Pasquale Pierimarchi

Stimulatory effect of Eucalyptus essential oil on innate cell-mediated immune response.

BackgroundBesides few data concerning the antiseptic properties against a range of microbial agents and the anti-inflammatory potential both in vitro and in vivo, little is known about the influence of Eucalyptus oil (E O) extract on the monocytic/macrophagic system, one of the primary cellular effectors of the immune response against pathogen attacks. The activities of this natural extract have mainly been recognized through clinical experience, but there have been relatively little scientific studies on its biological actions. Here we investigated whether E O extract is able to affect the phagocytic ability of human monocyte derived macrophages (MDMs) in vitro and of rat peripheral blood monocytes/granulocytes in vivo in absence or in presence of immuno-suppression induced by the chemotherapeutic agent 5-fluorouracil (5-FU).MethodsMorphological activation of human MDMs was analysed by scanning electron microscopy. Phagocytic activity was tested: i) in vitro in EO treated and untreated MDMs, by confocal microscopy after fluorescent beads administration; ii) in vivo in monocytes/granulocytes from peripheral blood of immuno-competent or 5-FU immuno-suppressed rats, after EO oral administration, by flow cytometry using fluorescein-labelled E. coli. Cytokine release by MDMs was determined using the BD Cytometric Bead Array human Th1/Th2 cytokine kit.ResultsE O is able to induce activation of MDMs, dramatically stimulating their phagocytic response. E O-stimulated internalization is coupled to low release of pro-inflammatory cytokines and requires integrity of the microtubule network, suggesting that E O may act by means of complement receptor-mediated phagocytosis. Implementation of innate cell-mediated immune response was also observed in vivo after E O administration, mainly involving the peripheral blood monocytes/granulocytes. The 5-FU/EO combined treatment inhibited the 5-FU induced myelotoxicity and raised the phagocytic activity of the granulocytic/monocytic system, significantly decreased by the chemotherapic.ConclusionOur data, demonstrating that Eucalyptus oil extract is able to implement the innate cell-mediated immune response, provide scientific support for an additional use of this plant extract, besides those concerning its antiseptic and anti-inflammatory properties and stimulate further investigations also using single components of this essential oil. This might drive development of a possible new family of immuno-regulatory agents, useful as adjuvant in immuno-suppressive pathologies, in infectious disease and after tumour chemotherapy.

Treatment of doxorubicin-resistant MCF7/Dx cells with nitric oxide causes histone glutathionylation and reversal of drug resistance

Acquired drug resistance was found to be suppressed in the doxorubicin-resistant breast cancer cell line MCF7/Dx after pre-treatment with GSNO (nitrosoglutathione). The effect was accompanied by enhanced protein glutathionylation and accumulation of doxorubicin in the nucleus. Among the glutathionylated proteins, we identified three members of the histone family; this is, to our knowledge, the first time that histone glutathionylation has been reported. Formation of the potential NO donor dinitrosyl-diglutathionyl-iron complex, bound to GSTP1-1 (glutathione transferase P1-1), was observed in both MCF7/Dx cells and drug-sensitive MCF7 cells to a similar extent. In contrast, histone glutathionylation was found to be markedly increased in the resistant MCF7/Dx cells, which also showed a 14-fold higher amount of GSTP1-1 and increased glutathione concentration compared with MCF7 cells. These results suggest that the increased cytotoxic effect of combined doxorubicin and GSNO treatment involves the glutathionylation of histones through a mechanism that requires high glutathione levels and increased expression of GSTP1-1. Owing to the critical role of histones in the regulation of gene expression, the implication of this finding may go beyond the phenomenon of doxorubicin resistance.

