Identification of a novel PARP4 gene promoter CpG locus associated with cisplatin chemoresistance.

The protein family of poly (ADP-ribose) polymerases (PARPs) is comprised of multifunctional nuclear enzymes. Several PARP inhibitors have been developed as new anticancer drugs to combat resistance to chemotherapy. Herein, we characterized PARP4 mRNA expression profiles in cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines. PARP4 mRNA expression was significantly upregulated in cisplatin-resistant ovarian cancer cell lines, and this upregulation was associated with the hypomethylation of specific cytosine-phosphate-guanine (CpG) sites (cg18582260 and cg17117459) on its promoter. Reduced PARP4 expression was restored by treating cisplatin-sensitive cell lines with a demethylation agent, implicating the epigenetic regulation of PARP4 expression by promoter methylation. Depletion of PARP4 expression in cisplatin-resistant cell lines reduced cisplatin chemoresistance and promoted cisplatin-induced DNA fragmentation. The differential mRNA expression and DNA methylation status at specific PARP4 promoter CpG sites (cg18582260 and cg17117459) according to cisplatin responses, was further validated in primary ovarian tumor tissues. The results showed significantly increased PARP4 mRNA expressions and decreased DNA methylation levels at specific PARP4 promoter CpG sites (cg18582260 and cg17117459) in cisplatin-resistant patients. Additionally, the DNA methylation status at cg18582260 CpG sites in ovarian tumor tissues showed fairly clear discrimination between cisplatin-resistant patients and cisplatin-sensitive patients, with high accuracy (area under the curve = 0.86, P = 0.003845). Our findings suggest that the DNA methylation status of PARP4 at the specific promoter site (cg18582260) may be a useful diagnostic biomarker for predicting the response to cisplatin in ovarian cancer patients. [BMB Reports 2023; 56(6): 347-352].

Enhanced repair processes and iron uptake by ischemic preconditioning in the brain during the recovery phase after ischemic stroke.

Ischemic preconditioning (IP) reduces brain damage after subsequent ischemic strokes by activating endogenous protective mechanisms in rodents. Transient ischemic attack (TIA) induces tolerance in the human brain after ischemic strokes; defining mechanisms of IP effects may provide therapeutic targets to improve recovery of patients with ischemic strokes. Iron transported across the blood-brain barrier (BBB) is required for brain functions, including myelination, and its levels should be finely regulated to avoid harmful effects. This study aimed to determine whether IP enhances repair processes by modulating iron metabolism during the post-stroke chronic phase. Male mice were divided into sham and IP groups, and IP was induced 24 h before a transient focal ischemic stroke. Sensorimotor recovery was observed over 8 weeks after the stroke, and brain volumes and levels of proteins related to repair processes and iron metabolism in the ischemic brains were examined 8 weeks after the stroke. There was significantly less ischemic brain atrophy in the IP group than in the sham group, with no differences in sensorimotor recovery between the groups. Levels of tight junction proteins of BBB, neurites outgrowth markers, and myelin sheath proteins and markers for mature oligodendrocytes were significantly increased in the IP group. Iron import proteins, transferrin receptor 1 and DMT1, were also increased in the IP group. These results indicate that IP increases brain repair processes and iron uptake during the chronic phase after an ischemic stroke, and provide new insights to understand the molecular mechanisms of TIA effects on post-stroke recovery.

Ahnak scaffolds p11/Anxa2 complex and L-type voltage-gated calcium channel and modulates depressive behavior.

