Caspase-like activity is associated with bacterial infection of the urine in urinary tract diseases.

Urinary tract infections (UTIs) are a serious public health problem. They can be caused by a number of pathogens, but the most common are Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis and Staphylococcus saprophyticus. Bacterial infection is diagnosed by examining a urine sample. The presence of bacteria or white blood cells is determined under a microscope or a urine culture is performed. In this study, we used a panel of chromogenic substrates for the qualitative determination of specific enzyme activity in the urine of patients with confirmed bacterial infection and/or urinary tract disease. Healthy patients were used as a control group. It turned out that in the case of Escherichia coli infection, we observed the activity of the caspase subunit of the human 20S proteasome. We did not observe similar correlations for infections with other types of bacteria.

Different RONS Generation in MTC-SK and NSCL Cells Lead to Varying Antitumoral Effects of Alpha-Ketoglutarate + 5-HMF.

BACKGROUND: Carbonylated proteins (CPs) serve as specific indicators of increased reactive oxygen and nitrogen species (RONS) production in cancer cells, attributed to the dysregulated mitochondrial energy metabolism known as the Warburg effect. The aim of this study was to investigate the potential of alpha-ketoglutarate (aKG), 5-hydroxymethylfurfural (5-HMF), and their combination as mitochondrial-targeting antioxidants in MTC-SK or NCI-H23 cancer cells. METHODS: MTC-SK and NCI-H23 cells were cultured in the absence or presence of varying concentrations (0-500 µg/mL) of aKG, 5-HMF, and the combined aKG + 5-HMF 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 MTC-SK and NCI-H23 cells. RESULTS: The mitochondrial activity of MTC-SK cells exhibited a concentration- and time-dependent reduction upon treatment with aKG, 5-HMF, or the combined aKG + 5-HMF. The half-maximal inhibitory concentration (IC50%) for mitochondrial activity was achieved at 500 µg/mL aKG, 200 µg/mL 5-HMF, and 200 µg/mL aKG + 66.7 µg/mL 5-HMF after 72 h. In contrast, NCI-H23 cells showed a minimal reduction (10%) in mitochondrial activity even at the highest combined concentration of aKG + 5-HMF. The CP levels in MTC-SK cells were measured at 8.7 nmol/mg protein, while NCI-H23 cells exhibited CP levels of 1.4 nmol/mg protein. The combination of aKG + 5-HMF led to a decrease in CP levels specifically in MTC-SK cells. The correlation between mitochondrial activity and CP levels in the presence of different concentrations of combined aKG + 5-HMF in MTC-SK cells demonstrated a linear and concentration-dependent decline in CP levels and mitochondrial activity. Conversely, the effect was less pronounced in NCI-H23 cells. Cell growth of MTC-CK cells was reduced to 60% after 48 h and maintained at 50% after 72 h incubation when treated with 500 µg/mL aKG (IC50%). Addition of 500 µg/mL 5-HMF inhibited cell growth completely regardless of the incubation time. The IC50% for 5-HMF on MTC-CK cell growth was calculated at 375 µg/mL after 24 h incubation and 200 µg/mL 5-HMF after 72 h. MTC-SK cells treated with 500 µg/mL aKG + 167 µg/mL 5-HMF showed no cell growth. The calculated IC50% for the combined substances was 250 µg/mL aKG + 83.3 µg/mL 5-HMF (48 h incubation) and 200 µg/mL aKG + 66.7 µg/mL 5-HMF (72 h incubation). None of the tested concentrations of aKG, 5-HMF, or the combined solution had any effect on NCI-H23 cell growth at any incubation time. Caspase-3 activity increased to 21% in MTC-CK cells in the presence of 500 µg/mL aKG, while an increase to 59.6% was observed using 500 µg/mL 5-HMF. The combination of 500 µg/mL aKG + 167.7 µg/mL 5-HMF resulted in a caspase-3 activity of 55.2%. No caspase-3 activation was observed in NCI-H23 cells when treated with aKG, 5-HMF, or the combined solutions. CONCLUSION: CPs may serve as potential markers for distinguishing between cancer cells regulated by RONS. The combination of aKG + 5-HMF showed induced cell death in high-RONS-generating cancer cells compared to low-RONS-generating cancer cells.

