Jieun Yun

Cross-talk between Notch and the Estrogen Receptor in Breast Cancer Suggests Novel Therapeutic Approaches

High expression of Notch-1 and Jagged-1 mRNA correlates with poor prognosis in breast cancer. Elucidating the cross-talk between Notch and other major breast cancer pathways is necessary to determine which patients may benefit from Notch inhibitors, which agents should be combined with them, and which biomarkers indicate Notch activity in vivo. We explored expression of Notch receptors and ligands in clinical specimens, as well as activity, regulation, and effectors of Notch signaling using cell lines and xenografts. Ductal and lobular carcinomas commonly expressed Notch-1, Notch-4, and Jagged-1 at variable levels. However, in breast cancer cell lines, Notch-induced transcriptional activity did not correlate with Notch receptor levels and was highest in estrogen receptor alpha-negative (ERalpha(-)), Her2/Neu nonoverexpressing cells. In ERalpha(+) cells, estradiol inhibited Notch activity and Notch-1(IC) nuclear levels and affected Notch-1 cellular distribution. Tamoxifen and raloxifene blocked this effect, reactivating Notch. Notch-1 induced Notch-4. Notch-4 expression in clinical specimens correlated with proliferation (Ki67). In MDA-MB231 (ERalpha(-)) cells, Notch-1 knockdown or gamma-secretase inhibition decreased cyclins A and B1, causing G(2) arrest, p53-independent induction of NOXA, and death. In T47D:A18 (ERalpha(+)) cells, the same targets were affected, and Notch inhibition potentiated the effects of tamoxifen. In vivo, gamma-secretase inhibitor treatment arrested the growth of MDA-MB231 tumors and, in combination with tamoxifen, caused regression of T47D:A18 tumors. Our data indicate that combinations of antiestrogens and Notch inhibitors may be effective in ERalpha(+) breast cancers and that Notch signaling is a potential therapeutic target in ERalpha(-) breast cancers.

Hepatoprotective Effect of Aged Black Garlic Extract in Rodents

In this study, we investigated the hepatoprotective effects of aged black garlic (ABG) in rodent models of liver injury. ABG inhibited carbon tetrachloride-induced elevation of aspartate transaminase (AST) and alanine transaminase (ALT), which are markers of hepatocellular damage, in SD rats. D-galactosamineinduced hepatocellular damage was also suppressed by ABG treatment. However, ABG does not affect the elevation of alkaline phosphatase (ALP), a marker of hepatobilliary damage, in rats treated with carbon tetrachloride or D-galactosamine. We also examined the effect of ABG on high-fat diet (HFD)-induced fatty liver and subsequent liver damage. ABG had no significant effect on body weight increase and plasma lipid profile in HFD-fed mice. However, HFD-induced increase in AST and ALT, but not ALP, was significantly suppressed by ABG treatment. These results demonstrate that ABG has hepatoprotective effects and suggest that ABG supplementation might be a good adjuvant therapy for the management of liver injury.

Widdrol induces apoptosis via activation of AMP-activated protein kinase in colon cancer cells

Widdrol, a natural sesquiterpene present in Juniperus sp., has been shown to exert anticancer and antifungal effects. Emerging evidence has suggested that AMP-activated protein kinase (AMPK), which functions as a cellular energy sensor, is a potential therapeutic target for human cancers. In this study, we found that AMPK mediates the anticancer effects of widdrol through induction of apoptosis in HT-29 colon cancer cells. We showed that widdrol induced the phosphorylation of AMPK in a dose- and time-dependent manner. The selective AMPK inhibitor compound C abrogated the inhibitory effect of widdrol on HT-29 cell growth. In addition, we demonstrated that widdrol induced apoptosis and this was associated with the activation of caspases, including caspase‑3/7 and caspase-9, in HT-29 cells. We also demonstrated that transfection of HT-29 cells with AMPK siRNAs significantly suppressed the widdrol-mediated apoptosis and the activation of caspases. However, cell cycle arrest induced by widdrol was not affected by transfection of HT-29 cells with AMPK siRNAs. Furthermore, widdrol inhibited HT-29 tumor growth in a human tumor xenograft model. Taken together, our results suggest that the anticancer effect of widdrol may be mediated, at least in part, by induction of apoptosis via AMPK activation.

