NF-κB Reporter Gene Assay Research Articles

Discovery of the Alternaria mycotoxins alterperylenol and altertoxin I as novel immunosuppressive and antiestrogenic compounds in vitro.

Alternaria mycotoxins may pose significant challenges to food safety and public health due to the wide spectrum of reported adverse effects. Despite this, critical information on the immunomodulatory and antiestrogenic properties of most of these contaminants is still lacking. The present study aimed to identify the mycotoxins responsible for the immunosuppressive and antiestrogenic effects of a complex extract of Alternaria mycotoxins (CE) obtained by growing an Alternaria alternata strain on rice. Through a toxicity-guided fractionation procedure involving the production of CE-fractions by supercritical fluid chromatography and mycotoxin quantification by LC-MS/MS, the mycotoxins alternariol (AOH), tenuazonic acid (TeA), altertoxin I (ATX-I), and alterperylenol (ALTP) were identified as potential toxicologically relevant constituents contributing to the in vitro effects exerted by the extract. The assessment of the immunomodulatory effects, performed by applying the NF-κB reporter gene assay in THP1-Lucia™ monocytes, revealed the scarce contribution of AOH to the effects exerted by the CE. TeA showed no effect on the NF-κB pathway up to 250µM, whereas ATX-I and ALTP suppressed the LPS-mediated pathway activation at concentrations ≥ 1µM. The evaluation of antiestrogenic effects, performed in Ishikawa cells by applying the alkaline phosphatase assay, revealed the ability of ALTP (≥ 0.4µM) and ATX-I (≥ 2µM) to suppress the estrogen-dependent expression of enzyme activity. Given the risk of detrimental impacts stemming from alterations in endocrine and systemic immune responses by the investigated mycotoxins, further studies are needed to elucidate their underlying mechanisms of action and comprehensively evaluate the health risks posed by these toxins.

Anti-metastatic function of triterpene phytochemicals from guggul by targeting tumor-intrinsic NF-kB activation in triple-negative breast cancer cells

BackgroundThe endogenous activity of NF-κB is often elevated in various types of cancer cells, particularly breast cancer, and is strongly correlated with the induction of target genes that can inhibit apoptosis, interact with cell-cycle regulation, promote cell invasion and contribute to tumorigenesis, inflammation, and metastatic growth as well as drug resistance. Thus, the downregulation of NF-κB is regarded as a promising therapeutic target to inhibit breast cancer growth and metastasis. PurposeSince no NF-κB inhibitor has yet been approved by the FDA, we aim to identify small molecules from natural resources that could potentially inhibit NF-κB and suppress breast cancer metastasis. Study designIn the current study, nineteen extracts from different valuable medicinal plants were screened for their prospective biological activity against NF-κB transcriptional activation in 4T1 breast cancer cells, followed by isolation and purification analysis of the major active compounds from guggul extract, as it exhibited the most inhibitory efficacy of NF-κB activation. ResultsA bioassay-guided isolation of active constituents from the promising candidate, guggul methanol extract (GU-M), using NF-κB reporter gene assay was performed to identify potential anti-metastatic agents for triple-negative breast cancer cells (TNBC) and resulted in the isolation of four known major compounds namely; guggulsterone M (1), guggulsterol Ι (2), myyrhanol A (3), and myyrhanone A (4). ConclusionAmong the isolated compounds, myrrhanol A and myrrhanone A at the concentration of 10 μM markedly inhibited the intrinsic NF-κB pathway and potently suppressed the proliferative and metastatic abilities of murine and human breast cancer cells; 4T1, MDA-MB-231, and MDA-MB-453.

Revealing the suppressive role of protein kinase C delta and p38 mitogen-activated protein kinase (MAPK)/NF-κB axis associates with lenvatinib-inhibited progression in hepatocellular carcinoma in vitro and in vivo.

