Regulation of PHLDA1 Expression by JAK2-ERK1/2-STAT3 Signaling Pathway.

Toll-like receptor 2 (TLR2)-mediated signaling cascades and gene regulation are mainly involved in diseases, such as immunity and inflammation. In this study, microarray analysis was performed using bone marrow-derived macrophages (BMDM) and Raw 264.7 cells to identify novel proteins involved in the TLR2-mediated cellular response. We found that pleckstrin homology-like domain family, member 1 (PHLDA1) is a novel gene up-regulated by TLR2 stimulation and determined the unique signaling pathway for its expression. Treatment with TLR2 agonist Pam3 CSK4 increased mRNA, protein, and fluorescence staining of PHLDA1. Induction of PHLDA1 by TLR2 stimulation disappeared from TLR2 KO mice-derived BMDM. Among janus kinase (JAK) family members, JAK2 was involved in TLR2-stimulated PHLDA1 expression. Signal transducer and activator of transcription 3 (STAT3) also participated in PHLDA1 expression downstream of the JAK2. Interestingly, ERK1/2 was an intermediate between JAK2 and STAT3. In silico analysis revealed the presence of highly conserved γ-activated sites within mouse PHLDA1 promoter and confirmed the JAK2-STAT3 pathway is important to Pam3 CSK4 -induced PHLDA1 transcription. These findings suggest that the JAK2-ERK1/2-STAT3 pathway is an important signaling pathway for PHLDA1 expression and that these proteins may play a critical role in eliciting TLR2-mediated immune and inflammatory response.

RGS2 suppresses breast cancer cell growth via a MCPIP1-dependent pathway.

Regulator of G protein signaling 2 (RGS2) is a member of a family of proteins that functions as a GTPase-activating protein (GAP) for Gα subunits. RGS2 mRNA expression is lower in breast cancerous tissues than in normal tissues. In addition, expression of RGS2 is also lower in MCF7 (cancerous breast cells) than in MCF10A (normal breast cells). Here we investigated whether RGS2 inhibits growth of breast cancer cells. RGS2 overexpression in MCF7 cells inhibited epidermal growth factor- or serum-induced proliferation. In HEK293T cells expressing RGS2, cell growth was also significantly suppressed (In addition, exogenous expression of RGS2 in HEK293T cells resulted in the significant suppression of cell growth). These results suggest that RGS2 may have a tumor suppressor function. MG-132 treatment of MCF7 cells increased endogenous or exogenous RGS2 levels, suggesting a post-transcriptional regulatory mechanism that controls RGS2 protein levels. RGS2 protein was degraded polyubiquitinated the K71 residue, but stabilized by deubiquitinase monocyte chemotactic protein-induced protein 1 (MCPIP1), and not affected by dominant negative mutant (C157A) of MCPIP1. Gene expression profiling study showed that overexpression of RGS2 decreased levels of testis specific Y encoded like protein 5 (TSPYL5), which plays a causal role in breast oncogenesis. TSPYL5 protein expression was low in MCF10A and high in MCF7 cells, showing the opposite aspect to RGS2 expression. Additionally, RGS2 or MCPIP1 overexpression in MCF7 cells decreased TSPYL5 protein level, indicating that RGS2 stabilized by MCPIP1 have diminished TSPYL5 protein levels, thereby exerting an inhibitory effect of breast cancer cell growth.

Role of JAK2-STAT3 in TLR2-mediated tissue factor expression.

