Apoptotic Cell Death Pathways Research Articles

Structural Rationale towards Designing Coordination Polymer Based Metallogels Displaying Anti‐Cancer and Anti‐Bacterial Properties

AbstractA new series of organic‐inorganic‐hybrid‐systems (OIHS) was synthesized with the aim of developing metallogels that might display ‘drug‐like’ function. Reaction of two dicarboxylate ligands namely 3‐carboxycinnamic acid (3‐cca) and 4‐carboxycinnamic acid (4‐cca) with various bivalent metal ions (Cu(II), Zn(II), Co(II), Cd(II), Mn(II)) produced a new series of OIHS which were crystallographically characterized; five of which were coordination polymers (CP1‐CP5) and one metal‐organic polyhedra (MOP‐1). Reaction of 3‐cca with various metal ions in presence of DMSO/water produced five metallogels (MG1‐MG5) of which the Zn(II)‐metallogel namely MG1 was evaluated for both anti‐cancer and anti‐bacterial properties against murine tumor cell line B16‐F10 (human skin cancer model) and gram positive bacterium S. aureus. While MTT and cell migration assays established the anti‐cancer activity of MG1 against B16‐F10, flow cytometry and laser scanning confocal microscopy (LSCM) images under various staining conditions supported apoptotic pathway of cancer cell death. Anti‐bacterial ability of MG1 was also established against S. aureus by zone inhibition, and flow cytometry and LSCM studies under various staining conditions. MG1 was also demonstrated to be effective in killing the intracellular bacterial cells when B16‐F10 cells were infected with S. aureus thereby suggesting its potential in treating post‐operative cancer surgery model system.

Inhibition of the Anti-Apoptotic Bcl-2 Family by BH3 Mimetics Sensitize the Mitochondrial Permeability Transition Pore Through Bax and Bak.

Mitochondrial permeability transition pore (MPTP)-dependent necrosis contributes to numerous pathologies in the heart, brain, and skeletal muscle. The MPTP is a non-selective pore in the inner mitochondrial membrane that is triggered by high levels of matrix Ca2+, and sustained opening leads to mitochondrial dysfunction. Although the MPTP is defined by an increase in inner mitochondrial membrane permeability, the expression of pro-apoptotic Bcl-2 family members, Bax and Bak localization to the outer mitochondrial membrane is required for MPTP-dependent mitochondrial dysfunction and subsequent necrotic cell death. Contrary to the role of Bax and Bak in apoptosis, which is dependent on their oligomerization, MPTP-dependent necrosis does not require oligomerization as monomeric/inactive forms of Bax and Bak can facilitate mitochondrial dysfunction. However, the relationship between Bax and Bak activation/oligomerization and MPTP sensitization remains to be explored. Here, we use a combination of in vitro and ex vivo approaches to determine the role of the anti-apoptotic Bcl-2 family members, which regulate Bax/Bak activity, in necrotic cell death and MPTP sensitivity. To study the role of each predominantly expressed anti-apoptotic Bcl-2 family member (i.e., Mcl-1, Bcl-2, and Bcl-xL) in MPTP regulation, we utilize various BH3 mimetics that specifically bind to and inhibit each. We determined that the inhibition of each anti-apoptotic Bcl-2 family member lowers mitochondrial calcium retention capacity and sensitizes MPTP opening. Furthermore, the inhibition of each Bcl-2 family member exacerbates both apoptotic and necrotic cell death in vitro in a Bax/Bak-dependent manner. Our findings suggests that mitochondrial Ca2+ retention capacity and MPTP sensitivity is influenced by Bax/Bak activation/oligomerization on the outer mitochondrial membrane, providing further evidence of the crosstalk between the apoptotic and necrotic cell death pathways.

