Alterations of Bax/Bcl-2 ratio contribute to NaAsO2 induced thyrotoxicity in human thyroid follicular epithelial cells and SD rats.

The environmental toxicant arsenic causes various human diseases and threatens millions of people worldwide. Recently, a limited number of studies have revealed that exposure to arsenic is associated with thyroid dysfunction, indicating its toxicological impact on the thyroid gland, however, its precise forms of damage and underlying mechanisms remain largely unknown. Here, we sought to observe the thyrotoxicity of sodium arsenite (NaAsO2) on human thyroid follicular epithelial cells (Nthy-ori 3-1) and SD rats, and explore the role of Bax/Bcl-2 ratio in the above process. Our results displayed that NaAsO2 exerted a dose-dependent inhibitory effect on the viability of Nthy-ori 3-1 cells. Alongside the increase doses of NaAsO2 exposure, morphological changes and elevated LDH levels were observed. Furthermore, apoptosis rates increased in a dose- and time-dependent manner, accompanied by a decrease in Bcl-2 and an opposite change in Bax expression. SD rats were treated with 0, 2.5, 5, and 10 mg/kg NaAsO2 for 36 weeks. Our findings revealed that NaAsO2 exposure resulted in arsenic accumulation in thyroid tissue, elevated ratio of Bax/Bcl-2, and histopathological changes of thyroid in rats, which accompanied by the decreased serum T3 and T4 levels and the increased serum TSH level. Furthermore, T3 and T4 levels were negatively correlated with Bax expression, whereas positively correlated with Bcl-2 expression. Collectively, our results suggest that NaAsO2 exposure induces cytotoxicity in Nthy-ori 3-1 cells, causes structural damages and dysfunction of thyroid in SD rats, in which the imbalance of Bax/Bcl-2 ratio may play a significant role.

High-intensity interval training attenuates the effects caused by arterial hypertension in the ventral prostate.

BACKGROUND: The prostatic effects induced by arterial hypertension is very controversial and its mechanism is unclear. High-intensity interval training (HIIT) is an exercise considered to be hypotensive. The objective of this work was to investigate the molecular, biochemical, and morphological effects of 8 weeks of HIIT in the prostatic tissue of spontaneously hypertensive rats (SHR). METHODS: Twenty male SHR rats, 51.4 weeks old, were used. The SHR animals were divided into two groups: spontaneously sedentary hypertensive and spontaneously hypertensive submitted to HIIT. We analyze androgens receptor and glucocorticoid receptors in the prostate. Still, we verify effects of the hypertension and HIIT on the physiopathology prostatic, for immunohistochemistry investigated BCL-2, BAX, IGF-1, FAS/CD95, data's inflammatory tumour necrosis factor α, nuclear factor kappa B and interleukin (IL)-6, anti-inflammatory IL-10. The echocardiographic evaluation was performed at the baseline and after the training period. RESULTS: Arterial hypertension promote high prostatic intraepithelial neoplasia incidence in the prostate, increases IGF-1, BCL-2 (p < 0.05), and inflammatory proteins (p < 0.05). Eight weeks of HIIT training reduced the arterial pressure and increase the concentration of tissue collagen and intracellular glycogen and showed a higher expression of BAX, FAS/CD95, and IL-10 proteins (p < 0.05), coinciding with a lower incidence of lesions and lower prostate weight (p < 0.05) and reduction of the BCL-2 and IGF-1. CONCLUSION: Our data suggested that arterial hypertension suppressed apoptosis and increased damage prostatic. On other hand, HIIT promotes morphology and function improves in the prostatic environment, inhibited inflammation, and increased apoptosis.

Nanoliposomal formulation of pistachio hull extract: preparation, characterization and anti-cancer evaluation through Bax/Bcl2 modulation.

