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1.  Anti-mullerian hormone is expressed by endometriosis tissues and induces cell cycle arrest and apoptosis in endometriosis cells 
Background
The anti-mullerian hormone (AMH) is a member of the transforming growth factor β (TGF-β) superfamily, which is responsible of the regression of the mullerian duct. AMH is expressed in the normal endometrium, where, acting in a paracrine fashion, negatively regulates cellular viability. Our objective was to evaluate the in vitro effects of the treatment with AMH of endometriosic cells.
Methods
AMH expression in human endometriosis glands was evaluated by immunohistochemistry. RT-PCR has been used to quantify the expression levels of AMH and AMH RII isoforms, as well as of cytochrome P450 in both endometriosis epithelial and stromal cells Effects of AMH and AMH-cleaved treatment in endometriosis cells were evaluated by flow-cytometry analysis. Finally, it has been evaluated the effect of plasmin-digested AMH on cytochrome P450 activity.
Results
AMH and AMH RII isoforms, as well as cytochrome P450, were expressed in both endometriosis epithelial and stromal cells. Treatment of endometriosis stromal and epithelial cell growth with AMH was able to induce a decrease in the percentage of cells in S phase and increase percentage of cells in G1 and G2 phase; coherently, decreased cell viability and increased percentage of cells death fraction was observed. The plasmin-digested AMH was able to suppress most of the cytochrome P450 activity, causing an increase of pre-G1 phase and of apoptosis induction treating with plasmin-digested AMH in both cell lines, most marked in the epithelial cells.
Conclusions
The data produced suggest a possible use of AMH as therapeutic agents in endometriosis.
doi:10.1186/1756-9966-33-46
PMCID: PMC4046500  PMID: 24886254
Endometriosis; Immunohistochemistry; AMH; Apoptosis
2.  Matrix detachment and proteasomal inhibitors diminish Sulf-2 expression in breast cancer cell lines and mouse xenografts 
Sulfatase 2 (Sulf-2) has been previously shown to be upregulated in breast cancer. Sulf-2 removes sulfate moieties on heparan sulfate proteoglycans which in turn modulate heparin binding growth factor signaling. Here we report that matrix detachment resulted in decreased Sulf-2 expression in breast cancer cells and increased cleavage of poly ADP-ribose polymerase. Silencing of Sulf-2 promotes matrix detachment induced cell death in MCF10DCIS cells. In an attempt to identify Sulf-2 specific inhibitor, we found that proteasomal inhibitors such as MG132, Lactacystin and Bortezomib treatment abolished Sulf-2 expression in multiple breast cancer cell lines. Additionally, we show that Bortezomib treatment of MCF10DCIS cell xenografts in mouse mammary fat pads significantly reduced tumor size, caused massive apoptosis and more importantly reduced Sulf-2 levels in vivo. Finally, our immunohistochemistry analysis of Sulf-2 expression in cohort of patient derived breast tumors indicates that Sulf-2 is significantly upregulated in autologous metastatic lesions compared to primary tumors (p < 0.037, Pearson correlation, Chi-Square analysis). In all, our data suggest that Sulf-2 might play an important role in breast cancer progression from ductal carcinoma in situ into an invasive ductal carcinoma potentially by resisting cell death.
doi:10.1007/s10585-012-9546-5
PMCID: PMC3619208  PMID: 23412907
Sulfatase 2; Growth factor; Breast cancer
3.  HtrA Serine Proteases as Potential Therapeutic Targets in Cancer 
Current cancer drug targets  2009;9(4):451-468.
The human HtrA family of serine proteases consists of four members: HtrA1, HtrA2, HtrA3 and HtrA4. Although prokaryotic HtrA proteins are well characterized in their dual roles as chaperones and proteases that degrade misfolded proteins in the periplasm, some members of mammalian HtrA proteins are described as potential modulators of programmed cell death and chemotherapy-induced cytotoxicity. Goal of this review article is to describe the molecular alterations associated with these HtrA serine proteases and how these alterations may be associated with tumor behavior and response to chemotherapy. We will also discuss evidence that chemotherapeutic drugs regulate the expression and activation of HtrA serine proteases and that these proteases contributes to programmed cell death. Finally, we will discuss the potential role of epigenetic therapy in targeting the expression and activation of HtrA serine proteases and the mechanisms by which these proteases enhance cytotoxic effect of conventional chemotherapy.
PMCID: PMC3905973  PMID: 19519315
4.  Modulation of Wolframin Expression in Human Placenta during Pregnancy: Comparison among Physiological and Pathological States 
BioMed Research International  2014;2014:985478.
