Phenobarbital inhibits osteoclast differentiation and function through NF-κB and MAPKs signaling pathway

The purpose of this study was to determine the direct effects of phenobarbital (PB) on receptor activator of nuclear factor kappa-B ligand (RANKL) induced osteoclast differentiation and function in vitro and in vivo. Here, PB significantly inhibited osteoclast formation and bone resorption ability induced by RANKL in vitro. Meanwhile, intracellular signaling transduction analysis revealed PB specifically decreasing the phosphorylation level of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK), respectively. Besides, oral administration of PB at the dose of 60 mg/kg/day for 6 weeks led to improve the bone loss and to decrease the activity on both osteoblast and osteoclast. This suppression effect is more obvious in osteoblast-induced bone formation than that on osteoclast-induced bone resorption. Taken together, our findings demonstrated that PB down-regulate osteoclast differentiation and activity through modulation of NF-κB and MAPKs signaling pathway. The direct suppression effect on osteoclast can induce bone loss after long term oral administration. This bone loss is due to reducing bone turnover rate on both sides of bone formation and bone resorption.     

Okadaic acid induces apoptosis through the PKR, NF-κB and caspase pathway in human osteoblastic osteosarcoma MG63 cells

Okadaic acid (OA) is the major component of diarrheic shellfish poisoning toxins and a potent inhibitor of protein phosphatase 1 and 2A. However, the underlying regulatory mechanisms involved in OA-induced cell death are not well understood. In the present study, we examined the effects of OA on apoptosis of MG63 cells by characterizing apoptotic morphological changes of the cells and DNA fragmentation. The roles of double-stranded RNA-dependent protein kinase (PKR), nuclear factor-κB (NF-κB) and caspase in OA-mediated apoptosis in MG63 cells were also examined. Results showed that OA induced cytotoxicity and apoptosis in MG63 cells at IC₅₀ of 75 nM. A functional PKR pathway is required to induce apoptosis in response to OA treatment. Blockade of NF-κB by ammonium pyrrolidinedithiocarbamate (PDTC) resulted in down-regulation of apoptosis. The caspase-3 and caspase-8 inhibitors blocked apoptosis in MG63 cells. In conclusion, our results imply that OA can induce MG63 cell apoptosis through the PKR, NF-κB and caspase pathway.     

Esculentoside B inhibits inflammatory response through JNK and downstream NF-κB signaling pathway in LPS-triggered murine macrophage RAW 264.7 cells

Natural compound esculentoside B (EsB), (2S,4aR,6aR,6aS,6bR,8aR,9R,10R,11S,12aR,14bS)-11-hydroxy-9-(hydroxymethyl)-2 methoxycarbonyl-2,6a,6b,9,12a-pentamethyl-10-[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxy-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid with molecular weight of 664.833, isolated from roots of Phytolacca acinosa Roxb has been widely used as a constituent of traditional Chinese medicine (TCM). However, the anti-inflammatory capacity of EsB has not been reported yet. Therefore, the objective of this study was to investigate anti-inflammatory activities of EsB in LPS-treated macrophage RAW 264.7 cells. EsB could inhibit nitric oxide (NO) production. EsB also suppressed gene and protein expression levels of inducible isoform of NO synthase (NOS) and cyclooxygenase-2 in a dose-dependent manner. In addition, EsB decreased gene expression and protein secretion levels of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6. EsB remarkably suppressed nuclear translocation of nuclear factor kappa-B (NF-κB) from cytosolic space. Phosphorylation of IκB was also inhibited by EsB. Moreover, EsB specifically down-regulated phospho-c-Jun N-terminal kinase (p-JNK), but not p-p38 or phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2). Taken together, these results suggest that EsB has inhibitory effect on inflammatory response by inactivating NF-κB and p-JNK. It could be used as a new modulatory drug for effective treatment of inflammation-related diseases.     

Simvastatin in combination with bergamottin potentiates TNF-induced apoptosis through modulation of NF-κB signalling pathway in human chronic myelogenous leukaemia

Context Simvastatin (SV) and bergamottin (BGM) are known to exhibit diverse anti-cancer and anti-inflammatory activities.