The effect of increased ambient temperature on Hsp70, superoxide dismutase, nitric oxide, malondialdehyde, and caspase activity in relation to the intrinsic and extrinsic apoptosis pathway of broiler blood cells.

The objective of this study was to examine the effect of increased ambient temperature on Hsp70 expression, superoxide dismutase (SOD) activity, nitric oxide (NO), malondialdehyde (MDA), caspase 8, caspase 9 and caspase 3 in relation to the intrinsic and extrinsic apoptosis pathway (IAP and EAP) of broiler blood cells (BBC). BBC were maintained at 42 °C and at 5 ranges of temperatures; 42-43, 44-45, 46-47, 48-49, and 50-51 °C. Then Hsp70 expression, SOD activity, NO, MDA, activity of caspase 8, caspase 9 and caspase 3, and living and apoptotic BBC were measured. It was found that Hsp70 expression and SOD were increased at 42-43 °C (P < 0.05), while the activity of caspase 9 and caspase 3 decreased (P < 0.05). At higher temperatures of 44-45 °C, caspase 9 and caspase 3 activities were increased and were higher than at 42 °C (P < 0.05). Apoptosis commenced in the temperature range 46-47 °C; during this range of temperature, NO increased (P < 0.05), but it decreased at 48-49 °C (P < 0.05). At the same time, activity of SOD in BBC was increased, achieving the highest levels (P < 0.05). In addition, activity of caspase 8 and caspase 3 at 48-49 °C was higher than that at 42 °C, and the apoptotic BBC was increased (P < 0.05). Therefore, temperature effects on BBC could be divided into three phases. The first phase was the temperature at 42 °C. The second phase, or pre-apoptotic phase, was in the temperature range from 42 to 45 °C. The third phase was the apoptosis phase, which manifested at 46 °C and above. These phenomena showed that in the pre-apoptosis phase, Hsp70 suppressed the IAP. On the other hand, in the apoptosis phase, apoptotic BBC is caused by the influence of NO, superoxide and MDA through the EAP.

Effect of Antibiotic Amphotericin B Combinations with Selected 1,3,4-Thiadiazole Derivatives on RPTECs in an In Vitro Model.

4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol (C1) and 4-[5-(naphthalen-1-ylmethyl)-1,3,4-thiadiazol-2-yl] benzene1,3-diol (NTBD) are representative derivatives of the thiadiazole group, with a high antimycotic potential and minimal toxicity against normal human fibroblast cells. The present study has proved its ability to synergize with the antifungal activity of AmB. The aim of this work was to evaluate the cytotoxic effects of C1 or NTBD, alone or in combination with AmB, on human renal proximal tubule epithelial cells (RPTECs) in vitro. Cell viability was assessed with the MTT assay. Flow cytometry and spectrofluorimetric techniques were used to assess the type of cell death and production of reactive oxygen species (ROS), respectively. The ELISA assay was performed to measure the caspase-2, -3, and -9 activity. ATR-FTIR spectroscopy was used to evaluate biomolecular changes in RPTECs induced by the tested formulas. The combinations of C1/NTBD and AmB did not exert a strong inhibitory effect on the viability/growth of kidney cells, as evidenced by the negligible changes in the apoptotic/necrotic rate and caspase activity, compared to the control cells. Both NTBD and C1 displayed stronger anti-oxidant activity when combined with AmB. The relatively low nephrotoxicity of the thiadiazole derivative combinations and the protective activity against AmB-induced oxidative stress may indicate their potential use in the therapy of fungal infections.

Alpha-Ketoglutarate or 5-HMF: Single Compounds Effectively Eliminate Leukemia Cells via Caspase-3 Apoptosis and Antioxidative Pathways.

