Mucinous cystadenocarcinoma of the breast harbours TRPS1 expressions and PIK3CA alterations.

AIMS: Breast mucinous cystadenocarcinoma (BMCA) is a rare tumour recently recognised as a distinct entity by the World Health Organisation Tumour Classification Series. BMCA is a triple-negative tumour that lacks specific immunohistochemical markers; therefore, distinguishing it from mimickers such as ovarian and pancreatic cystadenocarcinomas requires careful clinicopathological correlation. Due to its rarity, little is known about the molecular alterations that underlie BMCA. METHODS AND RESULTS: In this study, we used immunohistochemical staining methods to investigate TRPS1 (trichorhinophalangeal syndrome type 1) expression in BMCA and compare it to expression in ovarian and pancreatic mucinous cystadenocarcinomas. We also collected tumour samples from three BMCA patients for molecular analysis by MALDI-TOF mass spectrometry, real-time polymerase chain reaction, whole exome sequencing and fluorescence in-situ hybridisation. TRPS1 immunoreactivity was found only in BMCA tumour cells and not in the ovarian and pancreatic counterparts. One of the three BMCA tumours also showed a PIK3CA hot-spot mutation, which was confirmed by whole genome next-generation sequencing (NGS). No KRAS, NRAS, BRAF or AKT mutations were found. CONCLUSIONS: To our knowledge, this is the first demonstration of TRPS1 expression in BMCA patients and the first identification of a PIK3CA hotspot mutation in these tumours. These findings provide insights into the molecular mechanisms underlying BMCA tumorigenesis and suggest a potential drug target for this rare and poorly understood cancer.

High G9a Expression in DLBCL and Its Inhibition by Niclosamide to Induce Autophagy as a Therapeutic Approach.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is a malignant lymphoid tumor disease that is characterized by heterogeneity, but current treatment does not benefit all patients, which highlights the need to identify oncogenic genes and appropriate drugs. G9a is a histone methyltransferase that catalyzes histone H3 lysine 9 (H3K9) methylation to regulate gene function and expression in various cancers. METHODS: TCGA and GTEx data were analyzed using the GEPIA2 platform. Cell viability under drug treatment was assessed using Alamar Blue reagent; the interaction between G9a and niclosamide was assessed using molecular docking analysis; mRNA and protein expression were quantified in DLBCL cell lines. Finally, G9a expression was quantified in 39 DLBCL patient samples. RESULTS: The TCGA database analysis revealed higher G9a mRNA expression in DLBCL compared to normal tissues. Niclosamide inhibited DLBCL cell line proliferation in a time- and dose-dependent manner, reducing G9a expression and increasing p62, BECN1, and LC3 gene expression by autophagy pathway regulation. There was a correlation between G9a expression in DLBCL samples and clinical data, showing that advanced cancer stages exhibited a higher proportion of G9a-expressing cells. CONCLUSION: G9a overexpression is associated with tumor progression in DLBCL. Niclosamide effectively inhibits DLBCL growth by reducing G9a expression via the cellular autophagy pathway; therefore, G9a is a potential molecular target for the development of therapeutic strategies for DLBCL.

Emodin Ameliorates the Efficacy of Carfilzomib in Multiple Myeloma Cells via Apoptosis and Autophagy.

BACKGROUND: Carfilzomib, the proteasome inhibitor, can increase the overall survival rate of multiple myeloma (MM) patients undergoing targeted therapy. However, relapse and toxicity present great challenges for such treatment, so an urgent need for effective combination therapy is necessary. Emodin is a natural chemical compound that inhibits the proliferation of various cancers and can effectively combine with other treatments. In this study, we evaluated the sensitizing effect of emodin combined with carfilzomib on MM cells. METHODS: The cells were treated with emodin, carfilzomib, and a combination of drugs to determine their effects on cell proliferation and viability. The cell cycle distribution and reactive oxygen species (ROS) expression were measured by flow cytometry. The level of RNA and protein were analyzed through real-time qPCR and immunoblotting. RESULTS: Emodin acted synergistically with carfilzomib to reduce the proliferation and viability of MM cell lines in vitro. Furthermore, the combination of emodin and carfilzomib increased ROS production, inducing apoptosis and autophagy pathways via caspase-3, PARP, p62, and LC3B. CONCLUSIONS: These results provide a molecular target for combination therapy in MM patients.

