Comparative phytochemical studies on the roots of Polygala azizsancarii and P. peshmenii and neuroprotective activities of the two xanthones.

Six known sucrose mono-, di- and triesters and five xanthone derivatives were isolated from the roots of Polygala peshmenii Eren, Parolly, Raus & Kürschner which is a narrow species endemic to Türkiye. Among the xanthones, 1,7-dihydroxy-2,3-methylenedioxy-5,6-dimethoxy-xanthone is an undescribed compound isolated for the first time from a natural source. The studies on the roots of P. azizsancarii Dönmez have resulted in the isolation of four known compounds including sucrose mono-, di- and triesters. The structures of the sucrose esters and xanthones isolated from P. azizsancarii and P. peshmenii were established by spectroscopic methods, including 1D-NMR (1H NMR, 13C NMR, DEPT-135), 2D-NMR (COSY, NOESY, HSQC, HMBC). Neuroprotective activities of two xanthones, 1,3,6-trihydroxy-2,5,7-trimethoxyxanthone and 3-O-ß-D-glucopyranosyloxy-1,6-dihydroxy-2,5,7-trimethoxyxanthone isolated from the roots of P. azizsancarii were evaluated in vitro using in a cellular model of Alzheimer's disease. SKNAS human neuroblastoma cells were used in the study and treated with different consecrations of Aß25₋35 oligomer for up to 48 h. Cell viability was evaluated using MTT assay. The distribution of ß-amyloid, α-synuclein, tau, JAK2, STAT3, caspase 3 and BMP-2 were investigated using indirect immunoperoxidase staining. Our results suggested that both xanthones control tau aggregation with no effect on ß-amyloid plaque formation. In addition, for neuronal pathophysiology in AD cell model, decreased distributions of JAK/STAT3 and BMP2 signaling pathways were demonstrated, therefore they play a role in the protective effect on neurons in neurodegenerative disease. A significant decrease in caspase 3 immunoreactivity was detected after the administration of both compounds in AD cells. Therefore, both compounds control neuronal pathophysiology and rescue cell death in AD disease.

Linking Cell Health and Reactive Oxygen Species from Secondary Organic Aerosols Exposure.

Oxidative stress is a possible mechanism by which ambient fine particulate matter (PM) exerts adverse biological effects. While multiple biological effects and reactive oxygen species (ROS) production have been observed upon PM exposure, whether the biological effects are ROS-mediated remains unclear. Secondary organic aerosols (SOA) constitute a major fraction of fine PM and can contribute substantially to its toxicity. In this work, we measured three types of cell responses (mitochondrial membrane potential (MMP), caspase 3/7 activity, and ROS) and investigated their associations upon exposure to SOA formed from anthropogenic (naphthalene) and biogenic (α-pinene) precursors. MMP and caspase 3/7 activity (an early indicator of apoptosis) are key indicators of cell health, and changes of them could occur downstream of ROS-mediated pathways. We observed a significant increase in caspase 3/7 activity after SOA exposure, suggesting that apoptosis is an important pathway of cell death induced by SOA. We further found strong associations between a decrease in MMP and increase in caspase 3/7 activity with an increase in cellular ROS level. These results suggest that cell health is largely dependent on the cellular ROS level, highlighting oxidative stress as a key mechanism for biological effects from SOA exposure. Linear regression analyses reveal greater changes of the three cellular responses with increasing carbon oxidation state (OSc) of SOA, suggesting that SOA are more toxic when they are more oxidized. Overall, our work provides critical insights into the associations between cell health and ROS level upon SOA exposure and proposes that OSc could be a suitable proxy to assess the overall SOA toxicity.

Tear-derived exosomal biomarkers of Graves' ophthalmopathy.

