Number Of Ki67-positive Cells Research Articles

Differential effects of high-fat diets before pregnancy and/or during pregnancy on fetal growth development

AimsThe aim of the study was to investigate the effects of high-fat diets before pregnancy and/or during pregnancy on fetal development. Main methodsFemale mice were fed with standard diets (SD) or high-fat diets (HFD). After 12 weeks, females were mated. In the SD + SD and HFD + SD groups, pregnant mice were fed with standard diets. In the SD + HFD and HFD + HFD groups, pregnant mice were fed with high-fat diets. All pregnant mice were sacrificed on gestational day (GD) 16. Key findingsFetal weight and crown-rump length were increased in SD + HFD-fed mice, whereas were decreased in HFD + SD-fed mice. The levels of CRP and TNF-α in maternal serum and amniotic fluid were elevated in all HFD-fed mice. Placenta weight was elevated in SD + HFD-fed but not in HFD + SD-fed mice. Blood sinusoid areas, and the number of Ki67-positive cells, a marker of cell proliferation, were elevated in placental labyrinth layer of SD + HFD-fed mice, but decreased in HFD + SD-fed mice. Finally, placental Fatp1, a fatty acid transporter gene, was up-regulated in SD + HFD-fed mice. By contrary, placental Fatp1, and Snat2, an amino acid transporter, were down-regulated in HFD + SD-fed mice. Moreover, the levels of placental FATP4 and SNAT2 were up-regulated in SD + HFD-fed mice. SignificanceHFD before pregnancy and HFD during pregnancy differentially disturb fetal growth development. HFD before pregnancy-induced fetal SGA might be partially attributed to inflammatory cytokines and mediators derived from maternal adipose tissue. By contrary, HFD during pregnancy-induced fetal overweight may be partially attributed to the increase of placental nutrient transport capacity.

Abstract 052: Role of Dopamine D2 Receptors in The Regulation of Cell Proliferation in Renal Injury

The dopamine D2 receptor (D2R) in the kidney has a direct and significant role in blood pressure (BP) control and in negatively regulating the mechanisms involved in the development of inflammation and injury. Impaired D2R function results in renal inflammation and end organ damage. Injury to the kidney triggers a cell proliferation response that can be adaptive, leading to repair of the tubular epithelium, or maladaptive, leading to fibrosis and chronic kidney disease. We investigated the role of renal D2Rs in the cell proliferation response in models of renal injury in mice and human renal proximal tubule cells (hRPTCs). Renal selective silencing of the D2R in mice resulted in an increase in the mRNA expression of the proliferation marker Ki-67 (2.8±0.8 vs 1.0±0.1 fold; qRT-PCR; P<0.05; n=4-5) along with an increase in the number of proliferating cells in renal cortical slices determined by nuclear Ki-67 staining (36±3 vs 6±1; positive cells/ field; P<0.05) in comparison with mice with no silencing. Rescue of D2R function in these mice by the ureteral infusion of AAV carrying a D2R vector reduced the expression of cortical Ki-67 mRNA (42±3 vs 100±5 %; P<0.05; n=4) and the number of positive cells/field (16±2 vs 32±5; P<0.05) in comparison with mice treated with a control AAV. In mice subjected to renal ischemia reperfusion KI-67 mRNA expression was higher (1.5±0.03 vs 1.0±0.05 fold; P<0.05; n=3-4) than in mice with sham operation. Treatment with AAV carrying D2R after ischemic injury reduced Ki-67 expression (0.65±0.05 vs 1±0.1; P<0.05) and the number of Ki-67 positive cells in kidney sections. Rescue of function or D2R overexpression reduced tissue damage, fibrosis and the increase in BP in these models. Treatment of hRPTCs with the nephrotoxic, aristolochic acid (5 μg/ml, 24h), increased mRNA Ki-67 expression (2.8±0.2 fold; P<0.05) in comparison with cells treated with vehicle while the presence in the medium of a D2R agonist, quinpirole (1μM, 24h), reduced the increase by 42%. The D2R agonist also reduced the increase in fibronectin 1 and collagen 1a1 elicited by aristolochic acid. Our data suggest that regulation of cell proliferation is one of the mechanisms involved in the protective effect of D2R function on renal inflammation and injury.

