Long-chain n-3 PUFA against breast and prostate cancer: Which are the appropriate doses for intervention studies in animals and humans?

The potential antineoplastic effect of the long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) remains a highly controversial issue. Numerous animal studies have supported the anticancer role of these dietary fatty acids, whereas conflicting results have been obtained in population studies, and only a few intervention human trials have been so far performed. In view of the possibility that the anticancer effects may be maximally observed within a defined range of EPA and DHA doses, herein we critically review the results and doses used in both animal studies and human clinical trials focusing on the possible n-3 PUFA protective effects against breast and prostate cancer. Our main aim is to identify the EPA and/or DHA ranges of doses needed to obtain clear anticancer effects. This may be of great help in designing future animal studies, and also in understanding the most appropriate dose for further human intervention studies. Moreover, since the healthy effects of these fatty acids have been strictly related to their increased incorporation in plasma and tissue lipids, we also examine and discuss the incorporation changes following the administration of the effective anticancer EPA and/or DHA doses in animals and humans.

Omega-3 Polyunsaturated Fatty Acids: The Way Forward in Times of Mixed Evidence.

Almost forty years ago, it was first hypothesized that an increased dietary intake of omega-3 polyunsaturated fatty acids (PUFA) from fish fat could exert protective effects against several pathologies. Decades of intense preclinical investigation have supported this hypothesis in a variety of model systems. Several clinical cardiovascular studies demonstrated the beneficial health effects of omega-3 PUFA, leading medical institutions worldwide to publish recommendations for their increased intake. However, particularly in recent years, contradictory results have been obtained in human studies focusing on cardiovascular disease and the clinical evidence in other diseases, particularly chronic inflammatory and neoplastic diseases, was never established to a degree that led to clear approval of treatment with omega-3 PUFA. Recent data not in line with the previous findings have sparked a debate on the health efficacy of omega-3 PUFA and the usefulness of increasing their intake for the prevention of a number of pathologies. In this review, we aim to examine the controversies on the possible use of these fatty acids as preventive/curative tools against the development of cardiovascular, metabolic, and inflammatory diseases, as well as several kinds of cancer.

Antioxidant and anti-inflammatory effects of selected natural compounds contained in a dietary supplement on two human immortalized keratinocyte lines.

Several advantages may derive from the use of dietary supplements containing multiple natural antioxidants and/or anti-inflammatory agents. At present, however, there is scarce information on the properties and potential of combined supplements. To fill the gap, the antioxidant and anti-inflammatory activities exerted by a combination of seven natural components (coenzyme Q10, krill oil, lipoic acid, resveratrol, grape seed oil, α-tocopherol, and selenium) contained in a dietary supplement used for the prevention of skin disorders were investigated in vitro. Each component was administered, alone or in combination, to human keratinocytes, and the inhibition of Reactive Oxygen Species production and lipid peroxidation as well as the ability to reduce inflammatory cytokine secretion and to modulate Nuclear Factor-κB pathway was evaluated. The combination exhibited high antioxidant activity and in specific conditions the combination's efficiency was higher than that of the most powerful components administered individually. Moreover, the combination showed remarkable anti-inflammatory properties. It reduced more efficiently than each component the secretion of Monocyte Chemoattractant Protein-1, a crucial cytokine for the development of chronic inflammation in skin, and inhibited Nuclear Factor-κB molecular pathway. Overall, our findings suggest that the combined formulation may have the potential to powerfully inhibit oxidative stress and inflammation at skin level.

Potential of long-chain n-3 polyunsaturated fatty acids in melanoma prevention.

The possible antineoplastic activity of dietary long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFAs) has been supported by ample preclinical studies that have identified a number of molecular factors and pathways affected by these fatty acids and involved in cell growth, apoptosis, invasion, and angiogenesis. The aim of this critical review is to assess the current state of knowledge on the potential anticancer effects of LC n-3 PUFAs against malignant melanoma, one of the most common cancers among Western populations. The results of preclinical as well as human observational and interventional studies investigating the effects of LC n-3 PUFAs in melanoma were examined. Overall, the analysis of the literature reveals that, even though a large body of information is available, further effort is needed to identify the main molecular targets of LC n-3 PUFAs in melanoma. Moreover, additional well-designed human observational studies are essential to shed further light on the issue. The results of these studies could provide support and specific information for the development of clinical studies, especially those performed in combination with conventional or innovative antineoplastic therapies.

