Myeong-Sok Lee

Berberine-induced AMPK activation inhibits the metastatic potential of melanoma cells via reduction of ERK activity and COX-2 protein expression.

Berberine is clinically important natural isoquinoline alkaloid that affects various biological functions, such as cell proliferation, migration and survival. The activation of AMP-activated protein kinase (AMPK) regulates tumor cell migration. However, the specific role of AMPK on the metastatic potential of cancer cells remains largely unknown. The present study investigated whether berberine induces AMPK activation and whether this induction directly affects mouse melanoma cell migration, adhesion and invasion. Berberine strongly increased AMPK phosphorylation via reactive oxygen species (ROS) production. 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), a well-known AMPK activator, also inhibited tumor cell adhesion and invasion and reduced the expression of epithelial to mesenchymal transition (EMT)-related genes. Knockdown of AMPKα subunits using siRNAs significantly abated the berberine-induced inhibition of tumor cell invasion. Furthermore, berberine inhibited the metastatic potential of melanoma cells through a decrease in ERK activity and protein levels of cyclooxygenase-2 (COX-2) by a berberine-induced AMPK activation. These data were confirmed using specific MEK inhibitor, PD98059, and a COX-2 inhibitor, celecoxib. Berberine- and AICAR-treated groups demonstrated significantly decreased lung metastases in the pulmonary metastasis model in vivo. Treatment with berberine also decreased the metastatic potential of A375 human melanoma cells. Collectively, our results suggest that berberine-induced AMPK activation inhibits the metastatic potential of tumor cells through a reduction in the activity of the ERK signaling pathway and COX-2 protein levels.

Adiponectin Is a Negative Regulator of NK Cell Cytotoxicity

NK cells are a key component of innate immune systems, and their activity is regulated by cytokines and hormones. Adiponectin, which is secreted from white adipose tissues, plays important roles in various diseases, including hypertension, cardiovascular diseases, inflammatory disorders, and cancer. In this study the effect of adiponectin on NK cell activity was investigated. Adiponectin was found to suppress the IL-2-enhanced cytotoxic activity of NK cells without affecting basal NK cell cytotoxicity and to inhibit IL-2-induced NF-κB activation via activation of the AMP-activated protein kinase, indicating that it suppresses IL-2-enhanced NK cell cytotoxicity through the AMP-activated protein kinase-mediated inhibition of NF-κB activation. IFN-γ enhances NK cell cytotoxicity by causing an increase in the levels of expression of TRAIL and Fas ligand. The production of IFN-γ, one of the NF-κB target genes in NK cells, was also found to be suppressed by adiponectin, accompanied by the subsequent down-regulation of IFN-γ-inducible TRAIL and Fas ligand expression. These results clearly demonstrate that adiponectin is a potent negative regulator of IL-2-induced NK cell activation and thus may act as an in vivo regulator of anti-inflammatory functions.

Adiponectin-Activated AMPK Stimulates Dephosphorylation of AKT through Protein Phosphatase 2A Activation

Low serum levels of adiponectin are a high risk factor for various types of cancer. Although adiponectin inhibits proliferation and metastasis of breast cancer cells, the underlying molecular mechanisms remain obscure. In this study, we show that adiponectin-activated AMPK reduces the invasiveness of MDA-MB-231 cells by stimulating dephosphorylation of AKT by increasing protein phosphatase 2A (PP2A) activity. Among the various regulatory B56 subunits, B56gamma was directly phosphorylated by AMPK at Ser(298) and Ser(336), leading to an increase of PP2A activity through dephosphorylation of PP2Ac at Tyr(307). We also show that both the blood levels of adiponectin and the tissue levels of PP2A activity were decreased in breast cancer patients and that the direct administration of adiponectin into tumor tissues stimulates PP2A activity. Taken together, these findings show that adiponectin, derived from adipocytes, negatively regulates the invasiveness of breast cancer cells by activating the tumor suppressor PP2A.

Induction of thioredoxin is required for nodule development to reduce reactive oxygen species levels in soybean roots.

