interleukin-6 (IL-6) is an important cytokine that activates the signal transduction, promotes tumor cell metastasis, and induces malignancy, including in gastric cancer. However, the effects of piperine on IL-6 expression in gastric cancer cells have not yet been well defined. In this study, we investigated the effects of piperine on the IL-6 expression, and examined the underlying signaling pathways via RT-PCR, promoter studies and Western blotting in human gastric cancer TMK-1 cells. Our results showed that piperine inhibited interleukin-1ß (IL-1ß)-induced IL-6 expression in a dose-dependent manner. In addition, piperine also inhibited IL-6 promoter activity. Experiments with mitogen-activated protein kinase (MAPK) inhibitors and dominant negative mutant p38 MAPK indicated that p38 MAPK was essential for IL-6 expression in the TMK-1 cells. Additionally, signal transducer and activator of transcription 3 (STAT3) was also involved in the IL-1ß-induced IL-6 expression in gastric cancer cells. Piperine inhibited IL-1ß-induced p38 MAPK and STAT3 activation and, in turn, blocked the IL-1ß-induced IL-6 expression. Furthermore, gastric cancer cells pretreated with IL-1ß showed markedly enhanced invasiveness, which was partially abrogated by treatment with IL-6 siRNA, piperine, and inhibitors of p38 MAPK and STAT3. These results suggest that piperine may exert at least part of its anti-cancer effect by controlling IL-6 expression through the suppression of p38 MAPK and STAT3.

Cadmium induces urokinase-type plasminogen activator receptor expression and the cell invasiveness of human gastric cancer cells via the ERK-1/2, NF-κB, and AP-1 signaling pathways.

Cadmium exposure has been linked to human cancers, including stomach cancer. In this study, the effects of cadmium on urokinase-type plasminogen activator receptor (uPAR) expression in human gastric cancer cells and the underlying signal transduction pathways were investigated. Cadmium induced uPAR expression in a time- and concentration-dependent manner. Cadmium also induced uPAR promoter activity. Additionally, cadmium induced the activation of extracellular signal regulated kinase-1/2 (ERK-1/2), p38 mitogen-activated protein kinase (MAPK), and the activation of c-Jun amino terminal kinase (JNK). A specific inhibitor of MEK-1 (PD98059) inhibited cadmium-induced uPAR expression, while JNK and p38 MAPK inhibitors did not. Expression vectors encoding dominant-negative MEK-1 (pMCL-K97M) also prevented cadmium-induced uPAR promoter activity. Site-directed mutagenesis and electrophoretic mobility shift studies showed that sites for the transcription factors nuclear factor (NF)-κB and activator protein-1 (AP-1) were involved in cadmium-induced uPAR transcription. Suppression of the cadmium-induced uPAR promoter activity by a mutated-type NF-κB-inducing kinase and I-κB and an AP-1 decoy oligonucleotide confirmed that the activation of NF-κB and AP-1 are essential for cadmium-induced uPAR upregulation. Cells pretreated with cadmium showed markedly enhanced invasiveness and this effect was partially abrogated by uPAR-neutralizing antibodies and by inhibitors of ERK-1/2, NF-κB, and AP-1. These results suggest that cadmium induces uPAR expression via ERK-1/2, NF-κB, and AP-1 signaling pathways and, in turn, stimulates cell invasiveness in human gastric cancer AGS cells.

Comparative proteomic analysis of cysteine oxidation in colorectal cancer patients.

Oxidative stress promotes damage to cellular proteins, lipids, membranes and DNA, and plays a key role in the development of cancer. Reactive oxygen species disrupt redox homeostasis and promote tumor formation by initiating aberrant activation of signaling pathways that lead to tumorigenesis. We used shotgun proteomics to identify proteins containing oxidation-sensitive cysteines in tissue specimens from colorectal cancer patients. We then compared the patterns of cysteine oxidation in the membrane fractions between the tumor and non-tumor tissues. Using nano-UPLC-MS(E) proteomics, we identified 31 proteins containing 37 oxidation-sensitive cysteines. These proteins were observed with IAM-binding cysteines in non-tumoral region more than tumoral region of CRC patients. Then using the Ingenuity pathway program, we evaluated the cellular canonical networks connecting those proteins. Within the networks, proteins with multiple connections were related with organ morphology, cellular metabolism, and various disorders. We have thus identified networks of proteins whose redox status is altered by oxidative stress, perhaps leading to changes in cellular functionality that promotes tumorigenesis.

Tyr740 and Tyr751 residues of platelet-derived growth factor beta receptor are responsible for the redox regulation of phosphatase and tensin homolog in the cells stimulated with platelet-derived growth factor.

