Competing memories of mitogen and p53 signalling control cell-cycle entry.

Regulation of cell proliferation is necessary for immune responses, tissue repair, and upkeep of organ function to maintain human health. When proliferating cells complete mitosis, a fraction of newly born daughter cells immediately enter the next cell cycle, while the remaining cells in the same population exit to a transient or persistent quiescent state. Whether this choice between two cell-cycle pathways is due to natural variability in mitogen signalling or other underlying causes is unknown. Here we show that human cells make this fundamental cell-cycle entry or exit decision based on competing memories of variable mitogen and stress signals. Rather than erasing their signalling history at cell-cycle checkpoints before mitosis, mother cells transmit DNA damage-induced p53 protein and mitogen-induced cyclin D1 (CCND1) mRNA to newly born daughter cells. After mitosis, the transferred CCND1 mRNA and p53 protein induce variable expression of cyclin D1 and the CDK inhibitor p21 that almost exclusively determines cell-cycle commitment in daughter cells. We find that stoichiometric inhibition of cyclin D1-CDK4 activity by p21 controls the retinoblastoma (Rb) and E2F transcription program in an ultrasensitive manner. Thus, daughter cells control the proliferation-quiescence decision by converting the memories of variable mitogen and stress signals into a competition between cyclin D1 and p21 expression. We propose a cell-cycle control principle based on natural variation, memory and competition that maximizes the health of growing cell populations.

CDK4, CDK6/cyclin-D1 Complex Inhibition and Radiotherapy for Cancer Control: A Role for Autophagy.

The expanding clinical application of CDK4- and CDK6-inhibiting drugs in the managements of breast cancer has raised a great interest in testing these drugs in other neoplasms. The potential of combining these drugs with other therapeutic approaches seems to be an interesting work-ground to explore. Even though a potential integration of CDK4 and CDK6 inhibitors with radiotherapy (RT) has been hypothesized, this kind of approach has not been sufficiently pursued, neither in preclinical nor in clinical studies. Similarly, the most recent discoveries focusing on autophagy, as a possible target pathway able to enhance the antitumor efficacy of CDK4 and CDK6 inhibitors is promising but needs more investigations. The aim of this review is to discuss the recent literature on the field in order to infer a rational combination strategy including cyclin-D1/CDK4-CDK6 inhibitors, RT, and/or other anticancer agents targeting G1-S phase cell cycle transition.

Investigating the effect of radiosensitizer for Ursolic Acid and Kamolonol Acetate | on HCT-116 cell line.

PURPOSE: The aim of this study was evaluating the cytotoxic and radiosensitizing effects of Ursolic Acid (UA) and Kamolonol Acetate (KA) on HCT116 cell line and finally investigating the functional role of NF-κB and CCND1 genes in the radiosensitizing activity of UA and KA. MATERIALS AND METHOD: The cytotoxic effects of UA and KA by MTT assay was evaluated on HCT-116. Clonogenic assay was performed to investigate of radiosensitizing effects of UA and KA on HCT116. To assessment the expression levels of NF-κB and CCND1 genes, real-time PCR method was used. RESULTS: The results of MTT assay revealed that UA and KA have cytotoxic effects on HCT116 cell line. According to clonogenic assay, survival fraction of treated cells with UA and KA has been decreased compared to the survival fraction of untreated cells. UA and KA lead to the decrease in the expression level of NF-κB. Synergistic effect of radiosensitizing agents with radiation was only approved for UA and 2 Gy of radiation. CONCLUSION: Based on our study, UA and KA have cytotoxic effects on HCT116 cell line. Furthermore, UA may lead to radiosensitization of human colorectal tumor cells by NF-κB1 and CCND1signaling pathways.

MicroRNA-519d-3p inhibits cell proliferation and cell cycle G1/S transition in glioma by targeting CCND1.