Tissue factor (TF) is a core protein with an essential function in the coagulation cascade that maintains the homeostasis of the blood vessels. TF not only participates in neointima formation, but also causes the development of atherosclerosis. This study investigated the mechanism regulating TF expression in macrophages using Pam3 CSK4 , a TLR2 ligand. Pam3 CSK4 induced TF expression in two types of macrophages (Raw264.7 and BMDM), but not in TLR2 KO mice derived BMDM. Pam3 CSK4 induced TF expression was inhibited by pretreatment with pan-JAK inhibitor or JAK2 inhibitor AG490. JAK2 knock-down by siRNA inhibited Pam3 CSK4 induced TF expression. Pam3 CSK4 stimulated STAT3 phosphorylation (S727), while STAT3 knock-down by siRNA reduced Pam3 CSK4 induced TF expression. These results suggest that Pam3 CSK4 induced TF expression is regulated by the JAK2-STAT3 signaling pathway. Pam3 CSK4 , unlike increased TF expression, significantly decreased RGS2 expression, while RGS2 overexpression decreased Pam3 CSK4 induced TF expression. Inhibition of TF by RGS2 WT did not occur in mutants with flawed RGS domains. We also investigated the correlation between RGS2 and STAT3 phosphorylation. RGS2 knock-down elevated Pam3 CSK4 induced STAT3 phosphorylation, but RGS2 overexpression had the opposite effect on STAT3 phosphorylation. These results suggest that, while Pam3 CSK4 induced TF expression is regulated by JAK2-STAT3 signaling, RGS2 is a negative regulator targeted to STAT3.

TLR2 stimulates ABCA1 expression via PKC-η and PLD2 pathway.

ATP-binding cassette transporter A1 (ABCA1) is a membrane-bound protein that regulates cardiovascular disease including atherosclerosis by the efflux of excess cholesterol from cells and by suppression of inflammation. Using a mouse macrophage cell line Raw264.7, we studied the importance of toll-like receptor 2 (TLR2) on ABCA1 expression and the signaling pathway responsible for TLR2-mediated ABCA1 expression. Interestingly, our data demonstrated that treatment of macrophages with TLR2 agonist Pam(3)CSK(4) significantly increased ABCA1 mRNA and protein levels. We found that ABCA1 induction is myeloid differentiation primary response gene 88 (MyD88)-dependent as well as TLR2-dependent. ABCA1 induction upon Pam(3)CSK(4) is controlled by protein kinase C-η (PKC-η) and phospholipase D2 (PLD2). Furthermore, direct treatment of dioctanoyl phosphatidic acid (diC(8)PA) into cells also induced ABCA1 mRNA and protein indicating that PLD2-mediated PA involve in the TLR2-stimulated ABCA1 expression. Cumulatively, these results demonstrate for the first time that activation of PKC-η and PLD2 signaling pathway is an important mechanism for regulation of TLR2-induced ABCA1 expression.

Calcium-independent phospholipase A2beta-Akt signaling is involved in lipopolysaccharide-induced NADPH oxidase 1 expression and foam cell formation.

Foam cell formation is the most important process in atherosclerosis, and low density lipoprotein oxidation by reactive oxygen species (ROS) is the key step in the conversion of macrophages to foam cells. This study reveals the control mechanism of the gene for NADPH oxidase 1 (Nox1), which produces ROS in the formation of foam cells by stimulating TLR4. Treatment of macrophages by the TLR4 agonist LPS stimulated ROS production and ROS-mediated macrophage to foam cell conversion. This LPS-induced ROS production and foam cell formation could be abrogated by pretreatment of macrophages with N-acetyl cysteine or apocynin. LPS increased Nox1 promoter activity, and resultant expression of mRNA and protein. Small interfering RNA mediated inhibition of Nox1 expression decreased LPS-induced ROS production and foam cell formation. LPS-mediated Nox1 expression and the responses occurred in a calcium-independent phospholipase A(2) (iPLA(2))-dependent manner. The iPLA(2)beta-specific inhibitor S-BEL or iPLA(2)beta small interfering RNA attenuated LPS-induced Nox1 expression, ROS production, and foam cell formation. In addition, activation of iPLA(2)beta by LPS caused Akt phosphorylation and was followed by increased Nox1 expression. These results suggest that the binding of LPS and TLR4 increases Nox1 expression through the iPLA(2)beta-Akt signaling pathway, and control ROS production and foam cell formation.

