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// Giuliana Gatti 1, * , Valeria Lucini 2, * , Silvana Dugnani 2 , Angela Calastretti 1 , Gilberto Spadoni 3 , Annalida Bedini 3 , Silvia Rivara 4 , Marco Mor 4 , Gianfranco Canti 1 , Francesco Scaglione 2 and Annamaria Bevilacqua 1 1 Department of Medical Biotechnology and Translational Medicine, Universita degli Studi di Milano, Milan, Italy 2 Department of Oncology and Hemato-oncology, Universita degli Studi di Milano, Milan, Italy 3 Department of Biomolecular Sciences, Universita degli Studi di Urbino “Carlo Bo”, Urbino, Italy 4 Department of Food and Drug, Universita degli Studi di Parma, Parma, Italy * Authors contributed equally to this work Correspondence to: Annamaria Bevilacqua, email: annamaria.bevilacqua@unimi.it Keywords: melatonin analogues, melatonin receptors, anti-cancer drugs, melanoma, breast cancer Received: April 11, 2017 Accepted: July 29, 2017 Published: August 10, 2017 ABSTRACT Melatonin plays different physiological functions ranging from the regulation of circadian rhythms to tumor inhibition, owing to its antioxidant, immunomodulatory and anti-aging properties. Due to its pleiotropic functions, melatonin has been shown to elicit cytoprotective processes in normal cells and trigger pro-apoptotic signals in cancer cells. The therapeutic potential of melatonin analogues prompted us to investigate the in vitro and in vivo antitumor activity of new melatonin derivatives and explore the underlying molecular mechanisms. The experiments revealed that the new melatonin analogues inhibited the growth of melanoma and breast cancer cells in a dose- and time-dependent manner. In addition, our results indicated that melatonin derivative UCM 1037 could induce apoptosis in melanoma and breast cancer cells, as well as cell necrosis, in MCF-7. Together, apoptosis and necrosis could be two possible mechanisms to explain the cytotoxic effect of the melatonin analogue against cancer cells. The suppression of tumor growth by the melatonin analogues was further demonstrated in vivo in a xenograft mice model. A decrease in the activation of MAPK pathway was observed in all cancer cells following UCM 1037 treatment. Overall, this study describes a promising antitumor compound showing antiproliferative and cytotoxic activity in melanoma and breast cancer cells.

The aim of this review is to report the current evidence on immunogenicity of monoclonal antibodies (moAbs) used in cancer compared with autoimmune diseases, focusing on local microenvironment. English abstracts were identified in Medline and www.clinicaltrials.gov . A total of 82 papers were selected. The percentage of immunogenicity of moAbs used for cancer therapy, evaluated as the serum concentration of antidrug antibodies, is significantly lower than that of moAbs used for the treatment of autoimmune diseases. This condition may rely on a different immunologic background characterized by a hyperactivation of immune cells in autoimmune diseases. The formation of complexes between antidrug antibodies and non-neutralizing moAbs bound to neoplastic antigens may allow more efficient elimination of cancer cells, but additional studies are needed.

Melatonin has been indicated as a possible oncostatic agent in different types of cancer, its antiproliferative role being demonstrated in several in vitro and in vivo experimental models of tumors. Specifically, melatonin was proven to inhibit cell growth of both androgen-dependent and independent prostate cancer cells, through various mechanisms. A number of melatonin derivatives have been developed and tested for their role in the prevention and treatment of neoplastic diseases. We recently proved the in vitro and in vivo anticancer activity of UCM 1037, a newly-synthetized melatonin analogue, on melanoma and breast cancer cells. In this study we evaluated UCM 1037 effects on cell proliferation, cell cycle distribution, and cytotoxicity in LNCaP, PC3, DU145, and 22Rv1 prostate cancer cells. We demonstrated significant dose- and time-dependent UCM 1037 antiproliferative effects in androgen-sensitive LNCaP and 22Rv1 cells. Data from flow cytometric studies suggest that UCM 1037 is highly cytotoxic in androgen-sensitive prostate cancer cells, although no substantial increase in the apoptotic cell fraction has been observed. UCM 1037 cytotoxic effects were much less evident in androgen-insensitive PC3 and DU145 cells. Experiments performed to gain insights into the possible mechanism of action of the melatonin derivative revealed that UCM 1037 down-regulates androgen receptor levels and Akt activation in LNCaP and 22Rv1 cells.

Melatonin has been indicated as a possible oncostatic agent in different types of cancer, its antiproliferative role being demonstrated in several in vitro and in vivo experimental models of tumors. Specifically, melatonin was proven to inhibit cell growth of both androgen-dependent and independent prostate cancer cells, through various mechanisms. A number of melatonin derivatives have been developed and tested for their role in the prevention and treatment of neoplastic diseases. We recently proved the in vitro and in vivo anticancer activity of UCM 1037, a newly-synthetized melatonin analogue, on melanoma and breast cancer cells. In this study we evaluated UCM 1037 effects on cell proliferation, cell cycle distribution, and cytotoxicity in LNCaP, PC3, DU145, and 22Rv1 prostate cancer cells. We demonstrated significant dose- and time-dependent UCM 1037 antiproliferative effects in androgen-sensitive LNCaP and 22Rv1 cells. Data from flow cytometric studies suggest that UCM 1037 is highly cytotoxic in androgen-sensitive prostate cancer cells, although no substantial increase in the apoptotic cell fraction has been observed. UCM 1037 cytotoxic effects were much less evident in androgen-insensitive PC3 and DU145 cells. Experiments performed to gain insights into the possible mechanism of action of the melatonin derivative revealed that UCM 1037 down-regulates androgen receptor levels and Akt activation in LNCaP and 22Rv1 cells.

