Toxicological Evaluation of Low Molecular Weight Fucoidan in Vitro and in Vivo.

For a long time, fucoidan has been well known for its pharmacological activities, and recently low molecular weight fucoidan (LMF) has been used in food supplements and pharmaceutical products. In the present study, LMF was extracted from Laminaria japonica by enzyme hydrolysis. The toxicity of LMF in mouse and rat models was determined by many methods, such as total arsenic content, bacterial reverse mutation assay, chromosome aberration assay, and in vivo micronucleus assay. The present findings showed that LMF at 5000 µg/mL exhibited no mutagenicity. It also produced no formatting disruption of red blood cells in vivo. At 2000 mg/kg BW/day there were no toxicological indications. LMF is expected to be used as a safe food supplement.

Fucoidan Elevates MicroRNA-29b to Regulate DNMT3B-MTSS1 Axis and Inhibit EMT in Human Hepatocellular Carcinoma Cells.

Accumulating evidence has revealed that fucoidan exhibits anti-tumor activities by arresting cell cycle and inducing apoptosis in many types of cancer cells including hepatocellular carcinoma (HCC). Exploring its effect on microRNA expression, we found that fucoidan markedly upregulated miR-29b of human HCC cells. The induction of miR-29b was accompanied with suppression of its downstream target DNMT3B in a dose-dependent manner. The reduction of luciferase activity of DNMT3B 3'-UTR reporter by fucoidan was as markedly as that by miR-29b mimic, indicating that fucoidan induced miR-29b to suppress DNMT3B. Accordingly, the mRNA and protein levels of MTSS1 (metastasis suppressor 1), a target silenced by DNMT3B, were increased after fucoidan treatment. Furthermore, fucoidan also down-regulated TGF-ß receptor and Smad signaling of HCC cells. All these effects leaded to the inhibition of EMT (increased E-cadherin and decreased N-cadherin) and prevention of extracellular matrix degradation (increased TIMP-1 and decreased MMP2, 9), by which the invasion activity of HCC cells was diminished. Our results demonstrate the profound effect of fucoidan not only on the regulation of miR-29b-DNMT3B-MTSS1 axis but also on the inhibition of TGF-ß signaling in HCC cells, suggesting the potential of using fucoidan as integrative therapeutics against invasion and metastasis of HCC.

The role of Sp1 and EZH2 in the regulation of LMX1A in cervical cancer cells.

We have reported previously that LIM homeobox transcription factor 1α (LMX1A) is hypermethylated and functions as a metastasis suppressor in cervical cancer cells. However, the regulation of LMX1A in carcinogenesis has not been reported. We aim to clarify whether specificity protein 1 (Sp1) and enhancer of zeste homolog 2 (EZH2) are involved in the regulation of LMX1A in cervical cancer. First we characterized the LMX1A promoter and used overexpression, knockdown, and reporter assays to show that Sp1 increased LMX1A promoter activity. Next, we used site-directed mutagenesis and electrophoresis mobility shift assays (EMSAs) to demonstrate that Sp1-binding sites were important for Sp1-mediated activation of the LMX1A promoter. Chromatin immunoprecipitation data demonstrated that Sp1 could bind directly to the LMX1A promoter and activate endogenous LMX1A expression in cells pretreated with 5-aza-2'-deoxycytidine (5-aza-dC). Knockdown of EZH2 decreased H3K27me3 histone modification but was insufficient to restore LMX1A expression. To explore the effect of EZH2 on the endogenous LMX1A promoter, we treated EZH2-knockdown cells with 5-aza-dC and trichostatin A (TSA) and then depleted the cells of drugs for 3days. H3K14ac was enriched at the LMX1A promoter in EZH2-knockdown cells and LMX1A mRNA was still expressed. Taken together, these data imply that Sp1 may activate LMX1A expression upon oncogenic stress during cervical cancer development. Moreover, suppression of EZH2 may delay resilencing of LMX1A after the removal of 5-aza-dC and TSA.

