The effects of thymidylate synthase 3'UTR genotype on methylation of tumor-specific genes promoter in 22 colorectal cancer patients from southern Iran.

To investigate the effects of thymidylate synthase (TS) 3'UTR genotype on promotor methylation of tumor-related genes in 22 patients with sporadic colorectal cancer (CRC) from southern Iran. We evaluated the correlations of TS 3'UTR genotype with promoter methylation of hTERT, hMLH1, MSH2, MMP2, CDH1, p14, p16, and p21 genes in CRC patients. The polymorphism of TS 3'UTR was evaluated through mutagenically specific PCR. The genes promoter methylation was determined using methylation-specific PCR. For 10 patients, the gene expression profile of epigenetic regulating enzymes, histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), was also examined in both tumor and normal adjacent tissues by quantitative real time PCR. There was a significant association between the hMLH1 methylation and age of patients (P= 0.039) and also between MSH2 methylation and tumor site (P= 0.036). There was insignificant association between gene-specific methylation and TS 3'UTR genotype. However, all polymorphic genotypes of TS were associated with higher methylation of hMLH1 and CDH1 and lower methylation of MSH2. The -6bp/+6bp (heterozygous mutant) and [-6bp/+6bp, +6bp/+6bp] (homozygous mutant) genotypes resulted in higher methylation of p16, and -6bp/+6bp and [-6bp/+6bp, +6bp/+6bp] genotypes were correlated with lower methylation of MMP2. The overexpression of epigenetic enzymes, HDACs and DNMTs, was also demonstrated. There was no association between DNMTs transcript levels and gene-specific hypermethylation. The polymorphic TS genotypes, especially -6bp/+6bp, could affect methylation frequencies of studied genes. Moreover, promoter methylation status was not dependent on DNMTs gene expression. Large sample size studies may contribute to validate these findings.

Antiproliferative and Antitelomerase Effects of Silymarin on Human Colorectal and Hepatocellular Carcinoma Cells.

Silymarin, a widely-used hepatoprotective agent, has shown antitumor properties in both in vitro and animal studies. Currently, there is limited knowledge regarding silymarin's antitelomerase effects on human colorectal cancer and hepatocyte carcinoma cells. In this study, we investigated the antiproliferative and antitelomerase effects of silymarin on four human colorectal cancer and HepG2 hepatocyte carcinoma cell lines. The cell viability and telomerase activity were assessed using MTT and the telomerase repeat amplification protocol assay, respectively. We also investigated the effects of silymarin on the expression of human telomerase reverse transcriptase and its promoter methylation in HepG2 cells by real-time RT-PCR and methylation-specific PCR, respectively. Silymarin treatment inhibited cell proliferation and telomerase activity in all cancer cells. After 24 h of treatment, silymarin exhibited IC50 values ranging from 19 - 56.3 µg/mL against these cancer cells. A 30-min treatment with silymarin at the IC50 concentration effectively inhibited telomerase activity in cell-free extracts of both colorectal cancer and hepatocyte carcinoma cells. Treatment of HepG2 cells with 10 and 30 µg/mL of silymarin for 48 h resulted in a decrease in human telomerase reverse transcriptase expression to 75 and 35% of the level observed in the untreated control (p < 0.01), respectively. Treatment with silymarin (10, 30, and 60 µg/mL) for 48 h did not affect human telomerase reverse transcriptase promoter methylation in HepG2 cells. In conclusion, our findings suggest that silymarin inhibits cancer cell growth by directly inhibiting telomerase activity and downregulating its human telomerase reverse transcriptase catalytic subunit. However, silymarin did not affect human telomerase reverse transcriptase promoter methylation at the concentrations of 10 - 60 µg/mL used in this study.

Serum Copper and Zinc Levels Among Iranian Vitiligo Patients.