Characterization of in vitro and in vivo bioactivity of a ferulic acid-2-Hydroxypropyl-ß-cyclodextrin inclusion complex.

Ferulic acid (FA) belongs to the family of phenolic acids and exhibits a wide variety of biological activities. However, the bioavailability of FA is not optimal, owing to its limited aqueous solubility. Several methods have been developed to increase FA bioavailability and enhance its cytoprotective effects. Complexing FA with cyclodextrins (CDs) may provide an alternative method to approach these goals. In this study, we prepared an FA-2-hydroxypropyl-ß-CD (FA-HP-ß-CD) complex, at a 1:1 M ratio of FA to HP-ß-CD, which was characterized by 1H NMR, two-dimensional rotating frame spectroscopy and differential scanning calorimetry. Aqueous solubility of FA was improved after complexing with HP-ß-CD. Furthermore, in vitro and in vivo experimental results indicated that the FA-HP-ß-CD complex had greater bioactivity than FA alone. Therefore, we can conclude that the limitations of FA usage due to low aqueous solubility and bioavailability can be overcome by creating an HP-ß-CD inclusion complex with the hydrophobic FA.

The effects of laser acupuncture on the modulation of cartilage extracellular matrix macromolecules in rats with adjuvant-induced arthritis.

OBJECTIVES: Articular cartilage damage related to irreversible physical disability affects most patients with chronic rheumatoid arthritis (RA). Strategies targeting the preservation of cartilage function are needed. Laser acupuncture (LA) can be an emerging alternative therapy for RA; however, its molecular mechanism underlying the beneficial effect on cartilage has not been elucidated. This study aimed to examine the potential chondroprotective effects of LA on extracellular matrix (ECM) macromolecules and proinflammatory cytokines in the articular cartilage of adjuvant-induced arthritis (AIA) rats and explore its related mechanisms. DESIGN: Monoarthritis was induced in adult male Sprague-Dawley rats (250-300 g) via intraarticular injection of complete Freund's adjuvant (CFA) into the tibiotarsal joint. Animals were treated with LA at BL60 and KI3 acupoints three days after CFA administration with a 780 nm GaAlAs laser at 15 J/cm2 daily for ten days. The main outcome measures including paw circumference, paw withdrawal threshold, histopathology and immunoassays of tumor necrosis factor-α (TNF-α), collagen type II (CoII), cartilage oligomeric matrix protein (COMP) were analyzed. RESULTS: LA significantly reduced ankle edema and inflammation-induced hyperalgesia in AIA rats (P < 0.05). Moreover, the TNF-α levels were significantly decreased while CoII, COMP and proteoglycans proteins were significantly enhanced following LA stimulation of the AIA cartilage compared to those treated with sham-LA (P < 0.05). CONCLUSIONS: LA attenuates cartilage degradation in AIA rat by suppressing TNF-α activation and up-regulating ECM macromolecules, suggesting LA might be of potential clinical interest in RA treatment.

Hypoxia Augments Increased HIF-1α and Reduced Survival Protein p-Akt in Gelsolin (GSN)-Dependent Cardiomyoblast Cell Apoptosis.

Cytoskeleton filaments play an important role in cellular functions such as maintaining cell shape, cell motility, intracellular transport, and cell division. Actin-binding proteins (ABPs) have numerous functions including regulation of actin filament nucleation, elongation, severing, capping, cross linking, and actin monomer sequestration. Gelsolin (GSN) is one of the actin-binding proteins. Gelsolin (GSN) is one of the actin-binding proteins that regulate cell morphology, differentiation, movement, and apoptosis. GSN also regulates cell morphology, differentiation, movement, and apoptosis. In this study, we have used H9c2 cardiomyoblast cell and H9c2-GSN stable clones to understand the roles and mechanisms of GSN overexpression in hypoxia-induced cardiomyoblast cell death. The data show that hypoxia or GSN overexpression induces HIF-1α expression and reduces the expression of survival markers p-Akt and Bcl-2 in H9c2 cardiomyoblast cells. Under hypoxic conditions, GSN overexpression further reduces p-Akt expression and elevates total as well as cleaved GSN levels and HIF-1α levels. In addition, GSN overexpression enhances apoptosis in cardiomyoblasts under hypoxia. Hypoxic challenge further induced activated caspase-3 and cell death that was attenuated after GSN knock down, which implies that GSN is a critical therapeutic target against hypoxia-induced cardiomyoblast cell death.

