Number Of Ki67-positive Cells Research Articles

Recombinant thrombomodulin suppresses tumor growth of pancreatic cancer by blocking thrombin-induced PAR1 and NF-κB activation

Thrombomodulin, an anticoagulant that inhibits thrombin-induced growth factor promotion, also has an anti-inflammatory effect. Furthermore, thrombomodulin inhibits nuclear factor-kappa B activation, which plays a crucial role in cancer progression. We assessed the antitumor activity of recombinant thrombomodulin for pancreatic cancer. A xenograft orthotopic model was established in mice by implantation of human pancreatic cancer cells. The animals were treated with intraperitoneal injection of recombinant thrombomodulin 5 times a week for 4weeks. Nuclear factor-kappa B activation was evaluated by measuring nuclear localization of the p65. Efficacy of recombinant thrombomodulin on the signal transduction of nuclear factor-kappa B was measured invitro under preconditioning with thrombin or epidermal growth factor. Tumor growth was suppressed by recombinant thrombomodulin (P<.05). Recombinant thrombomodulin inhibited the expression of IκB kinase β (P<.05) and pIκBα (P<.01), as well as the activation of nuclear factor-kappa B NF-κB (P<.001). Furthermore, recombinant thrombomodulin inhibited thrombin-induced protease activate receptor 1 and nuclear factor-kappa B activation invitro (P<.05). The number of Ki67-positive cells was decreased by recombinant thrombomodulin (P<.01). Recombinant thrombomodulin also suppressed body weight loss associated with pancreatic cancer (P<.05). No obvious adverse effects were observed. Recombinant thrombomodulin significantly suppressed tumor growth against human pancreatic cancer by blocking thrombin-induced nuclear factor-kappa B activation without adverse effects.

Treatment with Sildenafil and Donepezil Improves Angiogenesis in Experimentally Induced Critical Limb Ischemia.

Objectives. In this study, we aimed to demonstrate the role of sildenafil (an antagonist of phosphodiesterase type 5 (PDE-5)) and donepezil (a specific and reversible inhibitor of acetylcholinesterase (Ach)) in increasing ischemia-induced angiogenesis. Method. Critical limb ischemia was induced by ligation of the common femoral artery followed by ligation of the common iliac artery. The operated animals were divided into 3 groups: receiving sildenafil, receiving donepezil, and surgery alone; the contralateral lower limb was used as a negative control. The results were controlled based on clinical score and Doppler ultrasound. Gastrocnemius muscle samples were taken from all animals, both from the ischemic and nonischemic limb and were used for histopathological and immunohistochemical examination for the evaluation of the number of nuclei/field, endothelial cells (CD31), dividing cells (Ki-67), and vascular endothelial growth factor (VEGFR-3). Results. An increasing tendency of the number of nuclei/field with time was observed both in the case of sildenafil and donepezil treatment. The formation of new capillaries (the angiogenesis process) was more strongly influenced by donepezil treatment compared to sildenafil or no treatment. This treatment significantly influenced the capillary/fiber ratio, which was increased compared to untreated ligated animals. Sildenafil treatment led to a gradual increase in the number of dividing cells, which was significantly compared to the negative control group and compared to the ligation control group. The same effect (increase in the number of Ki-67 positive cells) was more obvious in the case of donepezil treatment. Conclusion. Donepezil treatment has a better effect in ligation-induced ischemia compared to sildenafil, promoting angiogenesis in the first place, and also arteriogenesis.

Abstract A46: AdipoRon suppresses ERK and STAT3 to inhibit pancreatic cancer growth