Downregulation of miR-29 contributes to cisplatin resistance of ovarian cancer cells.

Drug resistance is an obstacle to the treatment of ovarian cancer. Using a unique cell model, we have proven previously that a subpopulation of ovarian cancer cells is more resistant to cisplatin than are the original cells. MicroRNAs (miRNAs), small noncoding RNAs, are involved in many biological events in cancer cells. In our study, we explored whether miRNAs are involved in cisplatin resistance of ovarian cancer cells. Cisplatin-resistant cells expressed a lower level of miR-29a/b/c. Manipulation of microRNA-29 (miR-29) expression modulated cisplatin sensitivity of CP70, HeyC2, SKOV3 and A2780 ovarian cancer cells. Knockdown of miR-29a/b/c increased the ability of cells to escape cisplatin-induced cell death partly through upregulation of collagen type I alpha 1 (COL1A1) and increased the activation of extracellular signal-regulated kinase 1/2 and inactivation of glycogen synthase kinase 3 beta. When combined with cisplatin treatment, knockdown of miR-29 decreased the amount of the active form of caspase-9 and caspase-3. Ectopic expression of miR-29 alone or in combination with cisplatin treatment efficaciously reduced the tumorigenicity of CP70 cells in vivo. Our data show that downregulation of miR-29 increases cisplatin resistance in ovarian cancer cells. Taken together, these data suggest that overexpression of miR-29 is a potential sensitizer to cisplatin treatment that may have therapeutic implications.

Frontiers and hotspots in comorbid epilepsy and depression: a bibliometric analysis from 2003 to 2023.

Background: Epilepsy ranks among the most common neurological disorders worldwide, frequently accompanied by depression as a prominent comorbidity. This study employs bibliometric analysis to reveal the research of comorbid epilepsy and depression over the past two decades, aiming to explore trends and contribute insights to ongoing investigations. Methods: We conducted a comprehensive search on the Web of Science Core Collection database and downloaded relevant publications on comorbid epilepsy and depression published from 2003 to 2023. VOSviewer and CiteSpace were mainly used to analyze the authors, institutions, countries, publishing journals, reference co-citation patterns, keyword co-occurrence, keyword clustering, and other aspects to construct a knowledge atlas. Results: A total of 5,586 publications related to comorbid epilepsy and depression were retrieved, with a general upward trend despite slight fluctuations in annual publications. Publications originated from 121 countries and 636 institutions, with a predominant focus on clinical research. The United States led in productivity (1,529 articles), while Melbourne University emerged as the most productive institution (135 articles). EPILEPSY & BEHAVIOR was the journal with the highest publication output (1,189 articles) and citation count. Keyword analysis highlighted emerging trends, including "recognitive impairment" and "mental health," indicating potential future research hotspots and trends. Conclusion: This study is one of the first to perform a bibliometric analysis of the 20-year scientific output of comorbid epilepsy and depression. While research has trended upwards, ambiguity in pathogenesis and the absence of standardized diagnostic guidelines remain concerning. Our analysis offers valuable guidance for researchers, informing that this might be a strong area for future collaborations.

Frontiers and hotspots in anxiety disorders: A bibliometric analysis from 2004 to 2024.

Objective: This study aimed to analyze research on anxiety disorders using VOSviewer and CiteSpace to identify research hotspots and future directions. Methods: We conduct ed a comprehensive search on the Web of Science Core Collection (WoSCC) for relevant studies about anxiety disorders published within the past two decades (from 2004 to 2024). VOSviewer and CiteSpace were mainly used to analyze the authors, institutions, countries, publishing journals, reference co-citation patterns, keyword co-occurrence, keyword clustering, and other aspects to construct a knowledge atlas. Results: A total of 22,267 publications related to anxiety disorders were retrieved. The number of publications about anxiety disorders has generally increased over time, with some fluctuations. The United States emerged as the most productive country, with Harvard University identified as the most prolific institution and Brenda W. J. H. Penninx as the most prolific author in the field. Conclusion: This research identified the most influential publications, authors, journals, institutions, and countries in the field of anxiety research. Future research directions are involved advanced treatments based on pharmacotherapy, psychotherapy and digital interventions, mechanism exploration to anxiety disorders based on neurobiological and genetic basis, influence of social and environmental factors on the onset of anxiety disorders.