Heat pipe devices, for their typical working mechanisms, are particularly suitable for zero gravity applications, and have also been considered for applications in space satellites with nuclear propulsors, thanks to the absence of mobile systems for the coolant fluid circulation. The present work reports the results of the experimental tests carried out on a conventional cylindrical heat pipe designed to investigate on possible thermal-hydraulic performance improvements and to evidence the importance of the droplets generation that determines the boiling limit and consequently the heat transport capability. Some of the tests showed that the droplets generation, due to the nucleate boiling regime, can cause a liquid recirculation and a consequent reduction of liquid evaporation with a heat transport capability lower (about 250%) than that estimated from capillary (wicking) limit conditions. For this reason, generally, to predict more realistically the capability for given wick size, it is not sufficient to know the tilt angle, the total length and the diameter of the tube. Some short grooves (cuts of 15 mm in length) made in the screens at the end of the evaporating zone demonstrate their efficiency to increase the heat flux values more than 2 or 3 W/cm 2 . The same heat flux increasing, concerning the wick-wall contact improvement, is obtained thickening the coils of the spring used to tight the wick against the internal tube wall.

Cancer has been proposed as an example of systems biology disease or network disease. Accordingly, tumor cells differ from their normal counterparts more in terms of intracellular network dynamics than single markers. Here we shall focus on a recently recognized hallmark of cancer, the deregulation of cellular energetics. The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been confirmed as an essential step toward cell transformation. We will consider how the effects of Akt activation are connected with cell metabolism; more precisely, we will review existing metabolic models and discuss the current knowledge available to construct a kinetic model of the most relevant metabolic processes regulated by the PI3K/Akt pathway. The model will enable a systems biology approach to predict the metabolic targets that may inhibit cell growth under hyper activation of Akt. (C) 2011 Elsevier Inc. All rights reserved.

Prostate cancer is characterized by resistance to chemotherapy and is often associated with loss of the tumor suppressor PTEN, a negative regulator of the PI3K/AKT/mTOR pathway. Currently, taxanes are the first-line chemotherapy for advanced prostate cancer; however, effective therapeutic strategies are still needed that extend survival or alter the outcome of the disease. The inhibition of PI3K/AKT/mTOR pathway has been reported to enhance the efficacy of chemotherapeutic agents in different experimental models. Moreover, it has been reported that the overexpression of oncogenes under nutrient stress can activate a death program. Therefore, metabolic stress can be a useful strategy to sensitize AKT upregulated prostate cancer cells to antitumor drugs. We have previously demonstrated that: • mTOR is involved not only in PI3K/AKT survival pathway, but also in apoptotic pathway, induced by anti-microtubule agents such as taxanes. • AKT downregulation enhances the antitumor effects of taxanes. • Metabolic starvation increases the sensitivity to death induced by taxanes. • Downregulating Bcl-2 levels raises the sensitization to cell death induced by association of starvation plus taxanes. Here different cancers’ capabilities, such as sustained proliferative signaling, deregulated cellular energetics, cell death resistance, have been targeted by various mechanism-directed treatments in cellular model systems of human prostate cancer. The specific aim of our research project is to overcome therapeutic resistance and restore the sensitivity to anticancer drugs in prostate cancer cells, through selective inhibition of different components of the PI3K/AKT/mTOR cascade, in association with treatment with taxanes and starvation. The PI3K/AKT/mTOR pathway has been targeted using either rapamycin or small interference RNAs (siRNAs). LNCaP, PC3 (both PTEN-null) and DU145 (wild-type PTEN) cell lines have been treated with rapamycin as a single agent or in combination with metabolic starvation and/or taxanes. Cell viability of LNCaP and PC3 prostate cells, with high levels of phosphorylated AKT, was not significantly affected by rapamycin treatment alone, but the percentage of growth inhibition significantly increased in combined treatments with starvation and taxanes. DU145 cells, with lower levels of activated AKT, showed high inhibition of cell growth following rapamycin treatment alone (45%), which increased to 75% when rapamycin was associated with starvation and/or taxanes. PTEN-null and PTEN-wild type prostate cancer cells were also treated with siRNAs directed towards mTOR (simTOR), as a single agent or in association with starvation and taxanes. The combination of treatments caused a dramatic reduction of viable cell number in DU145 (up to 90%) and, to a lesser extent, in LNCaP cells (about 50%). These results have been confirmed by Annexin V assays analyzing cell death in the same samples. Therefore, we have established that acting at the same time on different cancers’ capabilities is a promising strategy to induce the apoptotic program, initiated by anti-microtubule drugs, in prostate cancer cells. We demonstrated that it is possible to induce prostate cancer cell death through selective inhibition of specific components of PI3K/AKT/mTOR signaling cascade, in association with treatment with taxanes and metabolic starvation. The identification of the most effective and safe combination of targets to achieve a specific and maximal sensitization to antiproliferative drugs might provide new therapeutic models in clinical applications. Citation Format: Annamaria Bevilacqua, Angela Calastretti, Giuliana Gatti, Gianfranco Canti. Combining metabolic starvation and PI3K/AKT/mTOR pathway inhibition to chemosensitize prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A052.

One of the most investigated heat transfer fluid or heat storage medium in the field of thermal solar conversion is the “solar salt”, a mixture of nitrates NaNO 3 /KNO 3 , 60/40% by weight, respectively. The content of potassium in this fluid allows exploiting the self activity of the isotope 40 K to perform measurements useful for control or diagnostics of a solar plant. Here an experimental test on the non-invasive measurement of the salt level in a tank by means of gamma-ray spectrometry is shown.