Genetic polymorphisms of the L-type voltage-gated calcium channel (VGCC) are associated with psychiatric disorders including major depressive disorder. Alterations of S100A10 (p11) level are also implicated in the etiology of major depressive disorder. However, the existence of an endogenous regulator in the brain regulating p11, L-type VGCC, and depressive behavior has not been known. Here we report that Ahnak, whose function in the brain has been obscure, stabilizes p11 and Anxa2 proteins in the hippocampus and prefrontal cortex in the rodent brain. Protein levels of Ahnak, p11, and Anxa2 are highly and positively correlated in the brain. Together these data suggest the existence of an Ahnak/p11/Anxa2 protein complex. Ahnak is expressed in p11-positive as well as p11-negative neurons. Ahnak, through its N-terminal region, scaffolds the L-type pore-forming α1 subunit and, through its C-terminal region, scaffolds the ß subunit of VGCC and the p11/Anxa2 complex. Cell surface expression of the α1 subunits and L-type calcium current are significantly reduced in primary cultures of Ahnak knockout (KO) neurons compared to wild-type controls. A decrease in the L-type calcium influx is observed in both glutamatergic neurons and parvalbumin (PV) GABAergic interneurons of Ahnak KO mice. Constitutive Ahnak KO mice or forebrain glutamatergic neuron-selective Ahnak KO mice display a depression-like behavioral phenotype similar to that of constitutive p11 KO mice. In contrast, PV interneuron-selective Ahnak KO mice display an antidepressant-like behavioral phenotype. Our results demonstrate L-type VGCC as an effector of the Ahnak/p11/Anxa2 complex, revealing a novel molecular connection involved in the control of depressive behavior.

SPSB1 enhances ovarian cancer cell survival by destabilizing p21.

SPRY domain-containing SOCS box protein 1 (SPSB1) is an E3 ligase adaptor protein with unknown functions in cancer cells. In this study, we found that SPSB1 knockdown markedly decreased the viability and migration of ovarian cancer cells, while ectopic SPSB1 overexpression in IL-3-dependent Ba/F3 cells significantly increased their proliferation rate compared with empty vector-transfected cells. SPSB1 knockdown significantly elevated p21 protein and mRNA levels and induced apoptosis in ovarian cancer cells, as evidenced by increased levels of cleaved PARP and decreased levels of Bcl-2. Notably, mechanistic investigations revealed that SPSB1 accelerated p21 destabilization by directly interacting with p21 and promoting its ubiquitin-mediated proteasomal degradation. Taken together, our findings provide novel insights into the role of SPSB1 in ovarian cancer cells.

C16­ceramide and sphingosine 1­phosphate/S1PR2 have opposite effects on cell growth through mTOR signaling pathway regulation.

Recently, sphingolipid derivatives, such as ceramide and sphingosine­1­phosphate (S1P), have emerged as key modulators in apoptotic cell death and cell proliferation. This study aimed to clarify the underlying signaling pathways of ceramide and S1P involved in breast cancer cell proliferation. Ceramide acyl chain length is determined by six mammalian ceramide synthases (CerS). We overexpressed CerS1 to 6 in MCF­7 cells to examine whether ceramide signaling propagation varies as a function of acyl chain length. Among the six CerS, only CerS6 overexpression reduced phosphorylation of Akt, S6 kinase (S6K), and extracellular signal­regulated kinases (ERK) as shown by western blotting. In addition, CerS6 overexpression reduced MCF­7 cell proliferation. This effect was partially reversed by co­treatment with MHY1485, an activator of mammalian target of rapamycin (mTOR), demonstrating an important role for the mTOR pathway in the CerS6­mediated decrease in MCF­7 cell proliferation. ERK inhibition, but not Akt inhibition, along with mTOR inhibition synergistically reduced MCF­7 cell proliferation as measured by MTT assay. Notably, the expression of CerS6 and S1P receptor 2 (S1PR2), or CerS6 and sphingosine kinase 1 (SphK1), were negatively correlated according to the invasive breast carcinoma patient cohort in The Cancer Genome Atlas database. In addition, both SphK1 overexpression and S1P addition increased mTOR phosphorylation as shown by ELISA, while S1PR2 inhibition had the inverse effect. These data suggest that CerS6 and SphK1 regulate mTOR signaling in breast cancer cell proliferation. Moreover, mTOR activity can be regulated by the balance between S1P and C16­ceramide, which is generated by CerS6.

Epigenetic modification of α-N-acetylgalactosaminidase enhances cisplatin resistance in ovarian cancer.

Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α-Nacetylgalactosaminidase (NAGA) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, NAGA was significantly downregulated and hypermethylated at a promoter CpG site at position +251 relative to the transcriptional start site. Low NAGA expression in cisplatin-resistant cell lines was restored by treatment with a DNA demethylation agent, indicating transcriptional silencing by hyper-DNA methylation. Furthermore, overexpression of NAGA in cisplatin-resistant lines induced cytotoxicity in response to cisplatin, whereas depletion of NAGA expression increased cisplatin chemoresistance, suggesting an essential role of NAGA in sensitizing ovarian cells to cisplatin. These findings indicate that NAGA acts as a cisplatin sensitizer and its gene silencing by hypermethylation confers resistance to cisplatin in ovarian cancer. Therefore, we suggest NAGA may be a promising potential therapeutic target for improvement of sensitivity to cisplatin in ovarian cancer.

Effect of Necrosis on the miRNA-mRNA Regulatory Network in CRT-MG Human Astroglioma Cells.

PURPOSE: Glioblastoma multiforme (GBM) is the most common adult primary intracranial tumor. The remarkable features of GBM include central necrosis. MicroRNAs (miRNAs) have been considered as diagnostic/prognostic biomarkers for many cancers, including glioblastoma. However, the effect of necrosis on the miRNA expression profile and predicted miRNA-mRNA regulatory information remain unclear. The purpose of this study is to examine the effect of necrotic cells on the modulation of miRNA and mRNA expression profiles and miRNA-mRNA network in CRT-MG cells. MATERIALS AND METHODS: We used human astroglioma cells, CRT-MG, treated with necrotic CRT-MG cells to examine the effect of necrosis on the modulation of miRNA and mRNA by next-generation sequencing. For preparation of necrotic cells, CRT-MGcellswere frozen and thawed through cycle of liquid nitrogen-water bath. The putative miRNA-mRNA regulatory relationshipwas inferred through target information, using miRDB. RESULTS: The necrotic cells induced dysregulation of 106 miRNAs and 887 mRNAs. Among them, 11 miRNAs that had a negative correlation value of p < 0.05 by the hypergeometric test were screened, and their target mRNAs were analyzed by Gene Ontology enrichment analysis. Using the Kyoto Encyclopedia of Genes and Genomes database, we also found several necrotic cell treatment-activated pathways that were modulated by relevant gene targets of differentially expressed miRNAs. CONCLUSION: Our result demonstrated that dysregulation of miRNA and mRNA expression profiles occurs when GBM cells are exposed to necrotic cells, suggesting that several miRNAs may have the potential to be used as biomarkers for predicting GBM progression and pathogenesis.

Aberrant epigenetic regulation of GABRP associates with aggressive phenotype of ovarian cancer.

Metastasis is a major cause of therapeutic failure in ovarian cancer. To elucidate molecular mechanisms of ovarian cancer metastasis, we previously established a metastatic xenograft mouse model using human ovarian carcinoma SK-OV-3 cells. Using gene expression profiling, we found that γ-aminobutyric acid (GABA)A receptor π subunit (GABRP) expression was upregulated (>4-fold) in metastatic tissues from our xenograft mice compared with SK-OV-3 cells. Importantly, GABRP knockdown diminished the migration and invasion of SK-OV-3 cells, and reduced extracellular signal-regulated kinase (ERK) activation while overexpression of GABRP exhibited significantly increased cell migration, invasion and ERK activation. Moreover, treatment with the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126 similarly suppressed the migration and invasion of SK-OV-3 cells, implying that GABRP promotes these cellular behaviors by activating the MAPK/ERK pathway. Using genome-wide DNA methylation profiling, we identified hypomethylated CpG sites in the GABRP promoter in metastatic tissues from the xenograft mice compared with SK-OV-3 cells. Treatment with a DNA methyltransferase inhibitor demonstrated that methylation at -963 bp from the GABRP transcription start site (-963 CpG site) was critical for the epigenetic regulation of GABRP. Finally, we analyzed human ovarian cancer patient samples and showed DNA hypomethylation at the GABRP -963 CpG site in advanced stage, but not early-stage, primary tumors compared with their paired normal tissues. These findings suggest that GABRP enhances the aggressive phenotype of ovarian cancer cells, and that the DNA methylation status of the GABRP -963 CpG site may be useful for predicting the metastatic potential in ovarian cancer patients.