Introduction: Vitiligo is a chronic skin disease, which its etiopathogenesis is not fully understood. Numerous studies have suggested that oxidative stress may play a role in the pathophysiology of vitiligo. There are controversial reports as to the changes of serum trace elements, copper (Cu) and zinc (Zn) levels in vitiligo patients. Objectives: We evaluated the alterations in the level of serum Cu and Zn among a group of Iranian vitiligo patients. Methods: The levels of serum Cu and Zn were compared between 117 vitiligo patients and 137 healthy controls using atomic absorption spectrophotometry. Results: The mean Cu and Zn levels in the cases (113.57 ± 59.43 and 95.01 ± 58.95 µg/dl, respectively) were significantly lower than those of the controls (138.90 ± 38.14 and 121.83 ± 33.80 µg/dl, respectively) (P = 0.00). We also observed significantly lower serum Cu and Zn concentrations in young (< 50 years) than the elderly (≥ 50 years) patients (P = 0.00). The mean Cu and Zn levels in the patients with generalized vitiligo (111.63±54.18 and 93.11±59.33 µg/dl, respectively) were significantly lower than patients with localized vitiligo (120.74 ±71.64 and 98.69±58.63 µg/dl, respectively) and those in the control (P = 0.00). The serum Cu/Zn ratio obtained in the young and male patients was higher than those in their matched controls (P = 0.01). Conclusions: The current study has shown that the disturbance of serum Cu and Zn levels is associated with vitiligo, and may play an important role in the disease development of Iranian patients.

The Roles of microRNA miR-185 in Digestive Tract Cancers.

Digestive tract cancers represent a serious public health issue. In recent years, evidence has accumulated that microRNA miR-185 is implicated in the pathogenesis of this group of highly malignant tumors. Its expression variations correlate with clinical features, such as tumor size, lymph node metastasis, tumor node metastatic stage, survival, recurrence and response to adjuvant therapy, and have diagnostic and prognostic potential. In this review, we compile, evaluate and discuss the current knowledge about the roles of miR-185 in digestive tract cancers. Interestingly, miR-185 is apparently involved in regulating both tumor suppressive and oncogenic processes. We look at downstream effects as well as upstream regulation. In addition, we discuss the utility of miR-185 for diagnosis and its potential concerning novel therapeutic approaches.

The effect of benzo[alpha]pyrene on DNA methylation and telomerase activity in human normal and cancer cells.

Benzo[a]pyrene (BaP) exposure has been associated with an increased risk of carcinogenesis. We investigated the effects of BaP on cell viability, the promoter methylation of 11 tumor-associated genes, the global DNA methylation, and telomerase enzyme activity in 5 human cancer cell lines (HCT116, PC3, MDA-MB-231, A549, and HepG2) and normal human peripheral blood mononuclear cells (PBMCs) and adipose-derived mesenchymal stem cells (AD-MSCs). BaP inhibited the proliferation of cells in a dose-dependent manner, as measured by MTT assay. Human normal cells were more sensitive to BaP cytotoxicity than cancer cells. After treatment with the minimally toxic concentration of BaP (5 µM for 72 h), 3 differentially methylated genes (genes with different promoter methylation status) were identified between BaP-treated and untreated control cells, as verified by MSP analysis. BaP induced hypomethylation of COX-2 and MSH2 in normal PBMCs and hypermethylation of APC in HCT116 CRC cells. BaP also non-significantly decreased global methylation levels in 3 cancer cell lines (HCT16, PC3, and A549), as measured by ELISA assay. BaP also reduced telomerase enzyme activity in human AD-MSC cells in a dose-dependent manner. To our knowledge, this is the first report of BaP-effects on telomerase activity and DNA methylation in human normal and cancer cells.

Bilirubin and Epigenetic Modifications in Metabolic and Immunometabolic Disorders.

Bilirubin is the main waste product of heme catabolism. At high concentrations, bilirubin may cause toxicity, especially in the brain, kidney, and erythrocytes. Membrane and mitochondrial dysfunction, oxidative stress, apoptosis, necrosis, endoplasmic reticulum stress, excitotoxicity, inflammation, and epigenetic modifications are the main mechanisms of toxicity triggered by bilirubin in susceptible organs. Many studies have shown that there is an interaction between bilirubin and epigenetic modifications in metabolic and immune diseases. In this review, we first outline the toxicity mediated by bilirubin and then summarize the current knowledge linking bilirubin and epigenetic modifications in metabolic and immunometabolic disorders.

Anti-Proliferative and Anti-Telomerase Effects of Blackberry Juice and Berry- Derived Polyphenols on HepG2 Liver Cancer Cells and Normal Human Blood Mononuclear Cells.