AIM2 inflammasome mediates hallmark neuropathological alterations and cognitive impairment in a mouse model of vascular dementia

Chronic cerebral hypoperfusion is associated with vascular dementia (VaD). Cerebral hypoperfusion may initiate complex molecular and cellular inflammatory pathways that contribute to long term cognitive impairment and memory loss. Inflammasome is an intracellular multi-protein complex that initiates an innate immune response, and is involved in multiple acute and chronic neurological diseases such as ischemic stroke, Alzheimer's disease (AD), Parkinson's Disease (PD), and amyotrophic lateral sclerosis (ALS). While evidence for direct involvement of the inflammasome complex in VaD is lacking, a cytokine profile of plasma from VaD patients found IL-1β to be the most abundant.Here we used a bilateral common carotid artery stenosis (BCAS) mouse model of VaD to investigate the effect of chronic cerebral hypoperfusion on the inflammasome signaling pathway. To further examine the functional role of AIM2 inflammasome activation on injury following chronic cerebral hypoperfusion, mice with AIM2 deficiency (AIM2 KO) were studied.Comprehensive analyses revealed that chronic cerebral hypoperfusion induces a complex temporal expression and activation of inflammasome components and their products (IL-1β and IL-18) in different brain regions, and promotes activation of apoptotic and pyroptotic cell death pathways. Glial cell activation, white matter lesion formation and hippocampal neuronal loss also occurred in a spatiotemporal manner. Moreover, in AIM2 KO mice we observed attenuated inflammasome-mediated production of proinflammatory cytokines, apoptosis and pyroptosis, as well as resistance to chronic microglial activation, myelin breakdown, hippocampal neuronal loss, and behavioural and cognitive deficits following BCAS.Hence, we have demonstrated that activation of the AIM2 inflammasome substantially contributes to the pathophysiology of chronic cerebral hypoperfusion-induced brain injury and may therefore represent a promising therapeutic target for attenuating cognitive impairment in VaD.

Identification and Validation of Afatinib Potential Drug Resistance Gene BIRC5 in Non-Small Cell Lung Cancer.

IntroductionResistance to second-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), afatinib, is the most significant challenge in the clinical management of non-small cell lung cancer (NSCLC), and the underlying mechanisms remain unclear.MethodsGenomic signatures that may confer afatinib resistance in NSCLC were identified via data mining of public databases and integrative bioinformatic analyses. Furthermore, acquired afatinib-resistant lung adenocarcinoma cell lines (HCC827 AR) were established by long-term exposure under afatinib in vitro for stepwise escalation. The expression of baculovirus IAP repeat protein 5 (BIRC5) was detected by western blot, and cellular viability of HCC827 AR was determined by CCK8.ResultsThrough integrative bioinformatic analyses of public datasets, overexpression of baculovirus IAP repeat protein 5 (BIRC5) was identified in both afatinib-resistant NSCLC cells and tissues, and BIRC5 overexpression was positively correlated with lymph node metastasis as well as pathological stage in NSCLC. Furthermore, NSCLC patients with BIRC5 overexpression showed poor survival outcomes. Immune infiltration analysis suggested that BIRC5 expression was significantly inversely correlated with tumor-infiltrating cell numbers and immune biomarker expression in NSCLC. The functions of genes co-expressed with BIRC5 were mainly enriched in cell cycle mitotic phase transition, double-strand break repair, and negative regulation of the cell cycle process signaling pathway. In addition, overexpression of BIRC5 protein was detected in afatinib-resistant cells by western blot, while BIRC5-expressing cells treated with BIRC5 inhibitor, YM155, were sensitive to afatinib.ConclusionsIn this study, we showed that overexpression of BIRC5 resulted in resistance to afatinib in NSCLC and BIRC5-specific inhibitors may overcome the resistant phenotype, indicating that dysregulation of the apoptotic cell death pathway may be the key mechanism underlying TKI resistance in the development of NSCLC.

Antiproliferative and Antimicrobial Potentials of a Lectin from Aplysia kurodai (Sea Hare) Eggs.

