Lun Song

Nickel Compounds Render Anti-apoptotic Effect to Human Bronchial Epithelial Beas-2B Cells by Induction of Cyclooxygenase-2 through an IKKβ/p65-dependent and IKKα- and p50-independent Pathway

The carcinogenicity of nickel compounds has been well documented both in vitro and in vivo; however, the molecular mechanisms by which nickel compounds cause cancers are far from understood. Because suppression of apoptosis is thought to contribute to carcinogenesis, we investigated the mechanisms implicated in nickel-induced anti-apoptotic effect in human bronchial epithelial (Beas-2B) cells. We found that exposure of Beas-2B cells to nickel compounds resulted in increased cyclooxygenase-2 (COX-2) expression and that small interfering RNA (siCOX-2) knockdown of COX-2 expression resulted in increased cell sensitivity to nickel-triggered cell apoptosis, demonstrating that COX-2 induction has an anti-apoptotic effect on Beas-2B cells. Overexpression of IKKβ-KM, a kinase inactive mutant of IKKβ, blocked NF-κB activation and COX-2 induction by nickel compounds, indicating that activated NF-κB may be a mediator for COX-2 induction. To further explore the contribution of the NF-κB pathway in COX-2 induction and in protection from nickel exposure, mouse embryonic fibroblasts deficient in IKKβ, IKKα, p65, and p50 were analyzed. Loss of IKKβ impaired COX-2 induction by nickel exposure, whereas knockout of IKKα had a marginal effect. Moreover, the NF-κB p65, and not the p50 subunit, was critical for nickel-induced COX-2 expression. In addition, a deficiency of IKKβ or p65 rendered cells more sensitive to nickel-induced apoptosis as compared with those in wild type cells. Finally, it was shown that reactive oxygen species H2O2 were involved in both NF-κB activation and COX-2 expression. Collectively, our results demonstrate that COX-2 induction by nickel compounds occurs via an IKKβ/p65 NF-κB-dependent but IKKα- and p50-independent pathway and plays a crucial role in antagonizing nickel-induced cell apoptosis in Beas-2B cells.

IKKβ programs to turn on the GADD45α–MKK4–JNK apoptotic cascade specifically via p50 NF-κB in arsenite response

Cross talk between NF-κB and c-Jun N-terminal kinases (JNKs) has been implicated in the cell life and death decision under various stresses. Functional suppression of JNK activation by NF-κB has recently been proposed as a key cellular survival mechanism and contributes to cancer cells escaping from apoptosis. We provide a novel scenario of the proapoptotic role of IκB kinase β (IKKβ)–NF-κB, which can act as the activator of the JNK pathway through the induction of GADD45α for triggering MKK4/JNK activation, in response to the stimulation of arsenite, a cancer therapeutic reagent. This effect of IKKβ–NF-κB is dependent on p50 but not the p65/relA NF-κB subunit, which can increase the stability of GADD45α protein through suppressing its ubiquitination and proteasome-dependent degradation. IKKβ–NF-κB can therefore either activate or suppress the JNK cascade and consequently mediate pro- or antiapoptotic effects, depending on the manner of its induction. Furthermore, the NF-κB p50 subunit can exert a novel regulatory function on protein modification independent of the classical NF-κB transcriptional activity.

Black Raspberry Extracts Inhibit Benzo(a)Pyrene Diol-Epoxide–Induced Activator Protein 1 Activation and VEGF Transcription by Targeting the Phosphotidylinositol 3-Kinase/Akt Pathway