Glioma is the most common highly malignant primary brain tumor. MicroRNA-519d-3p exerts important effects in several tumors, but its functional role in glioma remained poorly understood. In this study, we found miR-519d-3p expression was significantly decreased in glioma tissues and cell lines. Moreover, the in vitro experiments showed that overexpression of miR-519d-3p suppressed cell proliferation and induced cell cycle G0/G1 phase arrest using MTT and flow cytometry assays in glioma cell lines, U87 and U251. Mechanistically, Cyclin D1 (CCND1) was predicted and confirmed as the direct target genes of miR-519d-3p using luciferase report assay. In addition, knockdown of CCND1 imitated the suppressive effects of miR-519d-3p on cell proliferation and cell cycle progression. Furthermore, restoration of CCND1 reversed the effects of miR-519d-3p overexpression in glioma cells. Taken together, these data demonstrate that suppression of CCND1 by miR-519d-3p might be a therapeutic target for glioma.Abbreviations miR-519d-3p: microRNA-519d-3p; CCND1: Cyclin D1; ATCC: American Type Culture Collection; MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PI: propidium iodide; WT: wild type; MUT: mutant type; SD: standard deviation.

Modification of the aminopyridine unit of 2'-deoxyaminopyridinyl-pseudocytidine allowing triplex formation at CG interruptions in homopurine sequences.

The antigene strategy based on site-specific recognition of duplex DNA by triplex DNA formation has been exploited in a wide range of biological activities. However, specific triplex formation is mostly restricted to homo-purine strands within the target duplex DNA, due to the destabilizing effect of CG and TA inversion sites where there is an absence of natural nucleotides that can recognize the CG and TA base pairs. Hence, the design of artificial nucleosides, which can selectively recognize these inversion sites with high affinity, should be of great significance. Recently, we determined that 2-amino-3-methylpyridinyl pseudo-dC (3MeAP-ΨdC) possessed significant affinity and selectivity toward a CG inversion site and showed effective inhibition of gene expression. We now describe the design and synthesis of new modified aminopyridine derivatives by focusing on small chemical modification of the aminopyridine unit to tune and enhance the selectivity and affinity toward CG inversion sites. Remarkably, we have newly found that 2-amino-4-methoxypyridinyl pseudo-dC (4OMeAP-ΨdC) could selectively recognize the CG base pair in all four adjacent base pairs and form a stable triplex structure against the promoter sequence of the human gene including multiple CG inversion sites.

Magnolol induces apoptosis in osteosarcoma cells via G0/G1 phase arrest and p53-mediated mitochondrial pathway.

Osteosarcoma is a highly invasive primary malignancy of bone. Magnolol is biologically active, which shows antitumor effects in a variety of cancer cell lines. However, it has not been elucidated magnolol's effects on human osteosarcoma cells (HOC). This study aimed to determine antitumor activity of magnolol and illustrate the molecular mechanism in HOC. Magnolol showed significant inhibition effect of growth on MG-63 and 143B cells and induced apoptosis and cell cycle arrest at G0/G1. In osteosarcoma cells, magnolol upregulated expressions of proapoptosis proteins and suppressed expressions of antiapoptosis proteins. Additionally, under the pretreatment of pifithrin-a (PFT-a, a p53 inhibitor), the magnolol-induced apoptosis was significantly reversed. The results above indicated that magnolol induces apoptosis in osteosarcoma cells may via G0/G1 phase arrest and p53-mediated mitochondrial pathway.

Differentiation-inducing factor-1 suppresses cyclin D1-induced cell proliferation of MCF-7 breast cancer cells by inhibiting S6K-mediated signal transducer and activator of transcription 3 synthesis.

