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Introduction: Follicular thyroid carcinoma (FTC) is more aggressive than the most common papillary thyroid carcinoma (PTC). However, the current research on FTC is less than PTC. Here, we investigated the effects of long noncoding RNA (lncRNA) GAS5 and miR-221-3p in FTC. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to detect GAS5 and miR-221-3p expression in the FTC tissues and cells. Cell proliferation was assessed by CCK8 and EdU assays. Flow cytometry was performed to determine the cell cycle. The dual-luciferase reporter assay was employed to validate the binding relationship of GAS5/miR-221-3p and miR-221-3p/cyclin-dependent kinase inhibitor 2B (CDKN2B). Western blot was conducted to measure the protein level of CDKN2B. Results: Our results displayed that GAS5 was downregulated, while miR-221-3p was upregulated in FTC tissues and cells. What’s more, overexpression of GAS5 or miR-221-3p inhibition induced G0/G1 phase arrest and inhibited cell proliferation of FTC cells. GAS5 acted as a sponge of miR-221-3p, and CDKN2B was a target gene of miR-221-3p. Additionally, GAS5 inhibited cell cycle and proliferation of FTC cells via reducing miR-221-3p expression to enhance CDKN2B expression. Conclusion: GAS5 induced G0/G1 phase arrest and inhibited cell proliferation via targeting miR-221-3p/CDKN2B axis in FTC. Thus, GAS5 may be a potential therapeutic target for the treatment of FTC.

The amyloid precursor protein (APP) intracellular domain (AICD) is a metabolic by-product of APP produced through sequential proteolytic cleavage by α-, β-, and γ-secretases. The interaction between AICD and Fe65 has been reported to impair adult neurogenesis in vivo. However, the exact role of AICD in mediating neural stem cell fate remains unclear. To identify the role of AICD in neuronal proliferation and differentiation, as well as to clarify the molecular mechanisms underlying the role of AICD in neurogenesis, we first generated a mouse model expressing the Rosa26-based AICD transgene. AICD overexpression did not alter the spatiotemporal expression pattern of full-length APP or accumulation of its metabolites. In addition, AICD decreased the newly generated neural progenitor cell (NPC) pool, inhibited the proliferation and differentiation efficiency of NPCs, and increased cell death both in vitro and in vivo. Given that abnormal neurogenesis is often associated with depression-like behavior in adult mice, we conducted a forced swim test and tail suspension test with AICD mice and found a depression-like behavioral phenotype in AICD transgenic mice. Moreover, AICD stimulated FOXO3a transcriptional activation, which in turn negatively regulated AICD. In addition, functional loss of FOXO3a in NPCs derived from the hippocampal dentate gyrus of adult AICD transgenic mice rescued neurogenesis defects. AICD also increased the mRNA expression of FOXO3a target genes related to neurogenesis and cell death. These results suggest that FOXO3a is the functional target of AICD in neurogenesis regulation. Our study reveals the role of AICD in mediating neural stem cell fate to maintain homeostasis during brain development via interaction with FOXO3a.

Background Ligustrazine, active ingredients extracted from the natural herb Ligusticum Chuanxiong Hort, has promising anti-tumor properties on tumor cell lines. However, the potential anti-tumor activity of ligustrazine on colorectal cancer (CRC) cells and the molecular mechanisms have not been elucidated. In this study, we explored the critical functions of ligustrazine on SW480 and CT26 cells at cellular levels. Methods CCK-8 assay was performed to analyze the cell viability. Flow cytometry analysis was applied to study cell apoptosis and cell cycle. The expressions of cell apoptosis and cell cycle-associated proteins were conducted by western blot and qRT-PCR analysis. Results Ligustrazine showed significant inhibitory effects on the proliferation of SW480 and CT26 cells. Ligustrazine induced cell apoptosis was associated with the up-regulation of pro-apoptotic protein and the down-regulation of anti-apoptotic protein in an activated mitochondrial-dependent pathway. And it indicated that ligustrazine induced cell cycle arrest by changing the cell cycle distribution, which leads to cell cycle arrest at the G0/G1 phase. Besides, the ligustrazine-induced cell apoptosis and cell cycle arrest were markedly reversed by pifithrin-α (p53 inhibitor), which suggested that ligustrazine-induced cell apoptosis was achieved by regulating p53-dependent mitochondrial pathway and cell cycle arrest at the G0/G1 phase. Conclusions These findings demonstrated that ligustrazine could induce SW480 and CT26 cells apoptosis via a p53-dependent mitochondrial pathway and cell cycle arrest at the G0/G1 phase. Ligustrazine may serve as a potential anti-cancer agent for CRC.

BACKGROUND Many findings have shown that pyruvate kinase type M2 (PKM2) plays crucial roles in regulating the occurrence and development of various human cancers; however, its roles in ovarian cancer oncogenesis remain to be determined. MATERIAL AND METHODS The expression intensity of PKM2 in ovarian cancer tissues was examined by immunohistochemistry (IHC), and was then correlated to patient clinicopathologic characteristics. The roles of PKM2 in ovarian cancer cell proliferation, growth, and survival were examined by CCK-8, colony forming, and flow cytometry assays. The potentially involved molecular were then investigated by Western blot analysis. RESULTS IHC results showed that PKM2 was overexpressed in 100 of 114 (87.7%) serous ovarian cancer tissues as compared with 50 cases of non-cancerous ovarian tissues, and was associated with tumor size ≥7.5 cm and

