Immunological changes in the ascites of cancer patients after intraperitoneal administration of the bispecific antibody catumaxomab (anti-EpCAM×anti-CD3).

OBJECTIVE: To explore the effects of intraperitoneal (i.p.) infusion of catumaxomab, a bispecific monoclonal antibody (anti-EpCAM×anti-CD3), on T cells, NK cells and macrophages in ascites of cancer patients and to understand how ascitic immune cells can be activated despite the pervasive immunosuppressive ability of ascites microenvironment. METHODS: Six patients with malignant ascites received i.p. catumaxomab infusion. Ascitic immune cells were profiled by flow cytometry and gene expression at baseline and after i.p. catumaxomab infusion. In vitro experiments enabled investigations on the adverse effect of ascites microenvironment on catumaxomab-stimulated immune cells. RESULTS: I.p. catumaxomab infusion enhanced the expression of the CD69 and CD38 activation molecules in CD4(+) and CD8(+) T cells, NK cells and macrophages, and favoured CD8(+) T cell accumulation into the peritoneal cavity. An analogous immune cell activation as well as IFN-γ and IL-2 production were induced by catumaxomab in vitro. In vitro experiments showed that the immunosuppressive milieu of ascites abrogated all the immunostimulatory activities of catumaxomab. Adding EpCAM(+) tumour cells to the culture permitted both catumaxomab Fab regions to engage cognate antigens and restored immunostimulatory catumaxomab activity. CONCLUSIONS: This is the first demonstration in a clinical setting that i.p. catumaxomab infusion activates NK cells and macrophages in addition to T cells in ascites and favours CD8(+) T cell accumulation into the peritoneal cavity. Moreover, our findings indicate that the concomitant binding of both catumaxomab Fab regions delivers an activation signal that is strong enough to activate immune cells despite the prevailing immunosuppressive environment of malignant ascites.

A diet supplemented with ALA-rich flaxseed prevents cardiomyocyte apoptosis by regulating caveolin-3 expression.

AIMS: n-3 polyunsaturated fatty acids (PUFAs) induce beneficial effects on the heart, but the mechanisms through which these effects are operated are not completely clarified yet. Among others, cardiac diseases are often associated with increased levels of cytokines, such as tumour necrosis factor-α (TNF), that cause degeneration and death of cardiomyocytes. The present study has been carried out to investigate (i) the potential anti-apoptotic effects induced by the n-3 polyunsaturated α-linolenic acid (ALA) in experimental models of cardiac diseases characterized by high levels of TNF, and (ii) the potential role of caveolin-3 (Cav-3) in the mechanisms involved in this process. METHODS AND RESULTS: An ALA-rich flaxseed diet, administered from weaning to hereditary cardiomyopathic hamsters, prevented the onset of myocardial apoptosis associated with high plasma and tissue levels of TNF preserving caveolin-3 expression. To confirm these findings, isolated neonatal mouse cardiomyocytes were exposed to TNF to induce apoptosis. ALA pre-treatment greatly enhanced Cav-3 expression hampering the internalization of the caveolar TNF receptor and, thus, determining the abortion of the apoptotic vs. survival cascade. CONCLUSION: This study unveiled the Cav-3 pivotal role in defending cardiomyocytes against the TNF pro-apoptotic action and the ALA capacity to regulate this mechanism preventing cardiac degenerative diseases.

Correction: Lycopene Inhibits NF-kB-Mediated IL-8 Expression and Changes Redox and PPARγ Signalling in Cigarette Smoke-Stimulated Macrophages.

[This corrects the article DOI: 10.1371/journal.pone.0019652.].

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.

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.

DHA induces apoptosis and differentiation in human melanoma cells in vitro: involvement of HuR-mediated COX-2 mRNA stabilization and ß-catenin nuclear translocation.