Differentiation-inducing factor-1 (DIF-1) has been reported to inhibit the proliferation of various mammalian cells by unknown means, although some possible mechanisms of its action have been proposed, including the activation of glycogen synthase kinase-3 (GSK-3). Here, we report an alternative mechanism underlying the action of DIF-1 in human breast cancer cell line MCF-7, on which the effects of DIF-1 have not been examined previously. Intragastric administration of DIF-1 reduced the tumor growth from MCF-7 cells injected into a mammary fat pad of nude mice, without causing adverse effects. In cultured MCF-7, DIF-1 arrested the cell cycle in G0 /G1 phase and suppressed cyclin D1 expression, consistent with our previous results obtained in other cell species. However, DIF-1 did not inhibit the phosphorylation of GSK-3. Investigating an alternative mechanism for the reduction of cyclin D1, we found that DIF-1 reduced the protein levels of signal transducer and activator of transcription 3 (STAT3). The STAT3 inhibitor S3I-201 suppressed cyclin D1 expression and cell proliferation and the overexpression of STAT3 enhanced cyclin D1 expression and accelerated proliferation. Differentiation-inducing factor-1 did not reduce STAT3 mRNA or reduce STAT3 protein in the presence of cycloheximide, suggesting that DIF-1 inhibited STAT3 protein synthesis. Seeking its mechanism, we revealed that DIF-1 inhibited the activation of 70 kDa and/or 85 kDa ribosomal protein S6 kinase (p70S6K /p85S6K ). Inhibition of p70S6K /p85S6K by rapamycin also reduced the expressions of STAT3 and cyclin D1. Therefore, DIF-1 suppresses MCF-7 proliferation by inhibiting p70S6K /p85S6K activity and STAT3 protein synthesis followed by reduction of cyclin D1 expression.

Evaluation of double heptamer-type sgRNA as a potential therapeutic agent against multiple myeloma.

Emergence of drug-resistant mutations in the course of myeloma cell evolution and subsequent relapse of myeloma appears to be currently inevitable in most patients. To remedy this situation, we are trying to develop therapeutic small guide RNAs (sgRNAs) based on tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing), an RNA-mediated gene expression control technology. We designed two sets of double heptamer-type sgRNA, which target the human BCL2 mRNA. Both sets of double heptamer-type sgRNA reduced viability of human myeloma cell lines, RPMI-8226 and KMM-1. We also performed a mouse xenograft experiment to examine how the double heptamer-type sgRNA DHa1(BCL2)/DHa2(BCL2) can reduce the growth of KMM-1 cells in vivo. Median survival periods of the sgRNA cohorts were greater than that of the control cohort by 11-43 days. Furthermore, we designed two sets of double heptamer-type sgRNA, which target the human CCND1 mRNA, and both sets synergistically reduced RPMI-8226 cell viability.

Blockade of Hedgehog Signaling Attenuates Biliary Cystogenesis in the Polycystic Kidney (PCK) Rat.

Caroli disease represents a hepatic manifestation of autosomal recessive polycystic kidney disease, and belongs to a class of cholangiociliopathies. The role of Hedgehog signaling, a major pathway regulated by primary cilia, in biliary cystogenesis in Caroli disease remains unknown. Using the polycystic kidney (PCK) rat as an animal model of Caroli disease, this study investigated the involvement of Hedgehog signaling in its pathogenesis. In vitro experiments revealed that PCK cholangiocytes overexpressed Smoothened, Gli1, and Gli1's target molecule cyclin D1. The nuclear expression of Gli1, Gli2, and Gli3 was observed in PCK cholangiocytes by immunocytochemistry. An immunohistochemical analysis using liver sections confirmed the overexpression of Smoothened and cyclin D1, and the nuclear expression of the Gli proteins in the biliary epithelium of PCK rats as well as human Caroli disease. The treatment of PCK cholangiocytes with cyclopamine inhibited cell proliferative activity that was associated with the inhibition of nuclear translocation of Gli1 and Gli2, and reduced cyclin D1 expression. The in vivo administration of cyclopamine to PCK rats decreased abnormally elevated serum liver enzymes, and significantly attenuated bile duct dilation as well as kidney cyst formation. These results suggest that cholangiocyte hyperproliferation is causally associated with the aberrant activation of Hedgehog signaling, and the inhibition of the signaling has potential as a therapeutic strategy for biliary cystogenesis in Caroli disease.