Graves' ophthalmopathy (GO), the most frequent extrathyroidal manifestation of Graves' disease (GD), can lead to a significant decline in the quality of life in patients. Exosomes, which contain proteins, lipids and DNA, play important roles in the pathological processes of various diseases. However, their roles in Graves' ophthalmopathy are still unclear. We aimed to isolate exosomes and analyze the different exosomal proteins. Tear fluids were collected from twenty-four GO patients, twenty-four GD patients and sixteen control subjects. The numbers of tear exosomes were assayed using nanoparticle tracking analysis. A Luminex 200 kit and ELISA kit were used to confirm the different cytokine concentrations in serum. Extraocular muscle from GO patients and controls was extracted, and western blotting was used to assay the levels of Caspase-3 and complement C4A. Our study demonstrated that the number of tear exosomes differ from GD patients and control. The expression levels of cytokines, including IL-1 and IL-18, were significantly increased in the tear exosomes and serum from GO patients compared with GD patients and controls. The levels of the exosomal proteins Caspase-3, complement C4A and APOA-IV were significantly increased in GO patients compared to GD patients and controls. Orbital fibroblasts from GO patients showed significantly higher levels of Caspase-3 and complement C4A than those from controls. The levels of serum APOA-IV in GO patients were significantly higher than those in GD patients and controls. Specific proteins showed elevated expression in tear exosomes from GO patients, indicating that they may play important roles in GO pathogenesis.

Modifying a bacterial tyrosinase zymogen for use in protease activity assays.

Current clinical laboratory assays are not sufficient for determining the activity of many specific human proteases yet. In this study, we developed a general approach that enables the determination of activities of caspase-3 based on the proteolytic activation of the engineered zymogen of the recombinant tyrosinase from Verrucomicrobium spinosum (Vs-tyrosinase) by detecting the diphenolase activity in an increase in absorbance at 475 nm. Here, we designed three different zymogen constructs of Vs-tyrosinase, including RSL-pre-pro-TYR, Pre-pro-TYR, and Pro-TYR. The active domain was fused to the reactive site loop (RSL) of α1-proteinase inhibitor and/or its own signal peptide (pre) and/or its own C-terminal domain (pro) via a linker containing a specific caspase-3 cleavage site. Further studies revealed that both RSL peptide and TAT signal peptide were able to inhibit tyrosinase diphenolase activity, in which RSL-pre-pro-TYR had the lowest background signals. Therefore, a specific protease activity such as caspase-3 could be detected when a suitable zymogen was established. Our results could provide a new way to directly detect the activities of key human proteases, for instance, to monitor the efficacy and safety of tumor therapy by determining the activity of apoptosis-related caspase-3 in patients. KEY POINTS: • RSL inhibited the activity of Verrucomicrobium spinosum tyrosinase. • N-pre and C-terminal domain exerted stronger dual inhibition on the Vs-tyrosinase. • The activity of caspase-3 could be measured by the zymogen activation system.

Unveiling Antioxidant and Antiproliferative Effects of Prosopis juliflora Leaves against Human Prostate Cancer LNCaP Cells.

Herbal medications or formulations are regularly recommended by clinicians as a potential therapeutic method for a variety of human ailments, including cancer. Although Prosopis juliflora extracts have shown promise in anticancer activity, the effects on prostate cancer and the accompanying molecular mechanisms of action are still unexplored. This research aims at the antioxidant, antiproliferative, and apoptosis-inducing properties of Prosopis juliflora methanolic leaves extract in human prostate cancer LNCaP cells. The antioxidant ability of the extract was assessed using the DPPH (2, 2-diphenyl-2-picrylhydrazyl) and two additional reducing power tests. Antitumor activity was determined using MTT cell viability tests and LDH cytotoxicity assays. The probable mechanism of apoptotic cell death was further investigated utilizing a caspase-3 activation assay and qRT-PCR mRNA expression investigations of apoptotic-related genes. The results revealed that the methanol extract of Prosopis juliflora leaves contains alkaloids, flavonoids, tannins, glycosides, and phenols, all of which have substantial antioxidant activity. In vitro anticancer tests demonstrated that extract therapy resulted in a dose-dependent reduction in cell viability of LNCaP prostate cancer cells, but normal HaCaT cells showed no cytotoxic effects. Furthermore, plant extract therapy increased caspase-3 activation and mRNA expression of apoptotic-related genes, suggesting that this could be a mechanism for cancer cell growth suppression. The significance of Prosopis juliflora as a source of new antioxidant compounds against prostate cancer was emphasized in the current study. However, more study is needed to demonstrate the efficacy of Prosopis juliflora leaves extract in the treatment of prostate cancer.

