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Breast cancer is a leading cause of cancer mortality in women worldwide. Using the Infinium MethylationEPIC BeadChip, we analyzed plasma sample methylation to identify the SRCIN1 gene in breast cancer patients. We assessed SRCIN1 -related roles and pathways for their biomarker potential. To verify the methylation status, quantitative methylation-specific PCR (qMSP) was performed on genomic DNA and circulating cell-free DNA samples, and mRNA expression analysis was performed using RT‒qPCR. The results were validated in a Western population; for this analysis, the samples included plasma samples from breast cancer patients from the USA and from The Cancer Genome Atlas (TCGA) cohort. To study the SRCIN1 pathway, we conducted cell viability assays, gene manipulation and RNA sequencing. SRCIN1 hypermethylation was identified in 61.8% of breast cancer tissues from Taiwanese patients, exhibiting specificity to this malignancy. Furthermore, its presence correlated significantly with unfavorable 5-year overall survival outcomes. The levels of methylated SRCIN1 in the blood of patients from Taiwan and the USA correlated with the stage of breast cancer. The proportion of patients with high methylation levels increased from 0% in healthy individuals to 63.6% in Stage 0, 80% in Stage I and 82.6% in Stage II, with a sensitivity of 78.5%, an accuracy of 90.3% and a specificity of 100%. SRCIN1 hypermethylation was significantly correlated with increased SRCIN1 mRNA expression ( p < 0.001). Knockdown of SRCIN1 decreased the viability of breast cancer cells. SRCIN1 silencing resulted in the downregulation of ESR1, BCL2 and various cyclin protein expressions. SRCIN1 hypermethylation in the blood may serve as a noninvasive biomarker, facilitating early detection and prognosis evaluation, and SRCIN1 -targeted therapies could be used in combination regimens for breast cancer patients.

Objective: To study the in vitro and in vivo antitumor effects of the polysaccharide of Alocasia cucullata (PAC) and the underlying mechanism., Methods: B16F10 and 4T1 cells were cultured with PAC of 40 µg/mL, and PAC was withdrawn after 40 days of administration. The cell viability was detected by cell counting kit-8. The expression of Bcl-2 and Caspase-3 proteins were detected by Western blot and the expressions of ERK1/2 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A mouse melanoma model was established to study the effect of PAC during long-time administration. Mice were divided into 3 treatment groups: control group treated with saline water, positive control group (LNT group) treated with lentinan at 100 mg/(kg·d), and PAC group treated with PAC at 120 mg/(kg·d). The pathological changes of tumor tissues were observed by hematoxylin-eosin staining. The apoptosis of tumor tissues was detected by TUNEL staining. Bcl-2 and Caspase-3 protein expressions were detected by immunohistochemistry, and the expressions of ERK1/2, JNK1 and p38 mRNA were detected by qRT-PCR., Results: In vitro, no strong inhibitory effects of PAC were found in various tumor cells after 48 or 72 h of administration. Interestingly however, after 40 days of cultivation under PAC, an inhibitory effect on B16F10 cells was found. Correspondingly, the long-time administration of PAC led to downregulation of Bcl-2 protein (P<0.05), up-regulation of Caspase-3 protein (P<0.05) and ERK1 mRNA (P<0.05) in B16F10 cells. The above results were verified by in vivo experiments. In addition, viability of B16F10 cells under long-time administration culture in vitro decreased after drug withdrawal, and similar results were also observed in 4T1 cells., Conclusions: Long-time administration of PAC can significantly inhibit viability and promote apoptosis of tumor cells, and had obvious antitumor effect in tumor-bearing mice., (© 2023. The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: To estimate the performance of the FibroTouch-based ultrasound attenuation parameter (UAP) for assessing hepatic steatosis in nonalcoholic fatty liver disease (NAFLD), with magnetic resonance imaging proton density fat fraction (MRI-PDFF) as the reference standard., Methods: This prospective, cross-sectional study included 275 individuals in the training group and 110 individuals in the validation group, all of whom completed a standardized research visit, laboratory tests, MRI-PDFF, and UAP measurements over 1 month. Pearson correlation coefficient and Bland-Altman analysis were used to assess the agreement between UAP and MRI-PDFF for the detection of hepatic steatosis. The diagnostic value of UAP was evaluated by the area under the receiver operating characteristic (ROC) curve (AUROC). Confounding factors to UAP performance were identified by ROC curves and regression analyses., Results: The AUROC of UAP for detecting MRI-PDFF at ≥5%, ≥10%, and ≥20% were 0.95 (95% confidence interval [CI] 0.92-0.97), 0.86 (95% CI 0.81-0.90), and 0.90 (95% CI 0.86-0.93), respectively, and their optimal thresholds were 259, 274, and 295 dB/m, respectively. The UAP measurements had higher diagnostic accuracy in participants with lower waist circumference (≤90 cm for men and ≤80 cm for women) compared to those with higher waist circumference (AUROC values: 0.97 vs 0.84, P < 0.05). Bland-Altman analysis showed good agreement between UAP and MRI-PDFF (bias 0.00021). According to established regression analyses, hepatic steatosis could be accurately diagnosed using UAP estimation., Conclusions: FibroTouch-UAP has a high diagnostic potential for hepatic steatosis in NAFLD patients and helps clinical assessment and monitoring., (© 2023 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.)

The study used clinical data to develop a prediction model for breast cancer survival. Breast cancer prognostic factors were explored using machine learning techniques. We conducted a retrospective study using data from the Taipei Medical University Clinical Research Database, which contains electronic medical records from three affiliated hospitals in Taiwan. The study included female patients aged over 20 years who were diagnosed with primary breast cancer and had medical records in hospitals between January 1, 2009 and December 31, 2020. The data were divided into training and external testing datasets. Nine different machine learning algorithms were applied to develop the models. The performances of the algorithms were measured using the area under the receiver operating characteristic curve (AUC), accuracy, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and F1-score. A total of 3914 patients were included in the study. The highest AUC of 0.95 was observed with the artificial neural network model (accuracy, 0.90; sensitivity, 0.71; specificity, 0.73; PPV, 0.28; NPV, 0.94; and F1-score, 0.37). Other models showed relatively high AUC, ranging from 0.75 to 0.83. According to the optimal model results, cancer stage, tumor size, diagnosis age, surgery, and body mass index were the most critical factors for predicting breast cancer survival. The study successfully established accurate 5-year survival predictive models for breast cancer. Furthermore, the study found key factors that could affect breast cancer survival in Taiwanese women. Its results might be used as a reference for the clinical practice of breast cancer treatment., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Epigenetic alterations play a pivotal role in cancer treatment outcomes. Using the methylation array data and The Cancer Genome Atlas (TCGA) dataset, we observed the hypomethylation and upregulation of thiosulfate sulfurtransferase-like domain containing 1 ( TSTD1 ) in patients with breast cancer. We examined paired tissues from Taiwanese patients and observed that 65.09% and 68.25% of patients exhibited TSTD1 hypomethylation and overexpression, respectively. A significant correlation was found between TSTD1 hypomethylation and overexpression in Taiwanese (74.2%, p = 0.040 ) and Western (88.0%, p < 0.001 ) cohorts. High expression of TSTD1 protein was observed in 68.8% of Taiwanese and Korean breast cancer patients. Overexpression of TSTD1 in tumors of breast cancer patients was significantly associated with poor 5-year overall survival ( p = 0.021) and poor chemotherapy response ( p = 0.008). T47D cells treated with TSTD1 siRNA exhibited lower proliferation than the control group, and transfection of TSTD1 in MDA-MB-231 induced the growth of MDA-MB-231 cells compared to the vector control. Additionally, overexpression of TSTD1 in MCF7 cells mediated a poor response to chemotherapy by epirubicin ( p < 0.001) and docetaxel ( p < 0.001) and hormone therapy by tamoxifen ( p =0.025). Circulating cell-free hypomethylated TSTD1 was detected in plasma of Taiwanese breast cancer patients with disease progression and poor chemotherapy efficacy. Our results indicate that promoter hypomethylation and overexpression of TSTD1 in patients with breast cancer are potential biomarkers for poor 5-year overall survival and poor treatment response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ansar, Thu, Hung, Su, Huang, Liao, Chung and Lin.)

