Laura Aimati

Alteration of expression levels of the oxidative phosphorylation system (OXPHOS) in breast cancer cell mitochondria

Mitochondria are dynamic intracellular organelles playing a central role in cell metabolism by generating ATP, through the oxidative phosphorylation system (OXPHOS). Altered mitochondrial functions have been identified as causative or contributing factors in some degenerative diseases and are becoming crucial to understanding cancer mechanisms. We report on distinct expression differences between mitochondria of normal and breast-infiltrating ductal carcinoma (IDC) cells. Mitochondria isolated from HMC (human mammary carcinoma) and HMEC (human mammary epithelial cell) cultures were assayed for expression levels of the multi-protein OXPHOS complexes using Western blot and densitometric analyses. Depressed expression levels were detected for all HMC OXPHOS complexes. Drastic signal reduction was observed for the succinate-dehydrogenase complex II iron-sulphur protein SDH-B (3.38%), while decreasing was reported for the NADH-ubiquinone oxidoreductase complex I Fe-S protein 3 NDUFS3 (32.78%) and the ubiquinol-cytochrome c reductase complex III protein 2 UQCRC2 (50.34%). A significant signal dropping was detected for the ATP-synthase complex V F1β subunit (18.07%). For the cytochrome-oxidase complex IV (CO), near-depletion of the mitochondrial-encoded COI (4.37%) and no apparent variation of the COIV (97.26%) subunits were observed. CO and ATP-synthase were also assayed by cryo-immunoelectron microscopy (CIEM) on unfractionated HMC and HEMC cell mitochondria. COI and F1β differential expression, invariance of COIV levels were corroborated, while HMC mitochondria morphology deterioration was highlighted. MitoTracker Red and fluorescence immunolabelling merging confirmed CIEM data. MitoTracker Red and Green co-staining showed mitochondria membrane property modulation. These data describe bioenergetic and phenotypic alterations of IDC cell mitochondria, possibly providing new cancer hallmarks.

Differential response to keratinocyte growth factor receptor and epidermal growth factor receptor ligands of proliferating and differentiating intestinal epithelial cells

The expression of the keratinocyte growth factor receptor (KGFR) has been analyzed on intestinal epithelial Caco‐2 cells upon confluence‐induced spontaneous differentiation. Western blot and immunofluorescence analysis showed that the expression of functional KGFRs, differently from that of epidermal growth factor receptor (EGFR), was up‐modulated in post‐confluent differentiated cultures compared with the pre‐confluent cells. Confocal microscopy and immunoelectron microscopy revealed that the up‐regulated KGFRs displayed a basolateral polarized distribution on the cell surfaces in the monolayer. In vivo immunohistochemical analysis on normal human colon tissue sections showed that KGFRs, differently from EGFRs, were mostly distributed on the more differentiated cells located on the upper portion of the intestinal crypt. Bromodeoxyuridine incorporation assay and Ki67 labeling indicated that the differentiated cells were able to proliferate in response to the two ligands of KGFR, KGF and FGF‐10, whereas they were not stimulated by the EGFR ligands TGFα and EGF. Western blot and quantitative immunofluorescence analysis of the expression of carcinoembryonic antigen (CEA) in post‐confluent cells revealed that incubation with KGF induced an increase of cell differentiation. Taken together these results indicate that up‐modulation of KGFR may be required to promote proliferation and differentiation in differentiating cells and that, among the cells componing the intestinal epithelial monolayer, the target cells for KGFR ligands appear to be different during differentiation from those responsive to EGFR ligands.

Endocytic pathways and biological effects induced by UVB-dependent or ligand-dependent activation of the keratinocyte growth factor receptor

UVB exposure of epidermal cells is known to trigger early and late molecular pathways dependent on receptor tyrosine kinases and reactive oxygen species (ROS). We have recently reported that UVB irradiation induces tyrosine phosphorylation, kinase activation, and internalization of the receptor for the keratinocyte growth factor (KGFR), a paracrine mediator of epithelial growth, differentiation, and survival. Here we analyzed in more detail the UVB‐induced endocytic pathway of KGFR and the role of KGFR activation and internalization in regulating UVB‐promoted apoptosis and cell cycle arrest. Immunogold electron microscopy and confocal analysis revealed that the UVB‐induced endocytosis of KGFR occurs through clathrin‐coated pits and that the internalized receptors are sorted to the degradative route and reach the lysosomal compartment with a timing similar to that induced by their ligand KGF. Treatment with the anti‐oxidant Nacetylcysteine inhibited KGFR endocytosis, suggesting that the receptor internalization is mediated by the intracellular production of ROS. The ligand‐independent KGFR endocytic pathway induced by UVB requires receptor kinase activity and tyrosine phosphorylation and involves transient receptor ubiquitination. Inhibition of KGFR activity reduces both the KGF‐mediated proliferative response and the UVB‐promoted apoptotic cell death, indicating a different effect of ligand‐induced and UVB‐induced KGFR triggering. In addition, receptor internalization leads to protection from apoptosis caused by UVB exposure. Finally, we compared directly the behavior of KGFR with that of the epidermal growth factor receptor (EGFR) upon UVB exposure. Surprisingly, biochemical and immunofluorescence analysis showed that EGFR, differently from KGFR, does not undergo UVB‐induced tyrosine phosphorylation and internalization. Taken together, our results suggest a differential role of KGFR and EGFR in the response of epidermal cells to UVB possibly because KGFR endocytosis could be crucial for attenuation of survival signals in the suprabasal layers of human skin.

