Investigating metal aberrations in cellular senescence

This study explored perturbations in iron and copper homeostasis in senescent cells that contribute to age-associated disease and dysfunction. Altered metal regulation was associated with impaired cellular autophagy and resulted in extreme resistance to ferroptosis, a novel iron mediated cell death process linked to various pathologies.<br /

Copper as a target for prostate cancer therapeutics: copper-ionophore pharmacology and altering systemic copper distribution

Copper-ionophores that elevate intracellular bioavailable copper display significant therapeutic utility against prostate cancer cells in vitro and in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice. However, the pharmacological basis for their anticancer activity remains unclear, despite impending clinical trails. Herein we show that intracellular copper levels in prostate cancer, evaluated in vitro and across disease progression in TRAMP mice, were not correlative with copper-ionophore activity and mirrored the normal levels observed in patient prostatectomy tissues (Gleason Score 7 & 9). TRAMP adenocarcinoma cells harbored markedly elevated oxidative stress and diminished glutathione (GSH)-mediated antioxidant capacity, which together conferred selective sensitivity to prooxidant ionophoric copper. Copper-ionophore treatments [CuII(gtsm), disulfiram & clioquinol] generated toxic levels of reactive oxygen species (ROS) in TRAMP adenocarcinoma cells, but not in normal mouse prostate epithelial cells (PrECs). Our results provide a basis for the pharmacological activity of copper-ionophores and suggest they are amendable for treatment of patients with prostate cancer. Additionally, recent in vitro and mouse xenograft studies have suggested an increased copper requirement by prostate cancer cells. We demonstrated that prostate adenocarcinoma development in TRAMP mice requires a functional supply of copper and is significantly impeded by altered systemic copper distribution. The presence of a mutant copper-transporting Atp7b protein (tx mutation: A4066G/Met1356Val) in TRAMP mice changed copper-integration into serum and caused a remarkable reduction in prostate cancer burden (64% reduction) and disease severity (grade), abrogating adenocarcinoma development. Implications for current clinical trials are discussed

Heterogeneous copper concentrations in cancerous human prostate tissues

&copy; 2015 Wiley Periodicals, Inc. &copy; 2015 Wiley Periodicals, Inc. Background Therapeutics that target copper for the treatment of prostate cancer are being evaluated in human clinical trials. Elevated intracellular copper is considered to sensitize prostate cancer cells to certain copper-coordination compounds, especially those with ionophoric properties. While there is compelling in vitro evidence that prostate cancer cells accumulate intracellular copper, a corresponding status for copper in patient tissues has not been corroborated. We therefore established whether copper concentrations increase in cancerous prostate tissues, and in sera, in patients throughout disease progression. Methods Human prostate tissue samples were obtained from patient prostatectomies (n=28), and together with patient-matched sera, were analyzed for copper content by inductively coupled plasma mass spectrometry. Results When grouped together, cancerous prostate tissues exhibiting moderate disease severity (Gleason Score 7) (n=10) had 1.6-fold more copper than age-matched normal tissues (n=10) (P&lt;0.05). Those with more aggressive disease (Gleason Score 9) (n=8) had 1.8-fold more copper (P&lt;0.05). In both disease stages however, the copper concentrations between individual samples were rather variable (0.55-3.02&mu;g/g), with many clearly within the normal range (0.52-1.28&mu;g/g). Additionally, we found that there was no change in serum copper concentrations in patients with either moderate or aggressive prostate cancer (Gleason Score 7 or 9), compared with reference intervals and to age-matched controls. Conclusions The heterogeneous nature of copper concentrations in cancerous prostate tissues, suggest that a small subset of patients may respond to treatments that target elevated intratumoral copper. Therefore, such approaches would likely require personalized treatment strategies. Prostate 75:1510-1517, 2015

Striking while the iron is hot: Iron metabolism and ferroptosis in neurodegeneration

