Mitochondrial-targeted curcumin inhibits T-cell activation via Nrf2 and inhibits graft-versus-host-disease in a mouse model.

Anti-inflammatory and immune suppressive agents are required to moderate hyper-activation of lymphocytes under disease conditions or organ transplantation. However, selective disruption of mitochondrial redox has not been evaluated as a therapeutic strategy for suppression of T-cell-mediated pathologies. Using mitochondrial targeted curcumin (MitoC), we studied the effect of mitochondrial redox modulation on T-cell responses by flow cytometry, transmission electron microscopy, transcriptomics, and proteomics, and the role of Nrf2 was studied using Nrf2- /- mice. MitoC decreased mitochondrial TrxR activity, enhanced mitochondrial ROS (mROS) production, depleted mitochondrial glutathione, and suppressed activation-induced increase in mitochondrial biomass. This led to suppression of T-cell responses and metabolic reprogramming towards Treg differentiation. MitoC induced nuclear translocation and DNA binding of Nrf2, leading to upregulation of Nrf2-dependent genes and proteins. MitoC-mediated changes in mitochondrial redox and modulation of T-cell responses are abolished in Nrf2- /- mice. Restoration of mitochondrial thiols abrogated inhibition of T-cell responses. MitoC suppressed alloantigen-induced lymphoblast formation, inflammatory cytokines, morbidity, and mortality in acute graft-versus-host disease mice. Disruption of mitochondrial thiols but not mROS increase inculcates an Nrf2-dependent immune-suppressive disposition in T cells for the propitious treatment of graft-versus-host disease.

Malabaricone C, a constituent of spice Myristica malabarica, exhibits anti-inflammatory effects via modulation of cellular redox.

The present study primarily focuses on the efficacy of Malabaricone C (Mal C) as an anti-inflammatory agent. Mal C inhibited mitogen-induced T-cell proliferation and cytokine secretion. Mal C significantly reduced cellular thiols in lymphocytes. N-acetyl cysteine (NAC) restored cellular thiol levels and abrogated Mal C-mediated inhibition of T-cell proliferation and cytokine secretion. Physical interaction between Mal C and NAC was evinced from HPLC and spectral analysis. Mal C treatment significantly inhibited concanavalin A-induced phosphorylation of ERK/JNK and DNA binding of NF-κB. Administration of Mal C to mice suppressed T-cell proliferation and effector functions ex vivo. Mal C treatment did not alter the homeostatic proliferation of T-cells in vivo but completely abrogated acute graft-versus-host disease (GvHD)-associated morbidity and mortality. Our studies indicate probable use of Mal C for prophylaxis and treatment of immunological disorders caused due to hyper-activation of T-cells.

Silencing of type II phosphatidylinositol 4-kinase ß stabilizes prostate apoptosis response-4 and induces apoptosis in cancer cells.

Type II phosphatidylinositol 4-kinase ß (PtdIns 4-kinase II ß) is an enigma among the phosphatidylinositol 4-kinase family. The role of PtdIns 4-kinase II ß in MCF-7 cells was addressed with the help of short hairpin RNA (shRNA). PtdIns 4-kinase II ß shRNA transfection increased pan-caspase activity and induced apoptosis in cancerous MCF-7 cells. Non-cancerous MCF-10A cells were resistant to PtdIns 4-kinase II ß shRNA-induced apoptosis. Caspase 8 and 9 inhibitors rescued MCF-7 cells from apoptosis. Shotgun proteomic studies with Flag-tagged PtdIns 4-kinase II ß immunoprecipitates showed tumor suppressor prostate apoptosis response-4 (Par-4) as one of the interacting proteins in HEK293 cells. In reciprocal experiments, Par-4 antibodies co-precipitated PtdIns 4-kinase II ß from MCF-7 cells. Deletion of membrane localization motif (ΔCCPCC) or a mutation in ATP-binding region (D304A) of PtdIns 4-kinase II ß did not affect its interaction with Par-4. Pull-down assays with GST-PtdIns 4-kinase II ß-truncated mutants showed that the region between 101 and 215 amino acid residues is essential for interaction with Par-4. At molecular level, PtdIns 4-kinase II ß shRNA transfection increased Par-4 stability, its nuclear localization and inhibition of NF-κB binding to target DNA. Knocking down of Par-4 with siRNA (small interfering RNA) rescued MCF-7 cells from PtdIns 4-kinase II ß shRNA-induced apoptosis. These results suggest that PtdIns 4-kinase II ß may be a novel regulator of Par-4 through protein-protein interactions. These studies have potential implications in cancer therapy.

