A Comparison of the Anti-Cancer Effects of Free and PLGA-PAA Encapsulated Hydroxytyrosol on the HT-29 Colorectal Cancer Cell Line.

BACKGROUND: Hydroxytyrosol is one of the phenolic compounds of olive oil and can induce anticancer effects on colorectal cancer cells. OBJECTIVE: The aim of the present study was to evaluate the free hydroxytyrosol and nano-capsulated hydroxytyrosol effects on the cell cycle arrest in HT-29 colorectal cancer cell line. METHODS: The nano-capsulated hydroxytyrosol was synthesized in poly lactide-co-glycolide-co-polyacrylic acid (PLGA-PAA) copolymer. MTT assay was performed to evaluate the anti-proliferative and anti-tumor effects of the free hydroxytyrosol and nano-capsulated hydroxytyrosol. Finally, the relative expression of CDKN1A, CDKN1B, and CCND1 genes was evaluated in control and treated colorectal cancer cells by using Real-Time PCR. RESULTS: The obtained results from the MTT assay showed that the cytotoxic effects of the nano-capsulated hydroxytyrosol on the colorectal cancer cell line (IC50= 6PPM) were significantly more than free hydroxytyrosol (IC50= 12PPM) after 72h. Also, nano-capsulated hydroxytyrosol showed more significant effects on the upregulation of CDKN1A and CDKN1B genes and down-regulation of the CCND1 gene in colorectal cancer cells. CONCLUSION: In conclusion, the present study showed that hydroxytyrosol led to the death of colorectal cancer cells through cell cycle arrest. Also, the PLGA-PAA copolymer dramatically caused to increase the cytotoxic effects of the hydroxytyrosol on the colorectal cancer cells.

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.

Kaempferol suppresses proliferation and induces apoptosis and DNA damage in human gallbladder cancer cells through the CDK4/CDK6/cyclin D1 pathway.

OBJECTIVE: Kaempferol has been reported to play an anti-tumor role in various human cancers, while its role in gallbladder cancer (GBC) is unclear. MATERIALS AND METHODS: We found that kaempferol significantly inhibited the growth, invasion and migration, meanwhile induced apoptosis through cells arrested at G0/G1 phase of GBC cell lines, including GBC-SD and SGC996 cells in vitro. RESULTS: Kaempferol promoted the release of cytochrome C from the mitochondria to cytoplasm, the activation of c-caspase-3 and c-caspase-9 and increased the expression levels of pro-apoptotic factor Bax, meanwhile decreased the expression levels of anti-apoptotic factor Bcl-2. In addition, the expression levels of CDK4, CDK6 and cyclin D1, which are members of the CDK4/CDK6/cyclin D1 signaling pathway, were also decreased by kaempferol. Moreover, kaempferol could efficiently prevent tumor progression of GBC in the xenograft in vivo. CONCLUSIONS: Our results demonstrated that kaempferol suppressed GBC progression through activation of the CDK4/CDK6/cyclin D1 signaling pathway, suggesting that it might be a potential anti-tumor agent for clinical treatment of GBC.

Paeoniflorin Sensitizes Breast Cancer Cells to Tamoxifen by Downregulating microRNA-15b via the FOXO1/CCND1/ß-Catenin Axis.