Exposure of cells to hydrogen peroxide or platelet-derived growth factor (PDGF) induced Akt phosphorylation and oxidation of phosphatase and tensin homolog (PTEN). The Cys124 and Cys71 residues of PTEN were critical for the formation of a disulfide bond and the intermediate glutathionylation in the process of reduction of the disulfide bond. To determine which specific tyrosine residues of the PDGF beta receptor (PDGFßR) is involved in PDGF-induced PTEN oxidation and Akt phosphorylation, we investigated a kinase activity-deficient mutant and PDGFßR mutants where the tyrosine residues in the binding site for phosphoinositide 3-kinase (PI3K), GTPase-activating protein of Ras, Src homology 2 domain containing protein-tyrosine phosphatase-2, and phospholipase C-1 were replaced by Phe. Both PTEN oxidation and Akt phosphorylation did not occur in response to PDGF in the kinase-deficient mutant and in the PDGFßR mutant with a mutation in the PI3K binding site (Tyr740 and Tyr751). Thus, the kinase activity and the constituent Tyr740 and Tyr751 residues of PDGFßR in the cells stimulated with PDGF are responsible for the oxidation of PTEN and the Akt phosphorylation.

Estrogen receptor α induces down-regulation of PTEN through PI3-kinase activation in breast cancer cells.

Estrogen receptor α (ERα) mediates most of the biological effects of estrogen in mammary epithelial cells and stimulates growth signals involving phosphoinositide-3-OH kinase (PI3K)/Akt in breast cancer cells. Phosphatase and tensin homologue (PTEN) is a critical counter-regulator of PI3K signaling and is thus one of the major tumor suppressors in breast cancer. Inhibition of PI3K with an inhibitor, wortmannin, increased the level of PTEN protein in ERα-positive MCF-7 cells, while levels in ERα-negative MDA-MB 231 cells were not altered. In addition, the level of PTEN protein in MCF-7 cells was significantly lower than that in MDA-MB 231 cells, which correlated with high levels of phospho-Akt and phosphatidylinositol-3,4,5,-trisphosphate (PIP3). However, PTEN mRNA expression as measured by real-time PCR showed no differences in either cell line. Notably, the levels of casein kinase 2 (CK2) and phospho-PTEN (Ser380/Thr382/383) in MCF-7 cells were lower than those in MDA-MB 231 cells, indicating that the down-regulation of PTEN protein in MCF-7 cells is caused by low levels of CK2 expression, leading to accelerated PTEN degradation. Collectively, these results suggest that ERα induces the down-regulation of PTEN through PI3K activation in breast cancer cells.

Redox regulation of the tumor suppressor PTEN by glutaredoxin 5 and Ycp4.

Human PTEN (phosphatase and tensin homolog deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) expressed in Saccharomyces cerevisiae was oxidized in a time- and H(2)O(2)-concentration-dependent manner. Oxidized hPTEN was reduced by cellular reductants as in human cells. The reduction rate of oxidized hPTEN was monitored in S. cerevisiae mutants in which the genes involved in redox homeostasis had been disrupted. Reduction of hPTEN was delayed in each of S. cerevisiae grx5Δ and ycp4Δ mutants. Expression of Grx5 and Ycp4 in each of the mutants rescued the reduction rate of oxidized hPTEN. Furthermore, an in vitro assay revealed that the human Grx5/GSH system efficiently catalyzed the reduction of oxidized hPTEN. These results suggest that the reduction of oxidized hPTEN is regulated by Grx5 and Ycp4.

Redox regulation of the tumor suppressor PTEN by glutathione.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expressed in Saccharomyces cerevisiae was reversibly oxidized by hydrogen peroxide and reduced by cellular reductants. Reduction of hPTEN was delayed in each of S. cerevisiae gsh1Delta and gsh2Delta mutants. Expression of gamma-glutamylcysteine synthetase Gsh1 in the gsh1Delta mutant rescued regeneration rate of hPTEN. Oxidized hPTEN was reduced by glutathione in a concentration- and time-dependent manner. Glutathionylated PTEN was detected. Incubation of 293T cells with BSO and knockdown expression of GCLc in HeLa cells by siRNA resulted in the delay of reduction of oxidized PTEN. Also, in HeLa cells transfected with GCLc siRNA, stimulation with epidermal growth factor resulted in the increase of oxidized PTEN and phosphorylation of Akt. These results suggest that the reduction of oxidized hPTEN is mediated by glutathione.

Taxol induces oxidative neuronal cell death by enhancing the activity of NADPH oxidase in mouse cortical cultures.

We examined the involvement of oxidative stress in neuronal cell death induced by taxol, a microtubule-stabilizing anti-cancer drug and investigated whether NADPH oxidase plays a role in taxol-induced neuronal cell death in mouse cortical cultures. Cell death was assessed by measuring lactate dehydrogenase in the bathing media after 24-h exposure to taxol. Taxol (30-1000 nM) induced the concentration-dependent neuronal death with apoptotic features. The neuronal death induced by taxol was significantly attenuated not only by anti-apoptotic drugs such as z-VAD-fmk and cycloheximide but also by antioxidants such as trolox, ascorbic acid and tempol. Vinblastine, a microtubule-depolymerizing anti-cancer drug, also induced neuronal death. The neuronal cell death induced by vinblastine was also attenuated by z-VAD-fmk, but not by antioxidants and NADPH oxidase inhibitors. Exposure the cortical cultures to taxol for 80 min formed neurite beadings visualized by fluorescence immunocytochemistry for tubulin. Treatment with either trolox or apocynin, an NADPH oxidase inhibitor, did not affect formation of the neurite beadings. RT-PCR and Western blot analysis revealed that exposure to taxol increased the expression of p47(phox) and gp91(phox) and induced translocation of the p47(phox) to the membrane in cortical cultures. Exposure to taxol markedly increased cellular 2,7-dichlorofluorescin diacetate fluorescence, an indicator for reactive oxygen species. Apocynin and trolox markedly inhibited the taxol-induced increase of the fluorescence. Moreover, treatment with NADPH oxidase inhibitors or suppression of gp91(phox) by siRNA significantly attenuated the taxol-induced neuronal death. These results indicate that taxol induces oxidative neuronal apoptosis by enhancing the activity of NADPH oxidase.

