Ameliorative effects of melatonin against hydrogen peroxide-induced oxidative stress on boar sperm characteristics and subsequent in vitro embryo development.

Melatonin, the major secretory product of the pineal gland, scavenges a variety of reactive oxygen and nitrogen species in vivo and in vitro, indicating that melatonin is a potent function as an antioxidant. The objective of this study was to investigate the effect of melatonin in the presence or absence of hydrogen peroxide (H(2)O(2)) on sperm characteristics (motility, viability, survival rate, membrane integrity, lipid peroxidation (LPO) and mitochondria activity) and also to examine the developmental rates to the blastocysts stage of porcine oocytes fertilized in vitro with semen treated with or without melatonin (100 nM) in the presence or absence of H(2)O(2) (250 µM). The sperm were treated with melatonin in the presence or absence of H(2)O(2) for 3, 6, 9 and 12 h at 37°C and then analysed for the sperm characteristics. The porcine embryos were produced by in vitro maturation and in vitro fertilization (IVM/IVF) using semen treated with or without melatonin (100 nM) in the presence or absence of H(2)O(2) (250 µM) for 6 h. The semen characteristics, including motility, viability, survival rate, membrane integrity and mitochondria activity, were higher in the groups that were treated with melatonin in comparison to other groups, irrespective of incubation periods. Malondialdehyde levels in control, melatonin and melatonin + H(2)O(2) groups were lower than H(2)O(2) only group. A positive correlation was shown among motility, viability, survival rate and membrane integrity, but a negative correlation was observed between LPO and the other evaluation methods. The developmental rates to blastocysts of IVM/IVF porcine oocytes fertilized by semen treated with melatonin were significantly increased compared with any other groups, with the cell number of blastocysts shown to have a similar trend to the developmental rates. These results demonstrate that melatonin can improve the semen characteristics during in vitro storage and support the developmental ability of IVM/IVF embryos in pigs.

Antioxidative effects of astaxanthin against nitric oxide-induced oxidative stress on cell viability and gene expression in bovine oviduct epithelial cell and the developmental competence of bovine IVM/IVF embryos.

The aim of the present study was to elucidate the fundamental mechanism of bovine oviduct epithelial cell (BOEC) co-culture on developmental capacity of bovine in vitro oocyte maturation/in vitro fertilization (IVM/IVF) embryos. We examined the effects of astaxanthin against nitric oxide-induced oxidative stress on cell viability by MTT assay, lipid peroxidation (LPO) by using thiobarbituric acid (TBA) reaction for malondialdehyde (MDA) and the expression of antioxidant genes (CuZnSOD, MnSOD and Catalase) or apoptosis genes (Bcl-2, Caspase-3 and Bax) by RT-PCR in BOEC. We also evaluated the developmental rates of bovine IVM/IVF embryos co-cultured with BOEC pre-treated with astaxanthin (500 µM) in the presence or absence of sodium nitroprusside (SNP, 1000 µM) for 24 h. Cell viability in BOEC treated with SNP (50-2000 µM) lowered, while astaxanthin addition (50-500 µM) increased it in a dose-dependent manner. Cell viability in astaxanthin plus SNP (1000 µM) gradually recovered according to the increase in astaxanthin additions (100-500 mM). The LPO in astaxanthin group (50-500 µM) gradually decreased in a dose dependent manner and among SNP or astaxanthin plus SNP group, SNP alone and astaxanthin (50 µM) plus SNP shown a significant increase than other groups (p < 0.05). Expression of apoptosis or antioxidant genes was detected by RT-PCR. Bcl-2 and antioxidant genes were detected in astaxanthin or astaxanthin plus SNP group, and Caspase-3 and Bax genes were only found in SNP group. When bovine IVM/IVF embryos were cultured for 6-7 days under co-culture system such as BOEC treated with astaxanthin in the presence or absence of SNP, the developmental ability to blastocysts in 500 µM astaxanthin group was the highest of all groups. These results suggest that astaxanthin has a antioxidative effect on cell viability and LPO of BOEC, and development of bovine IVM/IVF embryos due to the induction of antioxidant genes and suppression of apoptosis genes.