Objective Very little is known about the potential efficacy of combination of these two agents to potentiate TNF-induced apoptosis in human chronic myelogenous leukaemia (CML).

Materials and methods In the present study, we investigated whether SV combined with BGM mediates its effect through suppression of NF-κB-signalling pathway.

Results We found that the combination treatment enhanced cytotoxicity and potentiated the apoptosis induced by TNF as indicated by intracellular esterase activity, Annexin V staining and caspase activation. This effect of co-treatment correlated with down-regulation of various gene products that mediate cell proliferation (cyclin D1), cell survival (cIAP-1, Bcl-2, Bcl-xL and Survivin), invasion (MMP-9) and angiogenesis (VEGF); all known to be regulated by NF-κB. SV combined with BGM also produced TNF-induced cell-cycle arrest in S-phase and this arrest correlated with a concomitant increase in the levels of cyclin-dependent inhibitor p21 and p27. The combination therapy inhibited TNF-induced NF-κB activation, IκBα degradation and p65 translocation to the nucleus as compared with the treatment with individual agents alone. Besides, SV combined with BGM did not significantly potentiate apoptotic effect induced by TNF in p65^?/? cells, as compared with wild-type fibroblasts.

Discussion and conclusion Our results provide novel insight into the role of SV and BGM in potentially preventing and treating cancer through modulation of NF-κB signalling pathway and its regulated gene products.     

FKBP51 and the NF-κB regulatory pathway in cancer

Constitutive activation of NF-κB occurs in a significant percentage of human cancers. Genetic abnormalities of tumors often enhance normal NF-κB signaling. Chronic inflammation is also associated with constitutive NF-κB activation and increases the risk of cancer. Aberrant NF-κB activation favors cellular transformation, sustains cancer survival, and contributes to tumor invasion. Strategies to inhibit NF-κB represent a promising therapeutic option against cancer. In the last decade, several studies point to the large immunophilin FKBP51 as an important element for the control of NF-κB activation in human neoplasia. This article is an overview of the causes of aberrant NF-κB regulation in cancer and highlights recent papers that implicate FKBP51 in such deregulation.     

Resveratrol analog, 3,5,2′,4′-tetramethoxy-trans-stilbene, potentiates the inhibition of cell growth and induces apoptosis in human cancer cells

Resveratrol, a trihydroxystilbene found in grapes and several plants, has been shown to be active in inhibiting multistage carcinogenic process. Using resveratrol as the prototype, we synthesized several analogs and evaluated their growth inhibitory effect using cultured human cancer cells. In the present report we show that one of the resveratrol analogs, 3, 5,2',4'-tetramethoxy-trans-stilbene, potentiated the inhibition of cancer cell growth. Prompted by the strong growth inhibitory activity of the compound (IC50; 0.8 microg/ml) compared to resveratrol (IC50; 18.7 microg/ml) in cultured human colon cancer cells (Col2), we performed an action mechanism study using the compound. The compound induced the accumulation of cellular DNA contents in the sub-G0 phase DNA contents of the cell cycle by in a time-dependent manner. The morphological changes were also consistent with an apoptotic process. This result indicated that the compound induced apoptosis of cancer cells, and may be a candidate for use in the development of potential cancer chemotherapeutic or cancer chemopreventive agents.     

Aflatoxin B1 induces microglia cells apoptosis mediated by oxidative stress through NF-κB signaling pathway in mice spinal cords

Many studies have shown that aflatoxin B1 (AFB1) can cause cytotoxicity in numerous cells and organs induced by oxidative stress. However, the toxic effects and related mechanism of AFB1 on the microglia cells in the spinal cords have not been studied yet. Our results showed that AFB1 significantly reduced the number of microglia cells, increased the oxidants (malonaldehyde and hydrogen peroxide) but decreased the anti-oxidants (superoxide dismutase and total antioxidant capacity) in a dose dependent manner in mice spinal cords. In addition, AFB1 significantly increased the oxidative stress, promoted apoptosis and cell cycle arrest in G2-M phase, and activated NF-κB phosphorylation in BV2 microglia cells. However, the addition of active oxygen scavenger N-acetylcysteine can significantly reduce the ROS production, improve cell cycle arrest, reduce apoptosis, and the expression of phosphorylated NF-κB in BV2 microglia cells. These results indicate that AFB1 induces microglia cells apoptosis through oxidative stress by activating NF-κB signaling pathway.     

