Top PDF Cannabidiol (CBD) Prevents Palmitic Acid Induced Drop in Mitochondrial Membrane Potential

Cannabidiol (CBD) Prevents Palmitic Acid Induced Drop in Mitochondrial Membrane Potential

Cannabidiol (CBD) Prevents Palmitic Acid Induced Drop in Mitochondrial Membrane Potential

3.1. Cannabidiol (CBD) Prevents Palmitic Acid (PA)-Induced Drop in Mitochondrial Membrane Potential in Macrophages Palmitic acid (PA) has previously been shown to induce apoptosis of macro- phages [15] [16]. Here we show that a 24 hrs incubation with palmitic acid leads to strong reduction in mitochondrial membrane potential (Figure 1; compare D-F with A-C) with concomitant shrinkage and rounding up of cells with occa- sional appearance of blebbing cells indicative for apoptosis (Figure 2; compare D-F with A-C). ImageJ analysis of red fluorescence intensity/cell shows an 80% ± 4% reduction in MitoTracker staining of PA-treated macrophages (Figure 3(A); p = 0.02). However, when the macrophages were simultaneously treated with CBD, the PA-induced drop in ∆Ψ m was significantly prevented (Figure 1;
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Cannabidiol Attenuates Palmitic Acid-Induced Injury in Cultured Hepatocytes Through Promoting Autophagic Flux

Cannabidiol Attenuates Palmitic Acid-Induced Injury in Cultured Hepatocytes Through Promoting Autophagic Flux

Abstract: Objective: This work was designed to investigate the protection of cannabidiol (CBD) against palmitic acid (PA)-induced injury in cultured hepatocytes and the underlying mechanism associated with autophagic flux. Methods: Experiment 1: Primary cultured hepatocytes were stimulated with PA (800 µmol/L) and treated with CBD (5 µmol/L) and chloroquine (CQ, 50 nmol/L) or not for 24 hours (1: control group; 2: PA-stimulated group; 3: PA-stimulated group treated with CBD; 4: PA-stimulated group treated with CBD and CQ). Autophagic flux was evaluated by Western blot analysis. Apoptosis was measured by flow cytometry. The mRNA expression of genes involved in endoplasmic reticulum stress was determined by reverse transcription PCR. The mitochondrial function was determined by using fluorescent probe including Rh123 and lucigenin. Experiment 2: Primary cultured hepatocytes were treated with CBD alone for 24 h (1: control group; 2: lower-dose CBD-treated group; 3: higher-dose CBD-treated group). Then, the autophagic flux was evaluated by Western blot analysis. Results: When compared to control group, exposure to PA significantly led to impaired autopagic flux (evidenced by increased ratio of LC3-II/LC3-I and protein expression of p62), increased apoptosis, endoplasmic reticulum stress (evidenced by increased mRNA expression of C/EBP homologous protein, glucose-regulated protein 78, and X-box protein 1), and mitochondrial dysfunction (evidenced by reduced mitochondrial membrane potential and enhanced formation of mitochondrial reactive oxygen species). When compared to PA-stimulated group, CBD treatment significantly attenuated PA-induced impaired autophagic flux, apoptosis, endoplasmic reticulum stress, and mitochondrial dysfunction in cultured hepatocytes. The protection of CBD against PA was abolished by co-incubation with CQ. In addition, treatment with CBD alone had no significant effect on autophagic flux in cultured hepatocytes Conclusion: Cannabidiol attenuates palmitic acid-induced impaired autophagic flux, apoptosis, endoplasmic reticulum stress, and mitochondrial dysfunction in cultured hepatocytes through promoting autophagic flux.
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Apocynin prevents mitochondrial burdens, microglial activation, and pro-apoptosis induced by a toxic dose of methamphetamine in the striatum of mice via inhibition of p47phox activation by ERK

Apocynin prevents mitochondrial burdens, microglial activation, and pro-apoptosis induced by a toxic dose of methamphetamine in the striatum of mice via inhibition of p47phox activation by ERK