BACKGROUND: Previous studies have provided strong evidence for the anticancer activity of berry fruits. OBJECTIVE: In this study, we investigated the effects of blackberry juice and three berry- polyphenolic compounds on cell proliferation and telomerase activity in human hepatoma HepG2 and normal peripheral blood mononuclear cells (PBMCs). METHODS: The cell viability and telomerase activity were measured by MTT and TRAP assay, respectively. Berry effects on the expression of genes were determined by quantitative RT-PCR assay. RESULTS: Blackberry, gallic acid, and resveratrol inhibited proliferation of both HepG2 and PBMC cells in a dosedependent manner. Resveratrol was more effective than gallic acid for reducing the viability of HepG2 cells, but both showed the same level of growth inhibition in PBMC cells. Berry, resveratrol, and gallic acid significantly inhibited telomerase activity in HepG2 cells. The antiproliferative effect of berry was associated with apoptotic DNA fragmentation. Gallic acid was more effective for reducing telomerase activity than resveratrol, but anthocyanin moderately increased telomerase activity in cancer cells. Telomerase activity was induced by all three polyphenols in PBMCs. Overall, Krumanin chloride was more effective to induce telomerase than gallic acid and resveratrol in PBMC cells. There was no significant difference in hTERT, hTR, and Dnmts expressions between berry treated and the control untreated HepG2 cells. But, a significant downregulation of HDAC1 and HDAC2 and upregulation of SIRT1 were observed in berry-treated cells. CONCLUSION: These data indicate that the berry anticancer effect is associated with antitelomerase activity and changes in HDACs expression. The data also suggest that berry antitelomerase activity is mainly related to its gallic acid and resveratrol, but not anthocyanin content.

Epigenetic Effects of Blackberry Extract on Human Colorectal Cancer Cells.

Fruit-derived polyphenolic compounds have been shown to exert anticancer effects via epigenetic mechanisms. In this study, we investigated the effect of blackberry extract on the expression of DNMTs (Dnmt1, Dnmt3a, and Dnmt3b) and HDACs (HDAC1-4 and SIRT1) and its influence on the cellular differentiation and promoter DNA methylation of tumor-related genes using a panel of six human CRC cell lines. Treatment with IC20 and IC50 concentrations of blackberry extract for 72 h significantly reduced Dnmt1 and Dnmt3b transcript levels in HCT116, SW480, HT29/219, SW742, and LS180 cells in a dose-dependent manner. Blackberry also induced promoter DNA demethylation of SFRP2 and p16 genes in four tested CRC cell lines. Berry treatment, however, upregulated Dnmt3a genes in SW480, SW742, and HT29/219 cell lines. A dose-dependent and cell-type-specific reduction of HDAC1, HDAC2, and HDAC4 expressions were observed in CRC-treated cells. Treatment with berry extract induced the expression of SIRT1 gene in HCT116 and HT29/219 cells and increased the expression of two colonic epithelial cell differentiation markers, carcinoembryonic antigen (CEA) and alkaline phosphatase in LS180 cells in a time-dependent manner. This study is the first to report the epigenetic effects of blackberry in cancer cells.

A novel promising delivery system for cuminaldehyde using gelled lipid nanoparticles: Characterization and anticancer, antioxidant, and antibacterial activities.

This work aimed to develop a novel nanoencapsulation system for food colloidal formulations using gelled lipid nanoparticles (GLNs) to improve the functionality, stability, and bioactivity of cuminaldehyde as a highly volatile and poor hydrophilic food additive. Cuminaldehyde-loaded GLNs with diameters of 117-138 nm were fabricated through a hot emulsification process with monoglyceride (10 and 15 g/100 g lipid phase) as a lipid gelator at two concentrations of cuminaldehyde (500 and 1000 mg/L). All samples remained stable towards macroscopic phase separation and creaming during 28 days of storage at 4 °C, which could be related to the rigid structure of dispersed particles in the gelled state and retarding droplet movement. Moreover, all samples were stable to creaming after subjecting to the environmental changes including temperature (30, 60, and 90 °C for 30 min), ionic strength (100, 200, and 300 mM NaCl), and pH (3, 5, and 7). Measurement of apparent viscosity showed non-Newtonian shear thinning nature in all samples, which was more pronounced at higher concentrations of the gelator. Interestingly, higher cytotoxic effects of cuminaldehyde against human lung and colorectal cancer cells were observed after encapsulation within GLNs. However, weak toxicity was also found against normal peripheral blood mononuclearcells.On the other hand, the antioxidant activity and lipid oxidation stability were improved by increasing cuminaldehyde concentration, while it was reduced at higher monoglyceride concentration. All samples exhibited stronger antibacterial activity against Bacillus cereus than Eschershia coli. These findings suggest the significant potential benefits of GLNs as novel nanocarriers to enrich various food and beverage formulations with essential oils, flavors, and aromas.