Dietary chlorophyllin inhibits the canonical NF-κB signaling pathway and induces intrinsic apoptosis in a hamster model of oral oncogenesis

Chlorophyllin, a water-soluble, semi-synthetic derivative of the ubiquitous green pigment chlorophyll is shown to exert potent anticarcinogenic effects. In the present study, we investigated the chemopreventive effects of chlorophyllin on 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis by analyzing the expression of NF-κB family members and markers of intrinsic apoptosis. Dietary administration of chlorophyllin (4 mg/kg bw) suppressed the development of HBP carcinomas by inhibiting the canonical NF-κB signaling pathway by downregulating IKKβ, preventing the phosphorylation of IκB-α, and reducing the expression of nuclear NF-κB. Inactivation of NF-κB signaling by chlorophyllin was associated with the induction of intrinsic apoptosis as evidenced by modulation of Bcl-2 family proteins, enforced nuclear localization of survivin, upregulation of apoptogenic molecules, activation of caspases, and cleavage of PARP. The results of the present study demonstrate that chlorophyllin inhibits the development of DMBA-induced HBP carcinogenesis by targeting NF-κB and the intrinsic apoptotic pathway. Thus, dietary agents such as chlorophyllin that simultaneously target divergent pathways of cell survival and cell death are novel candidates for cancer chemoprevention.     

Theophylline inhibits NF-κB activation and IκBα degradation in human pulmonary epithelial cells

Previous studies demonstrated that theophylline modulates NF-kappaB activation in mast cells and pulmonary epithelial cells. We examined whether or not this modulation of NF-kappaB activation by theophylline is due to inhibition of the degradation of the IKBalpha protein, which suppresses NF-kappaB activation. TNF-alpha-induced NF-kappaB activation in a human pulmonary epithelial cell line (A549) was evaluated by Western blotting and a chloramphenicol acetyltransferase (CAT) assay. Expression of the IkappaBalpha protein was evaluated by Western blotting. Western blotting of nuclear extracts of A549 cells demonstrated that theophylline suppresses NF-kappaB-p65 nuclear translocation. The CAT assay indicated that NF-kappaB-dependent reporter gene expression is inhibited in A549 cells pretreated with theophylline. Western blotting of cytoplasmic extracts of A549 cells revealed that this inhibition was linked to theophylline-induced protection of expression of the IkappaBalpha protein. Moreover, theophylline inhibited interleukin-6 production induced by TNF-alpha in A549 cells. These findings are consistent with the idea that theophylline suppresses the production of proinflammatory cytokines via inhibition of NF-kappaB activation through protection of the IkappaBalpha protein.     

Ligustilide induces apoptosis and reduces proliferation in human bladder cancer cells by NFκB1 and mitochondria pathway

Ligustilide (LIG), the bioactive constituent of Angelica sinensis, may exert potential benefits in cancer treatment. However, the potential mechanism of LIG in the suppression of bladder cancer (BC) has not been reported yet. This study uncovered the inhibitory effect of LIG on the proliferation and cell cycle arrest of BC cells (T24 and EJ-1) along with unveiling the underlying molecular mechanism. The IC₅₀ values of LIG-treated T24 for 24 and 48 h are 39.91 μg/mL (209.8 μM) and 40.94 μg/mL (215.2 μM) separately. The same conditions, the IC₅₀ values of EJ-1 are 45.73 μg/mL (240.4 μM) and 43.81 μg/mL (230.3 μM), separately. Additionally, LIG induced apoptosis and cycle arrest of T24 and EJ-1 cells in sub-G1 phase. Further studies showed that LIG induced apoptosis of BC cells by upregulating Caspase-8, truncated BID (tBID) and BAX proteins, and downregulating NFκB1 (p50) protein. In conclusion, LIG significantly inhibits the growth of BC cells in vitro and in vivo by inducing apoptosis and is inexpensive, making it a promising candidate for novel anti-BC drugs.     