Effects of bed height on the performance of chest compressions.

OBJECTIVES: The correct chest compression technique was emphasised to enhance the result of cardiopulmonary resuscitation in the 2005 guidelines. The present study compared the effects of different bed heights, including a bed at knee height, on the performance of chest compressions. METHODS: Twenty-four healthcare providers participated in this study. Knee height was defined as the baseline bed height. Bed heights were adjusted to 10 and 20 cm above the baseline and 10 and 20 cm below the baseline. At the five bed heights, chest compressions were performed for 2 minutes, and the compression rate was maintained at 100 per minute, with audible feedback. RESULTS: The mean compression depths (MCD) were 28.3 mm (SD 10.7; knee height +20 cm), 32.3 mm (SD 9.2; knee height +10 cm), 32.7 mm (SD 8.5; knee height), 32.3 mm (SD 9.0; knee height -10 cm) and 31.1 mm (SD 8.5; knee height -20 cm). The MCD was significantly lower at knee height plus 20 cm (p<0.001). CONCLUSION: The performance of chest compressions decreased when the bed height was 20 cm higher than the knee height of the rescuer.

Comparison of the GlideScope video laryngoscope and Macintosh laryngoscope in simulated tracheal intubation scenarios.

OBJECTIVE: To compare the GlideScope video laryngoscope (GVL) with the classic Macintosh laryngoscope in simulated airway scenarios of varying difficulty. MATERIALS AND METHODS: A prospective, crossover and randomised study was performed. Four airway scenarios were simulated using the Airsim model as follows: normal; cervical spine immobilisation; tongue oedema and combined cervical spine immobilisation with tongue oedema. Emergency physicians performed tracheal intubations using both devices in each of the scenarios. The time required to intubate, the success rate and the number of intubation attempts were recorded. At the end of each scenario, participants scored vocal cord visualisation using the percentage of glottic opening (POGO) visible and the subjective ease of intubation on a visual analogue scale (VAS). RESULTS: All 25 participants successfully completed the study. There was no difference in the time required for successful tracheal intubation using the GVL compared with using the Macintosh laryngoscope in the four airway scenarios. Only one participant failed to intubate the trachea with the Macintosh laryngoscope for the combined scenario. There was a significant increase in POGO when using the GVL in the cervical spine immobilisation group (p = 0.027). The VAS score of the subjective ease of intubation was lower for the GVL than for the Macintosh laryngoscope device in difficult scenarios but this difference was not significant. CONCLUSION: This study suggests that the GVL could be an option for airway management even by emergency physicians with little experience and no training in its use.

Accuracy of Web-based recording program for in-hospital resuscitation: laboratory study.

OBJECTIVE: The purpose of this study was to assess the accuracy of a Web-based resuscitation recording program compared with the handwritten method. METHODS: A Web site was developed to record in-hospital resuscitation events and a mock resuscitation was recorded using both the Web site and handwritten method by emergency nurses. Accurate recorded events and times were compared between the two methods through the use of a video clip. Paired t tests were used to compare differences in absolute timing error, the number of omitted events out of 11 reference events and total recorded events. RESULTS: Twenty-one emergency nurses recorded simulated resuscitation events using both the handwritten and Web-based computerised recording system. The mean absolute timing errors were significantly lower using the computerised recording program (37.3 s (SD 17.1) versus 8.3 s (SD 5.3), p<0.001). The mean number of omissions for the computerised program was 1.8 (SD 0.8) compared with 1.4 (SD 1.1) for the handwritten method (p = 0.202). The mean number of total recorded events for the computerised program was 16.5 (SD 3.5) compared with 15.0 (SD 3.8) for the handwritten method (p = 0.063). CONCLUSIONS: This study suggests that a Web-based recording program decreased timing error while causing no differences in the number of recorded or omitted events in a laboratory setting.

A novel Bcl-2 related gene, Bfl-1, is overexpressed in stomach cancer and preferentially expressed in bone marrow.