Cancer stem cells in colorectal cancer from pathogenesis to therapy: controversies and perspectives.

Colorectal cancer remains one of the most common and lethal malignancies worldwide despite the use of various therapeutic strategies. A better understanding of the mechanisms responsible for tumor initiation and progression is essential for the development of novel, more powerful therapies. The traditional, so-called "stochastic model" of tumor development, which assumes that each cancer cell is tumorigenic, has been deeply challenged during the past decade by the identification of cancer stem cells (CSCs), a biologically distinct subset of cells within the bulk of tumor mass. This discovery led to the development of the hierarchical model of tumorigenesis which assumes that only CSCs have the ability to initiate tumor growth, both at primary and metastatic sites. This model implies that the elimination of all CSCs is fundamental to eradicate tumors and that failure to do so might be responsible for the occurrence of relapses and/or metastases frequently observed in the clinical management of colorectal cancer patients. Identification and isolation of CSCs is essential for a better understanding of their role in the tumorigenetic process and for the development of CSC-specific therapies. Several methods have been used for this purpose and many efforts have been focused on the identification of specific CSC-surface markers. This review provides an overview of the proposed roles of CSC in human colorectal tumorigenesis focusing on the most important molecules identified as CSC-specific markers in colorectal cancer and on the potential strategies for the development of CSC-targeted therapy.

Experimental evidence of ω-3 polyunsaturated fatty acid modulation of inflammatory cytokines and bioactive lipid mediators: their potential role in inflammatory, neurodegenerative, and neoplastic diseases.

A large body of evidence has emerged over the past years to show the critical role played by inflammation in the pathogenesis of several diseases including some cardiovascular, neoplastic, and neurodegenerative diseases, previously not considered inflammation-related. The anti-inflammatory action of ω-3 polyunsaturated fatty acids (PUFAs), as well as their potential healthy effects against the development and progression of the same diseases, has been widely studied by our and others' laboratories. As a result, a rethinking is taking place on the possible mechanisms underlying the beneficial effects of ω-3 PUFAs against these disorders, and, in particular, on the influence that they may exert on the molecular pathways involved in inflammatory process, including the production of inflammatory cytokines and lipid mediators active in the resolving phase of inflammation. In the present review we will summarize and discuss the current knowledge regarding the modulating effects of ω-3 PUFAs on the production of inflammatory cytokines and proresolving or protective lipid mediators in the context of inflammatory, metabolic, neurodegenerative, and neoplastic diseases.

M tuberculosis in the adjuvant modulates time of appearance of CNS-specific effector T cells in the spleen through a polymorphic site of TLR2.

DC deliver information regulating trafficking of effector T cells along T-cell priming. However, the role of pathogen-derived motives in the regulation of movement of T cells has not been studied. We hereinafter report that amount of M tuberculosis in the adjuvant modulates relocation of PLP139-151 specific T cells. In the presence of a low dose of M tuberculosis in the adjuvant, T cells (detected by CDR3 BV-BJ spectratyping, the so-called "immunoscope") mostly reach the spleen by day 28 after immunization ("late relocation") in the SJL strain, whereas T cells reach the spleen by d 14 with a high dose of M tuberculosis ("early relocation"). The C57Bl/6 background confers a dominant "early relocation" phenotype to F1 (SJL×C57Bl/6) mice, allowing early relocation of T cells in the presence of low dose M tuberculosis. A single non-synonymous polymorphism of TLR2 is responsible for "early/late" relocation phenotype. Egress of T lymphocytes is regulated by TLR2 expressed on T cells. Thus, pathogens engaging TLR2 on T cells regulate directly T-cell trafficking, and polymorphisms of TLR2 condition T-cell trafficking upon a limiting concentration of ligand.

From magnesium to magnesium transporters in cancer: TRPM7, a novel signature in tumour development.