Abstract Introduction: The increasing incidence of pancreatic cancer is associated with a rising prevalence of obesity, a documented risk factor for the disease. Obesity harbors a systemic chronic inflammatory disorder characterized by increased production and secretion of pro-inflammatory adipokines leptin, TNF-α, and IL-6; while exhibiting a decrease in the anti-inflammatory adipokine; adiponectin. Dysregulation of these factors is thought to be a key mechanism of obesity associated cancers, contributing to increased activation of mitogenic pathways including PI3K and MAPK. Adiponectin represents an important negative regulator of leptin, TNF-α and IL-6. We previously demonstrated that adiponectin inhibits pancreatic cancer proliferation and tumor growth, however, the molecular mechanisms by which adiponectin regulates these processes are unknown. We hypothesize that Adiponectin Receptor (AdipoR) agonists elicit anti-tumor effects through suppression of RAS-MAPK mediated pathways and its downstream signaling components in pancreatic cancer progression. Experimental Procedures: The anti-tumor effects of AdipoRon, a novel small molecule agonist of the AdipoR, were assessed in vitro on human (MiaPaca-2 and Panc-1) and murine (P-4313 and K8484) pancreatic cancer cell lines. Cells were treated with AdipoRon in a dose-dependent manner and then assayed for cellular proliferation, apoptosis, colony formation and anchorage-independent growth. The effect of AdipoRon on activation of key RAS-MAPK signaling regulators was investigated by immunoblot analysis. To determine whether AdipoRon could inhibit the effects of obesity associated pro-tumorigenic cytokines, human and mouse pancreatic cancer cells were exposed to plasma collected from obese mice or specifically with recombinant cytokines. To determine whether AdipoRon could inhibit tumor growth in vivo, mice were orthotopically injected in the pancreas with the murine KrasG12D mutant P-4313 cell line. Tumors were allowed to establish for two weeks and treated with either vehicle or AdipoRon. Tumor size and number of Ki67 positive cells were assessed. Results: Compared to vehicle treatment, in vitro assessment confirmed that AdipoRon was highly effective at inhibiting cell proliferation, increasing apoptosis and preventing colony formation for all pancreatic cell lines tested. Anchorage independent growth was drastically reduced for both Panc1 (3.8 fold) and MiaPaca-2 (5.1 fold) cell lines in the presence of AdipoRon. Treatment of both murine and human pancreatic cancer cell lines with AdipoRon caused a significant dose dependent decrease in pSTAT3, pERK1, and pERK2 with a simultaneous increase in pAMPK. Importantly, AdipoRon completely antagonized the stimulatory effects of obese plasma or recombinant IL-6 on the activation of pSTAT3. Administration of AdipoRon to P-4313 orthotopic pancreatic tumor bearing mice resulted in four fold decrease in tumor size and a 50% reduction in tumor cell proliferation. Conclusions: AdipoRon, an adiponectin receptor agonist, suppresses KRAS signaling mediators ERK and STAT3 while simultaneously increasing AMPK resulting in inhibition of pancreatic cancer proliferation and tumor growth. Targeting of adiponectin receptors can provide a viable therapeutic strategy for the treatment of pancreatic cancer. Citation Format: Fanuel Messaggio, Alisha M. Mendonsa, Jason A. Castellanos, Casey Roberts, Nagaraj S. Nagathihalli, Nipun B. Merchant, Lee D. Gorden, Michael N. VanSaun.{Authors}. AdipoRon suppresses ERK and STAT3 to inhibit pancreatic cancer growth. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A46.

Maternal administration of magnesium sulfate promotes cell proliferation in hippocampus dentate gyrus in offspring mice after exposing to prenatal stress

Prenatal stress (PS) inhibits cell proliferation in the hippocampal dentate gyrus (DG), which is related to hippocampal anatomy and function abnormality. The aim of the study was to investigate the effects of magnesium sulfate (MgSO4) on PS-induced cell proliferation suppression in offspring during embryonic stage and postnatal spatial learning. MgSO4 administration was performed after PS treatment on pregnant mice. Mice were randomly divided into four groups: non-PS or PS maternal mice injected with MgSO4 or saline (P+NS, P+MG, C+MG and C+NS group). Corticosterone was collected from amniotic fluid of mother mice on day 17 of embryonic stage (E17). The ability for spatial learning and memory of pups postnatal 3 week was evaluated using water maze assay. Additionally, cell proliferation was detected by assessing the expression of Ki67 using immunohistochemistry in mice fetuses or pups. PS significantly increased corticosterone level in amniotic fluid (P<0.05) and impaired the spatial learning and memory (P+NS vs C+NS of latency time and track path length: P<0.05) of offspring on postnatal day 21. However, MgSO4 administration could reverse PS-induced spatial learning and memory disability (P+MG vs P+NS, P<0.05). Additionally, PS reduced the number of Ki67-positive cell in hippocampal DG on E17, E19 and postnatal day 21 (P+NS vs C+NS, P<0.05), which were also abrogated by maternal administration of MgSO4 (P+MG vs P+NS, P<0.05). Collectively, prenatal administration of MgSO4 can reverse PS-induced reduction of cell proliferation in hippocampal DG during embryonic stage and postnatal spatial learning.