Akt activation assists tumor cell survival and promotes resistance to chemotherapy. Here we show that constitutively active Akt (CA-Akt) cells are highly sensitized to cell death induced by nutrient and growth factor deprivation, whereas dominant-negative Akt (DN-Akt) cells have a high rate of survival. The content of autophagosomes in starved CA-Akt cells was high, while DN-Akt cells expressed autophagic vacuoles constitutively, independently of nutrition conditions. Thus Akt down-regulation and downstream events can induce autophagosomes which were not directly determinants of cell death. Biochemical analysis in Akt-mutated cells show that (i) Akt and mTOR proteins were degraded more rapidly than the housekeeping proteins, (ii) mTOR phosphorylation at position Thr(2446) was relatively high in DN-Akt and low in CA-Akt cells, induced by starvation in mock cells only, which suggests reduced autoregulation of these pathways in Akt-mutated cells, (iii) both protein synthesis and protein degradation were significantly higher in starved CA-Akt cells than in starved DN-Akt cells or mock cells. In conclusion, constitutively active Akt, unable to control synthesis and wasting of proteins, accelerates the death of starved cells.

BACKGROUND In prostate cancer, mutations of the phosphatase PTEN can activate the kinase cascade PI3K/Akt/mTOR which induces drug resistance. METHODS Chemosensitization by siRNA targeting Akt was studied in HEK293 cells forced to express CA-Akt or kinase-dead DN-Akt. To decrease drug resistance, Akt was silenced with siRNA in human prostate DU-145 cell line expressing the normal PTEN or in LNCaP and PC3 cell lines expressing mutated-PTEN. Taxol was used for the chemosensitization studies. RESULTS Silencing Akt in the drug-resistant CA-Akt cells efficiently sensitized cells to antitubule agents, whereas silencing drug-responsive DN-Akt cells did not. Only minor effects were obtained in wild-type HEK293 cells. Potentiation by siRNA of taxol cytotoxicity was significantly greater in mutated-PTEN cells than in prostate cells expressing wild-type PTEN. The apoptotic program induced by taxol was preferentially potentiated by Akt siRNA in PTEN-mutated cell lines as regards the DU-145 cell line. CONCLUSIONS Silencing Akt in PTEN-mutated prostate cancer cells enhances the antitumor effects of taxol. No siRNA chemosensitization was obtained in prostate cells with wild type PTEN. Prostate 67: 782–789, 2007. © 2007 Wiley-Liss, Inc.

RNA has become a promising target for pharmacological purposes. Most current strategies are directed toward down-regulating its functions. In this study, we provide evidence of the up-regulation of messenger RNA in a sequence-specific manner. The bcl2 (b)-ARE (adenine-uridine-rich element) in the 3'-untranslated region of the b-RNA that regulates the rate of RNA degradation has been targeted with three chemically modified oligoribonucleotides designed in the antisense orientation (asORNs). The three asORNs were studied by a cell-free degradation assay. All three slowed the rate of RNA decay in a dose-response fashion, they were specific to the b-ARE, and two of them were individually effective. asORNs were then transfected into the malignant cells in culture and b-RNA half-life was measured by real-time reverse transcriptase-polymerase chain reaction. We showed that by stabilizing b-RNA the three asORNs increased the expression of b-RNA and of the relevant protein in a dose-response fashion.

Background and ObjectiveFluorescence techniques can provide powerful noninvasive means for medical diagnosis, based on the detection of either endogenous or exogenous fluorophores. The fluorescence of I´-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) has already shown promise for the diagnosis of tumors. The aim of the study was to investigate the localization of skin tumors after the topical application of ALA, by detecting the PpIX fluorescence either in the spectral or in the time domain.Study Design/Materials and MethodsTwo fluorescence imaging systems were used to identify basal cell carcinomas of the skin in humans, after topical application of 20% ALA ointment. Both systems rely on the comparison between the exogenous and the endogenous fluorescence, performed either in the spectral domain or in the time domain. The first system works by using three images acquired through different spectral filters, whereas the second one measures the spatial map of the average fluorescence lifetime of the sample.ResultsA clear demarcation of skin malignancies was successfully performed in vivo noninvasively with both fluorescence imaging systems.ConclusionThe two complementary approaches considered in the present study show promise for skin tumor detection and delineation based on specific fluorescence features. Lasers Surg. Med. 26:76–82, 2000. © 2000 Wiley-Liss, Inc. (Less)

Heat pipe design and manufacturing require the knowledge of the thermal hydraulic performance of the wicks. The aim of the present work is the thermal hydraulic characterization of stainless steel wicks (sintered porous media and gauzes) to be employed in our experimental water heat pipe. Commercial sintered porous media (able to capture 90 % of 90 μm particles and 99.9 % of 130 μm particles) and gauzes (nominal wire size 0.11 mm, square mesh opening 0.209 mm) have been used. Thermal hydraulic characterization of the wicks is obtained through the experimental measurement of: capillary height (through which the equivalent porous radius can be evaluated), liquid hydraulic head (through which the liquid pressure drop in the wick is evaluated) wick permeability is also evaluated from the hydraulic head (through Darcy's law), heat flux, wick mass flow rate during the evaporation (through which, from the knowledge of other measured wick parameters, the wick two-phase pressure drop is calculated) and wick porosity (through which the thermal conductivity of the wick saturated with liquid can be determined). Concerning the heat flux, it is found to be dependent on the distance between the liquid level and the evaporating zone, the evaporating zone length, the wall superheat and the water subcooling, the contact between the heater and the wick and the superficial boundary conditions of the wick.