Naturally evolved immune-escape PreS2 mutant is an oncogenic caveat of liver cirrhosis and hepatocellular carcinoma (HCC) during chronic hepatitis B virus (HBV) infection. PreS2 mutant is prevalent in above 50% of patients with HCC. In addition, intrahepatic expression of PreS2 mutant large surface antigen (PreS2-LHBS) induces endoplasmic reticulum stress, mitochondria dysfunction, cytokinesis failure, and subsequent chromosome hyperploidy. As PreS2-LHBS has no enzymatic activity, the development of PreS2-specific inhibitors can be challenging. In this study, we aim to identify inhibitors of PreS2-LHBS via the induction of protein-specific degradation. We set up a large-scale protein stability reporter platform and applied an FDA-approved drug library for the screening. We identified ABT199 as a negative modulator of PreS2-LHBS, which induced the degradation of PreS2-LHBS without affecting the general cell viability in both hepatoma and immortalized hepatocytes. Next, by affinity purification screening, we found that PreS2-LHBS interacted with HSC70, a microautophagy mediating chaperone. Simultaneously, inhibitions of lysosomal degradation or microautophagy restored the expression of PreS2-LHBS, suggesting microautophagy is involved in ABT199-induced PreS2-LHBS degradation. Notably, a 24-hr treatment of ABT199 was sufficient for the reduction of DNA damage and cytokinesis failure in PreS2-LHBS expressing hepatocytes. In addition, a persistent treatment of ABT199 for 3 weeks reversed chromosome hyperploidy in PreS2-LHBS cells and suppressed anchorage-independent growth of HBV-positive hepatoma cells. Together, this study identified ABT-199 as a negative modulator of PreS2-LHBS via mediating microautophagy. Our results indicate that long-term inhibition of PreS2-LHBS may serve as a novel strategy for the therapeutic prevention of HBV-mediated HCC., Competing Interests: Declaration of comprting interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

High-grade neuroendocrine tumors (NETs) of the lung consist of small-cell lung cancer (SCLC) and large-cell neuroendocrine carcinoma (LCNEC). Both exhibit aggressive malignancy with poor prognosis. The transformation of lung adenocarcinoma (ADC) to SCLC or LCNEC also contributes to acquired resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs). Despite initially being responsive to chemotherapy, high-grade NET patients inevitably develop drug resistance; thus, novel therapeutic targets are urgently needed for these patients. Our study reported that VGF (nerve growth factor inducible), a factor mainly expressed in neurons during neural development, is highly expressed in SCLC and LCNEC as well as in a subset of ADCs, whereas targeting VGF attenuates cancer cell growth and tumor formation. High VGF expression was associated with advanced stage SCLC and predicted poor prognosis in lung ADC. In addition, EGFR-TKI selection enriched VGF expression in TKI-resistant ADC under epigenetic control. The VGF locus possessed the HDAC1 binding site, and treatment of ADC cells with the HDAC1 inhibitor induced VGF expression. High VGF expression was associated with chemoresistance, and silencing VGF induced BMF and BCL2L11 expression and rendered lung cancer cells sensitive to chemotherapy drugs. These findings suggested the potential of VGF as a prognostic factor and therapeutic target in lung cancers with neuroendocrine feature., (© 2022 UICC.)

Psoriasis is a chronic inflammatory skin disease that can significantly impact the quality of human life. Various drug treatments are available; however, due to their long-term severe side effects the usage of these drugs is limited. Photodynamic therapy (PDT) has been clinically approved for skin diseases due to its non-invasive nature. We present novel NNO-tridentate vanadium(IV) complexes used in PDT for anti-inflammatory effects in an imiquimod-induced psoriasis-like skin disease mouse model. The vanadium(IV) complexes (1-4) were synthesized using the NNO-tridentate ligand with a benzo[ i ]dipyrido[3,2- a ;2',3'- c ]phenazine (dppn) moiety, and were characterized by UV/Visible spectroscopy, EPR spectroscopy, NMR ( 1 H, and 13 C) spectroscopy, electrospray ionization mass (ESI-MS) spectrometry and cyclic voltammetry (CV) studies. The photocytotoxicity of vanadium(IV) complexes (1-4) was low under dark conditions and complex (4) showed remarkable photocytotoxicity under blue light (430 nm, 8 W cm -2 , 30 min) irradiation. Moreover, [VO( t -butylL)(dppn)] (4)-mediated PDT down-regulated inflammatory cytokines IL-17A and IL-22 in the psoriasis-like mouse model, which could evidence the significant relieving of the psoriatic-like symptoms in the mouse model. Overall, these results suggested that [VO( t -butylL)(dppn)] (4) could be a potential candidate for the treatment of psoriasis both in vitro and in vivo .

Background: Approximately 25% of patients with early-stage breast cancer experience cancer progression throughout the disease course. Alterations in TMEM240 in breast cancer were identified and investigated to monitor treatment response and disease progression., Methods: Circulating methylated TMEM240 in the plasma of breast cancer patients was used to monitor treatment response and disease progression. The Cancer Genome Atlas (TCGA) data in Western countries and Illumina methylation arrays in Taiwanese breast cancer patients were used to identify novel hypermethylated CpG sites and genes related to poor hormone therapy response. Quantitative methylation-specific PCR (QMSP), real-time reverse transcription PCR, and immunohistochemical analyses were performed to measure DNA methylation and mRNA and protein expression levels in 394 samples from Taiwanese and Korean breast cancer patients. TMEM240 gene manipulation, viability, migration assays, RNA-seq, and MetaCore were performed to determine its biological functions and relationship to hormone drug treatment response in breast cancer cells., Results: Aberrant methylated TMEM240 was identified in breast cancer patients with poor hormone therapy response using genome-wide methylation analysis in the Taiwan and TCGA breast cancer cohorts. A cell model showed that TMEM240, which is localized to the cell membrane and cytoplasm, represses breast cancer cell proliferation and migration and regulates the expression levels of enzymes involved in estrone and estradiol metabolism. TMEM240 protein expression was observed in normal breast tissues but was not detected in 88.2% (67/76) of breast tumors and in 90.0% (9/10) of metastatic tumors from breast cancer patients. QMSP revealed that in 54.5% (55/101) of Taiwanese breast cancer patients, the methylation level of TMEM240 was at least twofold higher in tumor tissues than in matched normal breast tissues. Patients with hypermethylation of TMEM240 had poor 10-year overall survival (p = 0.003) and poor treatment response, especially hormone therapy response (p < 0.001). Circulating methylated TMEM240 dramatically and gradually decreased and then diminished in patients without disease progression, whereas it returned and its levels in plasma rose again in patients with disease progression. Prediction of disease progression based on circulating methylated TMEM240 was found to have 87.5% sensitivity, 93.1% specificity, and 90.2% accuracy., Conclusions: Hypermethylation of TMEM240 is a potential biomarker for treatment response and disease progression monitoring in breast cancer., (© 2022. The Author(s).)

Entry inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed to control the outbreak of coronavirus disease 2019 (COVID-19). This study developed a robust and straightforward assay that detected the molecular interaction between the receptor-binding domain (RBD) of viral spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor in just 10 min. A drug library of 1068 approved compounds was used to screen for SARS-CoV2 entry inhibition, and 9 active drugs were identified as specific pseudovirus entry inhibitors. A plaque reduction neutralization test using authentic SARS-CoV-2 virus in Vero E6 cells confirmed that 2 of these drugs (Etravirine and Dolutegravir) significantly inhibited the infection of SARS-CoV-2. With molecular docking, we showed that both Etravirine and Dolutegravir are preferentially bound to primary ACE2-interacting residues on the RBD domain, implying that these two drug blocks may prohibit the viral attachment of SARS-CoV-2. We compared the neutralizing activities of these entry inhibitors against different pseudoviruses carrying spike proteins from alpha, beta, gamma, and delta variants. Both Etravirine and Dolutegravir showed similar neutralizing activities against different variants, with EC50 values between 4.5 to 5.8 nM for Etravirine and 10.2 to 22.9 nM for Dolutegravir. These data implied that Etravirine and Dolutegravir may serve as general spike inhibitors against dominant viral variants of SARS-CoV-2.