ANOVA: analysis of variance; ASO: antisense oligonucleotide; Cleaved PKC δ : cleaved form of protein kinase C delta type; DA: dopamine; DMSO: dimethyl sulfoxide; DOPAC: 3,4-dihydroxyphenylacetic acid; EDTA: ethylenediaminetetraacetic acid; EGTA: ethylene glycol-bis(2-aminoethyl ether)-N,N,N ′ ,N ′ -tetraacetic acid; ERK: extracellular signal-regulated kinase; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HNE: 4-hydroxynonenal; HVA: homovanillic acid; Iba-1: ionized calcium-binding adaptor molecule 1; JNK: c-Jun N-terminal kinase; KO: knockout; LPS: lipopolysaccharides; MA: methamphetamine; MAPKs: mitogen-activated protein kinases; MMP: mitochondrial membrane potential; MnSOD: manganese-dependent superoxide dismutase; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; p38: p38 mitogen-activated protein kinase; PD: Parkinson ’ s disease; PHOX: NADPH oxidase; ROS: reactive oxygen species; RT-PCR: reverse transcription and polymerase chain reaction; SO: sense oligonucleotide; Tg: transgenic; TH: tyrosine hydroxylase; TH-IR: TH-immunoreactivity; TUNEL: terminal deoxynucleotidyl transferase dUDP nick-end labeling; WT: wild-type.
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Protection of palmitic acid-mediated lipotoxicity by arachidonic acid via channeling of palmitic acid into triglycerides in C2C12

Protection of palmitic acid-mediated lipotoxicity by arachidonic acid via channeling of palmitic acid into triglycerides in C2C12

indicative of apoptotic cell death, was checked after cells were loaded with PA or with PA and unsaturated fatty acids (Figure 2A). Treatment of C2C12 with PA induced obviously DNA cleavage, and AA but not ETYA pre- vented more effectively PA-caused DNA laddering than did POA or OA. Apoptosis is associated with a wide set of biochemical and physical changes in cytoplasm, nu- cleus and plasma membrane. However, the alteration in the mitochondrial permeability transition precedes cellu- lar apoptosis, that is, mitochondrial opening induces depolarization of the transmembrane potential with con- comitant release of apoptogenic factors and loss of oxi- dative phosphorylation. In this presentation, changes in mitochondrial potential of C2C12 cells exposed to PA were measured by using JC-1 (Figure 2B). As apoptotic progression undergoes, the electrochemical gradient across the mitochondrial membrane collapses and is aptly monitored by JC-1 dye. PA caused a decrease in mito- chondrial membrane potential, which was indicated by the increased ratio of fluorescence (485 nm/530 nm). Fur- thermore, PA-induced mitochondrial dysfunction was re- versed to the control level by AA. In contrast, ETYA did
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Apigenin prevents ultraviolet B radiation induced oxidative stress and dna damage in human dermal fibroblasts

Apigenin prevents ultraviolet B radiation induced oxidative stress and dna damage in human dermal fibroblasts

Mitochondrial changes are critical for the inductive effect phase of apoptosis. The loss of Δψm signifies metabolic cell death (Denning et al., 2002). In this study, rhodamine 123 was used to assess the effect of apigenin treatments on Δψm in UVB‑irradiated HDFa cells. The changes in mitochondrial membrane potential have been strongly linked with apoptotic signaling envents (Denning et al., 2002; Fumelli et al., 2000). In this study, it has been observed that there was a significant loss of mitochondrial membrane potential during UVB exposure. Conversely, apigenin treatment prevented UVB- induced loss of mitochondrial membrane potential in HDFa cells (Fig. 4). These results suggest that protection of mitochondria by apigenin could be implicated due to its antiapoptotic property. Previous studies suggest that the 30 mJ/cm 2 UVB led to significant apoptosis of HaCaT cells, accompanied by a marked reduction of Δψm. Kanagalakshmi et al. (2014) reported that ferulic acid treatment before UVB ‑ exposure significantly prevented UVB ‑ induced loss of Δψm. UVB radiation is the primary environmental agent that leads to apoptosis in human cells. UVB‑irradiation of cells elicits a complex cellular response via cell surface receptor aggregation (Ji et al., 2012; Brash et al., 1991) and upon prolonged exposure; it induces apoptosis in mammalian cells including keratinocytes and lymphocytes (Kanimozhi et al., 2011). In agreement with this, the present results showed the protective effect of apigenin on UVB radiation‑induced apoptotic morphological changes.
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Mitochondrial activity in gametes and transmission of viable mtDNA