BACKGROUND: We recently showed that a combined solution containing alpha-ketoglutarate (aKG) and 5-hydroxymethyl-furfural (5-HMF) has a solid antitumoral effect on the Jurkat cell line due to the fact of its antioxidative, caspase-3 and apoptosis activities, but no negative effect on human fibroblasts was obtained. The question arises how the single compounds, aKG and 5-HMF, affect peroxynitrite (ONOO-) and nitration of tyrosine residues, Jurkat cell proliferation and caspase-activated apoptosis. METHODS: The ONOO- luminol-induced chemiluminescence reaction was used to measure the ONOO- scavenging function of aKG or 5-HMF, and their protection against nitration of tyrosine residues on bovine serum albumin was estimated with the ELISA technique. The Jurkat cell line was cultivated in the absence or presence of aKG or 5-HMF solutions between 0 and 3.5 µM aKG or 0 and 4 µM 5-HMF. Jurkat cells were tested for cell proliferation, mitochondrial activity and caspase-activated apoptosis. RESULTS: aKG showed a concentration-dependent reduction in ONOO-, resulting in a 90% elimination of ONOO- using 200 mM aKG. In addition, 20 and 200 mM 5-HMF were able to reduce ONOO- only by 20%, while lower concentrations of 5-HMF remained stable in the presence of ONOO-. Nitration of tyrosine residues was inhibited 4 fold more effectively with 5-HMF compared to aKG measuring the IC50%. Both substances, aKG and 5-HMF, were shown to cause a reduction in Jurkat cell growth that was dependent on the dose and incubation time. The aKG effectively reduced Jurkat cell growth down to 50% after 48 and 72 h of incubation using the highest concentration of 3.5 µM, and 1, 1.6, 2, 3 and 4 µM 5-HMF inhibited any cell growth within (i) 24 h; 1.6, 2, 3 and 4 µM 5-HMF within 48 h (ii); 2, 3 and 4 µM 5-HMF within 72 h (iii). Furthermore, 4 µM was able to eliminate the starting cell number of 20,000 cells after 48 and 72 h down to 11,233 cells. The mitochondrial activity measurements supported the data on aKG or 5-HMF regarding cell growth in Jurkat cells, in both a dose- and incubation-time-dependent manner: the highest concentration of 3.5 µM aKG reduced the mitochondrial activity over 24 h (67.7%), 48 h (57.9%) and 72 h (46.8%) of incubation with Jurkat cells compared to the control incubation without aKG (100%). 5-HMF was more effective compared to aKG; the mitochondrial activity in the presence of 4 µM 5-HMF decreased after 24 h down to 68.4%, after 48 h to 42.9% and after 72 h to 32.0%. Moreover, 1.7 and 3.4 µM aKG had no effect on caspase-3-activated apoptosis (0.58% and 0.56%) in the Jurkat cell line. However, 2 and 4 µM 5-HMF increased the caspase-3-activated apoptosis up to 22.1% and 42.5% compared to the control (2.9%). A combined solution of 1.7 µM aKG + 0.7 µM 5-HMF showed a higher caspase-3-activated apoptosis (15.7%) compared to 1.7 µM aKG or 2 µM 5-HMF alone. In addition, 3.5 µM µg/mL aKG + 1.7 µM 5-HMF induced caspase-activated apoptosis up to 55.6% compared to 4.5% or 35.6% caspase-3 activity using 3.5 µM aKG or 4 µM 5-HMF. CONCLUSION: Both substances showed high antioxidative potential in eliminating either peroxynitrite or nitration of tyrosine residues, which results in a better inhibition of cell growth and mitochondrial activity of 5-HMF compared to aKG. However, caspase-3-activated apoptosis measurements revealed that the combination of both substances synergistically is the most effective compared to single compounds.

Topoisomerase poisoning by the flavonoid nevadensin triggers DNA damage and apoptosis in human colon carcinoma HT29 cells.