Differentiation of human melanoma cells induced by cyanidin-3-O-β-glucopyranoside

Great attention has been recently given to a flavonoid of the anthocyanin class, cyanidin‐3‐O‐β‐ glucopyranoside (C‐3‐G), which is widely spread throughout the plant kingdom, and is present in both fruits and vegetables of human diets. In this study, we investigated the effect of C‐3‐G on proliferation and differentiation of human melanoma cells. Both morphological and functional parameters were evaluated, using electron and confocal microscopy, cytofluorometric analysis, HPLC assay, Western blot analysis, and enzymatic assay, as appropriate. A treatment with a single dose of C‐3‐G decreased cell proliferation without affecting cell viability and without inducing apoptosis or necrosis. The mitotic index and cell percentage in S phase were significantly lower in C‐3‐G treated cells compared with untreated control. C‐3‐G treatment induced, in a dose‐ and time‐dependent manner, melanoma cell differentiation characterized by a strong increase in dendrite outgrowth accompanied with a remodeling of the microtubular network, a dramatic increase of focal adhesion and an increased expression of “brain specific” cytoskeletal components such as NF‐160 and NF‐200 neurofilament proteins. C‐3‐G treatment also induced increase of cAMP levels and up‐regulation of tyrosinase expression and activity resulting in an enhanced melanin synthesis and melanosome maturation. Up‐regulation of the melanoma differentiation antigen Melan‐A/MART‐1 in treated cells respect to the untreated control was also recorded. Data obtained provide evidence that a single treatment with C‐3‐G is able to revert the human melanoma cells from the proliferating to the differentiated state. We conclude that C‐3‐G is a very promising molecule to include in the strategies for treatment of melanoma; also because of its nutritional relevance.

Thymosin α1 and cancer: action on immune effector and tumor target cells

Since it was first identified, thymosin alpha 1 (Tα1) has been characterized to have pleiotropic effects on several pathological conditions, in particular as a modulator of immune response and inflammation. Several properties exerted by Tα1 may be attributable to a direct action on lymphoid cells. Tα1 has been shown to exert an immune modulatory activity on both T cell and natural killer cell maturation and to have an effect on functions of mature lymphocytes, including stimulating cytokine production and cytotoxic T lymphocyte–mediated cytotoxic responses. In previous studies we have shown that Tα1 increases the expression of major histocompatibility complex class I surface molecules in murine and human tumor cell lines and in primary cultures of human macrophages. In the present paper, we describe preliminary data indicating that Tα1 is also capable of increasing the expression of tumor antigens in both experimental and human tumor cell lines. This effect, which is exerted at the level of the target tumor cells, represents an additional factor increasing the antitumor activity of Tα1.

Thymosin alpha 1: from bench to bedside.

Abstract:  After the initial dramatic effects, observed in a Lewis lung carcinoma animal model, using a combination of thymosin alpha 1 (Tα1) and interferon (IFN) after cyclophosphamide, a number of other preclinical models in mice (Friend erythroleukemia and B16 melanoma) and in rats (DHD/K12 colorectal cancer liver metastasis) have confirmed the efficacy of the combination therapy with Tα1 and either IFN or IL‐2 plus chemotherapy. These results provided the scientific foundation for the first clinical trials using Tα1 in combination with BRMs and/or chemotherapy. Pivotal trials in advanced non‐small cell lung cancer (NSCLC) and melanoma with Tα1 and IFN‐α low doses after cis‐platinum or dacarbazine produced the first evidence of the high potentiality of this approach in the treatment of human cancer. The combination of Tα1 and IFN‐α was also used in patients affected by chronic B and C hepatitis including IFN‐nonresponders and infected by precore mutants or genotype 1b. Further studies demonstrated additional biological activities clarifying the mechanism of action of Tα1, partially explaining the synergism with IFN. It has been shown the capacity of activating infected dendritic cells through Toll‐like receptor signaling, thus influencing the inflammation balance, and of increasing the expression of tumor, viral, and major histocompatibility complex (MHC) I antigens. Dose–response studies suggested the possibility of improving the efficacy of this molecule reducing the overall toxic. Based on these information two clinical trials are ongoing: a large phase II on advanced melanoma patients treated with Tα1 at different doses after dacarbazine and a phase III one, on IFN‐resistant hepatitis C virus (HCV) patients treated with a triple combination (IFN, ribavirin, and Tα1).

