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Purpose: Alteration in cell death is a hallmark of cancer. A functional role regulating survival, apoptosis, and necroptosis has been attributed to RIP1/3 complexes.Experimental Design: We have investigated the role of RIP1 and the effects of MC2494 in cell death induction, using different methods as flow cytometry, transcriptome analysis, immunoprecipitation, enzymatic assays, transfections, mutagenesis, and in vivo studies with different mice models.Results: Here, we show that RIP1 is highly expressed in cancer, and we define a novel RIP1/3–SIRT1/2–HAT1/4 complex. Mass spectrometry identified five acetylations in the kinase and death domain of RIP1. The novel characterized pan-SIRT inhibitor, MC2494, increases RIP1 acetylation at two additional sites in the death domain. Mutagenesis of the acetylated lysine decreases RIP1-dependent cell death, suggesting a role for acetylation of the RIP1 complex in cell death modulation. Accordingly, MC2494 displays tumor-selective potential in vitro, in leukemic blasts ex vivo, and in vivo in both xenograft and allograft cancer models. Mechanistically, MC2494 induces bona fide tumor-restricted acetylated RIP1/caspase-8–mediated apoptosis. Excitingly, MC2494 displays tumor-preventive activity by blocking 7,12-dimethylbenz(α)anthracene–induced mammary gland hyperproliferation in vivo.Conclusions: These preventive features might prove useful in patients who may benefit from a recurrence-preventive approach with low toxicity during follow-up phases and in cases of established cancer predisposition. Thus, targeting the newly identified RIP1 complex may represent an attractive novel paradigm in cancer treatment and prevention. Clin Cancer Res; 24(12); 2886–900. ©2018 AACR.

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Purpose: Alteration in cell death is a hallmark of cancer. A functional role regulating survival, apoptosis, and necroptosis has been attributed to RIP1/3 complexes. Experimental Design: We have investigated the role of RIP1 and the effects of MC2494 in cell death induction, using different methods as flow cytometry, transcriptome analysis, immunoprecipitation, enzymatic assays, transfections, mutagenesis, and in vivo studies with different mice models. Results: Here, we show that RIP1 is highly expressed in cancer, and we define a novel RIP1/3–SIRT1/2–HAT1/4 complex. Mass spectrometry identified five acetylations in the kinase and death domain of RIP1. The novel characterized pan-SIRT inhibitor, MC2494, increases RIP1 acetylation at two additional sites in the death domain. Mutagenesis of the acetylated lysine decreases RIP1-dependent cell death, suggesting a role for acetylation of the RIP1 complex in cell death modulation. Accordingly, MC2494 displays tumor-selective potential in vitro, in leukemic blasts ex vivo, and in vivo in both xenograft and allograft cancer models. Mechanistically, MC2494 induces bona fide tumor-restricted acetylated RIP1/caspase-8–mediated apoptosis. Excitingly, MC2494 displays tumor-preventive activity by blocking 7,12-dimethylbenz(α)anthracene–induced mammary gland hyperproliferation in vivo. Conclusions: These preventive features might prove useful in patients who may benefit from a recurrence-preventive approach with low toxicity during follow-up phases and in cases of established cancer predisposition. Thus, targeting the newly identified RIP1 complex may represent an attractive novel paradigm in cancer treatment and prevention. Clin Cancer Res; 24(12); 2886–900. ©2018 AACR.

Increased cytokine expression contributes to the pathogenesis of cancer anorexia–cachexia syndrome. Carnitine may reduce inflammation in chronic diseases. We tested the effects of L-propionylcarnitine (PC group) or saline (C group) on food intake (FI), body composition, and inflammatory status of MCA-sarcoma-bearing rats. On tumor appearance, rats were randomly assigned to daily i.p. injection of L-propionylcarnitine (250 mg/kgBW/d; n = 8) or saline (equal volume; n = 8). FI and fat-free mass wasting improved in PC rats only (p < .01 vs. controls). Cytokines’ levels decreased in PC rats vs. controls (p < .02). Results suggest that carnitine may ameliorate cancer anorexia–cachexia, via reduction of the inflammatory status.

The cell cycle is the cascade of events that allows a growing cell to duplicate all its components and split into two daughter cells. Cell cycle progression is mediated by the activation of a highly conserved family of protein kinases, the cyclin-dependent kinases (CDKs). CDKs are also regulated by related proteins called cdk inhibitors grouped into two families: the INK4 inhibitors (p16, p15, p19, and p18) and the Cip/Kip inhibitors (p21, p27, and p53). Several studies report the importance of cell-cycle proteins in the pathogenesis and the prognosis of lung cancer. This paper will review the most recent data from the literature about the regulation of cell cycle. Finally, based essentially on the data generated in our laboratory, the expression, the diagnostic, and prognostic significance of cell-cycle molecules in lung cancer will be examined.

