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.

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.

Attenuation by l-thyroxine of oxidant-induced gut epithelial damage.

OBJECTIVES: Severe injuries are often associated with tissue hypothyroidism, elevated damaging mediators in circulation, and broken gut epithelial barrier. However, the relationships between the hypothyroid state and gut epithelial damage are largely unknown. Therefore, in this study, we investigated the effects of L-thyroxine (T4) on in vitro models of intact and compromised gut epithelium. MATERIALS AND METHODS: Gut epithelium equivalent was generated by cultivation of IEC-18 rat intestinal epithelial cells into transwell inserts. Confluent cultures were then compromised by scratching or H2O2 and traumatized rat sera (TUR sera) treatments. Macromolecules permeation and transepithelial electrical resistance (TEER) were evaluated by conventional methods. Morphology and scratch wound closure were assessed microscopically. Cell viability/proliferation was assessed by MTT assay. RESULTS: Both H2O2 and TUR sera induced marked yet different types of epithelial disintegration. While H2O2 significantly increased and decreased probe permeation and TEER, respectively, TUR sera was ineffective. Cultures treated with normal rat sera (sham sera) exhibited morphology, probe permeation, and TEER comparable to those of control cultures. Presence of T4 attenuated the H2O2-induced but not TUR sera-induced damages. T4 treatment accelerated, albeit marginally, wound closure but had virtually no effects on cell viability/proliferation. CONCLUSION: These data suggest that different mechanisms are involved in oxidant- and trauma-induced gut epithelial barrier breakdown. Besides, they show that T4 markedly attenuates oxidant-induced gut epithelial damage. Accordingly, one may also conclude that tissue hypothyroidism does not contribute to trauma-induced gut barrier breakdown.

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.

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.

Modulation by thyroid hormone of myosin light chain phosphorylation and aquaporin 5 protein expression in intact lung.

Myosin light chain kinase (MLCK) may play a key role in cellular contraction, paracellular permeability and lung water homeostasis. In vitro, thyroid hormones (THs) potently inhibit MLCK activation and, hence, MLC phosphorylation. Whether similar effect is exerted by THs in in vivo systems is not known. Therefore, we investigated the effects of hypothyroid (HO) and hyperthyroid (HR) states on the level of phospho-MLC, aquaporin 5 (AQP5) protein expression, and water holding capacity in the rat lung. Alterations in thyroid state were induced by adding methimazole or levothyroxine (L-T4) to animals' drinking water. Serum TH concentration and thyroid gland histomorphology were assessed to verify the onset of the thyroid state. Lung phospho-MLC and AQP5 proteins were assessed by Western blotting and immunohistochemistry. Lung extravascular water content was estimated by the tissue wet weight-to-dry weight (W/D) ratio. The HO state induced significant increases in the expression of lung phospho-MLC and AQP5 proteins. In contrast, the HR state caused moderate decreases in lung phospho-MLC and AQP5 proteins. While lung water holding capacity was significantly increased in HO animals, it was significantly reduced in HR animals. The data of this study show that THs are able to modulate MLC phosphorylation in in vivo systems. Besides, they suggest that the circulating level of THs may alter lung fluid balance not only through expression of water channels but also through regulation of cellular contraction and paracellular permeability.

Modulation by thyroid hormone of myosin light chain phosphorylation and aquaporin 5 protein expression in intact lung

Myosin light chain kinase (MLCK) may play a key role in cellular contraction, paracellular permeability and lung water homeostasis. In vitro, thyroid hormones (THs) potently inhibit MLCKactivation and, hence, MLC phosphorylation. Whether similar effect is exerted by THs in in vivo systems is not known. Therefore, we investigated the effects of hypothyroid (HO) and hyperthyroid (HR) states on the level of phospho-MLC, aquaporin 5 (AQP5) protein expression, and water holding capacity in the rat lung. Alterations in thyroid state were induced by adding methimazole or levothyroxine (L-T4) to animals’ drinking water. Serum TH concentration and thyroid gland histomorphology were assessed to verify the onset of the thyroid state. Lung phospho-MLC and AQP5 proteins were assessed by Western blotting and immunohistochemistry. Lung extravascular water content was estimated by the tissue wet weight-to-dry weight (W/D) ratio. The HO state induced significant increases in the expression of lung phospho-MLC and AQP5 proteins. In contrast, the HR state caused moderate decreases in lung phospho-MLC and AQP5 proteins. While lung water holding capacity was significantly increased in HO animals, it was significantly reduced in HR animals. The data of this study show that THs are able to modulate MLC phosphorylation in in vivo systems. Besides, they suggest that the circulating level of THs may alter lung fluid balance not only through expression of water channels but also through regulation of cellular contraction and paracellular permeability

Activation of mitosis and angiogenesis in diabetes-impaired wound healing by processed human amniotic fluid.

