Rosario Caltabiano

The Role of Intrinsic Pathway in Apoptosis Activation and Progression in Peyronie's Disease

Peyronies disease (PD) is characterized with formation of fibrous plaques which result in penile deformity, pain, and erectile dysfunction. The aim of this study was to investigate the activation of the intrinsic apoptotic pathway in plaques from PD patients. Tunica albuginea from either PD or control patients was assessed for the expression of bax, bcl-2 and caspases 9 and 3 using immunohistochemistry and by measurement of apoptotic cells using TUNEL assay. Bax overexpression was observed in metaplastic bone tissue, in fibroblasts, and in myofibroblast of plaques from PD patients. Little or no bcl-2 immunostaining was detected in samples from either patients or controls. Caspase 3 immunostaining was very strong in fibrous tissue, in metaplasic bone osteocytes, and in primary ossification center osteoblasts. Moderate caspase 9 immunostaining was seen in fibrous cells plaques and in osteocytes and osteoblasts of primary ossification centers from PD patients. Control samples were negative for caspase 9 immunostaining. In PD patients the TUNEL immunoassay showed intense immunostaining of fibroblasts and myofibroblasts, the absence of apoptotic cells in metaplasic bone tissue and on the border between fibrous and metaplastic bone tissue. Apoptosis occurs in stabilized PD plaques and is partly induced by the intrinsic pathway.

Specific Alterations of MicroRNA Transcriptome and Global Network Structure in Colorectal Carcinoma after Cetuximab Treatment

The relationship between therapeutic response and modifications of microRNA (miRNA) transcriptome in colorectal cancer (CRC) remains unknown. We investigated this issue by profiling the expression of 667 miRNAs in 2 human CRC cell lines, one sensitive and the other resistant to cetuximab (Caco-2 and HCT-116, respectively), through TaqMan real-time PCR. Caco-2 and HCT-116 expressed different sets of miRNAs after treatment. Specifically, 21 and 22 miRNAs were differentially expressed in Caco-2 or HCT-116, respectively (t test, P < 0.01). By testing the expression of differentially expressed miRNAs in CRC patients, we found that miR-146b-3p and miR-486-5p are more abundant in K-ras–mutated samples with respect to wild-type ones (Wilcoxon test, P < 0.05). Sixty-seven percent of differentially expressed miRNAs were involved in cancer, including CRC, whereas 19 miRNA targets had been previously reported to be involved in the cetuximab pathway and CRC. We identified 25 transcription factors putatively controlling these miRNAs, 11 of which have been already reported to be involved in CRC. On the basis of these data, we suggest that the downregulation of let-7b and let-7e (targeting K-ras) and the upregulation of miR-17* (a CRC marker) could be considered as candidate molecular markers of cetuximab resistance. Global network functional analysis (based on miRNA targets) showed a significant overrepresentation of cancer-related biological processes and networks centered on critical nodes involved in epidermal growth factor receptor internalization and ubiquitin-mediated degradation. The identification of miRNAs, whose expression is linked to the efficacy of therapy, should allow the ability to predict the response of patients to treatment and possibly lead to a better understanding of the molecular mechanisms of drug response. Mol Cancer Ther; 9(12); 3396–409.

Specific alterations of the microRNA transcriptome and global network structure in colorectal cancer after treatment with MAPK/ERK inhibitors