SOX1 functions as a tumor suppressor by antagonizing the WNT/ß-catenin signaling pathway in hepatocellular carcinoma.

UNLABELLED: Oncogenic activation of the Wnt/ß-catenin signaling pathway is common in hepatocellular carcinoma (HCC). Our recent studies have demonstrated that SRY (sex determining region Y)-box 1 (SOX1) and secreted frizzled-related proteins are concomitantly promoter-hypermethylated, and this might lead to abnormal activation of the Wnt signaling pathway in HCC. SOX1 encodes a transcription factor involved in the regulation of embryonic development and cell fate determination. However, the expression and functional role of SOX1 in HCC remains unclear. In this study, we confirmed via quantitative methylation-specific polymerase chain reaction that SOX1 was frequently downregulated through promoter hypermethylation in HCC cells and tissues. Overexpression of SOX1 by a constitutive or inducible approach could suppress cell proliferation, colony formation, and invasion ability in HCC cell lines, as well as tumor growth in nonobese diabetic/severe combined immunodeficiency mice. Conversely, knockdown of SOX1 by withdrawal of doxycycline could partially restore cell proliferation and colony formation in HCC cells. We used a T cell factor (TCF)-responsive luciferase reporter assay and western blot analysis to prove that SOX1 could regulate TCF-responsive transcriptional activity and inhibit the expression of Wnt downstream genes. Furthermore, we used glutathione S-transferase pull-down, co-immunoprecipitation, and confocal microscopy to demonstrate that SOX1 could interact with ß-catenin but not with the ß-catenin/TCF complex. Moreover, restoration of the expression of SOX1 induces significant cellular senescence in Hep3B cells. CONCLUSION: Our data show that a developmental gene, SOX1, may function as a tumor suppressor by interfering with Wnt/ß-catenin signaling in the development of HCC.

SOX1 suppresses cell growth and invasion in cervical cancer.

OBJECTIVE: Abnormal activation of the Wnt/ß-catenin signaling pathway is common in human cancers, including cervical cancer. Many papers have shown that SRY (sex-determining region Y)-box (SOX) family genes serve as either tumor suppressor genes (TSGs) or oncogenes by regulating the Wnt signaling pathway in different cancers. We have demonstrated recently that epigenetic silencing of SOX1 gene occurs frequently in cervical cancer. However, the possible role of SOX1 in cervical cancer remains unclear. This study aimed to explore whether SOX1 functions as a TSG in cervical cancer. METHODS: We established a constitutive and an inducible system that overexpressed SOX1 and monitored its function by in vitro experiments. To confirm SOX1 function, we manipulated SOX1 using an inducible expression approach in cell lines. The effect of SOX1 on tumorigenesis was also analyzed in animal models. RESULTS: Overexpression of SOX1 inhibited cell proliferation, anchorage independency, and invasion in vitro. SOX1 suppressed tumor growth in nonobese diabetic/severe combined immunodeficiency mice. After induction of SOX1 by doxycycline (DOX), SOX1 inhibited cell growth and invasion in the inducible system. Repression of SOX1 by withdrawal of DOX partially reversed the malignant phenotype in cervical cells. SOX1 inhibited TCF-dependent transcriptional activity and the Wnt target genes. SOX1 also repressed the invasive phenotype by regulating the expression of invasion-related genes. CONCLUSIONS: Taken together, these data suggest that SOX1 can function as a tumor suppressor partly by interfering with Wnt/ß-catenin signaling in cervical cancer.

Frequent concomitant epigenetic silencing of SOX1 and secreted frizzled-related proteins (SFRPs) in human hepatocellular carcinoma.