The WFS1 gene, encoding a transmembrane glycoprotein of the endoplasmic reticulum called wolframin, is mutated in Wolfram syndrome, an autosomal recessive disorder defined by the association of diabetes mellitus, optic atrophy, and further organ abnormalities. Disruption of the WFS1 gene in mice causes progressive β-cell loss in the pancreas and impaired stimulus-secretion coupling in insulin secretion. However, little is known about the physiological functions of this protein. We investigated the immunohistochemical expression of wolframin in human placenta throughout pregnancy in normal women and diabetic pregnant women. In normal placenta, there was a modulation of wolframin throughout pregnancy with a strong level of expression during the first trimester and a moderate level in the third trimester of gestation. In diabetic women, wolframin expression was strongly reduced in the third trimester of gestation. The pattern of expression of wolframin in normal placenta suggests that this protein may be required to sustain normal rates of cytotrophoblast cell proliferation during the first trimester of gestation. The decrease in wolframin expression in diabetic placenta suggests that this protein may participate in maintaining the physiologic glucose homeostasis in this organ.
doi:10.1155/2014/985478
PMCID: PMC3920918  PMID: 24588001
5.  Outcomes in Thread Lift for Facial Rejuvenation: a Study Performed with Happy Lift™ Revitalizing 
Dermatology and Therapy  2014;4(1):103-114.
Introduction
Barbed suture lifting is a minimally invasive surgical technique for facial rejuvenation. This study examined the efficacy and associated risks with this procedure, using a new synthetic, monofilament suspension thread named “Happy Lift™ Revitalizing” (Promoitalia International S.r.l, Naples, Italy).
Methods
All the patients had average aging signs and required a lifting of modest degree. A total of 37 thread lifts were performed over a 24-month period.
Results
In the majority of patients (89%), the results obtained were considered satisfactory. The incidence of complications was low. Only 6% of the patients had slight post-operation asymmetry that was easily corrected. Minor complications experienced by patients included small ecchymosis (62%), mild erythema (40%), small hemorrhage (25%), mild transitory esthesia (6%) and mild post-operation tumefaction (40%). Histopathological and ecographic analyses were performed on the treated skin of selected patients, demonstrating that the lifting effect is guaranteed and fortified by the cutaneous reaction that appears along the length of the thread.
Conclusion
Thread lift with “Happy Lift™ Revitalizing” is a safe procedure associated with minor complications, when performed on cohorts of patients requiring a facial lifting of modest degree.
Electronic supplementary material
The online version of this article (doi:10.1007/s13555-014-0041-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s13555-014-0041-6
PMCID: PMC4065274  PMID: 24436079
Barbed sutures; Dermatology; Facial ptosis; Facial rejuvenation; Happy Lift™ Revitalizing; Thread lift
6.  Electrochemotherapy for the treatment of recurring aponeurotic fibromatosis in a dog 
The Canadian Veterinary Journal  2013;54(6):606-609.
This paper reports the clinical findings, histopathology, and clinical outcome of a rare case of aponeurotic fibromatosis in a dog. The dog was treated with 4 courses of electrochemotherapy using the drugs cisplatin and bleomycin. There was complete remission and the dog was still disease-free after 18 months.
PMCID: PMC3659460  PMID: 24155455
7.  SOCS1 gene transfer accelerates the transition to heart failure through the inhibition of the gp130/JAK/STAT pathway 
Cardiovascular Research  2012;96(3):381-390.
Aims
The suppressors of cytokine signalling (SOCS) are identified inhibitors of cytokine and growth factor signalling that act via the Janus kinase (JAK) signal transducers and activators of transcription (STAT) pathways. Aberrant JAK/STAT signalling promotes progression from hypertrophy to heart failure. Little information is available concerning the role of SOCS in the transition from hypertrophy to heart failure. To this aim, we investigated the effects of SOCS1 overexpression obtained by in vivo adeno-associated gene transfer using an aortopulmonary cross-clamping technique in a chronic pressure-overload cardiac rat model.
Methods and results
Rats were randomized into four groups: sham-operated (n = 18), aortic banding (AB) (n = 18), AB + viral vector encoding for haemoagglutinin (AB + HA, n = 16), and AB + viral vector encoding for SOCS1 (AB + SOCS1, n = 18). Echocardiographic and haemodynamic measurements were performed 15 weeks after banding. While SOCS3 was upregulated during the hypertrophic phase, SOCS1 transcript levels increased significantly between 15 and 20 weeks. Remodelling was markedly worse in AB + SOCS1, showed larger left ventricular internal dimensions (+16%), higher end-diastolic pressures (+57%) and wall stress (+45%), and reduced fractional shortening (−32%) compared with AB + HA; apoptotic rate was increased three­fold and the gp130 pathway was inhibited. Ex vivo experiments showed that mechanical stretch upregulated SOCS1 expression, which was in turn attenuated by tumour necrosis factor-α (TNF-α) inhibition.