Sirt1 alleviates LPS induced inflammation of periodontal ligament fibroblasts via downregulation of TLR4

This study was aimed to investigate the role of Sirt1 in LPS-injured periodontal ligament fibroblast (PDLF), to evaluate potential functions of Sirt1 in the pathogenesis of periodontitis. Sirt1 overexpression significantly increased cell viability, Ki67 positive cell number and PCNA expression in LPS-injured PDLF, and vice versa. Cell apoptosis was significantly decreased, and the release of pro-inflammatory cytokines (IL-1α, IL-6, IL-8 and TNF-α) was significantly reduced when Sirt1 was overexpressed. Sirt1 overexpression down-regulated toll-like receptor 4 (TLR4), and inhibited the JNK/NF-κB pathways; while knockdown of Sirt1 up-regulated TLR4, and activated JNK/NF-κB pathways. To conclude, Sirt1 alleviates inflammation of PDLF induced by LPS through down-regulation of TLR4 and inactivation of JNK/NF-κB pathways.

Abstract 1388: Pharmacologic targeting of DNA methylation blocks breast cancer growth and metastasis

Abstract Abnormal DNA methylation is a hallmark of cancer which orchestrates changes in gene transcription seen in cancer. Both hypermethylation-mediated inactivation of tumor suppressor genes and hypomethylation-mediated activation of pro-metastatic genes are common attributes of cancer cells which make the methylome an attractive anti-cancer therapeutic target. Interestingly, in contrast to genetic changes, DNA methylation-mediated epigenetic changes are potentially reversible by either dietary supplements or therapeutic strategies. Consequently, a wide variety of epigenetic drugs primarily targeting DNA hypermethylation has been shown to be effective in preclinical and clinical settings. Pioneering works done by us have shown that treatment of various human cell lines (breast, prostate, osteosarcoma) with a methylating agent S-adenosyl methionine (SAM) can block tumor growth and metastasis in vivo. At the molecular level, SAM treatment induces hypermethylation of promoters of key pro-metastatic genes; and thereby inhibits their expression. However, the anti-cancer effect of SAM has never been examined. Based on the heterogeneity of tumor cells which are at different stages of tumor invasiveness, we hypothesized that treatment with demethylating (Decitabine) and methylating agents (SAM) would collectively lead to the activation of tumor suppressor genes and suppression of pro-metastatic genes to block cancer growth and metastasis. In the current study, we first investigated the effects of Decitabine and SAM alone and in combination to prevent breast cancer development, growth, and metastasis using the MDA-MB-231 xenograft model of breast cancer. Our data showed that treatment with Decitabine and SAM resulted in a significant delay in the progression of mammary tumors in experimental animals compared to controls, effects which were significantly more pronounced when Decitabine and SAM were administered in a combination setting. Gene expression analysis of the cancer cells revealed that SAM-treatment repressed the expression of several key genes involved in cancer progression. In addition, immunohistochemical analysis of primary tumors revealed that the combination treatment (Decitabine, SAM) reduced the number of Ki67 positive cells as well reduced the expression of angiogenesis marker CD-31. Further studies examining the effects of combined therapy on genome-wide gene expression changes as well as any potential side effects on animal behavior and toxicity will be presented and discussed. Results from this study will provide compelling evidence and rationale for the initiation of clinical trials with SAM alone as a monotherapy and in combination with currently approved epigenetic drugs (Decitabine) to reduce breast cancer-associated morbidity and mortality. Citation Format: Niaz Mahmood, Ani Arakelian, William J. Muller, Moshe Szyf, Shafaat A. Rabbani. Pharmacologic targeting of DNA methylation blocks breast cancer growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1388.

Involvement of Prokineticin 2–expressing Neutrophil Infiltration in 5-Fluorouracil–induced Aggravation of Breast Cancer Metastasis to Lung