Abstract 3942: Increased frequency of proliferating peripheral white blood cells in the blood of hepatocellular carcinoma patients compared with noncancer controls

Abstract Introduction Proliferation of peripheral blood cells, including activated CD8+ T-cells, has been reported in numerous pathologies including haematopoietic cancers and solid tumours. During development of a method to detect and characterise circulating tumour cells by imaging flow cytometry it became apparent that peripheral proliferating lymphocytes, while still rare, are substantially more common than CTCs in all cancer patients and are present in everybody. We investigated the frequency of these cells in patients with hepatocellular carcinoma (HCC) and healthy volunteers, and went on to assess their utility as a surrogate tissue for proof of mechanism studies of anti-proliferative agents. Methods Whole blood samples (4ml) from 7 patients with hepatocellular carcinoma and 14 healthy volunteers were collected into EDTA tubes. After RBC lysis and fixation WBCs were stained with fluorochrome conjugated antibodies to Ki67 and CD45 and the nuclei were stained with DAPI to assess relative DNA content. For each sample images of 100,000 cells were collected by imaging flow cytometry. Putative G2 cells were identified as single cells with DNA content equivalent to twice the amount of the majority of cells and the same as images of doublets. The frequency of cells with nuclear localised Ki67 reactivity was assessed independently of DNA content. Whole blood from healthy volunteers was also treated by ex vivo incubation with PMA ± the CDK inhibitor Dinaciclib. Results The mean frequency of Ki67 positive G1 peripheral WBCs in blood from patients with HCC was 0.44%, as compared to a frequency of 0.24% in the blood from healthy volunteers (p = 0.007). Incubation of healthy volunteer blood with PMA for 20 minutes at 37°C resulted in a small but reproducible increase in the number of Ki67 positive cells and this increase was abrogated by simultaneous incubation with Dinaciclib (141 ± 41% versus 92 ± 39%, expressed as a percentage of no PMA control, p = 0.0007). Additional staining of the ex vivo samples with an antibody to pMCM2, as a potential proof of mechanism marker for CDC7 inhibitor efficacy, was carried out. Results showed that pMCM2 expression was associated with ki67 and the frequency of pMCM2 positive cells also increased with exposure to PMA Conclusion The frequency of proliferating WBCs is increased in patients with HCC. These cells may have utility as a predictive/prognostic biomarker, or possibly in the pharmacodynamic monitoring of anti-proliferative pharmacological agents. Citation Format: Claire Hutton, Suriyon Uitrakul, Laura F. Ogle, Helen L. Reeves, Alastair Greystoke, Gareth J. Veal, David Jamieson. Increased frequency of proliferating peripheral white blood cells in the blood of hepatocellular carcinoma patients compared with noncancer controls. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3942.

Ameloblastic carcinoma: a Brazilian collaborative study of 17 cases.

Ameloblastic carcinoma (AMECA) is an odontogenic malignancy that combines the histological features of ameloblastoma and cytological atypia. Because of its rarity, it poses difficulties in diagnosis. The aim of this study was to investigate the socio-demographic data, histopathology, immunohistochemical features, treatment and outcomes of 17 cases. Descriptive statistical analyses were used to portray the clinicopathological data collected, retrospectively. Log-rank tests were performed to determine new prognostic factors. Lesions were immunostained for Ki67, p16, p53, and cytokeratins (CKs), and compared with solid/multicystic ameloblastomas (n=15). AMECA was mostly diagnosed at a late stage, affecting the posterior mandible of male patients in their fifth decade of life. Recurrence was diagnosed in nearly 90% of treated patients, and metastasis occurred in four patients. The mean number of Ki67-positive cells was 86.4±66 per field. Tumours were focally positive for CK7, CK8, CK14, and CK18, and diffusely positive for CK19, p53, and p16. AMECA showed increased immunoexpression of CK18, CK19, p16, p53 and Ki67 as compared with benign cases. Our study has contributed to the improved characterization of the epidemiology, prognostic markers, treatment options and outcomes of AMECA. Current criteria must be reviewed to simplify the diagnostic process for these neoplasms.

Enhanced Ghrelin Levels and Hypothalamic Orexigenic AgRP and NPY Neuropeptide Expression in Models of Jejuno-Colonic Short Bowel Syndrome.