Nuclear factor-kappa B (NF-κB), an oncogenic transcription factor, modulates tumor formation and progression by inducing the expression of oncogenes involved in proliferation, survival, angiogenesis, and metastasis. Oral multikinase inhibitors, such as sorafenib, regorafenib, and lenvatinib have been used for the treatment of hepatocellular carcinoma (HCC). Both sorafenib and regorafenib were shown to abolish the NF-κB-mediated progression of HCC. However, the effect of lenvatinib on NF-κB-mediated progression of HCC is ambiguous. Therefore, the primary purpose of the present study was to evaluate the inhibitory effect of lenvatinib and its inhibitory mechanism on the NF-κB-mediated progression of HCC in vitro and in vivo. Here, we used two HCC cell lines to identify the cytotoxicity, apoptosis and metastasis effect of lenvatinib. We also applied a Hep3B-bearing animal model to investigate the therapeutic efficacy of lenvatinib on in vivo model. An NF-κB translocation assay, NF-κB reporter gene assay, a Western blotting assay and immunohistochemistry staining were used to investigate the underlying mechanism by which lenvatinib acts on HCC. In this study, we demonstrated that lenvatinib induced extrinsic/intrinsic apoptosis and suppressed the metastasis of HCC both in vitro and in vivo. Lenvatinib may also suppress NF-κB translocation and activation. We also found both protein kinase C delta (PKC-δ) and p38 mitogen-activated protein kinase (MAPK) inactivation participated in lenvatinib-reduced NF-κB signaling. In conclusion, this study reveals that the suppression of PKC-δ, and the p38 MAPK/NF-κB axis is associated with the lenvatinib-inhibited progression of HCC in vitro and in vivo.

Suppression of EGFR/PKC-δ/NF-κB Signaling Associated With Imipramine-Inhibited Progression of Non-Small Cell Lung Cancer.

BackgroundAnti-depressants have been reported to own anti-tumor potential types of cancers; however, the role of imipramine in non-small cell lung cancer (NSCLC) has not been elucidated. Epidermal growth factor receptor (EGFR) was known to be one of the key regulators that control NSCLC progression. Whether EGFR would be the target of imipramine for suppressing tumor signaling transduction and results in anti-tumor potential is remaining unclear.MethodsWe used CL-1-5-F4 cells and animal models to identify the underlying mechanism and therapeutic efficacy of imipramine. Cytotoxicity, apoptosis, invasion/migration, DNA damage, nuclear translocation of NF-κB, activation of NF-κB, phosphorylation of EGFR/PKC-δ/NF-κB was assayed by MTT, flow cytometry, transwell, wound healing assay, comet assay, immunofluorescence staining, NF-κB reporter gene assay and Western blotting, respectively. Tumor growth was validated by CL-1-5-F4/NF-κB-luc2 bearing animal model.ResultsImipramine effectively induces apoptosis of NSCLC cells via both intrinsic and extrinsic apoptosis signaling. DNA damage was increased, while, invasion and migration potential of NSCLC cells was suppressed by imipramine. The phosphorylation of EGFR/PKC-δ/NF-κB and their downstream proteins were all decreased by imipramine. Similar tumor growth inhibition was found in imipramine with standard therapy erlotinib (EGFR inhibitor). Non-obvious body weight loss and liver pathology change were found in imipramine treatment mice.ConclusionImipramine-triggered anti-NSCLC effects in both in vitro and in vivo model are at least partially attributed to its suppression of EGFR/PKC-δ/NF-κB pathway.

Abstract 1838: LBL-024, an anti-PD-L1 and 4-1BB bispecific antibody with highly differentiated binding affinity, shows anti-tumor efficacy in a mouse tumor model

Abstract Objective: 4-1BB is an inducible costimulatory receptor expressed on activated T and NK cells. 4-1BB ligation on T cells triggers a signaling cascade that results in upregulation of antiapoptotic molecules, cytokine secretion, and enhanced effector function. PD-L1, a ligand of PD-1, is mainly expressed on tumor cells, and a key checkpoint inhibitor implicated in tumor immune evasion. A tetravalent bispecific antibody LBL-024, targeting both PD-L1 and 4-1BB, was generated to maximize anti-tumor immunity. Methods: The bispecific antibody was designed as 2:2 IgG-scFv format with an affinity ratio of approximately 500:1 between two molecules. The binding affinity of LBL-024 to human PD-L1 and 4-1BB was determined by ForteBio. The activity of LBL-024 was determined using several in vitro assays, including PD-1/NFAT, 4-1BB/NF-κB reporter gene and PBMC activation assays. The in vivo anti-tumor activity of LBL-024 was investigated in the PD-L1/4-1BB double knock-in mouse model (Biocytogen) implanted with MC38 cells expressing PD-L1. Results: The binding affinity of LBL-024 to PD-L1 and 4-1BB was designed with affinity differentiation (~500-fold) between two molecules, with 0.29 nM to PD-L1 and 146 nM to 4-1BB, respectively, which may restrict activating 4-1BB signal on T cells only in PD-L1 expressing tumor microenvironment, rather activating T cells systemically. In vitro LBL-024 could block PD-1-PD-L1 interaction, recovering NFAT reporter signal comparable to anti-PD-L1 antibody. In 4-1BB/NF-κB reporter gene assay, LBL-024 activated the reporter gene system, only in the presence of CHO-K1-PD-L1 cells, suggesting possibly more restricted activation of 4-1BB within tumor microenvironment and potentially less toxicity in patients. The potency of LBL-024 on 4-1BB/NF-κB reporter gene assay was much stronger than Urelumab, a 4-1BB antibody. In pre-activated human PBMC, co-cultured with CHO-K1-PD-L1 cells, IL-2 level was significantly elevated more in LBL-024 treated group than using an anti-PD-L1, anti-4-1BB, or even the combination of anti-PD-L1 and anti-4-1BB was applied. In a model of huPD-L1/hu4-1BB double knock-in mice implanted with MC38-PD-L1 cells. LBL-024 was shown a significant anti-tumor activity (p<0.001), with 81.8% of tumor growth inhibition (TGI), more efficacious than mice dosed with anti-PD-L1 (70.5% of TGI) and anti-4-1BB (46.1% of TGI). Conclusions: LBL-024, an anti-PD-L1 and 4-1BB bispecific antibody was shown a great synergetic anti-tumor efficacy in a mouse tumor model, more effective than the antibodies targeting either PD-L1 or 4-1BB alone. With highly differentiated binding affinity between two molecules, the efficacy and toxicity could be well balanced, to avoid the potential toxicity of systemic activation of 4-1BB, hence a promising bispecific antibody for further clinical development. Citation Format: Xiao Huang, Jianming Sun, Yurong Qin, Huan Lin, Jing Guan, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-024, an anti-PD-L1 and 4-1BB bispecific antibody with highly differentiated binding affinity, shows anti-tumor efficacy in a mouse tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1838.