Colorectal cancer (CRC) arises from chromosomal instability, resulting from aberrant hypermethylation in tumor suppressor genes. This study identified hypermethylated genes in CRC and investigated how they affect clinical outcomes. Methylation levels of specific genes were analyzed from The Cancer Genome Atlas dataset and 20 breast cancer, 16 esophageal cancer, 33 lung cancer, 15 uterine cancer, 504 CRC, and 9 colon polyp tissues and 102 CRC plasma samples from a Taiwanese cohort. In the Asian cohort, Eps15 homology domain-containing protein 3 ( EHD3 ) had twofold higher methylation in 44.4% of patients with colonic polyps, 37.3% of plasma from CRC patients, and 72.6% of CRC tissues, which was connected to vascular invasion and high microsatellite instability. Furthermore, EHD3 hypermethylation was detected in other gastrointestinal cancers. In the Asian CRC cohort, low EHD3 mRNA expression was found in 45.1% of patients and was connected to lymph node metastasis. Multivariate Cox proportional-hazards survival analysis revealed that hypermethylation in women and low mRNA expression were associated with overall survival. In the Western CRC cohort, EHD3 hypermethylation was also connected to overall survival and lower chemotherapy and antimetabolite response rates. In conclusion, EHD3 hypermethylation contributes to the development of CRC in both Asian and Western populations.

The early detection of cancer can reduce cancer-related mortality. There is no clinically useful noninvasive biomarker for early detection of breast cancer. The aim of this study was to develop accurate and precise early detection biomarkers and a dynamic monitoring system following treatment. We analyzed a genome-wide methylation array in Taiwanese and The Cancer Genome Atlas (TCGA) breast cancer (BC) patients. Most breast cancer-specific circulating methylated CCDC181 , GCM2 and ITPRIPL1 biomarkers were found in the plasma. An automatic analysis process of methylated ccfDNA was established. A combined analysis of CCDC181 , GCM2 and ITPRIPL1 (CGIm) was performed in R using Recursive Partitioning and Regression Trees to establish a new prediction model. Combined analysis of CCDC181 , GCM2 and ITPRIPL1 (CGIm) was found to have a sensitivity level of 97% and an area under the curve (AUC) of 0.955 in the training set, and a sensitivity level of 100% and an AUC of 0.961 in the test set. The circulating methylated CCDC181 , GCM2 and ITPRIPL1 was also significantly decreased after surgery (all p < 0.001). The aberrant methylation patterns of the CCDC181 , GCM2 and ITPRIPL1 genes means that they are potential biomarkers for the detection of early BC and can be combined with breast imaging data to achieve higher accuracy, sensitivity and specificity, facilitating breast cancer detection. They may also be applied to monitor the surgical treatment response.

The incidence and mortality rates of colorectal cancer (CRC) have been high in recent years. Prevention and early detection are crucial for decreasing the death rate. Therefore, this study aims to characterize the alteration patterns of mothers against decapentaplegic homolog 3 ( SMAD3 ) in patients with CRC and its applications in early detection by using a genome-wide methylation array to identify an aberrant hypomethylation site in the intron position of the SMAD3 gene. Quantitative methylation-specific polymerase chain reaction showed that hypomethylated SMAD3 occurred in 91.4% (501/548) of Taiwanese CRC tissues and 66.6% of benign tubular adenoma polyps. In addition, SMAD3 hypomethylation was observed in 94.7% of patients with CRC from The Cancer Genome Atlas dataset. A decrease in circulating cell-free methylation SMAD3 was detected in 70% of CRC patients but in only 20% of healthy individuals. SMAD3 mRNA expression was low in 42.9% of Taiwanese CRC tumor tissues but high in 29.4% of tumors compared with paired adjacent normal tissues. Hypomethylated SMAD3 was found in cancers of the digestive system, such as liver cancer, gastric cancer, and colorectal cancer, but not in breast cancer, endometrial cancer, and lung cancer. In conclusion, SMAD3 hypomethylation is a potential diagnostic marker for CRC in Western and Asian populations.

Background: Gene silencing by aberrant DNA methylation of promoter regions remains the most dominant phenomenon occurring during tumorigenesis. Improving the early diagnosis, prognosis, and recurrence prediction of colorectal cancer using noninvasive aberrant DNA methylation biomarkers has encouraging potential. The aim of this study is to characterize the DNA methylation of the promoter region of TMEM240, as well as gene expression and its effect on cell biological functions and its applications in early detection and outcome prediction., Results: Highly methylated CpG sites were identified in the TMEM240 gene by Illumina methylation 450K arrays in 26 Taiwanese patient paired samples and 38 paired samples from The Cancer Genome Atlas (TCGA) colorectal cancer dataset. Transient transfection and knockdown of TMEM240 were performed to demonstrate the role of TMEM240 in colorectal cancer cells. The data showed that TMEM240 could lead to G1 cell cycle arrest, repress cancer cell proliferation, and inhibit cancer cell migration. The quantitative methylation-specific real-time polymerase chain reaction (PCR) results revealed that 87.8% (480 of 547) of the colorectal cancer tumors had hypermethylated TMEM240, and this was also found in benign tubular adenomas (55.6%). Circulating cell-free methylated TMEM240 was detected in 13 of 25 (52.0%) Taiwanese colorectal cancer patients but in fewer (28.6%) healthy controls. In 72.0% (85/118) of tissue samples, TMEM240 mRNA expression was lower in Taiwanese CRC tumor tissues than in normal colorectal tissues according to real-time reverse transcription PCR results, and this was also found in benign tubular adenomas (44.4%). The TMEM240 protein was analyzed in South Korean and Chinese CRC patient samples using immunohistochemistry. The results exhibited low protein expression in 91.7% (100/109) of tumors and 75.0% (24/32) of metastatic tumors but exhibited high expression in 75.0% (6/8) of normal colon tissues. Multivariate Cox proportional hazards regression analysis found that mRNA expression of TMEM240 was significantly associated with overall, cancer-specific, and recurrence-free survival (p = 0.012, 0.007, and 0.022, respectively)., Conclusions: Alterations in TMEM240 are commonly found in Western and Asian populations and can potentially be used for early prediction and as poor prognosis and early-recurrence biomarkers in colorectal cancer.

In dye-sensitized solar cells (DSSCs), the TiO 2 /dye interface significantly affects photovoltaic performance. However, the adsorption and photoinduced behavior of dye molecules on the TiO 2 substrate remains unclear. Herein, shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) was used to study the adsorption and photoinduced behavior of dye (N719) molecules on different TiO 2 ( hkl ) surfaces. On TiO 2 (001) and TiO 2 (110) surfaces, the in situ SHINERS and mass spectrometry results indicate S[double bond, length as m-dash]C bond cleavage in the anchoring groups of adsorbed N719, whereas negligible bond cleavage occurs on the TiO 2 (111) surface. Furthermore, DFT calculations show the stability of the S[double bond, length as m-dash]C anchoring group on three TiO 2 ( hkl ) surfaces in the order TiO 2 (001) < TiO 2 (110) < TiO 2 (111), which correlated well with the observed photocatalytic activities. This work reveals the photoactivity of different TiO 2 ( hkl ) surface structures and can help with the rational design of DSSCs. Thus, this strategy can be applied to real-time probing of photoinduced processes on semiconductor single crystal surfaces., Competing Interests: The authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)

Objective: To investigate the mechanism of occurrence and development of zinc-alpha-2-glycoprotein (AZGP1) in the activated hepatic stellate cells (HSCs) and liver fibrosis. Methods: The activated human hepatic stellate cell line LX2 was induced by the stimulation of transforming growth factor - β1 to construct carbon tetrachloride liver fibrosis mice model. The situation expression of AZGP1 in liver cells and tissues were observed. Plasmid transfection method was used to detect the activation, proliferation, apoptotic functions and changes in related factors of LX2 cells, respectively, after the overexpression and inhibition of AZGP1expression. Univariate analysis of variance was used for multiple group comparison. Results: The results of immunofluorescence staining showed that AZGP1 protein was decreased and α-smooth muscle actin was increased in the activated LX2 cells, and the two were negatively correlated. AZGP1 gene and protein were significantly under-expressed in activated LX2 cells and liver tissues of mice with carbon tetrachloride liver fibrosis. Collagen I, matrix metalloproteinase-2, and α-smooth muscle actin genes and proteins were significantly down-regulated in LX2 cells after over-expression of AZGP1. Cell fluorescence showed that AZGP1-overexpressing cells were activated and α-smooth muscle actin protein was reduced. In addition, the proliferative activity and G1/S-specific cyclin D1 protein of LX2 cells were significantly reduced after overexpression of AZGP1, while cell cycle experiments showed that the proportion of cells overexpressing AZGP1 was significantly increased in the G0/G1 phase, and the proportion of S phase was significantly reduced. AZGP1 had no significant effect on the apoptosis of LX2 cells. Conclusion: AZGP1 can reverse liver fibrosis by inhibiting the activation and proliferation of hepatic stellate cells, and thereby overexpression of AZGP1 is expected to become a new target for liver fibrosis treatment.