Short bowel syndrome (SBS) patients developing hyperphagia have a better outcome. Gastrointestinal endocrine adaptations help to improve intestinal functions and food behaviour. We investigated neuroendocrine adaptations in SBS patients and rat models with jejuno-ileal (IR-JI) or jejuno-colonic (IR-JC) anastomosis with and without parenteral nutrition. Circulating levels of ghrelin, PYY, GLP-1, and GLP-2 were determined in SBS rat models and patients. Levels of mRNA for proglucagon, PYY and for hypothalamic neuropeptides were quantified by qRT-PCR in SBS rat models. Histology and immunostaining for Ki67, GLP-1 and PYY were performed in SBS rats. IR-JC rats, but not IR-JI, exhibited significantly higher crypt depths and number of Ki67-positive cells than sham. Fasting and/or postprandial plasma ghrelin and PYY concentrations were higher, or tend to be higher, in IR-JC rats and SBS-JC patients than in controls. Proglucagon and Pyy mRNA levels were significantly enhanced in IR-JC rats. Levels of mRNA coding hypothalamic orexigenic NPY and AgRP peptides were significantly higher in IR-JC than in sham rats. We demonstrate an increase of plasma ghrelin concentrations, major changes in hypothalamic neuropeptides levels and greater induction of PYY in SBS-JC rats and patients suggesting that jejuno-colonic continuity creates a peculiar environment promoting further gut-brain adaptations.

Hypoxic Conditioned Medium From Human Adipose-Derived Stem Cells Promotes Mouse Liver Regeneration Through JAK/STAT3 Signaling.

Adipose-derived stem cells (ASCs) mainly exert their function by secreting materials that are collectively termed the secretome. Despite recent attention to the secretome as an alternative to stem cell therapy, the culture conditions for generating optimal secretome contents have not been determined. Therefore, we investigated the role of hypoxic-conditioned media (HCM) from ASCs. Normoxic-conditioned media (NCM) and HCM were obtained after culturing ASCs in 20% O2 or 1% O2 for 24 hours, respectively. Subsequently, partially hepatectomized mice were infused with saline, control medium, NCM, or HCM, and then sera and liver specimens were obtained for analyses. Hypoxia (1% O2) significantly increased mRNA expression of mediators from ASCs, including interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF). HCM infusion significantly increased the number of Ki67-positive cells in the liver (p < .05). HCM infusion significantly increased phospho-signal transducer and activator of transcription 3 (STAT3) and decreased suppressor of cytokine signaling 3 (SOCS3) expression in the liver (p < .05). To determine the role of IL-6 in liver regeneration, we then performed IL-6 RNA interference study. Conditioned media (CM) obtained from ASCs, which were transfected with either siIL-6 or siControl, were administered to partially hepatectomized mice. The siIL-6 CM groups exhibited lower liver proliferation (Ki67-positive cells) and markers of regeneration (protein expression of proliferating cell nuclear antigen, p-STAT3, HGF, and VEGF and liver weights) than the siControl CM groups (p < .05). Taken together, hypoxic preconditioning of ASCs increased expression of mediators promoting anti-inflammatory and regenerative responses. The liver regenerative effects of HCM appear to be mediated by persistent and uninhibited expression of STAT3 in the liver, which results from decreased expression of SOCS3. In this study, it was found that treatment with the medium from hypoxic-preconditioned adipose-derived stem cells (ASCs) increased the viability of hepatotoxic hepatocytes and enhance liver regeneration in partially hepatectomized mice. In addition, the researchers first revealed that the hepatoprotective effects of hypoxic-conditioned media are mediated by persistent and uninhibited expression of signal transducer and activator of transcription 3 in the liver, which result from a decreased expression of suppressor of cytokine signaling 3. Therefore, the hypoxic preconditioning of ASCs is expected to play a crucial role in regenerative medicine by optimizing the production of a highly effective secretome from ASCs.

A therapeutic trial of human melanomas with combined small interfering RNAs targeting adaptor molecules p130Cas and paxillin activated under expression of ganglioside GD3