Nevadensin, an abundant polyphenol of basil, is reported to reduce alkenylbenzene DNA adduct formation. Furthermore, it has a wide spectrum of further pharmacological properties. The presented study focuses the impact of nevadensin on topoisomerases (TOPO) in vitro. Considering the DNA-intercalating properties of flavonoids, first, minor groove binding properties (IC50 = 31.63 µM), as well as DNA intercalation (IC50 = 296.91 µM) of nevadensin, was found. To determine potential in vitro effects on TOPO I and TOPO IIα, the relaxation and decatenation assay was performed in a concentration range of 1-500 µM nevadensin. A partial inhibition was detected for TOPO I at concentrations  ≥ 100 µM, whereas TOPO IIα activity is only inhibited at concentrations  ≥ 250 µM. To clarify the mode of action, the isolating in vivo complex of enzyme assay was carried out using human colon carcinoma HT29 cells. After 1 h of incubation, the amount of TOPO I linked to DNA was significantly increased by nevadensin (500 µM), why nevadensin was characterized as TOPO I poison. However, no effects on TOPO IIα were detected in the cellular test system. As a subsequent cellular response to TOPO I poisoning, a highly significant increase of DNA damage after 2 h and a decrease of cell viability after 48 h at the same concentration range were found. Furthermore, after 24 h of incubation a G2/M arrest was observed at concentrations ≥ 100 µM by flow cytometry. The analysis of cell death revealed that nevadensin induces the intrinsic apoptotic pathway via activation of caspase-9 and caspase-3. The results suggest that cell cycle disruption and apoptotic events play key roles in the cellular response to TOPO I poisoning caused by nevadensin in HT29 cells.

Tango7 directs cellular remodeling by the Drosophila apoptosome.

It is now well appreciated that the apoptosome, which governs caspase-dependent cell death, also drives nonapoptotic caspase activation to remodel cells. However, the determinants that specify whether the apoptosome acts to kill or remodel have yet to be identified. Here we report that Tango7 collaborates with the Drosophila apoptosome to drive a caspase-dependent remodeling process needed to resolve individual sperm from a syncytium. In these cells, Tango7 is required for caspase activity and localizes to the active apoptosome compartment via its C terminus. Furthermore, Tango7 directly stimulates the activity of this complex in vitro. We propose that Tango7 specifies the Drosophila apoptosome as an effector of cellular remodeling.

Chemical Composition, Apoptotic Activity, and Antiparasitic Effects of Ferula macrecolea Essential Oil against Echinococcus granulosus Protoscoleces.

BACKGROUND: Today, the present protoscolicidals used to minimize the serious risks during hydatid cyst surgery are not completely safe and have various adverse side effects. The present study aimed to evaluate the chemical composition and apoptotic activity of Ferula macrecolea essential oil (FMEO) as well as its in vitro and ex vivo protoscolicidal effects against hydatid cyst protoscoleces. METHODS: Gas chromatography/mass spectrometry (GC/MS) analysis was performed to determine the chemical composition of FMEO. Protoscoleces of hydatid cysts were collected from liver fertile hydatid cysts of infected sheep and were then treated with various concentrations of the essential oil (75, 150, and 300 µL/mL) for 5-60 min in vitro and ex vivo. Then, by using the eosin exclusion test, the viability of the protoscoleces was studied. The caspase-3-like activity of the FMEO-treated protoscoleces was also evaluated through the colorimetric protease assay Sigma Kit based on the manufacturer's instructions. RESULTS: According to GC/MS, the main constituents of the essential oil were terpinolene (77.72%), n-nonanal (4.47%), and linalool (4.35%), respectively. In vitro, the maximum protoscolicidal activity of FMEO was observed at the concentrations of 150 and 300 µL/mL, such that 100% of the protoscoleces were killed after 30 and 20 min of exposure, respectively. Based on the obtained findings, the results demonstrate that FMEO required a longer time to kill protoscoleces ex vivo; after 12 min of exposure to FMEO, only 13.4% of the protoscoleces remained alive. After 48 h of the treatment of protoscoleces, FMEO, in a dose-dependent manner and at doses of 75, 150, and 300 µL/mL, induced the activation of the caspase enzyme by 24.3, 35.3, and 48.3%, respectively. CONCLUSIONS: Our findings demonstrate the potent protoscolicidal effects of FMEO in vitro and ex vivo; however, further studies are required to assess the safety and the efficiency of FMEO as a promising scolicidal agent in a preclinical model and clinical setting.

A fluorescent toolkit for spatiotemporal tracking of apoptotic cells in living Drosophila tissues.