In recent years, there has been considerable interest in lectins from marine invertebrates. In this study, the biological activities of a lectin protein isolated from the eggs of Sea hare (Aplysia kurodai) were evaluated. The 40 kDa Aplysia kurodai egg lectin (or AKL-40) binds to D-galacturonic acid and D-galactose sugars similar to previously purified isotypes with various molecular weights (32/30 and 16 kDa). The N-terminal sequence of AKL-40 was similar to other sea hare egg lectins. The lectin was shown to be moderately toxic to brine shrimp nauplii, with an LC50 value of 63.63 µg/mL. It agglutinated Ehrlich ascites carcinoma cells and reduced their growth, up to 58.3% in vivo when injected into Swiss albino mice at a rate of 2 mg/kg/day. The morphology of these cells apparently changed due to AKL-40, while the expression of apoptosis-related genes (p53, Bax, and Bcl-XL) suggested a possible apoptotic pathway of cell death. AKL-40 also inhibited the growth of human erythroleukemia cells, probably via activating the MAPK/ERK pathway, but did not affect human B-lymphoma cells (Raji) or rat basophilic leukemia cells (RBL-1). In vitro, lectin suppressed the growth of Ehrlich ascites carcinoma and U937 cells by 37.9% and 31.8%, respectively. Along with strong antifungal activity against Talaromyces verruculosus, AKL showed antibacterial activity against Staphylococcus aureus, Shigella sonnei, and Bacillus cereus whereas the growth of Escherichia coli was not affected by the lectin. This study explores the antiproliferative and antimicrobial potentials of AKL as well as its involvement in embryo defense of sea hare.

Dependence of mitochondrial function on the filamentous actin cytoskeleton in cultured mesenchymal stem cells treated with cytochalasin B

Owing to their self-renewal and multi-lineage differentiation capability, mesenchymal stem cells (MSCs) hold enormous potential in regenerative medicine. A prerequisite for a successful MSC therapy is the rigorous investigation of their function after invitro cultivation. Damages introduced to mitochondria during cultivation adversely affect MSCs function and can determine their fate. While it has been shown that microtubules and vimentin intermediate filaments are important for mitochondrial dynamics and active mitochondrial transport within the cytoplasm of MSCs, the role of filamentous actin in this process has not been fully understood yet. To gain a deeper understanding of the interdependence between mitochondrial function and the cytoskeleton, we applied cytochalasin B to disturb the filamentous actin-based cytoskeleton of MSCs. In this study we combined conventional functional assays with a state-of-the-art oxygen sensor-integrated microfluidic device to investigate mitochondrial function. We demonstrated that cytochalasin B treatment at a dose of 16μM led to a decrease in cell viability with high mitochondrial membrane potential, increased oxygen consumption rate, disturbed fusion and fission balance, nuclear extrusion and perinuclear accumulation of mitochondria. Treatment of MSCs for 48h ultimately led to nuclear fragmentation, and activation of the intrinsic pathway of apoptotic cell death. Importantly, we could show that mitochondrial function of MSCs can efficiently recover from the damage to the filamentous actin-based cytoskeleton over a period of 24h. As a result of our study, a causative connection between the filamentous actin-based cytoskeleton and mitochondrial dynamics was demonstrated.

Trypan Blue - Adapting a Dye Used for Labelling Dead Cells to Visualize Pinocytosis in Viable Cells.