Mitochondrial activity in gametes and transmission of viable mtDNA

Veneridae), and up to 50% in freshwater mussels (family Unionidae)]. In the phylogeny of unionids (the other family, other than Mytilids, in which DUI has been extensively studied) the F mt genomes from all the species, genera and subfamilies cluster together, and so do the M genomes (gender-joining pattern); the occurrence of role-reversal events in unionids cannot be ruled out, but the non-fixation of masculinized mt genomes is almost certain (Zouros 2013). DUI is an evolutionary stable mechanism, F and M ge- nomes being separated for million years (>200 million years in Unionids, see Breton et al. 2011; for a thorough discussion about these aspects of DUI, see Zouros 2013). We think that in such a long evolutionary time, nuDNA must have coevolved with both mtDNA types. M-type mtDNA evolves faster, but we do not think that this is be- cause of a relaxed selection: M-type has to perform in very important contexts, such as gametogenesis and fertilization, so it has to be under selective constraints. Under this light, there is no need for role-reversal events to restore M-type viability and/or mito-nuclear match, and role-reversal is not needed for the existence of DUI. Supporting this viewpoint, a recent SNP analysis of the mitochondrial DNA population in R. philippinarum male and female gonads showed a lower amount of dele- terious polymorphism in M-type (see Ghiselli et al. 2013 for a detailed discussion). Consistently, although we have not yet found any traces of masculinization events in R. philippinarum, this species is highly invasive, therefore we think that the absence (or paucity) of role-reversal events does not affect the evolutionary success of this organism.
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Carbon black suppresses the osteogenesis of mesenchymal stem cells: the role of mitochondria

Carbon black suppresses the osteogenesis of mesenchymal stem cells: the role of mitochondria

ATP synthesis is coupled with oxygen consumption because the electron transport chain (ETC) in the inner membrane of mitochondria transfers electrons to oxygen resulting in ATP generation. Reduction of ATP by Printex 90 treatment led us to evaluate the oxygen consumption of osteogenic differentiated MSCs using a mitochondrial stress assay. We optimized the cell number and the concentration of each chemical used in the experiments (data not shown) and the optimized conditions were used (1 μ M oligomycin, 1.8 μ M FCCP, 0.5 μ M rotenone and 0.5 μ M Antimycin A). We found accumulated damages to mitochondrial func- tions in the process of MSCs osteogenic differentiation (Additional file 4: Figure S3). Figure 6b and c, show that both 3 ng/mL and 30 ng/mL Printex 90 produced significant declines in basal OCR. These were 67.5 ± 1.8 and 65.1 ± 1.7 pmol/min respectively, compared to the con- trol (74.8 ± 1.7 pmol/min). The basal OCR is composed of respiration linked to ATP production and proton leakage. ATP-linked respiration (Basal OCR - Oligomycin response) was significantly lower ( p < 0.05) in both 3 ng/mL and 30 ng/mL Printex 90 treated groups. These were 50.9 ± 1.5 and 46.2 ± 1.7 pmol/min respectively, compared with control (55.2 ± 1.2 pmol/min), which was consistent with the decreased ATP production demonstrated in Fig. 6a. Proton leak (Oligomycin response – Antimycin A & Rote- none response) in 3 ng/mL treated cells was significantly lower ( p < 0.05) than control, 16.2 ± 0.9 vs.19.6 ± 1.2 pmol/ min, but in 30 ng/mL Printex 90 treated cells, it was similar to the normal level (19.0 ± 0.8 pmol/min). The
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A Study of Mitochondrial Dynamics and Glycosylation Events in PC12 Cells and Neurons

A Study of Mitochondrial Dynamics and Glycosylation Events in PC12 Cells and Neurons

