D-Amino acids differentially trigger an inflammatory environment in vitro

To evade the immune system, the etiologic agent of plague, Yersinia pestis, injects an exceptionally active tyrosine phosphatase called YopH into host cells using a type III secretion system. We recently reported that YopH acutely inhibits T cell antigen receptor signaling by dephosphorylating the Lck tyrosine kinase. Here, we show that prolonged presence of YopH in primary T cells or Jurkat T leukemia cells causes apoptosis, detected by annexin V binding, mitochondrial breakdown, caspase activation, and internucleosomal fragmentation. YopH also causes cell death when expressed in HeLa cells, and this cell death was inhibited by YopH-specific small molecule inhibitors. Cell death induced by YopH was also prevented by caspase inhibition or co-expression of Bcl-xL. We conclude that YopH not only paralyzes T cells acutely, but also ensures that the cells will not recover to induce a protective immune response but instead undergo mitochondrially regulated programmed cell death.

Purpose: Cobiprostone is being clinically tested as a protectant against nonsteroidal anti-inflammatory drug (NSAID)-induced ulcers, and the present study is to determine the cellular and molecular basis of protection by cobiprostone against NSAID-induced cellular damage. Methods: T84 cells were grown to confluence on solid supports. The effects of cobiprostone on indomethacin-induced changes in intracellular Ca2+ levels ([Ca2+]i) using Indo-1/AM (a dye that measures [Ca2+]I), and the effects of cobiprostone on mitochondrial membrane potential using JC-1 (a dye that monitors mitochondrial membrane potential), were tested. The effects of cobiprostone on indomethacin-induced cytochrome c release, cyclic AMP, and cell death and regrowth were also investigated. Results: Five hundred (500) μM indomethacin caused an immediate increase in [Ca2+]i, which became irreversible (calcium deregulation) after approximately 30 minutes. One hundred (100) nM cobiprostone was shown to protect against indomethacin-induced increases in [Ca2+]i, and caused a reversal of indomethacin-induced calcium deregulation. However, cobiprostone did not prevent or reverse the increase in ([Ca2+]i caused by indomethacin in the presence of different Cl− channel blockers, including diphenylamine-2-carboxylate (DPC), niflumic acid or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), suggesting that the protective effects of cobiprostone are due to activation of Cl− channels. Cobiprostone has only a small effect on cAMP levels in T84 cells, precluding a role of cAMP in the protective action of cobiprostone. Indomethacin caused depolarization of the mitochondrial membrane potential. Cobiprostone protected against indomethacin-induced depolarization of mitochondrial membrane potential and reversed the loss of mitochondrial membrane potential. Cobiprostone was also shown to protect against cytochrome c relocation from mitochondria into the cytoplasm, which is associated with apoptosis. Finally, in a novel long-term assay using T84 cell cultures, cobiprostone reduced indomethacin-induced cell death and promoted cell regrowth. Conclusion: Activation of Cl− channels may underlie the protective effects of cobiprostone in protection against NSAID-induced damage. The results further indicate that cobiprostone has the potential to be of benefit against NSAID-induced cell death. This research was funded entirely by Sucampo Pharmaceuticals, Inc., Bethesda, MD.