Magnesium availability affects many cellular functions that are critical for tumour growth and spreading, such as proliferation, metabolism and angiogenesis. In vivo, magnesium deficiency, and the resulting inflammation, can trigger both anti- and pro-tumour effects. Recent experimental evidence indicates that altered expression of the transient receptor potential melastatin, type 7 (TRPM7) epithelial magnesium channel is a frequent finding in cancer cells and human tumour tissues, and correlates with cell proliferation and/or migration. We review the role of TRPM7 in tumour development, with particular regard to its channelling function mediating both Ca(2+) and Mg(2+) influx, as well as its kinase activity, likely regulating actomyosin contractility. The potential diagnostic and therapeutic applications based on TRPM7 detection and inhibition, are also discussed.

Comparative antioxidant effects of lycopene, apo-10'-lycopenoic acid and apo-14'-lycopenoic acid in human macrophages exposed to H2O2 and cigarette smoke extract.

Much of the beneficial effects of tomato lycopene in the prevention of chronic diseases has been attributed to its antioxidant properties, which could be mediated by its metabolites and/or oxidation products. However, the biological functions of these lycopene derivatives remain still unknown. In the present study, we evaluated and compared the antioxidant efficacy of the lycopene eccentric cleavage products apo-10'-lycopenoic acid and apo-14'-lycopenoic acid in counteracting the oxidative effects of H(2)O(2) and cigarette smoke extract (CSE) in THP-1 macrophages. Both apo-10'-lycopenoic acid and apo-14'-lycopenoic acid were able to inhibit spontaneous and H(2)O(2)-induced ROS production in a dose-dependent manner. Such an effect was accompanied by an inhibition of MAPK phosphorylation, by NF-κB inactivation, and by inhibition of hsp-70 and hsp-90 expressions. Both apo-lycopenoic acids also decreased CSE-induced ROS production, 8-OHdG formation and reduced the increase in NOX-4 and COX-2 expressions caused by CSE. However, in both the models of oxidative stress, apo-14'-lycopenoic acid was much more potent as an antioxidant than apo-10'-lycopenoic acid, showing antioxidant properties similar to lycopene. These data strongly suggest that apo-lycopenoic acids, and particularly apo-14'-lycopenoic acid, may mediate some of the antioxidant functions of lycopene in cells.

New Insights into the CD133 (Prominin-1) Expression in Mouse and Human Colon Cancer Cells.

Following its discovery as a cancer stem cell marker, CD133 has been widely studied for its role in colorectal tumorigenesis. Indeed, colon cancer remains one of the major causes of cancer-related disease and death worldwide, and there is a strong need for an improvement of current diagnostic, prognostic, and therapeutic strategies. Thus, efforts have been devoted to try to understand whether CD133 might play a role in human colorectal tumorigenesis and might contribute to a better management of colon cancer patients. This chapter reviews the current knowledge on CD133 expression in normal and cancer colon tissues, both in humans and mice, discussing apparently conflicting data reported in the two species. Moreover, a great attention is devoted to the available information regarding the functional role of CD133 in colon cancer cells. Finally, the proposed clinical applications of CD133, as a prognostic and/or predictive marker as well as a target for novel antineoplastic strategies in colorectal cancer, are discussed. Overall, the available data support a potential important role of CD133 as cancer stem cell marker in colon cancer cells and warrant future studies to verify its potential use in the routine clinical management of colon cancer patients.

Antioxidant capacity of tomato seed oil in solution and its redox properties in cultured macrophages.

The health benefits of tomato seed oil (TSO) have been suggested to be related to its antioxidant activity, although at the moment not much information is available on the antioxidant effects of TSO in biological systems. In this paper, we evaluated the antioxidant capacity of TSO using different spectrophotometrical antioxidant assays (LPSC, FRAP, αTEAC, DPPH). Moreover, we determined the ability of TSO in inhibiting oxidative stress in human cultured macrophages. The peroxyl radical scavenging LPSC assay was the most sensitive assay to detect the antioxidant capacity of the TSO, followed by the DPPH, FRAP, and αTEAC assay. TSO was able to counteract spontaneous and H2O2-induced oxidative stress in human macrophages, limiting intracellular ROS production and controlling oxidative stress signaling. In particular, TSO was able to decrease the phosphorylation of the MAPK ERK1/2, JNK, and p-38, activation of the redox-sensitive NF-kB, and expression of the heat shock proteins 70 and 90. When the antioxidant capacity of TSO was compared with that of purified lycopene, inhibition of ROS production by TSO was remarkably higher. This was due to the high content of other antioxidants in TSO, including (5Z)-, (9Z)-, (13Z)-, and (15Z)-lycopene isomers, ß-carotene, lutein, γ-tocopherol, and α-tocopherol.