Previous studies have shown that freeze-dried black raspberry extract fractions inhibit benzo(a)pyrene [B(a)P]-induced transformation of Syrian hamster embryo cells and benzo(a)pyrene diol-epoxide [B(a)PDE]-induced activator protein-1 (AP-1) activity in mouse epidermal Cl 41 cells. The phosphotidylinositol 3-kinase (PI-3K)/Akt pathway is critical for B(a)PDE-induced AP-1 activation in mouse epidermal Cl 41 cells. In the present study, we determined the potential involvement of PI-3K and its downstream kinases on the inhibition of AP-1 activation by black raspberry fractions, RO-FOO3, RO-FOO4, RO-ME, and RO-DM. In addition, we investigated the effects of these fractions on the expression of the AP-1 target genes, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). Pretreatment of Cl 41 cells with fractions RO-F003 and RO-ME reduced activation of AP-1 and the expression of VEGF, but not iNOS. In contrast, fractions RO-F004 and RO-DM had no effect on AP-1 activation or the expression of either VEGF or iNOS. Consistent with inhibition of AP-1 activation, the RO-ME fraction markedly inhibited activation of PI-3K, Akt, and p70 S6 kinase (p70(S6k)). In addition, overexpression of the dominant negative PI-3K mutant delta p85 reduced the induction of VEGF by B(a)PDE. It is likely that the inhibitory effects of fractions RO-FOO3 and RO-ME on B(a)PDE-induced AP-1 activation and VEGF expression are mediated by inhibition of the PI-3K/Akt pathway. In view of the important roles of AP-1 and VEGF in tumor development, one mechanism for the chemopreventive activity of black raspberries may be inhibition of the PI-3K/Akt/AP-1/VEGF pathway.

p85α Acts as a Novel Signal Transducer for Mediation of Cellular Apoptotic Response to UV Radiation

ABSTRACT Apoptosis is an important cellular response to UV radiation (UVR), but the corresponding mechanisms remain largely unknown. Here we report that the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI-3K) exerted a proapoptotic role in response to UVR through the induction of tumor necrosis factor alpha (TNF-α) gene expression. This special effect of p85α was unrelated to the PI-3K-dependent signaling pathway. Further evidence demonstrated that the inducible transcription factor NFAT3 was the major downstream target of p85α for the mediation of UVR-induced apoptosis and TNF-α gene transcription. p85α regulated UVR-induced NFAT3 activation by modulation of its nuclear translocation and DNA binding and the relevant transcriptional activities. Gel shift assays and site-directed mutagenesis allowed the identification of two regions in the TNF-α gene promoter that served as the NFAT3 recognition sequences. Chromatin immunoprecipitation assays further confirmed that the recruitment of NFAT3 to the endogenous TNF-α promoter was regulated by p85α upon UVR exposure. Finally, the knockdown of the NFAT3 level by its specific small interfering RNA decreased UVR-induced TNF-α gene transcription and cell apoptosis. The knockdown of endogenous p85α blocked NFAT activity and TNF-α gene transcription, as well as cell apoptosis. Thus, we demonstrated p85α-associated but PI-3K-independent cell death in response to UVR and identified a novel p85α/NFAT3/TNF-α signaling pathway for the mediation of cellular apoptotic responses under certain stress conditions such as UVR.

Coordination of JNK1 and JNK2 Is Critical for GADD45α Induction and Its Mediated Cell Apoptosis in Arsenite Responses

Arsenite is a well documented environmental pathogen, whereas it has also been applied as medication to treat various neoplasmas. The pathogenic and therapeutic effects of arsenite are associated with cellular apoptotic responses. However, the molecular mechanisms of arsenite-induced apoptosis are not very well understood. Our previous study has shown that arsenite exposure is able to activate JNKs, which subsequently mediate the apoptotic outcome. The present study further revealed that the coordination of JNK1 and JNK2 was critical for the arsenite-induced expression of GADD45α (growth arrest and DNA damage 45α), which in turn mediated the cellular apoptosis. The arsenite-induced apoptosis and GADD45α expression were significantly impaired in mouse embryonic fibroblasts deficient in either jnk1 (JNK1–/–) or jnk2 (JNK2–/–). Knockdown of GADD45α by its specific small interfering RNA also dramatically reduced the apoptotic responses, and overexpression of GADD45α in either JNK1–/– or JNK2–/– mouse embryonic fibroblasts partially resensitized the cell death. Furthermore, it was found that the regulation of GADD45α by JNK1 and JNK2 was achieved through mediating the activation of c-Jun, since in the JNK1–/– and JNK2–/– cells the c-Jun activation was impaired, and overexpression of the dominant negative mutant of c-Jun (TAM67) in wild type cells could also block GADD45α induction as well as cellular apoptosis. Our results demonstrate that the coordination of JNK1 and JNK2 is critical for c-Jun/GADD45α-mediated cellular apoptosis induced by arsenite.