Adjuvant chemotherapy is used for human breast cancer patients, even after curative surgery of primary tumor, to prevent tumor recurrence primarily as a form of metastasis. However, anticancer drugs can accelerate metastasis in several mouse metastasis models. Hence, we examined the effects of postsurgical administration with 5-fluorouracil (5-FU), doxorubicin, and cyclophosphamide, on lung metastasis process, which developed after the resection of the primary tumor arising from the orthotopic injection of a mouse triple-negative breast cancer cell line, 4T1. Only 5-FU markedly increased the numbers and sizes of lung metastasis foci, with enhanced tumor cell proliferation and angiogenesis as evidenced by increases in Ki67-positive cell numbers and CD31-positive areas, respectively. 5-FU-mediated augmented lung metastasis was associated with increases in intrapulmonary neutrophil numbers and expression of neutrophilic chemokines, Cxcl1 and Cxcl2 in tumor cells, with few effects on intrapulmonary T-cell or macrophage numbers. 5-FU enhanced Cxcl1 and Cxcl2 expression in 4T1 cells in a NFκB-dependent manner. Moreover, the administration of a neutrophil-depleting antibody or a Cxcr2 antagonist, SB225002, significantly attenuated 5-FU-mediated enhanced lung metastasis with depressed neutrophil infiltration. Furthermore, infiltrating neutrophils and 4T1 cells abundantly expressed prokineticin-2 (Prok2) and its receptor, Prokr1, respectively. Finally, the administration of 5-FU after the resection of the primary tumor failed to augment lung metastasis in the mice receiving Prokr1-deleted 4T1 cells. Collectively, 5-FU can enhance lung metastasis by inducing tumor cells to produce Cxcl1 and Cxcl2, which induced the migration of neutrophils expressing Prok2 with a capacity to enhance 4T1 cell proliferation. Mol Cancer Ther; 17(7); 1515-25. ©2018 AACR.

CRISPR/Cas9 nickase mediated targeting of urokinase receptor gene inhibits neuroblastoma cell proliferation.

Neuroblastoma is a tumor arising from pluripotent sympathoadrenal precursor cells of neural cell origin. Neuroblastoma is one of the most aggressive childhood tumors with highly invasive and metastatic potential. The increased expression of urokinase and its receptor is often associated with a negative prognosis in neuroblastoma patients.We have shown that targeting of the Plaur gene in mouse neuroblastoma Neuro 2A cells by CRISPR/Cas9n results in ~60% decrease in cell proliferation (p<0.05), reduction in the number of Ki-67 positive cells, caspase 3 activation and PARP-1 cleavage. Knockout of uPAR leads to downregulation of mRNA encoding full-length TrkC receptor, which is involved in p38MAPK and Akt signalling pathways. This finding provides a rationale to study a role of uPAR in neuroblastoma progression, since uPAR could be considered a potential therapeutic target in neuroblastoma treatment.

PO-196 Vav1 and Mutant K-Ras synergize in pancreatic ductal adenocarcinoma development: lessons from mouse models

Introduction The overall 5 year survival rate of Pancreatic Ductal Adenocarcinoma (PDAC) is less than 5% and has remained stubbornly unchanged long time ago, despite the efforts in preclinical and clinical science. PDAC, the main form of pancreatic cancer, develops via acinar-ductal metaplasia (ADM) and neoplastic precursor lesions, such as pancreatic intraepithelial neoplasia (PanIN). Mutant K-Ras is present in >90% of PDAC and is the most frequent and the earliest genetic alteration, being found in low-grade lesions. Identification of other molecular lesions that affect PDAC is of cardinal importance. One such potential protein is Vav1, a hematopietic specific signal transducer. Overexpression of wild-type Vav1 is implicated in human cancers, such as neuroblastoma, lung and PDAC. The expression of Vav1 in PDAC is indicative of a worse survival rate. Our goal was to determine whether Vav1 plays a causative or facilitatory role in-vivo in PDAC development. Material and methods We generated several transgenic mouse models that express Vav1, K-Ras G12D , or Vav1 and K-Ras G12D in the pancreas. K-Ras was induced by tamoxifen and Vav1 by Dox. Pancreata were analysed at different times post transgenes induction. Results and discussions Vav1 Expression together with K-Ras G12D in the pancreas has a dramatic synergistic effect enhancing ADM formation already at 3 months post transgene induction, resulting in at least 3 times the number of lesions in the pancreata of Vav1;K-Ras G12D mice compared to K-Ras G12 mice. No lesions were observed in the pancreas of Vav1 mice. The number of Ki-67 positive cells in Vav1;K-Ras G12D mice was significantly higher than in Vav1 or K-Ras G12D transgenic mice. The increase of pancreatic lesions was also accompanied with an increase in staining of Sox9 and Keratin and various pathways such as pERK, pEGFR and Rac1-GTP., highlighting the synergistic effect of Vav1 and K-Ras G12D in the development of PDAC. Notably, removal of Dox, thus ablating the expression of Vav1 in the pancreas of Vav1;K-Ras G12D led to a significant reduction in malignant lesions, thus further highlighting the necessity of expression of both oncogenes to cancer development in the pancreas. Conclusion Vav1 contributes to the development of malignant lesions in the pancreas when expressed with mutant K-Ras. Identification of Vav1 as a protein that synergizes with mutant K-Ras in PDAC development might pave the way to choosing good candidates for therapeutic drug design.