Autophagy induction in the skeletal myogenic differentiation of human tonsil-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation and are thus a valuable source for the replacement of diseased or damaged organs. Previously, we reported that the tonsils can be an excellent reservoir of MSCs for the regeneration of skeletal muscle (SKM) damage. However, the mechanisms involved in the differentiation from tonsil-derived MSCs (T-MSCs) to myocytes via myoblasts remain unclear. To clarify these mechanisms, we analyzed gene expression profiles of T-MSCs during differentiation into myocytes compared with human skeletal muscle cells (hSKMCs). Total RNA was extracted from T-MSCs, T-MSC-derived myoblasts and myocytes, and hSKMCs and was subjected to analysis using a microarray. Microarray analysis of the three phases of myogenic differentiation identified candidate genes associated with myogenic differentiation. The expression pattern of undifferentiated T-MSCs was distinguishable from the myogenic differentiated T-MSCs and hSKMCs. In particular, we selected FNBP1L, which among the upregulated genes is essential for antibacterial autophagy, since autophagy is related to SKM metabolism and myogenesis. T-MSCs differentiated toward myoblasts and skeletal myocytes sequentially, as evidenced by increased expression of autophagy-related markers (including Beclin-1, LC3B and Atg5) and decreased expression of Bcl-2. Furthermore, we reconfirmed that autophagy has an effect on the mechanism of skeletal myogenic differentiation derived from T-MSCs by treatment with 5-azacytidine and bafilomycin A1. These data suggest that the transcriptome of the T-MSC-derived myocytes is similar to that of hSKMCs, and that autophagy has an important role in the mechanism of myogenic differentiation of T-MSCs.

Aberrant Hypomethylation of Solute Carrier Family 6 Member 12 Promoter Induces Metastasis of Ovarian Cancer.

PURPOSE: Ovarian cancer (OC) is the most fatal of gynecological malignancies with a high rate of recurrence. We aimed to evaluate the expression of solute carrier family 6, member 12 (SLC6A12) and methylation of its promoter CpG sites in a xenograft mouse model of metastatic OC, and to investigate the regulatory mechanisms that promote aggressive properties during OC progression. MATERIALS AND METHODS: Expression of SLC6A12 mRNA was determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and DNA methylation status of its promoter CpGs was detected by quantitative methylation-specific PCR. The metastatic potential of SLC6A12 was evaluated by in vitro migration/invasion transwell assays. Gene expression and DNA methylation of SLC6A12 and clinical outcomes were further investigated from publicly available databases from curatedOvarianData and The Cancer Genome Atlas. RESULTS: SLC6A12 expression was 8.1-14.0-fold upregulated and its DNA methylation of promoter CpG sites was 41-62% decreased in tumor metastases. After treatment with DNA methyltransferase inhibitor and/or histone deacetylase inhibitor, the expression of SLC6A12 was profoundly enhanced (~8.0-fold), strongly supporting DNA methylation-dependent epigenetic regulation of SLC6A12. Overexpression of SLC6A12 led to increased migration and invasion of ovarian carcinoma cells in vitro, approximately 2.0-fold and 3.3-fold, respectively. The meta-analysis showed that high expression of SLC6A12 was significantly associated with poor overall survival [hazard ratio (HR)=1.07, p value=0.016] and that low DNA methylation levels of SLC6A12 at specific promoter CpG site negatively affected patient survival. CONCLUSION: Our findings provide novel evidence for the biological and clinical significance of SLC6A12 as a metastasis-promoting gene.

Synaptotagmin-like protein 2 gene promotes the metastatic potential in ovarian cancer.

Ovarian cancer (OC) metastasis has unique biological behavior and most commonly occurs via the transcoelomic route. Previously, we established a mouse xenograft model of human ovarian carcinoma and analyzed alterations in gene expression during metastasis. Among the genes that were differentially expressed more than 2-fold in the xenografts compared with the SK-OV-3 cells, we selected synaptotagmin-like protein 2 (SYTL2) and investigated the mechanisms regulating its expression and its gene function in OC. The mRNA expression of SYTL2 was significantly upregulated and the methylation of specific CpG sites within the SYTL2 promoter was decreased in the metastatic implants from the ovarian carcinoma xenografts compared to wild-type SK-OV-3 cells. Treatment with the demethylating agent 5-aza­2'-deoxycytidine and/or the histone deacetylase inhibitor Trichostatin A induced upregulation of SYTL2 in SK-OV-3 cells, implying that a DNA methylation-dependent epigenetic mechanism is involved in the regulation of SYTL2 expression. We also found that overexpression of SYTL2 promoted metastatic potential, including increased migration and invasiveness in the ovarian carcinoma cells. Furthermore, we utilized publicly available gene expression data to confirm the correlation between SYTL2 expression and poor prognosis in serous-type OC patients. Our findings provide novel evidence for the direct association of SYTL2 with the metastatic potential of ovarian carcinoma cells and its influence on metastatic recurrence of OC.

