Lipidomic Analyses Uncover Apoptotic and Inhibitory Effects of Pyrvinium Pamoate on Cholangiocarcinoma Cells via Mitochondrial Membrane Potential Dysfunction

Pyrvinium pamoate (PP), an FDA-approved anthelmintic drug, has been validated as a highly potent anti-cancer agent and patented recently as a potential chemotherapeutic drug for various cancers. The aims of this study were, therefore, to investigate the ability of PP in anti-proliferative activity and focused on the lipid profiles revealing the alteration of specific lipid species in the liver fluke Opisthorchis viverrini (Ov)-associated cholangiocarcinoma (CCA) cells. PP inhibited CCA cell viability through suppressing mitochondrial membrane potential (MMP) and ATP productions, leading to apoptotic cell death. Liquid chromatography-mass spectrometry combined with chemometrics was performed to investigate lipid alteration during PP-induced apoptosis. The lipidomic analyses showed the altered lipid signatures of CCA cell types including S-acetyldihydrolipoamide, methylselenopyruvate, and triglycerides that were increased in PP-treated CCA cells. In contrast, the levels of sphinganine and phosphatidylinositol were lower in the PP-treated group compared with its counterpart. The orthogonal partial-least squares regression analysis revealed that PP-induced MMP dysfunction, leading to remarkably reduced ATP level, was significantly associated with triglyceride (TG) accumulation observed in PP-treated CCA cells. Our findings indicate that PP could suppress the MMP function, which causes inhibition of CCA cell viability through lipid production, resulting in apoptotic induction in CCA cells. These findings provide an anti-cancer mechanism of PP under apoptotic induction ability that may serve as the alternative approach for CCA treatment.

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Lipidomic Analyses Uncover Apoptotic and Inhibitory Effects of Pyrvinium Pamoate on Cholangiocarcinoma Cells via Mitochondrial Membrane Potential Dysfunction

10.21203/rs.3.rs-110218/v1 ◽

2020 ◽

Author(s):

Yingpinyapat Kittirat ◽

Jutarop Phetcharaburanin ◽

Bundit Promraksa ◽

Thanaporn Kultawatsiri ◽

Arporn Wangwiwatsin ◽

...

Keyword(s):

Membrane Potential ◽

Cell Viability ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Apoptotic Cell ◽

Atp Production ◽

Lipid Species ◽

Anti Cancer ◽

Apoptotic Induction ◽

Pyrvinium Pamoate

Abstract Pyrvinium pamoate (PP), an FDA-approved anthelmintic drug, has been validated as a highly potent anti-cancer agent. PP inhibits several crucial functions of cancer cells, and it has been patented recently as a potential chemotherapeutic drug for various cancers. The current study investigated the ability of PP in anti-proliferative activity and focused on the lipidomic profiles revealing the alteration of specific lipid species in cholangiocarcinoma (CCA) cells. PP inhibited CCA cell viability by suppressing mitochondrial membrane potential (MMP) and ATP production, leading to apoptotic cell death. Liquid chromatography-mass spectrometry combined with multivariate statistical analysis were performed to investigate lipid alteration of PP-induced apoptosis in CCA cells. The lipidomic analyses showed the altered lipid signatures of CCA cell types including S-acetyldihydrolipoamide, methylselenopyruvate and triglycerides that were increased in PP-treated CCA cells. In contrast, the levels of sphinganine and phosphatidylinositol were lower in the PP-treated group compared with its counterpart. Moreover, the orthogonal partial-least squares for regression analysis revealed that PP-induced MMP dysfunction, leading to inhibition of ATP contents, was significantly associated with triglyceride production, a characteristic of apoptotic cells. These alterations indicated that PP could suppress the MMP function, which caused inhibition of CCA cell viability through apoptotic induction resulting in lipid accumulation in CCA cells. These findings can provide new insight on an anti-cancer mechanism of PP under apoptotic induction ability that could be a promising therapeutic strategy for CCA treatment.