Conclusion
Enhanced SOCS1 myocardial signalling is associated with accelerated transition from hypertrophy to failure in an established model of pressure overload. SOCS1 may represent an attractive target for the prevention of heart failure progression.
doi:10.1093/cvr/cvs261
PMCID: PMC3732068  PMID: 22875468
SOCS; Gene therapy; Cardiac hypertrophy; Heart failure
8.  The class I-specific HDAC inhibitor MS-275 modulates the differentiation potential of mouse embryonic stem cells 
Biology Open  2013;2(10):1070-1077.
Summary
Exploitation of embryonic stem cells (ESC) for therapeutic use and biomedical applications is severely hampered by the risk of teratocarcinoma formation. Here, we performed a screen of selected epi-modulating compounds and demonstrate that a transient exposure of mouse ESC to MS-275 (Entinostat), a class I histone deacetylase inhibitor (HDAC), modulates differentiation and prevents teratocarcinoma formation. Morphological and molecular data indicate that MS-275-primed ESCs are committed towards neural differentiation, which is supported by transcriptome analyses. Interestingly, in vitro withdrawal of MS-275 reverses the primed cells to the pluripotent state. In vivo, MS275-primed ES cells injected into recipient mice give only rise to benign teratomas but not teratocarcinomas with prevalence of neural-derived structures. In agreement, MS-275-primed ESC are unable to colonize blastocysts. These findings provide evidence that a transient alteration of acetylation alters the ESC fate.
doi:10.1242/bio.20135587
PMCID: PMC3798190  PMID: 24167717
Stem cell; Epigenetic; HDACi
9.  Low-Level Laser Therapy and Vibration Therapy for the Treatment of Localized Adiposity and Fibrous Cellulite 
Dermatology and Therapy  2013;3(1):41-52.
Introduction
In recent years, there has been an upsurge in the application of low-level laser therapy in various medical diseases. Additionally, vibration therapy is a new and effective measure to prevent muscular atrophy and osteoporosis, along with some general health-related beneficial effects of exercise on skeletal muscles such as improvement of endothelial function and an increased enzyme capacity of energy metabolism. The aim of this study was to evaluate the application of a 635 nm and 0.040 W exit power per multiple diode laser in combination with vibration therapy for the application of non-invasive reduction of circumference in patients with localized adiposity and cellulite.
Methods
The study enrolled men and women (N = 33) aged 18–64 years with localized adiposity or fibrous cellulite. The evaluation parameters were: photographic evaluation, perimetric evaluation, blood tests, ecographic evaluation, histological evaluation, and subjective and objective tests.
Results
The results produced were statistically analyzed and resulted in a significant reduction of fat thickness when compared to the measurement prior to the treatment (P < 0.0001). Moreover, subjective and objective tests, as well as ecographic and histological evaluations, confirmed the reduction of fat thickness.
Conclusion
In this study we have demonstrated the safety and efficacy of the combination between low-level laser therapy and vibration therapy for the resolution of localized adiposity and fibrous cellulite.
doi:10.1007/s13555-013-0026-x
PMCID: PMC3680639  PMID: 23888254
Body contouring; Fibrous cellulite; Localized adiposity; Low-level laser therapy; Ultrasound; Vibration therapy
10.  Low-Level Laser Therapy and Vibration Therapy for the Treatment of Localized Adiposity and Fibrous Cellulite 
Dermatology and Therapy  2013;3(1):41-52.
Introduction
In recent years, there has been an upsurge in the application of low-level laser therapy in various medical diseases. Additionally, vibration therapy is a new and effective measure to prevent muscular atrophy and osteoporosis, along with some general health-related beneficial effects of exercise on skeletal muscles such as improvement of endothelial function and an increased enzyme capacity of energy metabolism. The aim of this study was to evaluate the application of a 635 nm and 0.040 W exit power per multiple diode laser in combination with vibration therapy for the application of non-invasive reduction of circumference in patients with localized adiposity and cellulite.
Methods
The study enrolled men and women (N = 33) aged 18–64 years with localized adiposity or fibrous cellulite. The evaluation parameters were: photographic evaluation, perimetric evaluation, blood tests, ecographic evaluation, histological evaluation, and subjective and objective tests.
Results
The results produced were statistically analyzed and resulted in a significant reduction of fat thickness when compared to the measurement prior to the treatment (P < 0.0001). Moreover, subjective and objective tests, as well as ecographic and histological evaluations, confirmed the reduction of fat thickness.