Stroke and traumatic brain injury augment the production of reactive oxygen species (ROS) leading to apoptosis in cerebral arteries, and females are more resilient to vascular damage than males. Depolarization of mitochondrial membrane potential (ΔΨm) is a key event in apoptosis, however, when ΔΨm is suffciently depolarized, ATP synthase can reverse directions and act as a proton pump to limit ΔΨm depolarization. We hypothesized that ATP synthase attenuates depolarization of ΔΨm in cerebral arteries during acute oxidative stress thus limiting cell death. Posterior cerebral arteries (PCA; ~80 μm diameter) from male and female mice (age, 4-6 mo) were isolated, cannulated, and pressurized to 90 cm H2O at 36°C. Cell death was quantified with Hoechst 33342 (1 μM, labels all nuclei) and propidium iodide (2 μM, labels dead nuclei), and ΔΨm was evaluated with tetramethylrhodamine methyl ester (TMRM, 10 nM). Vessels were exposed to H2O2 (200 μM) in the presence/absence of ATP synthase inhibitor oligomycin (2 μM). H2O2 exposure (50 min) led to significantly (p<0.05) greater smooth muscle cell death in males compared to females (30±7.4% vs. 7±3%; n=8); there was a similar trend for endothelial cell death. Oligomycin greatly augmented apoptosis in PCAs from both males and females to ~80% and eliminated differences between sexes. Consistently, H2O2 evoked a more robust depolarization of ΔΨm in males vs. females and oligomycin enhanced ΔΨm depolarization to H2O2 leading to a similar response in both sexes. To directly depolarize ΔΨm, PCAs were treated with the protonophore FCCP (10 μM). Surprisingly, depolarization of ΔΨm to FCCP was greater in males vs. females, and oligomycin augmented depolarization and eliminated differences between sexes. FCCP initiated minimal (<5%) cell death at 50-min or 3-h exposure; oligomycin did not alter cell death to FCCP. We conclude that: 1) cerebral vessels from female mice possess greater resilience to H2O2 -induced apoptosis than males by limiting depolarization of ΔΨm; and 2) oxidative stress triggers cell death through pathways in addition to ΔΨm depolarization. Support: NIH R01NS134690. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

This study aims to evaluate the in vitro cytotoxic and anti-migratory effects of Marantodes pumilum Blume Kuntze plant extracts on prostate cancer cells, identify the active compound/s, and characterize their mechanism of action. The crude methanolic extract was partitioned into n-hexane (MPh), chloroform (MPc), and aqueous (MPa) extracts. Antiproliferative fractions (IC50 < 30 μg/mL based on SRB staining of LNCaP and PC3 cell lines) were further fractionated. Active compound/s were identified using spectroscopic methods. In vitro mechanistic studies on PC3 cells included: annexin V-FITC staining, mitochondrial membrane potential (MMP) depolarization measurements, the activity of caspases 3 and 7, nuclear DNA fragmentation, cell cycle analysis, modulation of Bax, Bcl-2, Smac/Diablo, Alox-5, VEGF-A, CXCR4, and CXCL12 mRNA gene expression via RT-PCR, 2D migration (scratch assay), and 3D invasion (Boyden chamber). MPc extract was the most active, inducing cell death (p < 0.05) via apoptosis, as evidenced by nuclear DNA fragmentation and an increase in MMP depolarization (p < 0.05) as well as the activation of caspases 3/7 (MPc p < 0.01) in both PC3 and LNCaP cell lines. In addition, MPc upregulated Bax and Smac/DIABLO, downregulated Bcl-2 (p < 0.05), and inhibited ALOX-5 mRNA gene expression (p < 0.001). MPc was not cytotoxic against normal human fibroblast cells (HDFa) at the tested concentrations. Moreover, MPc inhibited migration and invasion of PC3 cells (p < 0.01). These effects were accompanied by the downregulation of both VEGF-A and CXCL-12 gene expressions (p < 0.001). A monounsaturated 5-alkyl resorcinol was isolated as the active compound in the MPc extract and identified as 5-henicosene-1-yl-resorcinol.

To investigate the antioxidative effects of two cysteinyl leukotriene receptors antagonists (CysLT1R and CysLT2R) montelukast and HAMI 3379 on ischemic injury of rat cortical neurons in vitro. Cultured rat cortical neurons were pretreated with CysLT1R antagonist montelukast and CysLT2R antagonist HAMI 3379, and then exposed to oxygen-glucose deprivation/recovery (OGD/R）or H2O2. Reactive oxygen species (ROS) mitochondrial membrane potential (MMP) depolarization, neuronal viability and lactate dehydrogenase (LDH) release were determined. Meanwhile, RNA interference was used to inhibit the expression of CysLT1R and CysLT2R，and the effects were observed. ROS production in neurons was significantly increased after 1 h OGD, which reached the peak at 30 min and lasted for 1.5 h after recovery. Montelukast and HAMI 3379 at 0.01-1μmol/L moderately decreased OGD/R-induced ROS production (P<0.05). Montelukast mildly attenuated OGD/R-induced MMP depolarization (P<0.05)，but HAMI 3379 had no effect. H2O2 reduced neuronal viability and increased LDH release, namely inducing neuronal injury. Montelukast and HAMI 3379 at 0.1-1μmol/L moderately attenuated H2O2-induced neuronal injury (P<0.05). However, both CysLT1R siRNA and CysLT2R shRNA did not significantly affect the responses mentioned above. In ischemic neuronal injury, montelukast and HAMI 3379 exert a moderate antioxidative effect, and this effect may be receptor-independent.

