Long-term intermittent caloric restriction remodels the gut microbiota in mice genetically prone to breast cancer.

OBJECTIVES: Gut microbiota dysbiosis is among the risk factors for breast cancer development, together with genetic background and dietary habits. However, caloric restriction has been shown to remodel the gut microbiota and slow tumor growth. Here, we investigated whether the gut microbiota mediates the preventive effects of long-term chronic or intermittent caloric restriction on breast cancer predisposition. METHODS: 10-week-old transgenic breast cancer-prone mice were randomly assigned to dietary groups (ad libitum, chronic caloric restriction, and intermittent caloric restriction groups) and fed up to week 81. Stool samples were collected at weeks 10 (baseline), 17 (young), 49 (adult), and 81 (old). 16S rRNA gene sequencing was performed to identify the gut microbiota profile of the different groups. In order to investigate the breast cancer gut microbiota profile within genetically predisposed individuals regardless of diet, mammary tumor-bearing mice and mammary tumor-free but genetically prone mice were selected from the ad libitum group (n = 6). RESULTS: Intermittent caloric restriction increased the microbial diversity of adult mice and modified age-related compositional changes. A total of 13 genera were differentially abundant over time. Pathogenic Mycoplasma was enriched in the re-feeding period of the old intermittent caloric restriction group compared with baseline. Furthermore, mammary tumor-free mice showed shared gut microbiota characteristics with mammary tumor-bearing mice, suggesting an early link between genetic predisposition, gut microbiota, and breast cancer development. CONCLUSIONS: Our study revealed the role of gut microbes in the preventive effects of caloric restriction against breast cancer development, implying the significance of diet and microbiome interplay.

Label-Free Surface Enhanced Raman Spectroscopy for Cancer Detection.

Blood is a vital reservoir housing numerous disease-related metabolites and cellular components. Thus, it is also of interest for cancer diagnosis. Surface-enhanced Raman spectroscopy (SERS) is widely used for molecular detection due to its very high sensitivity and multiplexing properties. Its real potential for cancer diagnosis is not yet clear. In this study, using silver nanoparticles (AgNPs) as substrates, a number of experimental parameters and scenarios were tested to disclose the potential for this technique for cancer diagnosis. The discrimination of serum samples from cancer patients, healthy individuals and patients with chronic diseases was successfully demonstrated with over 90% diagnostic accuracies. Moreover, the SERS spectra of the blood serum samples obtained from cancer patients before and after tumor removal were compared. It was found that the spectral pattern for serum from cancer patients evolved into the spectral pattern observed with serum from healthy individuals after the removal of tumors. The data strongly suggests that the technique has a tremendous potential for cancer detection and screening bringing the possibility of early detection onto the table.

Magnetic Nanoparticle-Based Electrochemical Sensing Platform Using Ferrocene-Labelled Peptide Nucleic Acid for the Early Diagnosis of Colorectal Cancer.

Diagnostic biomarkers based on epigenetic changes such as DNA methylation are promising tools for early cancer diagnosis. However, there are significant difficulties in directly and specifically detecting methylated DNA regions. Here, we report an electrochemical sensing system based on magnetic nanoparticles that enable a quantitative and selective analysis of the methylated septin9 (mSEPT9) gene, which is considered a diagnostic marker in early stage colorectal cancer (CRC). Methylation levels of SEPT9 in CRC samples were successfully followed by the selective recognition ability of a related peptide nucleic acid (PNA) after hybridization with DNA fragments in human patients' serums and plasma (n = 10). Moreover, this system was also adapted into a point-of-care (POC) device for a one-step detection platform. The detection of mSEPT9 demonstrated a limit of detection (LOD) value of 0.37% and interference-free measurement in the presence of branched-chain amino acid transaminase 1 (BCAT1) and SRY box transcription factor 21 antisense divergent transcript 1 (SOX21-AS1). The currently proposed functional platform has substantial prospects in translational applications of early CRC detection.

Effects of carbendazim and astaxanthin co-treatment on the proliferation of MCF-7 breast cancer cells.

There has been a controversy in the oncology field about the use of antioxidants along with chemotherapeutics in cancer treatment. This study aimed to investigate the effects of a potent antioxidant (astaxanthin) co-treatment with a promising anti-cancer drug (carbendazim), which is in phase I clinical trials, on MCF-7 breast cancer cell proliferation. MCF-7 cells were treated with carbendazim, astaxanthin, or their combinations and incubated for 24 h. After the incubation, each treatment group was evaluated for proliferation, cell cycle progression, and production of reactive oxygen species (ROS) using WST-1, flow cytometry, and CM-H2DCFDA, respectively. All tested carbendazim and astaxanthin combinations increased the anti-proliferative effect of Carb treatment alone and increased the G2/M phase cell cycle arrest compared to the DMSO-treated control. Astaxanthin, at all concentrations tested, reduced the elevated intracellular ROS levels induced by the carbendazim treatment. Our data suggest that astaxanthin and carbendazim co-treatment enhances the anti-proliferative effect of carbendazim as a single agent, while alleviating the carbendazim treatment-associated ROS production in MCF-7 cells. These findings may contribute to the current debate on the use of antioxidants along with anti-cancer drugs in cancer chemotherapy.