Propionyl-L-carnitine hydrochloride for treatment of mild to moderate colonic inflammatory bowel diseases.

AIM: To assess clinical and endoscopic response to propionyl-L-carnitine hydrochloride (PLC) in colonic inflammatory bowel disease. METHODS: Patients suffering from mild to moderate ulcerative colitis (UC) or Crohn's disease (CD) colitis, with disease activity index (DAI) between 3 and 10 and under stable therapy with oral aminosalicylates, mercaptopurine or azathioprine, for at least 8 wk prior to baseline assessments, were considered suitable for enrollment. Fourteen patients were enrolled to assume PLC 2 g/d (two active tablets twice daily) orally. Clinical-endoscopic and histological activity were assessed by DAI and histological index (HI), respectively, following a colonoscopy performed immediately before and after 4 wk treatment. Clinical response was defined as a lowering of at least 3 points in DAI and clinical remission as a DAI score ≤ 2. Histological response was defined as an improvement of HI of at least 1 point. We used median values for the analysis. Differences pre- and post-treatment were analyzed by Wilcoxon signed rank test. RESULTS: All patients enrolled completed the study. One patient, despite medical advice, took deflazacort 5 d before follow-up colonoscopy examination. No side effects were reported by patients during the trial. After treatment, 71% (SE 12%) of patients achieved clinical response, while 64% (SE 13%) obtained remission. Separating UC from CD patients, we observed a clinical response in 60% (SE 16%) and 100%, respectively. Furthermore 60% (SE 16%) of UC patients and 75% (SE 25%) of CD patients were in clinical remission after therapy. The median DAI was 7 [interquartile range (IQR): 4-8] before treatment and decreased to 2 (IQR: 1-3) (P < 0.01) after treatment. Only patients with UC showed a significant reduction of DAI, from a median 6.5 (IQR: 4-9) before treatment to 2 (IQR: 1-3) after treatment (P < 0.01). Conversely, in CD patients, although displaying a clear reduction of DAI from 7 (IQR: 5.5-7.5) before therapy to 1.5 (IQR: 0.5-2.5) after therapy, differences observed were not significant (P = 0.06). Seventy-nine percent (SE 11%) of patients showed improvement of HI of at least 1 point, while only one CD and two UC patients showed HI stability; none showed HI worsening. Median HI decreased from 1 (IQR: 1-2), to 0.5 (IQR: 0-1) at the endoscopic control in the whole population (P < 0.01), while it changed from 1 (IQR: 1-2) to 0.5 (IQR: 0-1) in UC patients (P < 0.01) and from 1.5 (IQR: 1-2) to 0.5 (IQR: 0-1) in CD patients (P = not significant). The two sample tests of proportions showed no significant differences in clinical and histological response or in clinical remission between UC and CD patients. No side effects were reported during treatment or at 4 wk follow-up visit. CONCLUSION: PLC improves endoscopic and histological activity of mild to moderate UC. Further studies are required to evaluate PLC efficacy in colonic CD patients.

Increased expression of CD133 and reduced dystroglycan expression are strong predictors of poor outcome in colon cancer patients.

BACKGROUND: Expression levels of CD133, a cancer stem cell marker, and of the α-subunit of the dystroglycan (α-DG) complex, have been previously reported to be altered in colorectal cancers. METHODS: Expression levels of CD133 and α-DG were assessed by immunohistochemistry in a series of colon cancers and their prognostic significance was evaluated. RESULTS: Scattered cells positive for CD133 were rarely detected at the bases of the crypts in normal colonic mucosa while in cancer cells the median percentage of positive cells was 5% (range 0-80). A significant correlation was observed with pT parameter and tumor stage but not with tumor grade and N status. Recurrence and death from disease were significantly more frequent in CD133-high expressing tumors and Kaplan-Meier curves showed a significant separation between high vs low expressor groups for both disease-free (p = 0.002) and overall (p = 0.008) survival.Expression of α-DG was reduced in a significant fraction of tumors but low α-DG staining did not correlate with any of the classical clinical-pathological parameters. Recurrence and death from the disease were significantly more frequent in α-DG-low expressing tumors and Kaplan-Meier curves showed a significant separation between high vs low expressor tumors for both disease-free (p = 0.02) and overall (p = 0.02) survival. Increased expression of CD133, but not loss of α-DG, confirmed to be an independent prognostic parameters at a multivariate analysis associated with an increased risk of recurrence (RR = 2.4; p = 0.002) and death (RR = 2.3; p = 0.003). CONCLUSIONS: Loss of α-DG and increased CD133 expression are frequent events in human colon cancer and evaluation of CD133 expression could help to identify high-risk colon cancer patients.