Conclusion
In this study we have demonstrated the safety and efficacy of the combination between low-level laser therapy and vibration therapy for the resolution of localized adiposity and fibrous cellulite.
doi:10.1007/s13555-013-0026-x
PMCID: PMC3680639  PMID: 23888254
Body contouring; Fibrous cellulite; Localized adiposity; Low-level laser therapy; Ultrasound; Vibration therapy
11.  A New Minimally Invasive Mesotherapy Technique for Facial Rejuvenation 
Dermatology and Therapy  2013;3(1):83-93.
Introduction
This study describes a pivotal clinical trial of a new minimally invasive mesotherapy technique for facial rejuvenation.
Methods
The authors utilized two formulations: formulation A with hyaluronic acid, vitamins, amino acids, minerals, coenzymes, and antioxidant substances; formulation B with hyaluronic acid and idebenone. Fifty participants were enrolled in the study and divided in two groups. Group 1 (50–65 years) treated with formulation A. Group 2 (35–50 years) treated with formulation B. The groups underwent four sessions of mesotherapy involving multiple injections. Treatment was conducted at 15 day intervals. All participants had pre- and posttreatment photographs. Punch biopsies were taken from randomly selected participants, baseline and after 6 weeks, and stained for interleukin (IL)-6, IL-1β, matrix metalloproteinase (MMP)-1, and collagen 1. Clinical evaluation was based on the Global Aesthetic Scale (GAIS) and on the Wrinkle Severity Rating Scale (WSRS).
Results
The results produced were statistically analyzed and resulted in a significant and long-lasting effect on facial rejuvenation. Evaluation of photographs at 0, 1, and 2 months revealed significant clinical improvement: brightness, texture, and firmness of the skin. The analysis of the GAIS and WSRS scores in the two groups demonstrated statistically significant results after 2 months. The biopsies taken from randomly selected participants at baseline and after 3 months showed a decrease in IL-1β, IL-6, and MMP1, and an increase in collagen 1.
Conclusion
The new minimally invasive mesotherapy technique described can improve the clinical appearance of the skin in different age groups.
doi:10.1007/s13555-012-0018-2
PMCID: PMC3680640  PMID: 23888258
Biorevitalization; Cosmetic dermatology; Facial rejuvenation; Fillers; Mesotherapy; Soft-tissue augmentation
12.  A New Minimally Invasive Mesotherapy Technique for Facial Rejuvenation 
Dermatology and Therapy  2013;3(1):83-93.
Introduction
This study describes a pivotal clinical trial of a new minimally invasive mesotherapy technique for facial rejuvenation.
Methods
The authors utilized two formulations: formulation A with hyaluronic acid, vitamins, amino acids, minerals, coenzymes, and antioxidant substances; formulation B with hyaluronic acid and idebenone. Fifty participants were enrolled in the study and divided in two groups. Group 1 (50–65 years) treated with formulation A. Group 2 (35–50 years) treated with formulation B. The groups underwent four sessions of mesotherapy involving multiple injections. Treatment was conducted at 15 day intervals. All participants had pre- and posttreatment photographs. Punch biopsies were taken from randomly selected participants, baseline and after 6 weeks, and stained for interleukin (IL)-6, IL-1β, matrix metalloproteinase (MMP)-1, and collagen 1. Clinical evaluation was based on the Global Aesthetic Scale (GAIS) and on the Wrinkle Severity Rating Scale (WSRS).
Results
The results produced were statistically analyzed and resulted in a significant and long-lasting effect on facial rejuvenation. Evaluation of photographs at 0, 1, and 2 months revealed significant clinical improvement: brightness, texture, and firmness of the skin. The analysis of the GAIS and WSRS scores in the two groups demonstrated statistically significant results after 2 months. The biopsies taken from randomly selected participants at baseline and after 3 months showed a decrease in IL-1β, IL-6, and MMP1, and an increase in collagen 1.
Conclusion
The new minimally invasive mesotherapy technique described can improve the clinical appearance of the skin in different age groups.
doi:10.1007/s13555-012-0018-2
PMCID: PMC3680640  PMID: 23888258
Biorevitalization; Cosmetic dermatology; Facial rejuvenation; Fillers; Mesotherapy; Soft-tissue augmentation
13.  Retinal Phenotypes in Patients Homozygous for the G1961E Mutation in the ABCA4 Gene 
Purpose.
We evaluated the pathogenicity of the G1961E mutation in the ABCA4 gene, and present the range of retinal phenotypes associated with this mutation in homozygosity in a patient cohort with ABCA4-associated phenotypes.