Far from being passive, apoptotic cells influence their environment. For example, they promote tissue folding, myoblast fusion and modulate tumor growth. Understanding the role of apoptotic cells necessitates their efficient tracking within living tissues, a task that is currently challenging. In order to easily spot apoptotic cells in developing Drosophila tissues, we generated a series of fly lines expressing different fluorescent sensors of caspase activity. We show that three of these reporters (GFP-, Cerulean- and Venus-derived molecules) are detected specifically in apoptotic cells and throughout the whole process of programmed cell death. These reporters allow the specific visualization of apoptotic cells directly within living tissues, without any post-acquisition processing. They overcome the limitations of other apoptosis detection methods developed so far and, notably, they can be combined with any kind of fluorophore.

Design, synthesis and biological evaluation of some new 2-Pyrazoline derivatives as potential anticancer agents.

A new series of N-(4-(1-Phenyl-5-aryl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-4-substitutedbenzamide derivatives were designed and synthesized from new chalcone derivatives. All newly synthesized compounds were determined by using IR, 1H-NMR, 13C-NMR spectroscopic methods, elemental analysis and evaluated for their in vitro antiproliferative activities on HeLa, MCF-7, MKN-45 cancer cell lines and NIH-3T3 cell line using MTT assay. Expression of Bax and Bcl-2 proteins was detected by Western-blot analysis and caspase-3 enzyme activity was measured. Notably, compounds 1f and 2f showed a significant cytotoxic effect in all three cancer cells and did not display cytotoxicity on NIH-3T3 normal cells. (IC50 = 26.66 ± 2.73 µM on HeLa, IC50 = 9.41 ± 2.19 µM on MCF-7, IC50 = 5.17 ± 3.54 µM on MKN-45 for 1f. IC50 = 17.96 ± 3.34 µM on HeLa, IC50 = 0.69 ± 0.13 µM on MCF-7, IC50 = 0.88 ± 0.16 µM on MKN-45 for 2f.) Moreover, 1f and 2f upregulated protein expression level of Bax and downregulated protein expression level of Bcl-2 in cells. Similarly, caspase-3 activity was increased in cells via 1f and 2f. It can be concluded that 1f and 2f activated apoptosis by inducing mitochondrial apoptotic proteins in HeLa, MCF-7, MKN-45. This could be potentially new anti-cancer derivatives and used to contribute to new therapeutic development.

Synthesis and In Vitro Antitumor Activity of Naringenin Oxime and Oxime Ether Derivatives.

Naringenin is one of the most abundant dietary flavonoids exerting several beneficial biological activities. Synthetic modification of naringenin is of continuous interest. During this study our aim was to synthesize a compound library of oxime and oxime ether derivatives of naringenin, and to investigate their biological activities. Two oximes and five oxime ether derivatives were prepared; their structure has been elucidated by NMR and high-resolution mass spectroscopy. The antiproliferative activity of the prepared compounds was evaluated by MTT assay against human leukemia (HL-60) and gynecological cancer cell lines isolated from cervical (HeLa, Siha) and breast (MCF-7, MDA-MB-231) cancers. Tert-butyl oxime ether derivative exerted the most potent cell growth inhibitory activity. Moreover, cell cycle analysis suggested that this derivative caused a significant increase in the hypodiploid (subG1) phase and induced apoptosis in Hela and Siha cells, and induced cell cycle arrest at G2/M phase in MCF-7 cells. The proapoptotic potential of the selected compound was confirmed by the activation of caspase-3. Antioxidant activities of the prepared molecules were also evaluated with xanthine oxidase, DPPH and ORAC assays, and the methyl substituted oxime ether exerted the most promising activity.

Class I-Histone Deacetylase (HDAC) Inhibition is Superior to pan-HDAC Inhibition in Modulating Cisplatin Potency in High Grade Serous Ovarian Cancer Cell Lines.