A 22-year-old Camargue mare was referred for showing signs of discomfort during chewing, building up of food in the mouth, and foul odor from the mouth. On physical examination, the mare presented bilateral pigmented labial masses affecting the upper and lower lips, more in the form of plaques at the mucocutaneous junction as well as multiple perivulvar and perianal lumps. The skin lesions were biopsied for histopathologic examination and the clinical suspect of melanoma was confirmed. The owner elected the lesions to be treated with electrochemotherapy using the drug cisplatin. After two sessions, the nodules were shrunk by 50% and the horse’s ability to feed normally was restored: at that time, the owner had the treatment discontinued because of financial issues. The horse is still in partial remission after 1 year from the end of the treatment. Electrochemotherapy can be successfully used to palliate large melanomas in horses.

Background: Electrochemotherapy (ECT) couples the administration of anticancer drugs with the delivery of electric pulses that increase the drug uptake through the cell membranes, resulting in an improved efficacy. Hypothesis: To evaluate the tolerability and efficacy of cisplatin (CDDP) as an ECT agent to prevent recurrence of incompletely resected mast cell tumors (MCTs). Animals: Thirty-seven dogs. Methods: Prospective study recruiting dogs with incompletely excised MCTs as confirmed by surgeon and pathology reports. After debulking, the tumor bed and margins were infiltrated with CDDP, and then exposed to trains of biphasic electrical pulses under sedation. Five minutes after the injection of the chemotherapy agent, sequences of 8 biphasic pulses lasting 50 + 50 μs each, were delivered in bursts of 1,300 V/cm for sclerosed and of 800 V/cm for exposed lesions, with caliper or needle array electrodes, respectively. A second session was performed 1 or 2 weeks later based on clinical considerations. Results: The treatment was well tolerated with minimal adverse effects. Twenty-nine dogs had no evidence of recurrence over the 6-year study period, 6 had tumor recurrence, 1 died of multiple cutaneous MCTs, and 1 died of unrelated causes. The estimated median time to recurrence was 1,200 days. Recurrence was not observed among the long-term (>1 year) treated dogs. Conclusions and Clinical Importance: ECT with CDDP appears effective in the treatment of incompletely resected MCT in dogs and could be a useful addition to the current options based on its low cost, limited toxicity, and ease of administration.

The ability of skeletal stem cells (SSCs) to direct spinal fusion (SF) upon transplantation in conjunction with osteoconductive biomaterials was investigated in a rabbit model. When tested in a mouse heterotopic transplantation assay, rabbit SSCs and Pro-Osteon 500R™ was osteoconductive and supported osteogenesis. When used in a SF model, the same constructs induced bone formation in periapophyseal regions (PARs). In this respect, they proved to be superior to grafts of cell-free carrier or total uncultured bone marrow–carrier constructs, used as controls. However, interapophyseal regions (IARs) remained devoid of new bone, such that true bony bridging of adjacent transverse apophyses (true SF) could not be achieved. Interestingly, this could not be predicted from high-resolution radiography. A systematic histological survey of the entire graft harvested at 6 months was essential for proper assessment of the transplantation procedure outcome. Immunohistochemical analysis of microvessel density revealed that IARs remained undervascularized, as compared to PARs, suggesting that differential vascularization could account for the absence or presence of new bone formation in the same regions. SF is an extreme model of stem cell-directed bone regeneration, requiring a combination of orthotopic (PAR) and heterotopic (IAR) bone formation. Our data show that, in this setting, graft size can be critical with respect to the necessary neovascularization, a crucial variable independent of proper osteogenic and osteoconductive competence of the cells and materials employed. Furthermore, stringent histological studies are mandatory for proper assessment of outcomes in SF studies, in which the use of mineralized materials can make radiographic assessment misleading. Copyright © 2009 John Wiley & Sons, Ltd.

In this article we used immunohistochemistry and FACS analyses to show that cells expressing markers typical of human stem cells such as SSEA4, OCT-4, ALP, and CD117 are present within the cytotrophoblastic tissue of human fetal chorionic villus samples (CVSs). After immunoselection of CV cells for SSEA4, FACS analyses showed an increased number of cells positive for OCT-4 and ALP and a small percentage (around 4%) of side population (SP) cells. In the same cell population, RT-PCR indicated the presence of OCT-4, NANOG, and SOX2 transcripts, also typical of stem cells. Depending on the in vitro conditions, a subset of SSEA4+ cells formed colonies resembling hESCs, with limited self renewal ability. At the same time, these cells were able to differentiate in vitro into derivatives of all three germ layers. When inoculated into immunocompromised mice, SSEA4+ cells did not form teratomas but were able to populate depleted hematopoietic tissues. Moreover, after injection into mouse blastocysts, they were incorporated into the inner cell mass and could be traced into several tissues of the adult chimeric mice. Finally, we show that SSEA4+ cells isolated from fetuses affected by Spinal Muscular Atrophy (SMA) can be genetically corrected with high efficiency in culture by Small Fragment Homologous Recombination (SFHR), a gene targeting approach. Taken together, our results indicate that SSEA4+ cells obtained from human CVSs contain a subpopulation of multipotent cells that we propose to name Human Cytotrophoblastic-derived Multipotent Cells (hCTMCs). These cells may be a safe and convenient source of cells for cell-based therapy, as well as an ideal target for in utero fetal gene therapy.