Thyroid Function Modulates Lung Fluid and Alveolar Viscoelasticity in Mechanically Ventilated Rat.

BACKGROUND: Mechanical ventilation (MV) is life saving; yet it may induce severe lung injury and lead to multisystem organ failure and death. Thyroid hormones (THs) promote alveolar fluid clearance and alleviates hypoxia-induced lung injury. Given that the mechanism involved in hypoxia-induced lung injury is different from that of ventilator-induced lung injury, we examined the effects of thyroid function on lung extravascular fluid (LF), aquaporin 5 (AQP 5) expression, and alveolar viscoelasticity (AVE) in mechanically ventilated rat. METHODS: Hypothyroid (hypo) and hyperthyroid (hyper) animals were generated by administration of metimazole and L-thyroxine, respectively. Lung injury was induced by high-tidal volume MV. The LF was estimated by lung wet weight-to-dry weight ratio assessment. Expression of AQP 5 was evaluated by western blotting and in situ immunohistochemistry. The AVE was judged by elastic lung pressure/volume curve recording. RESULTS: Injurious MV significantly reduced lung AQP 5 expression and altered LF and AVE in a thyroid function-dependent manner. Regardless of animals' ventilation mode, hyper state caused significant reductions in LF and lung AQP 5 protein. It also improved AVE irrespective of animals' ventilation mode. The effects of hypo condition on LF, AQP 5 expression, and AVE were in contrast to that of hyper state. CONCLUSIONS: These data indicate that thyroid function has profound effects on LF, AQP 5, and AVE in mechanically ventilated lungs. Given that the effects of thyroidal status were as prominent as that of injurious MV, we suggest that thyroid function should be considered when patients are to be subjected to MV.

Evaluation of Alpha 1-Antitrypsin for the Early Diagnosis of Colorectal Cancer.

Previous proteomic studies have identified alpha 1-antitrypsin (A1AT) as a potential serum biomarker for colorectal cancer (CRC). In this case-control study, we evaluated plasma A1AT concentration and activity as a biomarker for the early diagnosis of colorectal cancer in a group of 113 sporadic CRC patients. We also analyzed A1AT gene promoter methylation, and genotypes in this group of CRC patients. The plasma A1AT and CEA concentrations were measured using the nephelometric and ELISA methods, respectively. A1AT activity was determined by Trypsin Inhibitor Capacity assay. The genomic DNA from blood samples were subjected to Z and S genotype analysis using PCR-RFLP method and the gene promoter methylation in tumors and their adjacent normal tissues was determined by methylation specific-PCR assay. The plasma levels of A1AT and CEA in patients (median, 2.3 g/L and 5.96 ng/ml, respectively) were significantly higher than those in healthy controls (medians, 1.43 g/L and 2.57 ng/ml, respectively) (p = 0.0001). The plasma A1AT activity and concentrations were positively correlated with the tumor stage and well-discriminated between early and advanced stages. The A1AT activity in plasma was the most useful marker for CRC diagnosis (median 4.8 mmol/min/ml in cases vs 1.91 mmol/min/ml in controls, p = 0.0001). No deficient Z or S alleles of A1AT was observed in patients' genotype and the gene promoter tends to be more methylated in normal mucosa than in tumor tissues. We conclude that plasma A1AT activity has better sensitivity and specificity than CEA measurement for the early detection of CRC. Promoter demethylation might play a role in increasing plasma A1AT levels in CRC patients.

An Experimental Study on Spinal Cord µ-Opioid and α2-Adrenergic Receptors mRNA Expression Following Stress-Induced Hyperalgesia in Male Rats.