Objectives: Fungus Cordyceps militaris has been used as a herbal tonic in traditional Chinese medicine, which could be surface liquid-cultured for mycelia production. To evaluate the potential of polysaccharides obtained from mycelia of Cordyceps militaris (PS-MCM) for attenuation of side-effects of chemotherapy., Methods: Doxorubicin was used to induce cytotoxicity in THP-1 monocytes and EL-4 T cells, and the effects of PS-MCM on cell viability and cytokine production were detected on doxorubicin-treated THP-1 and EL-4 cells., Results: PS-MCM reduced doxorubicin-induced cell death and promoted cell proliferation in THP-1 and EL-4 cells. Moreover, PS-MCM elevated the level of cytokines associated with immune-modulation of THP-1 and EL-4 cells., Conclusion: These findings indicate that PS-MCM has potential for development as a functional food to counteract side effects of chemotherapy., (© 2019 Lin et al.)

Lung and breast cancer are the leading causes of mortality in women worldwide. The discovery of molecular alterations that underlie these two cancers and corresponding drugs has contributed to precision medicine. We found that CCND2 is a common target in lung and breast cancer. Hypermethylation of the CCND2 gene was reported previously; however, no comprehensive study has investigated the clinical significance of CCND2 alterations and its applications and drug discovery. Genome-wide methylation and quantitative methylation-specific real-time polymerase chain reaction (PCR) showed CCND2 promoter hypermethylation in Taiwanese breast cancer patients. As compared with paired normal tissues and healthy individuals, CCND2 promoter hypermethylation was detected in 40.9% of breast tumors and 44.4% of plasma circulating cell-free DNA of patients. The western cohort of The Cancer Genome Atlas also demonstrated CCND2 promoter hypermethylation in female lung cancer, lung adenocarcinoma, and breast cancer patients and that CCND2 promoter hypermethylation is an independent poor prognostic factor. The cell model assay indicated that CCND2 expression inhibited cancer cell growth and migration ability. The demethylating agent antroquinonol D upregulated CCND2 expression, caused cell cycle arrest, and inhibited cancer cell growth and migration ability. In conclusion, hypermethylation of CCND2 is a potential diagnostic, prognostic marker and drug target, and it is induced by antroquinonol D.

Photodynamic therapy (PDT) is a promising and minimally invasive method for the treatment of superficial diseases, and photosensitizers with high phototoxicity indices (defined as (IC 50 dark )/(IC 50 irradiation )) are essential for the development of ideal photosensitizing properties for this technology. Herein, we report a series of photocytotoxic copper(II) complexes [Cu( R QYMP)(dppn)] ( R QYMP=N,N,O-tridentate Schiff-base derivatives, dppn=benzo[i]dipyrido[3,2-a;2',3'-c]phenazine), the structures of which have been confirmed by mass spectrometry and FTIR spectroscopy. X-ray crystallography revealed that the CuN 4 O core of the [Cu( cumyl QYMP)(dppn)](ClO 4 ) complex (3) has a distorted square-pyramidal geometry. Phototoxicity indices of 329 against human squamous cell carcinoma (SCC15) and 296 against basal cell carcinoma (BCC) cell lines have been determined with [Cu( 3-OMe QYMP)(dppn)](ClO 4 ) (4). This can be attributed to the formation of reactive oxygen species, cell apoptosis, and caspase-3 activation, indicating high potential of complex 4 as a photosensitizer candidate in PDT. Thus, copper complexes bearing suitable Schiff-base ligands with a dppn co-ligand may be considered for the design of efficient metal-based anticancer agents for PDT., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aberrant hypermethylation of CpG islands in tumor suppressor genes (TSGs) contributes to colorectal tumorigenesis. To identify new colorectal cancer (CRC) screening marker, we investigated DNA methylation alterations in novel TSGs. Using HumanMethylation450 BeadChip arrays, CpG regions in BEND5 were the most highly methylated among all genomic regions in 26 colorectal tumors compared to paired non-neoplastic tissues from a Taiwan cohort. Therefore, BEND5 was selected for further analysis. Quantitative methylation-specific real-time PCR revealed that 86.7% (117/135) of CRC patients exhibited hypermethylated BEND5 . Real-time reverse transcription PCR identified that BEND5 mRNA expression was downregulated in 68% (32/47) of the analyzed samples. BEND5 hypermethylation was associated with poor overall survival (OS) in Taiwan patients with early-stage CRC ( P = 0.037). In a CRC tissue set from South Korea, OS was higher in patients with high BEND5 protein expression than in those with low BEND5 protein expression ( P = 0.037) by using immunohistochemistry assays. Consistently, BEND5 hypermethylation was associated with poor OS in patients with early-stage CRC in The Cancer Genome Atlas (TCGA) data set ( P = 0.003). Multivariate Cox proportional hazards regression analysis further supported that hypermethylation of BEND5 genes was significantly associated with OS in Taiwan and TCGA CRC patients ( P = 0.023 and 0.033, respectively). Finally, the cell model assay with transient transfection of BEND5 or si-BEND5 knockdown indicated that BEND5 inhibited cancer cell proliferation. In conclusion, epigenetic alteration in the candidate TSG BEND5 contributes to colorectal cancer development and is a prognostic marker of CRC., Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

Background: Activation of type IIB activin receptor (ActRIIB) in skeletal muscle leads to muscle atrophy because of increased muscle protein degradation. However, the intracellular signalling mechanism that mediates ActRIIB-activated muscle catabolism is poorly defined., Methods: We investigated the role of p38β mitogen-activated protein kinases (MAPK) in mediating ActRIIB ligand activin A-activated muscle catabolic pathways in C2C12 myotubes and in mice with perturbation of this kinase pharmacologically and genetically., Results: Treatment of C2C12 myotubes with activin A or myostatin rapidly activated p38 MAPK and its effector C/EBPβ within 1 h. Paradoxically, Akt was activated at the same time through a p38 MAPK-independent mechanism. These events were followed by up-regulation of ubiquitin ligases atrogin1 (MAFbx) and UBR2 (E3α-II), as well as increase in LC3-II, a marker of autophagosome formation, leading to myofibrillar protein loss and myotube atrophy. The catabolic effects of activin A were abolished by p38α/β MAPK inhibitor SB202190. Using small interfering RNA-mediated gene knockdown, we found that the catabolic activity of activin A was dependent on p38β MAPK specifically. Importantly, systemic administration of activin A to mice similarly activated the catabolic pathways in vivo, and this effect was blocked by SB202190. Further, activin A failed to activate the catabolic pathways in mice with muscle-specific knockout of p38β MAPK. Interestingly, activin A up-regulated MuRF1 in a p38 MAPK-independent manner, and MuRF1 did not appear responsible for activin A-induced myosin heavy chain loss and muscle atrophy., Conclusions: ActRIIB-mediated activation of muscle catabolism is dependent on p38β MAPK-activated signalling., (© 2016 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.)

Six new eremophilane-type sesquiterpenes, namely, nigriterpenes A-F (1-6), and one new phenolic compound, named 2-hydroxymethyl-3-pentylphenol (7), along with fomannoxin alcohol, 3-butyl-7-hydroxyphthalide, scytalone, and fomannoxin were isolated from the ethyl acetate extracts of the fermented broths of termite nest-derived Xylaria nigripes, which has long been used as a traditional Chinese medicine for treating insomnia and depression. Their structures were elucidated on the basis of spectroscopic data analysis and compared with the literature. All the pure isolates were evaluated against lipopolysaccharide-induced inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) expression, and NO production in murine brain microglial BV-2 cells. Of the compounds tested, nigriterpene C (3) and fomannoxin alcohol exerted significant inhibitory effects on two induced enzymes and NO production without any significant cellular toxicity. The most potent compound, 3, exhibited concentration-dependent inhibition on NO production and iNOS and COX-2 expression with IC 50 values of 21.7 ± 4.9, 8.1 ± 2.3, and 16.6 ± 5.5 μM, respectively. These results indicated that the potential anti-inflammatory effects of nigriterpene C (3) and fomannoxin alcohol on murine brain microglial BV-2 cells may provide a rationale for the traditional medical uses of X. nigripes for treating insomnia and depression.