Increased expression of CD133 is a strong predictor of poor outcome in stage I colorectal cancer patients.

OBJECTIVE: Stage I colorectal carcinomas display a highly variable behavior which is not accurately predicted by the available prognostic markers. CD133 is considered a useful marker to identify the so-called cancer stem cells in colorectal cancers (CRCs) and its expression has been shown to have prognostic significance in CRC patients. This study aimed to verify whether immunohistochemical evaluation of CD133 might correlate with the progression risk of stage I CRC patients. MATERIAL AND METHODS: Expression levels of the CD133 molecule were analyzed and compared in two series of stage I surgically resected CRC patients showing disease progression and death for the disease and patients with no evidence of disease progression after at least 6 years after surgery. RESULTS: A positive staining for CD133 was detected in 52% of the cases with poor prognosis and only in 9% of the group with good prognosis, and this difference was highly significant (p < 0.001). A significant correlation was detected between CD133 expression and histological parameters, such as tumor budding, vascular invasion, and presence of lymph node micrometastases but not tumor grading, gender, and age. Disease-free survival and cancer-specific survival of CD133 negative tumors were significantly longer compared to positive cases. In multivariate analyses, CD133 staining confirmed to be a predictor of shorter survival independent from vascular invasion but not from lymph nodes micrometastases. CONCLUSIONS: These findings demonstrate that CD133 immunostaining is a useful predictor of high risk progression in stage I CRC patients and might help to identify patients eligible for adjuvant chemotherapy.

DHA induces apoptosis by altering the expression and cellular location of GRP78 in colon cancer cell lines.

n-3 polyunsaturated fatty acids exert growth-inhibitory and pro-apoptotic effects in colon cancer cells. We hypothesized that the anti-apoptotic glucose related protein of 78kDa (GRP78), originally described as a component of the unfolded protein response in endoplasmic reticulum (ER), could be a molecular target for docosahexaenoic acid (DHA) in these cells. GRP78 total and surface overexpression was previously associated with a poor prognosis in several cancers, whereas its down-regulation with decreased cancer growth in animal models. DHA treatment induced apoptosis in three colon cancer cell lines (HT-29, HCT116 and SW480), and inhibited their total and surface GRP78 expression. The cell ability to undergo DHA-induced apoptosis was inversely related to their level of GRP78 expression. The transfection of the low GRP78-expressing SW480 cells with GRP78-GFP cDNA significantly induced cell growth and inhibited the DHA-driven apoptosis, thus supporting the essential role of GRP78 in DHA pro-apoptotic effect. We suggest that pERK1/2 could be the first upstream target for DHA, and demonstrate that, downstream of GRP78, DHA may exert its proapoptotic role by augmenting the expression of the ER resident factors ERdj5 and inhibiting the phosphorylation of PKR-like ER kinase (PERK), known to be both physically associated with GRP78, and by activating caspase-4. Overall, the regulation of cellular GRP78 expression and location is suggested as a possible route through which DHA can exert pro-apoptotic and antitumoral effects in colon cancer cells.

The tumor suppressor HINT1 regulates MITF and ß-catenin transcriptional activity in melanoma cells.