Programmed cell death (apoptosis) is an active process which is genetically encoded and plays an important role in several cellular activities such as embryonic development, deletion of autoreactive T-cells and homeostasis. Several genes regulating apoptosis have been reported, including p53, one of the tumor suppressor genes, c-myc, one of the proto-oncogenes, and various kinds of Bcl-2 related genes. A new cDNA clone which is homologous to Bcl-2, named as Bfl-1 were isolated from a human fetal liver at 22 week of gestation. This clone was identified by computer analysis of random cDNA sequences that were obtained in an effort to expand the expressed sequence tag (EST) databases to be used for human genome analysis. The homology was recognized by 72% amino acid identity to the murine A1 gene, a member of the Bcl-2-related genes. The homology to the BH1 and BH2 domains of Bcl-2 was especially significant, suggesting that Bfl-1 is a new member of the Bcl-2-related genes. Bfl-1 is abundantly expressed in the bone marrow and at a low level in some other tissues. Interestingly, a correlation was noted between the expression level of Bfl-1 gene and the development of stomach cancer in eight sets of clinical samples. It is conceivable that Bfl-1 is involved in the promotion of the cell survival in the stomach cancer development or progression.

Increased susceptibility of the c-Myc overexpressing cell line, SNU-16, to TNF-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is a macrophage-derived multifunctional cytokine that acts as a cytostatic or cytotoxic agent in many tumor cells. However, the molecular mechanisms by which tumor cells become sensitive to the cytotoxic action of TNF-alpha are not clear. In this study we demonstrated that the cytotoxicity of TNF-alpha markedly increased in c-Myc overexpressing tumor cells. The stomach cancer cell line, SNU-16, in which c-Myc expression is high due to gene amplification, showed programmed cell death detected by DNA fragmentation and morphological changes. An antisense c-myc S-oligonucleotide specifically inhibited the TNF-alpha-induced apoptosis of SNU-16 cells, provided that the oligonucleotide was added 4 h prior to TNF-alpha treatment. Western immunoblot analysis of p53 and Bax showed that in this cell line, TNF-alpha increased the level of these proteins in a time-dependent manner and that this effect lasted for 12 h. Taken together these data indicate that the deregulation of c-Myc plays an important role in sensitizing tumor cells to TNF-alpha. Furthermore, TNF-alpha-induced apoptosis in the SNU-16 cell line showed increased expression of p53 and Bax protein levels following TNF-alpha treatment. Therefore, we suggest that TNF-alpha-induced apoptosis, which is cytotoxic to tumor cells, is coupled with a p53 and Bax apoptotic pathway.

Mitomycin C induces apoptosis in a caspases-dependent and Fas/CD95-independent manner in human gastric adenocarcinoma cells.

We investigated the mechanism of mitomycin C (MMC)-induced apoptosis in SNU-16 human gastric adenocarcinoma cells. Caspase-8 and caspase-3 were activated in MMC-treated cells whereas caspase-1 was not activated, and cytochrome c was released from mitochondrial membrane to cytosol suggesting that caspase-9 was activated during the MMC-induced apoptotic process. Protein kinase C (PKC) delta was cleaved to its characteristic 40 kDa fragment in a caspase-3-dependent manner; on the other hand PKC zeta was cleaved to approximately 40 kDa independently of caspase-3 in the drug-induced apoptosis of the cells. Incubation with z-DEVD-fmk and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) almost completely abrogated MMC-induced DNA fragmentation, indicating that activation of these caspases was crucially involved in MMC-induced apoptosis. Activation of caspase-8 in response to Fas triggering by recruitment of caspase-8 to the Fas has also been found, however, MMC did not induce FasL and Fas expression, as evidenced by reverse transcriptase-polymerase chain reaction and Western blotting. Taken together, these findings indicate that MMC-induced apoptosis in SNU-16 cells was mediated by caspase-8, caspase-9, and caspase-3 activation independently of FasL/Fas interactions.

Protein kinase C activation by PMA rapidly induces apoptosis through caspase-3/CPP32 and serine protease(s) in a gastric cancer cell line.