The underlying molecular mechanism of the increased bone mass phenotype in Tricho-dento-osseous (TDO) syndrome remains largely unknown. Our previous study has shown that the TDO point mutation c.533A>G, Q178R in DLX3 could increase bone density in a TDO patient and transgenic mice partially through delaying senescence in bone marrow mesenchymal stem cells (BMSCs). In the present study, we provided a new complementary explanation for TDO syndrome: the DLX3 (Q178R) mutation increased BMSCs proliferation through H19/miR-675 axis. We found that BMSCs derived from the TDO patient (TDO-BMSCs) had stronger proliferation ability than controls by clonogenic and CCK-8 assays. Next, experiments of overexpression and knockdown of wild-type DLX3 via lentiviruses in normal BMSCs confirmed the results by showing its negative role in cell proliferation. Through validated high-throughput data, we found that the DLX3 mutation reduced the expression of H19 and its coexpression product miR-675 in BMSCs. Function and rescue assays suggested that DLX3, long noncoding RNA H19, and miR-675 are negative factors in modulation of BMSCs proliferation as well as NOMO1 expression. The original higher proliferation rate and the expression of NOMO1 in TDO-BMSCs were suppressed after H19 restoration. Collectively, it indicates that DLX3 regulates BMSCs proliferation through H19/miR-675 axis. Moreover, the increased expression of NOMO1 and decreased H19/miR-675 expression in DLX3 (Q178R) transgenic mice, accompanying with accrual bone mass and density detected by micro-CT, further confirmed our hypothesis. In summary, we, for the first time, demonstrate that DLX3 mutation interferes with bone formation partially through H19/miR-675/NOMO1 axis in TDO syndrome.

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin’s lymphoma in the adult population, and treatment of DLBCL is still unfavorable. Therefore, there is an urgent requirement to investigate the molecular mechanisms underlying DLBCL tumorigenesis. To study the potential function of microRNA-155 (miR-155) involved in the regulation of lymphoma, we monitored lymphoma cell behavior including proliferation, cell cycle, and apoptosis using CCK-8 and flow cytometry analysis. Real-time PCR was used to detect the expression levels of miR-155 in 118 lymphoma patients’ tissues, and Western blot was also used to analyze the expression level of proteins correlated with cell cycle and apoptosis in lymphoma cells. miR-155 expression levels were higher in lymphoma tissues compared with adjacent tissues. Downregulation of miR-155 inhibited lymphoma cell progress by arresting cell cycle in the G0/G1 phase and promoting apoptosis. Cell cycle-correlated proteins (cyclin B1, cyclin D1, and CDK4) were inhibited by downregulation of miR-155. Apoptosis-correlated proteins level (Bax/Bcl-2 and caspase 3 activity) were increased by downregulation of miR-155. In addition, a significant inverse correlation between the level of miR-155 and transforming growth factor-β receptor 2 (TGFBR2) was observed, which has been demonstrated to be a novel tumor suppressor gene. A further in vivo tumor formation study in nude mice indicated that downregulation of miR-155 in lymphoma cells delayed the progress of tumor formation. These findings indicate that miR-155 may serve as a useful potential target for the treatment of lymphoma.

There is considerable interest in the use of drugs and other methods for protecting implanted neural stem cells (NSCs) from the adverse environment of injured tissue for successful cell therapy. Resveratrol can modify cardiac stem cells to enhance their survival and differentiation, however, its effect and the mechanism underlying its neuroprotective effect on NSCs following stroke remain to be fully elucidated. Nuclear factor erythroid 2-related factor 2 (Nrf-2) signaling is important in antioxidative stress, and the role of Nrf-2 signaling in the enhanced neuroprotection of NSCs by resveratrol following stroke also remains to be elucidated. In the present study, NSCs were pretreated with resveratrol prior to oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. The survival, apoptosis and proliferation of the NSCs were assessed using an MTT assay, Hoechst 33258 staining of nuclei and flow cytometry, respectively. In addition, the activity of superoxide dismutase (SOD), level of malondiadehyde (MDA) and content of glutathione (GSH) were determined. The protein expressions levels of Nrf-2, NAD(P)H:quinone oxidoreductase 1 (NQO-1), and heme oxygenase 1 (HO-1) were detected using western blot analysis. It was found that resveratrol markedly enhanced NSC survival and proliferation, decreased apoptosis and the levels of MDA, and increased the activity of SOD and content of GSH in a concentration-dependent manner following OGD/R injury in vitro. In addition, the protein expression levels of Nrf2, HO-1 and NQO1 were significantly upregulated. These findings suggested that resveratrol attenuated injury and promoted proliferation of the NSCs, at least in part, by upregulating the expression of Nrf2, HO-1 and NQO1 following OGD/R injury in vitro.

SIRT5 has a wide range of functions in different cellular processes such as glycolysis, TCA cycle and antioxidant defense, which mediates lysine desuccinylation, deglutarylation and demalonylation. Recent evidences have implicated that SIRT5 is a potential suppressor of gastric cancer (GC). However, the underlying mechanism of SIRT5 in gastric cancer is still unclear. Here, we show that SIRT5 expression is significantly decreased in human GC tissues. Functional analysis demonstrates that SIRT5 inhibits cell growth in vitro and in vivo, arrests the cell cycle in G1/S transition, and suppresses migration and invasion of GC cells via regulating epithelial-to-mesenchymal transition. Mechanistically, we demonstrate that there is the direct interaction between SIRT5 and 2-oxoglutarate dehydrogenase (OGDH), and desuccinylation of OGDH by SIRT5 inhibits the activity of OGDH complex. Further studies of the relationship between SIRT5 and OGDH show OGDH inhibition by succinyl phosphonate (SP) or siRNA suppresses the increase in cell growth and migration induced by SIRT5 deletion. Moreover, SIRT5 decreases mitochondrial membrane potential (ΔΨm), ATP products and increases the ROS levels and NADP/NADPH ratio in GC cells through the inhibition of OGDH complex activity. Therefore, SIRT5 suppresses GC cell growth and migration through desuccinylating OGDH and inhibiting OGDH complex activity to disturb mitochondrial functions and redox status.