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Grape Seed Proanthocyanidins Protect N2a Cells against Ischemic Injury via Endoplasmic Reticulum Stress and Mitochondrial-associated Pathways

CNS & Neurological Disorders - Drug Targets ◽

10.2174/1871527318666190212111650 ◽

2019 ◽

Vol 18 (4) ◽

pp. 334-341 ◽

Cited By ~ 3

Author(s):

Kun Fu ◽

Liqiang Chen ◽

Lifeng Miao ◽

Yan Guo ◽

Wei Zhang ◽

...

Keyword(s):

Membrane Potential ◽

Cell Viability ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Grape Seed ◽

Mrna Levels ◽

Dependent Manner ◽

Grape Seed Proanthocyanidins ◽

N2a Cells ◽

Caspase 12

Background/Objective: Grape seed proanthocyanidins (GSPs) are a group of polyphenolic bioflavonoids, which possess a variety of biological functions and pharmacological properties. We studied the neuroprotective effects of GSP against oxygen-glucose deprivation/reoxygenation (OGD/R) injury and the potential mechanisms in mouse neuroblastoma N2a cells. Methods: OGD/R was conducted in N2a cells. Cell viability was evaluated by CCK-8 and LDH release assay. Apoptosis was assessed by TUNEL staining and flow cytometry. Protein levels of cleaved caspase-3, Bax and Bcl-2 were detected by Western blotting. CHOP, GRP78 and caspase-12 mRNA levels were assessed by real-time PCR. JC-1 dying was used to detect mitochondrial membrane potential. ROS levels, activities of endogenous antioxidant enzymes and ATP production were examined to evaluate mitochondrial function. Results: GSP increased cell viability after OGD/R injury in a dose-dependent manner. Furthermore, GSP inhibited cell apoptosis, reduced the mRNA levels of CHOP, GRP78 and caspase-12 (ER stressassociated genes), restored mitochondrial membrane potential and ATP generation, improved activities of endogenous anti-oxidant ability (T-AOC, GXH-Px, and SOD), and decreased ROS level. Conclusion: Our findings suggest that GSP can protect N2a cells from OGD/R insult. The mechanism of anti-apoptotic effects of GSP may involve attenuating ER stress and mitochondrial dysfunction.

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NSAID-induced injury of gastric epithelial cells is reversible: roles of mitochondria, AMP kinase, NGF, and PGE2

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00192.2019 ◽

2019 ◽

Vol 317 (6) ◽

pp. G862-G871

Author(s):

Amrita Ahluwalia ◽

Neil Hoa ◽

Michael K. Jones ◽

Andrzej S. Tarnawski

Keyword(s):

Membrane Potential ◽

Nerve Growth Factor ◽

Epithelial Cells ◽

Cell Viability ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Cell Injury ◽

Prostaglandin E ◽

Gastric Epithelial Cells ◽

Amp Kinase

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DFN) and indomethacin (INDO) are extensively used worldwide. Their main side effects are injury of the gastrointestinal tract, including erosions, ulcers, and bleeding. Since gastric epithelial cells (GEPCs) are crucial for mucosal defense and are the major target of injury, we examined the extent to which DFN- and INDO-induced GEPC injury can be reversed by nerve growth factor (NGF), 16,16 dimethyl prostaglandin E2 (dmPGE2), and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), the pharmacological activator of the metabolic sensor AMP kinase (AMPK). Cultured normal rat gastric mucosal epithelial (RGM1) cells were treated with PBS (control), NGF, dmPGE2, AICAR, and/or NSAID (DFN or INDO) for 1–4 h. We examined cell injury by confocal microscopy, cell death/survival using calcein AM, mitochondrial membrane potential using MitoTracker, and phosphorylation of AMPK by Western blotting. DFN and INDO treatment of RGM1 cells for 2 h decreased mitochondrial membrane potential and cell viability. NGF posttreatment (initiated 1 or 2 h after DFN or INDO) reversed the dissipation of mitochondrial membrane potential and cell injury caused by DFN and INDO and increased cell viability versus cells treated for 4 h with NSAID alone. Pretreatment with dmPGE2 and AICAR significantly protected these cells from DFN- and INDO-induced injury, whereas dmPGE2 and AICAR posttreatment (initiated 1 h after NSAID treatment) reversed cell injury and significantly increased cell viability and rescued the cells from NSAID-induced mitochondrial membrane potential reduction. DFN and INDO induce extensive mitochondrial injury and GEPC death, which can be significantly reversed by NGF, dmPGE2, and AICAR. NEW & NOTEWORTHY This study demonstrated that mitochondria are key targets of diclofenac- and indomethacin-induced injury of gastric epithelial cells and that diclofenac and indomethacin injury can be prevented and, importantly, also reversed by treatment with nerve growth factor, 16,16 dimethyl prostaglandin E2, and 5-aminoimidazole-4-carboxamide ribonucleotide.