International audience; In recent years, an increasing effort has been devoted to the optimization of acquisition and reconstruction schemes for fluorescence molecular tomography (FMT). In particular, wide-field structured illumination and compression of the measured images have enabled significant reduction of the data set and, consequently, a decrease in both acquisition and processing times. FMT based on this concept has been recently demonstrated on a cylindrical phantom with a rotating-view scheme that significantly increases the reconstruction quality. In this work, we generalize the rotating-view scheme to arbitrary geometries and experimentally demonstrate its applicability to murine models. To the best of our knowledge this is the first time that FMT based on a rotating-view scheme with structured illumination and image compression has been applied to animals.

The absorption spectrum of aluminum phthalocyanine with an average disulphonation of 2.1 (hereafter called disulphonated aluminum phthalocyanine, A1S2Pc) was measured in vivo in a murine tumour model by means of time-resolved reflectance. Mice bearing the L1210 leukaemia were administered 2.5 or 5 mg/kg body weight (b.w.) of A1S2Pc intraperitoneally. Reflectance measurements were performed in the 650-695 nm range before and 1, 4 and 7 h after the drug administration. Fitting of the data with the diffusion theory allowed us to assess the absorption coefficient in both conditions (i.e. before and after). As a difference between the latter and the former data, the in vivo absorption spectrum of A1S2Pc was evaluated. 1 h after the administration of 2.5 mg/kg b.w. A1S2Pc, the absorption peak was centred at 685 nm, red-shifted about 15 nm with respect to the spectrum in aqueous solution. For the lower dose, the absorption line shapes 4 and 7 h after the administration remained very similar. The red shift of the absorption spectrum is consistent with the therapeutic efficacy of the photodynamic therapy which was measured at 672, 685 and 695 nm, and proved to be maximum at 685 nm for both the L1210 leukaemia and the MS-2 fibrosarcoma. With the higher drug dose, the absorption spectra taken from different animals showed significant differences. In particular, in some mice the line shape was similar to that measured with 2.5 mg/kg b.w., while in other subjects it showed a broadening or a second peak at shorter wavelengths. Measurements on some animals were performed also 18 and 24 h after the injection of 5 mg/kg b.w., leading to no time evolution or to a progressive line shape narrowing.

Signal transduction pathways and gene regulation determine a major reorganization of metabolic activities in order to support cell proliferation. Protein Kinase B (PKB), also known as Akt, participates in the PI3K/Akt/mTOR pathway, a master regulator of aerobic glycolysis and cellular biosynthesis, two activities shown by both normal and cancer proliferating cells. Not surprisingly considering its relevance for cellular metabolism, Akt/PKB is often found hyperactive in cancer cells. In the last decade, many efforts have been made to improve the understanding of the control of glucose metabolism and the identification of a therapeutic window between proliferating cancer cells and proliferating normal cells. In this context, we have modelled the link between the PI3K/Akt/mTOR pathway, glycolysis, lactic acid production and nucleotide biosynthesis. We used a computational model in order to compare two metabolic states generated by the specific variation of the metabolic fluxes regulated by the activity of the PI3K/Akt/mTOR pathway. One of the two states represented the metabolism of a growing cancer cell characterised by aerobic glycolysis and cellular biosynthesis, while the other state represented the same metabolic network with a reduced glycolytic rate and a higher mitochondrial pyruvate metabolism, as reported in literature in relation to the activity of the PI3K/Akt/mTOR. Some steps that link glycolysis and pentose phosphate pathway revealed their importance for controlling the dynamics of cancer glucose metabolism.

The potential of protoporphyrin IX fluorescence induced by the systemic administration of delta-aminolevulinic acid (ALA) for the detection of tumours was tested in three different murine models (MS-2 fibrosarcoma, L1210 leukaemia, and Lewis lung carcinoma). Time-gated fluorescence images were acquired up to 4 h after the intraperitoneal injection of ALA (200 mg (kg body mass (BM))-1). For comparison images were acquired also after the administration of 25 mg (kg BM)-1 of haematoporphyrin derivative. The latter drug was characterized by better localization in the tumour area, leading to higher fluorescence contrast between neoplastic mass and surrounding healthy tissue, and consequently was preferable for tumour diagnosis in all the models under study.

In the 3′-untranslated region, the destabilizing adenine-uridine (AU)-rich elements (AREs) control the expression of several transcripts through interactions with ARE-binding proteins (AUBPs) and RNA degradation machinery. Although the fundamental role for AUBPs and associated factors in eliciting ARE-dependent degradation of cognate mRNAs has been recently highlighted, the molecular mechanisms underlying the specific regulation of individual mRNA turnover have not yet been fully elucidated. Here we focused on the post-transcriptional regulation of bcl-2 mRNA in human cell lines under different conditions and genetic backgrounds. In the context of an AUBPs silencing approach, HuR knockdown reduced the expression of endogenous bcl-2, whereas unexpectedly, a bcl-2 ARE-reporter transcript increased significantly, suggesting that HuR expression has opposite effects on endogenous and ectopic bcl-2 ARE. Moreover, evidence was provided for the essential, specific and dose-dependent role of the Bcl-2 protein in regulating the decay kinetics of its own mRNA, as ascertained by a luciferase reporter system. Altogether, the data support a model whereby the Bcl-2 protein is the major determinant of its own ARE-dependent transcript half-life in living cells and its effect overcomes the activity of ARE-binding proteins.