Abstract 1000: Treatment efficiency of regorafenib combined with magnolol was associated with suppression of VEGF and Mcl-1 on hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) was known as a malignant tumor that has been reported with vascular endothelial growth factor (VEGF) and myeloid cell leukemia 1 (Mcl-1) overexpression. Both VEGF and Mcl-1 are the important downstream factors of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) which was also reported to highly associated with HCC progression. Regorafenib a multi-kinase inhibitor of HCC can inhibit part of the expression of VEGF and Mcl-1 in HCC. However, the single treatment efficiency of regorafenib on HCC still needs to be improved. Due to the important role of VEGF and Mcl-1 in HCC progression, we hypothesize that enhance the inhibition of VEGF and Mcl-1 could ameliorate regorafenib therapeutic effect. Magnolol is a natural compound extract from the bark of Magnolia officinalis. It has been demonstrated as a multifunctional bioactive compound by several studies, including anti-inflammation, antioxidation, anti-cancer⋯etc. In this study, we aim to combine magnolol with regorafenib to enhance the inhibition efficacy of VEGF and Mcl-1 and thus improve therapeutic efficiency on HCC. We used SK-Hep1 and Hep3B cells to perform this project. In in vitro study, recombination protein VEGF, VEGF interference RNA (siVEGF), Mcl-1 interference RNA (siMcl-1) and magnolol were used to investigate the mechanism of these reagents combine with regorafenib. Here we used MTT assay and NF-κB reporter gene assay to investigate the viability of cells and the regulation of NF-κB under VEGF induction condition and/or combined with regorafenib. To validate the alteration of apoptosis and migration after regorafenib combined with magnolol, we performed flow cytometry, immunofluorescence, comet assay, and transwell assay. For in vivo study, Hep3B cells were subcutaneously inject into nude mice and treated magnolol, regorafenib, and combination treatment. The protein regulation of regorafenib combined with magnolol on tumor tissues was assayed by Western blotting. The suppression of VEGF and Mcl-1 was markedly enhanced in regorafenib combined with magnolol as compare to single treatment. In addition, the apoptosis effect was effectively increased and the migration ability were effectively suppressed by regorafenib combined with magnolol as compare to single treatment. Greatest Hep3B tumor growth inhibition was found in the combination treatment group as compared to single treatments. IHC staining also revealed that NF-κB mediated tumor progression markers, such as VEGF and Mcl-1 were significantly reduced after combination treatment as compared to single therapy.Taken together, regorafenib combined with magnolol showed greater anti-HCC efficacy which may majorly correlate to the enhancing regorafenib-induced suppression of VEGF and Mcl-1. Citation Format: Po-Fu Yueh, Zhao-Lin Tan, Yuan Chiang, I-Tsang Chiang, Keng-Li Lan, Fei-Ting Hus. Treatment efficiency of regorafenib combined with magnolol was associated with suppression of VEGF and Mcl-1 on hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1000.