Thymosin α1 in combination with cytokines and chemotherapy for the treatment of cancer

Abstract Multiple therapeutic approaches have been tested in different experimental tumour models and in human cancers. Most part of them are based on the hypothesis that the inhibition of tumour growth requires a strong immune response in which a main role is played by CTLs. It is known, however, that an efficient CTL response requires expression of tumour antigens, MHC class I surface molecules presentation, expression of different co-stimulatory molecules and a sustained generation and proliferation of specific cytotoxic CD8+ cells with an efficient CD4+ cooperation. In this context, our group has extensively explored a protocol of combined therapy consisting of the use of chemotherapeutic agents associated with thymosin α 1 (Tα1) and different cytokines, whose efficacy has been demonstrated in experimental models as well as in human cancers. In this manuscript, the main data supporting a pivotal role of Tα 1 in such combination protocols are reviewed. In particular, a special mention of the molecular mechanisms underlying the effects of Tα 1 on immune effector cells as well as on target tumour cells is provided. These data contribute to explain the mechanism of action of Tα1, when used in combination therapy, for the treatment of cancer and provide new insights in predicting further possible applications of this peptide in other pathological conditions.

Anti-proliferative effect of atrial natriuretic peptide on colorectal cancer cells: evidence for an Akt-mediated cross-talk between NHE-1 activity and Wnt/β-catenin signaling.

Acidic tumor microenvironment and Wnt/β-catenin pathway activation have been recognized as two crucial events associated with the initiation and progression of cancer. The aim of this study was to clarify the molecular mechanisms underlying the anti-proliferative effects of atrial natriuretic peptide (ANP) as well as to investigate the relationship between the cellular pH and the Wnt/β-catenin signaling in cancer cells.To pursue our aims, we conducted investigations in DHD/K12/Trb rat colon adenocarcinoma cells. Intracellular pH was measured by Confocal Laser Scanning Microscopy (CLSM) using the lysosensor Green DND-189 probe. Expression of crucial molecules in the Wnt/β-catenin signaling pathway was analyzed by CLSM, western blot, and real time PCR. Measurements of activation (phosphorylation state) of Akt, ERK1/2, and p38MAPKinase were performed by Reverse-Phase Protein Microarray Analysis (RPMA).We showed that ANP triggered a NHE-1-mediated increase of the intracellular acidity, inhibiting the Wnt/β-catenin signaling simultaneously. Moreover, we observed that the Wnt1a, a Wnt signaling activator, affected the intracellular pH in an opposite fashion. Results from the comparative analysis of ANP and EIPA (a NHE-1 specific inhibitor) showed that these two molecules affect both the intracellular acidification and the Wnt/β-catenin signaling cascade. Specifically, ANP acts on the upstream of the cascade, through a Frizzled-mediated activation, while EIPA does on the downstream.We show for the first time that the Akt activity might be a relevant molecular event linking the NHE-1-regulated intracellular pH and the Wnt/β-catenin signaling. This provides evidence for a cross-talk between the intracellular alkalinization and the Wnt signaling in tumor cells.

Thymosin α1 activates complement receptor-mediated phagocytosis in human monocyte-derived macrophages

Thymosin α1 (Tα1) is a naturally occurring thymic peptide used worldwide in clinical trials for the treatment of infectious diseases and cancer. The immunomodulatory activity of Tα1 on innate immunity effector cells has been extensively described, but its mechanism of action is not completely understood. We report that Tα1-exposed human monocyte-derived macrophages (MDMs) assume the typical activated morphology also exhibited by lipopolysaccharide-activated MDMs, but show a comparatively higher ability of internalizing fluorescent beads and zymosan particles. Tα1 exposure also promptly and dramatically stimulates MDM phagocytosis and killing of Aspergillus niger conidia starting as soon as 30 min after challenge. The effect is dose dependent and early coupled to low transcription of the proinflammatory cytokines tumor necrosis factor α and interleukin-6 and unmodified Toll-like receptor expression. The Tα1-stimulated phagocytosis is strictly dependent on the integrity of the microtubule network and protein kinase C activity and occurs by a variation in the classic zipper model, with recruitment of vinculin and actin at the phagosome exhibiting a punctate distribution. These findings indicate that, in human mature MDMs, Tα1 implements pathogen internalization and killing via the stimulation of the complement receptor-mediated phagocytosis. Our observations document that Tα1 is an early and potent activator of innate immunity and reinforce the concept of its pleiotropy.