The effect of systemic administration on drug uptake at cellular level was evaluated using time-gated fluorescence spectroscopy performed on a murine ascitic tumour model. Mice bearing L1210 leukaemia were injected intraperitoneally or intravenously with 25 mg per kg body weight hematoporphyrin derivative (HpD), 12.5 mg per kg body weight photofrin II (PII), 25 or 5 mg per kg body weight disulphonated aluminium phthalocyanine (AlS2Pc). Every 2 h and for up to 22 or 30 h, mice were sacrificed, leukaemic cells extracted from the peritoneum, washed, and resuspended in buffer for fluorescence measurements. HpD and PII emission spectra were almost identical 12 h after intraperitoneal injection with main peaks at 630 nm and no appreciable changes afterwards. In the first 12 h, the PII fluorescence spectrum was constant, while in the case of HpD a shoulder at 615 nm was detectable. Similar fluorescence behaviour was observed after intravenous administration of porphyrin derivatives. These results seem to confirm that the tumour localizing fraction is the part actually retained by the cells. The AlS2Pc spectrum peaked at 685 nm and did not change in any of our experiments. AlS2Pc is incorporated more rapidly with respect to porphyrins, as was clearly observed in the case of intravenous administration, where the AlS2Pc fluorescence was readily detectable after 2 h, whereas the PII emission became apparent only after 4–6 h.

Adenine-uridine rich elements (AREs) play an important role in modulating mRNA stability, being the target site of many ARE-binding proteins (AUBPs) that are involved in the decay process. Three 26-mer 2'-O-methyl oligoribonucleotides (ORNs) homologous to the core region of ARE of bcl2 mRNA have been studied for decoy-aptamer activity in UV cross-linking assays. Sense-oriented ORNs competed with the ARE motif for the interaction with both destabilizing and stabilizing AUBPs in cell-free systems and in cell lines. Moreover, ORNs induced mRNA stabilization and up-regulated both Bcl2 mRNA and protein levels in the cells. Bcl2 ORNs stabilized other ARE-containing transcripts and up-regulated their expression. These results indicate that Bcl2 ORNs compete for AUBP-ARE interactions independently of ARE class and suggest that in the cell, the default labile status of ARE-containing mRNAs depends on the combined interaction of such transcripts with destabilizing AUBPs.

The serine/threonine kinase mTOR, the major sensor of cell growth along the PI3K/Akt pathway, can be activated by agents acting on microtubules. Damaged microtubules induce phosphorylation of the Bcl-2 protein and lower the threshold of programmed cell death, both of which are inhibited by rapamycin. In HEK293 cells expressing Akt mutants, the level of Bcl-2 phosphorylation and the threshold of apoptosis induced by taxol or by nocodazole are significantly modified. In cells expressing dominant-negative Akt (DN-Akt), Bcl-2 phosphorylation and p70S6KThr421/Ser424 phosphorylation induced by taxol or nocodazole were significantly enhanced as compared to cells expressing constitutively active Akt (CA-Akt) and inhibited by rapamycin. Moreover, DN-Akt cells were more sensitive to antitubule agents than CA-Akt cells. In nocodazole-treated HEK293 cells sorted according to cell cycle, the p70S6KThr421/Ser424 phosphorylation was associated to the G2/M fraction. More relevant, nocodazole inhibited, in a dose-response manner, mTOR phosphorylation at Ser2448. This activity, potentiated in DN-Akt cells, was not detectable in CA-Akt cells. Our results suggest that death signals originating from damaged microtubules in G2/M can compete with G1 survival pathways at the level of mTOR. These findings have implications for cancer therapy and drug resistance.

Photodynamic therapy (PDT) is a cancer treatment involving systemic administration of a tumor-localizing photosensitizer; this, when activated by the appropriate wavelength of light, interacts with molecular oxygen to form a toxic, short-lived species known as singlet oxygen, which is thought to mediate cellular death. Photofrin, a complex mixture of porphyrin oligomers has recently received FDA approval for the photodynamic treatment of esophageal and endobronchial carcinoma, but its photodynamic and toxicity profiles are far from ideal. In the present study we evaluated a series of porphyrin-based PSs, some of which newly synthesized by our group, with the aim to identify agents with more favorable characteristics. For the most effective compounds in the porphyrin series, chlorin analogs were also synthesized; for comparison, the screening also included Photofrin. Cytotoxicity studies were performed by the MTT assay on a cultured human colon adenocarcinoma cell line (HCT116); the results indicate that the 3,4,5-trimethoxyphenyl, 3OH- and 4OH-phenyl, and the sulfonamidophenyl derivatives are significantly more potent than Photofrin. Flow cytometric studies and fluorescence microscopy indicate that in PDT-treated HCT116 cells death occurs mainly by apoptosis. In summary, novel PSs described in the present study, belonging both to the porphyrin and chlorin series, have proven more effective than Photofrin in killing colon cancer cells in vitro; extending these observation to in vivo models, particularly regarding the deeper reaching chlorin derivatives, might lead to significant advances in the development of tumor PDT.

We have shown previously that the decay of human bcl-2 mRNA is mediated by an adenine/uridine-rich element (ARE) located in the 3′-untranslated region. Here, we have utilized a non-radioactive cell-free mRNA decay system to investigate the biochemical and functional mechanisms regulating the ARE-dependent degradation of bcl-2 mRNA. Using RNA substrates, mutants, and competitors, we found that decay is specific and ARE-dependent, although maximized by the ARE-flanking regions. In unfractionated extracts from different cell types and in whole cells, the relative enzymatic activity was related to the amount of Bcl-2 protein expressed by the cells at steady state. The degradation activity was lost upon Bcl-2 depletion and was reconstituted by adding recombinant Bcl-2. Ineffective extracts from cells that constitutively do not express Bcl-2 acquire full degradation activity by adding recombinant Bcl-2 protein. We conclude that Bcl-2 is necessary to activate the degradation complex on the relevant RNA target.