Amentoflavone Induces Cell-cycle Arrest, Apoptosis, and Invasion Inhibition in Non-small Cell Lung Cancer Cells.

Amentoflavone, an effective compound derived from medicinal plants, has been shown to boost therapeutic efficacy of chemotherapy in non-small cell lung cancer (NSCLC). However, anti-NSCLC effect of amentoflavone is ambiguous. The major purpose of the present study was to verify the inhibitory effects of amentoflavone in NSCLC cells. The effects of amentoflavone on growth and invasion of NSCLC CL-1-5-F4 cells were evaluated by cell viability assay, flow cytometry, colony formation assay, nuclear factor-kappa B (NF-κB) reporter gene assay, immunofluorescence staining, transwell invasion, and western blot assay. Amentoflavone effectively induced cell growth inhibition, G1 cell-cycle arrest, apoptosis, and suppression of invasion. Furthermore, amentoflavone not only triggered expression of p27, cleaved caspase-3, -8 also reduced NF-κB signaling, protein levels of matrix metalloproteinase (MMP)-2, -9, Cyclin-D1, and vascular endothelial growth factor (VEGF). Cell-cycle arrest, apoptosis induction, NF-κB signaling inhibition are associated with amentoflavone-inhibited growth and invasion of NSCLC cells.

Functional analysis of novel A20 variants in patients with atypical inflammatory diseases

BackgroundA20 haploinsufficiency (HA20) is an early-onset autoinflammatory disease caused by mutations in the TNFAIP3 gene, which encodes the protein A20. Numerous truncating mutations in the TNFAIP3 gene have been reported in HA20 patients, whereas fewer missense variants have had their pathogenicity confirmed. Here, we evaluated the pathogenic significance of three previously unreported missense variants of the TNFAIP3 gene in suspected cases of HA20.MethodsWe obtained the clinical features and immunological data of three patients with missense variants (Glu192Lys, Ile310Thr, and Gln709Arg) of unknown significance of TNFAIP3. We then performed in vitro functional assays including analysis of nuclear factor (NF)-κB reporter gene activity, detection of A20 expression and phosphorylation of A20 by IκB kinase β (IKKβ), and K63-deubiquitination assay using TNFAIP3-deficient HEK293 cells. Three known pathogenic missense mutations reported previously were also investigated.ResultsThe inhibitory effect on NF-κB reporter gene activity was significantly disrupted by A20 Glu192Lys and the three known mutations. The variants Ile310Thr and Gln709Arg did not show a difference from the wild type in any of the assays performed in this study.ConclusionsAmong the three variants in the TNFAIP3 gene, Glu192Lys was interpreted as being likely pathogenic, but Ile310Thr and Gln709Arg as being not pathogenic (uncertain significance and likely benign, respectively), based on the American College of Medical Genetics and Genomics standards and guidelines. Our study highlights the necessity of performing in vitro functional assays, notably, NF-κB reporter gene assay, to evaluate the pathogenicity of TNFAIP3 missense variants for the accurate diagnosis of HA20.

Lenvatinib Inhibits AKT/NF-κB Signaling and Induces Apoptosis Through Extrinsic/Intrinsic Pathways in Non-small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is a serious disease and the leading cause of death globally. Overexpression of protein kinase B/nuclear factor-kappa B (NF-κB) signaling transduction of NSCLC cells was recognized as a potential therapeutic target. Lenvatinib is a multiple kinase inhibitor against vascular endothelial growth factor receptor family. However, whether lenvatinib may affect AKT/NF-κB in NSCLC remains unknown. MTT assay, NF-κB reporter gene assay, flow cytometry, tranwell migration/invasion analysis and western blotting were used to identify the alteration of cell viability, NF-κB activation, apoptosis effect, migration/invasion potential and AKT/NF-κB related protein expression, respectively, in CL-1-5-F4 cells after lenvatinib treatment. The cell viability and NF-κB activity were suppressed by lenvatinib. Extrinsic and intrinsic apoptosis were activated by lenvatinib. Additionally, the metastatic potential of CL-1-5-F4 cells was also suppressed by lenvatinib. Altogether, lenvatinib induced extrinsic/intrinsic apoptosis and suppressed migration/invasion ability of NSCLC cells that was associated with AKT/NF-κB signaling inactivation.