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has a master control role in various cancer-related biological processes as cell growth, proliferation, differentiation, migration, and apoptosis. It also regulates many transcription factors that control microRNAs (miRNAs) and their biosynthetic machinery. To investigate on the still poorly characterised global involvement of miRNAs within the pathway, we profiled the expression of 745 miRNAs in three colorectal cancer (CRC) cell lines after blocking the pathway with three different inhibitors. This allowed the identification of two classes of post-treatment differentially expressed (DE) miRNAs: (1) common DE miRNAs in all CRC lines after treatment with a specific inhibitor (class A); (2) DE miRNAs in a single CRC line after treatment with all three inhibitors (class B). By determining the molecular targets, biological roles, network position of chosen miRNAs from class A (miR-372, miR-663b, miR-1226*) and class B (miR-92a-1*, miR-135b*, miR-720), we experimentally demonstrated that they are involved in cell proliferation, migration, apoptosis, and globally affect the regulation circuits centred on MAPK/ERK signaling. Interestingly, the levels of miR-92a-1*, miR-135b*, miR-372, miR-720 are significantly higher in biopsies from CRC patients than in normal controls; they also are significantly higher in CRC patients with mutated KRAS than in those with wild-type genotypes (Wilcoxon test, p < 0.05): the latter could be a downstream effect of ERK pathway overactivation, triggered by KRAS mutations. Finally, our functional data strongly suggest the following miRNA/target pairs: miR-92a-1*/PTEN-SOCS5; miR-135b*/LATS2; miR-372/TXNIP; miR-663b/CCND2. Altogether, these results contribute to deepen current knowledge on still uncharacterized features of MAPK/ERK pathway, pinpointing new oncomiRs in CRC and allowing their translation into clinical practice and CRC therapy.

MMP-13 (collagenase 3) localization in human temporomandibular joint discs with internal derangement.

The normal articular disc of the human temporomandibular joint (TMJ) consists of regularly arranged collagen fibers and fibroblast-like cells. Disc tissue is subjected to various types of loading. Adaptive remodelling entails microscopic changes in which different types of collagen and non-collagen proteins, as well as matrix metalloproteinases (MMPs), are involved. Collagenase-3 (MMP-13) seems to play a key role. The aim of the study was to evaluate its involvement in TMJ disc histopathology. We studied 10 discs from patients with arthropathy of the TMJ. Paraffin wax-embedded sections were processed for histological and immunohistochemical analysis. A well-characterized mouse monoclonal anti-MMP-13 antibody (anti-collagenase 3) was used. The results showed increased MMP-13 immunoreactivity in TMJ diseased disc tissue was correlated with the severity of the histopathological changes. The more severe the pathological changes, the greater the number of immunolabelled cells detected. Nearly all chondrocyte-like cells were immunopositive, whereas fibroblast-like cells and fibrochondrocytes were more rarely labelled. MMP-13 upregulation in disc tissue from patients with TMJ internal derangement suggests that MMP-13 is involved in disc histopathology, and that disc cells actively participate in the synthesis of extracellular matrix-degrading proteinases.

BENEFICIAL EFFECTS OF RUTIN AND L-ARGININE COADMINISTRATION IN A RAT MODEL OF LIVER ISCHEMIA/REPERFUSION INJURY

Reperfusion following liver ischemia results in oxidative stress leading to liver injury. The aim of this study was to investigate the combined effects of two antioxidant agents, rutin and L-arginine, in rat liver ischemia-reperfusion (I/R). Male Wistar rats were divided into five groups: 1) sham operated, 2) I/R, 3) I/R+rutin, 4) I/R+L-arginine, and 5) I/R+rutin+L-arginine. Plasmatic and hepatic levels of alanine transaminase (ALT), aspartate transaminase (AST), lipid peroxides (LOOH), and thiol groups (RSH) were examined, as well as DNA fragmentation and liver histopathology. Furthermore, to elucidate the pathophysiological processes involved in the antioxidant mechanism(s) of rutin and L-arginine, we assessed the expression of inducible (iNOS) and endothelial nitric oxide synthase (eNOS) isoforms and heme oxygenase-1 (HO-1), both playing key roles in the biochemical cascade of liver injury. Significant increase in plasmatic ALT and AST activities were observed in untreated I/R rats compared with sham-operated animals, whereas treatment with rutin or L-arginine in I/R rats reduced hepatic damage. Interestingly, combined therapy with rutin and L-arginine resulted in a further reduction of plasmatic ALT and AST activities compared with rutin or L-arginine alone. These results were further confirmed by the analysis of DNA fragmentation, LOOH, RSH groups, and liver histopathology, which showed the highest protective effects following the coadministration of rutin and L-arginine. Finally, the combined therapy protocol resulted in a significant induction of liver HO-1 and a concomitant reduction of iNOS expression that may both be responsible for the beneficial effects of the proposed pharmacological protocol.