BACKGROUND AND AIM: Except for genetic mutations, epigenetic changes are also involved in the development of human cancers. Recently, we have identified SOX1, SRY (sex determining region Y)-box 1, is hypermethylated in cervical cancer and ovarian cancer. Therefore, we investigated whether promoter hypermethylation of SOX1 is common in hepatocellular carcinoma (HCC). METHODS: We used methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing to analyze the methyaltion level of the SOX1 promoter in seven HCC cell lines, 54 clinical HCCs, 42 cirrhotic livers, 21 livers with chronic hepatitis, and 15 control livers. Then, we employed quantitative MS-PCR (QMSP) to validate in an independent set of samples (60 paired HCCs and 30 control livers). Finally, we used luciferase reporter and colony formation assay to check the effect of SOX1 in HCC. RESULTS: Promoter methylation of SOX1 was significantly frequent in HCC cell lines and clinical HCCs, cirrhotic livers, but not in control livers (P < 0.0001). There is a significant correlation between downregulation of SOX1 expression and promoter methylation. QMSP results confirmed that promoter hypermethylation of SOX1 is significantly more frequent in HCCs than control livers (P < 0.0001). The frequency of SOX1 methylation in patients with secreted frizzled-related proteins (SFRPs) methylation is significantly higher than in patients without SFRPs methylation (P < 0.0001). Furthermore, ectopic expression of SOX1 could suppress T-cell factor-dependent transcriptional activity and colony formation number in HCCs. CONCLUSIONS: Concomitant epigenetic silencing of SOX1 and SFRPs through promoter hypermethylation is frequent in HCCs, and this might contribute to abnormal activation of canonical Wnt signal pathway.

SFRP1 and SFRP2 suppress the transformation and invasion abilities of cervical cancer cells through Wnt signal pathway.

OBJECTIVES: Aberrant activation of the Wnt/beta-catenin signaling pathway is common in human cancers, including cervical cancer. The secreted frizzled-related proteins (SFRPs) function as Wnt antagonists and play important implications in carcinogenesis. Recently, we have shown that SFRP1 and SFRP2 are frequently downregulated through promoter hypermethylation. However, the function of SFRP1 and SFRP2 in cervical cancer remains unclear. METHODS: To improve our understanding of the role of SFRP1 and SFRP2 in cervical cancer cells, we use overexpression or shRNA approach in cervical cancer cell lines. RESULTS: Restoration of the expression of SFRP1 and SFRP2 attenuated Wnt signaling in CaSki cells, decreased abnormal accumulation of free beta-catenin in the nucleus, and suppressed cancer cell growth. In addition, different statuses of beta-catenin accumulation in the cytoplasm of CaSki or HeLa3rd cells were observed, suggesting that different Wnt pathways are executed. Furthermore, we demonstrated that SFRP1 and SFRP2 enhance the expression of the epithelial marker E-cadherin, through inhibition of the expression of SLUG, TWIST and SNAIL, three transcription factors involved in the epithelial mesenchymal transition (EMT) program. Finally, in a xenograft animal model, we showed that SFRP1 suppresses tumorigenicity of cancer cells in vivo. CONCLUSIONS: Taken together, these data strongly suggest that epigenetic silencing of SFRP genes leads to oncogenic activation of the Wnt pathway and contributes to cervical cancer progression through the EMT program.

Promoter methylation of SFRPs gene family in cervical cancer.