Effect of Gestational and Lactational Exposure to DEHP, DINP, and DEP on Intestinal Morphology, Disaccharidases, and Alkaline Phosphatase in Rats during Postnatal Development.

The diesters of 1,2-benzenedicarboxylic acid (phthalic acid), commonly known as phthalates, are used primarily as plasticizers of polyvinyl chloride and as additives in consumer and personal care products. This study was designed to evaluate the impact of in utero and postnatal exposure to diisononyl phthalate (DINP), diethylhexyl phthalate (DEHP), and diethyl phthalate (DEP) on gut maturation in a Wistar rat model. Pregnant females were gavaged from day 8 of gestation through postnatal day (pd) 30 with 0 (vehicle control), DEHP (380 mg/kg/d), DINP (380 mg/kg/d), or DEP (800 mg/kg/d) dissolved in corn oil. Intestinal samples have been collected at 0, 7, 14, 21, and 30 pd for histological and biochemical analysis. The mitotic index has been evaluated based on the expression of Ki-67 antigen. All tested phthalate treatments have significantly decreased the body as well as the organ's weight (p < 0.001). DINP exposure resulted in severe villous atrophy, while DEHP treated group was characterized by lymphoepithelial lesions. In addition, a significant decrease of the Ki-67 proliferation index was observed in the youngest rats (0 and 7 days) upon the various treatments (p < 0.0001): , whereas at day 30, an increased numbers of Ki-67 positive cells were observed in DEHP and DEP but bot DINP group. Lactase and sucrase activities were inhibited by DEP in contrast to DINP and DEHP which increased enzymes activity (p < 0.05). Our results suggest that exposure to phthalates during gestational and lactational phases negatively impacts the development of the small intestine.

Investigation of c-KIT and Ki67 expression in normal, preneoplastic and neoplastic canine prostate

Backgroundc-KIT expression has been related to bone metastasis in human prostate cancer, but whether c-KIT expression can be similarly classified in canine prostatic tissue is unknown. This study assessed c-KIT and Ki67 expression in canine prostate cancer (PC). c-KIT gene and protein expression and Ki67 expression were evaluated in forty-four canine prostatic tissues by immunohistochemistry, RT-qPCR and western blot. Additionally, we have investigated c-KIT protein expression by immunoblotting in two primary canine prostate cancer cell lines.ResultsEleven normal prostates, 12 proliferative inflammatory atrophy (PIA) prostates, 18 PC, 3 metastatic lesions and two prostate cancer cell cultures (PC1 and PC2) were analysed. The prostatic tissue exhibited varying degrees of membranous, cytoplasmic or membranous/cytoplasmic c-KIT staining. Four normal prostates, 4 PIA and 5 prostatic carcinomas showed positive c-KIT expression. No c-KIT immunoexpression was observed in metastases. Canine prostate cancer and PIA samples contained a higher number of Ki67-positive cells compared to normal samples. The median relative quantification (RQ) for c-KIT expression in normal, PIA and prostate cancer and metastatic samples were 0.6 (0.1-2.5), 0.7 (0.09-2.1), 0.7 (0.09-5.1) and 0.1 (0.07-0.6), respectively. A positive correlation between the number of Ki67-positive cells and c-KIT transcript levels was observed in prostate cancer samples. In the cell line, PC1 was negative for c-KIT protein expression, while PC2 was weakly positive.ConclusionThe present study identified a strong correlation between c-KIT expression and proliferative index, suggesting that c-KIT may influence cell proliferation. Therefore, c-KIT heterogeneous protein expression among the samples (five positive and thirteen negative prostate cancer samples) indicates a personalized approach for canine prostate cancer.

Determination of The Properties of Rat Brain Thermostable Protein Complex which Inhibit Cell Proliferation.