BACKGROUND: Intense stress can change pain perception and induce hyperalgesia; a phenomenon called stress-induced hyperalgesia (SIH). However, the neurobiological mechanism of this effect remains unclear. The present study aimed to investigate the effect of the spinal cord µ-opioid receptors (MOR) and α2-adrenergic receptors (α2-AR) on pain sensation in rats with SIH. METHODS: Eighteen Sprague-Dawley male rats, weighing 200-250 g, were randomly divided into two groups (n=9 per group), namely the control and stress group. The stress group was evoked by random 1-hour daily foot-shock stress (0.8 mA for 10 seconds, 1 minute apart) for 3 weeks using a communication box. The tail-flick and formalin tests were performed in both groups on day 22. The real-time RT-PCR technique was used to observe MOR and α2-AR mRNA levels at the L4-L5 lumbar spinal cord. Statistical analysis was performed using the GraphPad Prism 5 software (San Diego, CA, USA). Student's t test was applied for comparisons between the groups. P<0.05 was considered statistically significant. RESULTS: There was a significant (P=0.0014) decrease in tail-flick latency in the stress group compared to the control group. Nociceptive behavioral responses to formalin-induced pain in the stress group were significantly increased in the acute (P=0.007) and chronic (P=0.001) phases of the formalin test compared to the control group. A significant reduction was also observed in MOR mRNA level of the stress group compared to the control group (P=0.003). There was no significant difference in α2-AR mRNA level between the stress and control group. CONCLUSION: The results indicate that chronic stress can affect nociception and lead to hyperalgesia. The data suggest that decreased expression of spinal cord MOR causes hyperalgesia.

Effects of novel and conventional thermal treatments on the physicochemical properties of iron-loaded double emulsions.

In this work, changes in different physicochemical properties of iron-loaded double emulsions (DEs) were monitored under the influence of novel (microwave and ohmic) and conventional heat treatments. Microwave heating led to destabilization and obvious phase separation. As compared to control samples, heat treatment increased particle size, light absorbance, centrifugal instability, iron expulsion from the internal aqueous phase, color difference, chemical instability of the lipid phase, release of iron in simulated gastrointestinal fluids and iron bioavailability in cell line SW742. Emulsion viscosity and whiteness index decreased after heat treatment, whereas the zeta-potential remained unchanged. There was a negative correlation between physicochemical stability and heat treatment intensity. Conventional heating resulted in greater stability than ohmic heating. Yoghurt samples (as model systems) were fortified with either iron-loaded or iron-free DEs. Our results showed that the presence of iron in the aqueous phase resulted in significant lipid oxidation during storage.

Blackberry Extract Inhibits Telomerase Activity in Human Colorectal Cancer Cells.

Several mechanisms have been proposed to explain berries' anticancer effects. However, no previously published studies have investigated berries' anti-telomerase activity. In this study, the anti-telomerase activity of blackberry crude extract was analyzed in six human colorectal cancer (CRC) cell lines by TRAP assay. The peripheral blood mononuclear cells (PBMCs) from a healthy donor were used as a normal control. We also examined the effect of blackberry on the human telomerase RNA (hTR) mRNA level and on human telomerase reverse transcriptase (hTERT) expression and promoter methylation in CRC cells. Blackberry extract significantly inhibited the growth of six CRC cell lines in a dose-dependent manner. Telomerase activity of CRC cells incubated with the IC50 concentration of berry's extract for 48 and 72 h decreased by 15%-37.5% and 43.23%-62.5% (P < 0.05), respectively. In cell-free assays, treatment with as little as 7 µl/ml of berry juice completely blocked telomerase activity in CRC cell lysates. Berry was much less effective in inhibiting telomerase activity in normal PBMCs than CRC cells. Berry treatment reduced hTERT expression and its promoter methylation in CRC cell lines, but the expression of hTR was less influenced by the treatment. Our data indicate that telomerase inhibition is a key mechanism by which blackberry exerts its anticancer effects in CRC cells.

Effects of Dietary Polyunsaturated Fatty Acids on DNA Methylation and the Expression of DNMT3b and PPARα Genes in Rats.

BACKGROUND: Previous studies have suggested a protective role for Polyunsaturated Fatty Acids (PUFA) against cancer, cardiovascular, and other diseases. To provide new insights into the in vivo effects of PUFA on gene expression, the effects of dietary PUFA on DNMT3b and PPARα gene expression and global DNA methylation were investigated in selected rat tissues. METHODS: Thirty sprague-dawley rats were allotted into 3 dietary groups of ten animals each, received experimental diets containing PUFAs every day by gavages for 12 weeks as follows: control group fed a normal diet and water; n-3 PUFAs group received 300 mg/kg/day n-3 PUFAs supplementation; mixed-PUFAs group received 300 mg/kg/day of a mixture of n-3, -6, -9 PUFAs supplementations. The expressions of DNMT3b and PPARα genes were quantitated using real-time RT-PCR. The genome-wide 5-methylcytosine contents in rat tissues were determined by ELISA method. RESULTS: The average expression of the DNMT3b mRNA was 50% lower in the colon and liver of rats fed the n-3- or mixed-PUFAs supplemented diet than control group (p=0.00). However, PPARα expression was significantly upregulated both in the colon and liver of PUFAs-supplemented rats (p<0.001). No significant difference was observed in the blood, colon, and liver DNA methylation levels between PUFAs-supplemented and control animals. CONCLUSION: The results indicate that dietary PUFAs could modulate the expressions of PPARα and DNMT3b genes in various rat tissues. The findings of this study provide additional insights into the in vivo mechanism of PUFA-mediated regulation of gene expression and could provide an opportunity to develop personalized diets for related disease control.