Long intergenic non-coding RNA No.668 (LINC00668) is implicated in the development of various malignancies. However, the role of LINC00668 and underlying mechanism in colorectal cancer (CRC) remains totally unknown. The expression pattern of LINC00668 in CRC cells were determined by qRT-PCR. CCK-8, EdU incorporation, flow cytometry, Transwell, and wound-healing assays were run to evaluate the functions of LINC00668 in CRC cells. Bioinformatics analyses were used to identify the LINC00668-specific binding with miRNAs that were screened by RNA pull-down. RNA immunoprecipitation and luciferase gene report assay were performed to confirm the interaction between miR-188-5p and LINC00668 in CRC cells. LINC00668 was significantly upregulated in CRC tissues and cells. Knockdown of LINC00668 suppressed cell proliferation and migration potential and induced cell apoptosis, but inhibition of miR-188-5p which was predicted to bind with LINC00668 reversed these effects. Furthermore, USP47 was a direct target of miR-188-5p, and overexpression of USP47 attenuated LINC00668 knockdown-induced tumor suppressive effects in CRC cells. Conclusively, our findings demonstrated that lncRNA LINC00668 acted as an oncogenic role in CRC cells by sponging miR-188-5p and upregulating USP47 and may represent a potential marker for CRC patients.

The advantage of using in utero electroporation is that it can study the gene function during neurodevelopment in vivo. Using functional analysis of a microRNA (miRNA) gene as an example, this protocol describes a set of techniques that are crucial for the success of neurogenesis studies, including mice time mating, plasmid preparation, utero electroporation following miRNA injection into mice embryonic brain ventricle, labeling of proliferating cells with EDU (ethynyldeoxyuridine), cryosectioning, immunofluorescence staining, and confocal microscopic analysis. This chapter also provides detailed technical tips regarding experimental planning, mouse surgery, multi-embryo injection with different plasmids, electroporation, and maintenance of pregnant mother with post-electroporated embryo.

Purpose: Epidemiologic studies indicate that dietary factors, such as coffee, may influence breast cancer and modulate hormone receptor status. The purpose of this translational study was to investigate how coffee may affect breast cancer growth in relation to estrogen receptor-α (ER) status. Experimental Design: The influence of coffee consumption on patient and tumor characteristics and disease-free survival was assessed in a population-based cohort of 1,090 patients with invasive primary breast cancer in Sweden. Cellular and molecular effects by the coffee constituents caffeine and caffeic acid were evaluated in ER+ (MCF-7) and ER− (MDA-MB-231) breast cancer cells. Results: Moderate (2–4 cups/day) to high (≥5 cups/day) coffee intake was associated with smaller invasive primary tumors (Ptrend = 0.013) and lower proportion of ER+ tumors (Ptrend = 0.018), compared with patients with low consumption (≤1 cup/day). Moderate to high consumption was associated with lower risk for breast cancer events in tamoxifen-treated patients with ER+ tumors (adjusted HR, 0.51; 95% confidence interval, 0.26–0.97). Caffeine and caffeic acid suppressed the growth of ER+ (P ≤ 0.01) and ER− (P ≤ 0.03) cells. Caffeine significantly reduced ER and cyclin D1 abundance in ER+ cells. Caffeine also reduced the insulin-like growth factor-I receptor (IGFIR) and pAkt levels in both ER+ and ER− cells. Together, these effects resulted in impaired cell-cycle progression and enhanced cell death. Conclusions: The clinical and experimental findings demonstrate various anticancer properties of caffeine and caffeic acid against both ER+ and ER− breast cancer that may sensitize tumor cells to tamoxifen and reduce breast cancer growth. Clin Cancer Res; 21(8); 1877–87. ©2015 AACR.

Pathogenic leucine-rich repeat kinase 2 (LRRK2) mutations are recognized as the most common cause of familial Parkinson's disease in certain populations. Recently, LRRK2 mutations were shown to be associated with a higher risk of hormone-related cancers. However, how LRRK2 itself contributes to cancer risk remains unknown. DNA damage causes cancer, and DNA damage responses are among the most important pathways in cancer biology. To understand the role of LRRK2 in DNA damage response pathway, we induced DNA damage by applying genotoxic stress to the cells with Adriamycin. We found that DNA damage enhances LRRK2 phosphorylation at Serine 910, Serine 935 and Serine 1292. We further showed that LRRK2 phosphorylation is abolished in the absence of ATM, suggesting that LRRK2 phosphorylation requires ATM. It should also be noted that LRRK2 interacts with ATM. In contrast, overexpression or knockdown of LRRK2 does not affect ATM phosphorylation, indicating that LRRK2 is the downstream target of ATM in response to DNA damage. Moreover, we demonstrated that LRRK2 increases the expression of p53 and p21 by increasing the Mdm2 phosphorylation in response to DNA damage. Loss-of-function in LRRK2 has the opposite effect to that of LRRK2. In addition, FACS analysis revealed that LRRK2 enhances cell cycle progression into S phase in response to DNA damage, a finding that was confirmed by 5-bromo-2'-deoxyuridine immunostaining. Taken together, our findings demonstrate that LRRK2 plays an important role in the ATM-Mdm2-p53 pathway that regulates cell proliferation in response to DNA damage.

Although, 5-Fluorouracil (5-FU) remains widely used in adjuvant therapy in patients with colon cancer, resistance to 5-FU-based chemotherapy is an important reason for treatment failure. Recent studies have reported that an enhanced reactive oxygen species (ROS) scavenging system shows drug resistance to 5-FU. Peroxiredoxin-2 (PRDX2), is an important member of the ROS scavenging system, and may be a potential target that promotes chemosensitivity to 5-FU in colon cancer. Here, we depleted PRDX2 by PRDX2-shRNA-LV transduction in two colon cancer cell lines and found that in vitro PRDX2 knockdown facilitates cell death, and apoptosis in 5-FU-treated colon cancer cells. In addition, we found that PRDX2 depletion in mice treated with 5-FU resulted in, inhibition of tumor growth, compared with mice treated with 5-FU alone. Our data also suggested that the PI3K/AKT signaling pathway links PRDX2 with 5-FU-induced apoptosis in colon cancer. Furthermore, when PRDX2 was overexpressed in colon cancer cells, we found increased p-AKT protein expression and reduced Bcl-2/Bax protein expression. PRDX2 and p-AKT protein expression were analyzed by immunohistochemistry technology in human colon carcinoma tissues. Pearson correlation coefficient is 0.873 and P