Objective Cell proliferation is known to be controlled by many networks of regulatory proteins. These multiple and complicated mechanisms of control are still being investigated. The aim of the present study is to determine the different properties of adult rat brain thermostable protein complex (TPC) which affect cell proliferation. Materials and Methods This experimental study used brain, kidney and liver tissue from adult (150-170 g) and adolescent (7, 10, 21, 28 days) white rats, adult pigeons and mice. Brain TPC was isolated by alcohol extraction, and primary antibodies Ki67 and GAD65/67 were used for immunohistochemistry, evaluation of transcriptional activity of the tissues and determination of the mitotic index. Results The results show that brain TPC from rats reversibly decreases cell proliferation by inhibiting transcription. The evidence suggests that TPC is not species-specific, but expresses tissue specificity with regards to terminally differentiated cells. Rat brain TPC inhibits mitotic activity of the progenitor cells in the dentate gyrus of adolescent rats, and corresponding with this decrease in the mitotic index the number of Ki67 positive cells increases. Simultaneously, the number of GAD65/67-positive cells in the hippocampus decreases by approximately threefold. ConclusionThese results indicate that rat brain TPC causes the reversible suppression of cell proliferation through the inhibition of transcription. Inhibitory effects of rat brain TPC leads to an increase the number of cells in the cell cycle, in tissues of adolescent rats.

Injectable Mussel-Inspired Immobilization of Platelet-Rich Plasma on Microspheres Bridging Adipose Micro-Tissues to Improve Autologous Fat Transplantation by Controlling Release of PDGF and VEGF, Angiogenesis, Stem Cell Migration.

Platelets-rich plasma (PRP) can produce growth factors (GFs) to improve angiogenesis. However, direct injection of PRP does not lead to highly localized GFs. The current study employs a mussel-inspired polydopamine to immobilize PRP on gelatin microspheres (GMs) with the purpose of bridging adipose micro-tissues to help implanted fat survive (GM-pDA-PRP). Enhanced PRP adhesion leads to a prolonged and localized production of GFs, which is verified by platelet counting and by ELISA of vascular endothelial growth factors (VEGFs) and of platelet derived growth factors (PDGFs). The GM-pDA-PRP "hatches" a microenvironment for the proliferation of adipose-derived stem cells. After the adipose micro-tissue has bridged with GM-pDA-PRP after 16 weeks, triple-fluorescence staining reveals that the mature adipocytes, blood vessels, and capillaries are arranged like in normal adipose tissue. The survival fat increases significantly compared to that in control, PRP, and GM-PRP groups (84.8 ± 11.4% versus 47.8 ± 8.9%, 56.9 ± 9.7%, and 60.2 ± 10.5%, respectively). Both histological assessments and CD31 immunofluorescence indicate that the improvement of angiogenesis in GM-pDA-PRP is higher than in the fat graft group (6.4-fold in quantitative CD31 positive cells). The CD34 positive cells in the GM-pDA-PRP group are around 3.5-fold the amount in the fat graft group, which suggests that more stem cells migrate to the implant area. Cell proliferation staining shows that the number of Ki67 positive cells is around five times as high as that in the fat graft group.

Determination of optimized oxygen partial pressure to maximize the liver regenerative potential of the secretome obtained from adipose-derived stem cells

BackgroundA hypoxic-preconditioned secretome from stem cells reportedly promotes the functional and regenerative capacity of the liver more effectively than a control secretome. However, the optimum oxygen partial pressure (pO2) in the cell culture system that maximizes the therapeutic potential of the secretome has not yet been determined.MethodsWe first determined the cellular alterations in adipose tissue-derived stem cells (ASCs) cultured under different pO2 (21%, 10%, 5%, and 1%). Subsequently, partially hepatectomized mice were injected with the secretome of ASCs cultured under different pO2, and then sera and liver specimens were obtained for analyses.ResultsOf all AML12 cells cultured under different pO2, the AML12 cells cultured under 1% pO2 showed the highest mRNA expression of proliferation-associated markers (IL-6, HGF, and VEGF). In the cell proliferation assay, the AML12 cells cultured with the secretome of 1% pO2 showed the highest cell proliferation, followed by the cells cultured with the secretome of 21%, 10%, and 5% pO2, in that order. When injected into the partially hepatectomized mice, the 1% pO2 secretome most significantly increased the number of Ki67-positive cells, reduced serum levels of proinflammatory mediators (IL-6 and TNF-α), and reduced serum levels of liver transaminases. In addition, analysis of the liver specimens indicated that injection with the 1% pO2 secretome maximized the expression of the intermediate molecules of the PIP3/Akt and IL-6/STAT3 signaling pathways, all of which are known to promote liver regeneration.ConclusionsThe data of this study suggest that the secretome of ASCs cultured under 1% pO2 has the highest liver reparative and regenerative potential of all the secretomes tested here.