Diosgenin is a steroidal sapogenin with estrogenic and antitumor properties. In order to elucidate the mechanism of its antiproliferative activity, we investigated its effects on the cell cycle and apoptosis in human chronic myelogenous leukemia K562 cells. Cell viability was assessed via an MTT assay. Apoptosis was investigated in terms of nuclear morphology, DNA fragmentation, and phosphatidylserine externalization. Cell cycle analysis was performed via PI staining and flow cytometry (FCM). Western blotting and immunofluorescence methods were used to determine the levels of p53, cell cycle-related proteins and Bcl-2 family members. FCM was also used to estimate the changes in mitochondrial membrane potential (MMP), intracellular Ca2+ concentration and reactive oxygen species (ROS) generation. Cell cycle analysis showed that diosgenin caused G2/M arrest independently of p53. The levels of cyclin B1 and p21Cip1/Waf1 were decreased, whereas cdc2 levels were increased. Subsequent apoptosis was demonstrated with the dramatic activation of caspase-3. A dramatic decline in intracellular Ca2+ concentration was observed as an initiating event in the process of cell cycle arrest and apoptosis, which was followed by the hyperpolarization and depolarization of MMP. Generation of ROS was observed in the progression of apoptosis. The antiapoptotic Bcl-2 and Bcl-xL proteins were downregulated, whereas the proapoptotic Bax was upregulated. Diosgenin inhibits K562 cell proliferation via cell cycle G2/M arrest and apoptosis, with disruption of Ca2+ homeostasis and mitochondrial dysfunction playing vital roles.

Background/Aims: Hypercholesterolemia is recently considered a risk factor for Parkinson’s disease (PD), the most consistent neurodegenerative movement disorder. The study aimed to investigate the effect of exogenous cholesterol on 1-methyl-4-phenylpyridinium (MPP⁺) parkinsonian neurotoxin-induced cell death, loss of mitochondrial membrane potential, and dopaminergic deficiency in a cellular model of PD. Methods: Cholesterol (50 μM) when added in the culture media alone or in combination with MPP⁺ was studied in SH-SY5Y neuroblastoma cells. There were 4 groups that were studied; SH-SY5Y cells treated with vehicle (control), cells that were treated with 1 mM MPP⁺ (MPP⁺), or cholesterol (chol) or both (M + chol). The loss of cell survival was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Dopamine depletion, microtubule-associated protein 2 (MAP-2), and tyrosine hydroxylase (TH)-positive neuronal loss were determined by HPLC-electrochemical detection and TH immunocytochemistry respectively. Mitochondrial membrane potential in cells stained by tetramethylrhodamine methyl ester dye was analysed by flow cytometry. Results: Cholesterol treatment potentiated a reduction of neuronal viability with loss of TH-positive neurons in cultures. MPP⁺-induced depletion of dopamine level in the post-mitotic MAP-2 immunoreactive neurons and loss of mitochondrial membrane potential were also heightened by cholesterol. Conclusion: Apparently, changes in neuronal cholesterol content significantly influenced the neurotoxicity and the direct mitochondrial mechanisms involved in MPP⁺-induced cell death. Our observations demonstrate that high cholesterol incorporated into the differentiated human neuroblastoma cells worsened dopaminergic neuronal survivability through increased depolarization of mitochondrial membrane potential, which is a known mechanism of dopaminergic cell death by MPP⁺. The present findings support the hypothesis that hypercholesterolemia could be a risk factor for PD.