Isochamanetin is a Selective Inhibitor for CyclinD1 in SKOV3 Cell Lines.

The cyclinD1 is an emerging potent therapeutic drug target in the treatment of ovarian cancer. CyclinD1, Cdks phosphorylation regulates cell cycle and controls transcription. For this reason, CyclinD1 have been subject to extensive cell cycle- related research, and consequently various therapeutic inhibitor drugs have been developed to these protein targets. In the present study we identified that the expression levels of Bcl-2, Bax, caspase 8, 9 and cyclinD1 using Q-PCR method in SKOV3 cell lines treated with Isochamanetin. The viability and migratory inhibition ability also studied to know the mode of cell death. Further the expression levels of Bcl-2, caspase8,9, Cytochrome C and CyclinD1 were significantly down regulated in SKOV3 cancer cells treated with isochamanetin, a specific binding molecule to CyclinD1. The therapeutic molecules found by a high through put insilicoscreen of this pocket exhibit cytostatic nature and reduce protein levels of cell cycle. The novel structural site on CyclinD1, which is well conserved and inhibits the SKOV3 cells from G0-G1 to S phase cell cycle progression. The current result suggests that isochamanetin serves as potent binding agent to the cyclinD1.

Protective effect of epigenetic silencing of CyclinD1 against spinal cord injury using bone marrow-derived mesenchymal stem cells in rats.

This study focuses on the protective effect of epigenetic silencing of CyclinD1 against spinal cord injury (SCI) using bone marrow-derived mesenchymal stem cells (BMSCs) in rats. Eighty-eight adult female Wistar rats were randomly assigned into the sham group, the control group, the si-CyclinD1 + BMSCs group and the BMSCs group. CyclinD1 protein and mRNA expressions after siRNA transfection were detected by Western blotting and qRT-PCR. The siRNA-CyclinD1 BMSCs were transplanted into rats in the si-CyclinD1 + BMSCs group using stereotaxic method 6 hr after SCI. Hindlimb locomotor performance was determined using inclined plane test and Basso-Beattie-Bresnahan (BBB) locomotor rating scale. Expressions of glial fibrillary acidic protein (GFAP) and nerve growth factor (NGF) were detected by immunohistochemistry. Inclined plane and BBB scores in the control, si-CyclinD1 + BMSCs, and BMSCs groups were significantly lower than the sham group, but these scores were evidently decreased in the control group and increased in the si-CyclinD1 + BMSCs group compared with the BMSCs group. The repair degree of spinal cord tissues of rats in the si-CyclinD1 + BMSCs group was obvious than the BMSCs group. GFAP and NGF protein expressions were markedly decreased in the control, si-CyclinD1 + BMSCs and BMSCs groups when compared with the sham group. GFAP- and NGF-positive cells were significantly increased in the si-CyclinD1 + BMSCs group while decreased in the control group. Our study provides evidence that epigenetic silencing of CyclinD1 using BMSCs might accelerate the repair of SCI in rats.

Activation of cyclin D1 affects mitochondrial mass following traumatic brain injury.

Cell cycle activation has been associated with varying types of neurological disorders including brain injury. Cyclin D1 is a critical modulator of cell cycle activation and upregulation of Cyclin D1 in neurons contributes to the pathology associated with traumatic brain injury (TBI). Mitochondrial mass is a critical factor to maintain the mitochondrial function, and it can be regulated by different signaling cascades and transcription factors including NRF1. However, the underlying mechanism of how TBI leads to impairment of mitochondrial mass following TBI remains obscure. Our results indicate that augmentation of CyclinD1 attenuates mitochondrial mass formation following TBI. To elucidate the molecular mechanism, we found that Cyclin D1 interacts with a transcription factor NRF1 in the nucleus and prevents NRF1's interaction with p300 in the pericontusional cortex following TBI. As a result, the acetylation level of NRF1 was decreased, and its transcriptional activity was attenuated. This event leads to a loss of mitochondrial mass in the pericontusional cortex following TBI. Intranasal delivery of Cyclin D1 RNAi immediately after TBI rescues transcriptional activation of NRF1 and recovers mitochondrial mass after TBI.