BACKGROUND: Functional characterization of human amniotic fluid (AF) proteome, 845 proteins, has revealed that top three functions are cell proliferation, movement and differentiation, events fundamental to development, and tissue repair. Although these findings fortify the idea that AF components play roles in regeneration-like fetal wound healing, it is not known whether the components endure processing. Therefore, we processed AF and tested its effects on diabetes-impaired wound healing in an animal model. MATERIALS AND METHODS: Through a germfree procedure, mature and premature AF samples were collected, respectively, from the mothers of full-term and preterm infants. Excisional wounds were generated on the dorsum of diabetic rats. Wounds were treated on day 3 and harvested on day 7 postwounding. Proliferating cell nuclear antigen and alpha-smooth muscles actin, markers for mitosis and angiogenesis, respectively, were assessed by in situ immunodetection method. RESULTS: Significant increases in the rate of wound closure and proliferating cell nuclear antigen-expressing cells were observed in AF-treated wounds when compared with that of sham and control wounds. Likewise, the number of large vessels was significantly increased in the wounds treated with the AF. However, population of myofibroblasts was not affected by the treatment. The mature and premature AF were almost equally effective. CONCLUSIONS: Our data, for the first time, show that processed AF accelerates diabetes-impaired wound healing by activating mitosis and angiogenesis, indicating that bioactive molecules in AF may endure processing. We believe that processed forms of this naturally designed "Cocktail" of bioactive molecules may have multiple clinical applications.

Effects of hypo- and hyperthyroid states on herpes simplex virus infectivity in the rat.

OBJECTIVE: Available data from in vitro studies show that thyroid hormones (THs) regulate herpes simplex virus (HSV) gene expression and may modulate latency/reactivation of the virus. Whether infectivity of the virus is also affected by THs is not known. Using animal models (in vivo study) and Vero cell culture (in vitro study), we examined the effects of alterations in THs level on HSV-1 infectivity. METHODS: Rats were rendered hypo- and hyperthyroid by daily addition of methimazole and l-thyroxine into their drinking water, respectively. Euthyroid animals served as control. All animals were given a single dose of HSV-1 (10(7)TCID50, ip) and sacrificed 3 d later. The spleen of the animals was then removed and viral particles were recovered from the tissue extract through aseptic procedures. Serial dilution of the extracts was prepared and added to Vero cell culture. For the in vitro study, the cultures were pretreated with l-thyroxine and the viral particles were then added. Virus titration was determined by Reed-Muench quantal assay. RESULTS: The viral load of spleen in hyperthyroid rats was significantly lower (1000-fold) than that of the euthyroid rats. Similarly, in vitro presence of supraphysiologic levels of l-thyroxine in the culture media of Vero cells decreased virus infectivity. Interestingly, hypothyroid animals showed a significant increase (10-fold) in spleen viral load as compared to that of their euthyroid counterparts. CONCLUSIONS: These data clearly show that the HSV-1 infectivity is affected by THs, and suggest that THs or their analogs may have a potential application in prevention and/or treatment of viral infections.

Hyperthyroid state or in vitro thyroxine treatment modulates TH1/TH2 responses during exposure to HSV-1 antigens.

Increasingly in recent years, thyroid hormones (THs) have been considered to be important regulators of the immune system. However, their roles in host defense against viral infections are not clearly established. Therefore, this study was undertaken to examine proliferative activity and cytokine production by lymphocytes isolated from hyperthyroid and euthyroid Balb/c mice in response to herpes simplex virus-1 (HSV-1). Lymphocytes of hyperthyroid animals showed a significantly higher rate of proliferation and interferon (IFN)-γ production when compared with that by lymphocytes from euthyroid mice. In vitro thyroxine (T4) treatment was similarly effective in the potentiation of proliferation, but not IFNγ production, by euthyroid lymphocytes. Furthermore, the hyperthyroid state significantly attenuated ConA-, but not HSV-1-, induced interleukin (IL)-10 release; in vitro T4 treatment synergized this effect. These findings suggest that supra-physiologic TH levels (i.e. as occur in hyper-thyroid states) or in vitro TH treatment modulate T-helper (TH)1/TH2 lymphocyte responses and thereby amplifies host defenses against viral infections. One may also conclude that THs may have a potential application in viral immunization and/or treatment of viral infections.

Isolation of adipose tissue mesenchymal stem cells without tissue destruction: a non-enzymatic method.

The conventional enzymatic method is widely used for mesenchymal stem cells (MSCs) isolation from adipose tissue. The method holds major drawbacks; it is costly, time-consuming and results in a heterogeneous cell population. Besides, digestion of extracellular matrix causes cell injury and compromise proliferation and differentiation of the cells. Also, because of over handling the samples are also prone to contamination. Here, we introduce a non-enzymatic method for MSCs isolation without disturbing the cells habitat. Small pieces of adipose tissue obtained from animal or human liposuction were explanted into a culture flask, immobilized by fetal bovine serum (FBS) and incubated overnight. The explants were then irrigated with DMEM containing FBS. Within few days, the fibroblast-like cells migrated from the tissue and proliferated rapidly. When subconfluent, the cells were harvested, expanded through 3 passages and used for immunophenotyping and differentiation assays. As judged by flow cytometric analysis of surface markers (CD44(+), CD105(+), CD34(-), CD45(-)), Oil Red O and Alizarin Red staining, the MSCs isolated by our non-enzymatic method were pluripotent and exhibited the potential for differentiation into adipocyte and osteoblast. Great isolation yields, homogeneity of isolated cells, brief procedure, and high economy are the advantages of our method over the conventional protocol.