Cdk2 activity is associated with depolarization of mitochondrial membrane potential during apoptosis

Bcl-2 protects against FCCP-induced apoptosis and mitochondrial membrane potential depolarization in PC12 cells

Dillenia suffruticosa, which is locally known as Simpoh air, has been traditionally used to treat cancerous growth. The ethyl acetate extract of D. suffruticosa (EADs) has been shown to induce apoptosis in MCF-7 breast cancer cells in our previous study. The present study aimed to elucidate the molecular mechanisms involved in EADs-induced apoptosis and to identify the major compounds in the extract. EADs was found to promote oxidative stress in MCF-7 cells that led to cell death because the pre-treatment with antioxidants α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of the extract (P<0.05). DCFH-DA assay revealed that treatment with EADs attenuated the generation of intracellular ROS. Apoptosis induced by EADs was not inhibited by the use of caspase-inhibitor Z-VAD-FMK, suggesting that the cell death is caspase-independent. The use of JC-1 dye reflected that EADs caused disruption in the mitochondrial membrane potential. The related molecular pathways involved in EADs-induced apoptosis were determined by GeXP multiplex system and Western blot analysis. EADs is postulated to induce cell cycle arrest that is p53- and p21-dependent based on the upregulated expression of p53 and p21 (P<0.05). The expression of Bax was upregulated with downregulation of Bcl-2 following treatment with EADs. The elevated Bax/Bcl-2 ratio and the depolarization of mitochondrial membrane potential suggest that EADs-induced apoptosis is mitochondria-dependent. The expression of oxidative stress-related AKT, p-AKT, ERK, and p-ERK was downregulated with upregulation of JNK and p-JNK. The data indicate that induction of oxidative-stress related apoptosis by EADs was mediated by inhibition of AKT and ERK, and activation of JNK. The isolation of compounds in EADs was carried out using column chromatography and elucidated using the nuclear resonance magnetic analysis producing a total of six compounds including 3-epimaslinic acid, kaempferol, kaempferide, protocatechuic acid, gallic acid and β-sitosterol-3-O-β-D-glucopyranoside. The cytotoxicity of the isolated compounds was determined using MTT assay. Gallic acid was found to be most cytotoxic against MCF-7 cell line compared to others, with IC50 of 36 ± 1.7 μg/mL (P<0.05). In summary, EADs generated oxidative stress, induced cell cycle arrest and apoptosis in MCF-7 cells by regulating numerous genes and proteins that are involved in the apoptotic signal transduction pathway. Therefore, EADs has the potential to be developed as an anti-cancer agent against breast cancer.

Chlorpyrifos induces apoptosis and DNA damage in Drosophila through generation of reactive oxygen species

The fluorescent benzothiazole dye thioflavin T (ThT) is widely used as a marker for protein aggregates, most commonly in the context of neurodegenerative disease research and diagnosis. Recently, this same dye was shown to indicate membrane potential in bacteria due to its cationic nature. This finding prompted a question whether ThT fluorescence is linked to the membrane potential in mammalian cells, which would be important for appropriate utilization of ThT in research and diagnosis. Here, we show that ThT localizes into the mitochondria of HeLa cells in a membrane-potential-dependent manner. Specifically, ThT colocalized in cells with the mitochondrial membrane potential indicator tetramethylrhodamine methyl ester (TMRM) and gave similar temporal responses as TMRM to treatment with a protonophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). Additionally, we found that presence of ThT together with exposure to blue light (λ = 405 nm), but neither factor alone, caused depolarization of mitochondrial membrane potential. This additive effect of the concentration and blue light was recapitulated by a mathematical model implementing the potential-dependent distribution of ThT and its effect on mitochondrial membrane potential through photosensitization. These results show that ThT can act as a mitochondrial membrane potential indicator in mammalian cells, when used at low concentrations and with low blue light exposure. However, it causes dissipation of the mitochondrial membrane potential depending additively on its concentrations and blue light exposure. This conclusion motivates a re-evaluation of ThT’s use at micromolar range in live-cell analyses and indicates that this dye can enable future studies on the potential connections between mitochondrial membrane potential dynamics and protein aggregation.