B56δ subunit of protein phosphatase 2A decreases phosphorylation of endothelial nitric oxide synthase at serine 116: Mechanism underlying aphidicolin-stimulated NO production.

DNA damage is significant in endothelial cells (EC), particularly in anticancer chemotherapy. Here, we explored whether and how aphidicolin, a DNA-damaging chemical with a promising anticancer activity, alters NO production in bovine aortic endothelial cells (BAEC). In addition to increasing eNOS-Ser1179 phosphorylation, aphidicolin decreased eNOS-Ser116 phosphorylation with a concomitant increase in NO production in a time-dependent manner. The amino acid sequence around the eNOS-Ser116 residue was identified as the substrate site of the regulatory subunit B56δ of protein phosphatase 2A (PP2A). As expected, okadaic acid, a specific PP2A inhibitor, reversed aphidicolin-induced eNOS-Ser116 dephosphorylation in a dose-dependent manner. Aphidicolin also increased B56δ-Ser566 phosphorylation, although expression of neither the catalytic subunit Cα (PP2A Cα) nor B56δ was altered. Ectopic expression of dominant negative (dn)-B56δ reversed all of the observed effects of aphidicolin with respect to phosphorylation of eNOS-Ser116 and B56δ-Ser566. Lastly, aphidicolin-stimulated NO production was also partially attenuated by ectopic expression of dn-B56δ. Taken together, our results are the first to demonstrate that aphidicolin decreases phosphorylation of eNOS-Ser116, at least in part by activating PP2A B56δ, resulting in NO release in BAEC.

Estrogen receptor-mediated resveratrol actions on blood-brain barrier of ovariectomized mice.

To test whether resveratrol provides benefits via estrogen receptors (ERs) in the blood-brain barrier of estrogen-deficient females, ovariectomized mice were treated with resveratrol then were subjected to transient middle cerebral artery occlusion (MCAO). Compared with vehicle treatment, resveratrol reduced infarct volume and neurologic deficits after MCAO. Basal tight junction (TJ) protein levels in the brain were increased by resveratrol. After MCAO, blood-brain barrier breakdown reduced levels of TJ proteins, and induction of HIF-1α and VEGF were attenuated by resveratrol. These effects were reversed by the ERs antagonist, ICI182,780. In mouse brain, endothelial cells (bEnd.3) exposed to hypoxia, resveratrol treatment protected the cells against cytotoxicity, increases of paracellular permeability and changes in levels of TJ protein and HIF-1α/VEGF proteins. These effects were reversed by ICI182,780 but not by specific ERα or ERß antagonists, indicating nonspecific ER mediated effects. Altogether, these results showed that neuroprotective effects of resveratrol in ovariectomized mice were mediated by ERs and associated with tightening of blood-brain barrier, suggesting that resveratrol can be an alternative to estrogens to protect the brains of estrogen-deficient females against ischemic insult.

DNA hypomethylation-mediated overexpression of carbonic anhydrase 9 induces an aggressive phenotype in ovarian cancer cells.

PURPOSE: Both genetic and epigenetic alterations can lead to abnormal expression of metastasis-regulating genes in tumor cells. Recent studies suggest that aberrant epigenetic alterations, followed by differential gene expression, leads to an aggressive cancer cell phenotype. We examined epigenetically regulated genes that are involved in ovarian cancer metastasis. MATERIALS AND METHODS: We developed SK-OV-3 human ovarian carcinoma cell xenografts in mice. We compared the mRNA expression and DNA methylation profiles of metastatic tissues to those of the original SK-OV-3 cell line. RESULTS: Metastatic implants showed increased mRNA expression of the carbonic anhydrase 9 (CA9) gene and hypomethylation at CpG sites in the CA9 promoter. Treatment of wild-type SK-OV-3 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reduced methylation of the CA9 promoter and increased CA9 mRNA expression. Eight CpGs, which were located at positions -197, -74, -19, -6, +4, +13, +40, and +86, relative to the transcription start site, were hypomethylated in metastatic tumor implants, compared to that of wild-type SK-OV-3. Overexpression of CA9 induced an aggressive phenotype, including increased invasiveness and migration, in SK-OV-3 cells. CONCLUSION: Alterations in the DNA methylation profile of the CA9 promoter were correlated with a more aggressive phenotype in ovarian cancer cells.