Seven new polyketides, phomaketides A-E (1-5) and pseurotins A 3 (6) and G (7), along with the known compounds FR-111142, pseurotins A, A 1 , A 2 , D, and F 2 , 14-norpseurotin A, α-carbonylcarbene, tyrosol, cyclo(-l-Pro-l-Leu), and cyclo(-l-Pro-l-Phe), were purified from the fermentation broth and mycelium of the endophytic fungal strain Phoma sp. NTOU4195 isolated from the marine red alga Pterocladiella capillacea. The structures were established through interpretation of spectroscopic data. The antiangiogenic and anti-inflammatory effects of 1-7 and related analogues were evaluated using human endothelial progenitor cells (EPCs) and lipopolysaccharide (LPS)-activated murine macrophage RAW264.7 cells, respectively. Of the compounds tested, compound 1 exhibited the most potent antiangiogenic activity by suppressing the tube formation of EPCs with an IC 50 of 8.1 μM, and compound 3 showed the most selective inhibitory activity of LPS-induced NO production in RAW264.7 macrophages with an IC 50 value of 8.8 μM.

Some hydroxamate compounds induce cancer cell death by intracellular reactive oxygen species (ROS). This study introduced the hydroxamate core into lovastatin, a fungus metabolite clinically used for the treatment of hypercholesterolemia. The resulting compounds were evaluated for the activity for inducing ROS production. Most compounds exhibited higher activity than original lovastatin. Of these compounds, compound 3c had the most potent activity. Test of cytotoxicity in a panel of human cancer cell lines indicated compound 3c had activities superior to cisplatin in prostate cancer PC-3 cells and breast cancer T47D cells. In contrast, it in amounts up to 40μM had a much lower cytotoxic effect on normal human IMR-90 cells. Further profiling of cell cycle progression, cell apoptosis, and DNA damage activated checkpoint signaling pathway revealed the important role of compound 3c-mediated cytotoxicity in ROS generation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

One new sesquiterpenoid, namely coprinol (1), along with guanacastanes J (2), E (3) and N (4), were isolated from the ethyl acetate extracts of the fermented broths of the fungal strain Coprinellus radians ≠1168. Their structures were elucidated on the basis of spectroscopic data analysis. The growth inhibitory activities against A549 of 1-4 were evaluated, and only 4 exhibited moderate growth inhibitory activity with a GI₅₀ value of 18.2 μM compared with fluorouracil (GI₅₀ = 3.6 μM). All the compounds were also subjected to antifungal assay against Candida albicans ATCC 18804, C. albicans SC-5314, Cryptococcus neoformans ATCC 13690 and Saccharomyces cerevisiae ATCC 2345; all showed mild antifungal activity with MIC values of 128.0 μg/mL for 1-3 and 64.0 μg/mL for 4 in comparison with amphotericin B (MIC = 0.25 μg/mL) and ketoconazole (MIC = 1.0 μg/mL).

Background: Treatment resistance and metastasis are the major causes of death among patients with colorectal cancer (CRC). Approximately 20% of surgically treated patients ultimately develop metastases during the follow-up period. Currently, the TNM system is the only available prognostic test. Therefore, the identification of new markers for CRC remains important. Thrombomodulin (TM), a glycoprotein, is involved in angiogenesis and has been linked to many malignant diseases. However, the function of TM in CRC remains unclear., Methods: A total of 170 patients with CRC participated in this study. TM expression was analyzed via immunohistochemistry. Univariate (Kaplan-Meier) analysis was used to analyze patient outcomes, including overall survival (OS) and disease-free survival (DFS). TM expression was manipulated using shRNA or an overexpression system. Transwell migration assays, wound healing migration assays, and the xCELLigence biosensor system were used to detect cell proliferative and migratory capacities., Results: TM expression in the tumor tissues significantly and positively correlated with the DFS and OS of non-metastatic patients with CRC (ps = 0.036 and 0.0218, respectively). Suppression of TM expression increased the proliferation and migration of DLD-1 cells. TM overexpression reduced the cells' proliferative and migratory capacities. Cyclooxygenase (COX)-2 expression was up-regulated following TM silencing. Furthermore, the association between the migration of colon cancer cells and the levels of TM and epithelial-to-mesenchymal transition (EMT) markers (fibronectin, vimentin and ezrin) was confirmed in HT29 and DLD-1 cells., Conclusions: Our study demonstrates that patients with non-metastatic CRC display low TM expression in their tumors and exhibit reduced DFS and OS. The enhanced expression of mesenchymal markers and COX-2 may be involved in the mechanisms that underlie recurrence in patients with cancer displaying low TM expression.

Aims: Alterations in histone proteins can lead to breast tumorigenesis. Selective histone deacetylase 8 (HDAC8) inhibitors with fewer adverse effects have been developed. A more comprehensive study of alterations and its mechanisms in HDAC8 is required. In this study, we investigated mechanisms of dysregulation of HDAC8 expression and its biological role and pathways in breast cancer., Main Methods: Alterations in HDAC8 were analyzed in Taiwanese breast cancer patients; and in tissue samples from The Cancer Genome Atlas (TCGA) data set that were derived from Western countries. Knockdown by si-HDAC8, treatment with the HDAC8-specific inhibitor PCI-34051, SRB assays, wound healing, Transwell migration assays, Illumina BeadArray™ arrays and Ingenuity Pathway Analysis (IPA) were performed in breast cancer cells., Key Findings: HDAC8 mRNA expression was upregulated in paired breast cancer tissue from Taiwanese patients and in paired breast cancer tissues from the TCGA data set. Hypomethylation of promoter regions was significantly correlated with HDAC8 mRNA overexpression in 588 breast cancer patients from the TCGA data set and was associated with poor prognosis in early-stage breast cancer. HDAC8 mRNA overexpression was associated with late stages and tumor progression. Wound healing and Transwell migration assays revealed that knockdown by si-HDAC8 or PCI-34051 treatment significantly inhibited breast cancer cell migration. Knockdown by si-HDAC8, Illumina BeadArray™ arrays and IPA found that ID3 and PTP4A2 pathways were regulated by HDAC8 in cancer cell migration., Significance: Hypomethylation of the HDAC8 promoter is correlated with HDAC8 overexpression and breast cancer progression and is a potential prognosis marker and drug target., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Mitochondria play important roles in providing metabolic energy and key metabolites for synthesis of cellular building blocks. Mitochondria have additional functions in other cellular processes, including programmed cell death and aging. A previous study revealed Drosophila mitochondrial topoisomerase III alpha (Top3α) contributes to the maintenance of the mitochondrial genome and male germ-line stem cells. However, the involvement of mitochondrial Top3α in the mitochondrion-mediated aging process remains unclear. In this study, the M1L flies, in which Top3α protein lacks the mitochondrial import sequence and is thus present in cell nuclei but not in mitochondria, is used as a model system to examine the role of mitochondrial Top3α in the aging of fruit flies., Results: Here, we reported that M1L flies exhibit mitochondrial defects which affect the aging process. First, we observed that M1L flies have a shorter life span, which was correlated with a significant reduction in the mitochondrial DNA copy number, the mitochondrial membrane potential, and ATP content compared with those of both wildtype and transgene-rescued flies of the same age. Second, we performed a mobility assay and electron microscopic analysis to demonstrate that the locomotion defect and mitophagy of M1L flies were enhanced with age, as compared with the controls. Finally, we showed that the correlation between the mtDNA deletion level and aging in M1L flies resembles what was reported in mammalian systems., Conclusions: The results reported here demonstrate that mitochondrial Top3α ablation results in mitochondrial genome instability and its dysfunction, thereby accelerating the aging process.