Trypan blue is routinely used in cell culture experiments to distinguish between dead cells, which are labelled by trypan blue, and viable cells, which are apparently free of any staining. The assumption that trypan blue labelling is restricted to dead cells derives from the observation that rupture of the plasma membrane correlates with intense trypan blue staining. However, decades ago, trypan blue has been used to trace fluid uptake by viable macrophage-like cells in animals. These studies contributed to the concept of the reticuloendothelial system in vertebrates. Trypan blue itself does not show a fluorescence signal, but trypan blue-labelled proteins do. Therefore, intracellular localization of trypan blue-labelled proteins could give a clue to the entrance pathway of the dye in viable cells. We used fluorescence microscopy to visualize trypan blue positive structures and to evaluate whether the bactericide, silver, enhances cellular trypan blue uptake in the brain macrophage-like cell line, BV-2. The pattern of chromatin condensation, visualized by DAPI staining, was used to identify the cell death pathway. We observed that silver nitrate at elevated concentrations (≥ 10 µM) induced in most cells a necrotic cell death pathway. Necrotic cells, identified by pycnotic nuclei, showed an intense and homogenous trypan blue staining. Apoptotic cells, characterized by crescent-like nuclear chromatin condensations, were not labelled by trypan blue. At lower silver nitrate concentrations, most cells were viable, but they showed trypan blue labelling. Viable cells showed a cell-type specific distribution of heterochromatin and revealed a perinuclear accumulation of bright trypan blue-labelled vesicles and, occasionally, a faint homogenous trypan blue labelling of the cytoplasm and nucleus. Amiloride, which prevents macropinocytosis by blocking the Na+ / H+ exchange, suppressed perinuclear accumulation of dye-labelled vesicles. Swelling of cells in a hypotonic solution induced an intense intracellular accumulation of trypan blue. Cells exposed to a hypotonic solution in the presence of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), which blocks volume-regulated ion channels, prevented labelling of the cytoplasm and nucleus but did not affect labelling of perinuclear vesicles. In viable cells trypan blue-labelled vesicles indicate trypan blue uptake by macropinocytosis and trypan blue-labelled cytosol could indicate a further entry pathway for the dye, like activated volume-regulated channels. Accordingly, fluorescence microscopic analysis of trypan blue-labelled cells allows not only a discrimination between necrotic and apoptotic cell death pathway but also a discrimination between the mode of trypan blue uptake in viable cells - via pinocytosis or via activated volume-regulated ion channels - in the same preparation at the single cell level.

Intermittent fasting attenuates inflammasome-associated apoptotic and pyroptotic death in the brain following chronic hypoperfusion

Chronic cerebral hypoperfusion (CCH) has been shown to initiate several inflammatory pathways that can contribute to cognitive deficits and memory loss in vascular cognitive impairment (VCI). Multi-protein complexes termed inflammasomes that may be involved in the inflammatory response to CCH has already been shown to contribute to the inflammatory process and cell death following acute cerebral ischemia. Intermittent fasting (IF) has already been shown to decrease inflammasome activation and protect the brain from ischemic stroke; however, its effects during CCH remains unknown. The present study investigated the impact of IF (16 h of food deprivation daily) for four months on inflammasome-mediated cell death in the cerebellum following CCH in a mouse model of VCI using fourteen to sixteen-week-old male C57BL/6NTac mice. Here we demonstrated that IF decreased inflammasome activation, and initiation of apoptotic and pyroptotic cell death pathways as reflected by the reduction (20–30%) in the expression levels of key effector proteins and cell death markers in the cerebellum following CCH. In summary, our results indicate that IF can attenuate the inflammatory response and cell death pathways in the brain following chronic hypoperfusion in a mouse model of VCI.

Photocytotoxicity of Thiophene- and Bithiophene-Dipicolinato Luminescent Lanthanide Complexes.

New thiophene-dipicolinato-based compounds, K2nTdpa (n = 1, 2), were isolated. Their anions are sensitizers of lanthanide ion (LnIII) luminescence and singlet oxygen generation (1O2). Emission in the visible and near-infrared regions was observed for the LnIII complexes with efficiencies (ϕLn) ϕEu = 33% and ϕYb = 0.31% for 1Tdpa2- and ϕYb = 0.07% for 2Tdpa2-. The latter does not sensitize EuIII emission. Fluorescence imaging of HeLa live cells incubated with K3[Eu(1Tdpa)3] indicates that the complex permeates the cell membrane and localizes in the mitochondria. All complexes generate 1O2 in solution with efficiencies () as high as 13 and 23% for the GdIII complexes of 1Tdpa2- and 2Tdpa2-, respectively. [Ln(nTdpa)3]3- (n = 1, 2) are phototoxic to HeLa cells when irradiated with UV light with IC50 values as low as 4.2 μM for [Gd(2Tdpa)3]3- and 91.8 μM for [Eu(1Tdpa)3]3-. Flow cytometric analyses indicate both apoptotic and necrotic cell death pathways.