A switch-on molecular biosensor for detection of caspase-3 and imaging of apoptosis of cells.

Apoptosis is a form of programmed cell death that is essential for maintaining internal environmental stability. Disordered apoptosis can cause a variety of diseases; therefore, sensing apoptosis can provide help in study of mechanism of the relevant diseases and drug development. It is known that caspase-3 is a key enzyme involved in apoptosis and the expression of its activity is an indication of apoptosis. Here, we present a genetically encoded switch-on mNeonGreen2-based molecular biosensor. mNeonGreen2 is the brightest monomeric green fluorescent protein. The substrate of caspase-3, DEVD amino acid residues, is inserted in it, while cyclized by insertion of Nostoc punctiforme DnaE intein to abolish the fluorescence (inactive state). Caspase-3-catalyzed cleavage of DEVD linearizes mNeonGreen2 and rebuilds the natural barrel structure to restore the fluorescence (activated state). The characterization exhibited that the Caspase-3 biosensor has shortened response time, higher sensitivity, and prolonged functional shelf life in detection of caspase-3 amongst the existing counterparts. We also used the Caspase-3 biosensor to evaluate the effect of several drugs on the induction of apoptosis of HeLa and MCF-7 tumor cells and inhibition of Zika virus invasion.

Ameliorative Effects of Gallic Acid on Cisplatin-Induced Nephrotoxicity in Rat Variations of Biochemistry, Histopathology, and Gene Expression.

BACKGROUND: Cisplatin is a powerful chemotherapeutic drug mainly used in the treatment of solid tumors. Aggregation of the drug in renal proximal tubule cells causes nephrotoxicity and renal failure. Investigations showed nephrotoxicity as Cisplatin's dose-limiting side effect. One of the Cisplatin toxicity mechanisms is generation of reactive oxygen species, which leads to oxidative stress and renal damage. The purpose of this study was evaluation of the modulating effects of Gallic acid on Cisplatin-induced variations including Caspase-3 and Clusterin expression and histopathological and biochemical parameters in adult male Wistar rats. METHOD: Rats were kept under standard condition of temperature, light, and humidity. The animals were divided into 4 groups: GpI: control group (received distilled water for 10 days); GpII: Gallic acid (alone) (50 mg/kg bw, once a day for 10 days); GpIII: Cisplatin (alone), single dose (6 mg/kg bw, I.P. on 5th day of study); GpIV: Gallic acid (50 mg/kg bw, once a day for 10 days) and also injected with single dose of Cisplatin (6 mg/kg bw, I.P., on 5th day of study). After 10 days, all rats were anaesthetized and plasma collected to estimate urea, creatinine, and uric acid. The right kidneys were removed for the study of gene expression and biochemical parameters. The left kidneys were used for histopathological studies. RESULTS: The Cisplatin-induced nephrotoxicity was evident from the elevated levels of creatinine, urea, uric acid, and renal tissue MDA and also decreased levels of SOD, CAT, GPX, and GSH in renal tissue. Administration of Gallic acid significantly modulated nephrotoxicity markers, gene expression variations, and histopathological damage. CONCLUSION: Outcomes of the present investigation suggest that Gallic acid provides protection against CP-induced nephrotoxicity, but for application in people, further studies are needed.

Cell death and pathological findings of the spleen in COVID-19 patients.