Preliminary evidences on mitochondrial injury and impaired oxidative metabolism in breast cancer

Mitochondriopathy is emerging as a new cancer theory; however, the relevance of mitochondrial pathobiology in breast cancer has not yet been completely explored. Herein we report on altered expression levels of the oxidative phosphorylation system (OXPHOS) subunits, mitochondrial structural injury and impaired ATP content from a breast-infiltrating ductal carcinoma (IDC). With this purpose, a human mammary carcinoma (HMC-1) cell, referred to a human mammary epithelial cell (HMEC) line, was assayed for: a) OXPHOS levels by quantitative cryo-immunoelectron microscopy (CIEM) labeling; b) morphological characterization by a newly introduced damage grading (scale Mt-g1-3), calculated on the % of intact cristae carrying mitochondria; c) bioenergetic impairment by luminometric determinations of cellular ATP content and cytochemical visualization of COX activity. Drastic OXPHOS reduction was observed in HMC-1 cells for the succinate-dehydrogenase complex II SDH-B protein, while decreasing was reported for the NADH-ubiquinone oxidoreductase complex I NDUFS3 and the ubiquinol cytochrome c reductase complex III UQCRC2 subunits. A significant dropping was detected for the ATP-synthase complex V F1β protein. For the COX complex near-depletion of the mitochondrial-encoded COXI and no apparent variation of the COXIV subunits were observed. Injury grading was categorized assigning three levels of morphological damage in HMC-1 mitochondria: i) severe (4.6%), ii) moderate (23.1%), iii) slight (44.6%), corresponding to 0%, 1-50% and 51-75% of area occupied by intact cristae. ATP generation and COX activity appeared significantly reduced in HMC-1 cells. The structural damage grading here described could provide new insight on IDC mitochondrial impairment and represent hallmark in the breast cancer mitochondriopathy.

Nickel-induced keratinocyte proliferation and up-modulation of the keratinocyte growth factor receptor expression

Abstract: Keratinocytes play a key role in the pathogenesis of allergic contact dermatitis (ADC) induced by the sensitizing agent nickel. We analyzed here the effects of treatment with nickel and of the pretreatment with zinc on HaCaT cells and primary human keratinocytes. Cell counting, 5‐bromo‐2′‐deoxyuridine incorporation assay and adenosine triphosphate (ATP) bioluminescence detection showed that treatment with NiSO4 induced DNA synthesis and cell proliferation and that pretreatment with ZnSO4 was able to abrogate this proliferative effect. This nickel‐induced cell growth appeared enhanced when primary human keratinocytes were co‐cultured with fibroblasts. Western blot analysis demonstrated that nickel ions induced up‐modulation of the expression of the keratinocyte growth factor receptors (KGFR) without affecting the keratinocyte differentiation, whereas the protein levels of the epidermal growth factor receptor (EGFR) and of its ligand transforming growth factor‐alpha (TGF‐α) appeared unmodified by the treatment. Double immunofluorescence showed that the effect of nickel on DNA synthesis was mainly exerted on KGFR expressing cells, suggesting that KGFR up‐modulation could be required for the nickel‐induced cell proliferation. These results indicate that KGFR and its ligands may play a role in the mechanism of action of nickel ions and in the protective effect of zinc pretreatment.

Hemochromatosis-induced bipolar disorder: A case report

OBJECTIVE A patient presenting with Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, bipolar disorder was found to be affected by high iron hemochromatosis. This prompted us to explore the relation between bipolar disorder and iron overload. METHOD We report the case and review the peer-reviewed literature focusing on mood symptoms in patients with hemochromatosis or iron overload. Animal studies of brain effects of iron overload are summarized. High iron hemochromatosis was confirmed by genetic testing, and treatment was instituted to address iron overload. RESULTS Patients bipolar symptoms completely subsided after phlebotomic reduction of iron overload. CONCLUSION Clinicians should explore the possibility of iron overload and seek genetic confirmation of hemochromatosis in resistant bipolar disorder to avoid unnecessary medication.

Specific effects exerted by B-lymphoproliferative diseases on peripheral T-lymphocyte protein expression

A proteomic approach was applied to study the protein expression profile of peripheral T‐cells derived from patients at the onset of different B‐lymphoproliferative diseases, because a rising interest in specific actions played by T‐cells in such pathologies has emerged. Decreased levels of profilin‐1 and cofilin‐1 and increased levels of coronin1A and prohibitin were found in patients, compared with healthy controls. The protein‐protein interaction network of these proteins was studied using a web‐based bioinformatics tool, highlighting the actin cytoskeleton regulation as the main biological process involved in peripheral T‐cells of such patients. Unsupervised cluster analysis of protein expression data shows that the recorded alteration of T‐cell proteome was specifically induced by B‐cell pathologies.