Many in vitro and in vivo evidence have shown that the status of p53 is a key determinant in the response of tumor cells to anticancer treatment. Here we provide evidence that peptide-mediated targeting of the protein complex mutantp53/p73 enhances the response of mutant p53 tumor cells to commonly used anticancer drugs. Indeed, we show that the disruption of the protein complex mutantp53/p73 and the consequent restoration of p73 transcriptional effects, through the activity of short interfering peptides, render mutant p53 cells more prone to the killing of adriamycin and cisplatin. Of note, the activity of the short interfering peptides is mutant p53 specific and causes no effects on wt-p53 and p53 null cells. Our findings highlight the protein complex mutantp53/p73 as a molecular target, whose successful overriding through the selective activity of small interfering peptides, might contribute to the optimization of mutant p53 tumor treatments.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, is a scavenger receptor that plays a central role in the pathogenesis of atherosclerosis. We have recently identified a truncated naturally occurring variant of the human receptor LOX-1, named LOXIN, which lacks part of the C-terminus lectin-like domain. In vivo and in vitro studies support that the new splicing isoform is protective against acute myocardial infarction. The mechanism by which LOXIN exerts its protective role is unknown. In this paper we report studies on the heterologous expression and functional characterization of LOXIN variant in mammalian fibroblasts and human endothelial cells. We found that LOXIN, when expressed in the absence of LOX-1, shows diminished plasma membrane localization and is deficient in ox-LDL ligand binding. When co-transfected with the full-length counterpart LOX-1, the two isoforms interact to form LOX-1 oligomers and their interaction leads to a decrease in the appearance of LOX-1 receptors in the plasma membrane and a marked impairment of ox-LDL binding and uptake. Co-immunoprecipitation studies confirmed the molecular LOX-1/LOXIN interaction and the formation of non-functional hetero-oligomers. Our studies suggest that hetero-oligomerization between naturally occurring isoforms of LOX-1 may represent a general paradigm for regulation of LOX-1 function by its variants.

Electroporation (EP) has been widely employed in the past years as a safe and effective technique to drive drugs and DNA plasmids into target cells both for experimental and therapeutic purposes. Despite the large bulk of literature on this topic, often describing successful outcomes, there is a lack of knowledge about the intimate mechanism(s) controlling this phenomenon. In this paper, we describe a number of ultrastructural alterations in the cellular membranes following the exposure of orthotopic melanomas and red blood cells to trains of biphasic pulses. Specifically, melanoma xenografts grown in nude mice were subject to trains of eight biphasic pulses using an electric field of 1250 or 2450 V/cm, excised after 5 min and processed for electron microscopy. The freeze-fracturing analysis of both cell types evidenced defects in the dynamic assembly of lipids and proteins, which generate “areas with rough structure” and intensive clustering of intramembrane proteins. Such modifications could be the hallmarks of lipid and protein alterations, of protein cohesion reduction, and of changes in lipid orientation inside cell membranes, as postulated in several mathematical models applied to electroporation, and warrant further investigations. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc.

Purpose: Although survival of encapsulated thymomas is usually good, some patients present a higher incidence rate of recurrence and a shorter long-term survival. Abnormalities in the components of cell cycle checkpoints are extremely common among virtually all neoplasms. In this study, three components of the cell cycle machinery (i.e., p21, p27 and p53) were examined in a series of well-characterized encapsulated thymoma specimens to analyze coregulation and influence on recurrence and survival. Experimental Design: Sixty-eight consecutive patients with thymoma were operated in our center from 1987 to 2000. Expression of p53, p21, and p27 was studied in specimens from 25 encapsulated thymomas using immunohistochemistry. Generic factors and gene expression influencing the probability of recurrence were studied. Positive expression was dichotomized defining positive when present in more than 5% of tumor cells. Mean follow up was 85.9 months; clinical data about recurrence were recorded. Results: Univariate analysis suggests that positive p53 (P < 0.05), negative p21 (P = 0.01), and especially negative p27 expressions (P = 0.001) significantly correlate with poor prognosis for disease-free survival. Multivariate Cox regression analysis suggests that negative p27 immunohistology is the only significant variable for poor prognosis (P = 0.03; odds ratio, 0.08; 95% confidence interval, 0.01-0.88). Conclusions: These results show that loss of control of cell cycle checkpoints is a common occurrence in thymomas and support the idea that functional cooperation between different cell cycle inhibitor proteins constitutes another level of regulation in cell growth control and tumor suppression.

Background: Experimental evidence suggests that lung cancer development and progression can be linked to an increased proliferation rate. Aims/Methods: To evaluate the immunohistochemical expression of seven components of the cell cycle machinery in a series of well characterised non-small cell lung cancer (NSCLC) specimens (n = 105). Results: Multivariate analysis revealed that simultaneous loss of expression of three of these factors—cyclin D1, the cyclin dependent kinase inhibitor p16, and the tumour suppressor retinoblastoma protein Rb2/p130—correlated with survival, confirming the hypothesis that the cyclin D1–p16–retinoblastoma tumour suppressor pathway is inactivated in most lung cancer samples. Conclusions: These results suggest that loss of control of cell cycle checkpoints is a common occurrence in lung cancer and support the idea that functional cooperation between different cell cycle regulatory proteins constitutes another level of regulation in cell growth control and tumour suppression.

