Giovanna Bianchi

Increased level of extracellular ATP at tumor sites: In vivo imaging with plasma membrane luciferase

Background There is growing awareness that tumour cells build up a “self-advantageous” microenvironment that reduces effectiveness of anti-tumour immune response. While many different immunosuppressive mechanisms are likely to come into play, recent evidence suggests that extracellular adenosine acting at A2A receptors may have a major role in down-modulating the immune response as cancerous tissues contain elevated levels of adenosine and adenosine break-down products. While there is no doubt that all cells possess plasma membrane adenosine transporters that mediate adenosine uptake and may also allow its release, it is now clear that most of extracellularly-generated adenosine originates from the catabolism of extracellular ATP. Methodology/Principal Findings Measurement of extracellular ATP is generally performed in cell supernatants by HPLC or soluble luciferin-luciferase assay, thus it generally turns out to be laborious and inaccurate. We have engineered a chimeric plasma membrane-targeted luciferase that allows in vivo real-time imaging of extracellular ATP. With this novel probe we have measured the ATP concentration within the tumour microenvironment of several experimentally-induced tumours. Conclusions/Significance Our results show that ATP in the tumour interstitium is in the hundrends micromolar range, while it is basically undetectable in healthy tissues. Here we show that a chimeric plasma membrane-targeted luciferase allows in vivo detection of high extracellular ATP concentration at tumour sites. On the contrary, tumour-free tissues show undetectable extracellular ATP levels. Extracellular ATP may be crucial for the tumour not only as a stimulus for growth but also as a source of an immunosuppressive agent such as adenosine. Our approach offers a new tool for the investigation of the biochemical composition of tumour milieu and for development of novel therapies based on the modulation of extracellular purine-based signalling.

Starvation-dependent differential stress resistance protects normal but not cancer cells against high-dose chemotherapy

Strategies to treat cancer have focused primarily on the killing of tumor cells. Here, we describe a differential stress resistance (DSR) method that focuses instead on protecting the organism but not cancer cells against chemotherapy. Short-term starved S. cerevisiae or cells lacking proto-oncogene homologs were up to 1,000 times better protected against oxidative stress or chemotherapy drugs than cells expressing the oncogene homolog Ras2val19. Low-glucose or low-serum media also protected primary glial cells but not six different rat and human glioma and neuroblastoma cancer cell lines against hydrogen peroxide or the chemotherapy drug/pro-oxidant cyclophosphamide. Finally, short-term starvation provided complete protection to mice but not to injected neuroblastoma cells against a high dose of the chemotherapy drug/pro-oxidant etoposide. These studies describe a starvation-based DSR strategy to enhance the efficacy of chemotherapy and suggest that specific agents among those that promote oxidative stress and DNA damage have the potential to maximize the differential toxicity to normal and cancer cells.

Fasting Cycles Retard Growth of Tumors and Sensitize a Range of Cancer Cell Types to Chemotherapy

Short-term starvation increases the effectiveness of chemotherapy against a wide range of tumor cell types. Fasting: Good for You, Bad for Tumors Many promising cancer drugs being developed will require years to become approved by regulatory bodies and, in most cases, will only be effective for a fraction of patients with specific types of cancer. It is therefore important to develop broader, complementary strategies that can be translated rapidly into effective therapies. Two to 4 days of fasting before chemotherapy treatment is safe and protect animals, and possibly humans, against the side effects of chemotherapy. Here, cycles of fasting for 2 days in the absence of other treatments are shown to delay the progression of several tumor types in mice and, in some cases, to be as effective as toxic chemotherapy drugs. However, the combination of fasting and chemotherapy was much more effective than either alone and delayed the progression of a variety of tumors, including breast cancer and glioma, reduced the number of organs affected by melanoma metastases, and promoted long-term cancer-free survival in up to 40% of mice with neuroblastomas. In mice injected with human breast and ovarian cancer cells, fasting cycles promoted survival extension by protecting the mice from chemotherapy while causing a strong inhibition of tumor progression. Experiments in simple organisms, human cells, and mice indicated that these effects of fasting were caused by changes inside and outside cells that increased the death of tumor but not normal cells, a process termed differential stress sensitization. Although clinical trials testing the effect of fasting in cancer treatment are still in the early stages, they suggest that fasting cycles may boost the efficacy of chemotherapeutic agents and could be as effective as chemotherapy drugs in the killing of specific tumor cells. Short-term starvation (or fasting) protects normal cells, mice, and potentially humans from the harmful side effects of a variety of chemotherapy drugs. Here, we show that treatment with starvation conditions sensitized yeast cells (Saccharomyces cerevisiae) expressing the oncogene-like RAS2val19 to oxidative stress and 15 of 17 mammalian cancer cell lines to chemotherapeutic agents. Cycles of starvation were as effective as chemotherapeutic agents in delaying progression of different tumors and increased the effectiveness of these drugs against melanoma, glioma, and breast cancer cells. In mouse models of neuroblastoma, fasting cycles plus chemotherapy drugs—but not either treatment alone—resulted in long-term cancer-free survival. In 4T1 breast cancer cells, short-term starvation resulted in increased phosphorylation of the stress-sensitizing Akt and S6 kinases, increased oxidative stress, caspase-3 cleavage, DNA damage, and apoptosis. These studies suggest that multiple cycles of fasting promote differential stress sensitization in a wide range of tumors and could potentially replace or augment the efficacy of certain chemotherapy drugs in the treatment of various cancers.