Phosphorylation of paxillin tyrosines 31 and 118 controls polarization and motility of lymphoid cells and is PMA-sensitive.

Tyrosine phosphorylation of paxillin regulates actin cytoskeleton-dependent changes in cell morphology and motility in adherent cells. In this report we investigated the involvement of paxillin tyrosine phosphorylation in the regulation of actin cytoskeleton-dependent polarization and motility of a non-adherent IL-3-dependent murine pre-B lymphocytic cell line Baf3. We also assessed the effect of phorbol myristate acetate (PMA), a phorbol ester analogous to those currently in clinical trials for the treatment of leukemia, on paxillin phosphorylation. Using tyrosine-to-phenylalanine phosphorylation mutants of paxillin and phosphospecific antibody we demonstrated that IL-3 stimulated phosphorylation of paxillin tyrosine residues 31 and 118, whereas the tyrosines 40 and 181 were constitutively phosphorylated. Phosphorylation of paxillin residues 31 and 118 was required for cell polarization and motility. In the presence of IL-3, PMA dramatically reduced the phosphorylation of residues 31 and 118, which was accompanied by inhibition of cell polarization and motility. This PMA effect was partially recapitulated by expression of exogenous tyrosine 31 and 118 mutants of paxillin. We also demonstrated that PMA inhibited the IL-3-induced and activation-dependent tyrosine phosphorylation of focal adhesion kinase. Thus, our results indicate that phosphorylation of paxillin tyrosine residues 31 and 118 regulates actin-dependent polarization and motility of pre-B Baf3 cells, both of which could be inhibited by PMA. They also suggest that inhibition of upstream signaling by PMA contributes to the decrease of paxillin phosphorylation and subsequent changes in cell morphology.

KAI1 COOH-terminal interacting tetraspanin (KITENIN), a member of the tetraspanin family, interacts with KAI1, a tumor metastasis suppressor, and enhances metastasis of cancer.

We cloned recently an alternatively spliced variant of KAI1 mRNA that lacked exon 7 at the COOH-terminal region and showed differences in metastasis suppression when compared with the wild-type KAI1. These findings indicated that the COOH-terminal region of KAI1 is critical for its metastasis suppressor function. In this study, we isolated a cDNA clone of VANGL1, a member of the tetraspanin protein family, which interacted specifically with the COOH-terminal cytoplasmic domain of KAI1 in the yeast two-hybrid system. We renamed it KAI1 COOH-terminal interacting tetraspanin (KITENIN). We found that KITENIN-overexpressing CT-26 mouse colon cancer cells showed increased tumorigenicity and early hepatic metastasis in vivo, as well as increased invasiveness and adhesion to fibronectin in vitro compared with parental cells. Moreover, increased levels of KITENIN were observed in a human gastric tumor and its metastatic tissues, compared with the normal adjacent mucosa. Our results indicate that KITENIN promotes adhesion and invasion of cancer cells in vitro and in vivo, and suggest that KITENIN participates in the regulation of the tumor formation and metastasis by interacting with KAI1, a metastasis suppressor and antisense KITENIN strategy that can be used to inhibit metastasis in various cancers.

SB203580, a P38 MAPK Inhibitor, Blocks in vitro Invasion by Human Gastric SNU-638 Cells.

PURPOSE: The role of P38 mitogen-activated protein kinase (MAPK) in gastric cancer invasion has not yet been determined. In this study, we examined the effects of SB203580, a specific P38 MAPK inhibitor, on the in vitro invasion of gastric cancer and upon the molecules involved in this process. MATERIALS AND METHODS: Human gastric cancer SNU-638 cells were maintained in RPMI 1640 supplemented with 10% FBS. BIOCOAT matrigel invasion chambers were used to examine in vitro invasiveness, zymography for gelatinase activity, CAT assay for uPA promoter activity and Western and Northern blotting to determine protein and mRNA levels, respectively. RESULTS: Treatment of SNU-638 cells with SB203580, a specific P38 MAPK inhibitor, reduced in vitro invasiveness, dose-dependently. SB203580 treatment was found to decrease both mRNA expression and uPA promoter activity in gastric SNU-638 cells. In vitro invasion of SNU-638 cells was partially abrogated by uPA-neutralizing antibodies. The activities of MMPs were not significantly altered by SB203580. CONCLUSION: Our results suggest that P38 MAPK is a potential therapeutic target for inhibiting uPA-dependent gastric tumor invasiveness and metastasis.