The transcriptional coactivator Yes-associated protein (YAP) has been shown to interact with and to enhance p73-dependent apoptosis in response to DNA damage. Here, we show that YAP requires the promyelocytic leukemia gene (PML) and nuclear body localization to coactivate p73. YAP imparts selectivity to p73 by promoting the activation of a subset of p53 and/or p73 target promoters. Endogenous p73, YAP, and p300 proteins are concomitantly recruited onto the regulatory regions of the apoptotic target gene p53AIP1 only when cells are exposed to apoptotic conditions. Silencing of YAP by specific siRNA impairs p300 recruitment and reduces histone acetylation on the p53AIP1 target gene, resulting in delayed or reduced apoptosis mediated by p73. We also found that YAP contributes to the DNA damage-induced accumulation of p73 and potentiates the p300-mediated acetylation of p73. Altogether, our findings identify YAP as a key determinant of p73 gene targeting in response to DNA damage.

To explore the expression and gain more information on the function of transglutaminase 5 enzyme in normal and defective human epidermis, we generated a rat antihuman transglutaminase 5 antiserum elicited against a purified active recombinant protein expressed in the baculovirus system. By use of Western blotting and immunofluorescence methods, the immunospecificity of the antibodies for transglutaminase 5 was tested; no crossreactivity with other transglutaminases (types 1, 2, and 3) was observed, thus allowing histochemistry studies. By indirect immunofluorescence analysis the antibodies decorated the upper layers of normal human epidermis, with consistent staining in the spinous and granular layers. We evaluated transglutaminase 5 expression in comparison with proliferating (keratin 14) and differentiating (transglutaminase 3) markers in different diseases, such as psoriasis, ichthyosis vulgaris, lamellar ichthyosis, and Darier's disease. We observed that transglutaminase 5 contributes, as a secondary effect, to the hyperkeratotic phenotype in ichthyosis (both vulgaris and lamellar) and in psoriasis. In Darier's disease, transglutaminase 5 expression, as well as transglutaminase 3, is completely missregulated, being overexpressed or totally absent in different areas of the same lesion.

The aim of this study was twofold: to assess the relationship between c-Myb and Bcl-x expression and to evaluate the prognostic significance of their expression in colorectal carcinoma (CRC) patients. Analysis of tumors from 91 CRC patients for expression of c-Myb and Bcl-x revealed a significant relationship between these two proteins. Kaplan-Meier’s analysis showed an increased risk of relapse and death in patients whose tumor specimens displayed high c-Myb levels and Bcl-x positivity. Similar results were also observed excluding Dukes’ D patients. Molecular analysis using three c-Myb-overexpressing LoVo clones indicated that c-Myb overexpression was accompanied by up-regulation of Bcl-xL protein and mRNA. Tumors originating from these clones injected in nude mice were significantly larger than those formed in mice injected with parental or vector-transfected LoVo cells. Moreover, tumors derived from parental and control vector-transfected but not from c-Myb-overexpressing LoVo cells showed high frequency of apoptotic cells. These results provide direct evidence of an association between c-Myb and Bcl-x expression and suggest that expression of both molecules might be a useful prognostic marker in CRC.

The effect of cyclic nucleotide phosphodiesterase (PDE) inhibitors Zaprinast and DC-TA-46 has been tested on SK-N-MC neuroblastoma growth. Antiproliferative activity of the tested drugs was assayed both in vitro and in the xenograft model of nude mice. In clonal density experiments, the IC50 value was 3.3 μM for Zaprinast and 1.9 μM for DC-TA-46, while 7.5 μM BCNU alkylating agent was required to obtain the same effect. SK-N-MC cells xenografted in the nude mouse showed that the administration of Zaprinast and DC-TA-46 caused a significant 50% decrease of the tumour weight. These data demonstrate that PDE inhibitors may be useful for at least reducing tumour growth; they may be of interest for further evaluation as alternative molecules in the design of multiple agent protocols for neuroblastoma treatment.

We investigated the effect of c-myb antisense phosphorothioate oligodeoxynucleotides [(S)ODNs] and cisplatin (CDDP) combination on the human colon carcinoma cell line LoVo Dx both in vitro and in nude mice bearing LoVo Dx solid tumour. We show that antisense (S)ODN treatment decreases c-myb mRNA and protein expression, induces growth arrest in the G1 phase of the cell cycle, and inhibits cell proliferation. In vivo treatment with c-myb antisense (S)ODNs results in a reduction in tumour growth. A greater inhibition of cell proliferation in vitro and a higher increase of tumour growth inhibition and growth delay in vivo were obtained with the combination of (S)ODNs and CDDP than when the two agents were administered separately. This comparative study, using the same tumour cell line in vitro and in vivo, suggests that c-myb antisense (S)ODNs might be useful in the therapy of colon cancer in combination with antineoplastic drugs. Images Figure 1 Figure 4

Increased oxidative stress may contribute to cancer anorexia, which could be ameliorated by antioxidant supplementation. methylcholanthrene (MCA) sarcoma-bearing Fisher rats were studied. After tumour inoculation, rats were randomly assigned to standard diet (CTR group, n = 6), or to an antioxidant-enriched diet (AOX group, n = 8). Eight more rats (STD-AOX group) switched from standard to antioxidant diet when anorexia developed. At the end of the study, food intake (FI, g/d), body weight and tumour weight (g) were recorded, and plasma samples were obtained. On day 16, anorexia has appeared only in CTR and STD-AOX animals. At the end of the study, FI in AOX animals was still higher than in the other groups (p = 0.08). No differences in body and tumour weights were observed among groups. However, hydrogen peroxide and interleukin-1β levels were significantly reduced only in AOX rats. Data obtained suggest that early antioxidant supplementation improves cancer anorexia, ameliorates oxidative stress and reduces inflammation.