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Effect of Glyphaea brevis twigs extract on cell viability, apoptosis induction and mitochondrial membrane potential in TM3 Leydig cells

Andrologia ◽

10.1111/and.13312 ◽

2019 ◽

Vol 51 (7) ◽

Cited By ~ 3

Author(s):

Janet Olayemi Olugbodi ◽

Edwina Olohirere Uzunuigbe ◽

Oladipupo David ◽

Oluwafemi Adeleke Ojo

Keyword(s):

Membrane Potential ◽

Cell Viability ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Leydig Cells ◽

Apoptosis Induction

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Progesterone Maintains Basal Intracellular Adenosine Triphosphate Levels and Viability of Spontaneously Immortalized Granulosa Cells by Promoting an Interaction between 14-3-3σ and ATP Synthaseβ/Precursor through a Protein Kinase G-Dependent Mechanism

Endocrinology ◽

10.1210/en.2006-1603 ◽

2007 ◽

Vol 148 (5) ◽

pp. 2037-2044 ◽

Cited By ~ 13

Author(s):

John J. Peluso ◽

Xiufang Liu ◽

Jonathan Romak

Keyword(s):

Membrane Potential ◽

Protein Kinase ◽

Mitochondrial Membrane Potential ◽

Granulosa Cells ◽

Mitochondrial Membrane ◽

Apoptotic Cell ◽

Protein Kinase G ◽

Disulfonic Acid ◽

Causative Role ◽

Atp Content

The present studies were designed to 1) describe changes in both the mitochondrial membrane potential and ATP content of spontaneously immortalized granulosa cells as they undergo apoptosis, 2) identify some of the downstream events that are activated by progesterone (P4), and 3) relate these downstream events to changes in mitochondrial function and apoptotic cell death. These studies revealed that in response to serum deprivation, the mitochondrial membrane potential initially hyperpolarizes and ATP content increases. That this increase in ATP is required for apoptosis was demonstrated by the finding that oligomycin inhibited the increase in ATP and apoptosis. Piridoxalphosphate-6-azopeyl-2′-4′-disulfonic acid, an inhibitor of purinergic receptors, which are activated by ATP, also inhibited apoptosis due to serum withdrawal. This study provides additional support for ATP’s causative role in apoptosis. Moreover, 8-Br-cGMP, a protein kinase G (PKG) activator, mimicked P4’s action, whereas a PKG antagonist, DT-3, attenuated P4’s suppressive effect on ATP and apoptosis. Finally, DT-3 treatment was shown to attenuate P4-regulated phosphorylation of 14-3-3σ and its binding partner, ATP synthaseβ/precursor and the amount of ATP synthaseβ/precursor that bound to 14-3-3σ. Based on these data, it is proposed that P4 prevents apoptosis in part by activating PKG, which in turn maintains the interaction between ATP synthaseβ/precursor and 14-3-3σ. In the absence of P4-induced PKG activity, we further propose that some ATP synthaseβ precursor dissociates from 14-3-3σ, resulting in its activation and incorporation into the ATP synthase complex, which ultimately results in an increase in ATP and apoptosis.

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In Vitro Cardiotoxicity Potential Comparative Assessments of Chronic Myelogenous Leukemia Tyrosine Kinase Inhibitor Therapies: Dasatinib, Imatinib and Nilotinib.

Blood ◽

10.1182/blood.v110.11.4582.4582 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4582-4582 ◽

Cited By ~ 8

Author(s):

Wendy J. Freebern ◽

Hengsheng S. Fang ◽

Martin D. Slade ◽

Susan Wells ◽

Jennifer Canale ◽

...