Discovery of a novel anticancer agent with both anti-topoisomerase I and II activities in hepatocellular carcinoma SK-Hep-1 cells in vitro and in vivo: Cinnamomum verum component 2-methoxycinnamaldehyde.

Cinnamomum verum is used to make the spice cinnamon and has been used as a traditional Chinese herbal medicine for various applications. We evaluated the anticancer effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the bark of the plant, and its underlying molecular biomarkers associated with carcinogenesis in human hepatocellular carcinoma SK-Hep-1 cell line. The results show that 2-MCA suppressed proliferation and induced apoptosis as indicated by mitochondrial membrane potential loss, activation of caspase-3 and caspase-9, increase in the DNA content in sub-G1, and morphological characteristics of apoptosis, including blebbing of plasma membrane, nuclear condensation, fragmentation, apoptotic body formation, and long comet tail. In addition, 2-MCA also induced lysosomal vacuolation with increased volume of acidic compartments, suppressions of nuclear transcription factors NF-κB, cyclooxygenase-2, prostaglandin E2 (PGE2), and both topoisomerase I and II activities in a dose-dependent manner. Further study reveals the growth-inhibitory effect of 2-MCA was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of 2-MCA against SK-Hep-1 cells is accompanied by downregulations of NF-κB-binding activity, inflammatory responses involving cyclooxygenase-2 and PGE2, and proliferative control involving apoptosis, both topoisomerase I and II activities, together with an upregulation of lysosomal vacuolation and volume of acidic compartments. Similar effects (including all of the above-mentioned effects) were found in other tested cell lines, including human hepatocellular carcinoma Hep 3B, lung adenocarcinoma A549, squamous cell carcinoma NCI-H520, colorectal adenocarcinoma COLO 205, and T-lymphoblastic MOLT-3 (results not shown). Our data suggest that 2-MCA could be a potential agent for anticancer therapy.

Anti-apoptotic effect of San Huang Shel Shin Tang cyclodextrin complex (SHSSTc) on CCl4 -induced hepatotoxicity in rats.

The metabolic loading is heavier in liver especially when injured or inflammation. San Huang Shel Shin Tang (SHSST) was an old traditional herbal decoction, which composed with Rheum officinale Baill, Scutellaria baicalnsis Geprgi and Coptis chinensis Franch (1:1:2 in weight), can provide a liver protection effects. We used a beta-cyclodextrin (ß-CD) drug modification method in reduce of the necessary dose of the SHSST. As the results, the FAS-FADD expressions leaded apoptosis in CCl4 intraperitoneal (IP) injection induced acute liver injury in rats. Silymarin, baicalein, SHSST, and SHSST ß-CD complex (SHSSTc) pretreatments protected liver through the decreasing of the expressions of FAS-FADD and downstream caspase-3 and caspase-8. Particularly, SHSSTc (30 mg/kg day) treatment enhanced cell survival pathway activation through the PI3K, Akt and Bad phosphorylation. Compared with SHSST as well as silymarin and baicalein, SHSSTc provided a magnificent liver protection effect, especially in survival pathway activation/TUNEL-apoptotic cell reduction/serum cholesterol level suppression. All these data suggested that ß-CD complex modified the SHSST and promoted the bioavailability and liver protection effects. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 663-670, 2016.

Discovery of a novel anti-cancer agent targeting both topoisomerase I and II in hepatocellular carcinoma Hep 3B cells in vitro and in vivo: Cinnamomum verum component 2-methoxycinnamaldehyde.

Cinnamomum verum has been used as a traditional Chinese herbal medicine. We evaluated the anticancer effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the bark of the plant, in hepatocellular carcinoma Hep 3B cells. The results show that 2-MCA suppressed proliferation and induced apoptosis as indicated by an up-regulation of pro-apoptotic bax and bak genes and down-regulation of anti-apoptotic bcl-2 and bcl-XL genes, mitochondrial membrane potential loss, cytochrome c release, activation of caspase 3 and 9, increase in the DNA content in sub G1, and morphological characteristics of apoptosis. 2-MCA also induced lysosomal vacuolation with increased volume of acidic compartments (VAC), suppressions of nuclear transcription factors NF-κB, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and both topoisomerase I and II activities in a dose-dependent manner. Further study reveals the growth-inhibitory effect of 2-MCA was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of 2-MCA against Hep 3B cells is accompanied by downregulations of NF-κB binding activity, inflammatory responses involving COX-2 and PGE2, and proliferative control involving apoptosis, both topoisomerase I and II activities, together with an upregulation of lysosomal vacuolation and VAC. Our data suggest that 2-MCA could be a potential agent for anticancer therapy.