Early growth response-1 is involved in foam cell formation and is upregulated by the TLR9-MyD88-ERK1/2 pathway.

Early growth response-1 (Egr-1), a zinc finger transcription factor, plays a key regulatory role in pathological cardiovascular processes including atherosclerosis. Here, we investigate whether Egr-1 expression and foam cell formation require toll-like receptor 9 (TLR9) and myeloid differentiation factor 88 (MyD88). CpG DNA and its related synthetic CpG oligodeoxynucleotides (CpG ODN) play an important role in immunity and inflammation. CpG ODN increased expression of Egr-1 and formation of foam cells in Raw264.7 cells or bone marrow-derived macrophages. Egr-1 siRNA decreased foam cell formation by CpG ODN compared to that of control siRNA. In addition, when TLR9 or MyD88 was knocked down, CpG ODN-induced Egr-1 expression was also attenuated. CpG ODN increased ERK1/2 phosphorylation. U0126, a MEK pathway inhibitor, suppressed activation of Egr-1 expression by CpG ODN. CpG ODN-induced expression of tissue factor (TF) and NGFI-A binding protein 2 (Nab2), and the expression of both genes is blocked by knockdown of TLR9 or MyD88 siRNA or MEK inhibition. These results suggest that CpG ODN activates the TLR9-MyD88-ERK1/2 pathway causing expression of Egr-1 and its target genes such as TF and Nab2, thus inducing foam cell formation.

Resveratrol inhibits foam cell formation via NADPH oxidase 1- mediated reactive oxygen species and monocyte chemotactic protein-1.

Resveratrol is a polyphenolic compound in red wine that has anti-oxidant and cardioprotective effects in animal models. Reactive oxygen species (ROS) and monocyte chemotactic protein-1 (MCP-1) play key roles in foam cell formation and atherosclerosis. We studied LPS-mediated foam cell formation and the effect of resveratrol. Resveratrol pretreatment strongly suppressed LPS-induced foam cell formation. To determine if resveratrol affected the expression of genes that control ROS generation in macrophages, NADPH oxidase 1 (Nox1) was measured. Resveratrol treatment of macrophages inhibited LPS-induced Nox1 expression as well as ROS generation, and also suppressed LPS-induced MCP-1 mRNA and protein expression. We investigated the upstream targets of Nox1 and MCP-1 expression and found that Akt-forkhead transcription factors of the O class (FoxO3a) is an important signaling pathway that regulates both genes. These inhibitory effects of resveratrol on Nox1 expression and MCP-1 production may target to the Akt and FoxO3a signaling pathways.

Toll-like receptor 9-stimulated monocyte chemoattractant protein-1 is mediated via JNK-cytosolic phospholipase A2-ROS signaling.

Monocyte chemoattractant protein-1 (MCP-1) influences monocyte migration into sites of inflammation. This study highlights the importance of cytosolic phospholipase A2 (cPLA2)-mediated reactive oxygen species (ROS) signaling processes in the regulation of MCP-1 release as a result of toll-like receptor (TLR) activation. In macrophages, activation of TLR9 induced MCP-1 and cPLA2-phosphorylated arachidonic acid (AA) release. Inhibition of cPLA2 blocked CpG-induced MCP-1 and AA release. Although CpG stimulates phosphorylation of ERK, p38 and JNK, only inhibition of the JNK signaling pathways attenuated MCP-1 release, suggesting that the TLR9-mediated MCP-1 release was dependent upon the JNK pathway. TLR9 activation also stimulated ROS generation, while inhibition of NADPH oxidases (Noxs) blocked CpG-induced MCP-1 release. The CpG treatment increased macrophage Nox1 mRNA level, however it had no effect on macrophage Nox2 mRNA level. Overall, these results suggest that CpG enhances ROS generation through cPLA2-dependent pathways, which results in MCP-1 release.

Phosphatidic acid as a regulator of matrix metalloproteinase-9 expression via the TNF-alpha signaling pathway.