The pro-inflammatory phenotype accompanying melanoma progression includes an enhanced expression of cyclooxygenase-2 (COX-2), which plays an important role in the acquisition of apoptosis resistance, and is a suitable target for melanoma prevention and therapy. We observed that the WM266-4 metastatic melanoma cell line showed a constitutive COX-2 expression higher than that of the primary WM115 cells, an increased cytosolic level of the COX-2 messenger RNA (mRNA)-stabilizer human antigen R (HuR) and a lower susceptibility to basal apoptosis. The transfection of HuR siRNA induced apoptosis and reduced COX-2 protein abundance in both the cells. The same effects were observed treating the cells with the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA), which reduced the cytoplasmic location and expression of HuR and, correspondently, decreased COX-2 protein expression and induced apoptosis. DHA also decreased the expression and stability of COX-2 mRNA, increased the ß-catenin expression in the nuclei and reduced it in the cytosol, where it forms a complex with HuR and COX-2 mRNA. DHA had also a pro-differentiating effect, which is compatible with the nuclear translocation of ß-catenin. These findings allow us to associate for the first time the constitutive expression of COX-2 in melanoma cells to the HuR-mediated stabilization of its mRNA and suggest that also ß-catenin may play a role in HuR-mediated COX-2 stabilization in these cells. The data demonstrate that the HuR-mediated stabilization of COX-2 may represent a target of DHA action in melanoma cells and suggest the application of DHA in the prevention and therapy of melanoma.

Lycopene inhibits NF-kB-mediated IL-8 expression and changes redox and PPARγ signalling in cigarette smoke-stimulated macrophages.

Increasing evidence suggests that lycopene, the major carotenoid present in tomato, may be preventive against smoke-induced cell damage. However, the mechanisms of such a prevention are still unclear. The aim of this study was to investigate the role of lycopene on the production of the pro-inflammatory cytokine IL-8 induced by cigarette smoke and the possible mechanisms implicated. Therefore, human THP-1 macrophages were exposed to cigarette smoke extract (CSE), alone and following a 6-h pre-treatment with lycopene (0.5-2 µM). CSE enhanced IL-8 production in a time- and a dose-dependent manner. Lycopene pre-treatment resulted in a significant inhibition of CSE-induced IL-8 expression at both mRNA and protein levels. NF-kB controlled the transcription of IL-8 induced by CSE, since PDTC prevented such a production. Lycopene suppressed CSE-induced NF-kB DNA binding, NF-kB/p65 nuclear translocation and phosphorylation of IKKα and IkBα. Such an inhibition was accompanied by a decrease in CSE-induced ROS production and NOX-4 expression. Lycopene further inhibited CSE-induced phosphorylation of the redox-sensitive ERK1/2, JNK and p38 MAPKs. Moreover, the carotenoid increased PPARγ levels which, in turn, enhanced PTEN expression and decreased pAKT levels in CSE-exposed cells. Such effects were abolished by the PPARγ inhibitor GW9662. Taken together, our data indicate that lycopene prevented CSE-induced IL-8 production through a mechanism involving an inactivation of NF-kB. NF-kB inactivation was accompanied by an inhibition of redox signalling and an activation of PPARγ signalling. The ability of lycopene in inhibiting IL-8 production, NF-kB/p65 nuclear translocation, and redox signalling and in increasing PPARγ expression was also found in isolated rat alveolar macrophages exposed to CSE. These findings provide novel data on new molecular mechanisms by which lycopene regulates cigarette smoke-driven inflammation in human macrophages.

Lycopene regulation of cholesterol synthesis and efflux in human macrophages.

Hypercholesterolemia is one of the most important risk factors for atherosclerosis, and tomato lycopene has been suggested to have beneficial effects against such a disease, although the exact molecular mechanism is unknown. We tested the hypothesis that lycopene may exert its antiatherogenic role through changes in cholesterol metabolism. Incubation of THP-1 cells with lycopene (0.5-2 µM) dose-dependently reduced intracellular total cholesterol. Such an effect was associated with a decrease in reduction of 3-hydroxy-3-methylglutaryl coenzyme A reductase expression and with an increase in ABCA1 and caveolin-1 (cav-1) expressions. In addition, lycopene enhanced RhoA levels in the cytosolic fraction, activating peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor alpha expressions. Concomitant addition of lycopene and the PPARγ inhibitor GW9662 or lycopene and mevalonate blocked the carotenoid-induced increase in ABCA1 and cav-1 expressions. These results imply a potential role of lycopene in attenuating foam cell formation and, therefore, in preventing atherosclerosis by a cascade mechanism involving inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase, RhoA inactivation and subsequent increase in PPARγ and liver X receptor alpha activities and enhancement of ABCA1 and cav-1 expressions.