Violapyrones H and I, New Cytotoxic Compounds Isolated from Streptomyces sp. Associated with the Marine Starfish Acanthaster planci

Two new α-pyrone derivatives, violapyrones H (1) and I (2), along with known violapyrones B (3) and C (4) were isolated from the fermentation broth of a marine actinomycete Streptomyces sp. The strain was derived from a crown-of-thorns starfish, Acanthaster planci, collected from Chuuk, Federated States of Micronesia. The structures of violapyrones were elucidated by the analysis of 1D and 2D NMR and HR-ESIMS data. Violapyrones (1–4) exhibited cytotoxicity against 10 human cancer cell lines with GI50 values of 1.10–26.12 μg/mL when tested using sulforhodamine B (SRB) assay. This is the first report on the cytotoxicity of violapyrones against cancer cell lines and the absolute configuration of violapyrone C.

Diagnostic Value of Circulating Extracellular miR-134, miR-185, and miR-22 Levels in Lung Adenocarcinoma-Associated Malignant Pleural Effusion

Purpose The accurate and timely diagnosis of malignant pleural effusion (MPE) in lung cancer patients is important because MPE has a poor prognosis and is classified as stage IV disease. Molecular biomarkers for pleural effusion, such as circulating extracellular microRNAs (miRNAs) isolated from pleural fluid, may help in the diagnosis of MPE. The present study examined whether miRNAs that are deregulated in lung cancer (miR-134, miR-185, and miR-22) can serve as diagnostic markers for lung adenocarcinoma-associated MPE (LA-MPE). Materials and Methods Real-time reverse transcription quantitative polymerase chain reaction was used to measure the expression of the three miRNAs in samples from 87 patients with pleural effusion comprising 45 LA-MPEs and 42 benign pleural effusions (BPEs). The area under the receiver operating characteristic curve (AUC) was then used to evaluate the diagnostic performance of each of the three miRNAs and compare it with that of the common tumor marker, carcinoembryonic antigen (CEA). Results The expression of all three miRNAs was significantly lower in LA-MPE than in BPE (p <0.001). The AUCs for miR-134, miR-185, miR-22, and CEA were 0.721, 0.882, 0.832, and 0.898, respectively. Combining CEA with the three miRNAs increased the diagnostic performance, yielding an AUC of 0.942 (95% confidence interval, 0.864 to 0.982), with a sensitivity of 91.9% and a specificity of 92.5%. Conclusion The present study suggests that the expression levels of circulating extracellular miR-134, miR-185, and miR-22 in patients with pleural effusion may have diagnostic value when differentiating between LA-MPE and BPE.

miR-6734 Up-Regulates p21 Gene Expression and Induces Cell Cycle Arrest and Apoptosis in Colon Cancer Cells

Recently, microRNAs have been implicated in the regulation of gene expression in terms of both gene silencing and gene activation. Here, we investigated the effects of miR-6734, which has a sequence homology with a specific region of p21WAF1/CIP1 (p21) promoter, on cancer cell growth and the mechanisms involved in this effect. miR-6734 up-regulated p21 expression at both mRNA and protein levels and chromatin immunoprecipitation analysis using biotin-labeled miR-6734 confirmed the association of miR-6734 with p21 promoter. Moreover, miR-6734 inhibited cancer cell growth and induced cell cycle arrest and apoptosis in HCT-116 cells, which was abolished by knockdown of p21. The phosphorylation of Rb and the cleavage of caspase 3 and PARP were suppressed by miR-6734 transfection in HCT-116 cells and these effects were also reversed by p21 knockdown. In addition, miR-6734 transfection caused prolonged induction of p21 gene and modification of histones in p21 promoter, which are typical aspects of a phenomenon referred to as RNA activation (RNAa). Collectively, our results demonstrated that miR-6734 inhibits the growth of colon cancer cells by up-regulating p21 gene expression and subsequent induction of cell cycle arrest and apoptosis, suggesting its role as an important endogenous regulator of cancer cell proliferation and survival.

Development of Novel 1,2,3,4-Tetrahydroquinoline Scaffolds as Potent NF-κB Inhibitors and Cytotoxic Agents.

1,2,3,4-Tetrahydroquinolines have been identified as the most potent inhibitors of LPS-induced NF-κB transcriptional activity. To discover new molecules of this class with excellent activities, we designed and synthesized a series of novel derivatives of 1,2,3,4-tetrahydroquinolines (4a-g, 5a-h, 6a-h, and 7a-h) and bioevaluated their in vitro activity against human cancer cell lines (NCI-H23, ACHN, MDA-MB-231, PC-3, NUGC-3, and HCT 15). Among all synthesized scaffolds, 6g exhibited the most potent inhibition (53 times that of a reference compound) of LPS-induced NF-κB transcriptional activity and the most potent cytotoxicity against all evaluated human cancer cell lines.