Phosphatidic acid (PA) is implicated in pathophysiological processes associated with cellular signaling events and inflammation, which include the expressional regulation of numerous genes. Here, we show that PA stimulation increases matrix metalloproteinase-9 (MMP-9) expression in macrophages through tumor necrosis factor (TNF)-alpha signaling. We performed antibody array analysis on proteins from macrophages stimulated with PA. PA was found to induce the production of TNF-alpha, but not of TNF receptor (TNFR)1 and TNFR2 in a time-dependent manner and stimulated significant, though delayed, MMP-9 expression. PA induced the phosphorylations of both ERK1/2 and p38, but not of c-jun amino-terminal kinase. Moreover, only ERK1/2 inhibition by U0126 suppressed PA-induced TNF-alpha production and MMP-9 expression. Neutralizing TNF-alpha, TNFR1 or TNFR2 antibodies significantly suppressed PA-induced MMP-9 expression, suggesting that the production of TNF-alpha in response to PA preceded the expression of MMP-9. Moreover, lipopolysaccharide-induced PA also led to TNF-alpha release and resulted in MMP-9 expression. Taken together, these observations suggest that PA may play a role in MMP-9 regulation through ERKs/TNF-alpha/TNFRs-dependent signaling pathway.

Secretory phospholipase A2 induces apoptosis through TNF-alpha and cytochrome c-mediated caspase cascade in murine macrophage RAW 264.7 cells.

Phospholipase A2 (PLA2) is an esterase that cleaves the sn-2 ester bond in glycerophospholipids, thereby releasing free fatty acids and lysophospholipids. In addition to the apoptotic activity of cytosolic PLA2 and Ca2+-independent PLA2, recent studies showed that secretory PLA2 (sPLA2) also play a role in apoptosis. However, the details of molecular mechanism have not been fully elucidated. Our data demonstrated that group IB PLA (IB PLA2)-exposed murine macrophage 264.7 cells showed characteristic features of apoptosis such as morphological changes, DNA laddering, staining positive for propidium iodide (PI) as well as Annexin V and activation of caspases and subsequent cleavage of poly (ADP-ribose) polymerase (PARP) in dose- and time-dependent manner. Moreover, IB PLA2 was found to elicit tumor necrosis factor (TNF)-alpha production and release of cytochrome c, suggesting that IB PLA2 exerts its apoptotic activity via the induction of TNF-alpha production and cytochrome c release, which results in triggering the activation of caspase cascade and PARP cleavage.

TRIF is a regulator of TLR2-induced foam cell formation.

The activation of toll-like receptor 2 (TLR2) stimulates foam cell formation, which is a key early event in the process of atherosclerosis. In the present study, the role of toll/interleukin-1 receptor-domain-containing adaptor-inducing interferon-ß (TRIF) in TLR2-mediated foam cell formation was investigated, and the importance of monocyte chemoattractant protein­1 (MCP­1), tissue factor (TF) and lectin­like oxidized low­density lipoprotein receptor­1 (Lox­1) were examined. Treatment of Raw 264.7 cells with the TLR2 agonist. Pam3CSK4, increased the gene expression of TRIF in a time­dependent manner (RT­PCR). The induced gene expression of TRIF stimulated by TLR2 was not observed in TLR2­knockout mice­derived bone marrow­derived macrophages (BMDMs). Pam3CSK4 increased the mRNA expression of TRIF in the wild­type BMDMs, but not in the TLR2­knockout BMDMs. Knockdown of the expression of TRIF using small interfering RNA decreased Pam3CSK4­induced foam cell formation (combination of oil­red O and hematoxylin staining), suggesting a role of TRIF. Stimulation of TLR2 increased the expression levels of various genes, which are known to control atherosclerosis, including MCP­1, TF and Lox­1. The knockdown of TRIF also attenuated the Pam3CSK4­induced expression of these genes. In addition, a reduction in TRIF affected the Pam3CSK4­induced protein expression of MCP­1 (EIA). Taken together, the results of the present study suggested that TRIF regulated foam cell formation via regulation of the expression levels of MCP­1, TF and Lox­1.