Cannabinoids pharmacological effects are beyond the palliative effects: CB2 cannabinoid receptor agonist induced cytotoxicity and apoptosis in human colorectal cancer cells (HT-29)

Colorectal cancer (CRC) is between the top three occurring cancers worldwide. The anticancer effects of Cannabinoid receptor 2 (CB2) agonist (GW833972A) in the presence and absence of its inverse agonist (SR144528) on Human colorectal adenocarcinoma cells (HT-29) was investigated. Following cell viability assays on HT-29 and HFF cells, the molecular mechanism(s) of cytotoxicity and apoptotic pathways of cell death were analyzed. The anticancer effects of CB2 agonist were measured with tumor cell migration and colony-forming assays. Real-time PCR and Western blotting techniques were used to examine any alterations in the expression of apoptotic genes. A concentration and time-dependent cytotoxicity of CB2 agonist with IC50 value of 24.92 ± 6.99μM was obtained. The rate of lipid peroxidation was elevated, while the TNF-α concentration was declined, significantly (p < 0.05). CB2 agonist (50μM) reduced the colony-forming capability by 83% and tumor cell migration by 50%. Apoptotic effects of CB2 agonist were revealed with the increase of apoptotic cells in Acridine orange/Ethidium bromide staining, clear DNA fragmentation, pro-apoptotic genes and proteins upregulation (Caspase-3 and p53), and significant downregulation of anti-apoptotic Bcl-2. All assessments demonstrated that CB2 agonist-induced effects were reversed by CB2 inverse agonist. These data suggest that CB2 agonists at micro-molar concentrations might be considered in the CRC treatment, and their effectiveness attributes to the apoptosis induction via upregulation of caspase-3 and p53 and downregulation of Bcl-2.

Antitumor activity via apoptotic cell death pathway of water soluble copper(II) complexes: effect of the diamino unit on selectivity against lung cancer NCI-H460 cell line.

The cytotoxicity against five human tumor cell lines (THP-1, U937, Molt-4, Colo-205 and NCI-H460) of three water soluble copper(II) coordination compounds containing the ligands 3,3'-(ethane-1,2-diylbis(azanediyl))dipropanamide (BCEN), 3,3'-(piperazine-1,4-diyl)dipropanamide (BPAP) or 3,3'-and (1,4-diazepane-1,4-diyl)dipropanamide (BPAH) are reported in this work. The ligands contain different diamine units (ethylenediamine, piperazine or homopiperazine) and two propanamide units attached to the diamine centers, resulting in N2O2 donor sets. The complex containing homopiperazine unit presented the best antiproliferative effect and selectivity against lung cancer cell line NCI-H460, showing inhibitory concentration (IC50) of 58μmoldm-3 and Selectivity Index (SI) > 3.4. The mechanism of cell death promoted by the complex was investigated by Sub-G1 cell population analysis and annexin V and propidium iodide (PI) labeling techniques, suggesting that the complex promotes death by apoptosis. Transmission electron microscopy investigations are in agreement with the results presented by mitochondrial membrane potential analysis and also show the impairment of other organelles, including endoplasmic reticulum.

In vitro toxicity of indoor and outdoor PM10 from residential wood combustion

Particulate matter with aerodynamic diameter < 10 μm (PM10) was collected, indoors and outdoors, when wood burning appliances (open fireplace and woodstove) were in operation. The PM10 ecotoxicity was assessed with the Vibrio fischeri bioluminescence inhibition assay, while the cytotoxicity was evaluated by the WST-8 and lactate dehydrogenase (LDH) release assays using A549 cells. Extracts of PM10-bound polycyclic aromatic hydrocarbons (PAH) were tested for their mutagenicity through the TA98 and TA100 Ames test. The bioluminescent inhibition assay revealed that indoor particles released from the fireplace were the most toxic. Indoors, the reduction in A549 cell metabolic activity was over two times higher for the fireplace in comparison with the woodstove (32 ± 3.2% and 72 ± 7.6% at the highest dose, respectively). Indoor particles from the fireplace were found to induce greater cytotoxicity than the corresponding outdoor samples. Combined WST-8 and LDH results suggest that PM10 exposure induce apoptotic cell death pathway in which the cell membrane integrity is maintained. Indoor and outdoor samples lacked direct and indirect mutagenic activity in any of the tester strains. For indoor-generated PM10, organic carbon and PAH were significantly correlated with cell viability and bioluminescence reduction, suggesting a role of organic compounds in toxicity.