Keyword(s):

Membrane Potential ◽

Tyrosine Kinase ◽

Cell Viability ◽

Mitochondrial Membrane Potential ◽

Chronic Myelogenous Leukemia ◽

Mitochondrial Membrane ◽

Myelogenous Leukemia ◽

Rat Cardiomyocytes ◽

In Vivo Animal Model

Abstract Tyrosine kinase inhibitors (TKI) selective for Bcr-Abl, such as dasatinib, imatinib, and nilotinib have had remarkable success in the clinic, potentially shifting the prognosis of chronic myelogenous leukemia (CML) to a manageable chronic disease. With the increase in longevity of CML patients, there is rising concern of co-morbidities that may be influenced by chemotherapy (Force et al., Nature Rev.2007;7:332–340). Recently, congestive heart failure (CHF) and direct cellular cardiotoxicity have been reported in CML patients on imatinib therapy (Kerkela et al., Nature Medicine2006;12:908–916). Ultrastructural mitochondrial abnormalities in cardiomyocytes were observed in CML patients with severe CHF and, interestingly, similar abnormalities were observed in cardiomyocytes of imatinib-treated mice, thus providing a prospective in vivo animal model for imatinib-induced cardiotoxicity. Furthermore, correlative findings of mitochondrial membrane potential loss, decreased cell viability, and increased apoptosis resulted from an array of cell-based assays in imatinib-treated primary rat cardiomyocytes, consequentially affording a supportive, if not predictive, in vitro cardiomyocyte toxicity model. Since imatinib-induced inhibition of the native form of c-Abl kinase was speculated to cause the observed cardiotoxicity and c-Abl is a shared target of dasatinib, imatinib, and nilotinib, the in vitro cardiotoxicity potential of dasatinib and nilotinib at pharmacologically relevant concentrations (0.09 μM and 5 μM, respectively) and up to 10-fold higher concentrations were compared side-by-side with imatinib in primary rat cardiomyocytes. Dasatinib did not significantly affect mitochondrial membrane potential, cell viability, apoptosis, or cellular ultrastructure in vitro, whereas imatinib significantly affected these parameters. Nilotinib at pharmacologically relevant concentration demonstrated decreased cell viability, but differed from imatinib in that mitochondrial membrane potential integrity was not affected under identical experimental conditions. Results suggest that at pharmacologically relevant concentrations, dasatinib does not induce cardiotoxicity, as does imatinib and nilotinib, and the molecular mechanisms of the observed cardiotoxicities may differ between imatinib and nilotinib. Of indirect relation, results from assessing another cardiovascular liability, namely hERG K+ channel blockade, demonstrated that dasatinib, imatinib and nilotinib differentially inhibited the hERG currents in vitro with IC50 of 14.3, 15.6 and 0.66 μM, respectively. These in vitro findings occurred at concentration levels approximately 150, 3 and 0.1-fold the expected human Cmax for the three TKIs, respectively. Thus, although TKI therapies may share similar targeting and clinical indications, differentiating specific toxicity profiles may be predictive of differences in potential clinical adversities.

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Potential Crosstalk Of Ca2+-ROS Dependent Mechanism Involved In Kasumi cells’ Apoptosis Induced By Lentivirus-Mediated HO-1 siRNA

Blood ◽

10.1182/blood.v122.21.5558.5558 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5558-5558

Author(s):

Jishi Wang ◽

Wei Sixi ◽

Wang Yating ◽

Chai Qixiang

Keyword(s):