We previously demonstrated that focal adhesion kinase (FAK), p130Cas and paxillin are crucially involved in the enhanced malignant properties under expression of ganglioside GD3 in melanoma cells. Therefore, molecules existing in the GD3-mediated signaling pathway could be considered as suitable targets for therapeutic intervention in malignant melanoma. The aim of this study was to determine whether blockade of p130Cas and/or paxillin by RNAi suppresses melanoma growth. We found a suitable dose (40μM siRNA, 25μl/tumor) of the siRNA to suppress p130Cas in the xenografts generated in nu/nu mice. Based on these results, we performed intratumoral (i.t.) treatment with anti-p130Cas and/or anti-paxillin siRNAs mixed with atelocollagen as a drug delivery system in a xenograft tumor of a human melanoma cell line, SK-MEL-28. Mixture of atelocollagen (1.75%) and an siRNA (500 or 1000pmol/tumor) was injected into the tumors every 3days after the first injection. An siRNA against human p130Cas markedly suppressed tumor growth of the xenograft in a dose-dependent manner, whereas siRNA against human paxillin slightly inhibited the tumor growth. A control siRNA against firefly luciferase showed no effect. To our surprise, siRNA against human p130Cas (500 or 1000pmol/tumor) combined with siRNA against human paxillin dramatically suppressed tumor growth. In agreement with the tumor suppression effects of the anti-p130Cas siRNA, reduction in Ki-67 positive cell number as well as in p130Cas expression was demonstrated by immunohistostaining. These results suggested that blockade of GD3-mediated growth signaling pathways by siRNAs might be a novel and promising therapeutic strategy against malignant melanomas, provided signaling molecules such as p130Cas and paxillin are significantly expressed in individual cases. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

Comparison of proliferation, apoptosis and expression of syndecan-1 and α-SMA in edentulous ridge oral mucosa of successful and early failed submerged dental implants—An immunohistochemical study

AimsPathogenic mechanisms involved in early submerged implant failure are poorly understood. In this study we immunohistochemically analyse differences in proliferation, apoptosis and inflammation in edentulous ridge oral mucosa (ERM) of successful and early failed submerged implants. Materials and methods30 samples of ERM covering successful and early failed submerged implants were obtained at the end of osseointegration period along with control samples of healthy ERM. Sections were stained with Ki-67 (proliferation), caspase-3 (apoptosis) and syndecan-1 (epithelial marker). Percentage of positive cells was analysed by Kruskal-Wallis test and Dunn’s post hoc test. Co-localization of Ki-67 and caspase-3 with α-SMA, CD68 and TGF-β was done by double immunofluorescence. ResultsThere was no significant difference in number of Ki-67 positive cells within surface epithelium (SE) in all groups. Proliferation was significantly higher in underlying connective tissue (UCT) of ERM of early failed submerged implants (26%) compared to ERM of successful submerged implants (3%) and controls (4%). More apoptotic cells appeared in UCT of early failed submerged implants (8%) compared to UCT of successful submerged implants (4%) and UCT of control ERM (3%). Co-localization of Ki-67 and α-SMA in ERM of early failed submerged implants disclosed proliferating fibroblasts and pericytes of blood vessels. Macrophages and cells expressing TGF-β appeared in UCT of failed implants. Expression of syndecan-1 was significantly weaker in SE of early failed submerged implants. ConclusionsImbalance between proliferation and apoptosis, changes in syndecan-1 expression and inflammation are histopathological features of ERM of early failed submerged implants.

Bangle (Zingiber purpureum) Improves Spatial Learning, Reduces Deficits in Memory, and Promotes Neurogenesis in the Dentate Gyrus of Senescence-Accelerated Mouse P8.

Bangle (Zingiber purpureum) is a tropical ginger that is used as a spice in Southeast Asia. Phenylbutenoid dimers isolated from Bangle have exhibited neurotrophic effects in primary cultured rat cortical neurons and PC12 cells. Furthermore, chronic treatment with phenylbutenoid dimers enhances hippocampal neurogenesis in olfactory bulbectomized mice. In this study, we investigated the effects of Bangle extract on behavior and hippocampal neurogenesis in vivo. SAMP8 mice, which are an established model for accelerated aging, with age-related learning and memory impairments, were given a Bangle-containing diet for 1 month, and subsequent behavioral tests and immunohistochemistry for Ki67, a proliferating cell marker, were performed. We found that the Bangle-containing diet improved spatial learning and memory deficits in the Morris water maze and significantly increased the numbers of Ki67-positive cells in the dentate gyrus of the SAMP8 mice. In addition, the Bangle extract exhibited a neurotrophin-like activity as indicated by the induction of neurite sprouting in PC12 cells. Our results suggest that Bangle is beneficial for the prevention of age-related progression of cognitive impairment.

Survivin Modulates Squamous Cell Carcinoma-Derived Stem-Like Cell Proliferation, Viability and Tumor Formation in Vivo.