Phorbol 12-myristate 13-acetate (PMA) rapidly induced cell death in SNU-16 gastric adenocarcinoma cells. DNA ladder formation and caspase-3/CPP32 activation were observed in PMA treated cells indicating that PMA induces apoptosis. z-DEVD-fmk, specific inhibitor of caspase-3/CPP32, inhibited the induction of apoptosis by PMA, demonstrating that caspase/CPP32 are critically involved in PMA-induced apoptosis. The serine protein inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride effectively blocked apoptosis, and also prevented caspase-3/CPP32 activation. Go6983, a specific inhibitor of PKC, almost completely suppressed apoptosis and caspase-3/CPP32 activation. Furthermore, 1,2-dihexanoyl-sn-glycerol, an endogenous activator of PKC, induced apoptosis detected by DNA fragmentation and Hoechst 33258 nuclear staining. From these results, we conclude that PMA is not only a tumor promoter, but can also induce apoptosis in gastric cancer cells. PMA-induced apoptosis appears to be mediated through activation of protein kinase C, and the activation of serine protease(s) and caspase-3/CPP32 may be the molecular mechanisms by which PMA induces apoptosis.

TNF-alpha induces apoptosis mediated by AEBSF-sensitive serine protease(s) that may involve upstream caspase-3/CPP32 protease activation in a human gastric cancer cell line.

In the present study, we investigated the role of caspase-3/CPP32 and serine protease(s) in cell death induced by TNF-alpha in SNU-16 human gastric adenocarcinoma cells. Apoptosis induced in SNU-16 cells by TNF-alpha was accompanied by the activation of caspase-3/CPP32. After treatment with TNF-alpha, PKCdelta cleaved to its characteristic 40 kDa fragment in a caspase-3/CPP32 dependent manner. Incubation with z-DEVD-fmk completely abrogated TNF-alpha-induced DNA fragmentation, indicating that activation of caspase-3/CPP32 was crucially involved in TNF-alpha-induced apoptosis. In addition, serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), clearly inhibited all the features of apoptosis including DNA fragmentation and chromatin condensation. Furthermore, in the AEBSF treated SNU-16 cells, only intact PKCdelta was detected by immunoblot analysis, suggesting that activation of caspase-3/CPP32 was blocked. Thus, the AEBSF-sensitive step may involve an upstream caspase-3/CPP32 protease activation. Taken together, these results suggest that both caspase-3/CPP32 and serine protease(s) are activated and play an important role in TNF-alpha induced apoptosis in SNU-16 cells.

Isolation of csm1 encoding a class V chitin synthase with a myosin motor-like domain from the rice blast fungus, Pyricularia oryzae.

A gene encoding a chitin synthase with a myosin motor-like domain (csm1) was isolated from Pyricularia oryzae using a PCR fragment amplified from a fungal chitin synthase conserved region. The deduced amino acid sequence of csm1 is homologous to that of CsmA of Aspergillus nidulans (65% identity). The putative gene product of csm1 is consisted of the myosin motor-like domain and a chitin synthase domain as in A. nidulans csmA. The chitin synthase domain of its C-terminus was also homologous to Aspergillus fumigatus ChsE (61.4% identity) and Ustilago maydis Chs6 (48.6% identity) that encode class V chitin synthases. Northern analysis demonstrated that the csm1 was expressed throughout the mycelial growth of P. oryzae. This is the first report on the isolation of the gene encoding a class V chitin synthase with the myosin motor-like domain from P. oryzae.

Protein kinase C activation by phorbol ester increases in vitro invasion through regulation of matrix metalloproteinases/tissue inhibitors of metalloproteinases system in D54 human glioblastoma cells.