BACKGROUND: Paeoniflorin (Pae) possesses anti-tumor activity in various malignancies. However, it is unclear whether Pae plays a sensitizer role in breast cancer (BC) and the molecular mechanisms involved in this process. Our oligonucleotide microarray revealed that microRNA (miR)-15b is the most significantly downregulated miRNA in MCF-7/4-hydroxytamoxifen (4-OHT) cells treated with Pae. This paper summarized the relevance of Pae in BC cell endocrine resistance to tamoxifen (Tam) and the molecular mechanisms involved miR-15b expression. MATERIALS AND METHODS: 4-OHT-resistant BC cell lines were developed and treated with different concentrations of Pae. Flow cytometry, lactose dehydrogenase activity, caspase-3 activity, colony formation, and EdU assays were carried out to assess the impact of Pae on BC cells. Differentially expressed miRNAs in BC cells treated with Pae were analyzed by microarray. Targeting mRNAs of screened miR-15b as well as the binding of forkhead box O1 (FOXO1) to the cyclin D1 (CCND1) promoter sequence were predicted through bioinformatics analysis. Finally, the expression of ß-catenin signaling-related genes in cells was detected by Western blotting. RESULTS: Pae (100 µg/mL) inhibited the clonality and viability of BC cells, while enhancing apoptosis in vitro. Pae also repressed miR-15b expression. Overexpression of miR-15b restored the growth and resistance of BC cells to 4-OHT. Moreover, Pae promoted FOXO1 expression by downregulating miR-15b, thereby transcriptionally inhibiting CCND1 and subsequently blocking ß-catenin signaling. CONCLUSION: Pae inhibits 4-OHT resistance in BC cells by regulating the miR-15b/FOXO1/CCND1/ß-catenin pathway.

Organosulphur Compounds Induce Apoptosis and Cell Cycle Arrest in Cervical Cancer Cells via Downregulation of HPV E6 and E7 Oncogenes.

BACKGROUND: The quest for strong, safe and cost-effective natural antiproliferative agents that could reduce cancer has been the focus now a days. In this regard, the organosulfur compounds from garlic (Allium sativum L.), like Diallyl Sulfide (DAS) and Diallyl Disulfide (DADS), have been shown to exhibit potent antiproliferative and anticancer properties in many studies. However, the potential of these compounds against viral oncoproteins in cervical cancer has not been fully elucidated yet. OBJECTIVE: The objective of this study was to analyze the antiproliferative and apoptotic properties of DADS and DAS in HPV16+ human cervical cancer Caski cell line. METHODS: Caski (cervical cancer cells) were cultured and followed by the treatment of various concentrations of organosulphur compounds (DADS and DAS), cell viability was measured by MTT assay. The apoptotic assay was performed by DAPI and Hoechst3342 staining. Reactive Oxygen Species (ROS) was estimated by DCFDA staining protocol. The distributions of cell cycle and apoptosis (FITC-Annexin V assay) were analyzed by flow cytometry. Finally, gene expression analysis was performed via quantitative real time PCR. RESULTS: Our results showed that DAS and DADS exerted a significant antiproliferative effect on Caski cells by reducing the cell viability and inducing a dose-related increment in intracellular ROS production along with apoptosis in Caski cells. DAS and DADS also induced cell cycle arrest in G0/G1 phase, which was supported by the downregulation of cyclin D1 and CDK4 and upregulation of CDK inhibitors p21WAF1/CIP1 and p27KIP1 in Caski cells. Additionally, DAS and DADS lead to the downregulation of viral oncogene E6 and E7 and restoration of p53 function. CONCLUSION: Thus, this study confirms the efficacy of both the organosulfur compounds DADS and DAS against cervical cancer cells.

TAG-RNAi overcomes off-target effects in cancer models.

RNA interference offers therapeutic opportunities for the clinical targeting of otherwise undruggable oncogenes. However RNAi can have off-target effects that considerably increase treatment risks. To manage these side effects and allow an easy subtraction of their activity in healthy tissues, we present here the TAG-RNAi approach where cells that are not designated targets do not have the mRNA tag. Using TAG-RNAi we first established the off-target signatures of three different siRNAs specific to the Cyclin D1 oncogene by RNA-sequencing of cultured cancer cells expressing a FLAG-HA-tagged-Cyclin D1. Then, by symmetrical allografts of tagged-cancer cells and untagged controls on the left and right flanks of model mice, we demonstrate that TAG-RNAi is a reliable approach to study the functional impact of any oncogene without off-target bias. Finally we show, as examples, that mutation-specific TAG-RNAi can be applied to downregulate two oncogenic mutants, KRAS-G12V or BRAF-V600E, while sparing the expression of the wild-type proteins. TAG-RNAi will thus avoid the traditional off-target limitations of RNAi in future experimental approaches.