A JNK1/AP-1–Dependent, COX-2 Induction Is Implicated in 12-O-Tetradecanoylphorbol-13-Acetate–Induced Cell Transformation through Regulating Cell Cycle Progression

Cyclooxygenase-2 (COX-2) is reported to be one of the early-response gene products induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the relevance of COX-2 in TPA-induced cell transformation and the underlying mechanisms remains to be explored. Initially, we verified COX-2 induction after TPA treatment in mouse embryonic fibroblasts (MEF) and mouse epidermal cells Cl 41. More importantly, introduction of COX-2 small interfering RNA in MEFs or Cl 41 cells suppressed the cell transformation caused by TPA treatment. This inhibition could be reversed by overexpression of human full-length COX-2, indicating that COX-2 is at least one of the critical molecules involved in TPA-induced cell transformation. We further showed that TPA-promoted cell cycle progression was partially suppressed by COX-2 small interfering RNA, indicating that COX-2 also participated in TPA-associated cell cycle progression. Investigation of the upstream signaling pathways revealed that c-Jun-NH2-kinase 1 (JNK1), but not JNK2, played important roles in COX-2 induction, because knockout of JNK1 gene rather than JNK2 gene markedly impaired COX-2 induction. Furthermore, inhibition of c-Jun/activator protein 1 pathway or JNKs/c-Jun pathway by overexpression of dominant negative mutants of c-Jun, or MKK4 and MKK7 together, resulted in impairment of COX-2 induction, suggesting that JNK1/c-Jun/activator protein 1 pathway is involved in TPA-associated COX-2 induction. In contrast, IKK/p65 nuclear factor-κB pathway was not implicated because knockout of IKKα, IKKβ, or p65 gene did not affect COX-2 induction although nuclear factor-κB was activated by TPA. In addition, the TPA-promoted cell cycle progression was found impaired in JNK1-deficient, but not in JNK2-deficient, MEFs. Our results show that JNK1-associated COX-2 induction is implicated in TPA-associated cell transformation and cell cycle progression. (Mol Cancer Res 2008;6(1):165–74)

Activation of both nuclear factor of activated T cells and inhibitor of nuclear factor-κB kinase β-subunit-/nuclear factor-κB is critical for cyclooxygenase-2 induction by benzo[a]pyrene in human bronchial epithelial cells

The carcinogenic effect of benzo[a]pyrene (B[a]P), presenting mainly in cigarette smoke and air pollution, has been well demonstrated both in vitro and in vivo. However, it is still not well understood how B[a]P facilitates pulmonary carcinogenesis. To explore this, we investigated the effect of B[a]P on the induction of cyclooxygenase‐2 (COX‐2), a critical enzyme implicated in inflammation and cancer development, as well as upstream signaling pathways leading to its expression in human bronchial epithelial cells (Beas‐2B). We found that exposure of Beas‐2B to B[a]P caused significant COX‐2 induction at both the transcriptional and protein levels. B[a]P also switched on the nuclear factor of activated T cells (NFAT) and nuclear factor κB (NF‐κB) signaling pathways. B[a]P‐induced COX‐2 expression was significantly blocked by inhibition of the NFAT pathway, and impairment of the NF‐κB signaling pathway by ectopic expression of an inhibitor of nuclear factor‐κB kinase β‐subunit (IKKβ) kinase inactive mutant (IKKβ‐KM) also dramatically inhibited COX‐2 induction. The IKKβ/NF‐κB‐dependent COX‐2 induction was further confirmed in mouse embryonic fibroblasts with IKKβ deficiency (IKKβ−/–) and in those that expressed reconstituted IKKβ. However, activation of the NFAT and NF‐κB signaling pathways by B[a]P were independent of each other, as blocking one signaling pathway didnt interrupt the activation of the other one. Mutation of either NFAT or NF‐κB binding sites significantly blocked COX‐2 promoter induction by B[a]P. Taken together, these data indicate that exposure of Beas‐2B to B[a]P can upregulate COX‐2 expression by increasing its transcription, which requires activation of both the NFAT and NF‐κB signaling pathways. (Cancer Sci 2007; 98: 1323–1329)

Knockdown of NFAT3 blocked TPA-induced COX-2 and iNOS expression, and enhanced cell transformation in Cl41 cells.