Squamous Cell Carcinoma-derived Stem-like Cells (SCC-SC) originate from alterations in keratinocyte stem cells (KSC) gene expression and sustain tumor development, invasion and recurrence. Since survivin, a KSC marker, is highly expressed in SCC-SC, we evaluate its role in SCC-SC cell growth and SCC models. Survivin silencing by siRNA decreases clonal growth of SCC keratinocytes and viability of total, rapidly adhering (RAD) and non-RAD (NRAD) cells from primary SCC. Similarly, survivin silencing reduces the expression of stem cell markers (OCT4, NOTCH1, CD133, β1-integrin), while it increases the level of differentiation markers (K10, involucrin). Moreover, survivin silencing improves the malignant phenotype of SCC 3D-reconstruct, as demonstrated by reduced epidermal thickness, lower Ki-67 positive cell number, and decreased expression of MMP9 and psoriasin. Furthermore, survivin depletion by siRNA in RasG12V-IκBα-derived tumors leads to smaller tumor formation characterized by lower mitotic index and reduced expression of the tumor-associated marker HIF1α, VEGF and CD51. Therefore, our results indicate survivin as a key gene in regulating SCC cancer stem cell formation and cSCC development.

The expression and significance of p53 protein and Ki-67 protein in pterygium.

Pterygium is considered to be a degenerative disease of the conjunctiva, however, the presence of tumor markers in pterygium reinforces the hypothesis that this lesion is similar to tumor. Inactivation of p53 function removes an obstacle to increased proliferation. Factors affecting the prevalence of p53 expression in pterygium deserve investigation. The aim of the study was to investigate the expression of p53 and Ki-67 proteins in pterygium and normal conjunctiva, the effects of gender and age on p53 expression, and the relationship between the expression of p53 and Ki-67 proteins. A total of 34 samples of pterygium and 34 samples of the normal conjunctiva were analyzed. The samples were studied by immunohistochemistry using antibodies against p53 and Ki-67. Totally 15 (44%) samples of pterygia were p53 positive. Correlations between the expression of p53 protein and sex, and age were not established. The number of Ki-67 positive cells in pterygium (9.74%) was significantly higher than the number of Ki-67 positive cells in the normal conjunctiva (1.74%), (P = 0.001). Between the expression of p53 protein and Ki-67 protein in pterygium there was a significant positive correlation (p = 0.000). The prevalence of p53 positive samples of pterygium was 44%. The influence of sex and age on p53 protein expression in pterygium was not found. The increased proliferative acivity was present in the epithelium of pterygium. The expression of Ki-67 protein is associated with the expression of p53 protein in pterygium. The findings of our study support the thesis of pterygium as tissue growth disorder.

Sirtuin-2 inhibition affects hippocampal functions and sodium butyrate ameliorates the reduction in novel object memory, cell proliferation, and neuroblast differentiation.

We investigated the effects of the sirtuin-2 (SIRT2) inhibitor AK-7 on novel object memory, cell proliferation, and neuroblast differentiation in the dentate gyrus. In addition, we also observed the relationships with sodium butyrate, a histone deacetylase inhibitor, on the hippocampal functions. To investigate the effects of AK-7 on hippocampal functions, 10-week-old C57BL/6 mice were daily injected intraperitoneally with 20 mg/kg AK-7 alone or in combination with subcutaneous administration of 300 mg/kg sodium butyrate, a histone deacetylase inhibitor, for 21 days. A novel object recognition test was conducted on days 20 (training) and 21 (testing) of treatment. Thereafter, the animals were sacrificed for immunohistochemistry for Ki67 (cell proliferation) and doublecortin (DCX, neuroblast differentiation). AK-7 administration significantly reduced the time spent exploring new objects, while treatment in combination with sodium butyrate significantly alleviated this reduction. Additionally, AK-7 administration significantly reduced the number of Ki67-positive cells and DCX-immunoreactive neuroblasts in the dentate gyrus, while the treatment in combination with sodium butyrate ameliorated these changes. This result suggests that the reduction of SIRT2 may be closely related to age-related phenotypes including novel object memory, as well as cell proliferation and neuroblast differentiation in the dentate gyrus. In addition, sodium butyrate reverses SIRT2-related age phenotypes.

Microbiota and bile acid profiles in retinoic acid-primed mice that exhibit accelerated liver regeneration.

