Jong-Seok Lim

Functional and Clinical Evidence for NDRG2 as a Candidate Suppressor of Liver Cancer Metastasis

We searched for potential suppressors of tumor metastasis by identifying the genes that are frequently down-regulated in hepatocellular carcinomas (HCC) while being negatively correlated with clinical parameters relevant to tumor metastasis, and we report here on the identification of N-myc downstream regulated gene 2 (NDRG2) as a promising candidate. NDRG2 expression was significantly reduced in HCC compared with nontumor or normal liver tissues [87.5% (35 of 40) and 62% (62 of 100) at RNA and protein levels, respectively]. Reduction of NDRG2 expression was intimately associated with promoter hypermethylation because its promoter region was found to carry extensively methylated CpG sites in HCC cell lines and primary tumors. Immunohistochemical analysis of NDRG2 protein in 100 HCC patient tissues indicated that NDRG2 expression loss is significantly correlated with aggressive tumor behaviors such as late tumor-node-metastasis (TNM) stage (P = 0.012), differentiation grade (P = 0.024), portal vein thrombi (P = 0.011), infiltrative growth pattern (P = 0.015), nodal/distant metastasis (P = 0.027), and recurrent tumor (P = 0.021), as well as shorter patient survival rates. Ectopically expressed NDRG2 suppressed invasion and migration of a highly invasive cell line, SK-Hep-1, and experimental tumor metastasis in vivo, whereas small interfering RNA-mediated knockdown resulted in increased invasion and migration of a weakly invasive cell line, PLC/PRF/5. In addition, NDRG2 could antagonize transforming growth factor beta1-mediated tumor cell invasion by specifically down-regulating the expression of matrix metalloproteinase 2 and laminin 332 pathway components, with concomitant suppression of Rho GTPase activity. These results suggest that NDRG2 can inhibit extracellular matrix-based, Rho-driven tumor cell invasion and migration and thereby play important roles in suppressing tumor metastasis in HCC.

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.

Epigallocatechin-3-Gallate Enhances CD8+ T Cell–Mediated Antitumor Immunity Induced by DNA Vaccination

Immunotherapy and chemotherapy are generally effective against small tumors in animal models of cancer. However, these treatment regimens are generally ineffective against large, bulky tumors. We have found that a multimodality treatment regimen using DNA vaccination in combination with chemotherapeutic agent epigallocatechin-3-gallate (EGCG), a compound found in green tea, is effective in inhibiting large tumor growth. EGCG was found to induce tumor cellular apoptosis in a dose-dependent manner. The combination of EGCG and DNA vaccination led to an enhanced tumor-specific T-cell immune response and enhanced antitumor effects, resulting in a higher cure rate than either immunotherapy or EGCG alone. In addition, combined DNA vaccination and oral EGCG treatment provided long-term antitumor protection in cured mice. Cured animals rejected a challenge of E7-expressing tumors, such as TC-1 and B16E7, but not a challenge of B16 7 weeks after the combined treatment, showing antigen-specific immune responses. These results suggest that multimodality treatment strategies, such as combining immunotherapy with a tumor-killing cancer drug, may be a more effective anticancer strategy than single-modality treatments.

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.

Suppression of NF-κB activity by NDRG2 expression attenuates the invasive potential of highly malignant tumor cells

Downregulation of the N-myc downstream-regulated gene 2 (NDRG2) gene is involved in the progression of aggressive forms of cancer, along with the poor prognosis of cancer patients. In the current study, we examined the effect of NDRG2 expression on the metastatic potential of HT1080 human fibrosarcoma and B16F10 murine melanoma cells in both in vitro and in vivo systems. In gelatin zymography, NDRG2 expression remarkably suppressed the matrix metalloproteinase (MMP)-9 activity and slightly inhibited MMP-2 activity of both cell lines. Tumor migration and invasion in vitro were significantly reduced by NDRG2 expression, and NDRG2 inhibited tumor cell proliferation in an anchorage-independent semisolid agar assay. Specifically, we found that NDRG2 affects invasion through suppression of nuclear factor kappa B (NF-kappaB) activity. In animal experiments, subcutaneously injected B16F10-NDRG2 cells showed delayed tumor growth compared with B16F10-mock cells. Furthermore, severe metastasis from primary tumor mass into the draining lymph nodes was observed after injection of B16F10-mock cells, but not with B16F10-NDRG2 cells. Pulmonary metastasis after intravenous injection of B16F10 cells was also reduced by NDRG2 expression. Intra- and peritumoral angiogenesis that is critical for the tumor growth and metastasis was clearly found in tumors after injection with B16F10-mock cells, whereas it was impaired in tumors after injection with B16F10-NDRG2 cells. Collectively, our data show that NDRG2 expression significantly suppresses tumor invasion by inhibiting MMP activities, which are regulated through the NF-kappaB signaling. Moreover, results from animal experiments provide evidence for the regulatory role of the NDRG2 gene in metastatic tumors.