Prostate cancer is a highly penetrant disease among men in industrialized societies, but the factors regulating the transition from indolent to aggressive and metastatic cancer remain poorly understood. We found that men with prostate cancers expressing high levels of the G protein–coupled receptor LGR4 had a significantly shorter recurrence-free survival compared with patients with cancers having low LGR4 expression. LGR4 expression was elevated in human prostate cancer cell lines with metastatic potential. We therefore generated a novel transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model to investigate the role of Lgr4 in prostate cancer development and metastasis in vivo. TRAMP Lgr4−/− mice exhibited an initial delay in prostate intraepithelial neoplasia formation, but the frequency of tumor formation was equivalent between TRAMP and TRAMP Lgr4−/− mice by 12 weeks. The loss of Lgr4 significantly improved TRAMP mouse survival and dramatically reduced the occurrence of lung metastases. LGR4 knockdown impaired the migration, invasion, and colony formation of DU145 cells and reversed epithelial–mesenchymal transition (EMT), as demonstrated by up-regulation of E-cadherin and decreased expression of the EMT transcription factors ZEB, Twist, and Snail. Overexpression of LGR4 in LNCaP cells had the opposite effects. Orthotopic injection of DU145 cells stably expressing shRNA targeting LGR4 resulted in decreased xenograft tumor size, reduced tumor EMT marker expression, and impaired metastasis, in accord with our findings in TRAMP Lgr4−/− mice. In conclusion, we propose that Lgr4 is a key protein necessary for prostate cancer EMT and metastasis.

Objectives The purpose of this study was to investigate the role of a novel mutant DLX3 on the odontogenic differentiation of human dental pulp cells (hDPCs) in tricho-dento-osseous (TDO) syndrome. Design hDPCs were obtained from the healthy premolars, stably-expressing wild-type DLX3 (WT), novel mutant DLX3 (Mu) and control vector (NC) cells were generated using recombinant lentiviruses. The proliferation rates of WT-hDPCs and Mu-hDPCs were measured by CCK8 assay. Odonto-differentiation of hDPCs was assessed by alkaline phosphatase (ALP) activity assay, and mineralization ability was assessed by Alizarin red staining. Odontogenic markers, including DMP-1, DSPP, Nes, ALP, and DLX5, were analyzed using real-time polymerase chain reaction (qPCR). DMP-1 and DSPP expressions were further confirmed by Western blotting. Results CCK8 results showed that the novel mutant DLX3 decreased the proliferation rate of hDPCs compared with wild-type DLX3. qPCR showed that the novel mutant DLX3 weakened odontogenic differentiation by downregulating the expression of odontogenic genes. These results were further confirmed by Western blotting and ALP activity assay. Additionally, Alizarin red staining showed that the novel mutant DLX3 decreased the mineralization of hDPCs compared with wild-type DLX3. Conclusions Novel de novo mutation of DLX3 significantly decreases the proliferation rate and inhibits the odontogenic differentiation and mineralization of hDPCs, suggesting that this novel mutation of DLX3 can influence the dentinogenesis in TDO syndrome.

The BCR-ABL kinase inhibitor, imatinib mesylate, is the front-line treatment for chronic myeloid leukemia, but the emergence of imatinib resistance has led to the search for alternative drug treatments. There is a pressing need, therefore, to develop and test novel drugs. Natural products including plants, microorganisms, and halobios provide rich resources for discovery of anticancer drugs. In this article, we demonstrate that emodin inhibited the growth of K562 cells harboring BCR-ABL in vitro and in vivo, and induced abundant apoptosis, which was correlated with the inhibition of PETN/PI3K/Akt level and deletion of BCR-ABL. These findings suggest that emodin is a promising agent to kill K562 cells harboring BCR-ABL.

Breast cancer progression is associated with loss of estrogen receptor (ER-α), often due to epigenetic silencing. IGFBP genes have consistently been identified among the most common to be aberrantly methylated in tumours. To understand the impact of losing IGFBP-3 tumour expression via DNA methylation, we treated four breast cancer cell lines (MCF-7, T47D, Hs578T and MDA-MB-231) with a DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine (AZA) to determine IGFBP-3's role in the effects of AZA on total cell number and survival relative to changes in the ER. AZA induced cell growth inhibition, death and a reduction in the formation of colonies, despite increasing ER-α expression in ER-negative cells but reducing ER-α in ER-positive cells. Regardless of the differential effects on the ER-α, AZA consistently increased the abundance of IGFBP-3 and negating this increase in IGFBP-3 with siRNA reduced the AZA-induced growth inhibition and induction of cell death and virtually negated the AZA-induced inhibition of colony formation. With ER-α positive cells AZA increased the abundance of the tumour suppressor gene, p53 and induced demethylation of the IGFBP-3 promoter, whereas with ER negative cells, AZA epigenetically increased the transcription factor AP2-α, which when silenced prevented the increase in IGFBP-3. IGFBP-3 plays an important role in the anti-tumorigenic effects of AZA on breast cancer cells.

Summary Objective Pro-inflammatory cytokines play an important role in inducing cartilage degradation during osteoarthritis pathogenesis. Muscle is a tissue that lies near cartilage in situ . However, muscle's non-loading biochemical effect on cartilage has been largely unexplored. Here, we tested the hypothesis that muscle cells can regulate the response to pro-inflammatory cytokine-mediated damage in chondrocytes derived from human bone marrow-derived mesenchymal stem cells (hMSCs). Method hMSCs were allowed to undergo chondrogenic differentiation in porous silk scaffolds in the typical chondrogenic medium for 12 days. For the next 9 days, the cells were cultured in chondrogenic medium containing 50% conditioned medium derived from C2C12 muscle cells or fibroblast control cells, and were subject to treatments of pro-inflammatory cytokines IL-1β or TNFα. Results Both IL-1β and TNFα-induced strong expression of multiple MMPs and hypertrophic markers Runx2 and type X collagen. Strikingly, culturing hMSC-derived chondrocytes in C2C12 muscle cell-conditioned medium strongly inhibited the expression of all these genes, a result further confirmed by GAG content and histological evaluation of matrix protein. To determine whether these effects were due to altered chondrocyte growth and survival, we assayed the expression of cell proliferation marker Ki67, cell cycle arrest markers p21 and p53, and apoptosis marker caspase 3. Muscle cell-conditioned medium promoted proliferation and inhibited apoptosis, thereby suggesting a possible decrease in the cellular aging and death that typically accompanies cartilage inflammation. Conclusion Our findings suggest the role of muscle in cartilage homeostasis and provide insight into designing strategies for promoting resistance to pro-inflammatory cytokines in hMSC-derived chondrocytes.