Iron is the most abundant transition metal on Earth and essential for life. Iron availability inprimordial oceans allowed for its incorporation in living organisms. Metabolic processes catalysed byiron or by iron-sulfur clusters that could be generated in prebiotic settings may be among the first ofsuch processes to evolve on Earth and essential for the emergence of carbon-based life (Bonfio et al.,2017, Varma et al., 2018). The photolysis of water by the process of photosynthesis around 2.45billion years ago introduced a new global poison i.e. oxygen, causing what is described as the GreatOxygenation Event (Sessions et al., 2009). The resultant oxidising environment transformed iron intoa limiting factor for life processes due to the limited solubility of the oxidised iron cation.The ability of iron to cycle through its oxidation states and form coordination bonds is utilisedby many enzymes to carry out their catalytic function. Iron has thus emerged as an indispensable cofactorfor proteins involved in essential (respiration, DNA replication, cell division) and specialised(oxygen transport, neurotransmission) cellular functions. Iron can serve as a potent oxidant that canwreak havoc on biomolecules, ironically endangering the life that it helps facilitate. This conundrumnecessitated the evolution of homeostatic mechanisms to ensure the availability of this critical elementwhile mitigating potential oxidative damage. In the body iron levels are maintained through theprecise uptake of iron from the diet. However, the body has no specific physiological mechanism foriron excretion. Iron thus tends to accumulate in certain tissues with age.The brain is a major organ where iron accumulates with age, especially in regions ofpathological relevance. The study of monogenic genetic disorders that affect iron homeostasis, andindications from dietary studies, have established that brain iron homeostasis is mostly independent ofsystemic iron homeostasis (Belaidi and Bush, 2016). Furthermore, indicators of systemic iron levelsare weakly correlated with iron in the brain. Several neurodegenerative conditions includingAlzheimer's disease (AD) and Parkinson's disease (PD) are associated with increased iron levels inaffected region of the brain with levels of iron corresponding to disease severity (Belaidi and Bush,2016). However, the iron-mediated events that may promote neurodegeneration appear to be moreintricate than iron-associated oxidative damage. Here we review the development of the “iron4hypothesis” of neurodegeneration, shifting our focus beyond iron toxicity to consider the recently(re)discovered iron-dependent programmed cell death pathway called ferroptosis

Copper ionophores as novel antiobesity therapeutics

The therapeutic utility of the copper ionophore disulfiram was investigated in a diet-induced obesity mouse model (C57BL/6J background), both through administration in feed (0.05 to 1% (w/w)) and via oral gavage (150 mg/kg) for up to eight weeks. Mice were monitored for body weight, fat deposition (perigonadal fat pads), metabolic changes (e.g., glucose dyshomeostasis) and pathologies (e.g., hepatic steatosis, hyperglycaemia and hypertriglyceridemia) associated with a high-fat diet. Metal-related pharmacological effects across major organs and serums were investigated using inductively coupled plasma mass spectrometry (ICP-MS). Disulfiram treatments (all modes) augmented hepatic copper in mice, markedly moderated body weight and abolished the deleterious systemic changes associated with a high-fat diet. Likewise, another chemically distinct copper ionophore H2(gtsm), administered daily (oral gavage), also augmented hepatic copper and moderated mouse body weight. Postmortem histological examinations of the liver and other major organs, together with serum aminotransferases, supported the reported therapeutic safety of disulfiram. Disulfiram specifically altered systemic copper in mice and altered hepatic copper metabolism, perturbing the incorporation of copper into ceruloplasmin (holo-ceruloplasmin biosynthesis) and subsequently reducing serum copper concentrations. Serum ceruloplasmin represents a biomarker for disulfiram activity. Our results establish copper ionophores as a potential class of antiobesity agents

Cervical cancer stem cells selectively overexpress HPV oncoprotein E6 that controls stemness and self-renewal through upregulation of HES1

PURPOSE: Perturbation of keratinocyte differentiation by E6/E7 oncoproteins of high-risk human papillomaviruses that drive oncogenic transformation of cells in squamocolumnar junction of the uterine cervix may confer &quot;stem-cell like&quot; characteristics. However, the crosstalk between E6/E7 and stem cell signaling during cervical carcinogenesis is not well understood. We therefore examined the role of viral oncoproteins in stem cell signaling and maintenance of stemness in cervical cancer. EXPERIMENTAL DESIGN: Isolation and enrichment of cervical cancer stem-like cells (CaCxSLCs) was done from cervical primary tumors and cancer cell lines by novel sequential gating using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133) in defined conditioned media for assessing sphere formation and expression of self-renewal and stemness markers by FACS, confocal microscopy, and qRT-PCR. Differential expression level and DNA-binding activity of Notch1 and its downstream targets in CaCxSLCs as well as silencing of HPVE6/Hes1 by siRNA was evaluated by gel retardation assay, FACS, immunoblotting, and qRT-PCR followed by in silico and in vivo xenograft analysis. RESULTS: CaCxSLCs showed spheroid-forming ability, expressed self-renewal and stemness markers Oct4, Sox2, Nanog, Lrig1, and CD133, and selectively overexpressed E6 and HES1 transcripts in both cervical primary tumors and cancer cell lines. The enriched CaCxSLCs were highly tumorigenic and did recapitulate primary tumor histology in nude mice. siRNA silencing of HPVE6 or Hes1 abolished sphere formation, downregulated AP-1-STAT3 signaling, and induced redifferentiation. CONCLUSIONS: Our findings suggest the possible mechanism by which HPVE6 potentially regulate and maintain stem-like cancer cells through Hes1