Overexpression of mucin 13 due to promoter methylation promotes aggressive behavior in ovarian cancer cells.

PURPOSE: Recent discoveries suggest that aberrant DNA methylation provides cancer cells with advanced metastatic properties. However, the precise regulatory mechanisms controlling metastasis genes and their role in metastatic transformation are largely unknown. To address epigenetically-regulated gene products involved in ovarian cancer metastasis, we examined the mechanisms regulating mucin 13 (MUC13) expression and its influence on aggressive behaviors of ovarian malignancies. MATERIALS AND METHODS: We injected SK-OV-3 ovarian cancer cells peritoneally into nude mice to mimic human ovarian tumor metastasis. Overexpression of MUC13 mRNA was detected in metastatic implants from the xenografts by expression microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The DNA methylation status within the MUC13 promoter region was determined using bisulfite sequencing PCR and quantitative methylation-specific PCR. We evaluated the effects of exogenous MUC13 on cell invasion and migration using in vitro transwell assays. RESULTS: MUC13 mRNA expression was up-regulated, and methylation of specific CpG sites within the promoter was reduced in the metastatic implants relative to those in wild-type SK-OV-3 cells. Addition of a DNA methyltransferase inhibitor to SK-OV-3 cells induced MUC13 expression, thereby implying epigenetic regulation of MUC13 by promoter methylation. MUC13 overexpression increased migration and invasiveness, compared to control cells, suggesting aberrant up-regulation of MUC13 is strongly associated with progression of aggressive behaviors in ovarian cancer. CONCLUSION: We provide novel evidence for epigenetic regulation of MUC13 in ovarian cancer. We suggest that the DNA methylation status within the MUC13 promoter region may be a potential biomarker of aggressive behavior in ovarian cancer.

Aberrant hypomethylation-mediated AGR2 overexpression induces an aggressive phenotype in ovarian cancer cells.

The metastatic properties of cancer cells result from genetic and epigenetic alterations that lead to the abnormal expression of key genes regulating tumor phenotypes. Recent discoveries suggest that aberrant DNA methylation provides cancer cells with advanced metastatic properties; however, the precise regulatory mechanisms controlling metastasis-associated genes and their roles in metastatic transformation are largely unknown. We injected SK-OV-3 human ovarian cancer cells into the perineum of nude mice to generate a mouse model that mimics human ovarian cancer metastasis. We analyzed the mRNA expression and DNA methylation profiles in metastasized tumor tissues in the mice. The pro-oncogenic anterior gradient 2 (AGR2) gene showed increased mRNA expression and hypomethylation at CpG sites in its promoter region in the metastatic tumor tissues compared with the cultured SK-OV-3 cells. We identified crucial cytosine residues at CpG sites in the AGR2 promoter region. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reduced the level of CpG methylation in the AGR2 promoter and increased the level of AGR2 expression. Next, we explored the functional role of AGR2 in the metastatic transformation of SK-OV-3 cells. SK-OV-3 cells overexpressing AGR2 showed increased migratory and invasive activity. Our results indicate that DNA methylation within the AGR2 promoter modulates more aggressive cancer cell phenotypes.

Aberrant DNA methylation in the IFITM1 promoter enhances the metastatic phenotype in an intraperitoneal xenograft model of human ovarian cancer.