High grade serous ovarian cancer (HGSOC) is the most common and aggressive ovarian cancer subtype with the worst clinical outcome due to intrinsic or acquired drug resistance. Standard treatment involves platinum compounds. Cancer development and chemoresistance is often associated with an increase in histone deacetylase (HDAC) activity. The purpose of this study was to examine the potential of HDAC inhibitors (HDACi) to increase platinum potency in HGSOC. Four HGSOC cell lines with different cisplatin sensitivity were treated with combinations of cisplatin and entinostat (class I HDACi), panobinostat (pan-HDACi), or nexturastat A (class IIb HDACi), respectively. Inhibition of class I HDACs by entinostat turned out superior in increasing cisplatin potency than pan-HDAC inhibition in cell viability assays (MTT), apoptosis induction (subG1), and caspase 3/7 activation. Entinostat was synergistic with cisplatin in all cell lines in MTT and caspase activation assays. MTT assays gave combination indices (CI values) < 0.9 indicating synergism. The effect of HDAC inhibitors could be attributed to the upregulation of pro-apoptotic genes (CDNK1A, APAF1, PUMA, BAK1) and downregulation of survivin. In conclusion, the combination of entinostat and cisplatin is synergistic in HGSOC and could be an effective strategy for the treatment of aggressive ovarian cancer.

Structural and functional characterization of phosphomimetic mutants of cytochrome c at threonine 28 and serine 47.

Protein function is frequently modulated by post-translational modifications of specific residues. Cytochrome c, in particular, is phosphorylated in vivo at threonine 28 and serine 47. However, the effect of such modifications on the physiological functions of cytochrome c - namely, the transfer of electrons in the respiratory electron transport chain and the triggering of programmed cell death - is still unknown. Here we replace each of these two residues by aspartate, in order to mimic phosphorylation, and report the structural and functional changes in the resulting cytochrome c variants. We find that the T28D mutant causes a 30-mV decrease on the midpoint redox potential and lowers the affinity for the distal site of Arabidopsis thaliana cytochrome c1 in complex III. Both the T28D and S47D variants display a higher efficiency as electron donors for the cytochrome c oxidase activity of complex IV. In both protein mutants, the peroxidase activity is significantly higher, which is related to the ability of cytochrome c to leave the mitochondria and reach the cytoplasm. We also find that both mutations at serine 47 (S47D and S47A) impair the ability of cytoplasmic cytochrome c to activate the caspases cascade, which is essential for triggering programmed cell death.

Mitochondrial DNA oxidation induces imbalanced activity of NLRP3/NLRP6 inflammasomes by activation of caspase-8 and BRCC36 in dry eye.

The concept of innate immunity has been expanded to recognize environmental pathogens other than microbial components. However, whether and how the innate immunity is initiated by epithelium in response to environmental physical challenges such as low humidity and high osmolarity in an autoimmune disease, dry eye, is still largely unknown. Using two experimental dry eye models, primary human corneal epithelial cultures exposed to hyperosmolarity and mouse ocular surface facing desiccating stress, we uncovered novel innate immunity pathway by ocular surface epithelium, where oxidized mitochondrial DNA induces imbalanced activation of NLRP3/NLRP6 inflammasomes via stimulation of caspase-8 and BRCC36 in response to environmental stress. Activated NLRP3 with suppressed NLRP6 stimulates caspase-1 activation that leads to IL-1ß and IL-18 maturation and secretion. NLRP3-independent caspase-8 noncanonically activates caspase-1 via reciprocal regulation of NLRP3/NLRP6-mediated inflammasomes. Reactive oxygen species-induced mitochondrial DNA oxidative damage and BRCC36 deubiquitinating activity provide a missing link and mechanism by which innate immunity responds to environmental stress via caspase-8-involved NLRP3/NLRP6 inflammasomes.

Towards cytotoxic and selective derivatives of maslinic acid.

Several novel esters and amides of maslinic acid were prepared. Their evaluation for cytotoxic activity with a panel of human cancer cell lines using a sulforhodamine B (SRB) assay revealed for some of them a noteworthy activity. The results from annexinV-FITC and caspase-assays as well as from DNA laddering experiments provided evidence for an apoptotic cell death. A diacetylated benzylamide (15) induced a G1/G0 arrest in tumor cells. It also displayed an extraordinary cytotoxicity against human ovarian cancer cells but a 300 times lower toxicity for non-malignant primary human fibroblasts.