LncRNA HOTAIR Regulates CCND1 and CCND2 Expression by Sponging miR-206 in Ovarian Cancer.

BACKGROUND/AIMS: The long noncoding RNA homeobox (HOX) transcript antisense intergenic RNA (HOTAIR) has been demonstrated to be a vital modulator in the proliferation and metastasis of ovarian cancer cells, but its potential molecular mechanism remains to be elucidated. In the current study, we aimed to uncover the biological role of lncRNA HOTAIR and its underlying regulatory mechanism in the progression and metastasis of ovarian cancer. METHODS: HOTAIR expression was detected by quantitative RT-PCR (qRT-PCR) and northern blotting. The SKOV3 ovarian cancer cell line was chosen for the subsequent assays. In addition, the molecular mRNA and protein expression levels were examined by qRT-PCR and western blotting. The competitive endogenous RNA (ceRNA) mechanism was validated by bioinformatics analysis and a dual luciferase reporter gene assay. RESULTS: HOTAIR expression was significantly higher in ovarian carcinoma tissues and cell lines than in the control counterparts. Both CCND1 and CCND2 were downstream targets of miR-206. The inhibition of HOTAIR elevated the expression of miR-206 and inhibited the expression of CCND1 and CCND2. Moreover, CCND1 and CCND2 were highly expressed in ovarian cancer tissues, and their expression was positively correlated with HOTAIR expression. Finally, the functional assays indicated that the anticancer effects of miR-206 could be rescued by the simultaneous overexpression of either CCND1 or CCND2 in ovarian cancer. CONCLUSION: HOTAIR enhanced CCND1 and CCND2 expression by negatively modulating miR-206 expression and stimulating the proliferation, cell cycle progression, migration and invasion of ovarian cancer cells.

Iron chelator-induced up-regulation of Ndrg1 inhibits proliferation and EMT process by targeting Wnt/ß-catenin pathway in colon cancer cells.

Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), as the novel iron chelator, has been reported to inhibit the tumorigenesis and progression of various cancer cells. However, whether Dp44mT has anticancer effects in colon cancer cells is still unknown. Here, we investigated the antitumor action of Dp44mT in colon cancer and its underlying mechanisms, and the connections between Dp44mT and N-myc downstream-regulated genes 1(Ndrg1). We used cell viability, migration and invasion assay, flow cytometry, western blot and qRT-PCR to examine the anticancer effects of Dp44mT and Ndrg1. We found that Dp44mT suppressed cell viability, migration, invasion and induced apoptosis of colon cancer cells and over-expression of Ndrg1 also suppressed cell viability, migration, invasion and induced apoptosis of colon cancer cells. Dp44mT attenuated the TGF-ß1-induced EMT in colon cancer cells, and Dp44mT could up-regulate Ndrg1 expression level. Overexpression of Ndrg1 attenuates the TGF-ß1-induced EMT, Dp44mT and Ndrg1 suppressed EMT through activation of Wnt/ß catenin signaling pathway. In conclusion, our data demonstrated that Dp44mT/Ndrg1 have effective anticancer capability in colon cancer cells and that may represent a promising treatment strategy for human colon cancer.

Palbociclib has antitumour effects on Pten-deficient endometrial neoplasias.