Nodules are formed on legume roots as a result of signaling between symbiotic partners and in response to the activities of numerous genes. We cloned fragments of differentially expressed genes in spot-inoculated soybean (Glycine max) roots. Many of the induced clones were similar to known genes related to oxidative stress, such as thioredoxin and β-carotene hydroxylase. The deduced amino acid sequences of full-length soybean cDNAs for thioredoxin and β-carotene hydroxylase were similar to those in other species. In situ RNA hybridization revealed that the thioredoxin gene is expressed on the pericycle of 2-d-old nodules and in the infected cells of mature nodules, suggesting that thioredoxin is involved in nodule development. The thioredoxin promoter was found to contain a sequence resembling an antioxidant responsive element. When a thioredoxin mutant of yeast was transformed with the soybean thioredoxin gene it became hydrogen peroxide tolerant. These observations prompted us to measure reactive oxygen species levels. These were decreased by 3- to 5-fold in 7-d-old and 27-d-old nodules, coincident with increases in the expression of thioredoxin and β-carotene hydroxylase genes. Hydrogen peroxide-producing regions identified with cerium chloride were found in uninoculated roots and 2-d-old nodules, but not in 7-d-old and 27-d-old nodules. RNA interference-mediated repression of the thioredoxin gene severely impaired nodule development. These data indicate that antioxidants such as thioredoxin are essential to lower reactive oxygen species levels during nodule development.

Tumor necrosis factor-α and interleukin-1β increases CTRP1 expression in adipose tissue

CTRP1, a member of the CTRP superfamily, consists of an N‐terminal signal peptide sequence followed by a variable region, a collagen repeat domain, and a C‐terminal globular domain. CTRP1 is expressed at high levels in adipose tissues of LPS‐stimulated Sprague‐Dawley rats. The LPS‐induced increase in CTRP1 gene expression was found to be mediated by TNF‐α and IL‐1β. Also, a high level of expression of CTRP1 mRNA was observed in adipose tissues of Zucker diabetic fatty (fa/fa) rats, compared to Sprague‐Dawley rats in the absence of LPS stimulation. These findings indicate that CTRP1 expression may be associated with a low‐grade chronic inflammation status in adipose tissues.

The Molecular Mechanisms of Vitamin C on Cell Cycle Regulation in B16F10 Murine Melanoma

Vitamin C has inconsistent effects on malignant tumor cells, which vary from growth stimulation to apoptosis induction. It is well known that melanoma cells are more susceptible to vitamin C than any other tumor cells, but the precise mechanism remains to be elucidated. In the present study, the proliferation of B16F10 melanoma cells was suppressed by vitamin C, which induced growth arrest in a dose‐dependent manner without cytotoxic effects. Therefore, we investigated the changes in cell cycle distribution of B16F10 melanoma cells by staining DNAs with propidium iodide (PI). The growth inhibition of B16F10 melanoma by vitamin C was associated with an arrest of cell cycle distribution at G1 stage. In addition, the levels of p53‐p21Waf1/Cip1 increased during G1 arrest, which were essential for vitamin C‐induced cell cycle arrest. The increased p21Waf1/Cip1 inhibited CDK2. Moreover, the activity of p53‐p21Waf1/Cip1 pathway was closely related with the activation of checkpoint kinase 2 (Chk2). Inhibitor of the PI3K‐family, LY294002 and the ATM/ATR inhibitor, caffeine, blocked vitamin C‐induced growth arrest in B16F10 melanoma cells. These results suggest that vitamin C might be a potent agent to inhibit proliferative activity of melanoma cells via the regulation of Chk2‐p53‐p21Waf1/Cip1 pathway. J. Cell. Biochem. 102: 1002–1010, 2007.

The role of vimentin as a methylation biomarker for early diagnosis of cervical cancer

Multiple cytosine guanine dinucleotides (CpG island) are found in the VIM promoter region. The levels of VIM promoter methylation and VIM gene expression were investigated in 7 cervical cancer cell lines and 50 human tissue samples with a distinctive degree of malignant trans-formation. While multiple CpG sites in the VIM promoter were highly methylated in CIN III and invasive carcinoma cells, they were rarely methylated in normal cells. Our result shows that methylation in the VIM promoter appears to start from CIN I and CIN II, relatively early stages of multistep carcinogenesis. This epigenetic alteration in VIM promoter suggests the availability as a biomarker for the early diagnosis and prevention of cervical cancer. We also show that hypermethylation in the VIM promoter is responsible for transcriptional silencing of the VIM gene in cervical cancer cells. In addition, our result shows that exogenous overexpression of the VIM gene in SiHa cervical cancer cells slightly activated cell proliferation and migration as shown in soft agar colony formation and migration assays.