Triptolide inhibits human breast cancer MCF-7 cell growth via downregulation of the ERα-mediated signaling pathway

Aim:

To investigate the anticancer mechanisms of triptolide, a diterpenoid isolated from the plant Tripterygium wilfordii Hook F, against human breast cancer cells and the involvement of the estrogen receptor-α (ERα)-mediated signaling pathway in particular.

Methods:

Human breast cancer ERα-positive MCF-7 cells and ERα-negative MDA-MB-231 cells were tested. PrestoBlue assay was used to evaluate the cell viability. The levels of ERα mRNA and protein were detected with real-time PCR and immunoblotting, respectively. Mouse models of MCF-7 or MDA-MB-231 xenograft tumors were treated with triptolide (0.4 mg·kg⁻¹·d⁻¹, po) or a selective estrogen receptor modulator tamoxifen (mg·kg⁻¹·d⁻¹, po) for 3 weeks, and the tumor weight and volume were measured.

Results:

Triptolide (5–200 nmol/L) dose-dependently inhibited the viability of both MCF-7 and MDA-MB-231 cells, with a more potent inhibition on MCF-7 cells. Knockdown of ERα in MCF-7 cells by siRNA significantly attenuated the cytotoxicity of triptolide, whereas overexpression of ERα in MDA-MB-231 cells markedly enhanced the cytotoxicity. Triptolide dose-dependently decreased the expression of ERα in MCF-7 cells and MCF-7 xenograft tumors. Furthermore, treatment of MCF-7 cells with triptolide inhibited the phosphorylation of ERK1/2 in dose- and time-dependent manners. In the mice xenografted with MCF-7 cells, treatment with triptolide or tamoxifen resulted in significant reduction in the tumor weight and volume. Similar effects were not obtained in the mice xenografted with MDA-MB-231 cells.

Conclusion:

The anticancer activity of triptolide against ERα-positive human breast cancer is partially mediated by downregulation of the ERα-mediated signaling pathway.     

Hydrogen sulfide-releasing aspirin suppresses NF-κB signaling in estrogen receptor negative breast cancer cells in vitro and in vivo

Hormone-dependent estrogen receptor positive (ER+) breast cancers generally respond well to anti-estrogen therapy. Unfortunately, hormone-independent estrogen receptor negative (ER-) breast cancers are aggressive, respond poorly to current treatments and have a poor prognosis. New approaches and targets are needed for the prevention and treatment of ER- breast cancer. The NF-κB signaling pathway is strongly implicated in ER- tumor genesis, constituting a possible target for treatment. Hydrogen sulfide-releasing aspirin (HS-ASA), a novel and safer derivative of aspirin, has shown promise as an anti-cancer agent. We examined the growth inhibitory effect of HS-ASA via alterations in cell proliferation, cell cycle phase transitions, and apoptosis, using MDA-MB-231 cells as a model of triple negative breast cancer. Tumor xenografts in mice, representing human ER- breast cancer, were evaluated for reduction in tumor size, followed by immunohistochemical analysis for proliferation, apoptosis and expression of NF-κB. HS-ASA suppressed the growth of MDA-MB-231 cells by induction of G(0)/G(1) arrest and apoptosis, down-regulation of NF-κB, reduction of thioredoxin reductase activity, and increased levels reactive oxygen species. Tumor xenografts in mice, were significantly reduced in volume and mass by HS-ASA treatment. The decrease in tumor mass was associated with inhibition of cell proliferation, induction of apoptosis and decrease in NF-κB levels in vivo. HS-ASA has anti-cancer potential against ER- breast cancer and merits further study.     