To elucidate possible mechanisms of phorbol 12-myristate 13-acetate (PMA) induced in vitro invasiveness of glioblastoma cells, we examined expression levels of membrane-type 1 matrix metalloproteinase (MT1-MMP), MMP-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 using Western blotting and gelatin zymography assay, and found that PMA induced the secretion of MMP-9, activated MMP-2 proenzyme to fully active form of 59 kDa, down-regulated the TIMP-1 and TIMP-2 secretion, and increased MT1-MMP on the cell surface. However, PKC inhibitor Go 6983 reversed all of these effects brought about by PMA. We, therefore, conclude the activation of PKC by PMA in these cells plays a critical role in the regulation of MMPs/TIMPs system, which has a major role in tumor invasion and metastasis.

Growth inhibitory effect on glioma cells of adenovirus-mediated p16/INK4a gene transfer in vitro and in vivo.

The tumour suppressor gene p16/INK4a encodes a specific inhibitor of the cyclin D-dependent kinases CDK4 and CDK6. p16/INK4a prevents the association of CDK4 with cyclin D1, and subsequently inhibits phosphorylation of retinoblastoma tumour suppressor protein (pRb), thus preventing exit from the G1 phase. In human cancers, the estimated frequency of genetic alteration involving the p16/INK4a locus is believed to be second only to alteration of p53. A high frequency (greater than 50%) of homozygous p16/INK4a gene deletion has been demonstrated in glioblastoma tissues and p16/INK4a is altered in 80% of glioma cell lines. Therefore, restoration of p16/INK4a would suppress cell proliferation and induce cell growth arrest. We showed here that restoration of p16/INK4a expression in p16 negative U87MG, U251MG and partially deleted U373MG by Ad-CMV-p16/INK4a induced growth suppression in vitro and in vivo. Expression of p16 transferred by Ad-CMV-p16/INK4a in glioma cells was highly efficient and maintained for more than seven days. In addition, we found that the endogenous status of p16 and Rb might affect the expression of exogenous p16/INK4a gene and inhibitory effect of cell proliferation. Even though, there were several factors affecting the efficiency of Ad-CMV-p16/INK4 gene transfer, our results suggest that Ad-CMV-p16 gene therapy strategy is potentially useful and warrants further clinical investigation for the treatment of gliomas.

Expression of a novel Bcl-2 related gene, Bfl-1, in various human cancers and cancer cell lines.

The expression of Bfl-1 gene, a novel Bcl-2 related gene, was determined by Northern blot analysis using a radiolabeled cDNA specific for Bfl-1 gene in 82 surgically resected tissue specimens of 28 gastric cancers, 15 colon cancers, nine breast cancers, eight bone and soft tissue sarcomas, five ovarian cancers, nine colon adenomas and eight gastric adenomas. A high rate of expression was observed in gastric and colon cancer, at 86 and 93%, respectively. In breast cancer, bone and soft tissue sarcoma and ovarian cancer, the expression rate was 33, 25 and 40%, respectively. In stomach cancer, the expression rate of Bfl-1 gene in metastatic lymph nodes was 82%, which was higher than 50% of the primary sites (p < 0.02). The intensity of RNA bands of the gastric cancer specimens was compared according to the stage, demonstrating that there was no difference in the expression levels of Bfl-1 gene between the stages in both primary sites and metastatic lymph nodes. Bfl-1 gene was expressed in three (33%) out of nine adenomas of the colon, while it was not detected in all eight gastric adenomas, We also examined the RNA expression of Bfl-1 gene in 22 human cancer cell lines consisting of five stomach cancer, four squamous cell carcinoma, three lung cancer, three cervical cancer, two colon cancer, two brain cancer, two leukemia and one osteosarcoma cell lines. Bfl-1 gene band was detected in one (5%) cervical cancer cell line, SiHa. The results of cancer tissue specimens indicate that Bfl-1 gene may play an important role in carcinogenesis of human cancers and may be involved in a relatively early phase of the adenoma-carcinoma sequence in colon cancer development. However, the mechanism responsible for the very low rate of expression in established cell lines is not clearly understood and further investigation is necessary to clarify the mechanism involved.

Alterations of p53 gene in primary gastric cancer tissues.