Membrane Potential ◽

Cell Viability ◽

Mitochondrial Membrane Potential ◽

Cell Apoptosis ◽

Mitochondrial Membrane ◽

The Other ◽

Ros Generation ◽

Dependent Manner ◽

Realtime Pcr ◽

Protein Expressions

Abstract Aim Using lentivirus-mediated HO-1 siRNA (lenti-siHO-1-GFP) to silence the HO-1 gene in Kasumi cells so as to explore the role and mechanism of HO-1 on cell apoptosis. Methods To infect Kasumi cells with lenti-siHO-1-GFP and check the infection efficiency by using fluorescence microscopy and flow cytometry (FCM). Experimental group was divided into three groups: untreated Kasumi (K), infected Kasumi by empty vector (lenti-GFP-K) and infected Kasumi by lentivirus-mediated HO-1 siRNA (lenti-siHO-1-K). The HO-1 expression of each group was detected by realtime PCR. Fluo3-AM method was used to detect the intracellular Ca2+ accumulation. DCFH-DA was used for the measurement of intracellular ROS. The change of mitochondrial membrane potential was evaluated by JC-1 stainning by using FCM. After being treated with various concentrations of daunorubicin for 24, 48, and 72 h respectively, cell viability was determined by MTT assay. Cell apoptosis was determined by FCM following with cells dual-stained with Annexin-V-FITC and propidium iodide (PI). The mRNA of HO-1 and apoptosis-related genes were analyzed by realtime PCR and, the expressions of their corresponding protein were determined by western blot. Additionally, After treating with 10mM Ca2+chelator BAPTA-AM and 0.5mM NAC for 12h, Ca2+ accumulation, ROS generation, the expression of HO-1 and apoptosis-related genes were detected respectively. Result presented in mean±sd manner. Results After lenti-siHO-1-GFP infection for 48h, we could observe the fluorescence clear, the fluorescent intensity was 95.87% after 72 hours. The HO-1 silencing efficiency of lenti-siHO-1-K was 77.00%. MTT result showed that daunorubicin exerted moderate inhibitory effects on cell proliferation in a dose and time dependent manner. With the same treating conditions, the cell viability of lenti-siHO-1-K group was significantly lower than the other two groups(e.g 49.20±1.30% survival in lenti-siHO-1-K group, 72.40±1.90% in K group and 74.10±2.10% in lenti-GFP-K group after being treated by 5ug/ml DNR,respectively, p=0.014), while the apoptosis rate was higher than the other two groups(e.g 75.77±3.41% in lenti-siHO-1-K group, 23.72±2.03% in K group and 26.10±1.95% in lenti-GFP-K group after being treated by 5ug/ml DNR,respectively, p=0.011). Compared with other two groups, the lenti-siHO-1-K group showed a downregulation in the mRNA and protein expression of HO-1. The mRNA and protein expressions of cyto-C, caspase3, caspase8, caspase9 and caspase12 in lenti-siHO-1-K group were upregulated after exposure to 5ug/ml daunorubicin for 24 hours. Compared with K and lenti-GFP-K groups, Ca2+ accumulation in lenti-siHO-1-K group was increased significantly(e.g 40.35±2.10% in lenti-siHO-1-K group, 17.30±1.81% in K group and 14.15±1.75% in lenti-GFP-K group,respectively, p=0.041). The ROS generation was higher than the other two groups(e.g 47.65±2.05% in lenti-siHO-1-K group, 21.30±1.94% in K group and19.90±2.01% in lenti-GFP-K group,respectively, p=0.037). The ratio of Green/Red fluorescence intensity increased significantly in lenti-siHO-1-K group(e.g 0.704±0.06 in lenti-siHO-1-K group, 0.57±0.09 in K group and 0.527±0.05 in lenti-GFP-K group, respectively, p=0.042). After exposure to 10mM BAPTA-AM and 0.1mM NAC alone or combined with, both the intracellular Ca2+accumulation and the ROS level in lenti-siHO-1-K group reduced(17.59±1.01% of Ca2+acumulation and 19.78±1.3% of ROS production after BAPTA-AM treatment alone, 23.42±1.97% of Ca2+and 15.47±1.14% of ROS after being treated by NAC alone, 16.52±1.23% of Ca2+and 14.37±1.21% of ROS after treatment by both agent) , while the mRNA and protein expressions of cyto-C, caspase3, caspase8, caspase9 and caspase12, decreased significantly. Conclusion HO-1 gene silencing played a role in pro-apoptosis in Kasumi cells. The mechanism may be related to the endoplasmic reticulum stress and abnormal accumulation of intracellular Ca2+, ROS generation, descending of the mitochondrial membrane potential and release cyto-C, then further activated the caspases cascade and promoted apoptosis. However, it tended to be initiated by crosstalk in Ca2+-ROS pathway. Disclosures: No relevant conflicts of interest to declare.

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