Effects of different glucose concentrations on the leptin signaling pathway in MCF-7 and T47D breast cancer cells.

AIM: Leptin activates multiple intracellular signaling pathways, including JAK/STAT, by binding to its receptor. Leptin is also an important regulator of glucose homeostasis. Although both glucose and leptin increase breast cancer cell proliferation in vitro, whether the enhancing effect of glucose on the proliferation of breast cancer cells is mediated by the leptin signaling pathway is not known. The aim of this study was to investigate the effect of different glucose concentrations on the leptin signaling pathway in MCF-7 and T47D breast cancer cells. MATERIAL AND METHODS: MCF-7 and T47D cell proliferation in different glucose concentrations (2.5 mM, 5 mM, 25 mM, or 50 mM) were assayed using CCK-8 assay. Leptin, leptin receptors (ObR, ObRb) as well as STAT3 mRNA and protein levels in both cell lines in different glucose concentrations were examined by RT-PCR and western blot, respectively. RESULTS: Incubation in 2.5 mM, 5 mM, 25 mM, or 50 mM glucose for 72h significantly increased the proliferation of both MCF-7 and T47D cells compared to 0 mM glucose incubated cells (P<0.001). mRNA levels of leptin, ObR, ObRb or STAT3 in 2.5 mM, 5 mM, 25 mM, or 50 mM glucose incubated cells were not significantly different in both cell lines compared to 0 mM (p>0.05). However, ObR protein levels in MCF-7 cells incubated in 25 mM glucose was significantly lower compared to 0 mM glucose by western blot (p<0.05). CONCLUSION: Our data suggest that the enhancing effect of glucose on breast cancer cell proliferation is not mediated by the JAK/STAT pathway.

Roles of Adiponectin Signaling Related Proteins in Mammary Tumor Development.

OBJECTIVE: This study aims to investigate the expression levels of adiponectin signaling related proteins in mammary tissue, liver and breast cancer tissue in mice. Adiponectin, an adipocytokine, is secreted from adipose tissue and has been documented to have roles in diabetes, inflammation, and cancer development. In particular, levels of serum adiponectin are inversely associated with obesity and a decrease in serum adiponectin levels have been reported to be associated with breast cancer. There are two adiponectin receptor subtypes, AdipoR1 and AdipoR2, which have been identified in mammalian tissues, including human cancer cell lines and also in human mammary tumors. However, the role of adiponectin receptors in breast cancer development remains to be established. METHODS: In this study, MMTV-TGF-α transgenic mice were fed from week 10 up to week 74 of age. Expression levels of adiponectin, AdipoR1 and AdipoR2 proteins were measured in the mammary fat pad (MFP), mammary tumor (MT) and liver tissues from 74 weeks old MMTV-TGF-α transgenic mice with and without MT using Western Blot. Adiponectin levels were measured using ELISA assay. RESULTS: Protein expression levels of Adiponectin and AdipoR1 were significantly lower in MTs compared to control tissues. However, AdipoR2 protein expression levels were similar in MT and MFP tissues from MT-positive and MT-negative mice. The expression levels of adiponectin, AdipoR1 and AdipoR2 proteins in liver tissues were also similar in MT-positive and MT-negative mice. Serum adiponectin levels of the MT-positive and MT-negative mice were similar. CONCLUSION: These results indicate that adiponectin and its receptors are differentially regulated depending upon the specific tissue analyzed. AdipoR1 and adiponectin may play important roles in MT development.

Towards frailty biomarkers: Candidates from genes and pathways regulated in aging and age-related diseases.