SHSST-cyclodextrin complex inhibits TGF-ß/Smad3/CTGF to a greater extent than silymarin in a rat model of carbon tetrachloride-induced liver injury.

At present, cirrhosis is an incurable liver disease. Transforming growth factor ß (TGF­ß) is important in myofibroblast induction during the cirrhosis initiation process. The current approach in the development of hepatoprotective drugs depends on TGF­ß inhibition. San Huang Shel Shin Tang (SHSST) is a traditional herbal decoction able to exert a protective effect on the liver, however, similar to silymarin, it is limited by its hydrophobicity. In the present study, SHSST was modified with ß­cyclodextrin to form a hydrophilic complex, which improved its bioavailability. In the carbon tetrachloride­induced acute injury animal model, the effects of pretreatment with silymarin, baicalein, SHSST and the SHSST­ß­CD­complex (SHSSTc) at a low and high dose were assessed. The biopsy results revealed marked liver protection following treatment with silymarin, baicalein and SHSST and these effects were improved further following pretreatment with SHSSTc. Protein analysis demonstrated that the hepatoprotective effects of silymarin occurred through inhibition of the TGF­ß/Smad­3/connective tissue growth factor (CTGF) signaling pathway. SHSSTc exerted the same protective mechanism, however, SHSSTc suppressed CTGF level to a greater extent compared with the groups treated with SHSST or silymarin. Only pretreatment with SHSST and SHSSTc exhibited partial enhancement in the expression of proteins involved in the regulation of liver regeneration, including extracellular­signal­regulated kinase 5, phospho­nuclear factor of activated T cells 3 and phospho­GATA4.

Inhibitory effect of Angelica sinensis extract in the presence of 2-hydroxypropyl-ß-cyclodextrin.

Angelica sinensis (AS) is a traditional Chinese medicinal herb. Its ethanolic extract contains active ingredients, such as ferulic acid, ligustilide, and butylidenephthalide, which are hydrophobic and have inhibitory effects on hepatoma cells. To increase the aqueous solubility/dispersibility of AS extract and study the consequent inhibitory effect on hepatoma cells, the ethanolic extract of AS was complexed with 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a cyclic oligosaccharide that has a hydrophilic outer surface and a hydrophobic central cavity. The AS-HP-ß-CD complex (weight ratio of AS extract: HP-ß-CD=1:5) was prepared and characterized. The effect of complexing the AS extract with HP-ß-CD on Hep3B cell growth was investigated by analyzing cytotoxicity. Our results showed that cytotoxicity inhibition of AS-HP-ß-CD complex was up to 94% and higher than that of AS extract (about 68%). These observations suggested that the use of HP-ß-CD to stabilize AS extract in aqueous solution was possible for herbal medicine application.

Enhancement of rhubarb extract solubility and bioactivity by 2-hydroxypropyl-ß-cyclodextrin.

Rhubarb is a traditional Chinese medicinal herb, and the ethanolic extract of rhubarb consists of active anthraquinones, which are hydrophobic and have antiproliferative effects on hepatoma cell lines. To increase the aqueous solubility of rhubarb and study the consequent bioavailability, the ethanolic extract of rhubarb was complexed with 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a cyclic oligosaccharide that has a hydrophilic outer surface and a hydrophobic central cavity, to form a rhubarb-HP-ß-CD complex. This complex was characterized by performing nuclear magnetic resonance spectroscopy, two-dimensional rotating frame spectroscopy and thin layer chromatography to confirm the inclusion of anthraquinones from rhubarb extract in HP-ß-CD (weight ratio of rhubarb extract:HP-ß-CD=1:9). We investigated the effects of complexing rhubarb extract with HP-ß-CD on the growth of Huh7 and HepG2 cells by performing cytotoxicity analysis, cellular uptake test, and colony formation assay. Our results showed that complexation of rhubarb extract with HP-ß-CD increased the aqueous solubility and bioavailability of rhubarb and thus enhanced its effect on hepatoma cells.