The impact of polyunsaturated fatty acids on DNA methylation and expression of DNMTs in human colorectal cancer cells.

Growing evidence suggests a role of polyunsaturated fatty acids (PUFA) in the prevention of various types of malignancy, including colorectal cancer (CRC). No published studies have yet examined the direct effect of PUFA treatment on DNA methylation in CRC cells. In this study, 5 human CRC cells were treated with 100 µM DHA, EPA, and LA for 6 days and changes in their global- and gene-specific DNA methylation status as well as expression of DNA methyl transferases (DNMT) were investigated. Cell-type specific differences in DNA methylation and expression of DNMTs were observed in PUFA-treated cells. DHA and EPA treatment induced global hypermethylation in HT29/219 and HCT116 cells, but reduced methylation in Caco2 cells (p < 0.05). Among 10 tumor related genes tested in 5 CRC cell lines, DHA and EPA induced promoter demethylation of Cox2 in HT29/219, p14 and PPARγ in HCT116, and ECAD in SW742 cells. Cell-type specific differences in expression of DNMT1, DNMT3a, and 3b genes were also observed between PUFA-treated and control cells (p < 0.05). Overall, treatment of PUFAs coordinately induced the expression of DNMTs in HT29/219, but suppressed in other 4 cell lines investigated in this study.

Expression of Inflammatory-Related NFκB Genes in Iranian Patients with Pterygium: A Case-Control Study.

Pterygium is one of the most common eye conditions without any clear etiology. Some studies have suggested an association between sun exposure and pterygium, but others have proposed the role of genetic variations in its pathogenesis. To date, no study has investigated the association of inflammatory transcription factor, NFκB genes with pterygium in the Middle East. We examined the changes in expression of 3 inflammatory related NFκB1, NFκB2, and RELA genes in patients with pterygium. Thirty patients with pterygium and 30 age and sex-matched controls were enrolled in this case-control study. None of the participants showed any clinical signs of inflammation in their conjunctiva. Demographic information was obtained and the expression levels of three genes including NFκB1, NFκB2, and RELA were measured in their conjunctiva by real-time RT-PCR using gene-specific primers. Mean expression level of NFκB1, NFκB2 and RELA genes in patients were 2.4±0.3, 1.9± 0.5, and 1.8±0.4 times higher than normal subjects, respectively. Higher levels of gene expression were observed in individuals with more outdoor activity and sun exposure. Moreover, a significant correlation was observed between the expression levels of NFκB2 and RELA genes, suggesting a possible NFκB2- RELA heterodimer formation in patients with pterygium. This study has indicated a significant association between expressions of inflammatory-related NFκB1, NFκB2 and RELA genes, and pterygium. Further studies to verify the role of inflammation in the pathogenesis of pterygium, may provide new targets for managing pterygia.

Differential modulation of claudin 4 expression and myosin light chain phosphorylation by thyroid function in lung injury.