miRNA profiling in vitreous humor, vitreal exosomes and serum from uveal melanoma patients: Pathological and diagnostic implications.

Uveal melanoma (UM) represents approximately 5–6% of all melanoma diagnoses and up to 50% of patients succumb to their disease. Although several methods are available, accurate diagnosis is not always easily feasible because of potential accidents (e.g., intraocular hemorrhage). Based on the assumption that the profile of circulating miRNAs is often altered in human cancers, we verified whether UM patients showed different vitreous humor (VH) or serum miRNA profiles with respect to healthy controls. By using TaqMan Low Density Arrays, we analyzed 754 miRNAs from VH, vitreal exosomes, and serum of 6 UM patients and 6 healthy donors: our data demonstrated that the UM VH profile was unique and only partially overlapping with that from serum of the same patients. Whereas, 90% of miRNAs were shared between VH and vitreal exosomes, and their alterations in UM were statistically overlapped with those of VH and vitreal exosomes, suggesting that VH alterations could result from exosomal dysregulation. We report 32 miRNAs differentially expressed in UM patients in at least 2 different types of samples analyzed. We validated these data on an independent cohort of 12 UM patients. Most alterations were common to VH and vitreal exosomes (e.g., upregulation of miR-21,-34 a,-146a). Interestingly, miR-146a was upregulated in the serum of UM patients, as well as in serum exosomes. Upregulation of miR-21 and miR-146a was also detected in formalin-fixed, paraffin-embedded UM, suggesting that VH or serum alterations in UM could be the consequence of disregulation arising from tumoral cells. Our findings suggest the possibility to detect in VH and serum of UM patients “diagnostic” miRNAs released by the affected eye: based on this, miR-146a could be considered a potential circulating marker of UM.

A histochemical survey of the human temporomandibular joint disc of patients with internal derangement without reduction.

This study describes, microscopically, the pathologic findings correlated to malpositioning of the human temporomandibular joint disc. The specimens studied consisted of 21 discs removed from patients affected by temporomandibular joint arthropathy. The specimens were cut longitudinally and were fixed overnight in 10% neutral-buffered formalin. They were embedded in paraffin with anatomic orientation preserved. Three- to 4-μm thick sections were cut according to routine procedures and mounted on slides. Then they were stained with Goldner-modified-Mallory staining used for morphologic study at light microscopy. Abnormal collagen fiber arrangements, fragmentation of collagen fibrils, new vessel formation, meniscal tears, mucoid degeneration of the disc matrix, chondrocyte-like cells proliferation, sometimes with clonal aggregation, and hyalinization were detected in temporomandibular joint disc specimens with internal derangement, although with a different extent. From our observations, it seems that in most cases, the temporomandibular joint disc responded to internal derangement without reduction with a fragmentation of collagen fibers because hyalinization of disc matrix occurred very rarely. Moreover, disc derangement without reduction is characterized also by a change in cell population phenotype.

