Donatella Aldinucci

A Sensitive Spectrophotometric Method for the Determination of Superoxide Dismutase Activity in Tissue Extracts

Superoxide dismutase (EC 1.15.1.1) has been assayed by a spectrophotometric method based on the inhibition of a superoxide-driven NADH oxidation. The assay consists of a purely chemical reaction sequence which involves EDTA, Mn(II), mercaptoethanol, and molecular oxygen, requiring neither auxiliary enzymes nor sophisticated equipment. The method is very flexible and rapid and is applicable with high sensitivity to the determination of both pure and crude superoxide dismutase preparations. The decrease of the rate of NADH oxidation is a function of enzyme concentration, and saturation levels are attainable. Fifty percent inhibition, corresponding to one unit of the enzyme, is produced by approximately 15 ng of pure superoxide dismutase. Experiments on rat liver cytosol have shown the specificity of the method for superoxide dismutase. Moreover, common cellular components do not interfere with the measurement, except for hemoglobin when present at relatively high concentrations. The assay is performed at physiological pH and is unaffected by catalase.

The role of the EGFR signaling in tumor microenvironment

The epidermal growth factor receptor (EGFR) family comprehends four different tyrosine kinases (EGFR, ErbB‐2, ErbB‐3, and ErbB‐4) that are activated following binding to epidermal growth factor (EGF)‐like growth factors. It has been long established that the EGFR system is involved in tumorigenesis. These proteins are frequently expressed in human carcinomas and support proliferation and survival of cancer cells. However, activation of the EGFR in non‐malignant cell populations of the neoplastic microenvironment might also play an important role in cancer progression. EGFR signaling regulates in tumor cells the synthesis and secretion of several different angiogenic growth factors, including vascular endothelial growth factor (VEGF), interleukin‐8 (IL‐8), and basic fibroblast growth factor (bFGF). Overexpression of ErbB‐2 also leads to increased expression of angiogenic growth factors, whereas treatment with anti‐EGFR or anti‐ErbB‐2 agents produces a significant reduction of the synthesis of these proteins by cancer cells. EGFR expression and function in tumor‐associated endothelial cells has also been described. Therefore, EGFR signaling might regulate angiogenesis both directly and indirectly. In addition, activation of EGFR is involved in the pathogenesis of bone metastases. Within the bone marrow microenvironment, cancer cells stimulate the synthesis of osteoclastogenic factors by residing stromal cells, a phenomenon that leads to bone destruction. It has been shown that EGFR signaling regulates the ability of bone marrow stromal cells to produce osteoclastogenic factors and to sustain osteoclast activation. Taken together, these findings suggest that the EGFR system is an important mediator, within the tumor microenvironment, of autocrine and paracrine circuits that result in enhanced tumor growth. J. Cell. Physiol. 214: 559–567, 2008.

The classical Hodgkin's lymphoma microenvironment and its role in promoting tumour growth and immune escape.

It has become clear that cancer is not merely a growth of autonomously proliferating cells, but that other non‐malignant cell types are a functional part of the disease. Immune cells, fibroblasts, specialized mesenchymal cells and microvasculature together make up the tumour microenvironment and have functional interactions with tumour cells. Classical Hodgkins lymphoma (cHL) is characterized by only a few malignant cells and an abundance of inflammatory cells. Hodgkin and Reed–Sternberg (HRS) cells are surrounded by T and B cells admixed with plasma cells, macrophages, eosinophils and mast cells. A constitutive activity of NF‐κB and an altered JAK–STAT signalling pathway are part of the biological background associated with the increased expression of cytokines and cytokine receptors seen in HRS cells. Over‐expression of the members of the TNF receptor family, especially CD30 and CD40, is a hallmark of HRS cells. cHL is a tumour where aberrant cytokine production contributes not only to the proliferation of HRS cells but also to the maintenance of an appropriate environment for the tumour cells. In addition, several chemokines contribute to the composition of the inflammatory background in cHL. This review summarizes updated information on the complex interactions between the HRS cells and their tissue microenvironment and highlights the development of newer therapeutic strategies aimed at targeting the non‐malignant inflammatory/immune cellular components of HL that are involved in cancer cell growth and/or immune escape. Copyright

Expression of CCR5 receptors on Reed–Sternberg cells and Hodgkin lymphoma cell lines: Involvement of CCL5/Rantes in tumor cell growth and microenvironmental interactions