Histamine Iontophoresis as in vivo Model to Study Human Skin Inflammation with Minimal Barrier Impairment: Pilot Study Results of Application of the Model to a Sensitive Skin Panel

Background/Aims: Histamine iontophoresis is known to elicit itch and a wheal-and-flare reaction; however, its impact on the skin barrier and underlying compartments has not been thoroughly evaluated yet. The primary objective of this study was to characterize that using immunohistochemistry, biophysical measurements, and image analysis, and secondly, to explore whether skin reactions to this model differ in sensitive skin (SS). Methods: Eighteen healthy subjects, n = 9 with SS and n = 9 with non-sensitive skin (NSS), were included based on a perception-based questionnaire. Histamine iontophoresis was performed on the buttock, and skin reactions were evaluated up to 72 h after stimulation. Results: The wheal-and-flare peaked at 30 min; after 8 h, no clinical signs were visible. No signs of disruption of the stratum corneum, as well as no increase in the number of Ki67-positive cells emerged, whereas fewer tryptase-positive mast cells and increased epidermal thickness were observed at 1 and 72 h, respectively. SS subjects showed higher perception of itch compared to NSS subjects. Conclusion: Histamine iontophoresis is a well-standardized in vivo model to quantitatively study the early stages of cutaneous inflammation with minimal impact on the skin barrier. In line with previous studies, it highlighted increased sensory perceptions in SS.

Metformin suppresses cancer initiation and progression in genetic mouse models of pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-associated mortality worldwide with an overall five-year survival rate less than 7%. Accumulating evidence has revealed the cancer preventive and therapeutic effects of metformin, one of the most widely prescribed medications for type 2 diabetes mellitus. However, its role in pancreatic cancer is not fully elucidated. Herein, we aimed to further study the preventive and therapeutic effects of metformin in genetically engineered mouse models of pancreatic cancer.MethodsLSL-KrasG12D/+; Pdx1-Cre (KC) mouse model was established to investigate the effect of metformin in pancreatic tumorigenesis suppression; LSL-KrasG12D/+; Trp53fl/+; Pdx1-Cre (KPC) mouse model was used to evaluate the therapeutic efficiency of metformin in PDAC. Chronic pancreatitis was induced in KC mice by peritoneal injection of cerulein.ResultsFollowing metformin treatment, pancreatic acinar-to-ductal metaplasia (ADM) and mouse pancreatic intraepithelial neoplasia (mPanIN) were decreased in KC mice. Chronic pancreatitis induced a stroma-rich and duct-like structure and increased the formation of ADM and mPanIN lesions, in line with an increased cytokeratin 19 (CK19)-stained area. Metformin treatment diminished chronic pancreatitis-mediated ADM and mPanIN formation. In addition, it alleviated the percent area of Masson’s trichrome staining, and decreased the number of Ki67-positive cells. In KPC mice, metformin inhibited tumor growth and the incidence of abdominal invasion. More importantly, it prolonged the overall survival.ConclusionsMetformin inhibited pancreatic cancer initiation, suppressed chronic pancreatitis-induced tumorigenesis, and showed promising therapeutic effect in PDAC.