Electrochemotherapy is a tumor treatment that adapts the systemic or local delivery of anticancer drugs by the application of permeabilizing electric pulses with appropriate amplitude and waveforms. This allows the use of lipophobic drugs, which frequently have a narrow therapeutic index, with a decreased morbidity for the patient, while maintaining appropriate anticancer efficacy. Electrochemotherapy is used in humans for the treatment of cutaneous neoplasms or the palliation of skin tumor metastases, and a standard operating procedure has been devised. In veterinary oncology, the electrochemotherapy approach is gaining popularity, becoming a first-line treatment in consideration of its high efficacy and low toxicity. This review summarizes the state of the art in veterinary oncology as a preclinical model. © 2012 Future Medicine Ltd.

Background The aim of this study was to evaluate the potential association between single nucleotide polymorphisms related response to radiotherapy injury, such as genes related to DNA repair or enzymes involved in anti-oxidative activities. The paper aims to identify marker genes able to predict an increased risk of late toxicity studying our group of patients who underwent a Single Shot 3D-CRT PBI (SSPBI) after BCS (breast conserving surgery). Methods A total of 57 breast cancer patients who underwent SSPBI were genotyped for SNPs (single nucleotide polymorphisms) in XRCC1, XRCC3, GST and RAD51 by Pyrosequencing technology. Univariate analysis (ORs and 95% CI) was performed to correlate SNPs with the risk of developing ≥ G2 fibrosis or fat necrosis. Results A higher significant risk of developing ≥ G2 fibrosis or fat necrosis in patients with: polymorphic variant GSTP1 (Ile105Val) (OR = 2.9; 95%CI, 0.88-10.14, p = 0.047). Conclusions The presence of some SNPs involved in DNA repair or response to oxidative stress seem to be able to predict late toxicity. Trial Registration ClinicalTrials.gov: NCT01316328

Pregnant adult Balb-C mice were exposed daily to two different doses of Bisphenol A (BPA) by subcutaneous injection beginning on gestational day 1 through the seventh day after delivery. The mothers were sacrificed on postpartum day 21, and the offspring were sacrificed at 3 months of age. Control mice were subjected to the same experimental protocol but received saline injections. The liver, muscles, hindbrain and forebrain of the offspring were dissected and processed using HPLC to assess the level of BPA in the tissues and to determine its dependence on the exposure dose and gender. For comparison, the same tissues were dissected from the mothers and analysed. We report the following results: (1) the level of BPA that accumulated in a given tissue was dependent on the exposure dose; (2) the rank order of BPA accumulation in the various tissues was dependent on the gender of the offspring; (3) the average BPA concentrations in the liver and muscle of the female offspring were higher than in the males; and (4) the average BPA concentration in the central nervous system (i.e., the hindbrain and forebrain) of the male offspring was higher than in the females. (C) 2011 Elsevier B.V. All rights reserved.

The inhibitory effect of c-myb antisense oligodeoxynucleotides (ODNs) conjugated to folic acid (FA) on HL-60 cell proliferation was examined. Folic acid was covalently linked to a polylysine chain and purified by gel chromatography. Sterile FA-polylysine was complexed with c-myb sense and antisense. Exposure of HL-60 cells to the FA-polylysine-c-myb antisense ODN complex resulted in a down-regulation of c-myb expression and a greater inhibition of proliferation than that obtained using free ODNs. Moreover, FA-polylysine conjugate alone or complexed to c-myb sense ODN was not toxic to cells. The antigenic properties and uptake of the vitamin were not affected by the polylysine chain. These data suggest that this strategy is potentially useful for the selective delivery of anti-oncogene-targeted ODNs into cancer cells. Images Figure 2 Figure 3 Figure 4 Figure 6

Timed pregnant Balb-C mice were treated from day 1 of gestation to 7 days after delivery with the endocrine disruptor bisphenol a (BPA) (100, or 1,000 microg/kg/day). After delivery, pups were hold for three months; then, ovaries were analyzed in their entirety. We found that in the ovaries of BPA-treated animals the number of primordial follicles and of developing follicles was significantly lower than in the untreated animals. Moreover, the number of atretic follicles was significantly higher in the treated animals. Finally, we found that the animals displaying endometriosis-like phenotype had a more severe impairment of the ovaries in term of number of primordial and developing follicles in comparison with the other mice exposed to BPA. In conclusion, we describe for the first time a complex phenotype in mice, elicited by pre-natal exposition to BPA, that includes ovarian lesions and endometriosis. Considering the high incidence of endometriosis and of the premature ovarian failure associated to infertility in these patients, the data showed prompt a thoroughly reconsideration of the pathological framing of these lesions.