Methods.
Patients were enrolled from the ABCA4 disease database at Columbia University or by inquiry from collaborating physicians. Only patients homozygous for the G1961E mutation were enrolled. The entire ABCA4 gene open reading frame, including all exons and flanking intronic sequences, was sequenced in all patients. Phenotype data were obtained from clinical history and examination, fundus photography, infrared imaging, fundus autofluorescence, fluorescein angiography, and spectral domain-optical coherence tomography. Additional functional data were obtained using the full-field electroretinogram, and static or kinetic perimetry.
Results.
We evaluated 12 patients homozygous for the G1961E mutation. All patients had evidence of retinal pathology consistent with the range of phenotypes observed in ABCA4 disease. The latest age of onset was recorded at 64 years, in a patient diagnosed initially with age-related macular degeneration (AMD). Of 6 patients in whom severe structural (with/without functional) fundus changes were detected, 5 had additional, heterozygous or homozygous, variants detected in the ABCA4 gene.
Conclusions.
Homozygous G1961E mutation in ABCA4 results in a range of retinal pathology. The phenotype usually is at the milder end of the disease spectrum, with severe phenotypes linked to the presence of additional ABCA4 variants. Our report also highlights that milder, late-onset Stargardt disease may be confused with AMD.
There is still debate as to the pathogenicity of homozygous G1961E mutation in the ABCA4 gene. We present 12 patients, homozygous for G1961E mutation, with retinal disease. In 6 cases, additional mutations were detected in ABCA4 and tended to yield more severe disease phenotypes.
doi:10.1167/iovs.11-9166
PMCID: PMC3394687  PMID: 22661473
14.  HSulf-1 Modulates FGF-2 and Hypoxia Mediated Migration and Invasion of Breast Cancer Cells 
Cancer research  2011;71(6):2152-2161.
HSulf-1 modulates the sulfation states of heparan sulfate proteoglycans critical for heparin binding growth factor signaling. In the present study, we demonstrate that HSulf-1 is transcriptionally deregulated under hypoxia in breast cancer cell lines. Knockdown of HIF-1α rescued HSulf-1 downregulation imposed by hypoxia, both at the RNA and protein levels. Chromatin immunoprecipitation with HIF-1α and HIF-2α antibodies confirmed recruitment of HIF-α proteins to the two functional hypoxia responsive elements on the native HSulf-1 promoter. HSulf-1 depletion in breast cancer cells resulted in an increased and sustained bFGF2 signaling, promoted cell migration and invasion under hypoxic conditions. Additionally, FGFR2 depletion in HSulf-1 silenced breast cancer cells attenuated hypoxia mediated cell invasion. Immunohistochemical analysis of 53 invasive ductal carcinomas and its autologous metastatic lesions revealed an inverse correlation of expression of HSulf-1 to CAIX in both the primary tumors (p=>0.0198) and in metastatic lesions (p=>0.0067) respectively, by χ2 test. Finally, HSulf-1 expression levels in breast tumors by RNA in situ hybridization showed that high HSulf-1 expression is associated with increased disease-free and overall survival (p= >0.03 and p=>0.0001 respectively). Collectively, these results reveal an important link between loss of HSulf-1 under hypoxic microenvironment and increased growth factor signaling, cell migration and invasion.
doi:10.1158/0008-5472.CAN-10-3059
PMCID: PMC3059381  PMID: 21266348
15.  Lansoprazole as a rescue agent in chemoresistant tumors: a phase I/II study in companion animals with spontaneously occurring tumors 
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.
doi:10.1186/1479-5876-9-221
PMCID: PMC3264547  PMID: 22204495
chemotherapy; lansoprazole; mitoxantrone; carboplatin; proton pump
16.  Tumor Suppressors and Cell-Cycle Proteins in Lung Cancer 
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.
doi:10.4061/2011/605042
PMCID: PMC3189597  PMID: 22007345
17.  Electrochemotherapy with cisplatin enhances local control after surgical ablation of fibrosarcoma in cats: an approach to improve the therapeutic index of highly toxic chemotherapy drugs 
Background
Cancer is one of the most difficult current health challenges, being responsible for millions of deaths yearly. Systemic chemotherapy is the most common therapeutic approach, and the prevailing orientation calls for the administration of the maximum tolerated dose; however, considerable limitations exist including toxicities to healthy tissues and low achievable drug concentrations at tumor sites. Electrochemotherapy (ECT) is a tumor treatment that combines the systemic or local delivery of anticancer drugs with the application of permeabilizing electric pulses. In this article we evaluate the capability of ECT to allow the use of cisplatin despite its high toxicity in a spontaneous feline model of soft tissue sarcoma.