The immunosuppressant rapamycin, an immunophilin-binding antibiotic, has been studied in follicular B-cell lymphoma lines that express the highest level of the BCL-2 protein. The growth rate of human follicular B-cell lymphoma lines was slowed more efficiently than that of other human B-cell lines or non-B-cell lines. This effect was dependent on the arrest of cells in the G(1) phase; the number of apoptotic cells was not increased. Rapamycin inhibited apoptosis or caspase activation induced by cytotoxic drugs, whereas caspase activation by doxorubicin was not inhibited. The increase in the cellular concentration of BCL-2 protein was related to its concentration in the steady state and was unrelated to the amount of bcl-2 mRNA. The increase of BCL-2 level in the cells rather than its level in the steady state may be important for drug resistance. The biochemical target of rapamycin, the mTOR kinase, may be a candidate sensitising agent for chemotherapy. This effect of rapamycin shows that G(1) arrest and protection from apoptosis are combined events susceptible to regulation by pharmacological means.

Time-resolved reflectance spectroscopy was performed on tumor-bearing mice, administered with disulphonated aluminum phthalocyanine (AlS 2 Pc, 5 mg/kg body weight), before, during and after photodynamic therapy. This allowed us to evaluate the absorption spectrum of AlS 2 Pc in vivo from 610 to 700 nm, and to investigate how the therapeutic irradiation affects it. Two tumor locations (intraderma on the back and intramuscular in the leg), and two uptake times (3 and 12 h) were considered. As already observed previously, the absorption spectrum of AlS 2 Pc in vivo is centered at 680–685 nm. The irradiation causes a blue-shift of the measured line shape, more or less marked depending on the experimental conditions. A reduction in absorption is also often observed upon illumination with therapeutic light doses.

An imaging system was used to monitor the fluorescence of disulphonated Aluminum Phthalocyanine during the PDT of tumors to check whether an index for therapy effectiveness could be found out. Two excitation wavelengths were tested to discover a possible change in the absorption spectrum of the sensitizer induced by the treatment. Two opposite phenomena take place in the tumor area: a mild photobleaching in the region directly irradiated and a marked increase in the fluorescence signal at the tumor borders. The different behavior observed with the two excitation wavelengths demonstrates that the drug absorption spectrum shifts towards shorter wavelengths as a result of the irradiation.

Time-resolved reflectance spectroscopy was used to investigate the effects of photodynamic irradiation on the in vivo absorption properties of disulphonated Aluminum Phthalocyanine (AlS2Pc) in tumor-bearing mice. Mice bearing tumors injected either intramuscularly or intradermally were administered with 5 mg/kg body weight of AlS2Pc. Uptake times of 3 h and 12 h were examined. Absorption spectra were measured from 610 to 700 nm before, during and after the irradiation. The administration of a therapeutic light dose causes a blue-shift and a reduction in the drug absorption, which are more marked 3 h than 12 h after drug injection.

Since immune response of the host is important in the control of tumor growth and spreading, and the Photodynamic therapy (PDT) is able to increase the antitumor immunity, in our laboratory we examine the effect of PDT on immune compartment of tumor bearing mice. Lymphocytes and macrophages collected from tumor bearing mice pretreated with PDT are cytotoxic in vitro and in vivo against the parental tumor lines, in contrast the same immune cells population collected from tumor bearing mice pretreated only with laser light are unable to lyse the parental tumor cells. In adoptive immunotherapy experiments, treatment of mice bearing MS-2 tumor with adoptive transfer of immune lymphocytes collected from mice pretreated with PDT is able to significantly increase the survival time; in contrast the lymphocytes collected from mice pretreated only with laser light were not able to modify the survival time suggesting that the laser treatment alone did not increase the immune response of the host. In conclusion these results demonstrate that the PDT induce a strong immune response on the host and the stimulated lymphocytes generated could be used for an adoptive immunotherapy approach; moreover laser treatment alone (thermal effect) is unable to modulate the immune response of the host.

The estimate of the optimal light dose is a basic step in the optimization of the protocol for the photodynamic therapy (PDT) of tumors. It depends essentially on the optical properties of the tissue to be treated and on the changes induced by the administration of the photosensitizer (i.e., essentially, the absorption increase).

Multispectral and lifetime imaging systems have been used to detect skin cancer in humans after the topical application of δ-arninolevulinic acid ointment. Both systems allowed a clear detection of tumors based either on spectral or on temporal differences between the fluorescence signals in the neoplasia and in the healthy tissues nearby.

Photodynamic therapy (PDT) is based on the administration of tumor-localizing photosensitizers followed by light exposure of the tumor mass. The photocytotoxic effects are mainly caused by the generation of singlet oxygen. Recently, PDT has been proposed for use in combination with anticancer chemotherapy with a view to exploiting any additive antitumor effect. We investigated the effect of PDT with photoactivated aluminum disulfonated phthalocyanine (AlS2Pc) combined with the antiblastic drugs Adriamycin (ADR) and cisplatinum (CDDP) on murine tumors. Mice bearing L1210 leukemia and P388 lymphoma were treated with ADR or CDDP and subsequently treated with PDT. Low chemotherapy doses were ineffective, but the combination of antiblastic drugs + PDT had a significantly additive antitumor effect. In conclusion, with this combined therapy we were able to greatly reduce the effective doses of antiblastic drugs, thus lowering their toxic effects on normal host tissues.

In this paper we report on the experimental setup and the performances of a fluorescence lifetime imaging system designed by the authors for cancer detection. The experimental apparatus relies on a CCD video camera coupled to a light intensifier and on a pulsed laser. Two or more fluorescence images are acquired within suitable time windows at different delays, with respect to the excitation pulses. The images are processed using matrix algorithms implemented in the fast processor of an advanced frame grabber; then, the resulting lifetime map is displayed in pseudocolor or gray shade images. The instrumentation is fully computer controlled and the whole process takes about 1 second per lifetime image. Using a faster processor this time could be easily reduced to some milliseconds for true real time measurements. The system has been successfully applied to detect experimental tumors in mice sensitized with hematoporphyrin derivative, which is a clinically approved photosensitizing drug.