The coronavirus disease 2019(COVID-19) is recognized as systemic inflammatory response syndrome. It was demonstrated that a rapid increase of cytokines in the serum of COVID-19 patients is associated with the severity of disease. However, the mechanisms of the cytokine release are not clear. By using immunofluorescence staining we found that the number of CD11b positive immune cells including macrophages in the spleens of died COVID-19 patients, was significantly higher than that of the control patients. The incidence of apoptosis as measured by two apoptotic markers, TUNEL and cleaved caspase-3, in COVID-19 patients' spleen cells is higher than that in control patients. By double immunostaining CD11b or CD68 and SARS-CoV-2 spike protein, it was found that up to 67% of these immune cells were positive for spike protein, suggesting that viral infection might be associated with apoptosis in these cells. Besides, we also stained the autophagy-related molecules (p-Akt、p62 and BCL-2) in spleen tissues, the results showed that the number of positive cells was significantly higher in COVID-19 group. And compared with non-COVID-19 patients, autophagy may be inhibited in COVID-19 patients. Our research suggest that SARS-CoV-2 may result in a higher rate of apoptosis and a lower rate of autophagy of immune cells in the spleen of COVID-19 patients. These discoveries may increase our understanding of the pathogenesis of COVID-19.

Evaluation of Silver Nanoparticles Caffeic Acid Complex Compound as New Potential Therapeutic Agent against Cancer Incidence in Mice.

OBJECTIVE: The present work was designed to study the effect of new conjugated caffeic and folic acid with silver nanoparticles with definite molecular size applied with and without gamma radiation exposure, as an antitumor agent against experimentally induced Ehrlich tumor and attempted to identify their potential molecular mechanisms of action throughout determination of anti-tumor activities using MTT cytotoxic assay against two human carcinoma cell lines in vitro, such as apoptosis analysis by flow cytometry through caspase-8, caspase-3 and TNF determination in vivo. MATERIALS AND METHODS: Adult female albino mice were used and divided into five groups. Animals were sacrificed and the following parameters were estimated, glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD) in blood in addition to caspase8, caspase 3 and tumor necrosis factor (TNF) of tumor tissue, liver and kidney function also measured in plasma. The tumor specimens were processed for histopathological examination. RESULTS: Nano-silver folate caffeic (NSFC) complex compound treatment resulted in growth inhibition in Ehrlich solid tumor, Hep-G2, and MCF-7 cells (IC50 0.062 mg, 7.70 µM, and 14.50 µM, respectively). Flow cytometric analysis revealed that (NSFC) with radiation IR had apoptotic effects at caspases 8 (Mean±SD) (49.4±14), caspase3 (39.97±9.75), and TNF (40.1±3.4) more than any other groups. Those disturbances were found to be associated with a kinetic induction of apoptosis and showed modulation of the antioxidant system {glutathione (GSH), glutathione peroxidase (GPx) and superoxide dismutase (SOD) which were 60.70±0.80, 26.73±0.80, 39.52±0.58 respectively}at the group which took (NSFC+IR), besides its high percentage of necrotic cells by histopathological studies. In conclusion, the present study showed that the treatment of (NSFC) exhibits very efficient oncolytic activity in delaying tumor growth in mice bearing Ehrlich Solid Carcinoma (ESC) and the mechanisms underlying the inhibitory effect of the present compound involve both an apoptotic effect against Hep-G2 and MCF-7 cells and modulation of antioxidant system.

Functional and structural alternations in the choroid plexus upon methamphetamine exposure.

Choroid plexus (CP) is the principal source of cerebrospinal fluid. CP can produce and release a wide range of materials including growth factors, neurotrophic factors, etc. all of which play an important role in the maintenance and proper functioning of the brain. Methamphetamine (METH) is a CNS neurostimulant that causes brain dysfunction. Herein, we investigated the potential effects of METH exposure on CP structure and function. Stereological analysis revealed a significant alteration in CP volume, epithelial cells and capillary number upon METH treatment. Electron microscopy exhibited changes in ultrastructure. Moreover, the upregulation of neurotrophic factors such as BDNF and VEGF as well as autophagy and apoptosis gene following METH administration were observed. We also identified several signaling cascades related to autophagy. In conclusion, gene expression changes coupled with structural alterations of the CP in response to METH suggested METH-induced autophagy in CP.

Dipolar coupling-based electron paramagnetic resonance method for protease enzymatic characterization and inhibitor screening.