Resveratrol inhibits inflammation induced by heat-killed Listeria monocytogenes.

Resveratrol is a polyphenolic compound in red wine that has antioxidant and cardioprotective effects in animal models. Listeria monocytogenes is a pathogen that mainly affects immunocompromised individuals and is initially detected at the cell surface or in phagosomes by toll-like receptor 2. Many antioxidants also exert anti-inflammatory activities; therefore, we evaluated the anti-inflammatory properties of resveratrol by studying the various inflammatory responses induced by heat-killed L. monocytogenes (HKLM). Resveratrol strongly blocked HKLM-induced NADPH oxidase-1 mRNA and reactive oxygen species production by macrophages. Resveratrol also suppressed monocyte chemotactic protein-1 expression, cyclooxygenase-2 expression, prostaglandin production, inducible nitric oxide (NO) synthase expression, and NO production induced by HKLM. We investigated the signaling pathway involved in the resveratrol effect. HKLM stimulated glycogen synthase kinase 3ß (GSK3ß) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. The involvement of GSK3ß and ERK1/2 was tested using inhibitors. While the GSK3ß inhibitor LiCl potentiated the effect of HKLM, the MEK inhibitor U0126 blocked these responses. Additionally, pretreatment with resveratrol blocked phosphorylation of both kinases induced by HKLM. These results suggest that HKLM is strong inducer of inflammatory mediators, and that the inhibitory effect of resveratrol may be mediated by the GSK3ß and ERK1/2 pathways.

TRAIL is involved in CpG ODN-mediated anti-apoptotic signals.

Synthetic oligodeoxynucleotides (ODNs) with the CpG-motifs are recognized by toll-like receptor 9 (TLR9), which elicits an immune response. Serum starvation of Raw264.7 cells increased tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression. However, treatment with CpG ODN reduced TRAIL expression as well as apoptosis by serum starvation. In serum starved cells, TLR9 inhibitors recovered the decreasing TRAIL expression and sub-G1 accumulation by CpG ODN. CpG ODN-regulated anti-apoptotic signals which were dependent on the Akt-FoxO3a signaling pathway. CpG ODNs activated Akt and inactivated FoxO3a in serum starved cells. Knockdown of FoxO3a by siRNA decreased TRAIL expression and apoptosis in serum-starved cells. In contrast, FoxO3a overexpression increased apoptosis by serum starvation, and CpG ODNs blocked these effects through TRAIL expression. LY294002, a PI3K-Akt inhibitor, blocked the CpG ODN effect of TRAIL expression and the sub-G1 population in serum starved cells. In contrast, overexpression of wild-type Akt reduced additional sub-G1 cells both in non-CpG ODN- and CpG ODN-treated cells. Taken together, these results demonstrate the involvement of Akt-FoxO3a signaling in TLR9-mediated downregulation of TRAIL and anti-apoptotic signals.

The JAK2-Akt-glycogen synthase kinase-3ß signaling pathway is involved in toll-like receptor 2-induced monocyte chemoattractant protein-1 regulation.

Monocyte chemoattractant protein-1 (MCP-1) is an essential cytokine for the migration of monocytes into vessels, and is also involved in the pathogenesis of atherosclerosis. In this study, we investigated the importance of janus kinase 2 (JAK2) and the function of the Akt and glycogen synthase kinase-3ß (GSK3ß) pathway in toll-like receptor (TLR2)-mediated MCP-1 expression. The TLR2 agonist, Pam3CSK4, induced MCP-1 expression in the Raw264.7 cell line. The induction of MCP-1 was seen in the bone marrow-derived macrophages of wild-type mice but not in TLR2 knockout mice. The TLR2-mediated MCP-1 induction was myeloid differentiation primary response gene 88 (MyD88)-independent. By contrast, the inactivation of JAK2 attenuated TLR2-mediated MCP-1 expression. The JAK inhibitor suppressed the phosphorylation of GSK3ß as well as Akt by Pam3CSK4 stimulation. While the inactivation of Akt by LY294002 suppressed TLR2-mediated MCP-1 induction, the inactivation of GSK3ß by LiCl potentiated TLR2-mediated MCP-1 induction. Furthermore, Akt inhibitor suppressed TLR2-mediated phosphorylation of GSK3ß. Taken together, these results suggest that a MyD88-independent pathway exists in TLR2 signaling; the JAK2-Akt-GSK3ß pathway is a novel MyD88-independent pathway for MCP-1 induction.