Apoptosis-inducing factor (AIF) inhibits protein synthesis by interacting with the eukaryotic translation initiation factor 3 subunit p44 (eIF3g).

Apoptosis‐inducing factor (AIF) is a ubiquitous FAD‐binding flavoprotein comprised of 613 amino acids and plays an important role in caspase‐independent apoptosis. During apoptotic induction, AIF is translocated from the mitochondrial intermembrane space to the nucleus, where it interacts with DNA and activates a nuclear endonuclease. By performing a yeast two‐hybrid screen with mature AIF, we have isolated the eukaryotic translation initiation factor 3 subunit p44 (eIF3g). Our deletion mutant analysis revealed that the eIF3g N‐terminus interacts with the C‐terminal region of AIF. The direct interaction between AIF and eIF3g was confirmed in a GST pull‐down assay and also verified by the results of co‐immunoprecipitation and confocal microscopy studies. Using an in vitro TNT coupled transcription–translation system, we found that mature AIF could inhibit newly‐translated protein synthesis and this inhibition was significantly blocked by eIF3g competitively. These results were also confirmed in cells. In addition, mature AIF overexpression specifically resulted in the activation of caspase‐7, thereby amplifying the inhibition of protein synthesis including eIF3g cleavage. Our data suggest that eIF3g is one of the cytosolic targets that interacts with mature AIF, and provide insight into the AIFs cellular functions of the inhibition of protein synthesis during apoptosis.

N-myc downstream-regulated gene 2 (NDRG2) suppresses the epithelial–mesenchymal transition (EMT) in breast cancer cells via STAT3/Snail signaling

Although NDRG2 has recently been found to be a candidate tumor suppressor, its precise role in the epithelial-mesenchymal transition (EMT) is not well understood. In the present study, we demonstrated that NDRG2 overexpression in MDA-MB-231 cells down-regulated the expression of Snail, a transcriptional repressor of E-cadherin and a key regulator of EMT, as well as the phosphorylation of signal transducer and activator of transcription 3 (STAT3), an oncogenic transcription factor that is activated in many human malignancies including breast cancer. In addition, we confirmed that the expression of Snail and phospho-STAT3 was recovered when NDRG2 was knocked down by siRNA in MCF7 cells in which NDRG2 is endogenously expressed. Interestingly, MDA-MB-231-NDRG2 cells showed remarkably decreased Snail expression after treatment with JSI-124 (also known as cucurbitacin I) or Stattic, STAT3 inhibitors, compared to MDA-MB-231-mock cells. Moreover, STAT3 activation by EGF treatment induced higher Snail expression, and NDRG2 overexpression resulted in the inhibition of Snail expression in MDA-MB-231 cells stimulated by EGF in the absence or presence of STAT3 inhibitor. Treatment of MDA-MB-231 cells with STAT3 inhibitor led to a moderate decrease in wound healing and migration capacity, whereas STAT3 inhibitor treatment of MDA-MB-231-NDRG2 cells resulted in a significant attenuation of migration in both resting and EGF-stimulated cells. Collectively, our data demonstrate that the inhibition of STAT3 signaling by NDRG2 suppresses EMT progression of EMT via the down-regulation of Snail expression.

Carabrol suppresses LPS-induced nitric oxide synthase expression by inactivation of p38 and JNK via inhibition of I-κBα degradation in RAW 264.7 cells

Carabrol, isolated from Carpesium macrocephalum, showed anti-inflammatory potential in LPS-induced RAW 264.7 murine macrophages. In present study, carabrol demonstrated the inhibitory activity on pro-inflammatory cytokines such as IL-1beta, IL-6 and TNF-alpha. In addition, mRNA and protein levels of iNOS and COX-2 were reduced by carabrol. Molecular analysis revealed that these suppressive effects were correlated with the inactivation of p38 and JNK via inhibition of NF-kappaB activation. Immunoblotting showed that carabrol suppressed LPS-induced degradation of I-kappaBalpha and decreased nuclear translocation of p65. Taken together, these results suggest that carabrol can be a modulator of pro-inflammatory signal transduction pathway in RAW 264.7 cells.