Expression of P2X7 receptor increases in vivo tumor growth

The P2X7 receptor is an ATP-gated ion channel known for its cytotoxic activity. However, recent evidence suggests a role for P2X7 in cell proliferation. Here, we found that P2X7 exhibits significant growth-promoting effects in vivo. Human embryonic kidney cells expressing P2X7 exhibited a more tumorigenic and anaplastic phenotype than control cells in vivo, and the growth rate and size of these tumors were significantly reduced by intratumoral injection of the P2X7 inhibitor-oxidized ATP. The accelerated growth of P2X7-expressing tumors was characterized by increased proliferation, reduced apoptosis, and a high level of activated transcription factor NFATc1. These tumors also showed a more developed vascular network than control tumors and secreted elevated amounts of VEGF. The growth and neoangiogenesis of P2X7-expressing tumors was blocked by intratumoral injection of the VEGF-blocking antibody Avastin (bevacizumab), pharmacologic P2X7 blockade, or P2X7 silencing in vivo. Immunohistochemistry revealed strong P2X7 positivity in several human cancers. Together, our findings provide direct evidence that P2X7 promotes tumor growth in vivo.

Reduced levels of IGF-I mediate differential protection of normal and cancer cells in response to fasting and improve chemotherapeutic index.

Inhibitors of the insulin-like growth factor-I (IGF-I) receptor have been widely studied for their ability to enhance the killing of a variety of malignant cells, but whether IGF-I signaling differentially protects the host and cancer cells against chemotherapy is unknown. Starvation can protect mice, but not cancer cells, against high-dose chemotherapy [differential stress resistance (DSR)]. Here, we offer evidence that IGF-I reduction mediates part of the starvation-dependent DSR. A 72-hour fast in mice reduced circulating IGF-I by 70% and increased the level of the IGF-I inhibitor IGFBP-1 by 11-fold. LID mice, with a 70% to 80% reduction in circulating IGF-I levels, were protected against three of four chemotherapy drugs tested. Restoration of IGF-I was sufficient to reverse the protective effect of fasting. Sixty percent of melanoma-bearing LID mice treated with doxorubicin achieved long-term survival whereas all control mice died of either metastases or chemotherapy toxicity. Reducing IGF-I/IGF-I signaling protected primary glia, but not glioma cells, against cyclophosphamide and protected mouse embryonic fibroblasts against doxorubicin. Further, S. cerevisiae lacking homologs of IGF-I signaling proteins were protected against chemotherapy-dependent DNA damage in a manner that could be reversed by expressing a constitutively active form of Ras. We conclude that normal cells and mice can be protected against chemotherapy-dependent damage by reducing circulating IGF-I levels and by a mechanism that involves downregulation of proto-oncogene signals.

IMMUNOGENICITY OF HUMAN MESENCHYMAL STEM CELLS IN HLA-CLASS I RESTRICTED T CELL RESPONSES AGAINST VIRAL OR TUMOR-ASSOCIATED ANTIGENS

Human mesenchymal stem cells (MSC) are immunosuppressive and poorly immunogenic but may act as antigen‐presenting cells (APC) for CD4+ T‐cell responses; here we have investigated their ability to serve as APC for in vitro CD8+ T‐cell responses. MSC pulsed with peptides from viral antigens evoked interferon (IFN)‐γ and Granzyme B secretion in specific cytotoxic T lymphocytes (CTL) and were lysed, although with low efficiency. MSC transfected with tumor mRNA or infected with a viral vector carrying the Hepatitis C virus NS3Ag gene induced cytokine release but were not killed by specific CTL, even following pretreatment with IFN‐γ. To investigate the mechanisms involved in MSC resistance to CTL‐mediated lysis, we analyzed expression of human leukocyte antigen (HLA) class I‐related antigen‐processing machinery (APM) components and of immunosuppressive HLA‐G molecules in MSC. The LMP7, LMP10, and ERp57 components were not expressed and the MB‐1 and zeta molecules were downregulated in MSC either unmanipulated or pretreated with IFN‐γ. Surface HLA‐G was constitutively expressed on MSC but was not involved in their protection from CTL‐mediated lysis. MSC supernatants containing soluble HLA‐G (sHLA‐G) inhibited CTL‐mediated lysis, whereas those lacking sHLA‐G did not. The role of sHLA‐G in such inhibition was unambiguously demonstrated by partial restoration of lysis following sHLA‐G depletion from MSC supernatants. In conclusion, human MSC can process and present HLA class I‐restricted viral or tumor antigens to specific CTL with a limited efficiency, likely because of some defects in APM components. However, they are protected from CTL‐mediated lysis through a mechanism that is partly sHLA‐G‐dependent.