OGT is ubiquitously expressed within organisms and highly conserved across species. Many factors affect the regulation of OGT, including splice variants, PTMs and nutrient availability. The human OGT gene is located on the X chromosome near the Xist locus, a region which is associated with Parksinson’s disease (Shafi et al., 2000), and three different isoforms may be generated through alternative splicing all of which contain an identical C-terminal catalytic domain, a linker region and N-terminal tetratricopeptide repeats (TPRs) (Love et al., 2003). The most abundant isoform is nucleocytoplasmic OGT (ncOGT) which has twelve TPRs, while an intermediate form known as mitochondrial OGT (mOGT) has nine TPRs and a mitochondrial targeting sequence at its N-terminus, and finally a short isoform (sOGT) with only two TPRs (Lazarus et al., 2006). However, recent research has suggested that only ncOGT is required for O- GlcNAcylation of mitochondrial proteins meaning mOGT is redundant and indeed not expressed in many species (Trapannone et al., 2016). OGT activity is highly dependent on the concentration of UDP-GlcNAc highlighting its role as a sentinel for nutrient availability in the cell. As GlcNAc is transferred to its target protein, UDP is released and acts as negative regulator of OGT activity showing fluctuating levels of UDP-GlcNAc are vital for regulating OGT activity (Hart, 2014). It is an inverting glycosyltransferase so it converts the a-linked GlcNAc of its donor to a b-linked PTM on its substrates (Drula et al., 2014, Coutinho et al., 2003). The process of O-GlcNAc addition appears to work via a bi-bi mechanism where UDP- GlcNAc binds the catalytic site first, followed by the polypeptide acceptor (Lazarus et al., 2012). In one instance, OGT has also been shown to act as proteolytic enzyme on host cell factor-1 (HCF-1), a transcriptional co-regulator of human cell cycle progression that undergoes cleavage as a maturation step. UDP-GlcNAc is required as a co-substrate for this reaction and to date is the only known cleavage reaction catalysed in the active site of a glycosyltransferase (Kötzler and Withers, 2016).
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Anti-inflammatory effects of an injectable copolymer of fatty acids (Ara 3000 beta®) in joint diseases

Anti-inflammatory effects of an injectable copolymer of fatty acids (Ara 3000 beta®) in joint diseases

Certain polyunsaturated fatty acids are beneficial to the health of mammals. For instance, long chain n-3 fatty acids are present in fish and marine mammals, and epidemiological data indicate a correlation between fish- wealthy diets and reduced incidence of inflammatory diseases [6]. In addition, in a variety of conditions, in- cluding rheumatoid arthritis or osteoarthritis, these fatty acids have been shown to offer therapeutic activity [7-9]. This may be associated with increased collagen synthesis and decreased amounts of the inflammatory mediator prostaglandin E2 as reported in fibroblasts in vitro [10]. At contrary, n-6 fatty acids have been reported to induce IL-1, IL-6, TNF-α and PGE2. However, n-3 fatty acids inhibit n-6 fatty acid induced-cytokine expression [11]. More recently, it has been shown that veterinary thera- peutic diet rich in omega-3 fatty acids improved the locomotor disability and the performance in activities of daily living of OA dogs [12]. A beneficial effect was also shown in OA-prone Dunkin Hartley strain guinea pigs on a diet rich in long chain omega 3 fats with an im- provement of OA degree and severity [13].
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Alteration of fatty acid oxidation by increased CPT1A on replicative senescence of placenta-derived mesenchymal stem cells

Alteration of fatty acid oxidation by increased CPT1A on replicative senescence of placenta-derived mesenchymal stem cells