Bufalin induces G2/M phase arrest and triggers autophagy via the TNF, JNK, BECN-1 and ATG8 pathway in human hepatoma cells.

Liver cancer is the fifth most common cause of cancer death worldwide. The study of more effective anti-hepatoma drugs is urgently required. Bufalin has been isolated from a traditional Chinese medicine and possesses less toxicity to normal cells. However, it has been found to inhibit growth of cancer cells. In this study, we aimed to investigate the efficacy and mechanism of bufalin in Huh7, Hep3B and HA22T human hepatoma cells. The three cell lines were treated with bufalin, the proliferation was detected by WST-1 assay and cell cycle was detected by flow cytometry analysis. The results showed that bufalin inhibited the proliferation of hepatoma cells and regulated the hepatoma cell death program in a dose- and time-dependent manner without typical features of apoptosis. RT-PCR arrays were used to investigate the autophagy transcriptional response triggered by bufalin and 13 genes were altered and further confirmed by real-time PCR. The translation levels of selected genes were examined by western blot analysis to reveal the bufalin-induced autophagy cascade. Bufalin synergized with the JNK pathway to induce autophagy of hepatoma cells and is closely associated with the upregulation of TNF, BECN-1, MAPK and ATG8, together with the downregulation of Bcl-2 and Bid. Our study provided a multi-angle evaluation system for anti-hepatoma pharmacology for pre-clinical drug investigation. In this case, bufalin was capable of inducing hepatoma cell autophagy, suggesting a potential regimen for single or combined chemotherapy to overcome hepatoma in clinical practice.

The contribution of XRCC6/Ku70 to hepatocellular carcinoma in Taiwan.

BACKGROUND: Hepatocellular carcinoma (HCC) is a neoplasm for which the prevalence and mortality rates are very high in Taiwan. The DNA non-homologous end-joining repair gene XRCC6/Ku70 plays an important role in the repair of DNA double-strand breaks (DSBs) induced by both exogenous and endogenous DNA-damaging agents. Defects in overall DSB repair capacity can lead to genomic instability and carcinogenesis. In this study, we investigated the contribution of variant XRCC6 in relation to the risk of HCC, from the levels of DNA, RNA and protein. MATERIALS AND METHODS: In this hospital-based case-control study, we collected 298 patients with HCC and 298 cancer-free controls, with frequency matched by age and gender. Firstly, the associations of XRCC6 promoter T-991C (rs5751129), promoter G-57C (rs2267437), promoter A-31G (rs132770), and intron-3 (rs132774) polymorphisms with HCC risk in this Taiwanese population were evaluated. Secondly, 30 HCC tissue samples with variant genotypes were tested to estimate the XRCC6 mRNA expression by real-time quantitative reverse transcription. Finally, the HCC tissue samples of variant genotypes were examined by immunohistochemistry and western blotting to estimate their XRCC6 protein expression levels. RESULTS: Compared with the TT genotype, the TC and CC genotypes conferred a significantly increased risk of HCC [adjusted odds ratio (aOR)=2.43 and 3.52, 95% confidence interval (CI)=1.52-4.03 and 1.18-13.36, p=0.0003 and 0.0385, respectively]. The mRNA and protein expression levels in HCC tissues revealed statistically significantly lower XRCC6 mRNA and protein expressions in the HCC samples with TC/CC genotypes compared with those with the TT genotype (p=0.0037 and 0.0003, respectively). CONCLUSION: Our multi-approach findings at the DNA, RNA and protein levels suggested that XRCC6 may play an important role in HCC carcinogenesis in the Taiwanese population.