Histidine triad nucleotide-binding protein 1 (HINT1) is a haploinsufficient tumor suppressor gene that inhibits the Wnt/ß-catenin pathway in colon cancer cells and Microphthalmia-associated transcription factor (MITF) activity in human mast cells. MITF and ß-catenin play a central role in melanocyte and melanoma cell survival, and this study aimed to investigate the effects of HINT1 on the MITF and ß-catenin pathways in malignant melanoma cells. We found that HINT1 inhibits MITF and ß-catenin transcriptional activity, and both proteins can be co-immunoprecipitated with an anti-HINT1-specific antibody in melanoma cell lines. Stable, constitutive overexpression of the HINT1 protein in human melanoma cells significantly impaired cell proliferation in vitro and tumorigenesis in vivo. These effects were associated with a decreased expression of cyclin D1 and BCL2, well known MITF and ß-catenin transcription targets, respectively. We also demonstrated that BCL2 and cyclin D1 can partially rescue the HINT1-driven phenotype. Moreover, we found in ChIP assays that HINT1 binds the chromatin at MITF and ß-catenin sites in BCL2 and cyclin D1 promoters, respectively, and that mSIN3a and HDAC1, well known transcriptional repressors, can be co-immunoprecipitated with an anti-HINT1-specific antibody. These findings support the tumor suppressor activity of HINT1 gene in melanoma cells by promoting the formation of non-functional complexes with oncogenic transcription factors like MITF and ß-catenin.

Rhodium and iridium salts inhibit proliferation and induce DNA damage in rat fibroblasts in vitro.

Environmental concentration of the platinum group elements is increased in the last years due to their use in automobile catalytic converters. Limited data are available on the effects of such elements at a cellular level and on their toxicity, especially for rhodium and iridium which have been more recently introduced in use. The toxic effects of rhodium and iridium salts were analyzed on a normal diploid rat fibroblast cell line in vitro. Both salts halted cell growth in a dose- and time-dependent fashion by inhibiting cell cycle progression, inducing apoptosis and modulating the expression of cell cycle regulatory proteins. In fact, they both caused an accumulation of cells in the G2/M phase of the cell cycle and affected the expression levels of pRb, cyclins D1 and E, p21(Waf1) and p27(Kip1). DNA strand breaks, as assessed by comet test, and an increase in the intracellular levels of reactive oxygen species also occurred in exposed cell cultures. These findings suggest a potential toxicity of both iridium and rhodium salts and emphasize the need for further studies to understand their effects at a cellular level to enable a better assessment of their toxic effects and to identify ways for their modulation and/or prevention.

Lycopene as a guardian of redox signalling.

It has been suggested that lycopene, the major carotenoid found in tomato, exhibits health-beneficial effects by virtue of its antioxidant activity. However, recent literature suggests that lycopene can actually "perform" roles independent of such capacity and involving a direct modulation of redox signalling. Reactive oxygen species are known to act as second messengers in the modulation of cellular signalling leading to gene expression changes and pharmacological responses. Lycopene may control redox-sensitive molecular targets, affecting enzyme activities and expressions and modulating the activation of MAPKs and transcription factors, such as NF-κB and AP-1, Nrf2.

Modulation of MMP-9 pathway by lycopene in macrophages and fibroblasts exposed to cigarette smoke.

Matrix metalloproteinase-9 (MMP-9) has been implicated in both inflammation and fibrosis. It has been reported that cigarette smoke induced MMP-9 expression and that lycopene may act as an anti-inflammatory agent and may counteract several signal pathways affected by cigarette smoke exposure. However, at the moment, it is unknown if lycopene may inhibit cigarette smoke-induced MMP-9 expression. Presently, we examined the inhibitory mechanism of lycopene on MMP-9 induction in cultured human macrophages (THP-1 cells), in isolated rat alveolar macrophages (AMs) and in cultured RAT-1 fibroblasts, all cellular sources of MMP-9, exposed to cigarette smoke extract (CSE). CSE induced a marked increase in MMP-9 expression in cultured as well as in isolated cells. A 8 h-lycopene pre-treatment (0.5-2 µM) reduced CSE-mediated MMP-9 induction in a dose- and time-dependent manner. Lycopene attenuated CSE-mediated activation of Ras, enhancing the levels of this protein in the cytosolic fraction. Moreover, lycopene inhibited CSE-induced ERK1/2 and NF-κB activation in a dose-dependent manner. Lycopene-mediated inhibition of MMP-9 was reversed by mevalonate and associated with a reduced expression of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Taken together, these results suggest that lycopene may inhibit CSE-mediated MMP-9 induction, primarily by blocking prenylation of Ras in a signaling pathway, in which MEK1/2-ERK1/2 and NF-κB are involved.