Oxidative stress and apoptosis cell death are critical secondary damage mechanisms that lead to losing neighboring healthy tissue after cerebral ischemia. This study aims to characterize the type of interaction between dapsone (DDS) and cannabidiol (CBD) and its cytoprotective effect in an in vitro model of oxygen and glucose deprivation for 6 h followed by 24 h of reoxygenation (OGD/R), using the SH-SY5Y cell line. For the combined concentrations, an isobolographic study was designed to determine the optimal concentration-response combinations. Cell viability was evaluated by measuring the lactate dehydrogenase (LDH) release and 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assays. Also, the reactive oxygen species (ROS) and reduced glutathione (GSH) levels were analyzed as oxidative stress markers. Finally, caspase-3 activity was evaluated as a marker cell death by apoptosis. The results showed a decrease in cell viability, an increase in oxidant stress, and the activity of caspase-3 by the effect of OGD/R. Meanwhile, both DDS and CBD demonstrated antioxidant, antiapoptotic, and cytoprotective effects in a concentration-response manner. The isobolographic study indicated that the concentration of 2.5 µM of DDS plus 0.05 µM of CBD presented a synergistic effect so that in treatment, cell death due to OGD/R decreased. The findings indicate that DDS-CBD combined treatment may be a helpful therapy in cerebral ischemia with reperfusion.

UNASSIGNED: Earlier evidence suggests that extremely low frequency magnetic fields (ELF MFs) can modify the effects of carcinogenic agents. However, the studies conducted so far with ionizing radiation as the co-exposure agent are sparse and have provided inconclusive results. We investigated whether 50 Hz MFs alone, or in combination with ionizing radiation alter cell biological variables relevant to cancer and the biological effects of ionizing radiation.
UNASSIGNED: Human SH-SY5Y neuroblastoma cells were sham exposed or exposed to 100 or 500 µT MF for 24 h either before or after ionizing radiation exposure (0, 0.4 or 2 Gy). After the exposures, cells were assayed for viability, clonogenicity, reactive oxygen species, caspase-3 activity, and cell cycle distribution. Cell cycle distribution was assayed with propidium iodide staining followed by flow cytometry analysis and ROS levels were assayed together with cell viability by double staining with DeepRed and Sytox Blue followed by flow cytometry analysis.
UNASSIGNED: Increased caspase-3 activity was observed in cells exposed to 500 µT MF before or after ionizing radiation. Furthermore, exposure to the 500 µT MF after the ionizing radiation decreased the percentage of cells in S-phase. No changes in the ROS levels, clonogenicity, or viability of the cells were observed in the MF exposed groups compared to the corresponding sham exposed groups, and no MF effects were observed in cells exposed at 100 µT.
UNASSIGNED: Only the 500 µT magnetic flux density affected SH-SY5Y cells significantly. The effects were small but may nevertheless help to understand how MFs modify the effects of ionizing radiation. The increase in caspase-3 activity may not reflect effects on apoptosis, as no changes were observed in the subG1 phase of the cell cycle. In contrast to some earlier findings, 50 Hz MF exposure after ionizing radiation was not less effective than MF treatment given prior to ionizing radiation.

Cancer is one of the most important problems of modern societies. Recently, studies have reported the anticancer properties of rosiglitazone related to its ability to bind peroxisome proliferator receptor γ (PPARγ), which has various effects on cancer and can inhibit cell proliferation. In this study, we investigated the effect of new 4-thiazolidinone (4-TZD) hybrids Les-4369 and Les-3467 and their effect on reactive oxygen species (ROS) production, metabolic activity, lactate dehydrogenase (LDH) release, caspase-3 activity, and gene and protein expression in human foreskin fibroblast (BJ) cells and lung adenocarcinoma (A549) cells. The ROS production and caspase-3 activity were mainly increased in the micromolar concentrations of the studied compounds in both cell lines. Les-3467 and Les-4369 increased the mRNA expression of PPARG, P53 (tumor protein P53), and ATM (ATM serine/threonine kinase) in the BJ cells, while the mRNA expression of these genes (except PPARG) was mainly decreased in the A549 cells treated with both of the tested compounds. Our results indicate a decrease in the protein expression of AhR, PPARγ, and PARP-1 in the BJ cells exposed to 1 µM Les-3467 and Les-4369. In the A549 cells, the protein expression of AhR, PPARγ, and PARP-1 increased in the treatment with 1 µM Les-3467 and Les-4369. We have also shown the PPARγ modulatory properties of Les-3467 and Les-4369. However, both compounds prove weak anticancer properties evidenced by their action at high concentrations and non-selective effects against BJ and A549 cells.