BACKGROUND: Pistachio is one of the main crops in Iran. Pistachio green hull, as a by-product of this fruit, is obtained in large quantities after the processing of pistachios. This novel work was designed to examine the possible anti-cancer impact of the pistachio hull extract in the liposomal form (PHEL) on HepG2 cells. METHODS AND RESULTS: The thin-film hydration approach was used for preparing liposomes and the physicochemical features of the liposomes were subsequently characterized. Afterward, apoptosis and the expression of genes related to apoptosis were assessed using flow cytometry assay and quantitative real-time polymerase chain reaction (qPCR), respectively. According to the results, the size range of PHEL was between 198 and 201 nm with a negative surface charge of - 39.2 to - 42.9 mV. As revealed by the flow cytometry results, this liposomal extract exhibits good potential for the induction of apoptosis. Moreover, the qPCR results demonstrated the up-regulation of p53 and Bax expressions and the down-regulation of Bcl-2 expression with an associated Bax/Bcl-2 ratio up-regulation. CONCLUSION: The flow cytometry and real-time PCR results indicated the potential of this liposomal extract as an anti-cancer drug candidate for the treatment of liver cancer in the future, and the mitochondrial pathway involving the up-regulation of the Bax/Bcl-2 ratio can mediate its impact.

Cellular uptake and apoptotic properties of gemini curcumin in gastric cancer cells.

INTRODUCTION: Curcumin is a polyphenolic natural compound, which has demonstrated to possess antioxidant, anti-inflammatory, and anticancer effects in vitro & in vivo. However, its applicability in cancer therapy has been limited due to its poor cellular uptake. Here, we aimed to evaluate the anticancer effect of novel gemini curcumin (Gemini-Cur) on the gastric cancer AGS cells. METHOD: The AGS cancerous and HFF-2 non-cancerous cells were treated with Gemini-Cur and curcumin (Cur) in a time- and dose-dependent manner. Cellular toxicity was studied using MTT, fluorescence microscopy, annexin V/FITC, and cell cycle assays. Additionally, real-time PCR and western blotting were employed to evaluate the expression of Bax, Bcl-2 and survivin genes. RESULTS: Our data indicated that Gemini-Cur is significantly taken into AGS cells compared to Cur. Moreover, the viability of Gemini-Cur treated cells was significantly reduced in a time- and dose-dependent manner (p < 0.001). Gemini-Cur compound induced G2/M cell cycle arrest that was followed by apoptosis in a time-dependent manner (p < 0.0001). DISCUSSION: Taken together, our findings support the idea that Gemini-Cur has the potential to be considered as an anticancer agent.

Cardioprotective effects of hesperidin on carbon monoxide poisoned in rats.

Carbon monoxide (CO) poisoning causes cardiotoxicity and so far, no definite antidote has been proposed to overcome CO-induced adverse outcomes. Hesperidin, a citrus flavonoid, has shown cardio-protective effects in cardiac ischemia/reperfusion models. This study investigated the protective effects of hesperidin against CO-induced cardiac injury. To induce CO poisoning, rats were exposed to CO at 3000 ppm for 60 min. On the exposure day and the four following days, hesperidin (at three different doses of 25, 50, and 100 mg/kg/day) was administered intraperitoneally. A group of animals received normal saline and served as the control group. The electrocardiogram (ECG) was recorded and evaluated with special focus on S-T segment changes (depression or elevation), T-wave alterations, AV block and ventricular and supraventricular arrhythmias. On day 6 (i.e., the day after the last injection day), the animals were sacrificed and the hearts were harvested and evaluated for necrosis using hematoxylin and eosin staining. In addition, Akt protein expression levels and BAX/BCL2 ratio were determined by western blotting. Our results showed that hesperidin decreased cardiac necrosis. In animals treated with hesperidin 100 mg/kg, Akt protein expression was increased, while the BAX/BCL2 ratio was significantly decreased. ECG changes were reversed in all groups 2 h following CO exposure, regardless of hesperidin administration. Overall, hesperidin decreased the deleterious cardiac effects of CO poisoning in rats.

Reactive oxygen species generation and mitochondrial dysfunction for the initiation of apoptotic cell death in human hepatocellular carcinoma HepG2 cells by a cyclic dipeptide Cyclo(-Pro-Tyr).