OBJECTIVES: Oncogenic activation of the Wnt/beta-catenin signaling pathway is common in human cancers, including cervical cancer. The secreted frizzled-related proteins (SFRPs) function as negative regulators of Wnt signaling and play an important role in carcinogenesis. Frequent promoter hypermethylation of SFRPs has been identified in human cancers; however, the precise role of SFRPs in cervical cancer is not clear. METHODS: The methylation status of SFRPs gene family was analyzed in two cervical cancer cell lines and a full spectrum of cervical neoplasia, including 45 low-grade squamous intraepithelial lesions (LSIL), 49 high-grade squamous intraepithelial lesions (HSIL), 109 squamous cell carcinomas (SCC), and 45 normal controls. RESULTS: The SFRP1 promoter was hypermethylated in 33.9% of SCC, 8.2% of HSIL, 2.2% of LSIL, but not in normal tissues. The SFRP2 promoter was hypermethylated in 80.7% of SCC, 16.3% of HSIL, 15.6% LSIL and 4.4% normal tissues. The SFRP4 promoter was hypermethylated in 67.9% of SCC, 36.7% of HSIL, 4.4% of LSIL, but not in normal tissues. The SFRP5 promoter was hypermethylated in 10.1% of SCC, 4.1% of HSIL, 13.3% of LSIL and 4.4% normal tissues. The frequency of SFRP1, SFRP2 and SFRP4 promoter methylation in tumors was significantly higher than in normal, LSIL, and HSIL samples (P<0.0001). SFRP5 methylation was significantly different in patients with or without lymph-node metastases (0% vs 15.2%, respectively, P<0.05). CONCLUSIONS: Our data suggest that promoter hypermethylation of SFRP1, SFRP2 and SFRP4 is associated with cervical carcinogenesis, which could be used for molecular screening of cervical neoplasias in future.

The anti-tumor activity of brown seaweed oligo-fucoidan via lncRNA expression modulation in HepG2 cells.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death in Asia. HCC is less sensitive to chemotherapy and is known to express multidrug resistant genes to acquire resistance to chemotherapeutic agents, therefore the development of a potent HCC suppressor is essential in treating HCC. Our previous reports demonstrated that oligo-fucoidan from the brown seaweed Sargassum hemiphyllum elevates microRNA-29b to inhibit epithelial-mesenchymal transition in hepatoma cells. In this study, we aimed to examine in vitro effect of oligo-fucoidan in hepatocellular carcinoma through apoptosis and long noncoding RNA (lncRNA) pathway. Oligo-fucoidan was studied for its anti-hepatoma cells by MTT and DNA ladder analysis. And the mechanism was studied by flow cytometry, qPCR and western blot analysis. In this study, oligo-fucoidan induced sub-G1 phase cell cycle arrest and activation of caspases, indicating that the intrinsic and extrinsic apoptotic pathways were involved in the mechanism of oligo-fucoidan-induced cell death. Moreover, oligo-fucoidan significantly increased the expression of p53, p21, and p27, while cyclin-B1 and -D1 were decreased at the mRNA and protein levels. Finally, we showed that targeting apoptosis and cell cycle pathways could also contribute to the induction of the lncRNA-Saf and lncRNA-p21. Through human lncRNA profiler array analysis, the differential expression of lncRNAs in HCC cells following oligo-fucoidan exposure was further examined. These findings indicated that lncRNAs switched oligo-fucoidan-induced apoptosis, which might be potentially valuable in HCC adjuvant therapy.

Carbonic anhydrase III promotes transformation and invasion capability in hepatoma cells through FAK signaling pathway.

Carbonic anhydrase III (CAIII) is distinguished from the other members of the CA family by low carbon dioxide hydratase activity, resistance to the CA inhibitor acetazolamide, and a predominant expression in the liver of males. In this report the effects of CAIII expression on liver cancer cells invasiveness were explored. Overexpression of CAIII in the HCC cell line SK-Hep1 resulted in increased anchorage-independent growth and invasiveness. And siRNA-mediated silencing of CAIII expression decreased the invasive ability of SK-Hep1 cells. Furthermore, CAIII transfectants showed elevated focal adhesion kinase (FAK) and Src activity. Silencing of FAK expression in CAIII transfectants led to suppression of HCC cell invasion. More importantly, the CAIII transfectants acidified the culture medium at an accelerated speed than the control cells did. Taken together, these data suggest that the CAIII-promoted invasive ability of HCC cells may probably be mediated through, at least in part, the FAK signaling pathway via intracellular and/or extracellular acidification.