Three time-delayed fluorescence images were used to calculate the spatial distribution of the fluorescence decay time in tumor-bearing mice sensitized with Hematoporphyrin Derivative (HpD). This allowed us to observe that the drug has a significantly longer fluorescence lifetime when it is incorporated in neoplastic tissues with respect to healthy surrounding ones. The imaging technique can be applied noninvasively in vivo in real time. Its diagnostic potential was tested on two murine tumor models after sensitization with low doses of HpD. In all the experimental conditions considered, the tumor could be easily identified in the lifetime images. Images were acquired also after removal of the skin overlying the tumor mass. Removal of the skin led to higher values of the lifetime contrast and allowed an even easier tumor localization.© (1997) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Tumor detection has been carried out in mice sensitized with hematoporphyrin derivative (HpD) by measuring the spatial distribution of the fluorescence lifetime of the exogenous compound. This result has been achieved using a time-gated video camera and a suitable mathematical processing that led to the so-called "lifetime images." Extensive experimental tests have been performed on mice bearing the MS-2 fibrosarcoma or the L1210 leukemia. Lifetime images of mice show that the fluorescence decay of HpD is appreciably slower in the tumor than in healthy tissues nearby, allowing a reliable detection of the neoplasia. The lengthening of the lifetime in tumors depends little on the drug dose, which in our experiments could be lowered down to 0.1 mg/kg body weight, still allowing a definite tumor detection. In order to ascertain the results achieved with the imaging apparatus, high-resolution spectroscopy, based on a time-correlated single photon counting system, has also been performed to measure the fluorescence lifetime of the drug inside the tumor and outside. The outcomes obtained with two techniques are in good agreement.

A system for time-gated imaging has been used to measure the spatial distribution of the fluorescence decay time in tumor-bearing mice sensitized with Hematoporphyrin Derivative. Mice were injected with five doses of the sensitizer ranging from 0.1 to 10 mg/kg body weight. Several fluorescence images of each mouse were acquired at different delays after the excitation pulses, and a matrix processing algorithm was applied to calculate the average decay time corresponding to each pixel. It was found that, for any drug dose, the decay time of the exogenous fluorescence in the tumor is always significantly longer than in normal tissues. The images created by associating a gray scale to the decay time matrix of each sample allow a reliable and precise detection of the neoplasia, even with a very low dose of the sensitizer.

A time-gated fluorescence-imaging technique was applied to study the distribution of sensitizer in porphyrin-treated tumour-bearing mice. The animals were administered with either haematoporphyrin derivative (HpD) or Photofrin® and sacrificed 4 or 12 h later. Fluorescence images were acquired from tumour, skin, muscle, fat, brain, lymph node, bowel and bone of both treated and untreated mice. The results obtained with HpD and Photofrin® are similar. In images acquired 30 ns after excitation a bright exogenous fluorescence allows clear detection of the tumour. Nevertheless, the images show that porphyrins localize with different concentrations in all the examined tissues except the brain. Moreover, an appreciable long-living endogenous emission was observed in the bone.

Photodynamic therapy (PDT) is a relatively new cancer treatment modality that employs light excitation of a photosensitizer to yield cytotoxic oxygen-related species. In the present study we explored whether PDT would have therapeutic effect against doxorubicin-resistant murine tumors. We compared the efficacy of PDT with aluminium disulphonated phthalocyanine (A1S2Pc) and laser light on the doxorubicin-sensitive murine tumors, B16 melanoma (B16), L1210 leukemia (L1210), P388 lymphoma (P388) and the corresponding doxorubicin-resistant lines (B16/Dx, L1210/Dx and P388/Dx). Mice bearing L1210-L1210/Dx, P388-P388/Dx and B16-B16/Dx, were treated with 5 mg/kg of A1S2Pc and laser light (100 mW/cm2 x 10 min of exposure) or with doxorubicin (10 or 12 mg/kg i.v.). The results show that PDT is active versus all tumors while doxorubicin is effective only against the three sensitive tumor lines (L1210, P388 and B16). These observations suggest that PDT might be a beneficial alternative treatment for drug-resistant tumors.

The absorption spectrum of disulphonated Aluminum Phthalocyanine (AlS2Pc) was measured in vivo in a murine tumor model by time-resolved reflectance (TRR). Mice bearing the L1210 leukemia were administered intraperitoneally 2.5 mg/kg body weight of AlS2Pc. Reflectance measurements were performed in the 650-695 nm range before and 4 h after the drug administration. The absorption coefficient at each wavelength was obtained by fitting the experimental curve using the diffusion equation. The spectrum of the photosensitizer was obtained as a difference between the absorption of treated and untreated mice. The absorption spectrum of AlS2Pc was peaked at 685 nm, showing a red-shift of approximately 15 nm with respect to the spectrum in aqueous solution.© (1995) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

An intensified CCD video camera has been used to measure the spatial distribution of the fluorescence decay time in tumor-bearing mice sensitized with hematoporphyrin derivative. Mice were injected with five doses of sensitizer, ranging from 0.1 to 10 mg/kg body weight. For any drug dose the decay time of the exogenous fluorescence in the tumor is always significantly longer than in normal tissues. The image created by associating a gray-shade scale to the decay time matrix of each mouse permits a reliable and precise detection of the neoplasia.