Early antioxidant responses via the concerted activation of NF-κB and Nrf2 characterize the gamma-radiation-induced adaptive response in quiescent human peripheral blood mononuclear cells.

The radiation-induced adaptive response (RI-AR) is a non-targeted effect which is outside the scope of the classical Linear-No-Threshold (LNT) dose-response paradigm. However, the mechanisms of the RI-AR are not well understood. We have studied the RI-AR in quiescent human peripheral blood mononuclear cells (PBMCs). PBMCs in G0 phase were 'primed' with a low dose (100 mGy gamma radiation) and then, after an 'adaptive window' of 4 h, 'challenged' with a high dose (2 Gy). A small (5.7%) increase in viability and a decrease in DNA strand breaks were seen in primed cells, compared to non-primed cells. This was consistent with lower levels of reactive oxygen species, higher mitochondrial membrane potential, and increased activity of antioxidant enzymes such as catalase, superoxide dismutase, thioredoxin reductase, and glutathione peroxidase, in the primed cells. Reduced oxidative stress in primed PBMCs correlated with greater nuclear translocation of the redox-sensitive transcription factors Nuclear factor kappa B (NF-κB) and Nuclear factor E2-related factor 2 (Nrf2). Distinct differences in responses were seen in PBMCs irradiated with low dose (100 mGy) and high dose (2 Gy). These findings provide insight into the mechanisms of radioadaptation in human cells.

Ascorbyl Stearate Promotes Apoptosis Through Intrinsic Mitochondrial Pathway in HeLa Cancer Cells.

BACKGROUND: Ascorbic acid is proposed to have antitumor potential against certain cancer types but has the limitation of requiring high doses for treating cancer. Ascorbyl stearate (ASC-S) is a fatty acid ester derivative of ascorbic acid with comparable potent apoptotic activity. The present study was aimed at understanding the pathway involved in apoptotic activity of ASC-S in cervical cancer cells. MATERIALS AND METHODS: The effect of ASC-S on reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) was studied in HeLa cells. Furthermore, the dose-dependent effect of ASC-S on release of cytochrome c, pro-caspase-9, caspase-3, BH3 interacting-domain death agonist (BID), truncated BH3 interacting-domain death agonist (t-BID), FAS ligand (FASL) and transcription factors nuclear factor-kappa B (NF-ĸB), nuclear factor of activated T-cells (NFAT) and activator protein-1 (AP1) were studied in HeLa cells. RESULTS: Treatment of HeLa cells with ASC-S significantly increased the MMP. The modulation of MMP resulted in cleavage of BID, expression of FAS, cleavage of pro-caspase-9 and release of cytochrome c into cytosol. In addition, ASC-S treatment resulted in deregulation of transcription factors NF-ĸB, NFAT and AP1, which play an important role in the development of inflammation and cancer. CONCLUSION: Our data, for the first time, suggest that ASC-S has an apoptotic effect against HeLa cells by inducing change in mitochondrial membrane permeability, cytochrome c release and subsequent activation of caspase-3 and NF-ĸB.

Baicalein exhibits anti-inflammatory effects via inhibition of NF-κB transactivation.