Docosahexaenoic acid reverts resistance to UV-induced apoptosis in human keratinocytes: involvement of COX-2 and HuR.

The dramatic increase in the incidence of nonmelanoma skin cancer over the last decades has been related to the augmented exposure to ultraviolet (UV) radiation (UVR). It is known that apoptosis is induced as a protective mechanism after the acute irradiation of keratinocytes, whereas apoptotic resistance and carcinogenesis may follow the chronic exposure to UVR. We found that not all the human keratinocytes lines studied underwent apoptosis following acute exposure to UVR (10-60 mJ/cm(2)). Whereas UVR induced apoptosis in the HaCaT cells, NCTC 2544 and nr-HaCaT cells showed apoptosis resistance. The cytokeratin pattern of the apoptosis-resistant cells indicated that they possessed a degree of differentiation lower than that of HaCaT cells. They also showed an enhanced expression of cyclooxygenase-2 (COX-2), an early marker of carcinogenesis in various tissues, including skin. n-3 polyunsaturated fatty acids have drawn increasing interest as nutritional factors with the potential to reduce UVR carcinogenesis, and since they are apoptosis inducers and COX-2 inhibitors in cancer cells, we investigated the ability of n-3 polyunsaturated fatty acids to influence the resistance to UVR-induced apoptosis in keratinocytes. We observed that docosahexaenoic acid (DHA) reverted the resistance of nr-HaCaT cells to UVR-induced apoptosis, increasing the Bax/Bcl-2 ratio and caspase-3 activity, and reduced COX-2 levels by inhibiting the expression of the human antigen R (HuR), a known COX-2 mRNA stabilizer in keratinocytes. The transfection of nr-HaCaT cells with HuR siRNA mimicked the proapoptotic effect of DHA. Overall, our findings further support the role of DHA as a suitable anticarcinogenic factor against nonmelanoma skin cancers.

Lycopene prevention of oxysterol-induced proinflammatory cytokine cascade in human macrophages: inhibition of NF-κB nuclear binding and increase in PPARγ expression.

It is now well accepted that oxysterols play important roles in the formation of atherosclerotic plaque, involving cytotoxic, pro-oxidant and proinflammatory processes. It has been recently suggested that tomato lycopene may act as a preventive agent in atherosclerosis, although the exact mechanism of such a protection is not clarified. The main aim of this study was to investigate whether lycopene is able to counteract oxysterol-induced proinflammatory cytokines cascade in human macrophages, limiting the formation of atherosclerotic plaque. Therefore, THP-1 macrophages were exposed to two different oxysterols, such as 7-keto-cholesterol (4-16 µM) and 25-hydroxycholesterol (2-4 µM), alone and in combination with lycopene (0.5-2 µM). Both oxysterols enhanced pro-inflammatory cytokine [interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor α) secretion and mRNA levels in a dose-dependent manner, although at different extent. These effects were associated with an increased reactive oxygen species (ROS) production through an enhanced expression of NAD(P)H oxidase. Moreover, a net increment of phosphorylation of extracellular regulated kinase 1/2, p-38 and Jun N-terminal kinase and of nuclear factor kB (NF-κB) nuclear binding was observed. Lycopene prevented oxysterol-induced increase in pro-inflammatory cytokine secretion and expression. Such an effect was accompanied by an inhibition of oxysterol-induced ROS production, mitogen-activated protein kinase phosphorylation and NF-κB activation. The inhibition of oxysterol-induced cytokine stimulation was also mimicked by the specific NF-κB inhibitor pyrrolidine dithiocarbamate. Moreover, the carotenoid increased peroxisome proliferator-activated receptor γ levels in THP-1 macrophages. Taken all together, these data bring new information on the anti-atherogenic properties of lycopene, and on its mechanisms of action in atherosclerosis prevention.