RGS2 is a negative regulator of STAT3-mediated Nox1 expression.

NADPH oxidase 1 (Nox1) is essential for reactive oxygen species production in the innate immune responses mediated by toll-like receptor (TLR), but the mechanism regulating its expression remains uncertain. Here, we find that Nox1 induction is TLR2-dependent, but independent of myeloid differentiation primary response gene 88 (MyD88). We demonstrate the capacity of signal transducer and activator of transcription 3 (STAT3) to activate Nox1's transcription, as well as cooperative regulation by janus kinase 1 and 3 (JAK1 and JAK3). We find that regulator of G-protein signaling 2 (RGS2) inhibits STAT3-mediated Nox1 transcription, and can itself be repressed by TLR2; Nox1 induction upon RGS2 down-regulation is controlled by protein kinase C-η (PKC-η) and phospholipase D2 (PLD2). A GFP-tagged version of RGS2 concentrates in the nucleus; RGS2 additionally directly binds STAT3 to regulate its transcriptional activity through TLR2 stimulation. Cumulatively, these results suggest that TLR2 signaling enhances Nox1 expression through the JAK1/3-STAT3 pathway, and that RGS2, through its regulation by the PKC-η/PLD2 pathway, represses STAT3's transcriptional activation of Nox1.

Toll-like receptor 9-mediated inhibition of apoptosis occurs through suppression of FoxO3a activity and induction of FLIP expression.

Synthetic oligodeoxynucleotides (ODN) with a CpG-motif are recognized by Toll-like receptor 9 (TLR9) and pleiotropic immune responses are elicited. Stimulation of macrophages with TLR9 agonist prevented apoptosis induced by serum deprivation through increased expression of FLICE-like inhibitory protein (FLIP). CpG ODN-mediated anti-apoptosis depended on the TLR9-Akt-FoxO3a signaling pathway. Inhibition of TLR9 by small interfering (si) RNA or an inhibitor suppressed CpG ODN-mediated anti-apoptosis. Analysis of signaling pathways revealed that the anti-apoptotic effect of CpG ODN required phosphorylation of FoxO3a and its translocation from the nucleus to the cytosol. Overexpression of FoxO3a increased apoptosis induced by serum deprivation and CpG ODN blocked these effects through FLIP expression. In contrast, siRNA knock-down of FoxO3a decreased apoptosis by serum deprivation. In addition, Akt activation was involved in CpG ODN-induced phosphorylation of FoxO3a, expression of FLIP, and anti-apoptosis. Taken together, these results demonstrate the involvement of Akt-FoxO3a in TLR9-mediated anti-apoptosis and indicate that FoxO3a is a distinct regulator for FLIP expression.

A combination of Lox-1 and Nox1 regulates TLR9-mediated foam cell formation.