Peripherally Crowded Cationic Phthalocyanines as Efficient Photosensitizers for Photodynamic Therapy.

Photodynamic therapy is a treatment modality of cancer based on the production of cytotoxic species upon the light activation of photosensitizers. Zinc phthalocyanine photosensitizers bearing four or eight bulky 2,6-di(pyridin-3-yl)phenoxy substituents were synthesized, and pyridyl moieties were methylated. The quaternized derivatives did not aggregate at all in water and retained their good photophysical properties. High photodynamic activity of these phthalocyanines was demonstrated on HeLa, MCF-7, and EA.hy926 cells with a very low EC50 of 50 nM (for the MCF-7 cell line) upon light activation while maintaining low toxicity in the dark (TC50 ≈ 600 μM), giving thus good phototherapeutic indexes (TC50/EC50) above 1400. The compounds localized primarily in the lysosomes, leading to their rupture after light activation. This induced an apoptotic cell death pathway with secondary necrosis because of extensive and swift damage to the cells. This work demonstrates the importance of a bulky and rigid arrangement of peripheral substituents in the development of photosensitizers.

Capturing Dioclea Reflexa Seed Bioactives on Halloysite Nanotubes and pH Dependent Release of Cargo against Breast (MCF-7) Cancers In Vitro

In this work, optimization parameters were developed to capture plant metabolites from Dioclea Reflexa (DR) seed ex-tracts onto halloysites nanotubes (HNTs). A one-step pool of the crude extracts at neutral pH from the HNT lumen failed to elicit a reduction in breast cancer, Michigan Cancer Foundation-7 (MCF-7) cell viability. However, the pH-dependent elution of metabolites revealed that the acidic pH samples exhibited profound antiproliferative effects on the cancer cells compared to the basic pH metabolites using both trypan blue dye exclusion assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) viability test. pH~5.2 samples demonstrated by half-maximal inhibitory concentration (IC50) of 0.8 mg and a cyclic voltammetry oxidation peak potential and current of 234 mV and 0.45 µA, respectively. This indicates that the cancer cells death could be attributed to membrane polarization/depolarization effects of the sample. Fluorescence-activated cell sorting (FACS) studies confirmed that the plant metabolites affected breast cancer apoptotic signaling pathways of cell death. The studies proved that plant metabolites could be captured using simplified screening procedures for rapid drug discovery purposes. Such procedures, however, would require the integration of affordable analytical tools to test and isolate individual metabolites. Our approach could be an important strategy to create a library and database of bioactive plant metabolites based on pH values.

Transcriptional network constituted of CBP, Ku70, NOX2, and BAX prevents the cell death of necrosis, paraptosis, and apoptosis in human melanoma

CREB-binding protein (CBP) is an acetyltransferase known to play multiple roles in the transcriptions of genes involving oxidative metabolism, cell cycle, DNA damage checkpoints, and cell death. In this study, CBP was found to positively regulate the expression of Ku70, and both CBP and Ku70 were found to negatively regulate the expression of NOX2, therefore, mitigating the intracellular ROS in human melanoma. Knocking down CBP or Ku70 induced necrotic and paraptotic cell death as indicated by high-level intracellular ROS, cytoplasmic vacuolization, and cell cycle arrest in the S phase. In addition, chromosomal condensations were also observed in the cells proceeding necrotic and paraptotic cell death, which was found to be related to the BAX-associated intrinsic pathway of apoptotic cell death, when Ku70 was decreased either by CBP depletion or by Ku70 depletion directly. Our results, therefore, supported the idea that CBP, Ku70, BAX, and NOX2 have formed a transcriptional network in the prevention of cell death of necrosis, paraptosis, and apoptosis in human melanoma.