Herein, we report an EPR-based method for protease enzymatic characterization and inhibitor screening. This method utilizes dual paramagnetically-labeled probes consisting of a nitroxide spin probe and a Gd3+ ion flanking a peptide that could be specifically cleaved by protease caspase-3. Distance-dependent dipolar coupling between the two paramagnetic centers can be modulated by the protease cleavage activity, thus providing a straightforward and convenient method for protease activity detection using EPR spectroscopy under ambient conditions. Moreover, time-course monitoring of the protease-catalyzed cleavage reaction demonstrated that this EPR-based method could not only allow a direct quantitative enzymatic kinetic assessment, but also could be used for protease inhibitor screening, thus holding great potential in drug discovery studies.

Placental apoptotic markers are associated with placental morphometry.

INTRODUCTION: Preeclampsia is a hypertensive disorder affecting both mother and the fetus and is a major cause of maternal and neonatal morbidity and mortality. Abnormal placentation is a common feature in preeclampsia that contributes to placental dysfunction. It is likely that increased homocysteine and oxidative stress influence apoptosis in preeclampsia. Increased placental apoptosis may aggravate the symptoms of preeclampsia through disruption of the placental structure. The current study aims to examine the association between various placental apoptotic markers with placental dimensions and maternal and neonatal characteristics in women with preeclampsia. METHODS: A total of 80 pregnant women [preeclampsia (n = 40); normotensive control (n = 40)] were included in the study. Placental characteristics such as its major axis, minor axis, breadth, thickness (at centre, cord insertion and periphery) and trimmed placental weight were recorded.Placental protein levels of caspase-3, caspase-8, BAX and Bcl-2 were estimated by ELISA and gene expression were examined by real time quantitative PCR. RESULT: Protein levels of proapoptotic markers such as caspase-8 and 3 were higher (p < 0.01) in the preeclampsia group compared to control whereas, the level of antiapoptotic marker Bcl-2 (p < 0.05) was lower in the preeclampsia group. Caspase-3 and Bcl-2 protein levels were negatively associated with thickness of placenta at cord insertion (p < 0.01). Protein levels of caspase-8 and caspase-3 were positively associated with placental MDA levels (p < 0.01). Caspase-8 was negatively associated with baby length (p = 0.055). DISCUSSION: This study demonstrates the association of various apoptotic markers with oxidative stress and placental dimensions.

Effects of hyperbaric oxygen pretreatment on brain antioxidant capacity in rats with decompression sickness.

OBJECTIVE: Decompression sickness (DCS) causes serious brain hypoxic-ischemic injury. This experiment was designed to observe whether hyperbaric oxygen (HBO2) pretreatment played a neuroprotective effect in decompression sickness rat models and to explore the mechanism of protective effects. METHODS: Sprague-Dawley (SD) male rats were pretreated with HBO2 and then underwent decompression to establish the DCS rat model. Antioxidant capacities were evaluated by detecting peroxides (GPx), superoxide dismutase (SOD), catalase (CAT) activity and malondialdehyde (MDA) content in brains. The levels of metal elements manganese (Mn), zinc (Zn), iron (Fe) and magnesium (Mg) in brain tissues were assessed by flame atomic absorption spectrometry. Necrosis and apoptosis of neurons were assessed by H-E staining and immunohistochemical staining. RESULTS: HBO2 pretreatment reduced the degree of necrosis and apoptosis in brain tissues of decompression sickness rat models. In addition, HBO2 pretreatment increased GPx, SOD and CAT activities and reduced MDA accumulation. It also increased the content of Mn, Zn, Fe and Mg in brain tissue, which are all related to free radical metabolism. CONCLUSION: These results suggested that HBO2 pretreatment has protective effects on brain injury of rats with decompression sickness. The mechanism of the protective effects may be related to reducing oxidative damage by affecting metal elements in vivo.

Caspase-3 and gasdermin E detection in peri-implantitis.