NF-κB is a crucial mediator of inflammatory and immune responses and a number of phytochemicals that can suppress this immune-regulatory transcription factor are known to have promising anti-inflammatory potential. However, we report that inducer of pro-inflammatory transcription factor NF-κB functions as an anti-inflammatory agent. Our findings reveal that a plant derived flavonoid baicalein could suppress mitogen induced T cell activation, proliferation and cytokine secretion. Treatment of CD4+ T cells with baicalein prior to transfer in to lymphopenic allogenic host significantly suppressed graft versus host disease. Interestingly, addition of baicalein to murine splenic lymphocytes induced DNA binding of NF-κB but did not suppress Concanavalin A induced NF-κB. Since baicalein did not inhibit NF-κB binding to DNA, we hypothesized that baicalein may be suppressing NF-κB trans-activation. Thioredoxin system is implicated in the regulation of NF-κB trans-activation potential and therefore inhibition of thioredoxin system may be responsible for suppression of NF-κB dependent genes. Baicalein not only inhibited TrxR activity in cell free system but also suppressed mitogen induced thioredoxin activity in the nuclear compartment of lymphocytes. Similar to baicalein, pharmacological inhibitors of thioredoxin system also could suppress mitogen induced T cell proliferation without inhibiting DNA binding of NF-κB. Further, activation of cellular thioredoxin system by the use of pharmacological activator or over-expression of thioredoxin could abrogate the anti-inflammatory action of baicalein. We propose a novel strategy using baicalein to limit NF-κB dependent inflammatory responses via inhibition of thioredoxin system.

Amelioration of radiation-induced hematopoietic syndrome by an antioxidant chlorophyllin through increased stem cell activity and modulation of hematopoiesis.

Hematopoietic stem cells and progenitor cells (HSPC) are low in abundance and exhibit high radiosensitivity and their ability to divide dramatically decreases following exposure to ionizing radiation. Our earlier studies have shown antiapoptotic, immune-stimulatory, and antioxidant effects of chlorophyllin, a constituent of the over the counter drug derifil. Here we describe the beneficial effects of chlorophyllin against radiation-induced hematopoietic syndrome. Chlorophyllin administration significantly enhanced the abundance of HSPC in vivo. It induced a transient cell cycle arrest in lineage-negative cells in the bone marrow. However, the chlorophyllin-treated mice exposed to whole body irradiation (WBI) had a significantly higher proportion of actively dividing HSPC in the bone marrow as compared to only WBI-exposed mice. It significantly increased the number of colony forming units (CFUs) by bone marrow cells in vitro and spleen CFUs in irradiated mice in vivo. Pharmacokinetic study showed that chlorophyllin had a serum half-life of 141.8 min in mice. Chlorophyllin upregulated antiapoptotic genes and antioxidant machinery via activation of prosurvival transcription factors Nrf-2 and NF-κB and increased the survival and recovery of bone marrow cells in mice exposed to WBI. Chlorophyllin stimulated granulocyte production in bone marrow and increased the abundance of peripheral blood neutrophils by enhancing serum levels of granulocyte-colony stimulation factor (GCSF). Most importantly, prophylactic treatment of mice with chlorophyllin significantly abrogated radiation-induced mortality. Chlorophyllin mitigates radiation-induced hematopoietic syndrome by increasing the abundance of hematopoietic stem cells, enhancing granulopoiesis, and stimulating prosurvival pathways in bone marrow cells and lymphocytes.

1,4-Naphthoquinone, a pro-oxidant, suppresses immune responses via KEAP-1 glutathionylation.

Low levels of oxidative stress have been shown to activate Nrf-2, an important anti-inflammatory transcription factor, by us and also by several other investigators. Earlier we showed that pro-oxidants protect normal lymphocytes against radiation injury by activating Nrf-2. In the present study, we have investigated the effect of oxidative stress on immune responses and delineated the underlying mechanism. Hydrogen peroxide, tert-butylhydroquinone and 1,4-naphthoquinone (NQ) inhibited mitogen induced proliferation of lymphocytes. NQ also inhibited mitogen (Concanavalin A) induced cytokine secretion by murine T cells and lipopolysaccharide induced release of cytokines, nitric oxide and cyclooxygenase-2 expression by macrophages. NQ modulated cellular redox by decreasing GSH/GSSG ratio and the immunosuppressive effects of NQ were significantly abrogated by thiol containing antioxidants and not by non-thiol antioxidants. This redox perturbation led to activation of Nrf-2 pathway and inhibition of NF-κB. NQ treatment increased total protein S-thiolation, induced glutathionylation of KEAP-1 protein and decreased IKKß levels in lymphocytes. Molecular docking studies revealed that NQ can disrupt KEAP-1/Nrf-2 interaction by directly blocking the binding site of Nrf-2 in the KEAP-1 protein. Further, inhibitors of Nrf-2 and HO-1 abrogated the anti-inflammatory effects of NQ. T cells isolated from spleen and gut associated lymphoid tissue of NQ administered mice also showed suppression of NF-κB activation and were hyporesponsive to mitogenic stimulation. These results demonstrate that pro-oxidants modulate inflammatory and immune responses via oxidative stress mediated KEAP-1 glutathionylation and IKKß degradation.