Cyclic dipeptides are increasingly gaining importance as considering its significant biological and pharmacological activities. This study was aimed to investigate the anticancer activity of a dipeptide Cyclo(-Pro-Tyr) (DP) identified from marine sponge Callyspongia fistularis symbiont Bacillus pumilus AMK1 and the underlying apoptotic mechanisms in the liver cancer HepG2 cell lines. MTT assay was done to demonstrate the cytotoxic effect of DP in HepG2 cells and mouse Fibroblast McCoy cells. Initially, apoptosis inducing activity of DP was identified using propidium iodide (PI) and acridine orange/ethidium bromide (AO/EB) dual staining, then it was confirmed by DNA fragmentation assay and western blotting analysis of apoptosis related markers Bax, Bcl-2, cytochrome c, caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Rhodamine 123 staining was performed to observe DP effects on the mitochondrial membrane potential (MMP) and DCFH-DA (Dichloro-dihydro-fluorescein diacetate) staining was done to measure the intracellular reactive oxygen species (ROS) levels. The MTT results revealed that DP initiated dose-dependent cytotoxicity in HepG2 cells, but no significant toxicity in mouse Fibroblast McCoy cells treated with DP at the specified concentrations. DP induced apoptosis, which is confirmed by the appearance of apoptotic bodies with PI and AO/EB dual staining, and DNA fragmentation. DP significantly elevated the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), enhanced cytochrome c release from mitochondria, increased caspase-3 activation, the cleavage of PARP and increased intracellular reactive oxygen species (ROS) levels. Besides this, DP successfully inhibited the phosphorylation of PI3K, AKT and increased PTEN expression. These results suggested DP might have anti-cancer effect by initiating apoptosis through mitochondrial dysfunction and downregulating PI3K/Akt signaling pathway in HepG2 cells with no toxicity effect on normal fibroblast cells. Therefore, DP may be developed as a potential alternative therapeutic agent for treating hepatocellular carcinoma.

Proapoptotic Index Evaluation of Two Synthetic Peptides Derived from the Coneshell Californiconus californicus in Lung Cancer Cell Line H1299.

Lung cancer is one of the most common types of cancer, accounting for approximately 15% of all cancer cases worldwide. Apoptosis is the dominant defense mechanism against tumor development. The balance between pro- and antiapoptotic members of the Bcl-2 protein family can determine cellular fate. The venom of predatory marine snails Conus is estimated to have 100-400 toxins called conotoxins. The family of α-conotoxins is known to consist of selective antagonists of nicotinic acetylcholine receptors (nAChRs). Lung cancer cells overexpress several subunits of nAChRs and are considered as an excellent target for new anticancer drugs. We compared the cytotoxic effect of two synthetic peptides derived from Californiconus californicus, Cal14.1a, and Cal14.1b, which only differ by one amino acid in their sequence, and compared their proapoptotic balance by Bax and Bcl-2 mRNA expression. We determined the caspase-3 and -7 activation to demonstrate apoptosis induction. Results showed that Cal14.1a induces a high Bax/Bcl-2 ratio in H1299 (lung cancer cells). Although Cal14.1b has a cytotoxic effect on H1299 cells, reducing cell viability by 30%, it does not increase the Bax/Bcl-2 ratio, which could be explained by the Glu in the 15th residue, which is crucial for the ability of Cal14.1a to induce apoptosis.

The Protective Effect of Insulin on Rat Cortical Neurons in Oxidative Stress and Its Dependence on the Modulation of Akt, GSK-3beta, ERK1/2, and AMPK Activities.

Insulin is a promising drug for the treatment of diseases associated with brain damage. However, the mechanism of its neuroprotective action is far from being understood. Our aim was to study the insulin-induced protection of cortical neurons in oxidative stress and its mechanism. Immunoblotting, flow cytometry, colorimetric, and fluorometric techniques were used. The insulin neuroprotection was shown to depend on insulin concentration in the nanomolar range. Insulin decreased the reactive oxygen species formation in neurons. The insulin-induced modulation of various protein kinase activities was studied at eight time-points after neuronal exposure to prooxidant (hydrogen peroxide). In prooxidant-exposed neurons, insulin increased the phosphorylation of GSK-3beta at Ser9 (thus inactivating it), which resulted from Akt activation. Insulin activated ERK1/2 in neurons 5-30 min after cell exposure to prooxidant. Hydrogen peroxide markedly activated AMPK, while it was for the first time shown that insulin inhibited it in neurons at periods of the most pronounced activation by prooxidant. Insulin normalized Bax/Bcl-2 ratio and mitochondrial membrane potential in neurons in oxidative stress. The inhibitors of the PI3K/Akt and MEK1/2/ERK1/2 signaling pathways and the AMPK activator reduced the neuroprotective effect of insulin. Thus, the protective action of insulin on cortical neurons in oxidative stress appear to be realized to a large extent through activation of Akt and ERK1/2, GSK-3beta inactivation, and inhibition of AMPK activity increased by neuronal exposure to prooxidant.