N-acetyl-selenomethionine (NASeLM), a representative of the selenium compounds, failed to convince in clinical studies and cell cultures that it neither inhibits cancer growth nor has a chemoprotective effect. This study aims to find out whether NASeLM shows a growth-inhibiting property compared to the carrier substance N-Acetyl-L-methionine (NALM) on two different cancer cells, namely Jurkat cells and MTC-SK cells.
METHODS: Jurkat and MTC-SK cells were cultured in the absence or presence of varying concentrations (0-500 µg/mL) of NASeLM and NALM solutions. After 0, 24, 48, and 72 h, mitochondrial activity, cancer cell membrane CP levels, cell growth, and caspase-3 activity were assessed in aliquots of Jurkat and MTC-SK cells.
RESULTS: Both substances, NASeLM and NALM, were similarly able to inhibit cell growth and mitochondrial activity of Jurkat cells in a concentration-dependent and time-dependent manner up to 70%. Only the determination of caspase activity showed that only NASeLM was able to increase this to almost 40% compared to the control as well as the same lack of NALM. However, the experiments on MTC-SK cells showed a clear difference in favor of NASeLM compared to NALM. While NASeLM was able to reduce cell growth to up to 55%, the same amount of NALM was only at around 15%, which turned out to be highly significant (p < 0.001). The same could also be measured for the reduction in MTC-SK mitochondrial activity. Time dependence could also be recognized: the longer both substances, NASeLM and NALM, were incubated, the higher the effect on cell growth and mitochondrial activity, in favour of NASeLM. Only NASeLM was able to increase caspase-3 activity in MTC-SK cells: at 250 µg/mL NASeLM, caspase-3 activity increased significantly to 28% after 24 and 48 h compared to the control (14%) or the same NALM concentration (14%). After 72 h, this could still increase to 37%. A further increase in the NASeLM concentration did not result in higher caspase-3 activity.
CONCLUSIONS: NASeLM could clearly increase caspase-3 activity in both cell types, Jurkat or MTC-SK cells, and thus induce cell death. NALM and NASeLM showed a reduction in cell growth and mitochondrial activity in both cell lines: While NALM and NASeLM showed almost identical measurements on Jurkat cells, NASeLM was much more effective on MTC-SK than the non-selenium-containing carrier, indicating that it has additional anti-chemoprotective effects.

Many signaling pathways are involved in the mammalian target of rapamycin (mTOR), and this serine/threonine kinase regulates the most important cellular processes such as cell proliferation, autophagy, and apoptosis. The subject of this research was the effect of protein kinase inhibitors involved in the AKT, MEK, and mTOR kinase signaling pathways on the expression of pro-survival proteins, activity of caspase-3, proliferation, and induction of apoptosis in melanoma cells. The following inhibitors were used: protein kinase inhibitors such as AKT-MK-2206, MEK-AS-703026, mTOR-everolimus and Torkinib, as well as dual PI3K and mTOR inhibitor-BEZ-235 and Omipalisib, and mTOR1/2-OSI-027 inhibitor in single-mode and their combinations with MEK1/2 kinase inhibitor AS-703026. The obtained results confirm the synergistic effect of nanomolar concentrations of mTOR inhibitors, especially the dual PI3K and mTOR inhibitors (Omipalisib, BEZ-235) in combination with the MAP kinase inhibitor (AS-703026) in the activation of caspase 3, induction of apoptosis, and inhibition of proliferation in melanoma cell lines. Our previous and current studies confirm the importance of the mTOR signal transduction pathway in the neoplastic transformation process. Melanoma is a case of a very heterogeneous neoplasm, which causes great difficulties in treating this neoplasm in an advanced stage, and the standard approach to this topic does not bring the expected results. There is a need for research on the search for new therapeutic strategies aimed at particular groups of patients. Effect of three generations of mTOR kinase inhibitors on caspase-3 activity, apoptosis and proliferation in melanoma cell lines.

Diclazuril (DIC) is widely used in the poultry industry to control coccidiosis. However, drug resistance makes it less effective, and the underlying mechanism remains unclear. One DIC-resistant E. tenella (RE) isolate and one sensitive E. tenella (SE) isolate were used to compare the differences in their endogenous development, pathogenicity, invasion-related gene expression and apoptotic characteristics. Chickens were allocated into four groups to receive RE or SE strain and their corresponding DIC treatment or not. Caeca tissues were sampled at 96 h, 120 h and 144 h post-infection (PI) for pathological analysis. Meanwhile, second-generation merozoites (Mz2) were separated at 120 h PI to detect alterations in mitochondrial membrane potential (MMP), apoptotic rate and caspase-3 activity and mRNA expression of protein phosphatase 5 (PP5), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin depolymerizing factor (ADF) and microneme proteins (MICs). Haematoxylin and eosin staining revealed that DIC treatment strictly blocked the development of the SE strain but slightly affected the RE strain. Meanwhile, the number of SE Mz2 and their MMP decreased at the same time the apoptotic rate increased after DIC treatment. Real-time quantitative PCR and caspase-3 activity studies demonstrated that Mz2 from the RE strain had higher mRNA expression of ADF and MICs along with no significant changes in GAPDH and caspase-3 activity under DIC pressure compared to its control; in contrast, the mRNA expression of ADF, MICs and PP5 was markedly suppressed in Mz2 from SE with upregulated caspase-3 activity and GAPDH transcription. In addition, the mRNA expression of GAPDH and PP5 in Mz2 from RE was remarkably higher than that of SE. Taken together, the higher mRNA expression of invasion-related genes and almost unaffected endogenous development provide a better understanding of coccidian resistance to DIC.