All-trans Retinoic acid (RA) regulates hepatic lipid and bile acid homeostasis. Similar to bile acid (BA), RA accelerates partial hepatectomy (PHx)-induced liver regeneration. Because there is a bidirectional regulatory relationship between gut microbiota and BA synthesis, we examined the effect of RA in altering the gut microbial population and BA composition and established their relationship with hepatic biological processes during the active phases of liver regeneration. C57BL/6 mice were treated with RA orally followed by 2/3 PHx. The roles of RA in shifting gut microbiota and BA profiles as well as hepatocyte metabolism and proliferation were studied. RA-primed mice exhibited accelerated hepatocyte proliferation revealed by higher numbers of Ki67-positive cells compared to untreated mice. Firmicutes and Bacteroidetes phyla dominated the gut microbial community (>85%) in both control and RA-primed mice after PHx. RA reduced the ratio of Firmicutes to Bacteroidetes, which was associated with a lean phenotype. Consistently, RA-primed mice lacked transient lipid accumulation normally found in regenerating livers. In addition, RA altered BA homeostasis and shifted BA profiles by increasing the ratio of hydrophilic to hydrophobic BAs in regenerating livers. Accordingly, metabolic regulators fibroblast growth factor 21, Sirtuin1, and their downstream targets AMPK and ERK1/2 were more robustly activated in RA-primed than unprimed regenerating livers. Priming mice with RA resulted in a lean microbiota composition and hydrophilic BA profiles, which were associated with facilitated metabolism and enhanced cell proliferation.

Extracellular Matrix and Growth Factors Improve the Efficacy of Intramuscular Islet Transplantation.

BackgroundThe efficacy of intramuscular islet transplantation is poor despite being technically simple, safe, and associated with reduced rates of severe complications. We evaluated the efficacy of combined treatment with extracellular matrix (ECM) and growth factors in intramuscular islet transplantation.MethodsMale BALB/C mice were used for the in vitro and transplantation studies. The following three groups were evaluated: islets without treatment (islets-only group), islets embedded in ECM with growth factors (Matrigel group), and islets embedded in ECM without growth factors [growth factor-reduced (GFR) Matrigel group]. The viability and insulin-releasing function of islets cultured for 96 h were significantly improved in Matrigel and GFR Matrigel groups compared with the islets-only group.ResultsBlood glucose and serum insulin levels immediately following transplantation were significantly improved in the Matrigel and GFR Matrigel groups and remained significantly improved in the Matrigel group at postoperative day (POD) 28. On histological examination, significantly decreased numbers of TdT-mediated deoxyuridine triphosphate-biotin nick end labeling-positive islet cells and significantly increased numbers of Ki67-positive cells were observed in the Matrigel and GFR Matrigel groups at POD 3. Peri-islet revascularization was most prominent in the Matrigel group at POD 14.ConclusionsThe efficacy of intramuscular islet transplantation was improved by combination treatment with ECM and growth factors through the inhibition of apoptosis, increased proliferation of islet cells, and promotion of revascularization.

Effects of Asiatic Acid on Spatial Working Memory and Cell Proliferation in the Adult Rat Hippocampus.

Asiatic acid is a pentacyclic triterpene from Centella asiatica. Previous studies have reported that asiatic acid exhibits antioxidant and neuroprotective activities in cell culture. It also prevents memory deficits in animal models. The objective of this study was to investigate the relationship between spatial working memory and changes in cell proliferation within the hippocampus after administration of asiatic acid to male Spraque-Dawley rats. Control rats received vehicle (propylene glycol) while treated rats received asiatic acid (30 mg/kg) orally for 14 or 28 days. Spatial memory was determined using the novel object location (NOL) test. In animals administered asiatic acid for both 14 and 28 days, the number of Ki-67 positive cells in the subgranular zone of the dentate gyrus was significantly higher than in control animals. This was associated with a significant increase in their ability to discriminate between novel and familiar object locations in a novel object discrimination task, a hippocampus-dependent spatial memory test. Administration of asiatic acid also significantly increased doublecortin (DCX) and Notch1 protein levels in the hippocampus. These findings demonstrate that asiatic acid treatment may be a potent cognitive enhancer which improves hippocampal-dependent spatial memory, likely by increasing hippocampal neurogenesis.