Abstract 4041: SUV39H2 inhibition enhances sensitivity of breast cancer cells to doxorubicin through downregulation of γ-H2AX production

Abstract Accumulating evidence has demonstrated the biological importance of protein methyltransferases in human tumorigenesis, and several small molecular inhibitors targeting these enzymes have been developed. Moreover, inhibiting pathways involved in DNA repair has been shown to enhance the cytotoxicity of DNA-damaging agents. Herein, we report the development of a potent SUV39H2 inhibitor (OTS193320) that decreases global histone H3 lysine 9 tri-methylation (H3K9me3) levels in cancer cells and attenuates cancer cell proliferation, and suppresses the tumor growth in mouse xenograft models. SUV39H2 (Suppressor of variegation 3-9 homolog 2), is a protein methyltransferase known to methylate histone H3 at lysine 9 (H3K9), and recently reported to methylate histone H2AX at lysine 134, which enhances the accumulation of phosphorylated H2AX (γ-H2AX) and regulates the DNA repair pathway in human cancer. SUV39H2 is highly expressed in many cancer types, including lung and breast cancers, while its expression levels are restricted to testis in normal adult tissues. Exposure of OTS193320 to two triple negative breast cancer cell lines attenuated H3K9me3 levels in a dose-dependent manner and triggered apoptotic cell death. Combination of OTS193320 and doxorubicin (DOX) resulted in reduction of γ-H2AX levels compared to single agent DOX, as visualized on western blot and immunocytochemical analysis. Furthermore, combination therapy attenuated the levels of p53-binding protein 1, which is reported to co-localize with γ-H2AX foci, compared to single agent DOX. Cell viability assays demonstrated a significant growth suppressive effect when OTS193320 was combined with DOX, compared to single agent treatment of either drug, suggesting chemosensitization to DOX. Importantly, in a mouse xenograft model of A549 lung cancer cells, we observed a tumor growth inhibition of 60.8% at day 14 with 25mg/kg intravenous administration of the inhibitor, without significant body weight loss or toxicity. Immunohistochemical staining of tumors treated with the inhibitor demonstrated a significantly lower number of Ki-67 positive cells and attenuated distribution patterns of H3K9me3 compared to the control tumors. Collectively, our results demonstrate that SUV39H2 inhibition may be a promising approach to develop a novel class of anti-cancer treatment. Citation Format: Theodore Vougiouklakis, Vassiliki Saloura, Yo Matsuo, Yusuke Nakamura. SUV39H2 inhibition enhances sensitivity of breast cancer cells to doxorubicin through downregulation of γ-H2AX production [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4041. doi:10.1158/1538-7445.AM2017-4041

Deficiency of NALP3 Signaling Impairs Liver Regeneration After Partial Hepatectomy.

Inflammatory response is required to proceed the optimal liver regeneration after liver injury. Recent reports demonstrated that inflammasomes are involved in the innate immune response. Several NOD-Like receptors (NLRs) participated in the formation of the inflammasomes. NACHT, LRR, and PYD domain-containing protein 3 (NALP3) belongs to the NLR families and recognizes adenosine triphosphate (ATP), crystals, and reactive oxygen species. The present study examined the effect of inflammasomes on the process of liver regeneration using NALP3 knockout (KO) mice. The activation of inflammasomes in the liver was induced after 70% partial hepatectomy (PHx). The liver-to-body weight ratio was significantly decreased in NALP3-KO mice compared to that in WT mice after PHx. The number of Ki67-positive cells and the expression of Cyclin D1 and E1 after PHx were reduced in NALP3-KO mice compared to WT mice. The expression of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) were decreased in the remnant liver of NALP3-KO mice compared to WT mice. Flow cytometric analysis revealed that the expression of chemokines was decreased and the accumulation of CD11b-positive cells was suppressed in NALP3-KO mice after PHx. The treatment with ATP, which is a ligand to NALP3, increased the liver-to-body weight ratio in WT mice. These results indicate that NALP3 signaling is required for the induction of inflammatory response and the development of liver regeneration after PHx.

MiR-203 Interplays with Polycomb Repressive Complexes to Regulate the Proliferation of Neural Stem/Progenitor Cells.

SummaryThe polycomb repressive complexes 1 (PRC1) and 2 (PRC2) are two distinct polycomb group (PcG) proteins that maintain the stable silencing of specific sets of genes through chromatin modifications. Although the PRC2 component EZH2 has been known as an epigenetic regulator in promoting the proliferation of neural stem/progenitor cells (NSPCs), the regulatory network that controls this process remains largely unknown. Here we show that miR-203 is repressed by EZH2 in both embryonic and adult NSPCs. MiR-203 negatively regulates the proliferation of NSPCs. One of PRC1 components, Bmi1, is a downstream target of miR-203 in NSPCs. Conditional knockout of Ezh2 results in decreased proliferation ability of both embryonic and adult NSPCs. Meanwhile, ectopic overexpression of BMI1 rescues the proliferation defects exhibited by miR-203 overexpression or EZH2 deficiency in NSPCs. Therefore, this study provides evidence for coordinated function of the EZH2-miR-203-BMI1 regulatory axis that regulates the proliferation of NSPCs.