Neuronal progenitor cells (NPCs) derived from human embryonic stem cells (hESCs) are an excellent cell source for transplantation therapy due to their availability and ethical acceptability. However, the traditional method of expansion and differentiation of hESCs into NPCs in monolayer cultures requires a long time, and the cell yield is low. A microcarrier (MC) platform can improve the expansion of hESCs and increase the yield of NPCs. In this study, for the first time, we transplanted microcarrier-expanded hESC-derived NPCs into the striatum of adult NOD-SCID IL2Rgc null mice, either as single cells or as cell aggregates. The recipient mice were perfused, and the in vivo survival, differentiation, and targeted innervation of the transplanted cells were assessed by immunostaining. We found that both the transplanted single NPCs and aggregate NPCs were able to survive 1 month posttransplantation, as revealed by human-specific neural cell adhesion molecule (NCAM) and human nuclear antigen staining. Compared to the single cells, the transplanted cell aggregates showed better survival over a 3-month period. In addition, both the transplanted single NPCs and the aggregate NPCs were able to differentiate into DCX-positive immature neurons and Tuj1-positive neurons in vivo by 1 month posttransplantation. However, only the transplantation of aggregate NPCs was shown to result in mature neurons at 3 months posttransplantation. Furthermore, we found that the cell aggregates were able to send long axons to innervate their targets. Our study provides preclinical evidence that the use of MCs to expand and differentiate hESC-derived NPCs and transplantation of these cells as aggregates produce longer survival in vivo.

During the development, tight regulation of the expansion of neural progenitor cells (NPCs) and their differentiation into neurons is crucial for normal cortical formation and function. In this study, we demonstrate that microRNA (miR)-128 regulates the proliferation and differentiation of NPCs by repressing pericentriolar material 1 (PCM1). Specifically, overexpression of miR-128 reduced NPC proliferation but promoted NPC differentiation into neurons both in vivo and in vitro. In contrast, the reduction of endogenous miR-128 elicited the opposite effects. Overexpression of miR-128 suppressed the translation of PCM1, and knockdown of endogenous PCM1 phenocopied the observed effects of miR-128 overexpression. Furthermore, concomitant overexpression of PCM1 and miR-128 in NPCs rescued the phenotype associated with miR-128 overexpression, enhancing neurogenesis but inhibiting proliferation, both in vitro and in utero. Taken together, these results demonstrate a novel mechanism by which miR-128 regulates the proliferation and differentiation of NPCs in the developing neocortex. DOI: http://dx.doi.org/10.7554/eLife.11324.001, eLife digest The neurons that transmit information around the brain develop from cells called neural progenitor cells. These cells can either divide to form more progenitor cells or to become specific types of neurons. If these carefully regulated processes go wrong – for example, if progenitors fail to stop dividing in order to mature – a range of neurodevelopmental conditions may develop, including autism spectrum disorders. Small RNA molecules called microRNAs control gene activity and protein formation by targeting certain other RNA molecules for destruction. One such microRNA, called miR-128, helps newly formed neurons to move to the correct region of the cortex – the outer layer of the brain, which is essential for many cognitive processes including thought and language. However, it was not clear whether miR-128 plays any other roles in the development of neurons. Zhang, Kim et al. have now analysed the role of miR-128 in the developing cortex of mice. The findings suggest that miR-128 prevents cortical neural progenitor cells from dividing and supports their development into more specialized cells. Causing miR-128 to be over-produced in the progenitor cells caused the cells to divide less often and encouraged them to mature into neurons. Conversely, removing miR-128 from the progenitor cells caused them to divide more and resulted in fewer neurons forming. Further investigation revealed that miR-128 works by causing less of a protein called PCM1 to be produced. Without this protein, cells cannot divide properly. Future studies could now investigate in more detail how miR-128 and PCM1 affect how the neurons in the cortex develop and work. DOI: http://dx.doi.org/10.7554/eLife.11324.002

Statins are being widely used for the therapy and prevention of several types of tumors, including human chronic myelogenous leukemia, but the underlying molecular mechanisms still remain unknown. Therefore, inhibition of cell proliferation, apoptosis and involved molecules were investigated in K562 cells after incubation with simvastatin.The results showed that simvastatin diminished K562 cell proliferation and induced apoptosis. At the same time, the level of reactive oxygen species (ROS) and intracellular calcium concentration increased. Furthermore, nitric oxide (NO) content and inducible NO synthase (iNOS) mRNA expression were significantly higher in the simvastatin-treated group than in the corresponding control group. The elevated ROS level and intracellular calcium concentration, enhanced mRNA expression of iNOS and total NO content might be responsible for the apoptotic and anti-proliferative effects of simvastatin in K562 cells.

We investigated role of beta-endorphin (END), which is released by immobilization stress, on intimal fibromuscular proliferation in a rat model of arterial remodeling after intimal injury. The endothelium of the abdominal aorta of Wistar-Kyoto rats was denuded, and the rats were subjected to immobilization stress (6 h/d), which raised the serum concentration of END, and intraperitoneal administration of either END (20 ng/kg/d) or naltrexone (NAL: 4 mg/kg/d). The proliferative activity (PA) of medial smooth muscle cells (SMCs) and the intima/media area ratio (R) were determined at 3 and 14 d after denudation, respectively. PA and R were significantly reduced by immobilization (PA: 64.8%, R: 34.6%), and NAL treatment completely reversed the decreases in PA and R. On the other hand, END reduced both PA and R (PA: 21.7% and R: 24.9%), and NAL also reversed the decreases in PA and R. END (20 pg/mL) inhibited both the proliferation (79% at 96 h) and migration (26%) of SMCs cultured with 5% fetal bovine serum in vitro, and NAL (100 microg/mL) reversed the inhibition of both activities. Our results suggest that immobilization stress stimulates the release of endogenous END, which then prevents both proliferation and migration of medial SMCs after intimal injury.