Induction of Forkhead Class box O3a and apoptosis by a standardized ginsenoside formulation, KG-135, is potentiated by autophagy blockade in A549 human lung cancer cells.

BACKGROUND: KG-135, a standardized formulation enriched with Rk1, Rg3, and Rg5 ginsenosides, has been shown to inhibit various types of cancer cells; however, the underlying mechanisms are not fully understood. In this study, we explored its effects in A549 human lung cancer cells to investigate the induction of Forkhead Class box O3a (FOXO3a) and autophagy. METHODS: Cell viability was determined by sulforhodamine B staining. Apoptosis and cell cycle distribution were analyzed using flow cytometry. The changes of protein levels were determined using Western blot analysis. Autophagy induction was monitored by the formation of acidic vesicular organelles stained with acridine orange. RESULTS: KG-135 effectively arrested the cells in G1 phase with limited apoptosis. Accordingly, a decrease of cyclin-dependent kinase-4, cyclin-dependent kinase-6, cyclin D1, and phospho-retinoblastoma protein, and an increase of p27 and p18 proteins were observed. Intriguingly, KG-135 increased the tumor suppressor FOXO3a and induced the accumulation of autophagy hallmark LC3-II and acidic vesicular organelles without an increase of the upstream marker Beclin-1. Unconventionally, the autophagy adaptor protein p62 (sequestosome 1) was increased rather than decreased. Blockade of autophagy by hydroxychloroquine dramatically potentiated KG-135-induced FOXO3a and its downstream (FasL) ligand accompanied by the cleavage of caspase-8. Meanwhile, the decrease of Bcl-2 and survivin, as well as the cleavage of caspase-9, were also drastically enhanced, resulting in massive apoptosis. CONCLUSION: Besides arresting the cells in G1 phase, KG-135 increased FOXO3a and induced an unconventional autophagy in A549 cells. Both the KG-135-activated extrinsic FOXO3a/FasL/caspase-8 and intrinsic caspase-9 apoptotic pathways were potentiated by blockade of autophagy. Combination of KG-135 and autophagy inhibitor may be a novel strategy as an integrative treatment for cancers.

Quantitative methylation analysis reveals distinct association between PAX6 methylation and clinical characteristics with different viral infections in hepatocellular carcinoma.

BACKGROUND: Related to genetic alteration, frequent promoter hypermethylation is also a contributing factor in the development of human cancers. Recently, we discovered numerous novel genes that were aberrantly methylated in hepatocellular carcinoma (HCC) by using Infinium HumanMethylation27 BeadChip array. We utilized a quantitative methylation-specific PCR (Q-MSP) system for the evaluation of PAX6 methylation in 29 normal controls and 160 paired HCC tissues and their adjacent non-tumor tissues. We verified the correlation between the methylation status of PAX6 and clinical characteristics with different viral status. RESULTS: Paired-box 6 promoter methylation was observed in 39.4 %, 15.6 %, and 3.4 % in primary HCCs, adjacent non-tumors, and normal control tissues, respectively. Methylation of the PAX6 promoter region in HCCs significantly increased compared with control tissues. PAX6 was frequently methylated in HCV-positive HCC tissues (61.3 %) and rarely methylated in HBV-positive (22.1 %) and double-negative HCC tissues (33.3 %). CONCLUSIONS: Our data suggests that promoter hypermethylation of PAX6 is a common event in HCCs and the association of PAX6 methylation in clinicopathological features is divergent with different viral status.

Quantitative DNA methylation analysis of selected genes in endometrial carcinogenesis.