Plumbagin, a vitamin K3 analogue, abrogates lipopolysaccharide-induced oxidative stress, inflammation and endotoxic shock via NF-κB suppression.

Plumbagin has been reported to modulate cellular redox status and suppress NF-κB. In the present study, we investigated the effect of plumbagin on lipopolysaccharide (LPS)-induced endotoxic shock, oxidative stress and inflammatory parameters in vitro and in vivo. Plumbagin inhibited LPS-induced nitric oxide, TNF-α, IL-6 and prostaglandin-E2 production in a concentration-dependent manner in RAW 264.7 cells without inducing any cell death. Plumbagin modulated cellular redox status in RAW cells. Plumbagin treatment significantly reduced MAPkinase and NF-κB activation in macrophages. Plumbagin prevented mice from endotoxic shock-associated mortality and decreased serum levels of pro-inflammatory markers. Plumbagin administration ameliorated LPS-induced oxidative stress in peritoneal macrophages and splenocytes. Plumbagin also attenuated endotoxic shock-associated changes in liver and lung histopathology and decreased the activation of ERK and NF-κB in liver. These findings demonstrate the efficacy of plumbagin in preventing LPS-induced endotoxemia and also provide mechanistic insights into the anti-inflammatory effects of plumbagin.

Schisandrin B exhibits anti-inflammatory activity through modulation of the redox-sensitive transcription factors Nrf2 and NF-κB.

Schisandrin B (SB), a dibenzocyclooctadiene derivative isolated from Schisandra chinensis and used commonly in traditional Chinese medicine for the treatment of hepatitis and myocardial disorders, has been recently shown to modulate cellular redox balance. Since we have shown that cellular redox plays an important role in the modulation of immune responses, the present studies were undertaken to study the effects of SB on activation and effector functions of lymphocytes. SB altered the redox status of lymphocytes by enhancing the basal reactive oxygen species levels and altering the GSH/GSSG ratio in lymphocytes. It also induced nuclear translocation of redox sensitive transcription factor Nrf2 and increased the transcription of its dependent genes. SB inhibited mitogen-induced proliferation and cytokine secretion by lymphocytes. SB also significantly inhibited mitogen-induced upregulation of T cell costimulatory molecules and activation markers. It was observed that SB inhibited mitogen-induced phosphorylation of c-Raf, MEK, ERK, JNK, and p38. It suppressed IκBα degradation and nuclear translocation of NF-κB in activated lymphocytes. Anti-inflammatory effects of SB were significantly abrogated by the inhibitors of Nrf2 and HO-1, suggesting the involvement of this pathway. Similar anti-inflammatory effects of SB on lymphocyte proliferation and cytokine secretion were also observed in vivo. To our knowledge, this is the first report showing that the anti-inflammatory effects of SB are mediated via modulation of Nrf2 and NF-κB in lymphocytes.

Ras puts the brake on doxorubicin-mediated cell death in p53-expressing cells.