Alterations in microRNAs miR-21 and let-7a correlate with aberrant STAT3 signaling and downstream effects during cervical carcinogenesis

BACKGROUND: Present study provides clinical evidence of existence of a functional loop involving miR-21 and let-7a as potential regulators of aberrant STAT3 signaling recently reported by our group in an experimental setup (Shishodia et al. BMC Cancer 2014, 14:996). The study is now extended to a set of cervical tissues that represent natural history of human papillomavirus (HPV)-induced tumorigenic transformation. MATERIALS AND METHODS: Cervical tissues from histopathologically-confirmed pre-cancer (23) and cancer lesions (56) along with the normal control tissues (23) were examined for their HPV infection status, expression level of miR-21 &amp; let-7a and STAT3 &amp; pSTAT3 (Y705) by PCR-based genotyping, quantitative real-time PCR and immunoblotting. RESULTS: Analysis of cancer tissues revealed an elevated miR-21 and reduced let-7a expression that correspond to the level of STAT3 signaling. While miR-21 showed direct association, let-7a expression was inversely related to STAT3 expression and its activation. In contrast, a similar reciprocal expression kinetics was absent in LSIL and HSIL tissues which overexpressed let-7a. miR-21 was found differentially overexpressed in HPV16-positive lesions with a higher oncoprotein E6 level. Overexpression of miR-21 was accompanied by elevated level of other STAT3-regulated gene products MMP-2 and MMP-9. Enhanced miR-21 was found associated with decreased level of STAT3 negative regulator PTEN and negative regulator of MMPs, TIMP-3. CONCLUSION: Overall, our study suggests that the microRNAs, miR-21 and let-7a function as clinically relevant integral components of STAT3 signaling and are responsible for maintaining activated state of STAT3 in HPV-infected cells during cervical carcinogenesis

Copper accumulation in senescent cells: Interplay between copper transporters and impaired autophagy

Cellular senescence is characterized by irreversible growth arrest incurred through either replicative exhaustion or by pro-oncogenic cellular stressors (radioactivity, oxidative stress, oncogenic activation). The enrichment of senescent cells in tissues with age has been associated with tissue dyshomeostasis and age-related pathologies including cancers, neurodegenerative disorders (e.g. Alzheimer's, Parkinson's, etc.) and metabolic disorders (e.g. diabetes). We identified copper accumulation as being a universal feature of senescent cells [mouse embryonic fibroblasts (MEF), human prostate epithelial cells and human diploid fibroblasts] in vitro. Elevated copper in senescent MEFs was accompanied by elevated levels of high-affinity copper uptake protein 1 (Ctr1), diminished levels of copper-transporting ATPase 1 (Atp7a) (copper export) and enhanced antioxidant defence reflected by elevated levels of glutathione (GSH), superoxide dismutase 1 (SOD1) and glutaredoxin 1 (Grx1). The levels of intracellular copper were further increased in senescent MEFs cultured in copper supplemented medium and in senescent Mottled Brindled (Mobr) MEFs lacking functional Atp7a. Finally, we demonstrated that the restoration/preservation of autophagic-lysosomal degradation in senescent MEFs following rapamycin treatment correlated with attenuation of copper accumulation in these cells despite a further decrease in Atp7a levels. This study for the first time establishes a link between Atp7a and the autophagic-lysosomal pathway, and a requirement for both to effect efficient copper export. Such a connection between cellular autophagy and copper homeostasis is significant, as both have emerged as important facets of age-associated degenerative disease. Keywords: Senescence, Copper, Ageing, Homeostasis, Autophag