BACKGROUND: Trauma and ventilator-induced lung injury is often associated with endothelial-epithelial barriers breakdown, which may lead to multiple system organ failure (MSOF) and death in critically ill patients. Although molecular mechanism involved in MSOF is not known, junctional opening is believed to happen. In vitro, thyroid hormones inhibit myosin light chain (MLC) phosphorylation and may, thus, inhibit cellular contraction and junctional opening. Trauma is also associated with tissue hypo-thyroid state. Therefore, we examined the effects of thyroid function on expression of phospho-MLC (pp-MLC) and claudin 4 (Clud4), key proteins involved in regulation of junctional tightness, in lung injury. METHODS: Rats were rendered hypo-thyroid (Hypo) or hyperthyroid (Hyper) by adding methimazole or levo-thyroxine, respectively, to their drinking water. Untreated euthyroid (Eue) animals were used as control. Lung pp-MLC and Clud4 proteins were assessed by western blotting and in situ immunodetection, respectively. Lung injury was induced by high tidal volume mechanical ventilation. RESULTS: Lung injury was significantly enhanced in Hypo animals and attenuated in Hyper animals. Parallel changes in expression of lung pp-MLC were detected. Alterations in lung histomorphology correlated with the level of pp-MLC. Expression of alveolar and bronchiolar Clud4 protein was differentially affected by the state of thyroid gland. CONCLUSIONS: Our data suggest that thyroid function plays significant role in lung injury perhaps by modulating expression of the proteins involved in junctional tightness. Besides, they strongly support the idea that the tissue hypo-thyroid state may contribute to endothelial-epithelial barriers breakdown associated with trauma.

The Impact of Thymidylate Synthase and Methylenetetrahydrofolate Reductase Genotypes on Sensitivity to 5-Fluorouracil Treatment in Colorectal Cancer Cells.

5-fluorouracil (5-FU) is one of the major components of many standard regimens for chemotherapy of colorectal cancer (CRC) and some other malignancies. Given the known relationship between thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR) activity and 5-FU metabolism, this study investigated the impact of selected functional polymorphisms of the TS and MTHFR genes on chemotherapy resistance in 5 human CRC cell lines. HCT116, SW1116, HT29/219, LS180, and Caco-2 CRC cells were cultured as monolayer and their chemosensitivity to 5-FU, oxaliplatin, and irinotecan was determined by MTT assay. Genomic DNA was extracted from the cultured cells, and a 6-bp insertion or deletion (6-bp ins/del) polymorphism in 3´-UTR of the TS gene was determined by the PCR-RFLP method. Genotyping of MTHFR 677 C/T and 1298A/C single nucleotide polymorphism (SNP) was also performed by MS-PCR and PCR-RFLP, respectively. Caco-2 with the homozygous TS 6-bp ins/ins and MTHFR 677 T/T and 1298 C/C genotype, was the most 5-FU resistant cell line. HCT116 with the homozygous TS 6-bp del/del and MTHFR 1298 A/A and heterozygous MTHFR 677 C/T genotype was the least 5-FU resistant cell. LS180, the second most 5-FU resistant cell line, was heterozygous for all three polymorphic sits. HT29/219 and SW1116 cells with homozygous TS 6-bp ins/ins and heterozygous MTHFR 677 C/T and 1298 A/C genotypes had intermediate 5-FU sensitivity. The results indicate that TS 3´-UTR 6-bp insertion and MTHFR 677T and 1298C alleles increase 5-FU resistance in CRC cells. No relationship was observed between TS and MTHFR genotypes and oxaliplatin or irinotecan sensitivity in these cells.

Carcinoembryonic Antigen Expression and Resistance to Radiation and 5-Fluorouracil-Induced Apoptosis and Autophagy.

Understanding the mechanism of tumor resistance is critical for cancer therapy. In this study, we investigated the effect of carcinoembryonic antigen (CEA) overexpression on UV-and 5-fluorouracil (5-FU)-induced apoptosis and autophagy in colorectal cancer cells. We used histone deacetylase (HDAC) inhibitor, NaB and DNA demethylating agent, 5-azacytidine (5-AZA) to induce CEA expression in HT29/219 and SW742 colorectal cancer cell lines. MTT assay was used to measure IC50 value of the cells exposed to graded concentrations of 5- FU with either 0.1 mM NaB or 1 µM 5-AZA for 72 h . Using CHO- and SW742-CEA transfectants, we also investigated the effect of CEA expression on UV- and 5-FU-induced apoptosis and autophagy. Treatment of HT29/219 cell line with NaB and 5-AZA increased CEA expression by 29% and 31%, respectively. Compared with control cells, the IC50 value for 5-FU of NaB and 5-AZA-treated cells increased by 40% and 57%, respectively. Treatment of SW742 cells with NaB or 5-AZA increased neither CEA expression nor the IC50 value for 5-FU. In comparison to parental cells, CEA expression also significantly protected transfected cells against UV-induced apoptosis. Decreased proportions of autophagy and apoptosis were also observed in 5-FU treated SW742- and CHO-CEA transfectants. We conclude that CEA expression can effectively protect colorectal cancer cells against radiation and drug-induced apoptosis and autophagy.