Methods
A cohort of sixty-four cats with incompletely excised sarcomas were treated with cisplatin-based adjuvant ECT and monitored for side effects. Their response was compared to that of fourteen cats treated with surgery alone.
Results
The toxicities were minimal and mostly treated symptomatically. ECT resulted in increased local control (median not reached at the time of writing) with a mean time to recurrence of 666 days versus 180 of controls.
Conclusions
We conclude that ECT is a safe and efficacious therapy for solid tumors; its use may be considered as part of strategies for the reintroduction of drugs with a narrow therapeutic index in the clinical protocols.
doi:10.1186/1479-5876-9-152
PMCID: PMC3182914  PMID: 21917133
18.  Altered Oxido-Reductive State in the Diabetic Heart: Loss of Cardioprotection due to Protein Disulfide Isomerase 
Molecular Medicine  2011;17(9-10):1012-1021.
Diabetes is associated with an increased risk of heart failure, in part explained by endoplasmic reticulum stress and apoptosis. Protein disulfide isomerase (PDI) prevents stressed cardiomyocytes apoptosis. We hypothesized that diabetes impairs PDI function by an alteration in its oxido-reductive state. Myocardial biopsies harvested from the anterolateral left ventricular wall from diabetic (n = 7) and nondiabetic (n = 8) patients were used to assess PDI expression and cardiomyocyte death. A mouse model of diabetes (streptozotocin injection, 130 mg/mL) was used to study PDI expression and its redox state after ischemia/reperfusion injury induced by 30-min occlusion of the left anterior coronary artery followed by reperfusion. Transthoracic echocardiography was performed to assess cardiac remodeling after 1 wk. Western blot analysis was used to analyze PDI expression, and methoxy-polyethyleneglycol-maleimide was used to assess its redox state. Dehydroascorbate (DHA) administration was used to restore the PDI redox state. Diabetic patients had a greater number of transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells than nondiabetic patients despite a greater myocardial PDI expression suggesting altered PDI function. Diabetic mice had a worse postinfarction remodeling associated with an altered PDI redox state. DHA treatment restored functional PDI redox state and ameliorated post–myocardial infarction remodeling. An increase in PDI levels with a paradoxical decrease of its active form occurs in the diabetic heart after ischemia and may explain the lack of protective effects of PDI in diabetes. Restoration of PDI redox state prevents adverse remodeling. The potential significance of these findings deserves to be validated in a clinical setting.
doi:10.2119/molmed.2011.00100
PMCID: PMC3188861  PMID: 21637911
19.  Apoptosis Induced by Piroxicam plus Cisplatin Combined Treatment Is Triggered by p21 in Mesothelioma 
PLoS ONE  2011;6(8):e23569.
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.
doi:10.1371/journal.pone.0023569
PMCID: PMC3157425  PMID: 21858171
20.  Electroporation increases antitumoral efficacy of the bcl-2 antisense G3139 and chemotherapy in a human melanoma xenograft 
Background
Nucleic acids designed to modulate the expression of target proteins remain a promising therapeutic strategy in several diseases, including cancer. However, clinical success is limited by the lack of efficient intracellular delivery. In this study we evaluated whether electroporation could increase the delivery of antisense oligodeoxynucleotides against bcl-2 (G3139) as well as the efficacy of combination chemotherapy in human melanoma xenografts.
Methods
Melanoma-bearing nude mice were treated i.v. with G3139 and/or cisplatin (DDP) followed by the application of trains of electric pulses to tumors. Western blot, immunohistochemistry and real-time PCR were performed to analyze protein and mRNA expression. The effect of electroporation on muscles was determined by histology, while tumor apoptosis and the proliferation index were analyzed by immunohistochemistry. Antisense oligodeoxynucleotides tumor accumulation was measured by FACS and confocal microscopy.
Results
The G3139/Electroporation combined therapy produced a significant inhibition of tumor growth (TWI, more than 50%) accompanied by a marked tumor re-growth delay (TRD, about 20 days). The efficacy of this treatment was due to the higher G3139 uptake in tumor cells which led to a marked down-regulation of bcl-2 protein expression. Moreover, the G3139/EP combination treatment resulted in an enhanced apoptotic index and a decreased proliferation rate of tumors. Finally, an increased tumor response was observed after treatment with the triple combination G3139/DDP/EP, showing a TWI of about 75% and TRD of 30 days.