Efficacy of repeated cycles of chemo-immunotherapy with Thymosin α1 and interleukin-2 after intraperitoneal 5-fluorouracil delivery

Abstract We have used chemo-immunotherapy with 5-fluorouracil (5-FU), thymosin α1 (Tα1) and interleukin-2 (IL-2) to treat multiple liver metastases from colorectal cancer induced by DHD/K12 cells in syngeneic BDIX rats, comparing one and two cycles of treatment, and different treatment combinations. 5-FU was delivered loco-regionally as a continuous infusion via an intraperitoneal (i.p.) catheter from a subcutaneously implanted mini-pump, a method we developed for this study. We show here that two cycles of a triple chemo-immunotherapy regimen significantly increased the average survival time compared to one cycle, and compared to untreated controls or those treated with two cycles of 5-FU alone. At 150 days, two rats treated with two cycles of triple therapy were cured, showing no signs of cancer at autopsy; all the other rats died before this time. Triple chemo-immunotherapy resulted in significantly fewer extra-hepatic metastases than in the controls and in those treated with 5-FU only. Further, we found that two cycles of triple treatment significantly increased the absolute number of peripheral T cells expressing IL-2 receptor, CD4 and CD8 compared to controls. We conclude that two cycles of chemo-immunotherapy with 5-FU, Tα1 and IL-2 were superior to one cycle of treatment and to other treatments tested. Our results suggest that the triple therapy acts by increasing numbers of effector T cells. This method shows promise for the use of multi-cycle chemo-immunotherapy in the treatment of unresectable metastases of colorectal cancer in humans.

PDGF receptor alpha inhibition induces apoptosis in glioblastoma cancer stem cells refractory to anti-Notch and anti-EGFR treatment

BackgroundCancer stem cells (CSC) represent a rare fraction of cancer cells characterized by resistance to chemotherapy and radiation, therefore nowadays there is great need to develop new targeted therapies for brain tumors and our study aim to target pivotal transmembrane receptors such as Notch, EGFR and PDGFR, which are already under investigation in clinical trials setting for the treatment of Glioblastoma Multiforme (GBM).MethodsMTS assay was performed to evaluate cells response to pharmacological treatments. Quantitative RT-PCR and Western blots were performed to state the expression of Notch1, EGFR and PDGFRα/β and the biological effects exerted by either single or combined targeted therapy in GBM CSC. GBM CSC invasive ability was tested in vitro in absence or presence of Notch and/or EGFR signaling inhibitors.ResultsIn this study, we investigated gene expression and function of Notch1, EGFR and PDGFR to determine their role among GBM tumor core- (c-CSC) vs. peritumor tissue-derived cancer stem cells (p-CSC) of six cases of GBM. Notch inhibition significantly impaired cell growth of c-CSC compared to p-CSC pools, with no effects observed in cell cycle distribution, apoptosis and cell invasion assays. Instead, anti-EGFR therapy induced cell cycle arrest, sometimes associated with apoptosis and reduction of cell invasiveness in GBM CSC. In two cases, c-CSC pools were more sensitive to simultaneous anti-Notch and anti-EGFR treatment than either therapy alone compared to p-CSC, which were mostly resistant to treatment. We reported the overexpression of PDGFRα and its up-regulation following anti-EGFR therapy in GBM p-CSC compared to c-CSC. RNA interference of PDGFRα significantly reduced cell proliferation rate of p-CSC, while its pharmacological inhibition with Crenolanib impaired survival of both CSC pools, whose effects in combination with EGFR inhibition were maximized.ConclusionsWe have used different drugs combination to identify the more effective therapeutic targets for GBM CSC, particularly against GBM peritumor tissue-derived CSC, which are mostly resistant to treatments. Overall, our results provide the rationale for simultaneous targeting of EGFR and PDGFR, which would be beneficial in the treatment of GBM.