Preparation, Optimization, and In-Vitro Evaluation of Brusatol- and Docetaxel-Loaded Nanoparticles for the Treatment of Prostate Cancer.

Challenges to docetaxel use in prostate cancer treatment include several resistance mechanisms as well as toxicity. To overcome these challenges and to improve the therapeutic efficacy in heterogeneous prostate cancer, the use of multiple agents that can destroy different subpopulations of the tumor is required. Brusatol, a multitarget inhibitor, has been shown to exhibit potent anticancer activity and play an important role in drug response and chemoresistance. Thus, the combination of brusatol and docetaxel in a nanoparticle platform for the treatment of prostate cancer is expected to produce synergistic effects. In this study, we reported the development of polymeric nanoparticles for the delivery of brusatol and docetaxel in the treatment of prostate cancer. The one-factor-at-a-time method was used to screen for formulation and process variables that impacted particle size. Subsequently, factors that had modifiable effects on particle size were evaluated using a 24 full factorial statistical experimental design followed by the optimization of drug loading. The optimization of blank nanoparticles gave a formulation with a mean size of 169.1 nm ± 4.8 nm, in agreement with the predicted size of 168.333 nm. Transmission electron microscopy showed smooth spherical nanoparticles. The drug release profile showed that the encapsulated drugs were released over 24 h. Combination index data showed a synergistic interaction between the drugs. Cell cycle analysis and the evaluation of caspase activity showed differences in PC-3 and LNCaP prostate cancer cell responses to the agents. Additionally, immunoblots showed differences in survivin expression in LNCaP cells after treatment with the different agents and formulations for 24 h and 72 h. Therefore, the nanoparticles are potentially suitable for the treatment of advanced prostate cancer.

The ORF7a protein of SARS-CoV-2 initiates autophagy and limits autophagosome-lysosome fusion via degradation of SNAP29 to promote virus replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is closely related to various cellular aspects associated with autophagy. However, how SARS-CoV-2 mediates the subversion of the macroautophagy/autophagy pathway remains largely unclear. In this study, we demonstrate that overexpression of the SARS-CoV-2 ORF7a protein activates LC3-II and leads to the accumulation of autophagosomes in multiple cell lines, while knockdown of the viral ORF7a gene via shRNAs targeting ORF7a sgRNA during SARS-CoV-2 infection decreased autophagy levels. Mechanistically, the ORF7a protein initiates autophagy via the AKT-MTOR-ULK1-mediated pathway, but ORF7a limits the progression of autophagic flux by activating CASP3 (caspase 3) to cleave the SNAP29 protein at aspartic acid residue 30 (D30), ultimately impairing complete autophagy. Importantly, SARS-CoV-2 infection-induced accumulated autophagosomes promote progeny virus production, whereby ORF7a downregulates SNAP29, ultimately resulting in failure of autophagosome fusion with lysosomes to promote viral replication. Taken together, our study reveals a mechanism by which SARS-CoV-2 utilizes the autophagic machinery to facilitate its own propagation via ORF7a.Abbreviations: 3-MA: 3-methyladenine; ACE2: angiotensin converting enzyme 2; ACTB/ß-actin: actin beta; ATG7: autophagy related 7; Baf A1: bafilomycin A1; BECN1: beclin 1; CASP3: caspase 3; COVID-19: coronavirus disease 2019; GFP: green fluorescent protein; hpi: hour post-infection; hpt: hour post-transfection; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MERS: Middle East respiratory syndrome; MTOR: mechanistic target of rapamycin kinase; ORF: open reading frame; PARP: poly(ADP-ribose) polymerase; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; shRNAs: short hairpin RNAs; siRNA: small interfering RNA; SNAP29: synaptosome associated protein 29; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TCID50: tissue culture infectious dose; TEM: transmission electron microscopy; TUBB, tubulin, beta; ULK1: unc-51 like autophagy activating kinase 1.

Copper Oxide Nanoparticles Exhibit Cell Death Through Oxidative Stress Responses in Human Airway Epithelial Cells: a Mechanistic Study.