OBJECTIVE: Most endometrial carcinomas appear to develop from precursors (e.g., endometrial hyperplasia) that progress for several years. Patients who are ultimately diagnosed with carcinoma often present clinically with complaints of abnormal vaginal bleeding years before diagnosis, which offers an opportunity for early diagnosis and curative treatment. The analysis of DNA methylation may be used as a method for detecting endometrial cancer (EC). To test the potential clinical application of this method, we used quantitative methylation analysis of five genes in a full spectrum of endometrial lesions. MATERIALS AND METHODS: This hospital-based, prospective, case-controlled study was conducted on 68 patients, which included patients who had a normal endometrium (n = 18), hyperplasia of the endometrium (n = 24), and EC (n = 26). Methylation levels of the following genes were determined by using real-time methylation-specific polymerase chain reaction (PCR) amplification: zinc finger protein 177 (ZNF177), collagen type XIV α1 (COL14A1), dihydropyrimidinase-like 4 (DPYSL4), homeobox A9 (HOXA9), transmembrane protein with epidermal growth factor-like and two follistatin-like domains 2 (TMEFF2). The methylation index (MI) cutoff values for the different diagnoses were determined to test the sensitivity and specificity of the method and to generate the receiver operating characteristic (ROC) curves. The Mann-Whitney U test was used to test between-group differences in the MI. RESULTS: The MI of the five genes was significantly higher in EC than the MIs in specimens of hyperplasia of endometrium and normal appearance (p < 0.001). The ROC analysis demonstrated that the sensitivity, specificity, and accuracy for detecting EC were 92.3%, 94.4%, and 95.1%, respectively, for ZNF177; 92.3%, 94.4%, and 95.7%, respectively, for COL14A1; 80.8%, 94.4%, and 81.4%, respectively, for HOXA9; 65.4%, 94.4%, and 89.5%, respectively, for TMEFF2; and 61.5%, 94.4%, and 63.3%, respectively, for DPYSL4. The combined testing of ZNF177 and COL14A1 had the best specificity (100%), but compromised sensitivity (88.5%). CONCLUSION: Promoter methylation of ZNF177, COL14A1, HOXA9, DPYSL4, and TMEFF2 genes is a frequent epigenetic event in EC. Furthermore, the epigenetic hypermethylation of TMEFF2 may be a valuable marker for identifying undetected EC within endometrial hyperplasia.

Methylation of IRAK3 is a novel prognostic marker in hepatocellular carcinoma.

AIM: To examine the methylation levels of interleukin-1 receptor-associated kinase 3 (IRAK3) and GLOXD1 and their potential clinical applications in hepatocellular carcinoma (HCC). METHODS: mRNA expression and promoter methylation of IRAK3 and GLOXD1 in HCC cells were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and methylation-specific PCR (MSP), respectively. Using pyrosequencing results, we further established a quantitative MSP (Q-MSP) system for the evaluation of IRAK3 and GLOXD1 methylation in 29 normal controls and 160 paired HCC tissues and their adjacent nontumor tissues. We also calculated Kaplan-Meier survival curves to determine the applications of gene methylation in the prognosis of HCC. RESULTS: IRAK3 and GLOXD1 expression was partially restored in several HCC cell lines after treatment with 5-aza-2'-deoxycytidine (DNA methyltransferase inhibitor; 5DAC). A partial decrease in the methylated band was also observed in the HCC cell lines after 5DAC treatment. Using GLOXD1 as an example, we found a significant correlation between the data obtained from the methylation array and from pyrosequencing. The methylation frequency of IRAK3 and GLOXD1 in HCC tissues was 46.9% and 63.8%, respectively. Methylation of IRAK3 was statistically associated with tumor stage. Moreover, HCC patients with IRAK3 methylation had a trend toward poor 3-year disease-free survival (P < 0.05). CONCLUSION: IRAK3 and GLOXD1 were frequently methylated in HCC tissues compared to normal controls and nontumor tissues. IRAK3 methylation was associated with tumor stage and poor prognosis of patients. These data suggest that IRAK3 methylation is a novel prognostic marker in HCC.

Promoter methylation of SFRP3 is frequent in hepatocellular carcinoma.