Expression and regulation of NDRG2 (N-myc downstream regulated gene 2) during the differentiation of dendritic cells

We searched for genes with expressions specific to human monocyte‐derived dendritic cells (DCs) using differential display reverse transcription‐polymerase chain reaction, and found that N‐myc downstream regulated gene 2 (NDRG2), a member of a new family of differentiation‐related genes, was expressed in DCs. While DCs derived from CD34+ progenitor cells also showed strong NDRG2 expression, the corresponding mRNA expression was absent in other cell lines including monocytes, B cells, and NK cells. The inhibition of DC differentiation by dexamethasone or vitamin D3 treatment down‐regulated the expression of the NDRG2 gene in DCs. In addition, gene expression was induced in a myelomonocytic leukemia cell line, which is capable of differentiating into DCs in cytokine‐conditioned culture. The level of NDRG2 gene expression in DCs was significantly higher than that of other members of the NDRG gene family. Finally, in contrast to the stable NDRG2 expression in CD40‐stimulated DCs, the induction of DC maturation by lipopolysaccharide (LPS) resulted in the down‐regulation of NDRG2 gene expression. This down‐regulation is likely to be due to a modification and subsequent destabilization of NDRG2 mRNA, because co‐treating with actinomycin D and LPS significantly blocked this LPS effect. Taken together, our results indicate that NDRG2 is expressed during the differentiation of DCs, and that NDRG2 gene expression is differentially regulated by maturation‐inducing stimuli.

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.

Bone morphogenetic protein-4 induced by NDRG2 expression inhibits MMP-9 activity in breast cancer cells.

In the current study, we examined the function of N-myc downstream-regulated gene 2 (NDRG2) expression in breast cancer cells, especially focusing on the role of bone morphogenetic protein-4 (BMP-4) induced by NDRG2. NDRG2 expression in MDA-MB-231 cells inhibited the mRNA expression of several matrix metalloproteinases (MMPs) and the gelatinolytic activity of MMP-9. Interestingly, a specific induction of active BMP-4 was exclusively observed in MDA-MB-231-NDRG2 cells but not in MDA-MB-231-mock cells. Neutralization of BMP-4 in MDA-MB-231-NDRG2 cells resulted in the rescue of MMP-9 mRNA expression and migration capacity. In addition, treatment with recombinant BMP-4 dramatically suppressed MMP-9 mRNA expression, gelatinolytic MMP-9 activity, migration, and invasion capacity both in MDA-MB-231 and PMA-treated MCF-7 cells. Collectively, our data show that BMP-4 induced by NDRG2 expression inhibits the metastatic potential of breast cancer cells, especially via suppression of MMP-9 activity.

DNA Gyrase Is Involved in Chloroplast Nucleoid Partitioning

DNA gyrase, which catalyzes topological transformation of DNA, plays an essential role in replication and transcription in prokaryotes. Virus-induced gene silencing of NbGyrA or NbGyrB, which putatively encode DNA gyrase subunits A and B, respectively, resulted in leaf yellowing phenotypes in Nicotiana benthamiana. NbGyrA and NbGyrB complemented the gyrA and gyrB temperature-sensitive mutations of Escherichia coli, respectively, which indicates that the plant and bacterial subunits are functionally similar. NbGyrA and NbGyrB were targeted to both chloroplasts and mitochondria, and depletion of these subunits affected both organelles by reducing chloroplast numbers and inducing morphological and physiological abnormalities in both organelles. Flow cytometry analysis revealed that the average DNA content in the affected chloroplasts and mitochondria was significantly higher than in the control organelles. Furthermore, 4′,6-diamidino-2-phenylindole staining revealed that the abnormal chloroplasts contained one or a few large nucleoids instead of multiple small nucleoids dispersed throughout the stroma. Pulse-field gel electrophoresis analyses of chloroplasts demonstrated that the sizes and/or structure of the DNA molecules in the abnormal chloroplast nucleoids are highly aberrant. Based on these results, we propose that DNA gyrase plays a critical role in chloroplast nucleoid partitioning by regulating DNA topology.

Expression of NDRG2 is related to tumor progression and survival of gastric cancer patients through Fas-mediated cell death

Although N-myc downstream regulated gene 2 (NDRG2) has been known to be a tumor suppressor gene, the function of this gene has not been elucidated. In the present study, we investigated the expression and function of NDRG2 in human gastric cancer. Among seven gastric cancer and two non-cancer cell lines, only two gastric cancer cell lines, SNU-16 and SNU-620, expressed NDRG2, which was detected in the cytoplasm. Interestingly, NDRG2 was highly expressed in normal gastric tissues, but gastric cancer patients were divided into NDRG2-positive and -negative groups. The survival rate of NDRG2-negative patients was lower than that of NDRG2-positive patients. We confirmed that the loss of NDRG2 expression was a significant and independent prognostic indicator in gastric carcinomas by multivariate analysis. To investigate the role of NDRG2 in gastric cancer cells, we generated a NDRG2-silenced gastric cancer cell line, which stably expresses NDRG2 siRNA. NDRG2-silenced SNU-620 cells exhibited slightly increased proliferation and cisplatin resistance. In addition, inhibition of NDRG2 decreased Fas expression and Fas-mediated cell death. Taken together, these data suggest that inactivation of NDRG2 may elicit resistance against anticancer drug and Fas-mediated cell death. Furthermore, case studies of gastric cancer patients indicate that NDRG2 expression may be involved in tumor progression and overall survival of the patients.