Topical imiquimod treatment of superficial and nodular basal cell carcinomas in patients affected by basal cell nevus syndrome: a preliminary report

BACKGROUND: Imiquimod 5% cream has been shown to be effective in the treatment of superficial basal cell carcinomas (sBCCs). OBJECTIVE: To evaluate the efficacy, safety and compliance of imiquimod 5% cream for the treatment of sBCCs and nodular BCCs (nBCCs) in patients affected by basal cell nevus syndrome. PATIENTS/METHODS: Three patients (two male, one female) were enrolled in the study. Nine tumors (five sBCCs and four nBCCs), all ranging in size from 0.5 cm to 1 cm, were treated. Treatment consisted for sBCCs of three weekly applications and for nBCCs of five weekly applications for 8 weeks. Histological examination was performed at the beginning and at the end of the study. For small tumors, similar-appearing lesions were removed for baseline histological confirmation. Follow-up was performed at 1-week intervals in order to carefully detect any change. RESULTS: Three sBCCs cleared clinically after 4 weeks of treatment and two nBCCs after 8 weeks. The remaining four lesions showed excellent clinical responses with evident (>50%) size reduction at 6 weeks, but no further improvement. Histological examination with multiple-step sections confirmed complete clearing for those lesions showing clinical resolution, except for one nBCC that showed scant tumor remnants. Local adverse effects (itching, erythema and bleeding) were mild and did not prompt discontinuation of treatment. No systemic side effects were noted. CONCLUSIONS: Imiquimod 5% cream is an effective therapeutic option for both sBCC and nBCC in patients with basal cell nevus syndrome. The treatment was very well received by all patients engaged in the study, who stated their appreciation for a topical treatment rather than multiple surgical excisions. A higher number of applications and longer treatment periods are required for nBCCs.

The natural history of spinal neurofibromatosis: a critical review of clinical and genetic features.

Spinal neurofibromatosis (SNF) is a related form of neurofibromatosis 1 (NF1), characterized by bilateral neurofibromas (histologically proven) of all spinal roots (and, eventually, of all the major peripheral nerve branches) with or without other manifestations of classical NF1. By rigorous application of these criteria to the 98 SNF cases published, we developed: (i) a cohort of 49 SNF patients (21 males and 28 females; aged 4–74 years]: 9 SNF families (21/49), 1 mixed SNF/NF1 family (1/49) and 27 of 49 sporadic SNF patients (including 5 unpublished patients in this report); and (ii) a group of 49 non‐SNF patients including: (a) 32 patients with neurofibromas of multiple but not all spinal roots (MNFSR): 4 mixed SNF/MNFSR families (6/32); (b) 14 patients with NF1 manifestations without spinal neurofibromas, belonging to SNF (8/49) or MNFSR families (6/32); (c) 3 patients with neurofibromas in one spinal root. In addition to reduced incidence of café‐au‐lait spots (67% in SNF vs 56% in MNFSR), other NF1 manifestations were less frequent in either cohort. Molecular testing showed common NF1 gene abnormalities in both groups. The risk of developing SNF vs NF1 was increased for missense mutations [p = 0.0001; odds ratio (OR) = 6.16; confidence interval (CI) = 3.14–13.11], which were more frequent in SNF vs MNFSR (p = 0.0271).

Dysregulated miR-671-5p / CDR1-AS / CDR1 / VSNL1 axis is involved in glioblastoma multiforme

MiR-671-5p is encoded by a gene localized at 7q36.1, a region amplified in human glioblastoma multiforme (GBM), the most malignant brain cancer. To investigate whether expression of miR-671-5p were altered in GBM, we analyzed biopsies from a cohort of forty-five GBM patients and from five GBM cell lines. Our data show significant overexpression of miR-671-5p in both biopsies and cell lines. By exploiting specific miRNA mimics and inhibitors, we demonstrated that miR-671-5p overexpression significantly increases migration and to a less extent proliferation rates of GBM cells. Through a combined in silico and in vitro approach, we identified CDR1-AS, CDR1, VSNL1 as downstream miR-671-5p targets in GBM. Expression of these genes significantly decreased both in GBM biopsies and cell lines and negatively correlated with that of miR-671-5p. Based on our data, we propose that the axis miR-671-5p / CDR1-AS / CDR1 / VSNL1 is functionally altered in GBM cells and is involved in the modification of their biopathological profile.