Planarians are freshwater flatworms, well known for their ability to regenerate a complete organism from any piece of their body. Furthermore, planarians are constantly growing and degrowing throughout their lives, maintaining a functional and proportioned body. These properties rely on the presence of a population of adult stem cells and on the tight control of their cell renewal, which is based on the balance between the proliferation of new cells and their differentiation, and the death of unnecessary cells. Due to the importance of these two processes in planarian biology, over the years, researchers have optimized molecular techniques to detect both cell proliferation and cell death in planarians. Here, we present the two main protocols currently used for cell death detection and quantification in the planarian field: Caspase-3 activity quantification and TUNEL assay.

In biliary atresia (BA), apoptosis is part of the pathomechanism, which results in progressive liver fibrosis. There is increasing evidence suggesting that apoptotic liver injury can be non-invasively detected by measuring the caspase activity in the serum. The purpose of this study was to investigate whether serological detection of caspase activation mirrors apoptotic liver injury in the infective murine BA-model and represents a suitable biomarker for BA in humans. Analysis showed increased caspase-3 activity and apoptosis in the livers of cholestatic BALB/c mice, which correlated significantly with caspase activation in the serum. We then investigated caspase activation and apoptosis in liver tissues and sera from 26 BA patients, 23 age-matched healthy and 11 cholestatic newborns, due to other hepatopathies. Compared to healthy individuals, increased caspase activation in the liver samples of BA patients was present. Moreover, caspase-3 activity was significantly higher in sera from BA infants compared to patients with other cholestatic diseases (sensitivity 85%, specificity 91%). In conclusion, caspase activation and hepatocyte apoptosis play an important role in experimental and human BA. We demonstrated that serological detection of caspase activation represents a reliable non-invasive biomarker for monitoring disease activity in neonatal cholestatic liver diseases including BA.

With an aim to develop more effective and affordable anticancer agents possessing a unique mechanism of action, we designed and synthesized derivatives of spirooxindole-pyrrolidine heterocyclic hybrids in good yields through a one-pot three-component (3+2) cycloaddition strategy. The synthesized compounds were characterized thoroughly for the physicochemical properties by making use of FT-IR, NMR spectroscopy, and mass spectrometry. Further, these compounds have been evaluated for the influence of anticancer activity against HepG2 cells up to 200 µg/mL concentration. The highly active molecular scaffold was tested for the in-depth mechanistic studies, and it was found that the major pathway of cell death is apoptosis which occurs through the induction of reactive oxygen species followed by the involvement of caspases.

Adaptogens are substances that act nonspecifically to combat stress by regulating the key elements involved in stress-induced pathologies. D-Ribose-L-cysteine (DRLC), a potent glutathione (GSH) booster, has been recommended for relief of stress. Hence, we investigated its adaptogenic-like effect in mice subjugated to unpredictable chronic mild stress (UCMS). Thirty six male Swiss mice were assigned to 6 groups (n = 6): group 1 received saline (p.o, non-stress control), group 2 (stress-control) also had saline, groups 3 to 5 received DRLC (25, 50 and 100 mg/kg, p.o) whereas group 6 had ginseng (50 mg/kg, p.o). The animals in groups 2-6 were subjugated to UCMS 30 min later, daily for 21 days and afterwards, tested for memory and anxiety. Blood glucose, serum corticosterone concentrations and adrenal weight were determined. The brain tissues were processed for estimation of malondialdehyde (MDA), GSH, superoxide-dismutase (SOD), catalase, tumor necrosis factor-alpha (TNF-α), interleukin-6, acetyl-cholinesterase, and caspase-3 activities. The histomorphologic features and neuronal viability of the hippocampus, amygdala and prefrontal cortex were also determined. DRLC (25-100 mg/kg) reduces anxiety, memory deficit, adrenal gland enlargement, glucose, and corticosterone concentrations in UCMS-mice. The increased brain contents of MDA, TNF-α, interleukin-6, acetyl-cholinesterase and decreased antioxidant (GSH, SOD and catalase) status induced by UCMS were attenuated by DRLC. The DRLC increased caspase-3 activity and reduces histomorphological distortions of neuronal cells of the hippocampus, amygdala and prefrontal cortex of stressed-mice. These findings suggest that DRLC has adaptogenic-like effect which might be related to modulation of corticosterone-mediated oxido-inflammatory processes and altered caspase-3 activity.