Abstract 628: Imipramine induced apoptosis and inhibits invasion via suppression of EGFR/ERK/NF-kappa B activation in non-small cells lung cancer

Abstract Imipramine was known as an anti-depression agent; interestingly, recent studies indicated the potential of various tumors growth inhibition. However, the anticancer mechanism of imipramine on non-small cell lung cancer (NSCLC) is remaining ambiguous. Thus, the aim of the study was to determent the effects and underlying mechanism of imipramine on human NSCLC CL1-5/F4 cells. MTT assays were used for testing the cell viability of imipramine. The activation and expression of apoptosis, anti-apoptosis, DNA damage, cellular calcium, and metastasis-associated proteins were observed by flow cytometry, immunofluorescence stain, comet assay and Western blotting assay. The inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) after imipramine treatment was performed by NF-κB reporter gene assay and immunofluorescence nuclear translocation assay on CL1-5/F4/NF-κB/luc2 and CL1-5/F4 wild type cells. Moreover, the ability of migration and invasion after imipramine treatment were also tested by trans-well assay and wound healing assay. In animal experiment, CL1-5/F4/NF-κB/luc2 cells were subcutaneously injection into nude mice right flask and treated with imipramine by oral. In addition, the standard treatment erlotinib was also used as a positive control to compare treatment efficacy of imipramine. In vivo NF-κB activation was investigated by optical image. Mice were sacrificed and tumors were extracted for size and IHC staining evaluation on day 14. Our results demonstrated that imipramine significantly reduced cell viability, cell migration and invasion. Reporter gene assay and Western blotting revealed imipramine may decrease NF-κB activation through inhibition of EGFR/ERK/NF-κB pathway. Imipramine also diminished the phosphorylation of ERK, the expression of anti-apoptotic (c-FLIP and XIAP), angiogenesis (VEGF), and metastasis-associated proteins (MMP-9). Additionally, the activity of PARP-1 that induced by imipramine was associated with DNA damage. Furthermore, imipramine was also triggered extrinsic (Fas/FasL, and caspase-8), intrinsic [mitochondrial membrane potential (ΔΨm)] apoptosis pathways, and the accumulation of cellular calcium. Tumor growth and NF-κB activation were both suppressed by imipramine treatment. Tumors IHC stain showed p-EGFR, p-ERK and p-NF-κB expressions level were reduced in imipramine treatment group as compared to vehicle. Most importantly, although body weight was remained unchanged in three treatment group, the cellular necrosis nucleus was found in erlotinib treated liver pathology H&E stain which was not found in control and imipramine groups. Taken together, these results suggested that imipramine can induce NSCLC apoptosis by activating extrinsic/intrinsic apoptosis mechanisms, DNA damage effect and the accumulation of cellular calcium concentration. In addition, imipramine may also suppress tumor proliferation, invasion and migration ability. Most importantly, imipramine diminished tumor progression via inactivation of EGFR/ERK/NF-κB signaling on NSCLC in vitro and in vivo. Citation Format: Po-Fu Yueh, I-Tsang chiang, Fei-Ting Hsu. Imipramine induced apoptosis and inhibits invasion via suppression of EGFR/ERK/NF-kappa B activation in non-small cells lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 628.

Linalool Glycosides from Flowers of Lantana montevidensis with Promising AntiInflammatory Potentials

Background and objective: Inflammation has been studied for thousands of years in an effort to mitigate its negative effects on the body. Analgesic and anti-inflammatory drugs were the primary concern in monoterpenes. Monoterpenes have been recognized for their ability to suppress inflammatory process and modulate the vital chemical mediators of inflammation, such as pro-inflammatory and anti-inflammatory cytokines. This study aimed to isolate the secondary metabolites of Lantana montevidensis (Verbenaceae) flowers and evaluate their in vitro antiinflammatory activity. Material and methods: Anti-inflammatory activity was done using TNF-α ELISA and NF-κB reporter gene assays. Results and discussion: As a result, seven compounds were isolated and identified, in which three of them were reported for the first time from genus Lantana. For the TNF-α ELISA assay, compound 1 significantly reduced the level of TNF-α (83.5 pg/mL), while compounds 2 and 3 showed moderate inhibition to TNF-α level (144.7 ± 7.43 and146.2 ± 5.27 pg/mL respectively). In addition, the NF-κB reporter gene assay revealed that the tested compounds (1-2) inhibited the relative NF-κB activity which was induced by TNF-α (0.41 ± 0.01 and 0.47 ± 0.01, respectively). Staursporine was used as a positive control in both assays. Conclusion: Linalool glycosides isolated from flowers of L. montevidensis exhibited significant anti-inflammatory activity by reducing the TNF-α level and inhibition of NF-κB pathway.