Kawasaki disease (KD) is characterized by pediatric systemic vasculitis of an unknown cause. The low affinity immunoglobulin gamma Fc region receptor II-a (FCGR2A) gene was reported to be involved in the susceptibility of KD. DNA methylation is one of the epigenetic mechanisms that control gene expression; thus, we hypothesized that methylation status of CpG islands in FCGR2A promoter associates with the susceptibility and therapeutic outcomes of Kawasaki disease. In this study, 36 KD patients and 24 healthy subjects from out-patient clinic were recruited. Eleven potential methylation sites within the targeted promoter region of FCGR2A were selected for investigation. We marked the eleven methylation sites from A to K. Our results indicated that methylation at the CpG sites G, H, and J associated with the risk of KD. CpG sites B, C, E, F, H, J, and K were found to associate with the outcomes of IVIG treatment. In addition, CpG sites G, J, and K were predicted as transcription factors binding sites for NF-kB, Myc-Max, and SP2, respectively. Our study reported a significant association among the promoter methylation of FCGR2A, susceptibility of KD, and the therapeutic outcomes of IVIG treatment. The methylation levels of CpG sites of FCGR2A gene promoter should be an important marker for optimizing IVIG therapy.

A novel gold-catalyzed intermolecular oxidation of o-alkynylbiaryls has been developed. A variety of functionalized fluorenes are readily accessed by utilizing this non-diazo approach, thus providing a viable alternative to synthetically useful fluorenes.

Introduction: DNA methyltransferase 3B (DNMT3B) contributes to de novo DNA methylation and its overexpression promotes tumorigenesis. However, whether DNMT3B is upregulated by transcriptional deregulation remains unclear., Methods: We studied the transcriptional repression of DNMT3B by forkhead O transcription factor 3a (FOXO3a) in lung cancer cell, animal, and clinical models., Results: The results of luciferase reporter assay showed that FOXO3a negatively regulated DNMT3B promoter activity by preferentially interacting with the binding element FOXO3a-E (+166 to +173) of DNMT3B promoter. Ectopically overexpressed FOXO3a or combined treatment with doxorubicin to induce FOXO3a nuclear accumulation further bound at the distal site, FOXO3a-P (-249 to -242) by chromatin-immunoprecipitation assay. Knockdown of FOXO3a resulted in an open chromatin structure and high DNMT3B mRNA and protein expression. Abundant FOXO3a repressed DNMT3B promoter by establishing a repressed chromatin structure. Note that FOXO3a is a degradation substrate of MDM2 E3-ligase. Cotreatment with doxorubicin and MDM2 inhibitor, Nutlin-3, further enforced abundant nuclear accumulation of FOXO3a resulting in decrease expression of DNMT3B leading to synergistic inhibition of tumor growth and decrease of methylation status on tumor suppressor genes in xenograft specimens. Clinically, lung cancer patients with DNMT3B high, FOXO3a low, and MDM2 high expression profile correlated with poor prognosis examined by immunohistochemistry and Kaplan-Meier survival analysis., Conclusions: We reveal a new mechanism that FOXO3a transcriptionally represses DNMT3B expression and this regulation can be attenuated by MDM2 overexpression in human lung cancer model. Cotreatment with doxorubicin and Nutlin-3 is a novel therapeutic strategy through epigenetic modulation.

Cancer is a leading cause of death worldwide. Aberrant promoter hypermethylation of CpG islands associated with tumor suppressor genes can lead to transcriptional silencing and result in tumorigenesis. DNA methyltransferases (DNMTs) are the enzymes responsible for DNA methylation and have been reported to be over-expressed in various cancers. This review highlights the current status of transcriptional and post-translational regulation of the DNMT expression and activity with a focus on dysregulation involved in tumorigenesis. The transcriptional up-regulation of DNMT gene expression can be induced by Ras-c-Jun signaling pathway, Sp1 and Sp3 zinc finger proteins and virus oncoproteins. Transcriptional repression on DNMT genes has also been reported for p53, RB and FOXO3a transcriptional regulators and corepressors. In addition, the low expressions of microRNAs 29 family, 143, 148a and 152 are associated with DNMTs overexpression in various cancers. Several important post-translational modifications including acetylation and phosphorylation have been reported to mediate protein stability and activity of the DNMTs especially DNMT1. In this review, we also discuss drugs targeting DNMT protein expression and activation for therapeutic strategy against cancer.

Background: The DEP domain is a globular domain containing approximately 90 amino acids, which was first discovered in 3 proteins: Drosophila disheveled, Caenorhabditis elegans EGL-10, and mammalian Pleckstrin; hence the term, DEP. DEPDC1B is categorized as a potential Rho GTPase-activating protein. The function of the DEP domain in signal transduction pathways is not fully understood. The DEPDC1B protein exhibits the characteristic features of a signaling protein, and contains 2 conserved domains (DEP and RhoGAP) that are involved in Rho GTPase signaling. Small GTPases, such as Rac, CDC42, and Rho, regulate a multitude of cell events, including cell motility, growth, differentiation, cytoskeletal reorganization and cell cycle progression., Results: In this study, we found that it was a guanine nucleotide exchange factor and induced both cell migration in a cultured embryonic fibroblast cell line and cell invasion in cancer cell lines; moreover, it was observed to promote anchorage-independent growth in oral cancer cells. We also demonstrated that DEPDC1B plays a role in regulating Rac1 translocated onto cell membranes, suggesting that DEPDC1B exerts a biological function by regulating Rac1. We examined oral cancer tissue; 6 out of 7 oral cancer tissue test samples overexpressed DEPDC1B proteins, compared with normal adjacent tissue., Conclusions: DEPDC1B was a guanine nucleotide exchange factor and induced both cell migration in a cultured embryonic fibroblast cell line and cell invasion in cancer cell lines; moreover, it was observed to promote anchorage-independent growth in oral cancer cells. We also demonstrated that DEPDC1B exerts a biological function by regulating Rac1. We found that oral cancer samples overexpressed DEPDC1B proteins, compared with normal adjacent tissue. Suggest that DEPDC1B plays a role in the development of oral cancer. We revealed that proliferation was linked to a novel DEPDC1B-Rac1-ERK1/2 signaling axis in oral cancer cell lines.

DNA methyltransferase 1 (DNMT1) catalyzes DNA methylation and is overexpressed in various human diseases, including cancer. A rational approach to preventing tumorigenesis involves the use of pharmacologic inhibitors of DNA methylation; these inhibitors should reactivate tumor suppressor genes (TSGs) in tumor cells and restore tumor suppressor pathways. Antroquinonol D (3-demethoxyl antroquinonol), a new DNMT1 inhibitor, was isolated from Antrodia camphorata and identified using nuclear magnetic resonance. Antroquinonol D inhibited the growth of MCF7, T47D, and MDA-MB-231 breast cancer cells without harming normal MCF10A and IMR-90 cells. The SRB assay showed that the 50% growth inhibition (GI50) in MCF7, T47D, and MDA-MB-231 breast cancer cells following treatment with antroquinonol D was 8.01, 3.57, and 25.08 μM, respectively. d-Antroquinonol also inhibited the migratory ability of MDA-MB-231 breast cancer cells in wound healing and Transwell assays. In addition, antroquinonol D inhibited DNMT1 activity, as assessed by the DNMT1 methyltransferase activity assay. As the cofactor SAM level increased, the inhibitory effects of d-antroquinonol on DNMT1 gradually decreased. An enzyme activity assay and molecular modeling revealed that antroquinonol D is bound to the catalytic domain of DNMT1 and competes for the same binding pocket in the DNMT1 enzyme as the cofactor SAM, but does not compete for the binding pocket in the DNMT3B enzyme. An Illumina Methylation 450 K array-based assay and real-time PCR assay revealed that antroquinonol D decreased the methylation status and reactivated the expression of multiple TSGs in MDA-MB-231 breast cancer cells. In conclusion, we showed that antroquinonol D induces DNA demethylation and the recovery of multiple tumor suppressor genes, while inhibiting breast cancer growth and migration potential.

Topoisomerase IIbeta (Top2b) is an enzyme that modulates DNA supercoiling by catalyzing the passage of DNA duplexes through one another. It is ubiquitously expressed in postmitotic cells and known to function during the development of neuromuscular junctions in the diaphragm and the proper formation of laminar structure in the cerebral cortex. However, due to the perinatal death phenotype of the traditional constitutive and brain-specific Top2b knockout mice, the precise in vivo function of Top2b, especially during postnatal neural development, remains to be determined. Using both the constitutive and retina-specific knockout mouse models, we showed that Top2b deficiency resulted in delayed neuronal differentiation, degeneration of the plexiform layers and outer segment of photoreceptors, as well as dramatic reduction in cell number in the retina. Genome-wide transcriptome analysis by RNA sequencing revealed that genes involved in neuronal survival and neural system development were preferentially affected in Top2b-deficient retinas. Collectively, our findings have indicated an important function of Top2b in proper development and the maintenance/survival of postmitotic neurons in the retina.