Lycopene induces cell growth inhibition by altering mevalonate pathway and Ras signaling in cancer cell lines.

Several evidences suggest that cancer cells have abnormal cholesterol biosynthetic pathways and prenylation of small guanosine triphosphatase proteins. Tomato lycopene has been suggested to have beneficial effects against certain types of cancer, including that of prostate, although the exact molecular mechanism(s) is unknown. We tested the hypothesis that lycopene may exert its antitumor effects through changes in mevalonate pathway and in Ras activation. Incubation of the Ras-activated prostatic carcinoma LNCaP cells with a 24 h lycopene treatment (2.5-10 µM) dose dependently reduced intracellular total cholesterol by decreasing 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase expression and by inactivating Ras, as evidenced by its translocation from cell membranes to cytosol. Concomitantly, lycopene reduced the Ras-dependent activation of nuclear factor-kappaB (NF-κB). Such a reduction was parallel to an inhibition of reactive oxygen species production and to a decrease in the phosphorylation ofc-jun N-terminal kinase, extracellular signal-regulated kinase 1/2 and p38. These effects were also accompanied by an arrest of cell cycle progression and by apoptosis induction, as evidenced by a decrease in cyclin D1 and phospho-AKT levels and by an increase in p21, p27 and p53 levels and in Bax:Bcl-2 ratio. The addition of mevalonate prevented the growth-inhibitory effects of lycopene as well as its increase in Ras cytoplasmatic accumulation and the subsequent changes in NF-κB. The ability of lycopene in inhibiting HMG-CoA reductase expression and cell growth and in inactivating Ras was also found in prostate PC-3, colon HCT-116 and HT-29 and lung BEN cancer cells. These findings provide a novel mechanistic insight into the growth-inhibitory effects of lycopene in cancer.

Parathyroid hormone-related peptide and parathyroid hormone-related peptide receptor type 1 expression in human lung adenocarcinoma.

BACKGROUND: In many primary tumors, parathyroid hormone-related peptide (PTHrP) and PTHrP type 1 receptor (PTH1R) are coexpressed, supporting the possibility that PTHrP/PTH1R system can mediate important signals for tumor progression through paracrine/autocrine mechanisms. In non-small cell lung carcinoma the clinical relevance of the expression of PTH1R remains to be investigated. METHODS: Fifty-four lung adenocarcinomas of mixed histologic type from patients with stage I and II cancer were assayed by quantitative immunohistochemistry for the expression of PTHrP and PTH1R. RESULTS: PTHrP and PTH1R were expressed in a wide range of intensity in the cytoplasm of tumor cells, and their values showed a positive correlation. PTH1R, but not PTHrP, was expressed by plasma cells infiltrating the tumor stroma. PTHrP and PTH1R were not associated with age, tumor diameter, or histopathologic grading, whereas they were directly associated with lymph node involvement at presentation. Cox regression analysis, using PTHrP and PTH1R as continuous covariates, showed that the covariate levels were directly associated with the risk of death and metastasis. Patients whose tumors coexpressed high levels of PTHrP and PTH1R showed the highest risk of metastasis (relative risk, 5.89; 95% CI, 2.1-16.6; P = .0003) and death (relative risk, 6.24; 95% CI, 1.6-23.9; P = .0033). The presence of PTH1R-positive plasma cells in the tumor stroma was associated with a more favorable survival rate independently from the PTHrP status of the tumor. CONCLUSION: The paracrine/autocrine signaling through PTHrP/PTH1R could be important in early-stage lung adenocarcinoma progression.

Lycopene prevents 7-ketocholesterol-induced oxidative stress, cell cycle arrest and apoptosis in human macrophages.