Apoptosis induction and AKT/NF-κB inactivation are associated with regroafenib-inhibited tumor progression in non-small cell lung cancer in vitro and in vivo

Non-small cell lung cancer (NSCLC) is a malignant lung cancer type with poor prognosis. NF-κB, the oncogenic transcription factor, has been recognized as an important mediator in progression of NSCLC. Regorafenib, a multikinase inhibitor, was demonstrated to inhibit tumor progression through suppression of ERK/NF-κB signaling in hepatocellular carcinoma cells in vitro and in vivo. However, whether regorafenib inhibit progression of NSCLC is ambiguous. Thus, the major purpose of present study was to evaluate anticancer efficacy and underlying mechanism of regorafenib on tumor progression in NSCLC in vitro and in vivo. CL-1-5-F4 cells were treated with regorafenib, NF-κB (QNZ) or AKT (LY294002) inhibitor for 24 or 48 h. Then, we performed cell viability assay, NF-κB reporter gene assay, transwell invasion assay and apoptosis related flow cytometry assay on cellular level to verify anti-cancer effect and mechanism of regorafenib. CL-1-5-F4 bearing animal model was treated with vehicle or regorafenib for 28 days. The therapeutic efficacy and mechanism of regorafenib in CL-1-5-F4 bearing animal model were investigated by tumor size evaluation, whole body computer tomography (CT) scan, Haemotoxylin and Eosin (H&E) stain and immunohistochemistry (IHC) stain. Our results demonstrated regorafenib significantly inhibited tumor growth and induced apoptosis through extrinsic/intrinsic pathways in NSCLC in vitro and in vivo. Furthermore, we also found the suppression of AKT/NF-κB signaling was required for regorafenib inhibited expression of progression-related and invasion-related proteins. Our finding indicated apoptosis induction and suppression of AKT/NF-κB signaling were associated with regorafenib-inhibited progression of NSCLC in vitro and in vivo.

Fluoxetine Induces Apoptosis through Extrinsic/Intrinsic Pathways and Inhibits ERK/NF-κB-Modulated Anti-Apoptotic and Invasive Potential in Hepatocellular Carcinoma Cells In Vitro.

The aim of the present study was to verify the effects of fluoxetine on dysregulation of apoptosis and invasive potential in human hepatocellular carcinoma (HCC) SK-Hep1 and Hep3B cells. Cells were treated with different concentrations of fluoxetine for different times. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assays were used for testing the effects of fluoxetine on cell viability. The regulation of apoptosis signaling, and anti-apoptotic, proliferation, and metastasis-associated proteins after fluoxetine treatment were assayed by flow cytometry and Western blotting assay. The detection of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation after fluoxetine treatment was performed by NF-κB reporter gene assay. The results demonstrated that fluoxetine significantly reduced cell viability, cell migration/invasion, NF-κB, extracellular signal-regulated kinases (ERK) activation, and expression of anti-apoptotic (Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (C-FLIP), Myeloid cell leukemia-1 (MCL-1), X-Linked inhibitor of apoptosis protein (XAIP), and Survivin), proliferation (Cyclin-D1), angiogenesis (vascular endothelial growth factor (VEGF)), and metastasis-associated proteins (matrix metalloproteinase-9 (MMP-9)). Fluoxetine also significantly induced apoptosis, unregulated extrinsic (activation of first apoptosis signal protein and ligand (Fas/FasL), and caspase-8) and intrinsic (loss of mitochondrial membrane potential (ΔΨm) pathways and increased Bcl-2 homologous antagonist killer (BAK) apoptosis signaling. Taken together, these results demonstrated that fluoxetine induced apoptosis through extrinsic/intrinsic pathways and diminished ERK/NF-κB-modulated anti-apoptotic and invasive potential in HCC cells in vitro.

Effects of phytogenic feed additives on cellular oxidative stress and inflammatory reactions in intestinal porcine epithelial cells1.