The expression of CCL5/Rantes by Hodgkin (H) and Reed‐Sternberg (RS) cells has been recently documented. In the present study we demonstrated that the CCL5 receptor (CCR5) is constitutively expressed by Hodgkin Lymphoma (HL)‐derived cell lines (i.e. L‐428, KM‐H2, L‐1236 and L‐540) as shown by immunohistochemistry, flow cytometry and western blotting and also detected by immunohistochemistry on primary H‐RS cells from lymph node tissues. sCD40L never significantly affected CCR5 expression, whereas a short exposure to doxorubicin down regulated its expression. CCR5 receptors on HL cell lines were functionally active, since neutralizing anti‐CCL5 monoclonal antibodies inhibited basal proliferation of HL‐derived cell lines and recombinant CCR5 ligands (CCL3/Mip‐1α, CCL4/Mip1β and CCL5/Rantes) increased their clonogenic growth. CCL5 secretion by L‐1236, L‐428 and KM‐H2 cells was stimulated by CD40 engagement and also by coculturing L‐1236 cells on primary stromal fibroblasts from HL‐involved lymph nodes (HLF). Coculture experiments indicated that a direct contact of H‐RS cells induces HLF cells to produce CCL5. Supernatants from L‐1236, L‐428 and KM‐H2 cells stimulated migration of purified CD4+ T‐cells and eosinophils in vitro. The migratory response to HL‐cell lines supernatants was only partially neutralized (CD4+ cells: 70%; esinophils: 36%) by anti‐CCL5 antibodies, reinforcing the notion that multiple chemokines are involved in the recruitment of nonmalignant reactive cells in HL tissues. Taken together, our results indicate a possible involvement of the CCR5/CCR5‐ligands signaling in the regulation of H‐RS cells growth and in the formation/maintenance of the typical tissue microenvironment of HL.

The Inflammatory Chemokine CCL5 and Cancer Progression

Until recently, inflammatory chemokines were viewed mainly as indispensable “gate keepers” of immunity and inflammation. However, updated research indicates that cancer cells subvert the normal chemokine system and these molecules and their receptors become important constituents of the tumor microenvironment with very different ways to exert tumor-promoting roles. The CCR5 and the CCL5 ligand have been detected in some hematological malignancies, lymphomas, and a great number of solid tumors, but extensive studies on the role of the CCL5/CCR axis were performed only in a limited number of cancers. This review summarizes updated information on the role of CCL5 and its receptor CCR5 in cancer cell proliferation, metastasis, and the formation of an immunosuppressive microenvironment and highlights the development of newer therapeutic strategies aimed to inhibit the binding of CCL5 to CCR5, to inhibit CCL5 secretion, or to inhibit the interactions among tumor cells and the microenvironment leading to CCL5 secretion.

Expression pattern of MUM1/IRF4 in the spectrum of pathology of Hodgkin's disease

Summary.  Biological and clinical studies have shown that Hodgkins disease (HD) can be divided into two major categories, termed nodular lymphocyte predominance HD (NLP HD) and classic HD (CHD). Within CHD four subtypes have been distinguished: nodular sclerosis, mixed cellularity, lymphocyte rich and lymphocyte depletion. To refine the histogenesis of the pathological spectrum of HD, 75 CHD and 13 NLP HD were analysed for the expression pattern of MUM1/IRF4 (Multiple Myeloma‐1/Interferon Regulatory Factor‐4), a lymphocyte‐specific member of the IRF family, that is expressed by late centrocytes and post‐germinal centre (GC) B cells. MUM1 reacted with Hodgkins and Reed–Sternberg (HRS) cells of all CHD cases (75/75 cases), with a moderate to strong staining intensity. Conversely, lymphocyte and histiocyte (L & H) cells, the putative tumour cells of NLP HD, were negative for MUM‐1 expression (9/13 cases) or displayed a weak reactivity for the antigen in < 10% neoplastic cells (4/13 cases). With respect to HD microenvironment, NLP HD displayed numerous MUM1‐positive T lymphocytes located in close proximity to L & H cells whereas, in CHD, MUM1‐positive T lymphocytes appeared to be distributed randomly with no specific relationship with HRS cells. Overall, this study shows that MUM1 expression differs in L & H cells versus HRS cells, corroborating the notion that NLP HD and CHD represent different stages of B‐cell differentiation. As MUM1‐positive T lymphocytes form rosettes around tumour cells of NLP HD, but not of CHD, these data point also to differences in the microenvironment of NLP HD and CHD, and postulate an interactive role of MUM1‐positive T lymphocytes with L & H cells.

Antitumor activity of gold(III)-dithiocarbamato derivatives on prostate cancer cells and xenografts

Among the nonplatinum antitumor drugs, gold(III)‐dithiocarbamato derivatives have recently attracted considerable attention due to their strong in vitro and in vivo antiproliferative activity and reduced renal toxicity. Some of them, namely [AuCl2(DMDT)] (compound 1) and [AuBr2(ESDT)] (compound 2), have shown to be highly active against the androgen‐resistant prostate cancer cell lines PC3 and DU145, both inhibiting cell proliferation in a dose‐dependent way, and are more active than the reference drug cisplatin (cis‐[PtCl2(NH3)2]). In particular, [AuCl2(DMDT)] was proved cytotoxic against cisplatin‐resistant R‐PC3 cells, with activity levels comparable to those induced on the parent cisplatin‐sensitive PC3 cells, ruling out the occurrence of cross‐resistance phenomena. Moreover, it causes early cell damage, slightly affecting the cell cycle, thus suggesting a different mechanism of action from clinically established platinum‐based drugs. In fact, the investigated gold(III) complex alters mitochondrial functions, promoting mitochondrial membrane permeabilization and Cyt‐c release, stimulating ROS generation, and strongly inhibiting the activity of the selenoenzyme TrxR, which is overexpressed in prostate cancer and associated with the onset of drug resistance. In addition, it induces apoptosis, caspase activation, Bcl‐2 downregulation and Bax upregulation, reduces the expression of the phosphorylated form of the EGFR, and it inhibits PC3 cell migration. Finally, the treatment of PC3 prostate tumor‐bearing nude mice with [AuCl2(DMDT)] significantly inhibited tumor growth in vivo, causing minimal systemic toxicity. Altogether, our results confirm that these gold(III)‐dithiocarbamato derivatives have potential for the treatment of prostate cancer.