The present study was undertaken to examine whether lycopene is able to counteract 7-ketocholesterol (7-KC)-induced oxidative stress and apoptosis in human macrophages. Human THP-1 macrophages were exposed to 7-KC (10-25 microM) alone and in combination with lycopene (0.5-2 microM), and we monitored changes in cell oxidative status [reactive oxygen species (ROS) production, NOX-4, hsp70 and hsp90 expressions, 8-OHdG formation] and in cell proliferation and apoptosis. After 24 h of treatment, lycopene significantly reduced the increase in ROS production and in 8-OHdG formation induced by the oxysterol in a dose-dependent manner. Moreover, the carotenoid strongly prevented the increase of NOX-4, hsp70 and hsp90 expressions as well as the phosphorylation of the redox-sensitive p38, JNK and ERK1/2 induced by the oxysterol. The attenuation of 7-KC-induced oxidative stress by lycopene coincided with a normalization of cell growth in human macrophages. Lycopene prevented the arrest in G0/G1 phase of cell cycle induced by the oxysterol and counteracted the increased expression of p53 and p21. Concomitantly, it inhibited 7-KC-induced apoptosis, by limiting caspase-3 activation and the modulatory effects of 7-KC on AKT, Bcl-2, Bcl-xL and Bax. Comparing the effects of lycopene, beta-carotene and (5Z)-lycopene on ROS production, cell growth and apoptosis show that lycopene and its isomer were more effective than beta-carotene in counteracting the dangerous effects of 7-KC in human macrophages. Our study suggests that lycopene may act as a potential antiatherogenic agent by preventing 7-KC-induced oxidative stress and apoptosis in human macrophages.

Ischemic injury activates PTHrP and PTH1R expression in human ventricular cardiomyocytes.

The PTHrP/PTH1R signalling system induces calciotropic and myorelaxant effects on the vascular system and plays critical roles in the development of the cardiovascular system. In animal models, PTHrP exerts important effects on heart rate and contractility, particularly under ischemic conditions, while, in human hearts, the expression of PTHrP by cardiomyocytes remains to be defined in both normal and ischemic conditions. The present study has been conducted on 101 autoptical cases and confirmed on nine explanted hearts in order to analyze the expression of the PTHrP/PTH1R system by ventricular myocardium in respect to morphological aspects of the myocardial ischemic damage, myofiber hypertrophy and disarray, coronarosclerosis, age and sex. Immunohistochemistry showed positive cytoplasmic immunostaining for both PTHrP and PTH1R in ventricular cardiomyocytes. The expression levels of the PTHrP/PTH1R system resulted significantly increased (P = 0.0008 and P < 0.0001, respectively) in association with the myocardial ischemic damage and the presence of cardiomyocyte hypertrophy (P = 0.02 and P = 0.009 respectively). Conversely, increased expression levels of PTHrP alone were observed in myofiber disarray (P = 0.04), whereas PTH1R was augmented in coronarosclerosis (P = 0.004) and age (P = 0.001). Taken together, these results demonstrate that human ventricular cardiomyocytes express PTHrP and PTH1R and suggest that the activation of the PTHrP/PTH1R system could represent an aspect of the embryonic gene program typically reactivated by the myocardium when subjected to ischemia and/or hypertrophy.

Docosahexaenoic acid induces apoptosis in lung cancer cells by increasing MKP-1 and down-regulating p-ERK1/2 and p-p38 expression.

Different agents able to modulate apoptosis have been shown to modify the expression of the MAP-kinase-phosphatase-1 (MKP-1). The expression of this phosphatase has been considered a potential positive prognostic factor in lung cancer, and smoke was shown to reduce the levels of MKP-1 in ferret lung. Our aim was to assess whether the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA), known to inhibit the growth of several cancer cells mainly inducing apoptosis, may exert pro-apoptotic effect in lung cancer cells by modifying MKP-1 expression. We observed that DHA increased MKP-1 protein and mRNA expression and induced apoptosis in different lung cancer cell lines (mink Mv1Lu adenocarcinoma cells, human A549 adenocarcinoma and human BEN squamous carcinoma cells). We inhibited the pro-apoptotic effect of DHA by treating the cells with the phosphatase inhibitor Na(3)VO(4) or by silencing the MKP-1 gene with the specific siRNA. This finding demonstrated that the induction of apoptosis by DHA involved a phosphatase activity, specifically that of MKP-1. DHA reduced also the levels of the phosphorylated MAP-kinases, especially ERK1/2 and p38. Such an effect was not observed when the MKP-1 gene was silenced. Altogether, the data provide evidence that the DHA-induced overexpression of MKP-1 and the resulting decrease of MAP-kinase phosphorylation by DHA may underlie the pro-apoptotic effect of this fatty acid in lung cancer cells. Moreover, they support the hypothesis that DHA may exert chemopreventive action in lung cancer.