We investigated fatty acid oxidation (FAO)-related factors to confirm FAO pathway in PD-MSCs during long-term cultivation. In skeletal muscle cells, it is well established that AMP-activated protein kinase (AMPK) inhibits acetyl-CoA carboxylase (ACC) through phosphorylation, which reduces intracellular malonyl-CoA levels and stim- ulates carnitine palmitoyl transferase 1 (CPT1) and then increases the influx of long-chain fatty acids into the mito- chondria where they are oxidized [21]. Quantitative RT- PCR revealed that late passage PD-MSCs increased dra- matically the mRNA levels of ACC and CPT1A compared to early cells (Fig. 5a). In addition, Western blot analysis showed that the increase of CPT1A (P value = 0.06) was followed by the increase of expression of p-ACC versus total-ACC by long-term cultivation in PD-MSCs (P value = 0.053), while the phosphorylation level of AMPK, an upstream of ACC, was significantly decreased suggest- ing activation of AMPK is cell type-specific. Peroxisome proliferator-activated receptor (PPARα) is another major regulator of FAO as a transcription factor, which is pre- dominantly expressed in tissues that oxidize fatty acids at a rapid rate, such as the liver, brown adipose tissue, heart, and kidney [22]. As expected, increased passages of PD- MSCs induced a significant increase of PPARα (P value = 0.02) (Fig. 5b). To further clarify the role of CPT1A
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Hepatitis C Virus Triggers Mitochondrial Permeability Transition with Production of Reactive Oxygen Species, Leading to DNA Damage and STAT3 Activation

Hepatitis C Virus Triggers Mitochondrial Permeability Transition with Production of Reactive Oxygen Species, Leading to DNA Damage and STAT3 Activation

ized in the nucleus of HCV-infected cells, while most of it was formed in the cytoplasm of the mock-infected cells (Fig. 4C, top two panels). In the infected cells treated with NAC, STAT3 was found predominantly in the cytoplasm. In Huh7 cells tran- siently expressing core or E1, STAT3 was also localized mainly in the nucleus. However, in the NS3-expressing cells, some of STAT3 was found in the cytoplasm. One possibility is that the expression level of NS3 was too low. Another possibility is that NS3 protein caused substantial effects on other signaling path- ways (12) which may affect the STAT3 nuclear translocation. In Huh7 cells harboring a subgenomic HCV replicon, which expresses HCV nonstructural proteins (NS3 through NS5B), STAT3 was also predominantly in the nucleus (Fig. 4C). In contrast, in Huh7 cells expressing the neomycin resistance or HCV NS4B protein or transfected with the vector plasmid, STAT3 was localized mainly in the cytoplasm (Fig. 4C). In the HEK293 cell line stably expressing the core protein, STAT3 was also found predominantly localized in the nucleus, in con- trast to cells expressing the neomycin resistance gene. These results combined demonstrate that HCV infection constitu- tively activated STAT3, resulting in its nuclear transloca- tion. This activation was mediated by core, E1, and NS3 proteins. When these cells were treated with NAC, most of STAT3 reverted back to the cytoplasm. Therefore, we con- cluded that the nuclear translocation of STAT3 induced by HCV proteins was mediated by ROS.
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Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis

Another issue which deserves some discussion is whether the above- mentioned antioxidant and antiinflam- matory effects of CBD would interfere with the antitumor activity of DOX. The answer is that it is not likely to do so. For example, mitochondrial-targeted antioxidants appear to have synergistic effects with various chemotherapeutic agents in tumor cell killing (43). Activa- tion of MMPs may be involved in tumor progression and metastasis for- mation, which is inhibited by CBD, likewise the NF-κB signaling. Further- more, CBD by itself has been reported to have antitumor effects in a large va- riety of cancer cell lines, as well as in some explanted tumor models (44–46), which would rather predict a synergis- tic effect with antineoplastic drugs. In fact, Insys Therapeutics just received FDA orphan drug designation for CBD as a potential treatment for glioblas- toma multiforme in humans.
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Oleic Acid Attenuates Palmitic Acid-Induced Impairments in Mouse Blastocyst Development

Oleic Acid Attenuates Palmitic Acid-Induced Impairments in Mouse Blastocyst Development