Oncogenic activation of the Wnt/ ß -catenin signaling pathway is common in human cancers. The secreted frizzled-related proteins (SFRPs) function as negative regulators of Wnt signaling and have important implications in carcinogenesis. Because there have been no reports about the role of SFRP3 in hepatocellular carcinoma (HCC), we investigated the level of methylation and transcription of SFRP3. Four HCC cell lines, 60 HCCs, 23 cirrhosis livers, 37 chronic hepatitis livers, and 30 control livers were prescreened for SFRP3 promoter methylation by methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing. SFRP3 promoter methylation was observed in 100%, 60%, 39.1%, 16.2%, and 0% in HCC cell lines, primary HCCs, cirrhosis livers, chronic hepatitis livers, and control livers, respectively. Demethylation treatment with 5-aza-2'-deoxycytidine in HCC cells restored or increased the SFRP3 mRNA expression. We next used quantitative MS-PCR (QMSP) to analyze the methylation level of SFRP3 in 60 HCCs and their corresponding nontumor tissues. Methylation of SFRP3 promoter region in HCCs increased significantly compared with control tissues. There is a positive correlation between promoter hypermethylation and SFRP3 mRNA downregulation. Our data suggest that promoter hypermethylation of SFRP3 is a common event in HCCs and plays an important role in regulation of SFRP3 mRNA expression.

Honokiol-induced apoptosis and autophagy in glioblastoma multiforme cells.

Honokiol, a hydroxylated biphenyl compound isolated from the Chinese herb Magnolia officinalis, has been reported to have anticancer activities in a variety of cancer cell lines. The present study aimed to evaluate the anticancer effect and possible molecular mechanisms of honokiol in a glioblastoma multiforme (GBM) cell line. The anticancer activities of honokiol were investigated in the DBTRG-05MG GBM cell line. The effect of honokiol on cell growth was determined using a sulforhodamine B assay. Flow cytometry and immunoblotting were used to measure honokiol-induced apoptosis (programmed cell death type I) and autophagy (programmed cell death type II). Honokiol was observed to reduce DBTRG-05MG cell viability in a dose-dependent manner. At a dose of 50 µM, honokiol markedly decreased the expression of Rb protein and led to the cleavage of poly(ADP-ribose) polymerase and Bcl-xL to promote apoptosis in the cancer cells. In addition, markers of autophagy, including Beclin-1 and LC3-II, were also significantly increased. In addition to apoptosis, honokiol was also able to induce autophagy in the DBTRG-05MG cells. The mechanisms that are responsible for the correlation between honokiol-induced apoptosis and autophagy require further investigation. Such efforts may provide a potential strategy for improving the clinical outcome of GBM treatment.

Oxidative stress enhances Axl-mediated cell migration through an Akt1/Rac1-dependent mechanism.

Persistent oxidative stress is common in cancer cells because of abnormal generation of reactive oxygen species (ROS) and has been associated with malignant phenotypes, such as chemotherapy resistance and metastasis. Both overexpression of Axl and abnormal ROS elevation have been linked to cell transformation and increased cell migration. However, the relationship between Axl and ROS in malignant cell migration has not been previously evaluated. Using an in vitro human lung cancer model, we examined the redox state of lung adenocarcinoma cell lines of low metastatic (CL1-0) and high metastatic (CL1-5) potentials. Here we report that Axl activation elicits ROS accumulation through the oxidase-coupled small GTPase Rac1. We also observed that oxidative stress could activate Axl phosphorylation to synergistically enhance cell migration. Further, Axl signaling activated by H2O2 treatment results in enhancement of cell migration via a PI3K/Akt-dependent pathway. The kinase activity of Axl is required for the Axl-mediated cell migration and prolongs the half-life of phospho-Akt under oxidative stress. Finally, downregulation of Akt1, but not Akt2, by RNAi in Axl-overexpressing cells inhibits the amount of activated Rac1 and the ability to migrate induced by H2O2 treatment. Together, these results show that a novel Axl-signaling cascade induced by H2O2 treatment triggers cell migration through the PI3K/Akt1/Rac1 pathway. Elucidation of redox regulation in Axl-related malignant migration may provide new molecular insights into the mechanisms underlying tumor progression.