Proteome profiling of different rat brain regions reveals the modulatory effect of prolonged maternal separation on proteins involved in cell death-related processes

BackgroundEarly-life stress in the form of maternal separation can be associated with alterations in offspring neurodevelopment and brain functioning. Here, we aimed to investigate the potential impact of prolonged maternal separation on proteomic profiling of prefrontal cortex, hippocampus and cerebellum of juvenile and young adult rats. A special attention was devoted to proteins involved in the process of cell death and redox state maintenance.MethodsLong-Evans pups were separated from their mothers for 3 h daily over the first 3 weeks of life (during days 2–21 of age). Brain tissue samples collected from juvenile (22-day-old) and young adult (90-day-old) rats were used for label-free quantitative (LFQ) proteomic analysis. In parallel, selected oxidative stress markers and apoptosis-related proteins were assessed biochemically and by Western blot, respectively.ResultsIn total, 5526 proteins were detected in our proteomic analysis of rat brain tissue. Approximately one tenth of them (586 proteins) represented those involved in cell death processes or regulation of oxidative stress balance. Prolonged maternal separation caused changes in less than half of these proteins (271). The observed alterations in protein expression levels were age-, sex- and brain region-dependent. Interestingly, the proteins detected by mass spectrometry that are known to be involved in the maintenance of redox state were not markedly altered. Accordingly, we did not observe any significant differences between selected oxidative stress markers, such as the levels of hydrogen peroxide, reduced glutathione, protein carbonylation and lipid peroxidation in brain samples from rats that underwent maternal separation and from the corresponding controls. On the other hand, a number of changes were found in cell death-associated proteins, mainly in those involved in the apoptotic and autophagic pathways. However, there were no detectable alterations in the levels of cleaved products of caspases or Bcl-2 family members. Taken together, these data indicate that the apoptotic and autophagic cell death pathways were not activated by maternal separation either in adolescent or young adult rats.ConclusionProlonged maternal separation can distinctly modulate expression profiles of proteins associated with cell death pathways in prefrontal cortex, hippocampus and cerebellum of juvenile rats and the consequences of early-life stress may last into adulthood and likely participate in variations in stress reactivity.

Co-Chaperone Bag-1 Plays a Role in the Autophagy-Dependent Cell Survival through Beclin 1 Interaction

Expression levels of the major mammalian autophagy regulator Beclin 1 and its interaction with Bcl-2 regulate the switch between autophagic cell survival and apoptotic cell death pathways. However, some of the regulators and the precise mechanisms of these processes still remain elusive. Bag-1 (Bcl-2 associated athanogene-1), a member of BAG family proteins, is a multifunctional pro-survival molecule that possesses critical functions in vital cellular pathways. Herein, we report the role of Bag-1 on Bcl-2/Beclin 1 crosstalk through indirectly interacting with Beclin 1. Pull-down experiments suggested a molecular interaction between Bag-1 and Beclin 1 in breast cancer cell lines. On the other hand, in vitro binding assays showed that Bag-1/Beclin 1 interaction does not occur directly but occurs through a mediator molecule. Bag-1 interaction with p-Beclin 1 (T119), indicator of early autophagy, is increased during nutrient starvation suggesting involvement of Bag-1 in the autophagic regulation. Furthermore, CRISPR/Cas9-mediated Bag-1 knock-out in MCF-7 cells hampered cell survival and proliferation and resulted in decreased levels of total LC3 under starvation. Collectively, we suggest that Bag-1 modulates cell survival/death decision through maintaining macroautophagy as a component of Beclin 1-associated complexes.

Optimization of Benzothiazole and Thiazole Hydrazones as Inhibitors of Schistosome BCL-2.