Synthesis, Anticancer Activity, and Apoptosis Induction of Novel 3,6-Diazaphenothiazines.

New 10-substituted derivatives of 3,6-diazaphenothiazine, containing the triple bond linker terminated with tertiary cyclic and acyclic amine groups, were synthesized and screened for their anticancer action. The compounds exhibited varied anticancer activities against human glioblastoma SNB-19, melanoma C-32, and breast cancer MDA-MB231 cell lines, depending on the nature of the substituents. The most active 3,6-diazaphenothiazine, 4, was the derivative with the N,N-diethylamino-2-butynyl substituent against glioblastoma SNB-19, and was ten times more potent than cisplatin. For this compound, the expression of H3, TP53, CDKN1A, BCL-2, and BAX genes was detected by the RT-qPCR method. The gene expression ratio BAX/BCL-2 indicated the induction of mitochondrial apoptosis in cancer cell lines. The transformation of the propynyl substituent into amino-2-butynyl can be a method applicable to the search for more anticancer-active azaphenothiazines.

Design, Synthesis, and Structural Characterization of Novel Diazaphenothiazines with 1,2,3-Triazole Substituents as Promising Antiproliferative Agents.

A series of novel 1,2,3-triazole-diazphenothiazine hybrids was designed, synthesized, and evaluated for anticancer activity against four selected human tumor cell lines (SNB-19, Caco-2, A549, and MDA-MB231). The majority of the synthesized compounds exhibited significant potent activity against the investigated cell lines. Among them, compounds 1d and 4c showed excellent broad spectrum anticancer activity, with IC50 values ranging from 0.25 to 4.66 µM and 0.25 to 6.25 µM, respectively. The most promising compound 1d, possessing low cytotoxicity against normal human fibroblasts NHFF, was used for gene expression analysis using reverse transcription-quantitative real-time PCR (RT-qPCR). The expression of H3, TP53, CDKN1A, BCL-2, and BAX genes revealed that these compounds inhibited the proliferation in all cells (H3) and activated mitochondrial events of apoptosis (BAX/BCL-2).

Supplementation of Adiantum capillus-veneris Modulates Alveolar Apoptosis under Hypoxia Condition in Wistar Rats Exposed to Exercise.

Background and Objectives: Several studies have reported that some conditions such as exercise and hypoxia induce DNA damage and dysfunction and apoptosis. Some plant foods contain numerous bioactive compounds and anti-inflammatory properties that can help fight DNA damage. Therefore, the current study evaluated the effect of supplementation of Adiantum capillus-veneris (ACV) extract on Bax/B-cell lymphoma 2 (Bcl-2) ratio apoptotic index and remodeling of pulmonary alveolar epithelial cells in lung tissue of healthy Wistar rats during stressful conditions (hypoxia). Materials and Methods: Twenty-seven Wistar male rats (four-week old, 72 ± 9 g) were randomly assigned into three groups: normoxic, sedentary, and not-supplemented (NG, n = 9); exercise and hypoxia and not-supplemented (HE, n = 9); and exercise and hypoxia and supplemented group (HS, n = 9). The NG remained sedentary in the normoxia environment for nine weeks. The HE group participated in a high-intensity (IT) program for six weeks, then remained sedentary in the hypoxia environment for three weeks. The low-pressure chamber simulated a ~2800 M altitude 24 h/d. HS participated in IT, then entered and remained sedentary in the hypoxia environment for three weeks, and they consumed 500 mg per kg of body weight ACV extract. Results: The Bax/Bcl-2 ratio of the HE group increased significantly (+50.27%, p ≤ 0.05), the average number of type I pneumocytes was reduced significantly (-18.85%, p ≤ 0.05), and the average number of type II pneumocytes was increased significantly (+14.69%, p ≤ 0.05). Also, after three weeks of consuming the ACV extract, the HS group in comparison with the HE group had their Bax/Bcl-2 ratio reduced significantly (-24.27%, p ≤ 0.05), the average number of type I pneumocytes increased significantly (+10.15%, p ≤ 0.05), and the average number of type II pneumocytes reduced significantly (-7.18%, p ≤ 0.05). Conclusion: The findings show that after three weeks of hypoxia following six weeks of high-intensity interval training in Wistar rats, the Bax/Bcl-2 ratio and the number of type II pneumocytes were increased and the number of type I pneumocytes was reduced significantly. These results strongly suggest that an apoptosis state was induced in the lung parenchyma, and consuming ACV extract modulated this state.