All of these procedures while commonly applied today, were developed and put into practice as a result of extensive amounts of research globally. Studies deciphered the key mechanisms controlling mammalian reproduction in various animal models, which were translated and applied to humans over the past 100 years of scientific inquiry. These efforts culminated in 2010 when Dr. Bob Edwards received the Nobel prize in Physiology for his efforts with Dr. Patrick Steptoe that resulted in the birth of the world’s very first IVF child, Louise Brown in 1978 10 . However, despite all of its successes, human fertility medicine only results in a 50-65% pregnancy rate of all patients seeking fertility treatment 11 . The ART process can be broken down into several stages: oocyte maturation and collection, fertilization, preimplantation development, embryo transfer and implantation, and pregnancy. Each of these stages and procedures still require significant improvement if we are to achieve the goal of helping everyone who seeks fertility treatment succeed in their quest of having a family. The research in this thesis is directed at specifically improving our ability to support preimplantation development in vitro so that only blastocysts with high developmental competence are transferred during SET. The potential implications of this thesis are to further explore the struggles faced by the obese patient as these patients are increasing dramatically in number in the global population, and their struggle to conceive is much greater than that of healthy BMI patients 12–14 . Although this
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Berberine Protects Glomerular Podocytes via Inhibiting Drp1-Mediated Mitochondrial Fission and Dysfunction

Berberine Protects Glomerular Podocytes via Inhibiting Drp1-Mediated Mitochondrial Fission and Dysfunction

FFA: free fatty acid; DKD: diabetic kidney disease; BBR: berberine; PA: palmitic acid; ROS: reactive oxygen species; Drp1: dynamin-related protein 1; mtDNA: mitochondrial DNA; ATP: adeno- sine triphosphate; MMP: mitochondrial membrane potential; MOM: mitochondrial outer membrane; GFB: glomerular filtration barrie; mtROS: mitochon- drial ROS; DM: diabetes mellitus; SDs: slit diaphragm proteins; MMP-9: matrix metalloproteinase-9; TEM: transmission electron microscope; mtBax: mitochon- dria Bax; DCFH-DA: 2′,7′-Dichlorofluorescein diacet- ate; NAC: antioxidant N-acetylcysteine; NDGA: nordihydroguaiaretic acid; MDA: malondialdehyde; MID51 and MID49: mitochondrial dynamics proteins of 51 and 49 kDa; FIS1: mitochondrial fission protein 1; MFF: mitochondrial fission protein; PGC1α: per- oxisome proliferator-activated receptor-γ co-activator 1α; TFAM: mitochondrial transcription factor A; NRF1 and NRF2: nuclear respiratory factors 1 and 2; ND1: NADH dehydrogenase subunit 1; GBM: glom- erular basement membrane; DHE: dihydroethidium; HE: hematoxylin-eosin; PAS: periodic acid–schiff; FBG: fasting blood glucose; TG: triglyceride; ACR: microalbumin-to-creatinine ratios; NF-κB: nuclear factor-κB; TLR4: toll-like receptor 4; PI3K: phosphati- dylinositol 3 kinase; AKT: protein kinase B; TGFβ: transforming growth factor-β; AGEs: advanced glyca- tion end products; RAGE: receptor of advanced glycation end products.
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Original Article Ulinastatin mediates protection against vascular hyperpermeability following hemorrhagic shock

Original Article Ulinastatin mediates protection against vascular hyperpermeability following hemorrhagic shock

filters (0.4 mm pore size) and were used until full confluence. Resistance values of multiple Transwell inserts of an experimental group were measured sequentially and the mean was Figure 1. UTI decreases HS-induced vascular hyperpermeability in rat mesentery postcapillary venules. Vascular permeability is expressed as change in fluorescent intensity inside the vessel compared with the intensity outside the vessel. Data are presented as mean ± SD (n = 6 in each group). *P < 0.05 versus the sham group; #P < 0.05 versus the shock+ NS group.