OBJECTIVE: Use of the frailty index to measure an accumulation of deficits has been proven a valuable method for identifying elderly people at risk for increased vulnerability, disease, injury, and mortality. However, complementary molecular frailty biomarkers or ideally biomarker panels have not yet been identified. We conducted a systematic search to identify biomarker candidates for a frailty biomarker panel. METHODS: Gene expression databases were searched (http://genomics.senescence.info/genes including GenAge, AnAge, LongevityMap, CellAge, DrugAge, Digital Aging Atlas) to identify genes regulated in aging, longevity, and age-related diseases with a focus on secreted factors or molecules detectable in body fluids as potential frailty biomarkers. Factors broadly expressed, related to several "hallmark of aging" pathways as well as used or predicted as biomarkers in other disease settings, particularly age-related pathologies, were identified. This set of biomarkers was further expanded according to the expertise and experience of the authors. In the next step, biomarkers were assigned to six "hallmark of aging" pathways, namely (1) inflammation, (2) mitochondria and apoptosis, (3) calcium homeostasis, (4) fibrosis, (5) NMJ (neuromuscular junction) and neurons, (6) cytoskeleton and hormones, or (7) other principles and an extensive literature search was performed for each candidate to explore their potential and priority as frailty biomarkers. RESULTS: A total of 44 markers were evaluated in the seven categories listed above, and 19 were awarded a high priority score, 22 identified as medium priority and three were low priority. In each category high and medium priority markers were identified. CONCLUSION: Biomarker panels for frailty would be of high value and better than single markers. Based on our search we would propose a core panel of frailty biomarkers consisting of (1) CXCL10 (C-X-C motif chemokine ligand 10), IL-6 (interleukin 6), CX3CL1 (C-X3-C motif chemokine ligand 1), (2) GDF15 (growth differentiation factor 15), FNDC5 (fibronectin type III domain containing 5), vimentin (VIM), (3) regucalcin (RGN/SMP30), calreticulin, (4) PLAU (plasminogen activator, urokinase), AGT (angiotensinogen), (5) BDNF (brain derived neurotrophic factor), progranulin (PGRN), (6) α-klotho (KL), FGF23 (fibroblast growth factor 23), FGF21, leptin (LEP), (7) miRNA (micro Ribonucleic acid) panel (to be further defined), AHCY (adenosylhomocysteinase) and KRT18 (keratin 18). An expanded panel would also include (1) pentraxin (PTX3), sVCAM/ICAM (soluble vascular cell adhesion molecule 1/Intercellular adhesion molecule 1), defensin α, (2) APP (amyloid beta precursor protein), LDH (lactate dehydrogenase), (3) S100B (S100 calcium binding protein B), (4) TGFß (transforming growth factor beta), PAI-1 (plasminogen activator inhibitor 1), TGM2 (transglutaminase 2), (5) sRAGE (soluble receptor for advanced glycosylation end products), HMGB1 (high mobility group box 1), C3/C1Q (complement factor 3/1Q), ST2 (Interleukin 1 receptor like 1), agrin (AGRN), (6) IGF-1 (insulin-like growth factor 1), resistin (RETN), adiponectin (ADIPOQ), ghrelin (GHRL), growth hormone (GH), (7) microparticle panel (to be further defined), GpnmB (glycoprotein nonmetastatic melanoma protein B) and lactoferrin (LTF). We believe that these predicted panels need to be experimentally explored in animal models and frail cohorts in order to ascertain their diagnostic, prognostic and therapeutic potential.

Hydrogen peroxide prolongs mitotic arrest in a dose dependent manner and independently of the spindle assembly checkpoint activity in Saccharomyces cerevisiae.

Oxidative stress and chromosome missegregation are important factors that are linked to aneuploidy. A major reason for chromosome missegragation is the inappropriate activity of the spindle assembly checkpoint (SAC), a conserved surveillance mechanism that monitors the state of kinetochore-microtubule attachments to ensure equal chromosome segregation in mitosis. SAC-activation induces a prolonged mitotic arrest. Mitosis is considered the most vulnerable cell cycle phase to several external signals, therefore increasing the time cells spent in this phase via mitotic arrest induction by SAC-activating agents is favorable for cancer therapy. Cancer cells also display elevated oxidative stress due to abnormally high production of reactive oxygen species (ROS). However, the effect of increased oxidative stress on the duration of mitotic arrest remains largely unknown. In this study, we investigated the effect of H2O2-induced oxidative stress on the mitotic arrest induced by a SAC-activating agent (nocodazole) in Saccharomyces cerevisiae. Our data suggest that oxidative stress prolongs SAC-activation induced mitotic arrest in a dose dependent manner. We, in addition, investigated the effect of H2O2 treatment on the mitotic arrest induced independently of SAC-activation by using a conditional mutant (cdc23) and showed that the effect of H2O2-induced oxidative stress on mitotic arrest is independent of the SAC activity.

Transglutaminase activity regulates atherosclerotic plaque composition at locations exposed to oscillatory shear stress.

OBJECTIVE: Atherosclerosis preferentially develops at sites of disturbed blood flow. We tested the hypothesis that transglutaminase activity plays a role in plaque development at these locations. METHODS AND RESULTS: Exposure of endothelial cells to steady flow (7 dynes/cm(2)) was associated with relatively low transglutaminase activity, whereas under low oscillatory flow (1.3 ± 2.6 dynes/cm(2)) endothelial cells showed a >4-fold higher level of transglutaminase activity. Under oscillatory flow, transglutaminase activity increased the expression of the chemokine MCP-1 (CCL2). In vivo, oscillatory flow was induced by placement of a tapered perivascular cast around the carotid artery of type 2 transglutaminase (TGM2) knockout mice and WT counterparts. After 2 days, significantly less monocytes adhered to the endothelium in TGM2 knockout mice as compared to WT. In a more chronic setting, ApoE knockout mice that were equipped with the flow-modifying cast developed lesions proximal to the cast (low shear stress), and distal to the cast (oscillatory shear stress). Inhibition of transglutaminase induced a marked reduction in macrophage and fat content in distal lesions only. In addition, lesion size was increased in this area, which was attributed to an increase in smooth muscle content. CONCLUSION: Oscillatory shear stress increases endothelial transglutaminase activity. In turn, transglutaminase activity affects the expression of MCP-1 in vitro and monocyte recruitment in vivo. In a mouse model of atherosclerosis, transglutaminase activity has a major effect on plaque composition under oscillatory shear stress.