The contribution of caveolin-1 genotype and phenotype to hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common types of malignant tumors worldwide, for which the prevalence and mortality rates are very high in Taiwan. Caveolin-1 (CAV-1) is a main structural protein of caveolae and plays a regulatory role in signaling pathways and tumorigenesis. High expression of Cav-1 in mouse HCC is positively correlated with higher cell invasive capacity, but the contribution of CAV-1 genetic variants during HCC progression is still largely unknown. In this study, we investigated the contribution of CAV-1 variant to the risk of HCC from the analyses of DNA, RNA and proteins. MATERIALS AND METHODS: We enrolled 298 patients with HCC and 298 cancer-free controls, frequency-matched by age and gender in this case-control study. Firstly, the associations of six single nucleotide polymorphisms (SNPs) of the Cav-1 gene at C521A (rs1997623), G14713A (rs3807987), G21985A (12672038), T28608A (rs3757733), T29107A (rs7804372), and G32124A (rs3807992) with HCC risk in a Taiwanese population were evaluated. Secondly, thirty HCC tissue samples with variant genotypes were tested to estimate for CAV-1 mRNA expression by real-time quantitative reverse transcription. Finally, the HCC tissue samples of variant genotypes were examined by western blotting to estimate their CAV-1 protein expression patterns. RESULTS: There were significant differences between the HCC and control groups in the distributions of the CAV-1 G14713A genotypes (p=0.0124), and these carrying AG and AA genotypes had a higher risk for HCC, compared with those with the GG genotype (odds ratio=1.51 and 1.94, respectively). Patients with CAV-1 G14713A AG or AA genotype had higher levels of mRNA (p=0.0001) and protein (p=0.0019) than those with the GG genotype. CONCLUSION: Our multi-approach findings at the DNA, RNA and protein levels suggest that CAV-1 may play a critical role in HCC carcinogenesis, and serve as a target for HCC therapy.

Analysis of urinary nucleosides as potential tumor markers in human breast cancer by high performance liquid chromatography/electrospray ionization tandem mass spectrometry.

BACKGROUND: Breast cancer is the most common female cancer and the fourth leading cause of cancer death among women in Taiwan. We measured urinary nucleoside levels in female breast cancer patients (n=36) to evaluate the diagnostic value of nucleosides as potential tumor markers. METHODS: Purification of urinary nucleosides was performed using a 96-well solid phase extraction (SPE, cation-exchange column) procedure to decrease the variation between the single column preparations and to shorten the pretreatment time. Cation-exchange allows for the comprehensive purification of modified nucleosides, such as 2-deoxynucleosides, that are not purifiable by phenylboronic acid-based SPE. High-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) in selected reaction monitoring (SRM) mode was used to quantify multiple nucleosides. Tubercidin was used as an internal standard. The qualitative parameters, retention time, and the parent and daughter ions used revealed that the method was more specific and sensitive than traditional UV detection. RESULTS: Urinary levels of 3 nucleosides, cytidine, 3-methylcytidine, and inosine were significantly higher in breast cancer patients than in normal controls (p<0.01). The discriminative powers of cytidine, 3-methylcytidine, and inosine were 58%, 58%, and 62%, respectively. CONCLUSIONS: LC/MS/MS is a highly specific and sensitive method for rapidly screening a large number of urinary nucleosides that may be potential cancer markers. The 3-methylcytidine may be a candidate marker for breast cancer.

Emodin inhibits the growth of hepatoma cells: finding the common anti-cancer pathway using Huh7, Hep3B, and HepG2 cells.

Emodin--a major component of Rheum palmatum L.-exerts antiproliferative effects in cancer cells that are regulated by different signaling pathways. Hepatocellular carcinoma has high-incidence rates and is associated with poor prognosis and high mortality rates. This study was designed to evaluate the effects of emodin on human hepatocarcinoma cell viability and investigate its mechanisms of action in Huh7, Hep3B, and HepG2 cells. To define the molecular changes associated with this process, expression profiles were compared in emodin-treated hepatoma cells by cDNA microarray hybridization, quantitative RT-PCRs, and Western blot analysis. G2/M phase arrest was observed in all 3 cell lines. Cell cycle regulatory gene analysis showed increased protein levels of cyclin A, cyclin B, Chk2, Cdk2, and P27 in hepatoma cells after time courses of emodin treatment, and Western blot analysis showed decreased protein levels of Cdc25c and P21. Microarray expression profile data and quantitative PCR revealed that 15 representative genes were associated with emodin treatment response in hepatoma cell lines. The RNA expression levels of CYP1A1, CYP1B1, GDF15, SERPINE1, SOS1, RASD1, and MRAS were upregulated and those of NR1H4, PALMD, and TXNIP were downregulated in all three hepatoma cells. Moreover, at 6h after emodin treatment, the levels of GDF15, CYP1A1, CYP1B1, and CYR61 were upregulated. Here, we show that emodin treatment caused G2/M arrest in liver cancer cells and increased the expression levels of various genes both in mRNA and protein level. It is likely that these genes act as biomarkers for hepatocellular carcinoma therapy.