Diabetic patients treated with metformin have a reduced incidence of cancer and cancer-related mortality. Here we show that metformin affects engraftment and growth of breast cancer tumours in mice. This correlates with the induction of metabolic changes compatible with clear anticancer effects. We demonstrate that microRNA modulation underlies the anticancer metabolic actions of metformin. In fact, metformin induces DICER expression and its effects are severely impaired in DICER knocked down cells. Conversely, ectopic expression of DICER recapitulates the effects of metformin in vivo and in vitro. The microRNAs upregulated by metformin belong mainly to energy metabolism pathways. Among the messenger RNAs downregulated by metformin, we found c-MYC, IRS-2 and HIF1alpha. Downregulation of c-MYC requires AMP-activated protein kinase-signalling and mir33a upregulation by metformin. Ectopic expression of c-MYC attenuates the anticancer metabolic effects of metformin. We suggest that DICER modulation, mir33a upregulation and c-MYC targeting have an important role in the anticancer metabolic effects of metformin.

Here we show that pemetrexed-treated mesothelioma cells undergo accelerated senescence. This is characterized by the secretion of proinflammatory and mitogenic cytokines, reminiscent of an SASP (senescence-associated secretory phenotype). Conditioned media from senescent MPM (malignant pleural mesothelioma) cells trigger the emergence of EMT (epithelial-to-mesenchymal)-like, clonogenic and chemoresistant cell subpopulations, expressing high levels of ALDH (aldehyde dehydrogenase) activity (ALDH(bright) cells). We show by fluorescence-activated cell sorting of purified ALDH(bright) and ALDH(low) cells, that both cell-autonomous and cell-non-autonomous mechanisms converge to maintain the SASP-induced, EMT-like cell subpopulations. Chemoresistant ALDH(bright) cells exist within primary MPM specimens and enrichment for ALDH(bright) cells correlates with an earlier tumor onset into NOD/SCID mice. We show that RAS(v12) expression induces SASP-like changes in untransformed human mesothelial cells, and that p53 ablation increases the effect of RAS(v12) expression. We identify STAT3 activation as a crucial event downstream to SASP signaling. In fact, small hairpin RNA-mediated ablation of STAT3 deeply attenuates the induction of EMT genes and the increase of ALDH(bright) cells induced by SASP-cytokines. This strongly affects the chemoresistance of MPM cells in vitro and leads to anticancer effects in vivo.

Few articles in the literature have focused on electroporation as a strategy to reverse multidrug resistance (MDR) of tumour cells and they are mostly limited to the improved efficacy of bleomycin. We tested the application of trains of biphasic pulses to cell suspensions and to murine xenografts as a strategy to increase the uptake of doxorubicin (DOX) and to enhance its cytotoxicity against chemoresistant cells. The human colon adenocarcinoma cell line LoVo DX, expressing MDR phenotype with high levels of P-glycoprotein (P-gp), has been used. The in vitro and in vivo studies gave the following results: (i) the application of the electric pulses to the cell suspension, immediately before DOX administration, induced a significant increase of drug retention; (ii) confocal microscopy observations showed a remarkable increase of intranuclear accumulation of DOX induced by electroporation; (iii) cell survival assay revealed a decrease of cell viability in the cultures treated with the combination of electroporation and doxorubicin; (iv) scanning electron microscopy observations revealed consistent morphological changes after the combined exposure to electroporation and doxorubicin; (v) in implanted mice the combined treatment induced an evident slowdown on the tumour growth when compared to treatment with DOX alone; (vi) histopathological analysis evidenced tumour destruction and its replacement by scar tissue in the tumours treated with the combination of doxorubicin and electroporation. © 2011 Elsevier Ltd. All rights reserved.

Breast cancer is the commonest form of female malignancy among women in Western countries. The advent of genomic technologies has enhanced the diagnosis and the biological classification of such pathology. It has been demonstrated that cancer takes many years to be fully established. This long dormancy could represent a potential window for intervening with chemoprevention studies. Cancer chemoprevention is by definition the use of natural, synthetic, or biological chemical agents to reverse, suppress, or delay the genetic or other alterations that culminate in the appearance of the tumor phenotype. An important step for the success of chemoprevention is the identification of molecularly targeted agents to prevent cancer development. Currently, only two chemoprevention agents, raloxifene and tamoxifen, are used in clinical practice to prevent breast cancer. In this review, we will mainly focus on: (1) the application of genomic technologies for the identification and validation of molecular targets for chemoprevention; (2) the role of vitamin D and its cognate receptor VDR (vitamin D receptor) as a model for the molecularly targeted chemoprevention of breast cancer.