Copper oxide nanoparticles (CuONPs) are purposefully used to inhibit the growth of bacteria, algae, and fungi. Several studies on the beneficial and harmful effects of CuONPs have been conducted in vivo and in vitro, but there are a few studies that explain the toxicity of CuONPs in human airway epithelial cells (HEp-2). As a result, the purpose of this study is to look into the dose-dependent toxicity of CuONPs in HEp-2 cells. After 24 h of exposure to 1-40 µg/ml CuONPs, the MTT and neutral red assays were used to test for cytotoxicity. To determine the mechanism(s) of cytotoxicity in HEp-2 cells, additional oxidative stress assays (LPO and GSH), the amount of ROS produced, the loss of MMP, caspase enzyme activities, and apoptosis-related genes were performed using qRT-PCR. CuONPs exhibited dose-dependent cytotoxicity in HEp-2 cells, with an IC50 value of ~ 10 µg/ml. The morphology of HEp-2 cells was also altered in a dose-dependent manner. The involvement of oxidative stress in CuONP-induced cytotoxicity was demonstrated by increased LPO levels and ROS generation, as well as decreased levels of GSH and MMP. Furthermore, activated caspase enzymes and altered apoptotic genes support CuONPs' ability to induce apoptosis in HEp-2 cells. Overall, this study demonstrated that CuONPs can cause apoptosis in HEp-2 cells via oxidative stress; therefore, CuONPs may pose a risk to human health and should be handled and used with caution.

Regulation of innate immune responses by cell death-associated caspases during virus infection.

Viruses are obligate intracellular pathogens that rely on cellular machinery for successful replication and dissemination. The host cells encode a number of different strategies to sense and restrict the invading viral pathogens. Caspase-mediated programmed cell death pathways that are triggered by virus infection, such as apoptosis and pyroptosis, provide a means for the infected cells to limit viral proliferation, leading to suicidal cell death (apoptosis) or lytic cell death and alerting uninfected cells to mount anti-viral responses (pyroptosis). However, some viruses can employ activated caspases to dampen the anti-viral responses and facilitate viral replication through cleavage of critical molecules of the innate immune pathways. The regulation of innate immune responses by caspase activation during virus infection has recently become an important topic. In this review, we briefly introduce the characteristics of different classes of caspases and the cell death pathways regulated by these caspases. We then describe how viruses trigger or dampen caspase activation during infection and how these activated caspases regulate three major innate immune response pathways of viral infections: the retinoic acid-inducible gene I-like receptor, toll-like receptor and cyclic GMP-AMP synthase-stimulator of interferon genes pathways.

Induction of cell apoptosis by biliverdin reductase inhibitor in MCF-7 and MDA-MB-468 breast cancer cell lines: Experimental and in silico studies.

Biliverdin reductase, biliverdin and bilirubin are known as important components of cellular signaling pathways that play major roles in cell proliferation and apoptosis, although their physiological relevance is still under evaluation. This study was designed to investigate the expression and activity of BVR-A and its apoptotic effect in the breast cancer cell lines, MCF-7 and MDA-MB-468. The expression of BVR-A was examined by real-time PCR and western blot analysis. Bilirubin concentration was measured by HPLC and molecular docking was performed to identify an appropriate inhibitor for BVR-A. To detect cell apoptosis, annexin V-PI staining, caspase-3, -8, and -9 activities were evaluated. Cell viability was reduced by biliverdin, in a dose-dependent manner, and an intrinsic apoptotic response occurred which was evidenced by caspase-3 and -9 activities. The intra- and extracellular concentrations of bilirubin were higher in MCF-7 cells than those of MDA-MB-468 cells. The expression of BVR-A, at mRNA and protein levels, in MCF-7 was also higher than that of MDA-MB-468 cells. Treatment of both cell lines with biliverdin plus DTNB, a BVR-A inhibitor, increased the cell death significantly when compared with biliverdin alone. Using annexin V-PI staining and assessment of caspase-3 activity, it was confirmed that biliverdin together with DTNB increases apoptosis in breast cancer cells. In conclusion, biliverdin has an important role in cell apoptosis and inhibition of biliverdin reductase increases the apoptotic effect of biliverdin.