Thiamine and benfotiamine prevent stress-induced suppression of hippocampal neurogenesis in mice exposed to predation without affecting brain thiamine diphosphate levels.

Thiamine is essential for normal brain function and its deficiency causes metabolic impairment, specific lesions, oxidative damage and reduced adult hippocampal neurogenesis (AHN). Thiamine precursors with increased bioavailability, especially benfotiamine, exert neuroprotective effects not only for thiamine deficiency (TD), but also in mouse models of neurodegeneration. As it is known that AHN is impaired by stress in rodents, we exposed C57BL6/J mice to predator stress for 5 consecutive nights and studied the proliferation (number of Ki67-positive cells) and survival (number of BrdU-positive cells) of newborn immature neurons in the subgranular zone of the dentate gyrus. In stressed mice, the number of Ki67- and BrdU-positive cells was reduced compared to non-stressed animals. This reduction was prevented when the mice were treated (200mg/kg/day in drinking water for 20days) with thiamine or benfotiamine, that were recently found to prevent stress-induced behavioral changes and glycogen synthase kinase-3β (GSK-3β) upregulation in the CNS. Moreover, we show that thiamine and benfotiamine counteract stress-induced bodyweight loss and suppress stress-induced anxiety-like behavior. Both treatments induced a modest increase in the brain content of free thiamine while the level of thiamine diphosphate (ThDP) remained unchanged, suggesting that the beneficial effects observed are not linked to the role of this coenzyme in energy metabolism. Predator stress increased hippocampal protein carbonylation, an indicator of oxidative stress. This effect was antagonized by both thiamine and benfotiamine. Moreover, using cultured mouse neuroblastoma cells, we show that in particular benfotiamine protects against paraquat-induced oxidative stress. We therefore hypothesize that thiamine compounds may act by boosting anti-oxidant cellular defenses, by a mechanism that still remains to be unveiled. Our study demonstrates, for the first time, that thiamine and benfotiamine prevent stress-induced inhibition of hippocampal neurogenesis and accompanying physiological changes. The present data suggest that thiamine precursors with high bioavailability might be useful as a complementary therapy in several neuropsychiatric disorders.

Diallyl trisulfide suppresses tumor growth through the attenuation of Nrf2/Akt and activation of p38/JNK and potentiates cisplatin efficacy in gastric cancer treatment.

Diallyl trisulfide (DATS), a garlic organosulfide, has shown excellent chemopreventive potential. Cisplatin (DDP) is widely used to treat solid malignant tumors, but causing serious side effects. In the current study, we attempted to elucidate the chemopreventive mechanisms of DATS in human gastric cancer BGC-823 cells in vitro, and to investigate whether DATS could enhance the anti-tumor efficacy of DDP and improve quality of life in BGC-823 xenograft mice in vivo. Treatment with DATS (25-400 μmol/L) dose-dependently inhibited the viability of BGC-823 cells in vitro with an IC50 of 115.2±4.3 μmol/L after 24 h drug exposure. DATS (50-200 μmol/L) induced cell cycle arrest at G2/M phase in BGC-823 cells, which correlated with significant accumulation of cyclin A2 and B1. DATS also induced BGC-823 cell apoptosis, which was accompanied by the modulation of Bcl-2 family members and caspase cascade activation. In BGC-823 xenograft mice, administration of DATS (20-40 mg·kg-1·d-1, ip) dose-dependently inhibited tumor growth and markedly reduced the number of Ki-67 positive cells in tumors. Interestingly, combined administration of DATS (30 mg·kg-1·d-1, ip) with DDP (5 mg/kg, every 5 d, ip) exhibited enhanced anti-tumor activity with fewer side effects. We showed that treatment of BGC-823 cells with DATS in vitro and in vivo significantly activated kinases such as p38 and JNK/MAPK and attenuated the Nrf2/Akt pathway. This study provides evidence that DATS exerts anticancer effects and enhances the antitumor efficacy of DDP, making it a novel candidate for adjuvant therapy for gastric cancer.

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.