PTEN is one of the most frequently mutated genes in human cancers. The frequency of PTEN alterations is particularly high in endometrial carcinomas. Loss of PTEN leads to dysregulation of cell division, and promotes the accumulation of cell cycle complexes such as cyclin D1-CDK4/6, which is an important feature of the tumour phenotype. Cell cycle proteins have been presented as key targets in the treatment of the pathogenesis of cancer, and several CDK inhibitors have been developed as a strategy to generate new anticancer drugs. Palbociclib (PD-332991) specifically inhibits CDK4/6, and it has been approved for use in metastatic breast cancer in combination with letrazole. Here, we used a tamoxifen-inducible Pten knockout mouse model to assess the antitumour effects of cyclin D1 knockout and CDK4/6 inhibition by palbociclib on endometrial tumours. Interestingly, both cyclin D1 deficiency and palbociclib treatment triggered shrinkage of endometrial neoplasias. In addition, palbociclib treatment significantly increased the survival of Pten-deficient mice, and, as expected, had a general effect in reducing tumour cell proliferation. To further analyse the effects of palbociclib on endometrial carcinoma, we established subcutaneous tumours with human endometrial cancer cell lines and primary endometrial cancer xenografts, which allowed us to provide more translational and predictive data. To date, this is the first preclinical study evaluating the response to CDK4/6 inhibition in endometrial malignancies driven by PTEN deficiency, and it reveals an important role of cyclin D-CDK4/6 activity in their development. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Ginger extract adjuvant to doxorubicin in mammary carcinoma: study of some molecular mechanisms.

PURPOSE: The present study aimed to investigate the molecular mechanisms underlying the anticancer properties of ginger extract (GE) in mice bearing solid Ehrlich carcinoma (SEC) and to evaluate the use of GE in combination with doxorubicin (DOX) as a complementary therapy against SEC. METHODS: SEC was induced in 60 female mice. Mice were divided into four equal groups: SEC, GE, DOX and GE + DOX. GE (100 mg/kg orally day after day) and DOX (4 mg/kg i.p. for 4 cycles every 5 days) were given to mice starting on day 12 of inoculation. On the 28th day, blood samples were collected, mice were scarified, tumor volume was measured, and tumor tissues were excised. RESULTS: The anti-cancer effect of GE was mediated by activation of adenosine monophosphate protein kinase (AMPK) and down-regulation of cyclin D1 gene expression. GE also showed pro-apoptotic properties as evidenced by elevation of the P53 and suppression of nuclear factor-kappa B (NF-κB) content in tumor tissue. Co-administration of GE alongside DOX markedly increased survival rate, decreased tumor volume, and increased the level of phosphorylated AMPK (PAMPK) and improved related pathways compared to DOX group. In addition, the histopathological results demonstrated enhanced apoptosis and absence of multinucleated cells in tumor tissue of GE + DOX group. CONCLUSION: AMPK pathway and cyclin D1 gene expression could be a molecular therapeutic target for the anticancer effect of GE in mice bearing SEC. Combining GE and DOX revealed a greater efficacy as anticancer therapeutic regimen.

Evaluation of the interaction between proliferation, oxidant-antioxidant status, Wnt pathway, and apoptosis in zebrafish embryos exposed to silver nanoparticles used in textile industry.

Antimicrobial textile products are developing rapidly as an important part of functional textiles. Silver nanoparticles (AgNPs) are nanotechnology products with antimicrobial properties. However, exposure to nanoparticles in daily life is an important issue for public health, still being updated. Aim was to evaluate the effects of AgNPs on the development of zebrafish embryos focusing on Wnt pathway, proliferation, oxidant-antioxidant status, and apoptosis. The expressions of ccnd1 and gsk3ß were determined by RT-PCR, whereas ß-catenin and proliferative cell antigen (PCNA) expressions were determined immunohistochemically. Lipid peroxidation, superoxide dismutase, and glutathione-S-transferase activities were determined spectrophotometrically. Apoptosis was determined using acridine orange staining. Oxidant status, apoptosis, immunohistochemical PCNA, and ß catenin staining increased, whereas ccnd1 and antioxidant enzyme activities decreased in AgNPs-exposed embryos in a dose-dependent manner. Our results indicate the interaction of possible mechanisms that may be responsible for the toxic effects of AgNPs in zebrafish embryos.

MicroRNA-424 Predicts a Role for ß-1,4 Branched Glycosylation in Cell Cycle Progression.