The sensitivity to beta-carotene growth-inhibitory and proapoptotic effects is regulated by caveolin-1 expression in human colon and prostate cancer cells.

Although several mechanisms have been proposed to explain the putative role of beta-carotene in cancer, no studies have investigated a possible influence of beta-carotene on caveolin-1 (cav-1) pathway, an important intracellular signaling deregulated in cancer. Here, different human colon and prostate cancer cell lines, expressing (HCT-116, PC-3 cells) or not (Caco-2, LNCaP cells) cav-1, were treated with varying concentrations of beta-carotene (0.5-30 muM) for different periods of time (3-72 h) and the effects on cell growth were investigated. The results of this study show that (i) beta-carotene acted as a growth-inhibitory agent in cav-1-positive cells, but not in cav-1-negative cells; (ii) in cav-1-positive cells, the carotenoid downregulated in a dose- and time-dependent manner the expression of cav-1 protein and messenger RNA levels and inhibited AKT phosphorylation which, in turn, stimulated apoptosis by increasing the expression of beta-catenin and c-myc and the activity of caspases-3, -7, -8 and -9; when the carotenoid was removed from culture medium, a progressive increase in cell growth was observed with respect to beta-carotene-treated cells and (iii) the transfection of cav-1 in cav-1-negative cells increased cell sensitivity to beta-carotene by inducing apoptosis. This effect was accompanied by a reduction of both cav-1 and AKT phosphorylation and by an increase of c-myc and beta-catenin expression. Silencing of c-Myc attenuated beta-carotene-induced apoptosis and beta-catenin expression. All together, these data suggest that the modulation of cav-1 pathway by beta-carotene could be a novel mechanism by which the carotenoid acts as a potent growth-inhibitory agent in cancer cells.

Neuropilin-1 expression identifies a subset of regulatory T cells in human lymph nodes that is modulated by preoperative chemoradiation therapy in cervical cancer.

We examined the phenotype and function of CD4+ T cells expressing the semaphorin III receptor neuropilin-1 (Nrp1) in human lymph nodes and peripheral blood. In lymph nodes, Nrp1 identified a small regulatory CD4+ CD25(high) T-cell subpopulation (Nrp1+ Treg) that expressed higher levels of Forkhead box P3 (Foxp3) message and protein than Nrp1- Treg, and various molecular markers of activated Treg, i.e. CD45RO, human leucocyte antigen (HLA)-DR and glucocorticoid-induced tumour necrosis factor receptor (GITR). Similarly to conventional Treg, Nrp1+ Treg proliferated poorly in vitro, and exerted contact-dependent in vitro suppression of T-cell proliferation and cytokine secretion. However, Nrp1+ Treg were more efficient than Nrp1- Treg at inducing suppression. Nrp1 was also expressed on a small subpopulation of CD25(int) and CD25- CD4+ T cells that expressed more Foxp3, CD45RO, HLA-DR and GITR than their Nrp1- counterparts. In contrast, in peripheral blood Nrp1 identified a minor CD4+ T-cell subset that did not display the phenotypic features of Treg lacking Foxp3 expression and marginally expressing CD25. Hence, the function of Nrp1+ CD4+ T cells seemingly depends on their anatomical location. In a previous report, we proposed that Treg may curb the anti-tumour T-cell response in cervical cancer. We show here that Treg and Nrp1+ Treg levels dropped in the tumour-draining lymph nodes of patients with cervical cancer following preoperative chemoradiotherapy in a direct relationship with the reduction of tumour mass, suggesting that suppressor cell elimination facilitated the generation of T cells mediating the destruction of the neoplastic cells left behind after cytotoxic therapy.