Fasting and differential chemotherapy protection in patients

Chronic calorie restriction has been known for decades to prevent or retard cancer growth, but its weight-loss effect and the potential problems associated with combining it with chemotherapy have prevented its clinical application. Based on the discovery in model organismsthat short term starvation (STS or fasting) causes a rapid switch of cells to a protected mode, we described a fasting-based intervention that causes remarkable changes in the levels of glucose, IGF-I and many other proteins and molecules and is capable of protecting mammalian cells and mice from various toxins, including chemotherapy. Because oncogenes prevent the cellular switch to this stress resistance mode, starvation for 48 hours or longer protects normal yeast and mammalian cells and mice but not cancer cells from chemotherapy, an effect we termed Differential Stress Resistance (DSR). In a recent article, 10 patients who fasted in combination with chemotherapy, reported that fasting was not only feasible and safe but caused a reduction in a wide range of side effects accompanied by an apparently normal and possibly augmented chemotherapy efficacy. Together with the remarkable results observed in animals, these data provide preliminary evidence in support of the human application of this fundamental biogerontology finding, particularly for terminal patients receiving chemotherapy. Here we briefly discuss the basic, pre-clinical, and clinical studies on fasting and cancer therapy.

ATP/P2X7 axis modulates myeloid-derived suppressor cell functions in neuroblastoma microenvironment.

Tumor microenvironment of solid tumors is characterized by a strikingly high concentration of adenosine and ATP. Physiological significance of this biochemical feature is unknown, but it has been suggested that it may affect infiltrating immune cell responses and tumor progression. There is increasing awareness that many of the effects of extracellular ATP on tumor and inflammatory cells are mediated by the P2X7 receptor (P2X7R). Aim of this study was to investigate whether: (i) extracellular ATP is a component of neuroblastoma (NB) microenvironment, (ii) myeloid-derived suppressor cells (MDSCs) express functional P2X7R and (iii) the ATP/P2X7R axis modulates MDSC functions. Our results show that extracellular ATP was detected in NB microenvironment in amounts that increased in parallel with tumor progression. The percentage of CD11b+/Gr-1+ cells was higher in NB-bearing mice compared with healthy animals. Within the CD11b/Gr-1+ population, monocytic MDSCs (M-MDSCs) produced higher levels of reactive oxygen species (ROS), arginase-1 (ARG-1), transforming growth factor-β1 (TGF-β1) and stimulated more potently in vivo tumor growth, as compared with granulocytic MDSCs (G-MDSCs). P2X7R of M-MDSCs was localized at the plasma membrane, coupled to increased functionality, upregulation of ARG-1, TGF-β1 and ROS. Quite surprisingly, the P2X7R in primary MDSCs as well as in the MSC-1 and MSC-2 lines was uncoupled from cytotoxicity. This study describes a novel scenario in which MDSC immunosuppressive functions are modulated by the ATP-enriched tumor microenvironment.

Structure-activity relationships of novel substituted naphthalene diimides as anticancer agents.

Novel 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) derivatives were synthesized and evaluated for their antiproliferative activity on a wide number of different tumor cell lines. The prototypes of the present series were derivatives 1 and 2 characterized by interesting biological profiles as anticancer agents. The present investigation expands on the study of structure-activity relationships of prototypes 1 and 2, namely, the influence of the different substituents of the phenyl rings on the biological activity. Derivatives 3-22, characterized by a different substituent on the aromatic rings and/or a different chain length varying from two to three carbon units, were synthesized and evaluated for their cytostatic and cytotoxic activities. The most interesting compound was 20, characterized by a linker of three methylene units and a 2,3,4-trimethoxy substituent on the two aromatic rings. It displayed antiproliferative activity in the submicromolar range, especially against some different cell lines, the ability to inhibit Taq polymerase and telomerase, to trigger caspase activation by a possible oxidative mechanism, to downregulate ERK 2 protein and to inhibit ERKs phosphorylation, without acting directly on microtubules and tubuline. Its theoretical recognition against duplex and quadruplex DNA structures have been compared to experimental thermodynamic measurements and by molecular modeling investigation leading to putative binding modes. Taken together these findings contribute to define this compound as potential Multitarget-Directed Ligands interacting simultaneously with different biological targets.

Immunosuppressive microenvironment in neuroblastoma

According to the cancer immunoediting model, the interplay between tumor cells and the host immune system is crucial for the control of tumor growth. NB is a pediatric tumor that presents with metastatic disease at diagnosis in about 50% of the cases, the majority of which have poor prognosis. In this Review article, immune escape pathways adopted by human neuroblastoma (NB) cells are reviewed. These include intrinsic defects of tumor cells such impaired expression of the HLA class I related antigen processing machinery and functional alterations of the tumor microenvironment (TM) induced by NB cell-derived immunosuppressive molecules as MICA and HLA-G. Finally, examples of therapeutic interventions targeting the TM are discussed to emphasize the concept that successful cancer treatment may be achieved using this strategy.