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Sensitivity Evaluation of Two Human Breast Cancer Cell Lines to Tamoxifen through Apoptosis Induction

Sensitivity Evaluation of Two Human Breast Cancer Cell Lines to Tamoxifen through Apoptosis Induction

In this investigation, we studied two widely used human breast cancer cell lines, namely estrogen-positive MCF-7 and estrogen-negative MDA-MB-231, to evaluate if these two cell lines differ in their sensitivity to TAM in vitro. Both cell lines are invasive breast ductal carcinoma and grow as adherent cells. The generation time for MCF-7 is longer than that of MDA-MB-231 and MCF-7 grows in clustered clumps (luminal epithelium phenotype) compared to MDA-MB-231, which grows as flattened epithelial layers. While MCF-7 is an estrogen receptor-positive cell line, MDA-MB-231 lacks both estrogen and progesterone receptors, and contains a mu- tated form of the P53 tumor suppressor gene [22]. Earlier studies involving these two human breast cancer cell lines have shown enhanced sensitivity of estrogen receptor-positive MCF-7 cancer cells to TAM compared to the estrogen receptor-negative MDA-MB-231 cells [12] [13]. However, the difference in their cytotoxic sensi- tivity measured through TAM-induced programmed cell death, or apoptosis, is not firmly established. The pur- pose of this study was to investigate the cytotoxic sensitivity of ER-positive MCF-7 and ER-negative MDA- MB-231 breast cancer cells exposed to different concentrations of TAM using a fluorescence-based assay to de- termine the functional status of mitochondria. Since mitochondria are generally known to be associated with early stages of apoptosis, in the present investigation we used a cationic dye purchased from Trevigen, Inc. (Gaithersburg, MD) to evaluate the status of mitochondrial membrane potential in healthy and apoptotic cells under a fluorescence microscope [23]. Our results revealed that ERα+ MCF-7 cells were statistically more sen- sitive to TAM treatment compared to ERα-MDA-MB-231 cells as evidenced from collated data obtained by enumerating early, mid- and late apoptotic cells. This difference, however, was more pronounced at the lower concentration (2.5 µg/mL). Although TAM-induced programmed cell death pointed towards a dose and time dependence, the differences, however, were not uniformly significant in this mitochondrial based assay.
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The Rcs Regulated Colanic Acid Capsule Maintains Membrane Potential in Salmonella enterica serovar Typhimurium

The Rcs Regulated Colanic Acid Capsule Maintains Membrane Potential in Salmonella enterica serovar Typhimurium

obtain mechanistic insights into the mechanism of cell death, a microarray analysis was performed to analyze the transcriptional response of SFZP (sit feo zup psp) mutant S. Typhimurium treated with dipyridyl for 2 h. As expected, induction of the Psp operon in an SFZP mutant was observed in the absence of the negative regulator PspA (see Table S1 in the supplemental material). Expression of the pspA gene could still be detected in the pspA deletion mutant, as the oligonucleotide probe contains sequences outside the deleted region. In addition to the psp genes, the most strongly induced loci were those comprising the colanic acid capsule operon (see Table S1), regulated by the RcsA-RcsB heterodimer (16). Other genes in the Rcs regulon, including rcsA and the yjbEFGH operon (18, 46), were also strongly induced. The induction of rcsA, yjbG, and genes from the colanic acid capsule operon was confirmed by quantitative PCR (qPCR) analysis (Fig. 1A) and observed only under conditions of iron depletion (see Fig. S1). As an SFZP mutant exhibited induction of the auxiliary regulator rcsA, we determined whether an rcsA-expressing plasmid could restore growth in chelated medium. Wild- type (WT) or mutant strains containing either a vector control or pRcsA were grown in chelated Luria-Bertani (LB) medium, and growth was monitored by measurement of optical density at 600 nm (OD 600 ) (Fig. 1B). As previously observed (40), the viability of
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Extraction and GC-MS analysis of the fatty acids in commonly consumed melon seed varieties in Nigeria

Extraction and GC-MS analysis of the fatty acids in commonly consumed melon seed varieties in Nigeria