Inflammation contributes to the pathogenesis of cancer anorexia-cachexia syndrome. Nicotine administration reduces cytokine levels and mortality during sepsis. Therefore, nicotine administration may result in improved anorexia-cachexia. Sixteen male Fischer rats inoculated with MCA sarcoma were assigned to random injections of nicotine (NIC; 200 mg/kg BW/d) or saline (C). Food intake (FI), body weight, body composition, interleukin (IL)-1, IL-6 levels were evaluated. Data were analyzed via Student's t-test for paired and unpaired data and ANOVA. FI started declining 12 days after tumor inoculation both in C and NIC rats, but the decline was significantly attenuated by nicotine administration. At the end of the study, lean body mass wasting was more severe in C rats than in NIC rats (P

Background: Malignant mesothelioma (MM) is a rare, highly aggressive tumor, associated to asbestos exposure. To date no chemotherapy regimen for MM has proven to be definitively curative, and new therapies for MM treatment need to be developed. We have previously shown in vivo that piroxicam/cisplatin combined treatment in MM, specifically acts on cell cycle regulation triggering apoptosis, with survival increase. Methodology/Principal Findings: We analyzed, at molecular level, the apoptotic increase caused by piroxicam/cisplatin treatment in MM cell lines. By means of genome wide analyses, we analyzed transcriptional gene deregulation both after the single piroxicam or cisplatin and the combined treatment. Here we show that apoptotic increase following combined treatment is mediated by p21, since apoptotic increase in piroxicam/cisplatin combined treatment is abolished upon p21 silencing. Conclusions/Significance: Piroxicam/cisplatin combined treatment determines an apoptosis increase in MM cells, which is dependent on the p21 expression. The results provided suggest that piroxicam/cisplatin combination might be tested in clinical settings in tumor specimens that express p21. © 2011 Baldi et al.

Niemann-Pick Type C Disease (NPCD) is a progressive neurodegenerative disorder characterized by accumulation of free cholesterol, sphingomyelin, glycosphingolipids (GSLs) and sphingosine in lysosomes, mainly due to a mutation in the NPC1 gene. One of the main symptoms in NPCD patients is hyperexcitability leading to epileptic activity, however, the pathophysiological basis of this neural disorder is not yet well understood. Here we studied the excitatory neurotransmission in the hippocampus of BALB/c NPC1NIH (NPC1-/-) mice, a well-described animal model of the disease. We report that hippocampal field potential population spike (fPS), as well as paired pulse ratio, is enhanced in NPC1-/- with respect to Wild Type (WT). To evaluate the contribution of glutamate receptor activity in the enhanced fPS observed in mutant mice, we recorded slices treated with glutamate receptor agonists alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and Kainate (KA). We found that a prolonged application of KA and AMPA in NPC1-/- mice do not induce the dramatic decrease of synaptic transmission observed in WT hippocampal slices suggesting a functional impairment of presynaptic KA receptors and an imbalance of AMPA receptor exo/endocytosis. In line with electrophysiological data, we also found notable differences in calcium influx during KA and AMPA bath application in NPC1-/- hippocampal culture as compared with WT. Nevertheless in synaptosomal membranes, Western Blot analysis didn't reveal any modification in protein expression levels of KA and AMPA receptor subunits. All together these data indicate that in mutant mice the hyperexcitability, that is at the basis of the insurgence of seizures, might be due to the enhanced glutamatergic neurotransmission caused by an altered KA and AMPA receptor functioning.

Background To evaluate the association between polymorphisms involved in DNA repair and oxidative stress genes and mean dose to whole breast on acute skin reactions (erythema) in breast cancer (BC) patients following single shot partial breast irradiation (SSPBI) after breast conservative surgery. Materials and Methods Acute toxicity was assessed using vers.3 criteria. single nucleotides polymorphisms(SNPs) in genes: XRCC1(Arg399Gln/Arg194Trp), XRCC3 (A4541G-5'UTR/Thr241Met), GSTP1(Ile105Val), GSTA1 and RAD51(untranslated region). SNPs were determined in 57 BC patients by the Pyrosequencing analysis. Univariate(ORs and 95% CI) and logistic multivariate analyses (MVA) were performed to correlate polymorphic genes with the risk of developing acute skin reactions to radiotherapy. Results After SSPBI on the tumour bed following conservative surgery, grade 1 or 2 acute erythema was observed in 19 pts(33%). Univariate analysis indicated a higher significant risk of developing erythema in patients with polymorphic variant wt XRCC1Arg194Trp, mut/het XRCC3Thr241Met, wt/het XRCC3A4541G-5'UTR. Similarly a higher erythema rate was also found in the presence of mut/het of XRCC1Arg194Trp or wt of GSTA1. Whereas, a lower erythema rate was observed in patients with mut/het of XRCC1Arg194Trp or wt of XRCC1Arg399Gln. The mean dose to whole breast(p = 0.002), the presence of either mut/het XRCC1Arg194Trp or wt XRCC3Thr241Met (p = 0.006) and the presence of either mut/het XRCC1Arg194Trp or wt GSTA1(p = 0.031) were confirmed as predictors of radiotherapy-induced erythema by MVA. Conclusions The Whole breast mean dose together with the presence of some polymorphic genes involved in DNA repair or oxidative stress could explain the erythema observed after SSPBI, but further studies are needed to confirm these results in a larger cohort. Trial Registration ClinicalTrials.gov Identifier: NCT01316328