Conclusions
These results demonstrate that electroporation is an effective strategy to improve the delivery of antisense oligodeoxynucleotides within tumor cells in vivo and it may be instrumental in optimizing the response of melanoma to chemotherapy. The high response rate observed in this study suggest to apply this strategy for the treatment of melanoma patients.
doi:10.1186/1479-5876-9-125
PMCID: PMC3163203  PMID: 21798045
21.  Radiofrequency waves with filling and peeling substances: An innovative minimally invasive technique for facial rejuvenation 
Dermatology and Therapy  2011;1(1):2-10.
Introduction
This study describes a pivotal clinical trial of a new minimally invasive cosmetic procedure for facial rejuvenation and for the treatment of scars and wrinkles.
Methods
The procedure consisted of a combination of techniques such as fillers, biorevitalization, peeling, and intradermal radiofrequency, emitted from a new device denominated by Spherofill Medical Plus (SMP; Spherofill MD, PromoItalia Group S.p.A., Pozzuoli, Italy), for treating cutaneous regeneration, depressions, and striae. One hundred and twelve patients, divided into five groups, were treated.
Results
The results produced were statistically analyzed and resulted in significant and long-lasting effects for facial rejuvenation. Indeed, the analysis of the Global Aesthetic Improvement Scale (GAIS) scores in the five groups demonstrated statistically significant results between 3 and 9 months after the treatments.
Conclusion
Evaluating the patients included in the study, it is possible to conclude that the treatment with SMP represents a safe and efficient solution for the treatment of wrinkles, acne lesions, striae, and of degenerated tissues caused by aging.
doi:10.1007/s13555-011-0001-3
PMCID: PMC3437644  PMID: 22984657
biorevitalization; facial rejuvenation; fillers; peeling; radiofrequency; soft-tissue augmentation
22.  Radiofrequency waves with filling and peeling substances: An innovative minimally invasive technique for facial rejuvenation 
Dermatology and Therapy  2011;1(1):2-10.
Introduction
This study describes a pivotal clinical trial of a new minimally invasive cosmetic procedure for facial rejuvenation and for the treatment of scars and wrinkles.
Methods
The procedure consisted of a combination of techniques such as fillers, biorevitalization, peeling, and intradermal radiofrequency, emitted from a new device denominated by Spherofill Medical Plus (SMP; Spherofill MD, PromoItalia Group S.p.A., Pozzuoli, Italy), for treating cutaneous regeneration, depressions, and striae. One hundred and twelve patients, divided into five groups, were treated.
Results
The results produced were statistically analyzed and resulted in significant and long-lasting effects for facial rejuvenation. Indeed, the analysis of the Global Aesthetic Improvement Scale (GAIS) scores in the five groups demonstrated statistically significant results between 3 and 9 months after the treatments.
Conclusion
Evaluating the patients included in the study, it is possible to conclude that the treatment with SMP represents a safe and efficient solution for the treatment of wrinkles, acne lesions, striae, and of degenerated tissues caused by aging.
doi:10.1007/s13555-011-0001-3
PMCID: PMC3437644  PMID: 22984657
biorevitalization; facial rejuvenation; fillers; peeling; radiofrequency; soft-tissue augmentation
23.  Right Ventricular Cardiomyocyte Apoptosis in Patients with Acute Myocardial Infarction of the left Ventricular Wall 
The American journal of cardiology  2008;102(6):658-662.
Cardiac remodeling after acute myocardial infarction (AMI) is characterised by molecular and cellular mechanisms involving both left (LV) and right ventricular (RV) walls. Cardiomyoycte apoptosis in the peri-infarct and remote LV myocardium plays a central role in cardiac remodeling. Whether apoptosis also occurs in the right ventricle of patients with ischemic heart disease has not been investigated. Aim of the current study was to investigate the presence of cardiomyocyte apoptosis in the right ventricle in patients with AMI. We assessed the number of apoptotic cardiomyocytes by multiple samplings in the LV and RV walls of 12 patients selected at autopsy who died 4 to 42 days after AMI. Five patients without cardiac disease were also selected at autopsy as controls. Apoptotic rates were calculated from the number of cardiomyocytes showing double positive staining for in situ end-labeling of DNA fragmentation – TUNEL – and for activated caspase-3. Potentially false positive results (DNA synthesis and RNA splicing) were excluded from the cell counts. The apoptotic rate in the RV in patients with AMI was significantly higher than in control hearts (0.8% [0.3–1.0] vs 0.01% [0.01–0.03], P<0.001). RV apoptosis was significantly correlated with parameters of global adverse remodeling such as cardiac diameter-to-LV free wall thickness (R=+0.57, P=0.050). RV apoptosis was significantly higher in 5 cases (42%) with infarct involving the ventricular septum and an adjacent a small area of the RV walls (1.0% [0.8–2.2] vs 0.5% [0.2–1.0], P=0.048; P<0.001 vs controls). The association between apoptotic rate in RV and cardiac remodeling was apparent even after exclusion of cases with RV AMI involvement (R=+0.82, P=0.023 for diameter-to-LV wall thickness ratio; and R=−0.91, P=0.002 for RV free wall thickness). In conclusion, patients with cardiac remodeling after AMI have a significant increase in RV apoptosis even when ischemic involvement of the RV wall is not apparent.