Limited therapeutic options are available for the treatment of human schistosomiasis caused by the parasitic Schistosoma flatworm. The B cell lymphoma-2 (BCL-2)-regulated apoptotic cell death pathway in schistosomes was recently characterized and shown to share similarities with the intrinsic apoptosis pathway in humans. Here, we exploit structural differences in the human and schistosome BCL-2 (sBCL-2) pro-survival proteins toward a novel treatment strategy for schistosomiasis. The benzothiazole hydrazone scaffold previously employed to target human BCL-XL was repurposed as a starting point to target sBCL-2. We utilized X-ray structural data to inform optimization and then applied a scaffold-hop strategy to identify the 5-carboxamide thiazole hydrazone scaffold (43) with potent sBCL-2 activity (IC50 30 nM). Human BCL-XL potency (IC50 13 nM) was inadvertently preserved during the optimization process. The lead analogues from this study exhibit on-target activity in model fibroblast cell lines dependent on either sBCL-2 or human BCL-XL for survival. Further optimization of the thiazole hydrazone class is required to exhibit activity in schistosomes and enhance the potential of this strategy for treating schistosomiasis.

Formulation of a Semisolid Emulsion Containing Leptospermum scoparium Essential Oil and Evaluation of In Vitro Antimicrobial and Antibiofilm Efficacy.

Manuka oil, an essential oil derived from the Leptospermum scoparium, has been traditionally used for wound care and as a topical antibacterial, antifungal, and anti-inflammatory. However, the essential oil is not well retained at mucosal sites, such as the oral cavity, where the benefits of the aforementioned properties could be utilized toward the treatment of persistent biofilms. Within this study, L.scoparium essential oil was incorporated into a semisolid emulsion for improved delivery. The safety profile of L.scoparium essential oil on human gingival fibroblasts was determined via cell viability, cytotoxicity, and caspase activation. The minimal bactericidal concentration of L.scoparium essential oil was determined, and the emulsion's antibiofilm effects visualized using confocal laser scanning microscopy. L.scoparium essential oil demonstrated a lower IC50 (0.02% at 48 h) when compared to the clinical control chlorhexidine (0.002% at 48 h) and displayed lower cumulative cytotoxicity. Higher concentrations of L.scoparium essential oil (≥ 0.1%) at 6 h resulted in higher caspase 3/7 activation, suggesting an apoptotic pathway of cell death. A minimal bactericidal concentration of 0.1% w/w was observed for 6 oral bacteria and 0.01% w/v for Porphyromonas gingivalis. Textural and rheometric analysis indicated increased stability of emulsion with a 1 : 3 ratio of L.scoparium essential oil: Oryza sativa carrier oil. The optimized 5% w/w L.scoparium essential oil emulsion showed increased bactericidal penetrative effects on Streptococci gordonii biofilms compared to oil alone and to chlorhexidine controls. This study has demonstrated the safety, formulation, and antimicrobial activity of L.scoparium essential oil emulsion for potential antibacterial applications at mucosal sites.

Prodigiosin induced the caspase-dependent apoptosis in human chronic myelogenous leukemia K562 cell.

Background and purpose:Chronic myeloid leukemia (CML) as a myeloproliferative disease is characterized by increased cellularity of bone marrow. Implementing the latest treatment protocols is currently accompanied by serious and life-threatening side effects. There are worldwide attempts to find new effective and potent therapeutic agents with minimal side effects on CML patients. This in vitro study was carried out to discover the potential antiproliferative and apoptotic effects of naturally produced prodigiosin (PDG) on K562 cells as an accepted model of CML.Experimental approach:The anti-proliferative effect of PDG was measured by MTT assay. To highlight the mechanism of cytotoxicity, the apoptotic cell death pathway was investigated by morphological and biochemical assessments. The dual acridine orange/ethidium bromide staining technique and western blotting method were applied to assess the mechanism of the potential apoptotic impact of PDG on K562 cells.Findings/Results:PDG-induced time- and concentration-dependent anti-proliferative effects were revealed with an estimated IC50 value of 54.06 μM. The highest cell viability reduction (60%) was recorded in cells, which were exposed to 100 μM concentration. Further assays demonstrated that in the dual acridine orange/ethidium bromide staining method the cell population in the late apoptosis phase was increased in a concentration-dependent manner, which was confirmed with remarkable DNA fragmentation.Conclusion and implications:We found that the PDG-induced apoptosis in K562 cells is mediated through the caspase-3 activation both in mRNA and protein levels. Our results suggest that PDG could be a potent compound for further pharmacokinetic and pharmacodynamics studies in the in vivo model of CML.