Naringin inhibits growth potential of human triple-negative breast cancer cells by targeting β-catenin signaling pathway

Triple-negative (ER-/PR-/HER2-) breast cancer (TNBC) is a severe clinical problem because of its relatively poorer prognosis, aggressive behavior and lack of targeted therapies. Naringin, a major flavonoid extracted from citrus fruits, has been reported to exert promising anticancer activities. However, the detailed antitumor mechanism of naringin still remains enigmatic. In this study, TNBC cell lines-based in vitro and in vivo models were used to explore the anticancer effect and mechanism of naringin. Our data demonstrated that naringin inhibited cell proliferation, and promoted cell apoptosis and G1 cycle arrest, accompanied by increased p21 and decreased survivin. Meanwhile, β-catenin signaling pathway was found to be suppressed by naringin. In contrast, over-expressing β-catenin by adenoviral vector system in TNBC cells reversed the antitumor activity of naringin, and regulated p21 and survivin. Correspondingly, the antitumor potential of naringin was also observed in naringin-treated MDA-MB-231 xenograft mice, while immunohistochemical analysis of tumors from naringin-treated mice showed higher expression of p21 and lower expression of survivin and active β-catenin. Taken together, these results indicate that naringin could inhibit growth potential of TNBC cells by modulating β-catenin pathway, which suggests naringin might be used as a potential supplement for the prevention and treatment of breast cancer.     

CP-25 inhibits the functions of activated human B cells through regulating BAFF-TRAF2-NF-κB and TNF-alpha-TRAF2-NF-κB signaling

OBJECTIVE This study was to investigate the effects of CP-25 on the functions of activated human B cells through regulating BAFF and TNF-alpha signaling.METHODS B cells from peripheral blood mononuclear cells(PBMCs) of normal human were isolated using magnetic cell separation(MACS) by a positive selection.B cells(107 cells·mL~(-1)) were stimulated by BAFF(100 ng·mL~(-1))or TNF-alpha(100 ng·mL~(-1)) for two hours,and then were treated with CP-25(10-5 mol·L~(-1)) or Rituximab(5 μg·mL~(-1)) or Etanercept(10 μg·mL~(-1)).B cell proliferation was detected by CCK-8.B cell subsets and BAFF receptors(BAFFR,BCMA and TACI) were analyzed by flow cytometry.The expression of TNFR1 and TNFR2 on B cells was analyzed by flow cytometry.The expression of MKK3,MKK6,P-p38,P-p65,TRAF2 and p100/52 was analyzed by Western blotting.RESULTS CP-25 inhibited B cells proliferation stimulated by BAFF or TNF-alpha.CP-25,Rituximab and Etanercept reduced the percentage and numbers of CD19~+B cells,CD19~+CD20~+B cells,CD19~+CD27~+B cells and CD19~+CD20~+CD27~+B cells induced by BAFF or TNF-alpha.CP-25 down-regulated the high expression of BAFFR,BCMA and TACI stimulated by BAFF or TNF-alpha.CP-25,Rituximab and Etanercept down-regulated significantly the expression of TNFR1 and TNFR2 on B cell stimulated by BAFF or TNF-alpha.CP-25,Rituximab and Etanercept down-regulated the expression of MKK3,P-p38,P-p65,TRAF2 and p52 in B cells stimulated by BAFF and the expression of TRAF2 and P-p65 in B cells stimulated by TNF-alpha.CONCLUSION CP-25 regulated moderately activated B cells function by by regulating the classical and alternative NF-κB signaling pathway mediated by BAFF and TNF-alpha-TRAF2-NF-κB signaling pathway.This study suggests that CP-25 may be a promising anti-inflammatory immune and soft regulation drug.     

IL-38 alleviates the inflammatory response and the degeneration of nucleus pulposus cells via inhibition of the NF-κB signaling pathway in vitro