A lack of reliable biomarkers for the early detection and risk of metastatic recurrences makes ovarian cancer the most lethal gynecological cancer. To understand the molecular mechanisms involved in ovarian cancer metastasis in vivo, we analyzed the transcriptional expression pattern in metastatic implants of human ovarian carcinoma xenografts in mice. The expression of 937 genes was significantly different, by at least 2-fold, in the xenografts compared with that in SK-OV-3 cells. We investigated the mechanisms that regulate the expression of one of the profoundly upregulated genes, interferon-induced transmembrane protein 1 (IFITM1), in the metastatic implants. Specific CpG sites within the IFITM1 promoter were hypomethylated in the metastatic implants relative to those in the wild-type SK-OV-3 cells. Treating wild-type SK-OV-3 cells with the demethylating agent 5-aza-2'-deoxycytidine enhanced IFITM1 expression in a dose-dependent manner, implying transcriptional regulation by promoter methylation. We also found that IFITM1 overexpression caused increased migration and invasiveness in SK-OV-3 cells. Our results demonstrate that IFITM1 could be a novel metastasis-promoting gene that enhances the metastatic phenotype in ovarian cancer via epigenetic transcriptional regulation. Our findings also suggest that the status of DNA methylation within the IFITM1 promoter region could be a biomarker indicating metastatic progression in ovarian cancer.

Protopanaxatriol ginsenoside Rh1 inhibits the expression of matrix metalloproteinases and the in vitro invasion/migration of human astroglioma cells.

Malignant gliomas are the most common and fatal brain tumors in adults. In particular, the strong invasiveness of glioma cells into the normal brain tissue makes eradication of glioma very difficult. Matrix metalloproteinases (MMPs) play a pivotal role in glioma invasion, and thus controlling MMP expression has been suggested as an important therapeutic target for brain tumors. In the present study, we investigated the effect of protopanaxatriol ginsenoside Rh1 on MMP expressions in human astroglioma U87MG and U373MG cells. RT-PCR analysis showed that Rh1 inhibits the mRNA expressions of MMP-1, -3, and -9 in PMA-stimulated U87MG and U373MG cells. Rh1 also suppressed the promoter activities of MMP-1, -3 and -9. The ELISA, Western blot, and zymographic analyses revealed that Rh1 inhibits the protein expression and/or enzymatic activity of MMP-1, -3 and -9. In accordance with the strong inhibitory effects of Rh1 on MMPs, Rh1 efficiently inhibited the invasion and migration of U87MG and U373MG glioma cells as demonstrated by Matrigel invasion assay and wound healing assay. Further mechanistic studies revealed that Rh1 inhibits MAPK and PI3K/Akt signaling pathways and downstream transcription factors such as NF-κB and AP-1, which play an important role in MMP gene expressions. The data collectively suggest that ginsenoside Rh1 may have a therapeutic potential for malignant gliomas.

Amyloid protein-mediated differential DNA methylation status regulates gene expression in Alzheimer's disease model cell line.

The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer's disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterations in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the -435, -295, and -271 CpG sites of CTIF, and at the -505 to -341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at -432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory mechanism may contribute to the pathogenesis of AD.

Swedish mutation within amyloid precursor protein modulates global gene expression towards the pathogenesis of Alzheimer's disease.

The Swedish mutation (K595N/M596L) of amyloid precursor protein (APP-swe) has been known to increase abnormal cleavage of cellular APP by Beta-secretase (BACE), which causes tau protein hyperphosphorylation and early-onset Alzheimer's disease (AD). Here, we analyzed the effect of APP-swe in global gene expression using deep transcriptome sequencing technique. We found 283 genes were down-regulated and 348 genes were up-regulated in APP-swe expressing H4-swe cells compared to H4 wild-type cells from a total of approximately 74 million reads of 38 base pairs from each transcriptome. Two independent mechanisms such as kinase and phosphatase signaling cascades leading hyperphosphorylation of tau protein were regulated by the expression of APP-swe. Expressions of catalytic subunit as well as several regulatory subunits of protein phosphatases 2A were decreased. In contrast, expressions of tau-phosphorylating glycogen synthase kinase 3ß (GSK-3ß), cyclin dependent kinase 5 (CDK5), and cAMP-dependent protein kinase A (PKA) catalytic subunit were increased. Moreover, the expression of AD-related Aquaporin 1 and presenilin 2 expression was regulated by APP-swe. Taken together, we propose that the expression of APP-swe modulates global gene expression directed to AD pathogenesis.