This study was conducted to investigate the p53 gene alterations in 25 surgically-resected gastric adenocarcinomas in the Korea Cancer Center Hospital by polymerase chain reaction single-strand conformation polymorphism analysis (PCR-SSCP) for exons 4-8 and immunohistochemical staining (IHCS) with anti-p53 antibody, DO-7. p53 mutations were detected in nine (36%) out of 25 cancer tissues by PCR-SSCP in exon 4-8: 0,1,1,6 and 1 mutations in exons 4,5,6,7 and 8, respectively. All tissues were also tested by IHCS, and positive staining was observed in 11 cases (44%). A discrepancy of the results between the two methods was observed in four cases. In one which showed positivity by PCR-SSCP a negative reaction by IHCS, the two base deletion was observed in exon 7. On the other hand, in three cases the mutation was detected only in IHCS but not in PCR-SSCP. The exact mechanism by which this discrepancy develops is not clear at present, although it may be due to the mutation of other exons not tested in this study or the relatively low sensitivity of the PCR-SSCP method. The incidence of p53 gene mutations was analysed according to pathologic stage and histological differentiation, but no significant difference was observed between the p53 alterations and these factors. By combined use of PCR-SSCP and IHCS, 48% of the 25 primary gastric cancer were considered to have mutations of the p53 gene. These results suggest that p53 mutation is not an infrequent event in primary gastric cancer and the p53 gene plays an important role in the carcinogenesis process of gastric cancer.

Metastatic intracavitary cardiac aortic body tumor in a dog.

A mobile right-ventricular mass dynamically occluding the right ostium atrioventriculare in the systolic phase was detected in a 3-year-old male Tosa dog by echocardiography. At necropsy, multiple tumor masses of various sizes were observed in the heart base right ventricular lumen, myocardium, lung and liver. Dysplasia of tricuspid valve characterized by irregular shape of leaflets, upward malposition of large papillary muscles, and shortened and stout chordae tendineae was also detected. Histopathologically, the tumor cells, arranged in sheets or nests, were polyhedral with lightly eosinophilic and finely granular cytoplasm, and contained a hyperchromatic round or oval nucleus. By Grimelius' silver stain, tumor cells had cytoplasmic positive granules. Ultrastructurally, tumor cells contained characteristic small membrane-limited granules. This is the first report of metastatic intracavitary cardiac aortic body tumor in a dog.

Establishment of the prediction table of parturition day with ultrasonography in small pet dogs.

To establish a prediction table of parturition day the real-time B-mode ultrasonographic examinations were performed in the 8 pregnant Malteses and 10 Yorkshire terriers (total pups, 25 and 38 pups, respectively) from 18 days of gestation until the parturition. Ovulation was designated the first day of gestation (day 0). Extra fetal and fetal structures were measured from all conceptues. The parameters that exhibited the best correlation to parturition were used to compile a prediction table of parturition day. To testify the precision of the prediction table of parturition day, the 15 pregnant Malteses (48 pups) and 13 pregnant Yorkshire terriers (42 pups) with unknown mating time were examined using ultrasonography. Inner chorionic cavity diameter on days 18 to 37 and fetal head diameter on day 38 to parturition that showed the best correlation to gestational age were the most pertinent to the estimation of gestational age and the prediction of parturition day. The two parameters were used to compile a prediction table of parturition with averaged regression equations. In verificational examinations, with the exception of I Yorkshire terrier (3.6%) having 1 fetus, 18 of 28 bitches (64.3%) delivered exactly on the date predicted and 9 of 28 bitches (32.1%) delivered within I day of the date predicted. Therefore, the prediction table of parturition day seems to be a useful tool of the prediction of parturition day in practice.

[Effects of para- and diamagnetic materials in a 1.5 Tesla high field magnetic resonance tomography system]. / Effekte von para- und diamagnetischen Materialien in einem 1,5-Tesla-Hochfeld-Magnetresonanztomographen (MRT).