Topoisomerase IIβ (Top2β)-DNA cleavage complexes are known to arrest elongating RNA polymerase II (RNAPII), triggering a proteasomal degradation of the RNAPII large subunit (RNAPII LS) and Top2β itself as a prelude to DNA repair. Here, we demonstrate that the degradation of Top2β occurs through a novel ubiquitin-independent mechanism that requires only 19S AAA ATPases and 20S proteasome. Our results suggest that 19S AAA ATPases play a dual role in sensing the Top2β cleavage complex and coordinating its degradation by 20S proteasome when RNAPII is persistently stalled by the Top2β protein roadblock. Clarification of this transcription-associated proteasome pathway could shed light on a general role of 19S AAA ATPases in processing tight protein-DNA complexes during transcription elongation.

The N-end rule pathway contributes significantly to accelerated muscle proteolysis mediated by the ubiquitin-proteasome pathway in various catabolic conditions. UBR2 (aka E3α-II) is the only known E3 ubiquitin ligase of the N-end rule pathway that is up-regulated by cachectic stimuli including proinflammatory cytokines and tumors. However, the signaling mechanism through which UBR2 is up-regulated remains undetermined. Here we identify a signaling pathway that mediates tumor cell-induced up-regulation of UBR2. UBR2 expression in C2C12 myotubes was up-regulated by conditioned medium from Lewis lung carcinoma cells or C26 colon adenocarcinoma cells, which was blocked by a pharmacological inhibitor of p38α/β mitogen-activated protein kinase (MAPK), SB202190. Similarly, SB202190 administration (i.p.) abolished UBR2 up-regulation in the tibialis anterior of LLC tumor-bearing mice. Genetic gain and loss of function assays in C2C12 myotubes indicated that tumor-induced activation of the p38β isoform is sufficient and necessary for UBR2 up-regulation. In addition, UBR2 up-regulation required p38β-mediated phosphorylation of CCAAT/enhancer binding protein (C/EBP)-β Thr-188, which was critical to C/EBPβ binding to the UBR2 promoter. Furthermore, luciferase reporter assay revealed that the C/EBPβ binding motif in the UBR2 promoter is a functional C/EBPβ-responsive cis-element that enhances the promoter activity on activation by p38β. Finally, genetic ablation of C/EBPβ blocked UBR2 up-regulation in LLC tumor-bearing mice. These results suggest that UBR2 up-regulation in cachectic muscle is mediated by the p38β-C/EBPβ signaling pathway responsible for the bulk of tumor-induced muscle proteolysis.

Camptothecin (CPT), a topoisomerase (Top) I-targeting drug that stabilizes Top1-DNA covalent adducts, can induce S-phase-specific cytotoxicity due to the arrest of progressing replication forks. However, CPT-induced non-S-phase cytotoxicity is less well characterized. In this study, we have identified topoisomerase IIβ (Top2β) as a specific determinant for CPT sensitivity, but not for many other cytotoxic agents, in non-S-phase cells. First, quiescent mouse embryonic fibroblasts (MEFs) lacking Top2β were shown to be hypersensitive to CPT with prominent induction of apoptosis. Second, ICRF-187, a Top2 catalytic inhibitor known to deplete Top2β, specifically sensitized MEFs to CPT. To explore the molecular basis for CPT hypersensitivity in Top2β-deficient cells, we found that upon CPT exposure, the RNA polymerase II large subunit (RNAP LS) became progressively depleted, followed by recovery to nearly the original level in wild-type MEFs, whereas RNAP LS remained depleted without recovery in Top2β-deficient cells. Concomitant with the reduction of the RNAP LS level, the p53 protein level was greatly induced. Interestingly, RNAP LS depletion has been well documented to lead to p53-dependent apoptosis. Altogether, our findings support a model in which Top2β deficiency promotes CPT-induced apoptosis in quiescent non-S-phase cells, possibly due to RNAP LS depletion and p53 accumulation.

Tumor suppressor gene silencing through cytosine methylation contributes to cancer formation. Whether DNA demethylation enzymes counteract this oncogenic effect is unknown. Here, we show that TET1, a dioxygenase involved in cytosine demethylation, is downregulated in prostate and breast cancer tissues. TET1 depletion facilitates cell invasion, tumor growth, and cancer metastasis in prostate xenograft models and correlates with poor survival rates in breast cancer patients. Consistently, enforced expression of TET1 reduces cell invasion and breast xenograft tumor formation. Mechanistically, TET1 suppresses cell invasion through its dioxygenase and DNA binding activities. Furthermore, TET1 maintains the expression of tissue inhibitors of metalloproteinase (TIMP) family proteins 2 and 3 by inhibiting their DNA methylation. Concurrent low expression of TET1 and TIMP2 or TIMP3 correlates with advanced node status in clinical samples. Together, these results illustrate a mechanism by which TET1 suppresses tumor development and invasion partly through downregulation of critical gene methylation., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Overexpression of DNA 5'-cytosine-methyltransferase 3A (DNMT3A), which silences genes including tumor suppressor genes (TSG), is involved in many cancers. Therefore, we examined whether the transcriptional deregulation of RB/MDM2 pathway was responsible for DNMT3A overexpression and analyzed the therapeutic potential of MDM2 antagonist for reversing aberrant DNA methylation status in lung cancer., Experimental Design: The regulation of DNMT3A expression and TSG methylation status by RB/MDM2 was assessed in cancer cell lines and patients. The effects of Nutlin-3, an MDM2 antagonist, on tumor growth in relation to DNMT3A expression and TSG methylation status were examined by xenograft model., Results: We found that RB suppressed DNMT3A promoter activity and mRNA/protein expression through binding with E2F1 protein to the DNMT3A promoter, leading to the decrease of methylation level globally and TSG specifically. In addition, MDM2 dramatically induced DNMT3A expression by negative control over RB. In clinical study, MDM2 overexpression inversely correlated with RB expression, while positively associating with overexpression of DNMT3A in samples from patients with lung cancer. Patients with high MDM2 and low RB expression showed DNMT3A overexpression with promoter hypermethylation in TSGs. Treatment with Nutlin-3, an MDM2 antagonist, significantly suppressed tumor growth and reduced DNA methylation level of TSGs through downregulation of DNMT3A expression in xenograft studies., Conclusions: This study provides the first cell, animal, and clinical evidence that DNMT3A is transcriptionally repressed, in part, by RB/E2F pathway and that the repression could be attenuated by MDM2 overexpression. MDM2 is a potent target for anticancer therapy to reverse aberrant epigenetic status in cancers.

A dearth of protein isoform-based clinical diagnostics currently hinders advances in personalized medicine. A well-organized protein biomarker validation process that includes facile measurement of protein isoforms would accelerate development of effective protein-based diagnostics. Toward scalable protein isoform analysis, we introduce a microfluidic "single-channel, multistage" immunoblotting strategy. The multistep assay performs all immunoblotting steps: separation, immobilization of resolved proteins, antibody probing of immobilized proteins, and all interim wash steps. Programmable, low-dispersion electrophoretic transport obviates the need for pumps and valves. A three-dimensional bulk photoreactive hydrogel eliminates manual blotting. In addition to simplified operation and interfacing, directed electrophoretic transport through our 3D nanoporous reactive hydrogel yields superior performance over the state-of-the-art in enhanced capture efficiency (on par with membrane electroblotting) and sparing consumption of reagents (ca. 1 ng antibody), as supported by empirical and by scaling analyses. We apply our fully integrated microfluidic assay to protein measurements of endogenous prostate specific antigen isoforms in (i) minimally processed human prostate cancer cell lysate (1.1 pg limit of detection) and (ii) crude sera from metastatic prostate cancer patients. The single-instrument functionality establishes a scalable microfluidic framework for high-throughput targeted proteomics, as is relevant to personalized medicine through robust protein biomarker verification, systematic characterization of new antibody probes for functional proteomics, and, more broadly, to characterization of human biospecimen repositories.