Sclera-related gene polymorphisms in high myopia.

PURPOSE: Transforming growth factor-beta2 (TGF-beta2), basic fibroblast growth factor (bFGF), and fibromodulin (FMOD) are important extracellular matrix components of the sclera and have been shown to be associated with the development of high myopia. Our aim was to examine the association between myopia and the polymorphisms within TGF-beta2, bFGF, and FMOD. METHODS: The study group comprised of patients (n=195; age range: 17-24 years) with a spherical equivalent of -6.5 diopters (D) or a more negative refractive error. The control group comprised of individuals (n=94; age range: 17-25 years) with a spherical equivalent ranging from -0.5 D to +1.0 D. The subjects with astigmatism over -0.75 D were excluded from the study. High resolution melting (HRM) genotyping and restriction fragment length polymorphism (RFLP) genotyping were used to detect single nucleotide polymorphisms (SNPs). The polymorphisms detected were TGF-beta2 (rs7550232 and rs991967), bFGF (rs308395 and rs41348645), and FMOD (rs7543418). Moreover, a stepwise logistic regression procedure was used to detect which of the significant SNPs contributed to the main effects of myopia development. RESULTS: There were significant differences in the frequency of the A allele and A/A genotype in TGF-beta2 (rs7550232; p=0.0178 and 0.03, respectively). Moreover, the haplotype distribution of haplotype 2 (Ht2; A/A) of TGF-beta2 differed significantly between the two groups (p=0.014). The results of the stepwise logistic regression procedure revealed that TGF-beta2 (rs7550232) contributed significantly to the development of high myopia. CONCLUSIONS: TGF-beta2 is an important structure of sclera and might contribute to the formation of myopia. TGF-beta2 (rs7550232) polymorphisms, A allele and A/A genotype, had a protective role against the development of high myopia.

Analysis of urinary nucleosides as potential tumor markers in human colorectal cancer by high performance liquid chromatography/electrospray ionization tandem mass spectrometry.

BACKGROUND: Increased levels of modified nucleosides have been observed in urine from patients suffering from several cancers. In this study, we evaluated whether urinary nucleosides can serve as potential tumor markers for colorectal cancer by high performance liquid chromatography-electrospray/tandem mass spectrometry (HPLC/ESI-MS/MS). METHODS: A simple and specific method based on HPLC/ESI-MS/MS was developed to determine the urinary nucleosides from patients with colorectal cancer. We studied the excretion patterns of nucleosides in urine from 26 patients with colorectal cancer and 18 healthy controls. RESULTS: The LC/MS/MS approach with selective reaction monitoring (SRM) allowed for the sensitive determination of nucleosides in human urine samples with colorectal cancer. The mean levels of 5 urinary nucleosides (adenosine, cytidine, N(2),N(2)-dimethylguanine, 8-hydroxy-2'-deoxyguanosine and uridine) were significantly higher in the patients with colorectal cancer than in the healthy adults. CONCLUSIONS: The results indicate that urinary nucleosides determined by LC/MS/MS may be useful as biological markers for colorectal cancer. Our findings suggest that LC/MS/MS is a highly specific and sensitive method for rapidly screening a large number of nucleoside that may be useful as markers for cancer in humans.

Th1 and Th2 cytokines are elevated in HCV-infected SVR(-) patients treated with interferon-alpha.

Interferon-alpha-based treatment is a standard therapy to cure hepatitis C virus-infected patients. However, the reasons for the failure of interferon-alpha treatment in some patients have not been fully elucidated. We evaluated the differences in the expression levels of various cytokines among patients with and without sustained viral response (SVR). We found that the chemokines (MIG and IP-10) and inflammation-related cytokines (IL-6) were transiently elevated in patients with SVR(+) before interferon-alpha treatment and in the early phase of treatment (week 2), indicating that these cytokines may be related to viral clearance. Furthermore, higher serum levels of Th1 and Th2 cytokines (IL-2, IL-4, IL-5, IL-10, tumor necrosis factor, and IFN-gamma) were observed in SVR(-) than in SVR(+) patients, indicating that they may be associated with ineffective anti-HCV immune response. Our data revealed that the patterns of cytokines varied greatly between SVR(+) and SVR(-) patients before and after IFN-alpha treatment.