Role of the EGFR ligand/receptor system in the secretion of angiogenic factors in mesenchymal stem cells.

Increasing evidence suggests that bone marrow‐derived mesenchymal stem cells (MSCs) are recruited into the stroma of developing tumors where they contribute to cancer progression. MSCs produce different growth factors that sustain tumor‐associated neo‐angiogenesis. Since the majority of carcinomas secrete ligands of the epidermal growth factor receptor (EGFR), we assessed the role of EGFR signaling in regulating the release of angiogenic factors in MSCs. Treatment of human primary MSCs and of the human osteoblastic cell line hFOB with transforming growth factor α (TGF‐α), one of the main ligands of the EGFR, significantly induced activation of this receptor and of different intracellular signaling proteins, including the PI3K/AKT and the MEK/MAPK pathways. TGF‐α induced a significant increase in the levels of secretion of vascular endothelial growth factor in both MSCs and hFOB. Conditioned medium from TGF‐α treated MSCs showed an higher in vivo angiogenic effect as compared with medium from untreated cells. Treatment of MSCs with TGF‐α also produced a significant increase in the secretion of other angiogenic growth factors such as angiopoietin‐2, granulocyte‐colony stimulating factor, hepatocyte growth factor, interleukin (IL)‐6, IL‐8, and platelet‐derived growth factor‐BB. Using selective MEK and PI3K inhibitors, we found that both MEK/MAPK and the PI3K/AKT signaling pathways mediate the ability of TGF‐α to induce secretion of angiogenic factors in MSCs. Finally, stimulation with TGF‐α increased the ability of MSCs to induce migration of MCF‐7 breast cancer cells. These data suggest that EGFR signaling regulates the ability of MSCs to sustain cancer progression through the release of growth factors that promote neo‐angiogenesis and tumor cell migration. J. Cell. Physiol. 226: 2131–2138, 2011.

Functional coexpression of Interleukin (IL)-7 and its receptor (IL-7R) on Hodgkin and Reed-Sternberg cells: Involvement of IL-7 in tumor cell growth and microenvironmental interactions of Hodgkin's lymphoma.

The clinical and pathological features of classical Hodgkin lymphoma (cHL) mirror an abnormal tissue and systemic immune response due to the production of a variety of cytokines and chemokines by the malignant Hodgkin‐Reed‐Sternberg (H‐RS) cells and/or surrounding reactive cells. Here, we demonstrate that HL‐derived cell lines (L‐428, KM‐H2, HDLM‐2, L‐1236 and L‐540) and primary H‐RS cells from lymph node tissues of HL patients express the IL‐7(R) receptor. IL‐7 appears to be involved in autocrine circuitries of HL because L‐1236, HDLM‐2 and KM‐H2 cells display the constitutive production of IL‐7 and neutralizing anti‐IL‐7 antibodies induces a statistically significant inhibition of their basal proliferation. In addition, IL‐7, either exogenous or fibroblasts‐derived, promotes the clonogenic growth and reduces apoptosis of cultured H‐RS cells, being also able to partially protect these cells from the cytotoxic effects of doxorubicin. We also provide evidence that IL‐7 stimulates IL‐6 secretion from IL‐7R‐expressing fibroblasts from HL‐involved lymph nodes (HLFs), and that a striking increase in IL‐6 secretion can be observed in cocultures of HLFs with L1236 cells. Finally, we show that L‐1236 cells‐derived IL‐7 represents a costimulator for proliferation of purified CD4+CD25+CD127dim/− regulatory T cells (Tregs). Taken together, our data indicates that the IL‐7/IL‐7R axis constitutes an additional signaling pathway between H‐RS cells and their reactive cellular background, thereby affecting proliferation and survival of tumor cells, acting as a cofactor for Tregs expansion and enhancing the microenviromental production of IL‐6, a cytokine associated with the presence of “B” symptoms and a poor outcome in HL patients.

Latest Insights into the Anticancer Activity of Gold(III)-Dithiocarbamato Complexes

In this review paper we aim at giving a detailed overview on our research work devoted to the design of gold-based anticancer agents. In particular, during the last decade, we have been developing some gold(III)-dithiocarbamato derivates showing outstanding in vitro and in vivo antitumor properties and reduced, or even no, systemic and renal toxicity, compared to the reference clinically-established anticancer drug cisplatin. Starting from the rationale behind our investigations, we here summarize the results achieved so far, focusing on the latest in-depth mechanistic studies that have recently provided insights into their mechanism of action, thus opening up new prospects for further pharmacological testing and, hopefully, to enter clinical trials.