A polyherbal medicine, Habb-e-Ustukhuddus (HU), is used for its anti-inflammatory properties. However, the anticancer and chemopreventive properties of HU were not known, and Therefore, investigated in the present study. Cancer cells were treated with 50-400 µg/ml HU and MTT, trypan blue, and clonogenic assays were performed. Propidium iodide (PI) staining, annexin V-FITC assay, and JC-1 staining were done for cell cycle progression, apoptosis, and mitochondrial membrane potential, respectively, using flow cytometry. Immunoblotting, cell migration and invasion assays were performed. Chemical characterization of HU was done through GC-MS and HPLC analyses. C57BL/6 mice were used to assess the in vivo toxicity of HU. While evaluating the anticancer activity, the methanolic extract of HU (50-400 µg/ml) strongly inhibited the growth and survival (P<0.05-0.001) of lung and breast cancer cells and increased the cell population in the sub-G1 phase of the cell cycle. HU caused apoptotic death of cancer cells (P<0.05-0.001), which was associated with the depolarization of mitochondrial membrane potential (Δψ) (P<0.001) and an increase in Bax to Bcl-2 protein ratio. Further, HU inhibited the invasion and migration of cancer cells, which was accompanied by an increase in the epithelial marker, E-cadherin, and a decrease in the mesenchymal marker, vimentin. The HU characterization by GC-MS and HPLC analyses showed the abundance of bioactive compounds including flavonoids and alkaloids. In the chemopreventive study, the oral administration of methanolic extract of the formulation HU (50 and 100 mg/kg body weight) to mice did not cause any toxicity and significantly increased the specific activities of hepatic drug metabolizing phase I and phase II enzymes, which suggested for its detoxification potential of xenobiotic compounds. Together, these results demonstrated the anticancer potential HU, without any apparent toxicity in mice, and thus HU could be further explored for its clinical utility in cancer control.

Candida biofilms play an important role in infections associated with medical devices and are resistant to antifungals. We hypothesized that the echinocandin micafungin (MICA) exerts an enhanced antifungal activity against caspofungin (CAS)-susceptible (CAS-S) and CAS–non-susceptible (CAS-NS) Candida albicans and Candida parapsilosis which is at least in part through apoptosis, even in the biofilm environment. Apoptosis was characterized by detecting reactive oxygen species (ROS) accumulation, depolarization of mitochondrial membrane potential (MMP), DNA fragmentation, lack of plasma membrane integrity, and metacaspase activation following exposure of Candida biofilm to MICA for 3h at 37°C in RPMI 1640 medium. The minimum inhibitory concentration was higher for CAS (2.0–16.0 μg/mL) than for MICA (1.0–8.0 μg/mL) for Candida biofilms. Elevated intracellular ROS levels and depolarization of MMP was evident in CAS-S C. albicans (3.0–4.2 fold) and C. parapsilosis (4.8–5.4 fold) biofilms compared with CAS-NS (1.2 fold) after exposure to MICA (0.25x-1xMIC). Elevated intracellular ROS levels and depolarization of MMP was evident in CAS-S C. albicans (3.0–4.2 fold) and C. parapsilosis (4.8–5.4 fold) biofilms compared with CAS-NS (1.2 fold) after exposure to MICA (0.25x-1xMIC). Finally higher ß-1, 3 glucan levels were seen in sessile cells compared to planktonic cells, especially in CAS-NS strains. MICA treatment might induce a metacaspase-dependent apoptotic process in biofilms of both CAS-S C. albicans and C. parapsilosis, and to some degree in CAS-NS strains.

The present study was undertaken to determine the molecular mechanism by which kaempferol induces cell death in human glioma cells. Kaempferol resulted in loss of cell viability and inhibition of proliferation in a dose- and time-dependent manner, which were largely attributed to cell death. Kaempferol caused an increase in reactive oxygen species (ROS) generation and the kaempferol-induced cell death was prevented by antioxidants, suggesting that ROS generation is involved in kaempferol-induced cell death. Kaempferol caused depolarization of mitochondrial membrane potential. Western blot analysis showed that kaempferol treatment caused a rapid reduction in phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. The ERK inhibitor U0126 and the Akt inhibitor LY984002 increased the kaempferol-induced cell death and overexpression of MEK, the upstream kinase of ERK, and Akt prevented the cell death. The expression of anti-apoptotic proteins XIAP and survivin was down-regulated by kaempferol and its effect was prevented by overexpression of MEK and Akt. Kaempferol induced activation of caspase-3 and kaempferol-induced cell death was prevented by caspase inhibitors. Taken together, these findings suggest that kaempferol results in human glioma cell death through caspase-dependent mechanisms involving down-regulation of XIAP and survivin regulating by ERK and Akt.