Due to increasing concerns about the use of antibiotic growth promoters (AGP) in livestock production and their complete ban in the European Union in 2006, suitable alternatives are urgently needed. Among others, anti-inflammatory activities of AGP are discussed as their putative mode of action. As numerous phytochemicals are known to modulate the cellular antioxidant capacity and immune response, we studied the antioxidative and anti-inflammatory properties of a phytogenic (plant-derived) feed additive (PFA) in intestinal porcine epithelial cells (IPEC-J2). The effects of the PFA were compared with those of selected phytogenic ingredients (grape seed extract [GRS], licorice extract [LIC], menthol [MENT], methyl salicylate [MES], oak bark extract [OAK], oregano essential oil [ORE], and a plant powder mix [PLA]), and with the effects of the AGP tylosin (TYL). Oxidative or inflammatory stress was induced by stimulating IPEC-J2 with hydrogen peroxide (H2O2; 0.5 mM) or tumor necrosis factor alpha (TNF-α; 10 ng/mL), respectively. The antioxidative effects of feed additives were assessed with a reactive oxygen species (ROS)-sensitive probe and by measuring the expression of 6 antioxidative target genes via quantitative real-time PCR (RT-qPCR). Anti-inflammatory potential was analyzed using a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) reporter gene assay. Moreover, the expression levels of 6 NF-κB target genes were measured using RT-qPCR analysis, and the release of IL-6 was analyzed via ELISA. Significant decreases in cellular ROS upon H2O2 treatment were observed for the PFA (P < 0.001), LIC (P < 0.001), ORE (P < 0.05), and GRS (P < 0.01). No significant changes in the expression of antioxidative genes were found. NF-κB activation upon TNF-α treatment was significantly inhibited by the PFA (P < 0.05) and by ORE (P < 0.001). Moreover, the PFA and ORE significantly reduced the gene expression of IL-6 (P < 0.001), IL-8 (P < 0.001), and C-C motif chemokine ligand 2 (CCL2; P < 0.05), as well as the release of IL-6 (P < 0.05). The other phytogenic compounds as well as the AGP TYL did not significantly affect any of the inflammatory parameters. In summary, we revealed the antioxidative properties of the PFA, LIC, ORE, and GRS, as well as anti-inflammatory properties of the PFA and ORE in IPEC-J2, providing a better understanding of the mode of action of this PFA under our experimental conditions.

Ribosomal Protein S3 Gene Silencing Protects Against Cigarette Smoke-Induced Acute Lung Injury

Chronic obstructive pulmonary disease (COPD) is estimated to be the third leading cause of death by 2030. Transcription factor NF-κB may play a critical role in COPD pathogenesis. Ribosomal protein S3 (RPS3), a 40S ribosomal protein essential for executing protein translation, has recently been found to interact with the NF-κB p65 subunit and promote p65 DNA-binding activity. We sought to study whether RPS3 gene silencing could protect against cigarette-smoke (CS)-induced acute lung injury in a mouse model. Effects of an intratracheal RPS3 siRNA in CS-induced lung injury were determined by measuring bronchoalveolar lavage (BAL) fluid cell counts, levels of inflammatory and oxidative damage markers, and NF-κB translocation. Lung RPS3 level was found to be upregulated for the first time with CS exposure, and RPS3 siRNA blocked CS-induced neutrophil counts in BAL fluid. RPS3 siRNA suppressed CS-induced lung inflammatory mediator and oxidative damage marker levels, as well as nuclear p65 accumulation and transcriptional activation. RPS3 siRNA was able to disrupt CS extract (CSE)-induced NF-κB activation in an NF-κB reporter gene assay. We report for the first time that RPS3 gene silencing ameliorated CS-induced acute lung injury, probably via interruption of the NF-κB activity, postulating that RPS3 is a novel therapeutic target for COPD.

Amentoflavone Inhibits ERK-modulated Tumor Progression in Hepatocellular Carcinoma In Vitro.

A previous study indicated that amentoflavone inhibits tumor growth of breast cancer. However, the anti-cancer effects and mechanism of amentoflavone in hepatocellular carcinoma (HCC) have not been elucidated. The aim of the present study was to verify the effect of amentoflavone on tumor progression in HCC. HCC SK-Hep1 cells were treated with different concentrations of amentoflavone or 10 μM PD98059 (extracellular signal-regulated kinases (ERK) inhibitor) for 48 h, respectively, and then cell viability, NF-κB activation, levels of tumor progression-associated proteins, and cell invasion were evaluated with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), NF-κB reporter gene assay, western blotting, and cell invasion assay. The results demonstrated that both amentoflavone and PD98059 not only significantly reduced cell viability, NF-κB activation, and cell invasion, but also inhibited the expression of tumor progression-associated proteins. In addition, we found that amentoflavone suppresses ERK phosphorylation. The results of the present study suggest that amentoflavone down-regulates ERK-modulated tumor progression in HCC.

Hyperforin Suppresses Tumor Growth and NF-κB-mediated Anti-apoptotic and Invasive Potential of Non-small Cell Lung Cancer.