Pleurotus highking Mushroom Induces Apoptosis by Altering the Balance of Proapoptotic and Antiapoptotic Genes in Breast Cancer Cells and Inhibits Tumor Sphere Formation.

Background and objectives: Mushrooms that have medicinal properties are part of many traditional diets. The aim of the present study was to use the human breast cancer cell line MCF-7 to investigate the anticancer activity of Pleurotus highking mushroom purified extract fraction-III (PEF-III) and to elucidate the possible mechanism of that activity. Materials and Methods: The effects of PEF-III on cell proliferation and viability were evaluated by a colony formation assay and an MTT assay, respectively. Cell morphological changes, annexin-V phycoerythrin and propidium iodide (PI) staining, DNA fragmentation, and caspase 3/7 activity assays were performed to determine the induction of apoptosis by PEF-III. The genes responsible for regulation of apoptosis were analyzed by means of Western blot analysis. In vitro tumor sphere formation assay was performed using a 3D sphere culture system. Results: PEF-III significantly reduced the proliferation and viability of MCF-7 cells. Cell shrinkage and rounding, and annexin-V phycoerythrin and PI staining followed by flow cytometry indicated that the cell death was due to apoptosis. Additionally, a laddering DNA pattern and increased levels of caspase-3/7 enzyme also corroborated the notion of apoptosis-mediated cell death. This incidence was further confirmed by upregulation of proapoptotic genes (p53 and its target gene, Bax) and downregulation of the expression of an antiapoptotic gene (Bcl-2). PEF-III also reduced the size and number of the tumor spheres in 3D culture conditions. Conclusions: The anticancer activity of PEF-III is due to induction of apoptosis by a shift in the balance of proapoptotic and antiapoptotic genes. Therefore, the findings of the present study may open a path to exploring potential drug candidates from the P. highking mushroom for combating breast cancer.

Ginsenoside Rb1 protects against spinal cord ischemia-reperfusion injury in rats by downregulating the Bax/Bcl-2 ratio and caspase-3 and p-Ask-1 levels.

The aim of this study was to confirm the effects of ginsenoside Rb1 on neural cell apoptosis in the spinal cord of rats with spinal cord ischemia-reperfusion injury (SCII) and to explore its potential mechanisms. A total of 100 healthy adult Sprague-Dawley (SD) rats were randomly divided into four groups: normal control (n = 10), sham-operated (n = 10), SCII model (n = 40), and ginsenoside Rb1-treated groups (n = 40). Basso, Beattie, Bresnahan (BBB) scale was used to examine rat hindlimb locomotor function. Nissl and Tunnel staining were used to observe neural cell injury and apoptosis, respectively, in the spinal cord of rats with SCII. Immunofluorescence staining was performed to detect the expression of Bax and Bcl-2. The levels of caspase-3 and phosphorylated Ask-1 (p-Ask-1) were detected by western blotting. Ginsenoside Rb1 prevented neural cell apoptosis in the spinal cord and improved hindlimb locomotor dysfunction of rats (P < .05). Moreover, SCII-induced upregulation of caspase-3 and p-Ask-1 levels and the Bax/Bcl-2 ratio were significantly decreased by ginsenoside Rb1 (P < .05). The protective effects of ginsenoside Rb1 on neural cells in the spinal cord of rats with SCII were mediated by the ginsenoside Rb1-induced downregulation of caspase-3 and p-Ask-1 levels and the Bax/Bcl-2 ratio.