doi:10.1016/j.amjcard.2008.05.007
PMCID: PMC3021317  PMID: 18773983
apoptosis; heart failure; right ventricle; myocardial infarction; remodeling
24.  Expression of apoptosis‐related markers in malignant epithelial tumours of the lacrimal gland and their relation to clinical outcome 
The British Journal of Ophthalmology  2007;91(9):1239-1243.
Objective
To investigate the correlation between the expression of apoptosis‐related markers and prognosis in malignant epithelial tumours of the lacrimal gland.
Materials and Methods
Series of cases.
Participants
Twenty one cases with malignant epithelial tumours of the lacrimal gland. Histological diagnosis was re‐examined and blocks selected were evaluated for the following parameters: incidence of apoptosis with TUNEL assay, expression of p53 and Bcl‐2 using monoclonal antibody. Predictors factors for survival, local recurrence and cumulative probability of death were statistically evaluated.
Results
Re‐eximination of the 21 specimens was as follow: 11 adenoid cystic carcinomas, 4 mucoepidermoid carcinomas, 3 squamous cell carcinomas and 3 adenocarcinomas. Eleven of the 21 patients (53%) died during the follow‐up period (4–192 months; mean 71). Bcl‐2 staining >6% was significantly correlated with the death of patients. A statistically significant positive relationship for TUNEL and p53, and an inverse correlation for Bcl‐2 staining, was demonstrated with overall survival.
Conclusion
The correlation with survival of apoptotic index, p53 and Bcl‐2 expression suggest the more tumour cells go in apoptosis, upregulating p53 and down‐regulating Bcl‐2, the better the survival of patients. This study establishes a role of apoptosis‐regulatory proteins in the pathogenesis of malignant epithelial lacrimal gland tumours, and supports the hypothesis that evaluation of the expression of apoptosis‐related markers in these tumours may provide a prognostic tool.
doi:10.1136/bjo.2007.118661
PMCID: PMC1954920  PMID: 17431014
25.  Molecular, cellular and physiological characterization of the cancer cachexia-inducing C26 colon carcinoma in mouse 
BMC Cancer  2010;10:363.
Background
The majority of cancer patients experience dramatic weight loss, due to cachexia and consisting of skeletal muscle and fat tissue wasting. Cachexia is a negative prognostic factor, interferes with therapy and worsens the patients' quality of life by affecting muscle function. Mice bearing ectopically-implanted C26 colon carcinoma are widely used as an experimental model of cancer cachexia. As part of the search for novel clinical and basic research applications for this experimental model, we characterized novel cellular and molecular features of C26-bearing mice.
Methods
A fragment of C26 tumor was subcutaneously grafted in isogenic BALB/c mice. The mass growth and proliferation rate of the tumor were analyzed. Histological and cytofluorometric analyses were used to assess cell death, ploidy and differentiation of the tumor cells. The main features of skeletal muscle atrophy, which were highlighted by immunohistochemical and electron microscopy analyses, correlated with biochemical alterations. Muscle force and resistance to fatigue were measured and analyzed as major functional deficits of the cachectic musculature.
Results
We found that the C26 tumor, ectopically implanted in mice, is an undifferentiated carcinoma, which should be referred to as such and not as adenocarcinoma, a common misconception. The C26 tumor displays aneuploidy and histological features typical of transformed cells, incorporates BrdU and induces severe weight loss in the host, which is largely caused by muscle wasting. The latter appears to be due to proteasome-mediated protein degradation, which disrupts the sarcomeric structure and muscle fiber-extracellular matrix interactions. A pivotal functional deficit of cachectic muscle consists in increased fatigability, while the reported loss of tetanic force is not statistically significant following normalization for decreased muscle fiber size.
Conclusions
We conclude, on the basis of the definition of cachexia, that ectopically-implanted C26 carcinoma represents a well standardized experimental model for research on cancer cachexia. We wish to point out that scientists using the C26 model to study cancer and those using the same model to study cachexia may be unaware of each other's works because they use different keywords; we present strategies to eliminate this gap and discuss the benefits of such an exchange of knowledge.
doi:10.1186/1471-2407-10-363
PMCID: PMC2912868  PMID: 20615237

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