Among frequent events in prostate cancer are loss of the tumor-suppressor phosphatase and tensin homologue (PTEN) and overexpression of prostate-specific G-protein-coupled receptor (PSGR), but the potential tumorigenic synergy between these lesions is unknown. Here, we report a new mouse model (PSGR-Pten(Δ/Δ)) combining prostate-specific loss of Pten with probasin promoter-driven PSGR overexpression. By 12 months PSGR-Pten(Δ/Δ) mice developed invasive prostate tumors featuring Akt activation and extensive inflammatory cell infiltration. PSGR-Pten(Δ/Δ) tumors exhibited E-cadherin loss and increased stromal androgen receptor (AR) expression. PSGR overexpression increased LNCaP proliferation, whereas PSGR short hairpin RNA knockdown inhibited proliferation and migration. In conclusion, we demonstrate that PSGR overexpression synergizes with loss of PTEN to accelerate prostate cancer development, and present a novel bigenic mouse model that mimics the human condition, where both PSGR overexpression and loss of PTEN occur concordantly in the majority of advanced prostate cancers, yielding an environment more relevant to studying human prostate cancer.

To construct a single-chain human anti-EGFR antibody (scFv) and truncated protamine (tP) fusion protein, ScFv/tP, carrying small interfering (si)RNA directed against the heat shock protein Hsp47, a collagen-binding glycoprotein, in order to evaluate the role Hsp47 in apoptosis of hepatic stellate cells.A single chain of the human variable fragment was obtained by phage display and fused with the tP gene and with or without (negative control) the Hsp47 siRNA sequences. Following expression and purification of the scFv/tP fusion protein and the scFv/tPHsp47 siRNA fusion protein, internalization capabilities were tested in isolated human hepatic stellate cells and the QSG-7701 human hepatocyte cells with visualization by immunofluorescent staining. The DNA binding ability of the fusion proteins were verified by gel shift assay.Following ScFv/tP-Hsp47 siRNA fusion protein transfection into the human hepatic stellate cells, the levels of Hsp47 mRNA and protein expression were tested by RT-PCR and Western blotting; in addition, effects of siRNA-mediated silencing of Hsp47 on cell proliferation and apoptosis were analyzed by the cell counting kit (CCK)-8, flow cytometry and Western blot detection of the apoptosis marker poly (ADP-ribose) polymerase (PARP).Indirect immunofluorescence revealed that the ScFv/tP fusion proteins were internalized into human hepatic stellate cells but not into the QSG-7701 cells.The ScFv/tP-Hsp47 siRNA fusion protein caused reduced expression of Hsp47 mRNA and protein expression in the human hepatic stellate cells, as well as increased the cells' apoptosis remarkably.The ScFv/tP fusion protein can be used as a transfection reagent to deliver Hsp47 siRNA into hepatic stellate cells and to mediate apoptosis via blockade of Hsp47 expression.

The aim of this study is to investigate the growth-inhibitory and apoptosis-inducing effect of deferoxamine (DFO) combined with arsenic trioxide (ATO) on the human HL-60 xenografts in nude mice and its mechanism.The highly tumorigenic leukemia cell line HL-60 cells were inoculated subcutaneously into nude mice to establish a human leukemia xenograft model. The HL-60 xenograft nude mice models were randomly divided into four groups: control (Normal saline, NS), 50mg/kg DFO, 3mg/kg ATO, the combined treatment (50mg/kg DFO+1.5mg/kg ATO) once HL-60 cells were inoculated. Tumor sizes, growth curves, inhibitory rates, cell apoptosis, and the expression of apoptosis related markers were measured to evaluate the tumor growth.Xenografted tumors were observed in all nude mice since the 5th day of inoculation. The inhibitory rates of tumor weight were 2.67%, 10.69%, and 25.57% in DFO, ATO and combination therapy groups, respectively. The combination of DFO with ATO induces significantly more tumor cell apoptosis than either agent alone (p0.05). The expression of NF-κBp65 and survivin proteins decreased significantly while the expression of Caspase-3 and Bax increased in the combination therapy group (p0.05). Double immunofluorescence for Caspase-3 and NFκBp65 demonstrated an inverse relationship between Caspase-3-positive areas and NFκBp65-positive areas, as well as the co-localization of Bax and survivin in xenografted tumor cells.Combination of DFO and ATO has synergistic effects on tumor growth inhibition and apoptosis-inducing in vivo with no significant side effects. The DFO and ATO can up-regulate the expression of Caspase-3 and Bax, and down-regulate the expression of NF-κBp65 and survivin, especially for their combination.

Drug resistance is a major reason for therapy failure in cancer. Clitocine is a natural amino nucleoside isolated from mushroom and has been shown to inhibit cancer cell proliferation in vitro. In this study, we observed that clitocine can effectively induce drug-resistant human cancer cell apoptosis in vitro and inhibit tumor xenograft growth in vivo. Clitocine treatment inhibited drug-resistant human cancer cell growth in vitro in a dose- and time-dependent manner. Biochemical analysis revealed that clitocine-induced tumor growth inhibition is associated with activation of caspases 3, 8 and 9, PARP cleavage, cytochrome c release and Bax, Bak activation, suggesting that clitocine inhibits drug-resistant cancer cell growth through induction of apoptosis. Analysis of apoptosis regulatory genes indicated that Mcl-1 level was dramatically decreased after clitocine treatment. Over-expression of Mcl-1 reversed the activation of Bax and attenuated clitocine-induced apoptosis, suggesting that clitocine-induced apoptosis was at least partially by inducing Mcl-1 degradation to release Bax and Bak. Consistent with induction of apoptosis in vitro, clitocine significantly suppressed the drug-resistant hepatocellular carcinoma xenograft growth in vivo by inducing apoptosis as well as inhibiting cell proliferation. Taken together, our data demonstrated that clitocine is a potent Mcl-1 inhibitor that can effectively induce apoptosis to suppress drug-resistant human cancer cell growth both in vitro and in vivo, and thus holds great promise for further development as potentially a novel therapeutic agent to overcome drug resistance in cancer therapy.