Low spontaneous apoptosis index at diagnosis predicts a high-risk phenotype in paediatric acute lymphoblastic leukaemia.

Background & objectives: Significance of apoptosis as a prognostic marker is less well studied in paediatric acute lymphoblastic leukaemia (ALL) cases. Hence, a prospective study, involving 30 paediatric ALL cases, was done to assess the clinical relevance of in vivo apoptosis. Methods: Peripheral blood mononuclear cells from all patients were subjected to annexin V/propidium iodide staining to detect the degree of apoptosis [apoptotic index (AI)] at day 0 and day 35 post-induction chemotherapy. In addition, Bax and Bcl2 apoptotic protein expressions were studied at day 0 and their relative fluorescence mean intensity (RFMI) ratios were calculated. Results: Mean age of patients was 5.1 years. Of the 30 cases, 21 (70%) were at standard-risk, five (17%) at intermediate and four (13%) at high risk. Majority (83%) were B-ALL. Day 8 absolute blast count was >1000/µl in seven (23%) and <1000/µl in 23 of 30 (77%) cases. Day 35 marrow was M1 in 23 (92%) and M2 in two of 25 (8%) cases. AI at day 0 and day 35 ranged from 0.9 to16.6 per cent and 1.4 to 62.8 per cent with a mean of 5.90 and 19.64 per cent, respectively. The Bax/Bcl2 ratio ranged from 0.2 to 3.5 with a mean of 0.83. The ratio was predominantly anti-apoptotic, i.e. <1 (77%). A significant association was noted between low AI at day 0 and high total leucocyte count (P=0.02), T-cell phenotype (P=0.043) and high-risk as per NCI category (P=0.025). Significant increase (>30%) in day 35 AI was seen in only six cases. Interpretation & conclusions: Our study showed that low AI at day 0 was associated with a high-risk clinical phenotype in paediatric ALL. However, studies on larger group, especially with longer follow up or study of relapse cases, will help draw conclusions regarding apoptosis assessment in paediatric ALL.

Neuro-Protective Effects of Resveratrol on Carbon Monoxide-Induced Toxicity in Male Rats.

Acute carbon monoxide (CO) poisoning causes neurotoxicity through induction of necrosis, apoptosis, lipid peroxidation and oxidative stress. Resveratrol (RES) is a natural polyphenolic phytoalexin that exhibits neuroprotective effects in ischemia/reperfusion due to its anti-apoptotic, anti-necrotic and strong anti-oxidant properties as well as its ability to activate pro-survival pathways. In this study, rats were exposed to CO 3000 ppm for 1 h. Immediately after poisoning and on the next four consecutive days, RES (1, 5 and 10 mg/kg) was administered intraperitoneally. On the fifth day, animals' brains were excised, and necrosis, lipid peroxidation level and the level of Akt, BAX and BCL2 expression were evaluated. The results showed that RES 10 mg/kg significantly reduced lipid peroxidation, but RES 1 and 5 mg/kg had no significant effect on this parameter. Furthermore, RES 5 and 10 mg/kg significantly increased Akt expression level, while BAX/BCL2 ratio was reduced by RES 1, 5 and 10 mg/kg. Moreover, RES reduced necrotic foci in the brain, but the best results were seen following treatment with RES 10 mg/kg. In summary, RES showed neuroprotective effect in CO-poisoned rats as it decreased necrosis and BAX/BCL2 ratio and increased Akt expression levels. Copyright © 2017 John Wiley & Sons, Ltd.

α-Tocopherol at Nanomolar Concentration Protects Cortical Neurons against Oxidative Stress.