This article shows the results of an experimental investigation of the interference by paramagnetic and diamagnetic materials on imaging in a closed 1.5 Tesla high field magnetic resonance imaging system (MRI). For different types of sequences (SE, GE, EPI) the effects of metal and non-metal profiles in producing artefacts were investigated. A phantom (plastic trunk) filled with Gd-Mn-solution was used for representation of the artefacts. The materials analysed were placed parallel to the phantom at predetermined distances. The images were obtained in transverse and sagittal planes and analysed with respect to the resulting artefacts. The results show that aluminum and polymer profiles produce the weakest artefacts, even when the material is positioned close to the phantom. A comparison of the sequence types shows that the SE-sequence has a low sensitivity to artefacts, despite the great profile variation in size and shape. The SE-sequence accordingly showed a higher imaging stability as compared with the GE- and EPI-sequences. Steel and copper produced the strongest artefacts. The examination was begun after an intensive study of the literature (Internet, Medline, Meditec). So far have been few publications on this subject.

Epigenetic modification of Nrf2 in 5-fluorouracil-resistant colon cancer cells: involvement of TET-dependent DNA demethylation.

5-Fluorouracil (5-FU) is a widely used anticancer drug for the treatment of colorectal cancer (CRC). However, resistance to 5-FU often prevents the success of chemotherapy. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcriptional regulator and a possible target to overcome 5-FU resistance. The present study examined epigenetic changes associated with Nrf2 induction in a human CRC cell line (SNUC5) resistant to 5-FU (SNUC5/5-FUR). Nrf2 expression, nuclear translocation, and binding to promoter were higher in SNUC5/5-FUR cells than in SNUC5 cells. The activated Nrf2 in SNUC5/5-FUR cells led to an increase in the protein expression and activity of heme oxygenase-1 (HO-1), an Nrf2-regulated gene. SNUC5/5-FUR cells produced a larger amount of reactive oxygen species (ROS) than SNUC5 cells. The siRNA- or shRNA-mediated knockdown of Nrf2 or HO-1 significantly suppressed cancer cell viability and tumor growth in vitro and in vivo, resulting in enhanced 5-FU sensitivity. Methylation-specific (MS) or real-time quantitative MS-PCR data showed hypomethylation of the Nrf2 promoter CpG islands in SNUC5/5-FUR cells compared with SNUC5 cells. Expression of the DNA demethylase ten-eleven translocation (TET) was upregulated in SNUC5/5-FUR cells. ROS generated by 5-FU upregulated TET1 expression and function, whereas antioxidant had the opposite effect. These results suggested that the mechanism underlying the acquisition of 5-FU resistance in CRC involves the upregulation of Nrf2 and HO-1 expression via epigenetic modifications of DNA demethylation.

Fabrication of a biodegradable xenoantigen-free rat liver scaffold for potential drug screening applications.

BACKGROUND: The increasing market in biological pharmaceuticals raises the demand for human test systems. Although 2-dimensional (2D) models are mostly used for these purposes, these models not mimic responses of 3-dimensional (3D) native tissue. METHODS: After generation of a rat liver scaffold using 0.1% sodium dodecyl sulfate, we characterized the histology, blood vessel integrity, and residual DNA as well as retained amounts of collagen and glycosaminoglycan (GAG). Then, we examined the susceptibility of extracellular matrix (ECM) to enzymatic remodeling. Finally, a mixed lymphocyte reaction (MLR) was performed to evaluate the in vitro immunogenicity of the ECM against human peripheral blood mononuclear cells (PBMCs). RESULTS: Histologic examination of decellularized liver revealed the removal of nuclear and cytoplasmic materials with preservation of architecture. The vascular network was intact after decellularization. Biochemical analysis of ECM components revealed that only a negligible amount of DNA was retained compared with the native liver with preservation of large amounts of GAG and collagen. Scaffolds were degraded in response to collagenase treatment. MLR demonstrated that decellularized matrices did not exert any xenostimulatory response against human PBMCs. CONCLUSION: Our findings suggested that naturally derived rat liver scaffolds show natural biocompatibility besides the ability to preserve the intact 3D structure and components. Because of these characteristics, the whole decellularized rat liver can retain many aspects of native tissue structure and function upon recellularization enabling it to be used for drug screening.