Previous studies have suggested that the inflammatory response contributes to the onset of intervertebral disc degeneration (IVDD). Interleukin (IL)-38, a newly discovered cytokine of the IL-1 family, has been demonstrated to play an anti-inflammatory role in autoimmune diseases, such as Crohn’s disease, rheumatoid arthritis and psoriasis. However, whether IL-38 participates in the pathogenesis of IVDD remains unknown. In this study, human disc tissues from IVDD patients and rat disc tissues from an IVDD model were collected to measure the expression of IL-38 in the IVDD groups and the control groups by western blot and immunohistochemical staining. To further determine the role of IL-38 in IVDD, human nucleus pulposus cells (HNPCs) were stimulated with TNF-α to generate an in vitro model of inflammation to mimic the local inflammatory environment of the lumbar disc. The inflammatory response and HNPC degeneration markers were measured after stimulation with TNF-α and IL-38. IL-38 was upregulated in both the human and rat degenerated disc tissues compared with the control tissues. In vitro, IL-38 significantly decreased the TNF-α-induced expression of IL-1β, IL-6, COX-2, MMP-13 and ADAMTS-5 in the HNPCs, and IL-38 also alleviated the TNF-α-induced reductions in type II collagen and aggrecan. Moreover, IL-38 inhibited the activation of the NF-κB signaling pathway in the HNPC-based model of inflammation by reducing the expression level of the NF-κB P-P65 protein. In conclusion, IL-38 could alleviate the inflammatory response and HNPC degeneration in vitro via the inhibition of the NF-κB signaling pathway. These results suggest that IL-38 may be a new strategy for the treatment of IVDD.     

α-Tocopheryl succinate induces apoptosis in erbB2-expressing breast cancer cell via NF-κB pathway

Aim:

To study the molecular mechanisms underlying α-tocopheryl succinate (α-TOS)-induced apoptosis in erbB2-positive breast cancer cells and to determine whether α-TOS and the human recombinant TNF-related apoptosis-inducing ligand (hrTRAIL) act synergically to induce cell death of erbB2-expressing breast cancer cells.

Methods:

The annexin V binding method was used to measure apoptosis induced by α-TOS and/or hrTRAIL. RT-PCR and Western blotting were performed to detect gene and protein expression. A colorimetric assay was performed to detect caspase activity. The TransAM^TM NF-κB p65 kit was used to assess NF-κB activation.

Results:

α-TOS (100 μmol/L) significantly inhibited NF-κB nuclear translocation in erbB2-expressing breast cancer cells; this inhibition is expected to result in the inactivation of NF-κB. α-TOS (50 and 100 μmol/L) inhibited the expression of Flice-like inhibitory protein (FLIP) and cellular inhibitor of apoptosis protein 1 (c-IAP1) in erbB2-positive cells. α-TOS (100 μmol/L) inhibited Akt activation and augmented the activity of caspase 3 and caspase 8 in breast cancer cells expressing erbB2. α-TOS (50 μmol/L) and hrTRAIL (30 mg/mL) acted synergically to induce apoptosis in breast cancer cells. α-TOS also decreased the hrTRAIL-induced transient activation of NF-κB .

Conclusion:

Our results suggest that α-TOS mediates the apoptosis of erbB2-positive breast cancer cells and acts synergically with hrTRAIL via the NF-κB pathway.     

Mitochondrial division inhibitor (mdivi-1) inhibits proliferation and epithelial-mesenchymal transition via the NF-κB pathway in thyroid cancer cells

Excessively fragmented mitochondria have been reported in thyroid cancer (TC). Mitochondrial division inhibitor (mdivi-1), a putative inhibitor of dynamin-related protein 1 (Drp1), prevents mitochondrial fission and thereby restricts cell proliferation across several types of primary cancer. However, the role of mdivi-1 on TC has not been sufficiently studied. This research is intended to explore the therapeutic effect of mdivi-1 in TC cells. Results demonstrated that highly invasive TC cells displayed excessive mitochondrial fission with more fragmented mitochondria. Treatment with mdivi-1 inhibited mitochondrial fission in 8505C cells as indicated by transmission electron microscope (TEM). It also impaired the proliferation and increased apoptosis in 8505C and K1 cells as shown by plate cloning assay, cell viability assay, and apoptosis assay. Mdivi-1 treatment also attenuated migratory and invasive abilities in 8505C and K1 cells as shown by the transwell assay and the wound healing assay. And we noticed the same inhibition of mdivi-1 in cell migration and cell viability after the knockdown of Drp1 in 8505C cells. This demonstrated that mdivi-1 exerted an anti-tumor effect independently of Drp1 in 8505C cells. Moreover, mdivi-1 treatment reversed epithelial-mesenchymal transition (EMT) by inhibiting the NF-κB pathway in 8505C cells. The present findings demonstrate that mdivi-1 has a therapeutic role in thyroid carcinoma.