Peri-implantitis could lead to progressive bone loss and implant failure; however, the mechanism of peri-implantitis remains unclear. Based on emerging evidence, pyroptosis, a novel proinflammatory programmed death, contributes to different oral infectious diseases. In the present study, we investigated the involvement of cleaved caspase-3 and gasdermin E (GSDME) in peri-implantitis and established a pyroptosis model in vitro. By collecting and examining the inflamed biopsies around peri-implantitis, we found that the pyroptosis-related markers (caspase-3, GSDME, and IL-1ß) were enhanced relative to levels in control individuals. Furthermore, human gingival epithelium cells (HGECs) induced by tumor necrosis factor-α (TNF-α) exhibited pyroptosis morphological changes (cell swelling and balloon-shaped bubbles) and upregulated expression of pyroptosis-related markers. Pretreated with Ac-DEVD-CHO (a caspase-3 inhibitor) or GSDME small interference RNA (siRNA) were found to attenuate pyroptosis in HGECs. In conclusion, our findings revealed a high expression of caspase-3 and GSDME in the inflamed biopsies of peri-implantitis and confirmed that the caspase-3/GSDME pathway mediates TNF-α-triggered pyroptosis in human gingival epithelium cells, which provides a new target for peri-implantitis treatment.

Neuroprotective Effect of Taurine against Cell Death, Glial Changes, and Neuronal Loss in the Cerebellum of Rats Exposed to Chronic-Recurrent Neuroinflammation Induced by LPS.

The present study investigated the neuroprotective effect of taurine against the deleterious effects of chronic-recurrent neuroinflammation induced by LPS in the cerebellum of rats. Adult male Wistar rats were treated with taurine for 28 days. Taurine was administered at a dose of 30 or 100 mg/kg, by gavage. On days 7, 14, 21, and 28, the animals received LPS (250 µg/kg) intraperitoneally. The vehicle used was saline. The animals were divided into six groups: vehicle, taurine 30 mg/kg, taurine 100 mg/kg, LPS, LPS plus taurine 30 mg/kg, and LPS plus taurine 100 mg/kg. On day 29, the animals were euthanized, and the cerebellum was removed and prepared for immunofluorescence analysis using antibodies of GFAP, NeuN, CD11b, and cleaved caspase-3. LPS group showed a reduction in the immunoreactivity of GFAP in the arbor vitae and medullary center and of NeuN in the granular layer of the cerebellar cortex. LPS increased the immunoreactivity of CD11b in the arbor vitae and in the medullary center. Taurine protected against these effects induced by LPS in immunoreactivity of GFAP, NeuN, and CD11b, with the 100 mg/kg dose being the most effective. LPS induced an increase in the number of positive cleaved caspase-3 cells in the Purkinje cell layers, granular layer, arbor vitae, and medullary center. Taurine showed its antiapoptotic activity by reducing the cleaved caspase-3 cells in relation to the LPS group. Here, a potential neuroprotective role of taurine can be seen since this amino acid was effective in protecting the cerebellum of rats against cell death and changes in glial and neuronal cells in the face of chronic-recurrent neuroinflammation.

Apoptosis in platelets from adult patients with chronic idiopathic thrombocytopenic purpura.

Adult chronic idiopathic thrombocytopenic purpura (cITP) is a chronic and usually life-long haemorrhagic disorder in which enhanced platelet destruction and weakened platelet production lead to thrombocytopenia. Platelets were isolated from blood samples collected from 40 adult patients with cITP and 40 healthy volunteers. Mitochondrial membrane potential (ΔΨm) and plasma membrane phosphatidylserine externalization were determined by flow cytometry, and activation of caspase-3 and expressions of Bax, Bak and Bcl-xL were analysed by western blotting. Flow cytometry showed increased mitochondrial depolarization and lower ΔΨm in platelets from adult patients with cITP. In addition, plasma membrane phosphatidylserine externalization was observed on platelets from adult patients with cITP, but rarely from healthy volunteers. Western blot analysis of platelet proteins revealed that, in adult cITP patients, caspase-3 was activated, which cleaved gelsolin and to release a 47-kDa fragment. Moreover, the expressions of Bax and Bak were elevated, and Bcl-xL was decreased markedly in platelets from adult patients with cITP. Our findings reveal, based on loss of mitochondrial membrane potential (Δψm), phosphatidylserine exposure, caspase-3 activation, enhanced expression of Bax and Bak, and attenuated expression of Bcl-xL, that platelet death in the pathogenesis of thrombocytopenia in chronic ITP in adults is apoptotic.