Although Lagenaria siceraria is composed of 16.86% of 2,4-decadienal, Citrullus vulgaris contained 0.30% of this compound not found in Cucumis melo. 2, 4-decadienal is an aromatic substance with deep fat flavour. While 2,4- decadienal is generated from polyunsaturated fatty acids by the action of plant lipoxygenases and used as a synthetic flavoring and fragrance material, its presence in higher percentage in Lagenaria siceraria, not commonly consumed, may boost its economic value and enhance its use as a food condiment over and above other types of melons. Shi and Ho [43] and Mottram [44] indicated that 2,4-decadienal is responsible for the inviting aroma of deep-fat-fried food. In a descending order, palmitic acid esters were present in Citrullus lanatus (21.20%), Citrullus vulgaris (2.89%), Cucumis melo (1.09%) and Lagenaria siceraria (0.63%). Palmitic acid is one of the most common saturated fatty acids which can increase unhealthy low density lipoprotein (LDL) cholesterol levels [45]. Being one of the most prevalent saturated fatty acids in body lipids, it could constitute a major risk factor for heart attacks and strokes. Diets high in saturated fatty acids increase the production of acetate fragments in the body which, in turn, leads to an increase in the production of cholesterol. When consumed, saturated fats tend to clump together and form deposits in the body, along with protein and cholesterol. They get lodged in blood cells and organs, leading to many health problems, including obesity, heart diseases and cancers of the breast and colon. However, since the dietary effects of high-fat diet, mainly in saturated fatty acids, have been focused on the reduction of cardiovascular diseases [46,47], obesity-related diseases and, recently, cancer prevention [48], consuming Cucumis melo, Citrullus vulgaris and Lagenaria siceraria having very low levels of saturated fatty acid compared to unsaturated fatty acids and less of Citrullus lanatus having high levels of saturated fatty acids would be of significant benefit. Oluba et al. [49], also, reported that egusi oil could improve serum and liver lipid profiles and offer better protection against resultant lipid peroxides from consumption of high fat diet.
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Cancer stem cell metabolism

Cancer stem cell metabolism

Many other unresolved issues need to be addressed. Elucidating the differences in metabolism between CSCs and non-stem cancer cells, and between CSCs and nor- mal progenitor stem cells, will be crucial to develop new therapies and may reveal new ways to distinctively target these TICs. Whereas normal stem cells rely more on glycolysis, CSCs might depend more on mitochondrial oxidative metabolism. If this is the case, why would the stemness state of cancer cells require a different metabolic state than normal stem cells? In contrast with normal physiological development, tumourigenesis tends to be highly disorganized and cancer metabolic malleability could provide a niche more prone to CSC survival. Never- theless, the stability or plasticity of the CSCs phenotype needs to be verified. Are CSCs really able to metabolically adapt depending on microenvironmental fluctuations? Is the CSC population metabolically heterogeneous or does it exhibit different degrees of stemness-related pheno- types? During tumourigenesis, characteristics of the CSCs might mutate, and distinct CSC populations could eventu- ally emerge in what it would be a metabolically changeable or versatile target. In such a scenario, future therapies de- signed to eradicate CSCs via targeting their metabolism might need to simultaneously block glycolysis and mito- chondrial respiration.
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Distinct intracellular signaling in Tumor Necrosis Factor-related Apoptosis-inducing Ligand- and CD95 Ligand-mediated apoptosis

Distinct intracellular signaling in Tumor Necrosis Factor-related Apoptosis-inducing Ligand- and CD95 Ligand-mediated apoptosis

Receptor Expression—A prerequisite for comparing the in- tracellular routes leading to apoptosis after TRAIL or CD95L exposure is the presence of CD95 and all four TRAIL receptors on the target cell. Therefore, the expression of the receptors was determined on CC531s, the rat colon carcinoma cell line that we found to be sensitive toward both ligands. Immunoblots stained for CD95 and all TRAIL receptors clearly show that all receptors were present (Fig. 1). There seemed to be slightly more DR5 than DR4 at the protein level. The same was true for DcR2 as compared with DcR1. Thus, CD95 and the four TRAIL receptors were present in the CC531s cell line which made it a suitable target cell to investigate TRAIL and CD95L signaling. TRAIL- and CD95L-induced Apoptosis in CC531s Cells—To study the effect of TRAIL and CD95L signaling, we used 2PK3 cells that had been transfected with either mTRAIL or mCD95L as effector cells, and 2PK3-mock cells served as con- trol (34). This provided us with a tool to compare TRAIL to CD95L using the same effector and target cell line. First, we F IG . 4. Mitochondrial membrane
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