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.

Inactivating NF1 Mutations Are Enriched in Advanced Breast Cancer and Contribute to Endocrine Therapy Resistance.

PURPOSE: Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC. EXPERIMENTAL DESIGN: Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients. RESULTS: We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro. Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant. CONCLUSIONS: Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.

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.

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.

Ara-c induces cell cycle G1/S arrest by inducing upregulation of the INK4 family gene or directly inhibiting the formation of the cell cycle-dependent complex CDK4/cyclin D1.

Cytosine arabinoside (Ara-c) is a pyrimidine anti-metabolite that is capable of interfering with cellular proliferation by inhibiting DNA synthesis. Each inhibitor of cyclin-dependent kinase 4 (INK4) family member has the ability to bind to cyclin-dependent kinase 4 (CDK4) and inhibit the formation of the cell cycle-dependent CDK4/cyclin D1 complex, subsequently leading to cell cycle arrest in the G1/S phase. In this study, the expression of INK4 family genes in kidney cancer and the impact of these genes on patient prognosis were examined. Additionally, the effects of INK4 family genes and Ara-c on cell proliferation and tumor formation and development were examined. Finally, a potential association between Ara-c-induced cell cycle arrest and INK4-associated gene expression was evaluated. An upregulation of INK4 family genes was found to be positively correlated with the prognosis of patients with kidney cancer. Both the INK4 family genes and Ara-c were shown to induce cell cycle arrest and inhibit tumor formation and development. Moreover, Ara-c-induced cell cycle arrest was found to be associated with an Ara-c-induced upregulation of INK4 family gene expression, which ultimately inhibited the formation of the CDK4/cyclin D1 complex. These findings suggested that an upregulation of INK4 family genes has a positive effect on kidney cancer prognosis and can inhibit the formation and development of tumors. Moreover, Ara-c was shown to promote the upregulation of INK4 family genes, at the same time, Ara-c could directly regulate the cell cycle-dependent genes CDK4 and cyclin D1 (CCND1), independent of the INK4 family genes.

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.

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.

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.

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.

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.

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.

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.

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.

Steroid sulfatase inhibition success and limitation in breast cancer clinical assays: An underlying mechanism.

Steroid sulfatase is detectable in most hormone-dependent breast cancers. STX64, an STS inhibitor, induced tumor reduction in animal assay. Despite success in phase І clinical trial, the results of phase II trial were not that significant. Breast Cancer epithelial cells (MCF-7 and T47D) were treated with two STS inhibitors (STX64 and EM1913). Cell proliferation, cell cycle, and the concentrations of estradiol and 5α-dihydrotestosterone were measured to determine the endocrinological mechanism of sulfatase inhibition. Comparisons were made with inhibitions of reductive 17ß-hydroxysteroid dehydrogenases (17ß-HSDs). Proliferation studies showed that DNA synthesis in cancer cells was modestly decreased (approximately 20%), accompanied by an up to 6.5% in cells in the G0/G1 phase and cyclin D1 expression reduction. The concentrations of estradiol and 5α-dihydrotestosterone were decreased by 26% and 3% respectively. However, supplementation of 5α-dihydrotestosterone produced a significant increase (approximately 35.6%) in the anti-proliferative effect of sulfatase inhibition. This study has clarified sex-hormone control by sulfatase in BC, suggesting that the different roles of estradiol and 5α-dihydrotestosterone can lead to a reduction in the effect of sulfatase inhibition when compared with 17ß-HSD7 inhibition. This suggests that combined treatment of sulfatase inhibitors with 17ß-HSD inhibitors such as the type7 inhibitor could hold promise for hormone-dependent breast cancer.