Xanthohumol-Induced Rat Glioma C6 Cells Death by Triggering Mitochondrial Stress.

AIM: To investigate the underlying mechanisms of xanthohumol (XN) on the proliferation inhibition and death of C6 glioma cells. METHODS: To determine the effects of XN on C6 cells, cell proliferation and mortality after XN treatment were assessed by SRB assay and trypan blue assay respectively. Apoptotic rates were evaluated by flowcytometry after Annexin V-FITC/PI double staining. The influence of XN on the activity of caspase-3 was determined by Western blot (WB); and nuclear transposition of apoptosis-inducing factor (AIF) was tested by immunocytochemistry and WB. By MitoSOXTM staining, the mitochondrial ROS were detected. Mitochondrial function was also tested by MTT assay (content of succinic dehydrogenase), flow cytometry (mitochondrial membrane potential (MMP)-JC-1 staining; mitochondrial abundance-mito-Tracker green), immunofluorescence (MMP-JC-1 staining; mitochondrial morphology-mito-Tracker green), WB (mitochondrial fusion-fission protein-OPA1, mfn2, and DRP1; mitophagy-related proteins-Pink1, Parkin, LC3B, and P62), and high-performance liquid chromatography (HPLC) (energy charge). Finally, mitochondrial protein homeostasis of C6 cells after XN treatment with and without LONP1 inhibitor bortezomib was investigated by trypan blue assay (proliferative activity and mortality) and WB (mitochondrial protease LONP1). All cell morphology images were taken by a Leica Microsystems microscope. RESULTS: XN could lead to proliferation inhibition and death of C6 cells in a time- and dose-dependent manner and induce apoptosis of C6 cells through the AIF pathway. After long incubation of XN, mitochondria of C6 cells were seriously impaired, and mitochondria had a diffuse morphology and mitochondrial ROS were increased. The content of succinic dehydrogenase per cell was significantly decreased after XN insults of 24, 48, and 72 h. The energy charge was weakened after XN insult of 24 h. Furthermore, the MMP and mitochondrial abundance were significantly decreased; the protein expression levels of OPA1, mfn2, and DRP1 were down-regulated; and the protein expression levels of Pink1, Parkin, LC3B-II/LC3B-I, and p62 were up-regulated in long XN incubation times (24, 48, and 72 h). XN incubation with bortezomib for 48 h resulted in lower proliferative activity and higher mortality of C6 cells and caused the cell to have visible vacuoles. Moreover, the protein expression levels of LONP1 was up-regulated gradually as XN treatment time increased. CONCLUSION: These data supported that XN could induce AIF pathway apoptosis of the rat glioma C6 cells by affecting the mitochondria.

Electrochemical determination of caspase-3 using signal amplification by HeLa cells modified with silver nanoparticles.

An electrochemical sensor capable of quantitative determination of caspase-3 activities was developed. A thiolated peptide whose sequence contained a caspase-3 cleaved site and a cell penetration sequence was preimmobilized onto an electrode. The quantification of caspase-3 was accomplished after cell penetration and the subsequent adsorption of silver nanoparticles (AgNPs). The oxidation current of AgNPs was found to be inversely proportional to the concentration of caspase-3 between 0.02 and 0.2 U/mL. A detection limit of 0.02 U/mL for caspase-3 was achieved due to the large number of positively charged AgNPs adsorbed onto the negatively charged cells. The proof of concept was demonstrated by monitoring the cleavage of surface-confined peptide substrates by caspase-3 in cell lysates. The current sensor could be extended to detect cells by replacing the surface-confined peptide with aptamers that recognize cells. Thus, the use of a cell as a matrix for AgNPs shows excellent potential for constructing electrochemical sensors and provides a useful alternative for sensor development in the future. Cells modified with silver nanoparticles were utilized as the electrochemical readout of an electrochemical assay.