Background The treatment of human cancer has been seriously hampered for decades by resistance to chemotherapeutic drugs. Mechanisms underlying this resistance are far from being entirely known. A very efficient mechanism of tumor resistance to drugs is related to the modification of tumour microenvironment through changes in the extracellular and intracellular pH. The acidification of tumor microenvironment depends on proton pumps that actively pump protons outside the cells, mostly to avoid intracellular acidification. In fact, we have shown in pre-clinical settings as pre-treatment with proton-pumps inhibitors (PPI) increase tumor cell and tumor responsiveness to chemotherapeutics. In this study pet with spontaneously occurring cancer proven refractory to conventional chemotherapy have been recruited in a compassionate study. Methods Thirty-four companion animals (27 dogs and 7 cats) were treated adding to their chemotherapy protocols the pump inhibitor lansoprazole at high dose, as suggested by pre-clinical experiments. Their responses have been compared to those of seventeen pets (10 dogs and 7 cats) whose owners did not pursue any other therapy than continuing the currently ongoing chemotherapy protocols. Results The drug was overall well tolerated, with only four dogs experiencing side effects due to gastric hypochlorhydria consisting with vomiting and or diarrhea. In terms of overall response twenty-three pets out of 34 had partial or complete responses (67.6%) the remaining patients experienced no response or progressive disease however most owners reported improved quality of life in most of the non responders. On the other hand, only three animals in the control group (17%) experienced short lived partial responses (1-3 months duration) while all the others died of progressive disease within two months. Conclusions high dose proton pump inhibitors have been shown to induce reversal of tumor chemoresistance as well as improvement of the quality of life in pets with down staged cancer and in the majority of the treated animals PPI were well tolerated. Further studies are warranted to assess the efficacy of this strategy in patients with advanced cancers in companion animals as well as in humans.

Shift of three Thiobacillus ferrooxidans strains from Fe(II) to S0 or thiosulphate liquid medium caused distinctive changes in the outer membrane protein profile. In addition to a new 55-kDa protein which was synthesized only in the presence of sulphur compounds, a higher expression of a 47-kDa protein was observed. This latter protein appeared to be constitutively synthesized, since it was detectable in small amounts even in tile presence of ferrous iron as sole energy source, but its expression was greatly enhanced when elemental sulphur or thiosulphate were present in the growth medium.

The lack of efficient and specific delivery to target cells still limits the potential application of antisense oligodeoxynucleotides as therapeutic agents in cancer disease. We have covalently linked a polylysine chain (10,000-20,000 mw) to compounds as folic acid, retinoic acid, transferrin, insulin and estradiol, to deliver c-myb antisense oligonucleotide into tumor cells. Using these complexes as carriers for the oligodeoxynucleotides can be achieved an increase in their uptake into target cells through a natural endocytosis pathway.

The vacuolar ATPases are ATP-dependent proton pumps whose functions include the acidification of intracellular compartments and the extrusion of protons through the cell cytoplasmic membrane. These pumps play a pivotal role in the regulation of cell pH in normal cells and, to a much greater extent, in tumor cells. In fact, the glucose metabolism in hypoxic conditions by the neoplasms leads to an intercellular pH drift towards acidity. The acid microenvironment is modulated through the over-expression of H+ transporters that are also involved in tumor progression, invasiveness, distant spread and chemoresistance. Several strategies to block/downmodulate the efficiency of these transporters are currently being investigated. Among them, proton pump inhibitors have shown to successfully block the H+ transporters in vitro and in vivo, leading to apoptotic death. Furthermore, their action seems to synergize with conventional chemotherapy protocols, leading to chemosensitization and reversal of chemoresistance. Aim of this article is to critically revise the current knowledge of this cellular machinery and to summarize the therapeutic strategies developed to counter this mechanism.

Endometriosis is a chronic gynecological disease characterized by the growth of endometrial tissue outside the uterine cavity. Exposure to endocrine disruptors during critical period of development causes long-lasting effects, being the genital system one of the targets. This study describes the effects on female genital system caused by developmental exposure to the endocrine-disrupting chemical bisphenol A (BPA) during pre- and pen-natal development in mice. To this end, timed pregnant Balb-C mice were treated from day 1 of gestation to 7 days after delivery with BPA (100, or 1000 mu g/kg/day). After delivery, pups were held for 3 months; then, pelvic organs were analyzed in their entirety and livers of both pups and moms were studied for the presence of BPA. We found in the adipose tissue surrounding the genital tracts of a consistent number of treated animals, endometriosis-like structure with the presence of both glands and stroma and expressing both estrogen receptor and HOXA-10. Moreover, cystic ovaries, adenomatous hyperplasia with cystic endometrial hyperplasia and atypical hyperplasia were significantly more frequent in treated animals respect to the controls. Finally, BPA was found in the livers of exposed moms and female offspring. In conclusion, we describe for the first time an endometriosis-like phenotype in mice, elicited by pre-natal exposition to BPA. This observation may induce to thoroughly reconsider the pathogenesis and treatment of endometriosis, considering the high incidence of endometriosis and the problems caused by associated infertility. (C) 2010 Elsevier Inc. All rights reserved.