The nuclear factor of activated‐T‐cells (NFAT) family is a ubiquitous transcription factor that mediates regulation on various gene expressions. Recent studies indicate that NFAT may implicate in cancer process, mainly through its direct regulation on the cyclooxygenase‐2 (COX‐2) gene expression. There is also evidence suggesting another aspect of NFAT in tumor suppression. However, the according mechanism remains unknown. In this study, we used a small interfering RNA (siRNA) expression construct to study the role of NFAT3 in the 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced cell transformation with the tumor promotion‐sensitive mouse epidermal Cl41 cells. Our results showed that TPA was able to induce NFAT3 activation in Cl41 cells. Stable transfection of NFAT3 siRNA specifically reduced endogenous NFAT3 expression. At the same time, TPA‐induced expression of both COX‐2 and inducible nitric oxide synthase (iNOS) were blocked. However, anchorage‐independent transformation in response to TPA was significantly enhanced in NFAT3 siRNA stable transfectants as compared with vector transfectants. Moreover, treatment with the iNOS specific inhibitor aminoguanidine (AG) also enhanced Cl41 cells transformation induced by TPA. As COX‐2 expression is proved to be required for cell transformation in Cl41 cells in our recent studies, our results demonstrate that the inducible NFAT3‐mediated iNOS upregulation represents a novel potent tumor‐suppressing pathway and may contribute to the tumor suppressor functions of NFAT protein. J. Cell. Biochem. 99: 1010–1020, 2006.

GADD45α mediates arsenite-induced cell apoptotic effect in human hepatoma cells via JNKs/AP-1-dependent pathway

Arsenite (As(III)), an effective chemotherapeutic agent for the acute promyelocytic leukemia (APL) and multiple myeloma (MM), might be also a promise for the therapy of other cancers, including the solid tumors. However, the molecular bases of arsenite‐induced cytotoxicity in the tumor cells have not been fully defined. In this study, we have disclosed that arsenite effectively induces the apoptotic response in the HepG2 human hepatoma cells by triggering GADD45α induction and the subsequent activation of JNKs/AP‐1 cell death pathway. However, signaling events relating to GADD45α/JNKs/AP‐1 pathway activation have not been observed in HL7702 human diploid hepatic cells under the same arsenite exposure condition. Our results thus have illustrated the selective pro‐apoptotic role of arsenite in the hepatoma cells by activating GADD45α‐dependent cell death pathway whereas with little effect on the normal hepatic cells. The approaches to up‐regulate GADD45α levels might be helpful in improving the chemotherapeutic action of arsenite on certain solid tumors including hepatoma. J. Cell. Biochem. 109: 1264–1273, 2010.

Bid Mediates Anti-Apoptotic COX-2 Induction Through the IKKβ/NFκB Pathway Due to 5-MCDE Exposure

Although Bid is considered to be a cell apoptotic mediator, current studies suggest that it has a possible role in cell survival for mouse embryonic fibroblasts (MEFs) in response to low doses of anti-(±)-5- methylchrysene-1,2-diol- 3,4-epoxide (≤0.25μM) (5-MCDE). We found that the exposure of MEFs to 0.25 μM 5-MCDE resulted in a slight apoptotic induction, while this apoptotic response was substantially increased in the Bid knockout MEFs (Bid-/-), suggesting that there is a Bid-mediated anti-apoptotic function in this response. This notion was further supported by the findings that re-constitution expression of Bid into Bid-/- cells could inhibit the increased apoptosis. Further studies show that the antiapoptotic function of Bid was associated with its mediation of COX-2 expression. This conclusion was based the reduction of COX-2 expression in Bid-/- cells, the restoration of low sensitivity to 5-MCDE-induced apoptosis by the introduction of Bid into Bid-/- cells, and increased sensitivity of WT MEFs to 5-MCDE-induced apoptosis by the knockdown of COX-2 expression. Furthermore, we found that Bid mediated COX-2 expression through the IKKβ/NFκB pathway because the deficiency of Bid in Bid-/- MEFs resulted in the blockade of IKK/NFκB activation and knockout of IKKβ caused abrogation of COX-2 expression induced by 5-MCDE. Collectively, our results demonstrate that Bid is critical for COX-2 induction through the IKKβ/NFκB pathway, which mediates its anti-apoptotic function, in cell response to low doses of 5-MCDE exposure.