The aim of the present work is to study the mechanism of the α-tocopherol (α-T) protective action at nanomolar and micromolar concentrations against H2O2-induced brain cortical neuron death. The mechanism of α-T action on neurons at its nanomolar concentrations characteristic for brain extracellular space has not been practically studied yet. Preincubation with nanomolar and micromolar α-T for 18 h was found to increase the viability of cortical neurons exposed to H2O2; α-T effect was concentration-dependent in the nanomolar range. However, preincubation with nanomolar α-T for 30 min was not effective. Nanomolar and micromolar α-T decreased the reactive oxygen species accumulation induced in cortical neurons by the prooxidant. Using immunoblotting it was shown that preincubation with α-T at nanomolar and micromolar concentrations for 18 h prevented Akt inactivation and decreased PKCδ activation induced in cortical neurons by H2O2. α-T prevented the ERK1/2 sustained activation during 24 h caused by H2O2. α-T at nanomolar and micromolar concentrations prevented a great increase of the proapoptotic to antiapoptotic proteins (Bax/Bcl-2) ratio, elicited by neuron exposure to H2O2. The similar neuron protection mechanism by nanomolar and micromolar α-T suggests that a "more is better" approach to patients' supplementation with vitamin E or α-T is not reasonable.

Inhibitory Effects of Probiotic Lactobacillus on the Growth of Human Colonic Carcinoma Cell Line HT-29.

This study was conducted to investigate the inhibitory effect of Lactobacillus cells and supernatants on the growth of the human colon cancer cell line HT-29. Our study results indicated that the PM153 strain exhibits the best adhesion ability and the highest survival in the gastrointestinal tract simulation experiment. Furthermore, after an 8-h co-culture of PM153 and HT-29 cells, the PM153 strain can induce the secretion of nitric oxide from the HT-29 cells. In addition, after the co-culture of the BCRC17010 strain (108 cfu/mL) and HT-29 cells, the Bax/Bcl-2 ratio in the HT-29 cells was 1.19, which showed a significant difference from the other control and LAB groups (p < 0.05), which therefore led to the inference that the BCRC17010 strain exerts a pro-apoptotic effect on the HT-29 cells. Upon co-culture with HT-29 cells for 4, 8 and 12 h, the BCRC14625 strain (108 cfu/mL) demonstrated a significant increase in lactate dehydrogenase (LDH) activity (p < 0.05), causing harm to the HT-29 cell membrane; further, after an 8-h co-culture with the HT-29 cells, it induced the secretion of nitric oxide (NO) from the HT-29 cells. Some lactic acid bacteria (LAB) strains have ability to inhibit the growth of the colorectal cancer cell line HT-29 Bax/Bcl-2 pathway or NO production. In summary, we demonstrated that the BCRC17010 strain, good abilities of adhesion and increased LDH release, was the best probiotic potential for inhibition of HT-29 growth amongst the seven LAB strains tested in vitro.

Negative impact of AQP-4 channel inhibition on survival of retinal ganglion cells and glutamate metabolism after crushing optic nerve.

The purpose of this study was to determine whether inhibition of aquaporin 4 (AQP4) is neuroprotective or neurodestructive after crushing the optic nerve of rats. The left optic nerves of rats were crushed, and TGN-020 (5.0 mg/kg, crush TGN-020) or its vehicle (DMSO, crush placebo) was injected intraperitoneally just after the crushing. As controls, the left optic nerves were exposed but not touched in other rats (sham controls). The retinal damages were determined by the density of retinal ganglion cells (RGCs) and the ratio of BAX/Bcl-2 on day 7. The glutamate level in the optic nerve on day 1 after the crushing was determined. The expressions of glutamine synthetase, glutamate-aspartate transporter (GLAST), and AQP4 were determined on day 3 by immunoblotting. The effects of AQP4 inhibition on the glutamate-induced changes of AQP4 expression and on the glutamate uptake were determined for optic nerve astrocytes in culture. The results showed that the density of RGCs was 2040 ± 91.3 cells/mm(2) (n = 6) in the sham control, and it was significantly decreased to 1072 ± 134.3 cells/mm(2) after crushing the optic nerve (P < 0.0001, crush placebo, n = 7; Fisher). An intraperitoneal injection of TGN-020 led to a further significant (P = 0.02, Fisher) decrease of the density of RGCs to 743 ± 371 cells/mm(2) (crush TGN-020, n = 7). The mRNA level of BAX/Bcl-2 ratio was 0.37 ± 0.05 in the sham control (n = 6) which was significantly increased to 0.88 ± 0.10 after crushing the optic nerve (placebo crush, n = 7; P = 0.0001, Scheffe). TGN-020 also significantly increased the BAX/Bcl-2 ratio to 1.29 ± 0.4 (n = 6) from the crush placebo group (P = 0.04, Scheffe). Immunoblotting showed similar changes in the protein levels. The glutamate level in the optic nerve was significantly increased to 53.7 ± 6.0 µM/mg/protein on day 1 (n = 4) from the sham control level of 45.9 ± 3.1 µM/mg/protein (n = 4; P = 0.04, t test). TGN-020 significantly (P < 0.05, Scheffe) depressed the expression of glutamate metabolism-related proteins on day 3. Exposure of cultured optic nerve astrocytes to glutamate (1.0 mM, n = 4) significantly increased the expression of AQP4 (P < 0.001, Scheffe) that was depressed by TGN-020 (100 nM, n = 4). In addition, glutamate uptake was inhibited by TGN-020 at 10 nM or higher. These results indicate that an inhibition of AQP4 enhances the loss of RGCs and retinal damages after crushing the optic nerve. Inhibition of AQP4 impairs glutamate metabolism which may account in part for these neurodestructive events.