Lipid-soluble ginseng extract inhibits invasion and metastasis of B16F10 melanoma cells.

This study was performed to elucidate the effect of a lipid-soluble ginseng extract (LSGE) on cancer invasion and metastasis. The LSGE, even at noncytotoxic concentrations, potently inhibited invasion and migration of B16F10 mouse melanoma cells in a dose-dependent manner. In the presence of 3 μg/mL of LSGE, the invasion and migration of B16F10 cells were significantly inhibited by 98.1% and 71.4%, respectively. Furthermore, the LSGE decreased mRNA and protein levels of matrix metalloproteinase (MMP)-2 in B16F10 cells, leading to a decrease in MMP-2 activity. After B16F10 cells were intravenously injected in the tail vein of C57BL/6 mice, 1000 mg/kg/day of LSGE was orally administered for 13 days, after which lung metastasis of cancer cells was inhibited by 59.3%. These findings indicate that LSGE inhibits cancer cell invasion and migration in vitro and lung metastasis of melanoma cells in vivo by inhibiting MMP-2 expression.

p53 Modulates Notch Signaling in MCF‐7 Breast Cancer Cells by Associating With the Notch Transcriptional Complex Via MAML1

p53 and Notch‐1 play important roles in breast cancer biology. Notch‐1 inhibits p53 activity in cervical and breast cancer cells. Conversely, p53 inhibits Notch activity in T‐cells but stimulates it in human keratinocytes. Notch co‐activator MAML1 binds p53 and functions as a p53 co‐activator. We studied the regulation of Notch signaling by p53 in MCF‐7 cells and normal human mammary epithelial cells (HMEC). Results show that overexpression of p53 or activation of endogenous p53 with Nutlin‐3 inhibits Notch‐dependent transcriptional activity and Notch target expression in a dose‐dependent manner. This effect could be partially rescued by transfection of MAML1 but not p300. Standard and quantitative co‐immunoprecipitation experiments readily detected a complex containing p53 and Notch‐1 in MCF‐7 cells. Formation of this complex was inhibited by dominant negative MAML1 (DN‐MAML1) and stimulated by wild‐type MAML1. Standard and quantitative far‐Western experiments showed a complex including p53, Notch‐1, and MAML1. Chromatin immunoprecipitation (ChIP) experiments showed that p53 can associate with Notch‐dependent HEY1 promoter and this association is inhibited by DN‐MAML1 and stimulated by wild‐type MAML1. Our data support a model in which p53 associates with the Notch transcriptional complex (NTC) in a MAML1‐dependent fashion, most likely through a p53‐MAML1 interaction. In our cellular models, the effect of this association is to inhibit Notch‐dependent transcription. Our data suggest that p53‐null breast cancers may lack this Notch‐modulatory mechanism, and that therapeutic strategies that activate wild‐type p53 can indirectly cause inhibition of Notch transcriptional activity. J. Cell. Physiol. 230: 3115–3127, 2015.

Agelasine D Suppresses RANKL-Induced Osteoclastogenesis via Down-Regulation of c-Fos, NFATc1 and NF-κB

In the present study, we investigated the effect of agelasine D (AD) on osteoclastogenesis. Treatment of bone marrow macrophages (BMMs) with receptor activator of nuclear factor κB ligand (RANKL) resulted in a differentiation of BMMs into osteoclasts as evidenced by generation of tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated cells and formation of pits in calcium phosphate-coated plates. However, RANKL-induced osteoclastogenesis was significantly suppressed by AD treatment. We also confirmed the increased mRNA and protein expression of osteoclastic markers, such as TRAP, cathepsin K and matrix metalloproteinase-9, during RANKL-induced osteoclast differentiation and this was down-regulated by AD treatment. Moreover, AD treatment significantly suppressed RANKL-induced mRNA expression of DC-STAMP and OC-STAMP and cell fusion of TRAP-positive mononuclear osteoclast precursors. In addition, AD suppressed RANKL-induced expression of transcription factors, c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are important transcription factors involved in differentiation of BMMs into osteoclasts. Furthermore, RANKL-induced phosphorylation of extracellular signal-related kinase (ERK) and activation of NF-κB were also inhibited by AD treatment. Collectively, these results suggest that AD inhibits RANKL-induced osteoclastogenesis by down-regulation of multiple signaling pathways involving c-Fos, NFATc1, NF-κB and ERK. Our results also suggest that AD might be a potential therapeutic agent for prevention and treatment of osteoporosis.