The absorption spectrum of disulphonated aluminum phthalocyanine (AlS2Pc) between 650 nm and 695 nm was measured in vivo by means of time-resolved reflectance. The experiments were performed on mice bearing the L1210 leukemia 1, 4, and 7 hr after the i.p. administration of 2.5 mg/kg body weight (b.w.) of AlS2Pc. The absorption peak is centered at 685 nm, red-shifted of 10 - 15 nm with respect to the spectra obtained in solution in various environments. Measurements performed in vitro confirm the results in vivo and seem to suggest that the extracellular environment can cause the shift in the absorption line shape.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Since immunity is important in the control of tumor growth and spreading, in our laboratory we examine the effect of photoactivated phthalocyanines on the immune compartment. Spleen hyperplasia as well as spleen and marrow hypercellularities were observed in mice bearing MS-2 fibrosarcoma treated at different times with Aluminum disulfonated Phthalocyanine and laser light. Results suggest a potential `tumor immunity' induced by PDT with photoactivated phthalocyanines and that the dyes used in PDT could play an immunopharmacological role in cancer treatment.

A time-gated fluorescence imaging technique was applied on tumor-bearing porphyrin-treated mice to study the sensitizer distribution in different organs and tissue types, and to establish whether false positives in the diagnosis of tumors (based on porphyrin fluorescence) could be generated by this localization in healthy tissues. Mice were administered 25 mg/kg body weight (b.w.) of HpD or 5 mg/kg b.w. of PII, and sacrificed 8 hr later. Time- gated fluorescence images were acquired from tumor, skin, muscle, fat, brain, heart, lung, lymph nodes, liver, bowel, spleen, and bone of both treated and untreated animals. Similar results were obtained with HpD and PII. The presence of porphyrins clearly helps the localization of the neoplastic area, which is characterized by the strongest fluorescence in delayed images. An appreciable long-living emission was observed also in bones. With the exception of the bowel, the fluorescence of other organs was weaker and, in untreated mice, short-living.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The photosensitizer of choice in photodynamic therapy (PDT) is the hematoporphyrin derivative (Hpd). However, Hpd has many characteristics which make it less than an ideal photosensitizer. The sulfonated phthalocyanines represent a new group of interesting compounds that have a strong absorption in the red part of the spectrum at 675 nm. In our laboratory we compare the efficacy of two phthalocyanines, the zincum tetrasulfonated (ZnS4Pc) and the aluminum disulfonated (AlS2Pc), on a murine tumor. Mice bearing MS-2 fibrosarcoma were treated with 5 or 25 mg/kg of ZnS4Pc or AlS2Pc and then the tumor mass was exposed to a laser light (100 mW for 10'). The results show that the treatment with AlS2Pc is significantly more therapeutically active in respect to the treatment with the same dose of ZnS4Pc. Moreover, resistance to rechallenge with the MS-2 tumor was evidenced by surviving animals. Studies are in progress with other murine tumors with different biological properties.© (1993) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The action spectrum of disulfonated aluminum phthalocyanine (AIS2-Pc) as a photosensitizer for photodynamic therapy (PDT) was evaluated in vivo from 640 to 710 nm on two murine tumors with different biological properties. Mice bearing MS-2 fibrosarcoma and mice bearing the pigmented tumor B16 melanoma were injected with 5 mg/kg of AIS2Pc and irradiated with a light dose of 50 mW for 10 min for the MS-2 fibrosarcoma and of 100 mW for 10 min for the B16 melanoma. The action spectrum for both tumors presents a red shift with respect to the absorption spectrum of AIS2Pc in saline. The effectiveness is limited for short wavelengths (less than or equal to 655 nm for MS-2 and less than or equal to 660 for B16), whereas it increases at longer wavelengths and reaches its maximum at a peak (672 nm). For wavelengths beyond 672 nm the photodynamic activity remains up to 710 nm despite the significant decrease in absorption. The results obtained for both murine tumors seem therefore to indicate that an appreciable modification of the absorption spectrum takes place when AIS2Pc is incorporated into tissues following systematic administration.

Time-gated fluorescence spectroscopy provides a very useful tool to evaluate the photophysical properties and the incorporation mechanisms of drugs interacting with biological substrates. In particular, taking into account that different fluorophores, even if overlapped in fluorescence spectrum, present different fluorescence lifetimes, it is possible to evidence the emission of a single molecular species by choosing a suitable observation window in the time domain. Using this technique, the effect of systemic administration on the uptake of Hematoporphyrin Derivative (HpD), its tumor localizing fraction (TLF), and disulphonated Aluminum Phthalocyanine (AlS2Pc) at the cellular level was evaluated on a murine ascitic tumor. The results obtained indicate that the TLF is the part of HpD actually retained by the cells and that AlS2Pc is incorporated more rapidly with respect to porphyrins. The observation of the gated spectra of HpD also evidenced the possibility of improving the contrast between the fluorescence of the cells and that of the drug. Thus, an imaging system has been developed which utilizes a gated, intensified, CCD camera synchronized with a subnanosecond laser- pulse excitation. The gate can be set to a minimum width of 5 ns and arbitrarily delayed with respect to the laser pulse. By optimization of the gate parameters, porphyrin fluorescence images in single cells and microscopy sections of tumor were obtained with a valuable signal- to-noise ratio.© (1991) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The cytocidal activity of light-activated mitoxantrone in mice bearing B16 melanoma was investigated. Mice inoculated with 10(6) tumor cells on day 0 were i.p. injected with 1 mg/kg body weight of mitoxantrone on days 1, 5 and 9 and exposed to 108 J/cm2 of suitably filtered red light from a halogen lamp on days 2, 6 and 10. The treatment significantly prolonged the median survival time compared to both therapy with mitoxantrone and with red light alone.

After a decade of controversy over the reliability of identification and quantification of the melatonin binding sites, a highly potent melatonin analogue (2-iodomelatonin) was synthesized (Vakkuri et al., 1984). In the years that followed the introduction of the iodinated melatonin the field has developed rapidly (for reviews see Morgan and Williams, 1989; Stankov and Reiter, 1990).