Previous studies have indicated that hyperforin inhibits tumor growth of hepatocellular carcinoma. However, the anticancer effects of hyperforin in non-small cell lung cancer (NSCLC) are ambiguous. The aim of the present study was to investigate the anticancer effect of hyperforin in NSCLC. NSCLC CL1-5-F4 cells were treated with different concentrations of hyperforin or NF-κB inhibitor (QNZ) for different time periods. Change of cell viability, NF-κB activation, apoptotic signaling pathways, expression of anti-apoptotic proteins, and cell invasion were detected using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, NF-κB reporter gene assay, flow cytometry, western blotting, and cell invasion assay. The results demonstrated that hyperforin significantly promotes extrinsic and intrinsic apoptotic pathways, and inhibits cell viability and NF-κB activation. In addition, results also indicated that blockage of NF-κB activation reduces the levels of anti-apoptotic proteins and cell invasion in CL1-5-F4 cells. These results suggested hyperforin induces apoptosis and inhibits NF-κB-modulated anti-apoptotic and invasive potential in NSCLC.

Amentoflavone Inhibits Metastatic Potential Through Suppression of ERK/NF-κB Activation in Osteosarcoma U2OS Cells.

The study goal was to investigate effect of amentoflavone on nuclear factor-κB (NF-κB)-modulated metastatic mechanism in osteosarcoma U2OS cells. U2OS cells were treated with amentoflavone, NF-κB inhibitor, protein kinase B (PKB or AKT) inhibitor or mitogen-activated protein kinase (MAPK) inhibitor. Change of cell viability, NF-κB activation, expression of metastasis-associated proteins, signal transduction, and cell migration and invasion were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, NF-κB reporter gene assay, western blotting, and cell migration and invasion assays. The results demonstrated that inhibition of activation of extracellular signal-regulated kinases (ERK) was a key point for suppression of NF-κB-modulated metastatic mechanism. Amentoflavone significantly inhibited NF-κB activation, ERK phosphorylation, expression of metastasis-associated proteins, and cell migration and invasion. Our findings indicate that amentoflavone reduces metastatic potential through suppression of ERK and NF-κB activation in osteosarcoma U2OS cells.

First Bispecific Inhibitors of the Epidermal Growth Factor Receptor Kinase and the NF-κB Activity As Novel Anticancer Agents

The activation of the NF-κB transcription factor is a major adaptive response induced upon treatment with EGFR kinase inhibitors, leading to the emergence of resistance in nonsmall cell lung cancer and other tumor types. To suppress this survival mechanism, we developed new thiourea quinazoline derivatives that are dual inhibitors of both EGFR kinase and the NF-κB activity. Optimization of the hit compound, identified in a NF-κB reporter gene assay, led to compound 9b, exhibiting a cellular IC50 for NF-κB inhibition of 0.3 μM while retaining a potent EGFR kinase inhibition (IC50 = 60 nM). The dual inhibitors showed a higher potency than gefitinib to inhibit cell growth of EGFR-overexpressing tumor cell lines in vitro and in a xenograft model in vivo, while no signs of toxicity were observed. An investigation of the molecular mechanism of NF-κB suppression revealed that the dual inhibitors depleted the transcriptional coactivator CREB-binding protein from the NF-κB complex in the nucleus.

Ribosomal protein S3 gene silencing protects against experimental allergic asthma.

Ribosomal protein S3 (RPS3) is a 40S ribosomal protein of the S3P family essential for implementing protein translation. RPS3 has recently been found to interact with the p65 subunit of the NF-κB complex and promote p65 DNA-binding activity. Persistent activation of the NF-κB pathway is evident in allergic asthma. We hypothesized that gene silencing of lung RPS3 can ameliorate allergic airway inflammation. The gene silencing efficacy of RPS3 siRNA was screened in three different mouse cell lines by real-time PCR and immunoblotting. Protective effects of intratracheal RPS3 siRNA in a house dust mite (HDM) mouse asthma model were determined by measuring cell counts in lung lavage fluid and lung sections, lung cytokine profiles and airway hyperresponsiveness (AHR). RPS3 siRNA markedly knocked down RPS3 levels in all mouse cell lines tested, and in mouse lung tissues, blocked TNF-α- or HDM-induced release of mediators by the cultured cells and reduced eosinophil counts in lung lavage fluid from the HDM mouse asthma model. RPS3 siRNA lessened HDM-induced airway mucus hypersecretion, cytokine production and serum IgE elevation. Moreover, RPS3 knockdown significantly suppressed methacholine-induced AHR in experimental asthma. RPS3 siRNA disrupted TNF-α-induced NF-κB activation in a NF-κB reporter gene assay in vitro and prevented the nuclear accumulation of p65 subunit and p65 transcriptional activation in HDM-challenged lungs and cells. RPS3 gene silencing ameliorates experimental asthma, probably by disrupting NF-κB activity. RPS3 could be a novel therapeutic target for allergic airway inflammation.