Doxorubicin is one of the most effective molecules used in the treatment of various tumors. Contradictory reports often open windows to understand the role of p53 tumor suppressor in doxorubicin-mediated cell death. In this report, we provide evidences that doxorubicin induced more cell death in p53-negative tumor cells. Several cells, having p53 basal expression, showed increase in p53 DNA binding upon doxorubicin treatment. Doxorubicin induced cell death in p53-positive cells through expression of p53-dependent genes and activation of caspases and caspase-mediated cleavage of cellular proteins. Surprisingly, in p53-negative cells, doxorubicin-mediated cell death was more aggressive (faster and intense). Doxorubicin increased the amount of Fas ligand (FasL) by enhancing activator protein (AP) 1 DNA binding in both p53-positive and p53-negative cells, but the basal expression of Fas was higher in p53-negative cells. Anti-FasL antibody considerably protected doxorubicin-mediated cell death in both types of cells. Activation of caspases was faster in p53-negative cells upon doxorubicin treatment. In contrast, the basal expression of Ras oncoprotein was higher in p53-positive cells, which might increase the basal expression of Fas in these cells. Overexpression of Ras decreased the amount of Fas in p53-negative cells, thereby decreasing doxorubicin-mediated aggressive cell death. Overall, this study will help to understand the much studied chemotherapeutic drug, doxorubicin-mediated cell signaling cascade, that leads to cell death in p53-positive and -negative cells. High basal expression of Fas might be an important determinant in doxorubicin-mediated cell death in p53-negative cells.

Azadirachtin interacts with retinoic acid receptors and inhibits retinoic acid-mediated biological responses.

Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies.

Late phase activation of nuclear transcription factor kappaB by doxorubicin is mediated by interleukin-8 and induction of apoptosis via FasL.

Doxorubicin is one of the most effective molecules used in the treatment of various tumors. Contradictory reports often open windows to understand the doxorubicin-mediated signaling to exert its apoptosis effect. In this report, we provide evidences that doxorubicin induced biphasic induction of nuclear factor kappaB (NF-kappaB) of immediate activation followed by decrease in the amount of RelA (p65) subunit possibly by inducing the activity of proteasome, but not proteases. Further induction of NF-kappaB was observed through interleukin 8 (IL-8), expressed by doxorubicin treatment. Increased amount of IL-8 induced apoptosis via increase in the releases of intracellular Ca(2+), activation of calcineurin, nuclear translocation of nuclear factor activated T cell (NF-AT), and NF-AT-dependent FasL expression. Anti-IL-8 or -FasL antibody, dominant negative TRAF6 (TRAF6-DN), or TRAF6 binding peptide (TRAF6-BP) inhibited doxorubicin-mediated late phase induction of NF-kappaB and diminished cell death. Thus, our study clearly demonstrated that doxorubicin-mediated cell death is obtained through expression of IL-8. IL-8-mediated calcification is required for enhancement of doxorubicin-mediated cell death. Overall, this study will help to understand the much studied chemotherapeutic drug, doxorubicin-mediated cell signaling cascade to exert its effect during chemotherapy.

Azadirachtin interacts with the tumor necrosis factor (TNF) binding domain of its receptors and inhibits TNF-induced biological responses.

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor kappaB (NF-kappaB) and also expression of NF-kappaB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-kappaB (IkappaB alpha) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IkappaB alpha kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-kappaB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.

Inhibition of constitutive activity of nuclear transcription factor kappaB sensitizes doxorubicin-resistant cells to apoptosis.

Doxorubicin is one of the most effective agents used in the treatment of various tumors. Its use is restricted by the development of resistance to apoptosis, the mechanism of which is not fully understood. Nuclear transcription factor kappaB (NF-kappaB) has been shown both to block apoptosis and to promote cell proliferation, and hence has been considered as an important target for anticancer drug development. We found that in wild type and Dox-revertant MCF-7 cells, Doxorubicin induced NF-kappaB was transient and Dox-resistant cells showed high basal activity of NF-kappaB and expression of genes dependent on it. Moreover, in resistant cells Doxorubicin was unable to induce apoptosis as detected by assays for reactive oxygen intermediates generation, lipid peroxidation, cytotoxicity, PARP degradation and Bcl-2 expression. High basal expressions of multi-drug resistant protein and transglutaminase were found in Dox-resistant cells and inhibition of NF-kappaB decreased those amounts and also sensitized these cells by Doxorubicin. These observations collectively suggest that high NF-kappaB activity confers resistance to Doxorubicin and its inhibition potentiates apoptosis. This study indicates that NF-kappaB plays an important role in chemoresistance and establishes the fact that inhibition of NF-kappaB will be a novel approach in chemotherapy.