Management and prognostic factors of recurrent pleomorphic adenoma of the parotid gland: personal experience and review of the literature

The aim of this study was to investigate the management and prognostic determinants of recurrent pleomorphic adenoma (RPA). A retrospective analysis was performed to examine the clinical features, the prevalence of surgical complications, and new recurrences of RPA. Tumor recurrence rate was estimated by the Kaplan–Meier method, and the prognostic value of some of the variables was tested by univariate analysis using the log rank test. The study focused on 33 patients, 18 female (54.5%) and 15 male (45.5%), aged 12–71 years (median 41). A total or extended total parotidectomy was performed in 16 cases (48.5%), a superficial parotidectomy in 10 cases (30.3%), and a local excision in 7 cases (21.2%). In ten patients (30.3%), a branch or the trunk of the facial nerve was deliberately sacrificed. Major complications included one unexpected definitive paralysis of the marginal mandibular branch of the facial nerve and 14 cases of Frey syndrome. Follow-up varied from 2 to 25 years (median 10.5 years), and there were 11 new recurrences (33.3%) within a period varying from 1 to 16 years (median 6 years). The estimated tumor recurrence rates were 14.1 ± 6.6% at 5 years, 31.4 ± 9.4% at 10 years, 43.0 ± 10.8% at 15 years, and 57.2 ± 14.8% at 20 years. Presence of a multinodular lesion and the type of intervention performed were significantly associated with a higher probability of recurrence. RPAs are prone to new recurrences, especially when multinodular and treated with a local excision. Surgical treatment should include facial nerve resection in selected cases. Follow-up for the patient’s lifetime is warranted

Suppression of adenine nucleotide translocase-2 by vector-based siRNA in human breast cancer cells induces apoptosis and inhibits tumor growth in vitro and in vivo

INTRODUCTION: Adenine nucleotide translocator (ANT) 2 is highly expressed in proliferative cells, and ANT2 induction in cancer cells is known to be directly associated with glycolytic metabolisms and carcinogenesis. In addition, ANT2 repression results in the growth arrest of human cells, implying that ANT2 is a candidate for cancer therapy based on molecular targeting. METHODS: We utilized an ANT2-specific RNA interference approach to inhibit ANT2 expression for evaluating its antitumor effect in vitro and in vivo. Specifically, to investigate the therapeutic potential of ANT2 repression, we used a DNA vector-based RNA interference approach by expressing shRNA to knockdown ANT2 in breast cancer cell lines overexpressing ANT2. RESULTS: ANT2 shRNA treatment in breast cancer cell line MDA-MB-231 repressed cell growth as well as proliferation. In addition, cell cycle arrest, ATP depletion and apoptotic cell death characterized by the potential disruption of mitochondrial membrane were observed from the ANT2 shRNA-treated breast cancer cells. Apoptotic breast cancer cells transfected with ANT2 shRNA also induced a cytotoxic bystander effect that generates necrotic cell death to the neighboring cells. The intracellular levels of TNFalpha and TNF-receptor I were increased in ANT2 shRNA transfected cells and the bystander effect was partly blocked by anti-TNFalpha antibody. Ultimately, ANT2 shRNA effectively inhibited tumor growth in vivo. CONCLUSION: These results suggest that vector-based ANT2 RNA interference could be an efficient molecular therapeutic method for breast cancer with high expression of ANT2.This work was supported in part by the grants from the Cancer Research Center, and the Korean Science & Engineering Foundation through the Tumor Immunity Medical Research Center at Seoul National University College of Medicine

In Vivo Analysis of the Role of O-Glycosylations of Von Willebrand Factor

The objective of this project was to study the function of O-glycosylations in von Willebrand factor (VWF) life cycle. In total, 14 different murine Vwf cDNAs mutated on one or several O-glycosylations sites were generated: 9 individual mutants, 2 doublets, 2 clusters and 1 mutant with all 9 murine glycosylation sites mutated (Del-O-Gly). We expressed each mutated cDNA in VWF deficient-mice by hydrodynamic injection. An immunosorbent assay with Peanut Agglutinin (PNA) was used to verify the O-glycosylation status. Wild-type (WT) VWF expressed by hepatocytes after hydrodynamic injection was able to bind PNA with slightly higher affinity than endothelial-derived VWF. In contrast, the Del-O-Gly VWF mutant did not bind PNA, demonstrating removal of O-linked glycans. All mutants displayed a normal multimeric pattern. Two mutants, Del-O-Gly and T1255A/T1256A, led to expression levels 50% lower than those induced by WT VWF and their half-life in vivo was significantly reduced. When testing the capacity of each mutant to correct the bleeding time of VWF-deficient mice, we found that S1486A, T1255A, T1256A and the doublet T1255A/T1256A were unable to do so. In conclusion we have shown that O-glycosylations are dispensable for normal VWF multimerization and biosynthesis. It also appears that some O-glycosylation sites, particularly the T1255 and T1256 residues, are involved in the maintenance of VWF plasma levels and are essential for normal haemostasis. As for the S1486 residue, it seems to be important for platelet binding as demonstrated in vitro using perfusion experiments

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field