Abstract B23: Pushing Myc inhibition towards the clinic by direct delivery of cell-penetrating peptides

Abstract Inhibiting Myc has long been regarded as a promising cancer treatment. However, clinical Myc inhibition was considered unfeasible due to its central role in normal proliferation and the difficulties of targeting a nuclear transcription factor. The expression of Omomyc (a Myc inhibitor derived from the dimerization and DNA-binding domain of Myc) in the KRasG12D non-small cell lung cancer (NSCLC) mouse model challenged these assumptions, as it resulted in dramatic tumor clearance with only limited and well tolerated side effects in normal tissues (Soucek et al., 2008 and 2013). Omomyc expression proved equally potent in several other mouse models of cancer, revealing the huge potential of this inhibitory approach against multiple cancer types including papilloma, pancreas and glioma (Soucek et al., 2004; Sodir et al., 2011; Annibali et al., 2014). Recently, Max*, a b-HLH-LZ peptide derived from Myc's obligate protein partner Max, was shown to spontaneously enter cells (Montagne et al., 2012). As Omomyc and Max* display high structural homology, we hypothesized that Omomyc could also behave as a cell-penetrating peptide and thus recapitulate the effects of its transgenic counterpart. Our preliminary results show that the Omomyc peptide is well folded in solution; it transduces into cancer cells and effectively stops their proliferation in a dose-dependent manner.In vivo, nasal instillation of fluorescently-labeled Omomyc peptide leads to its rapid distribution to lungs and brain, as well as to other organs (G.I. tract, liver), as observed by IVIS® imaging and immunohistochemistry. Finally, a short treatment with the Omomyc peptide reduces the tumor size and number of Ki67 positive cells in the KRasG12D-induced NSCLC mouse model. In summary, the Omomyc cell penetrating peptide represents a new opportunity to pharmacologically inhibit Myc in a variety of malignant diseases. Citation Format: Marie-Eve Beaulieu, Toni Jauset, Daniel Massó-Valles, Jonathan R. Whitfield, Erika Serrano del Pozo, Cynthia Tremblay, Loïka Maltais, Martin Montagne, Pierre Lavigne, Laura Soucek. Pushing Myc inhibition towards the clinic by direct delivery of cell-penetrating peptides. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B23.

Expression of Cell Membrane Antigens in Cells Excreted in the Urinary Sediment Predicts Progression of Renal Disease in Patients with Focal Segmental Glomerulosclerosis

Background/Aims: A link between the number of podocytes excreted in the urine and activity of glomerular disease has been established. The aim of this study was to investigate possible correlations between urinary cells' phenotype and the progression of focal segmental glomerulosclerosis (FSGS). Methods: Forty patients with newly diagnosed FSGS were included. Cells were isolated from urine by adherence to collagen-coated cover slips and assessed for the expression of podocalyxin (PDX), CD68 and Ki67 antigens by indirect immunofluorescence. In addition, double-staining procedures were performed in combinations of the above antigens plus cytokeratin, WT1 and CD-105. Twenty-two patients in whom urinary protein to creatinine ratio exceeded 2.0 at diagnosis were followed for 36 months, with assessments of renal function and proteinuria every 3 months. During observation, patients were subjected to standard therapy. Results: Significantly higher numbers of Ki67 positive cells at the onset of the study were observed in patients who have doubled serum creatinine (SCr) in follow-up, than in those who have not (p = 0.0149). By logistic regression analysis, both CD68 and Ki67, but not anti-PDX positive cell numbers at diagnosis were found to be predictors of doubling SCr concentration in 36 months' follow-up. Results of double staining indicate that PDX positive cells could be identified as podocytes or their precursors and parietal epithelial cells. Conclusion: Urinary sediment PDX positive cell numbers do not predict the progression of FSGS, whereas CD68 and Ki67 phenotype of urinary podocytic lineage clearly has a prognostic significance in 36 months' observation of primary FSGS.

Mitophagy defects arising from BNip3 loss promote mammary tumor progression to metastasis.

BNip3 is a hypoxia-inducible protein that targets mitochondria for autophagosomal degradation. We report a novel tumor suppressor role for BNip3 in a clinically relevant mouse model of mammary tumorigenesis. BNip3 delays primary mammary tumor growth and progression by preventing the accumulation of dysfunctional mitochondria and resultant excess ROS production. In the absence of BNip3, mammary tumor cells are unable to reduce mitochondrial mass effectively and elevated mitochondrial ROS increases the expression of Hif-1α and Hif target genes, including those involved in glycolysis and angiogenesis—two processes that are also markedly increased in BNip3-null tumors. Glycolysis inhibition attenuates the growth of BNip3-null tumor cells, revealing an increased dependence on autophagy for survival. We also demonstrate that BNIP3 deletion can be used as a prognostic marker of tumor progression to metastasis in human triple-negative breast cancer (TNBC). These studies show that mitochondrial dysfunction—caused by defects in mitophagy—can promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.