Studies in animal models have indicated that dietary isothiocyanates (ITCs) exhibit cancer preventive activities through carcinogen detoxification-dependent and -independent mechanisms. The carcinogen detoxification-independent mechanism of cancer prevention by ITCs has been attributed at least in part to their ability to induce apoptosis of transformed (initiated) cells (e.g. through suppression of IκB kinase and nuclear factor κB as well as other proposed mechanisms). In the current studies we show that ITC-induced apoptosis of oncogene-transformed cells involves thiol modification of DNA topoisomerase II (Top2) based on the following observations. 1) siRNA-mediated knockdown of Top2α in both SV40-transformed MEFs and Ras-transformed human mammary epithelial MCF-10A cells resulted in reduced ITC sensitivity. 2) ITCs, like some anticancer drugs and cancer-preventive dietary components, were shown to induce reversible Top2α cleavage complexes in vitro. 3) ITC-induced Top2α cleavage complexes were abolished by co-incubation with excess glutathione. In addition, proteomic analysis revealed that several cysteine residues on human Top2α were covalently modified by benzyl-ITC, suggesting that ITC-induced Top2α cleavage complexes may involve cysteine modification. Interestingly, consistent with the thiol modification mechanism for Top2α cleavage complex induction, the thiol-reactive selenocysteine, but not the non-thiol-reactive selenomethionine, was shown to induce Top2α cleavage complexes. In the aggregate, our results suggest that thiol modification of Top2α may contribute to apoptosis induction in transformed cells by ITCs.

Genistein is a bioflavonoid enriched in soy products. However, high levels of maternal soy consumption have been linked to the development of infant leukemia ALL and AML. The majority of infant leukemia is linked to mixed lineage leukemia gene (MLL) translocations. Previous studies have implicated topoisomerase II (Top2) in genistein-induced infant leukemia. In order to understand the roles of the two Top2 isozymes in and the molecular mechanism for genistein-induced infant leukemia, we carried out studies in vitro using purified recombinant human Top2 isozymes, as well as studies in cultured mouse myeloid progenitor cells (32Dc13) and Top2beta knockout mouse embryonic fibroblasts (MEFs). First, we showed that genistein efficiently induced both Top2alpha and Top2beta cleavage complexes in the purified system as well as in cultured mouse cells. Second, genistein induced proteasomal degradation of Top2beta in 32Dc13 cells. Third, the genistein-induced DNA double-strand break (DSB) signal, gamma-H2AX, was dependent on the Top2beta isozyme and proteasome activity. Fourth, the requirement for Top2beta and proteasome activity was mirrored in genistein-induced DNA sequence rearrangements, as monitored by a DNA integration assay. Together, our results suggest a model in which genistein-induced Top2beta cleavage complexes are processed by proteasome, leading to the exposure of otherwise Top2beta-concealed DSBs and subsequent chromosome rearrangements, and implicate a major role of Top2beta and proteasome in genistein-induced infant leukemia., (2010 Elsevier Inc. All rights reserved.)

Hypermethylation-mediated tumor suppressor gene silencing plays a crucial role in tumorigenesis. Understanding its underlying mechanism is essential for cancer treatment. Previous studies on human N-alpha-acetyltransferase 10, NatA catalytic subunit (hNaa10p; also known as human arrest-defective 1 [hARD1]), have generated conflicting results with regard to its role in tumorigenesis. Here we provide multiple lines of evidence indicating that it is oncogenic. We have shown that hNaa10p overexpression correlated with poor survival of human lung cancer patients. In vitro, enforced expression of hNaa10p was sufficient to cause cellular transformation, and siRNA-mediated depletion of hNaa10p impaired cancer cell proliferation in colony assays and xenograft studies. The oncogenic potential of hNaa10p depended on its interaction with DNA methyltransferase 1 (DNMT1). Mechanistically, hNaa10p positively regulated DNMT1 enzymatic activity by facilitating its binding to DNA in vitro and its recruitment to promoters of tumor suppressor genes, such as E-cadherin, in vivo. Consistent with this, interaction between hNaa10p and DNMT1 was required for E-cadherin silencing through promoter CpG methylation, and E-cadherin repression contributed to the oncogenic effects of hNaa10p. Together, our data not only establish hNaa10p as an oncoprotein, but also reveal that it contributes to oncogenesis through modulation of DNMT1 function.

Overexpression of DNA 5'-cytosine-methyltransferases (DNMT), which are enzymes that methylate the cytosine residue of CpGs, is involved in many cancers. However, the mechanism of DNMT overexpression remains unclear. Here, we showed that wild-type p53 negatively regulated DNMT1 expression by forming a complex with specificity protein 1 (Sp1) protein and chromatin modifiers on the DNMT1 promoter. However, the stoichiometry between p53 and Sp1 determined whether Sp1 acts as a transcription activator or corepressor. Low level of exogenous Sp1 enhanced the repressive activity of endogenous p53 on the DNMT1 promoter whereas high level of Sp1 upregulated DNMT1 gene expression level in A549 (p53 wild-type) cells. In H1299 (p53 null) cells, exogenous Sp1 induced DNMT1 expression in a dose-dependent manner. We also discovered a new mechanism whereby high level of Sp1, via its COOH-terminal domain, induced interaction between p53 and MDM2, resulting in degradation of p53 by MDM2-mediated ubiquitination. Clinical data from 102 lung cancer patients indicated that overexpression of DNMT1 was associated with p53 mutation (P = 0.014) and high expression of Sp1 protein (P = 0.006). In addition, patients with overexpression of both DNMT1 and Sp1 proteins showed poor prognosis (P = 0.037). Our cell and clinical data provided compelling evidence that deregulation of DNMT1 is associated with gain of transcriptional activation of Sp1 and/or loss of repression of p53. DNMT1 overexpression results in epigenetic alteration of multiple tumor suppressor genes and ultimately leads to lung tumorigenesis and poor prognosis., ((c)2010 AACR.)

Objectives: To investigate the effects of astilbin on the expressions of TNF alpha and IL-10 during liver warm ischemia-reperfusion injury., Methods: C57BL/ 6 mice were randomly divided into 4 groups (n = 8): sham-operated group (Sham), model control group(I/R), low dosage of astilbin treatment group (10 mg/kg) and high dosage of astilbin (40 mg/kg) treatment group. The treatment group mice were intraperitoneally injected with 10 or 40 mg/kg astilbin 24 hours and one hour before Ischemia, the hepatic ischemia-reperfusion model were thus established. After jn90 of min ischemia and 6 h reperfusion of the partial hepatic lobe, the expressions of TNF alpha and IL-10 in liver tissues collected from the experimental groups were detected by Western blot and semiquantitative RT-PCR., Results: The expression of TNF alpha protein in liver tissues gradually decreased in treatment groups (low and high dosages of astilbin treatment groups) as compared to the I/R model control group. Similar results were observed in the mRNA expressions of these genes as determined by semiquantitative RT-PCR (P less than 0.05 for low dosage group; P less than 0.01 for high dosage group). Compared with the I/R model control group, the expression of IL-10 was increased in both treatment groups (low dosage group P less than 0.05; large dosage group P less than 0.01)., Conclusion: Treatment with astilbin decreases TNF alpha expression but induces IL-10 expression in liver during warm ischemia-reperfusion injury.

Remnant livers will be regenerated in live donors after a large volume resection for transplantation. How the structures and hemodynamics of portal vein will evolve with liver regeneration remains unknown. This prompts the present hemodynamic simulation for a 25 year-old man who received a right donor lobectomy. According to the magnetic resonance imaging/computed tomography images taken prior to the operation and one month after the operation, three sequential models of portal veins (pre-op, immediately after the operation, and one-month post-op) were constructed by AMIRA and HYPERMESH, while the immediately after the operation model was generated by removing the right branch in the pre-op model. Hemodynamic equations were solved subject to the sonographically measured inlet velocity. The simulated branch velocities were compared with the measured ones. The predicted overall pressure in the portal vein after resection was found to increase to a magnitude that has not reached to an extent possibly leading to portal hypertension. As expected, blood pressure has a large change only in the vicinity of the resection region. The branches grew considerably different from the original one as the liver is regenerated. Results provide useful evidence to justify the current computer simulation.