Imperatorin is a small molecule nature compound isolated from the root of Angelica dahurica , and has been shown to exhibit multiple bioeffector functions, including anti-cancer activity. However, the molecular mechanism underlying imperatorin in suppression of tumor growth is unknown. In this study, we aimed at elucidating the molecular mechanisms underlying imperatorin function and determining the efficacy of imperatorin in suppression of drug-resistant human liver cancer. We observed that imperatorin suppresses tumor cell growth through inducing apoptosis, and imperatorin is more effective in induction of multidrug-resistant human liver cancer cells in vitro . We further determined that imperatorin induces apoptosis through both extrinsic and intrinsic apoptosis pathway. At the molecular level, we identified Mcl-1 as the molecular target of imperatorin and determined that imperatorin induces protesome-dependent Mcl-1 degradation to release Bak and Bax to trigger apoptosis in liver cancer cells. Consistent with its in vitro apoptosis induction activity, imperatorin exhibited potent activity against multidrug-resistant liver cancer xenograft growth in vivo . Taken together, we determined that imperatorin is a Mcl-1 degradation inducer that can effectively suppress multidrug-resistant human liver cancer growth in vivo , and thus holds great promise for development as an effective small molecule anti-cancer agent in human liver cancer therapy to overcome drug resistance.

In breast tumors IGF binding protein-2 (IGFBP-2) is elevated, and the presence of IGFBP-2 has been shown to correlate with malignancy. However, how IGFBP-2 contributes to the malignant state is still unclear. Silencing IGFBP-2 blocked cell proliferation and in MCF-7 cells increased cell death, indicating that IGFBP-2 was acting in both a mitogenic and a survival capacity. Exogenous IGFBP-2 acting via integrin receptors to reduce phosphatase and tensin homolog deleted from chromosome 10 (PTEN) levels protected these cells against death induced by various chemotherapeutic agents. This was dependent on a functional estrogen receptor (ER)-α because silencing ER-α blocked the ability of IGFBP-2 to confer cell survival. Loss of IGFBP-2 increased levels of PTEN and improved chemosensitivity of the cells, confirming its role as a survival factor. Silencing IGFBP-2 had no effect on the response to IGF-II, but responses to estrogen and tamoxifen were no longer observed due to loss of ER-α, which could be prevented by the inhibition of PTEN. Conversely, exogenous IGFBP-2 increased ER-α mRNA and protein in both normal and cancer cells via its interaction with integrin receptors. These actions of IGFBP-2 on ER-α involved the IGF-I receptor and activation of phosphatidylinositol 3-kinase in the cancer cells but were independent of this in normal breast cells. The production of IGFBP-2 by breast cancer cells enhances their proliferative potential, increases their survival, and protects them against chemotherapy-induced death. IGFBP-2 not only modulates IGFs and directly regulates PTEN but also has a role in maintaining ER-α expression.

To establish a method to isolate the CD4+CD25+ regulatory T cells (Tregs) and to identify the purity and function of these cells.The peripheral blood (8 mL) were collected from the great saphenous vein of 10 rhesus monkeys (4 females and 6 males, aged 4-5 years, and weighing 5-8 kg). The mononuclear cells were isolated with density gradient centrifugation. CD4+ T cells were separated by the Magnetic cell sorting (MACS) negative selection and MACS positive selection. The cell yield rate, the cell viability, and the cell purity were compared between MACS negative selection and MACS positive selection. In CD4+ MACS negative selection, the anti-biotin MicroBeads and biotin-antibody cocktai in CD4+CD25+ Tregs isolation kit non-human primate were used, and in MACS positive selection, the anti-APC MicroBeads in CD4+CD25+ Tregs isolation kit non-human primate and CD4-APC were used. The CD4+ T cells separated by positive selection were selected to obtain CD4+CD25 Tregs with CD25 MicroBeads. The purity, activity, the FoxP3 level, and the suppressive function to concanavalin A (ConA) activated autologous CD4+CD24 effective T cells (Teffs) of CD4+CD25+ Tregs were detected by flow cytometry.After CD4+ T cells were separated by MACS negative selection and MACS positive selection, the cell viabilities were all up to 95%, showing no significant difference (P0.05). The cell yield rate and purity of CD4+ T cells by positive selection were significantly higher than those of CD4+ T cells by negative selection (P0.05). CD4+CD25+ Tregs can be successfully isolated by MACS double positive selection. The classifying purity was 76.2% +/- 8.6%; the cell activity was 93.3% +/- 4.7%; and the level of FoxP3 was 74.2% +/- 6.9%. The CD4+CD25+ Tregs had suppressive effect on ConA activated autologous CD4+CD25 Teffs.MACS double positive selection can be used to isolate high-purity CD4+CD25+ Tregs from the peripheral blood of rhesus monkeys and the process does not influence the activity of CD4+CD25+ Tregs.

This study is to investigate the anti-tumor effect in vitro of methotrexate modified by LH-RH peptide (LH-RH-MTX). LH-RH receptors highly expressing MCF-7 human breast carcinoma cell line and lowly expressing K562 human erythroleukemia cell line were served as the tested cells. The cell proliferation inhibition rates of LH-RH-MTX were detected by MTT colorimetric assay. The effects of LH-RH-MTX on the cell cycle and apoptosis rates were detected by flow cytometry. The inhibition rate of LH-RH-MTX on MCF-7 cells was much higher than that on K562 cells, and the inhibition rate of LH-RH-MTX on MCF-7 cells was much higher than that of free MTX at the same concentration. The inhibition rate of LH-RH-MTX on rat bone marrow mononuclear cells was less than that of free MTX. The number of MCF-7 cells in S phase increased after administration of LH-RH-MTX. The apoptosis rate of LH-RH-MTX group significantly increased compared with that of the control group and MTX group. The relative expression of LHRHR mRNA of LH-RH-MTX group markedly decreased compared with that of the control group and MTX group. LH-RH-MTX is realizable to reduce drug side effects, increase the therapeutic index and achieve tumor-targeted therapy.