Lack of association between oxidative stress-related gene polymorphisms and chronic migraine in an Italian population

Migraine patients present increased risks of vascular diseases such as high blood pressure, insulin resistance, metabolic syndrome, stroke and coronary heart disease. Oxidative stress (OS) is increasingly being studied in relation to the pathophysiology of migraine, stimulated by the described association with the most frequent migraine comorbidities. Because many of the gene-encoded players of the OS balance are characterized by functional polymorphisms, it is supposed that the individual genomic profile could affect susceptibility to OS and to related pathophysiological conditions. This study aimed to characterize a panel of 10 polymorphisms in 8 OS-related genes in a chronic migraine (CM) population and healthy controls, to recognize a genetic risk in the process of migraine chronification. The sample consisted of 45 healthy women and 96 women diagnosed with CM. No deviations from the Hardy-Weinberg equilibrium were detected, or in the overall population, or in the CM group or in the control group.

Screening for neurotropic viruses in cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases.

The paper by Virtanen et al.1 strengthens the association of herpes viruses with multiple sclerosis (MS). The correlation between the presence of Epstein Barr virus (EBV)/Herpes Virus 6 (HHV-6) DNA in cerebrospinal fluid (CSF) and number of contrast enhancing lesions (CELs) on magnetic resonance imaging (MRI) is of interest and mirrors our results from a screening for neurotropic viruses in cerebrospinal fluid of patients with MS and other neurological diseases. We performed PCR for herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), cytomegalovirus (CMV), varicella zoster virus (VZV), HHV6, JC virus (JCV) and EBV on CSF samples from 159 patients who had had a lumbar puncture for diagnostic purposes. All CSF samples were analyzed using a commercial RHA CNS kit (Labo Bio-medical Products) for the detection of cell-free and cell-associated viral nucleic acids. Patients were then grouped according to their final diagnosis: 60 with MS (58 relapsing–remitting [RR] and two primary progressive [PP]; mean age 37±13 years, 40 females, 20 males), 48 with other inflammatory diseases (OIND; mean age 57±17 years, 23 females, 25 males), and 51 with other non-inflammatory diseases (NIND; mean age 56±18 years, 43 females, 8 males). Gadolinium-enhanced MRI was performed in all patients within 10 days of CSF sampling. In the MS group, 14 patients had a positive PCR for EBV, two for HSV1, three for HSV2 and one for HHV6. In OIND patients, 12 had positive PCR for EBV, two for HSV1, three for HSV2, four for CMV, one for JCV and four for HHV6. In NIND patients, two had positive PCR for EBV, one for HHV6 and one for VZV. The frequency of viral DNA in CSF was comparable among MS and OIND groups. A significant bias in the distribution of EBV was observed in MS and OIND groups with respect to NIND (p = 0.011 at Fisher’s exact test), while CMV infection was more frequent in OIND patients (p = 0.036 at Fisher’s exact test). In patients with MS we observed an excess of large T2 lesions at MRI in cases with EBV DNA in CSF: T2 lesions larger than 10 mm were present in six out 14 EBVpositive and five out 46 EBV-negative patients (p = 0.01 at Fisher’s exact test). This finding was not found in OIND patients; it was also independent of the cell count in CSF, which was higher in OIND than in MS patients (p = 0.02 at Mann–Whitney U test), but was comparable between EBV-positive and EBV-negative MS patients. Thus, the relationship between lesion size and EBV-positive PCR seems not to reflect a non-specific recruitment of leucocytes in the central nervous system in the context of amplified inflammation. The increased chance of detecting EBV DNA in MS patients with more active lesions is instead in accord with Jacobson’s group’s finding1 (at least as far as EBV is concerned) and supports evidence of intrathecal reactivation and virus-driven immunopathogenic response in MS.2,3

Effect of MTHFR Polymorphisms on Gastrointestinal Cancer Risk in Italy

Background The aim of the study was to assess the association of single nucleotide polymorphisms (SNPs) C677T and A1298C in the methylenetetrahydrofolate reductase gene with colorectal, esophageal/gastric and pancreatic cancer in a cohort of Italian patients. Methods A total of 790 cancer patients and 202 healthy controls were genotyped and distributions in genotype and allele frequencies were compared by Chi-squared analysis and logistic regression analysis. Results According to most of previous findings, we found an effect of the C677T variant, but no effect of the A1298C, in colorectal and esophageal/gastric, whereas no association was evidenced with pancreatic cancer. We found that only homozygous TT carriers of the C677T variant had an increased risk for onset of cancer. Conclusion This result could be related to dietary and behavioral habits of the analyzed population, which could mitigate the deleterious effect of the T allele in heterozygosity and it highlights the importance to validate genetic determinant of cancer risk in different population and geographical areas.