MicroRNA regulation of protein expression plays an important role in mediating many cellular processes, from cell proliferation to cell death. The human microRNA miR-424 is up-regulated in response to anti-proliferative cytokines, such as transforming growth factor ß (TGFß), and directly represses cell cycle progression. Our laboratory recently established that microRNA can be used as a proxy to identify biological roles of glycosylation enzymes (glycogenes). Herein we identify MGAT4A, OGT, and GALNT13 as targets of miR-424. We demonstrate that MGAT4A, an N-acetylglucosaminyltransferase that installs the ß-1,4 branch of N-glycans, is directly regulated by miR-424 in multiple mammary epithelial cell lines and observe the loss of MGAT4A in response to TGFß, an inducer of miR-424. Knockdown of MGAT4A induces cell cycle arrest through decreasing CCND1 levels. MGAT4A does not affect levels of ß-1,6 branched N-glycans, arguing that this effect is specific to ß-1,4 branching and not due to gross changes in overall N-linked glycosylation. This work provides insight into the regulation of cell cycle progression by specific N-glycan branching patterns.

Hypoxia-induced microRNA-146a represses Bcl-2 through Traf6/IRAK1 but not Smad4 to promote chondrocyte autophagy.

It has been shown that hypoxia stimulation promotes chondrocytes autophagy partly through HIF-1α, miR-146a and Bcl-2 progressively, and this mechanism represented the connection among hypoxia, miR-146a and autophagy, and provides a possible therapeutic strategy for osteoarthritis. However, the interaction between miR-146a and Bcl-2 is still unclear. Here in a hypoxic environment, we quantified the three reported miR-146a targets: two inflammation related targets Traf6, IRAK1; and the only reported target in chondrocytes Smad4. We confirmed the regulative function of miR-146a between hypoxia and these genes, and explored the Bcl-2 expression and autophagy level under extrinsic up-regulation of these three gene separately. All the three genes were down-regulated by hypoxia. Surprisingly, Traf6 and IRAK, but not the unique Smad4 in chondrocytes, were restored by antagomiR-146a. Both Ad-Traf6 and Ad-IRAK1 reinstated hypoxia or miR-146a repressed Bcl-2. However, Ad-Smad4 did not affect Bcl-2 in hypoxia or normoxia. The autophagy level showed a reverse variability compared to Bcl-2. Taken together, our results provided evidence that Smad4, the unique reported target for miR-146a in chondrocytes is unusually not involved in the chondrocytes autophagy, while the Traf6 and IRAK1 are the new targets for miR-146a in chondrocytes during autophagy.

Hydroxytyrosol Induces Apoptosis and Cell Cycle Arrest and Suppresses Multiple Oncogenic Signaling Pathways in Prostate Cancer Cells.

SCOPE: Hydroxytyrosol (HT), a polyphenol from olives, is a potential anticancer agent. This study was designed to evaluate the anticancer activity of HT against prostate cancer cells, and the mechanism thereof. METHODS AND RESULTS: Treatment of LNCaP and C4-2 prostate cancer cells with HT resulted in a dose-dependent inhibition of proliferation. This was in contrast to HT's ineffectiveness against normal prostate epithelial cells RWPE1 and PWLE2, suggesting cancer-cell-specific effect. HT induced G1/S cell cycle arrest, with inhibition of cyclins D1/E and cdk2/4 and induction of inhibitory p21/p27. HT also induced apoptosis, as confirmed by flow cytometry, caspase activation, PARP cleavage, and BAX/Bcl-2 ratio. It inhibited the phosphorylation of Akt/STAT3, and induced cytoplasmic retention of NF-κB, which may explain its observed effects. Finally, HT inhibited androgen receptor (AR) expression and the secretion of AR-responsive prostate-specific antigen. CONCLUSION: Castration-resistant prostate cancers retain AR signaling and are often marked by activated Akt, NF-κB, and STAT3 signaling. Our results establish a pleiotropic activity of HT against these oncogenic signaling pathways. Combined with its nontoxic effects against normal cells, our results support further testing of HT for prostate cancer therapy.