The formation of foam cells is the hallmark of early atherosclerotic lesions, and the uptake of modified low-density lipoprotein (LDL) by macrophage scavenger receptors is thought to be a key process in their formation. In this study, we examined the role of lectin-like oxLDL receptor-1 (Lox-1) and NADPH oxidase 1 (Nox1) in toll-like receptor 9 (TLR9)-mediated foam cell formation. TLR9 activation of Raw264.7 cells or mouse primary peritoneal macrophages by CpG ODN treatment enhanced Lox-1 gene and protein expression. In addition, CpG ODN-induced Nox1 mRNA expression, which in turn increased foam cell formation. The inhibition of CpG ODN-induced reactive oxygen species (ROS) generation by treatment with antioxidants, as well as with knockdown of Nox1 using siRNA, suppressed the formation of foam cells. The induction of Lox-1 and Nox1 by CpG ODN was regulated via the TLR9-p38 MAPK signaling pathway. CpG ODN also increased NFkappaB activity, and a potent inhibitor of NFkappaB that significantly blocked CpG-induced Nox1 expression, suggesting that Nox1 regulation is mediated through an NFkappaB-dependent mechanism. Taken together, these results suggest that a combination of Lox-1 and Nox1 plays a key role in the TLR9-mediated formation of foam cells via the p38 MAPK pathway.

Activation of toll-like receptor 4 modulates vascular endothelial growth factor synthesis through prostacyclin-IP signaling.

In this study, we show that activation of toll-like receptor (TLR)4 by lipopolysaccharide (LPS) induces cyclooxygenase-2 (COX-2) expression, which results in prostaglandin (PG)I2 formation in macrophages. The LPS-stimulated COX-2 expression and PGI2 release were accompanied by production of the potent angiogenic cytokine, vascular endothelial growth factor (VEGF), and these effects were suppressed by NS-398, which is a COX-2 inhibitor. Direct addition of iloprost (an analogue of PGI2) for IP receptor also induced the production of VEGF, whereas DP, FP, and TP receptor agonists did not. Inhibition of IP protein expression by micro interfering RNA blocked LPS-induced VEGF production. Additionally, macrophages transiently caused Akt phosphorylation after stimulation with LPS, and inhibition of Akt phosphorylation blocked the production of VEGF and COX-2 expression in response to LPS. Overall, this study demonstrated that engagement of TLR4 with LPS induces production of PGI2 via Akt and generates VEGF through IP receptor.

Arsenic trioxide induces apoptosis in human colorectal adenocarcinoma HT-29 cells through ROS.

PURPOSE: Treatment with arsenic trioxide (As(2)O(3)) results in a wide range of cellular effects that includes induction of apoptosis, inhibition of cell growth, promotion or inhibition of cellular differentiation, and inhibition of angiogenesis through a variety of mechanisms. The mechanisms of As(2)O(3)-induced cell death have been mainly studied in hematological cancers, and those mechanisms in solid cancers have yet to be clearly defined. In this study, the mechanisms by which As(2)O(3) induces apoptosis in human colorectal adenocarcinoma HT-29 cells were investigated. MATERIALS AND METHODS: To examine the levels of apoptosis, HT-29 cells were treated with As(2)O(3) and then we measured the percentage of Annexin V binding cells, the amount of ROS production and the mitochondrial membrane potential. Western blot analysis was performed to identify the activated caspases after As(2)O(3) exposure, and we compared the possible target molecules of apoptosis. As(2)O(3) treatment induced the loss of the mitochondrial membrane potential and an increase of ROS, as well as activation of caspase-3, -7, -9 and -10. RESULTS: As(2)O(3) induced apoptosis via the production of reactive oxygen species and the loss of the mitochondrial membrane potential. As(2)O(3) induced the activation of caspase-3, -7, -9 and -10. Furthermore, As(2)O(3) treatment downregulates the Mcl-1 and Bcl-2 expressions, and the release of cytochrome c and an apoptosis-inducing factor (AIF). Pretreating the HT-29 cells with N-acetyl-L-cysteine, which is a thiol-containing antioxidant, inhibited the As(2)O(3)-induced apoptosis and caspase activation. CONCLUSION: Taken together, these results suggest that the generation of reactive oxygen species (ROS) by As(2)O(3) might play an important role in the regulation of As(2)O(3)-induced apoptosis. This cytotoxicity is mediated through a mitochondria-dependent apoptotic signal pathway in HT-29 cells.