DNA damage triggers a network of signaling events that leads to cell cycle arrest or apoptosis. This DNA damage response acts as a mechanism to prevent cancer development. It has been reported that fatty acids (FAs) synthesis is increased in many human tumors while inhibition of fatty acid synthase (FASN) could suppress tumor growth. Here we report that saturated fatty acids (SFAs) play a negative role in DNA damage response. Palmitic acid, as well as stearic acid and myristic acid, compromised the induction of p21 and Bax expression in response to double stranded breaks and ssDNA, while inhibition or knockdown of FASN enhanced these cellular events. SFAs appeared to regulate p21 and Bax expression via Atr-p53 dependent and independent pathways. These effects were only observed in primary mouse embryonic fibroblasts and osteoblasts, but not in immortalized murine NIH3T3, or transformed HCT116 and MCF-7 cell lines. Accordingly, SFAs showed some positive effects on proliferation of MEFs in response to DNA damage. These results suggest that SFAs, by negatively regulating the DNA damage response pathway, might promote cell transformation, and that increased synthesis of SFAs in precancer/cancer cells might contribute to tumor progression and drug resistance.

Background: Statins, a family of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase inhibitors, are being investigated for the therapy and prevention of cancers. Here we aimed to investigate the effects of simvastatin on chronic myelogenous leukemia (CML) cells in vitro and in vivo, and to elucidate the mechanisms. Methods: Cell proliferation and cell cycle were measured after K562 cells were incubated with simvastatin, and differentially expressed genes were determined by oligonucleotide microarray. Changes of 2 genes obtained by oligonucleotide microarray were validated by real-time RT-PCR, and immunohistochemistry was performed to determine expression of proliferating cell nuclear antigen (PCNA). Finally, a xenograft tumor model was constructed to evaluate the effects of simvastatin in vivo. Results: Simvastatin could inhibit K562 cell proliferation, and the inhibition rate was approximately 30% after treatment with 20 µmol/l simvastatin for 48 h. Cell cycle was arrested in G1 phase, as shown by flow cytometry results. Fifteen downregulated, 9 upregulated cell cycle-related genes and decreased PCNA protein were observed in the presence of simvastatin. Furthermore, simvastatin exhibited impairment of xenograft tumor growth in nude mice and also blocked cell cycle in G1 phase. Conclusion: Simvastatin can inhibit CML cell proliferation in vitro and in vivo, and its mechanisms might be involved in cell cycle regulation.

The effects of N-linked oligosaccharide chains (TM), an inhibitor of N-glycosylation of proteins, on the expression of beta1 integrin and the apoptosis of glomerular mesangial cells (GMC) were observed for exploring the role of inhibitors of N-linked oligosaccharide chains in cell apoptosis, proliferation, and expression of beta1 integrin and cyclin D1, and hence finding a new approach to the synteresis of proliferative nephropathy.Cultured rat mesangial cells were divided into 4 groups: control group, and 0.5, 1.0 and 2.0 microg/mL TM-treatment groups. The expression of beta1 integrin and apoptosis rate were measured by flow cytometry; the expression of cyclin D, was measured by immunohistochemistry, and proliferation of GMCs was measured by MTT.The expression of beta1 integrin and cyclin D1 were decreased notably by inhibitors of N-linked oligosaccharide chains-TM. TM increased apoptosis and decreased the proliferative abilitiy of cells, and all the effects of TM were dose-dependent.Through repressed glycosylation of glycoprotein, TM can suppress the expression of beta1, integrin, affect the adhesion of cells, and increase the apoptosis rate; at the same time, the expression of cyclin D1 and the proliferative ability of cells were decreased, and all these were dose-dependent.

Up to now, searching for non-toxic and natural origin substances that induced the differentiation of cancer cells is a key for anticancer therapy. Ginseng is one of the most widely used natural tonics in oriental countries for thousands of years and has been reported to have various biological effects. Ginsenosides are thought to be the major effective ingredients in ginseng. Among them, ginsenoside Rh2(G-Rh2) has been suggested to have a cell-growth suppressive effect on various cancer cells, but the mechanism is unclear. This study was to investigate the induced differentiative effects of G-Rh2 on SMMC-7721 hepatocarcinoma cells.Effects of G-Rh2 on cell viability was analyzed by MTT assay. Cell morphology was examined by a light and electronic microscope. Alpha-fetoprotein (AFP) in plasma was determined qualitatively and quantitatively with immunohistochemistry and ELISA. The specific activities of alkaline phosphatase (ALP) and heat-resistant ALP in plasma were assayed by ALP kit based on Bessey method. The specific activity of gamma-glutamyltranspeptidase (gamma-GT) was measured with gamma-GT kit.The secretory amount of AFP or albumin was detected with radioimmunoassay kit.G-Rh2 inhibited the proliferation of SMMC-7721 cells in dose and time-dependent manners. The inhibition rate was 50.87% after 6-day treatment with 10 microg/ml G-Rh2 while 46.84% after 4-day treatment with 20 microg/ml G-Rh2. Twenty microg/ml G-Rh2 induced the mature and normality of morphology and ultrastructure in SMMC-7721 cells. After treated with 10 microg/ml or 20 microg/ml G-Rh2, the production of AFP was significantly reduced (P0.05), and the secretory amount of AFP was reduced from 6.60+/-0.30 to 2.35+/-0.06 (P0.01), and the specific activities of gamma-GT and heat-resistant ALP were remarkably declined (P0.01); while the secretory amount of albumin and ALP activity were remarkably enhanced (P0.01).G-Rh2 could induce the SMMC-7721 cell differentiation tending to normal.