The effects of pyrrolo[1,2-b][1,2,5]benzothiadiazepines in MEC1 cells.

Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia and is currently incurable. To expand the therapeutic armamentarium, we investigated antitumor activity of pyrrolo[1,2-b][1,2,5]benzothiadiazepine (PBTDs) in MEC1 cells. We found that PBTD (RS2778) treatment enhanced the activation of pro-apoptotic members, such as caspase-9, 3, poly (ADP-ribose) polymerase (PARP), and bax, but suppressed the activation of anti-apoptotic molecule BCL-2 in these cells. Furthermore, PBTD (RS2778)-induced autophagic cell death was verified by LC3-II conversion, and upregulation of Beclin-1 and ATG5. In addition, such compound impeded hyper phosphorylation of AKT as were determined by Western blot. In summary, PBTD (RS2778) inhibited viability and induced multiple cellular events including apoptosis, autophagic cell death, in human MEC1 cells. This distinct activity of PBTD (RS2778) against these cells suggests potential for PBTDs as a therapeutic agent for treatment of CLL.

Extremely low-frequency electromagnetic fields enhance the survival of newborn neurons in the mouse hippocampus.

In recent years, much effort has been devoted to identifying stimuli capable of enhancing adult neurogenesis, a process that generates new neurons throughout life, and that appears to be dysfunctional in the senescent brain and in several neuropsychiatric and neurodegenerative diseases. We previously reported that in vivo exposure to extremely low-frequency electromagnetic fields (ELFEFs) promotes the proliferation and neuronal differentiation of hippocampal neural stem cells (NSCs) that functionally integrate in the dentate gyrus. Here, we extended our studies to specifically assess the influence of ELFEFs on hippocampal newborn cell survival, which is a very critical issue in adult neurogenesis regulation. Mice were injected with 5-bromo-2'-deoxyuridine (BrdU) to label newborn cells, and were exposed to ELFEFs 9 days later, when the most dramatic decrease in the number of newly generated neurons occurs. The results showed that ELFEF exposure (3.5 h/day for 6 days) enhanced newborn neuron survival as documented by double staining for BrdU and doublecortin, to identify immature neurons, or NeuN labeling of mature neurons. The effects of ELFEFs were associated with enhanced spatial learning and memory. In an in vitro model of hippocampal NSCs, ELFEFs exerted their pro-survival action by rescuing differentiating neurons from apoptotic cell death. Western immunoblot assay revealed reduced expression of the pro-apoptotic protein Bax, and increased levels of the anti-apoptotic protein Bcl-2, in the hippocampi of ELFEF-exposed mice as well as in ELFEF-exposed NSC cultures, as compared with their sham-exposed counterparts. Our results may have clinical implications for the treatment of impaired neurogenesis associated with brain aging and neurodegenerative diseases.