5-Fluorouracil disrupts ovarian preantral follicles in young C57BL6J mice.

PURPOSE: 5-Fluorouracil (5-FU), an anti-cancer drug, has been used for hepatoblastoma (HB) chemotherapy in children, who may have impaired  ovarian follicle pool reserve with lasting effects to reproduction. Therefore, this study aimed to investigate 5-FU effects on survival, growth, and morphology of ovarian preantral follicles from C57BL6J young mice. METHODS: Experiments were carried-out both in vivo and in vitro. Mice were treated with 5-FU injection (450 mg/kg i.p) or saline and sacrificed 3 days after to obtain ovaries for histology and molecular biology. Ovaries for in vitro studies were obtained from unchallenged mice and cultured under basic culture medium (BCM) or BCM plus 5-FU (9.2, 46.1, 92.2 mM). Preantral follicles were classified according to developmental stages, and as normal or degenerated. To assess cell viability, caspase-3 immunostaining was performed. Transcriptional levels for apoptosis (Bax, Bcl2, p53, Bax/Bcl2) and Wnt pathway genes (Wnt2 and Wnt4) were also analyzed. Ultrastructural analyses were carried-out on non-cultured ovaries. In addition, ß-catenin immunofluorescence was assessed in mouse ovaries. RESULTS: The percentage of all-types normal follicles was significantly lower after 5-FU challenge. A total loss of secondary normal follicles was found in the 5-FU group. The highest 5-FU concentrations reduced the percentage of cultured normal primordial follicles. Large vacuoles were seen in granulosa cells and ooplasm of preantral follicles by electron microscopy. A significantly higher gene expression for Bax and Bax/Bcl2 ratio was seen after 5-FU treatment. A marked reduction in ß-catenin immunolabeling was seen in 5-FU-challenged preantral follicles. In the in vitro experiments, apoptotic and Wnt gene transcriptions were significantly altered. CONCLUSION: Altogether, our findings suggest that 5-FU can deleteriously affect the ovarian follicle reserve by reducing preantral follicles survival.

Enhanced Intracellular Delivery of Curcumin by Chitosan-Lipoic Acid as Reduction-Responsive Nanoparticles.

AIMS: Enhancement of anti-tumor activity of the chemotherapeutic agent CUR by redoxsensitive nanoparticle to get a deeper insight into cancer therapy. BACKGROUND: Tumor targetability and stimulus are widely used to study the delivery of drugs for cancer diagnosis and treatment because poor cellular uptake and inadequate intracellular drug release lead to inefficient delivery of anticancer agents to tumor tissue. OBJECTIVE: Studies distinguishing between tumor and normal tissues or redox-sensitive systems using glutathione (GSH) as a significant signal. METHODS: In this study, we designed Chitosan-Lipoic acid Nanoparticles (CS-LANPs) to improve drug delivery for breast cancer treatment by efficient delivery of Curcumin (CUR). The properties of blank CS-LANPs were studied in detail. The size and the Polydispersity Index (PDI) of the CS-LANPs were optimized. RESULTS: The results indicate the mean size and PDI of the blank CS-LANPs were around 249 nm and 0.125, respectively. However, the Drug Loading (DL) and Encapsulation Efficiency (EE) of the CSLANPs were estimated to be about 18.22% and 99.80%, respectively. Compared to non-reductive conditions, the size of reduction-sensitive CS-LANPs increased significantly under reductive conditions. Therefore, the drug release of CS-LANPs in the presence of glutathione was much faster than that of non-GSH conditions .Moreover, the antitumor effect of CS-LANPs on MCF-7 cells was determined in vitro by MTT assay, cell cytotoxicity, Caspase-3 Assay, detection of mitochondrial membrane potential and quantification of apoptosis incidence. CONCLUSION: CS-LANPs showed a remarkably increased accumulation in tumor cells and had a better tumor inhibitory activity in vitro. CS-LANPs could successfully deliver drugs to cancer cells and revealed better efficiency than free CUR.