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1.
Toxicol In Vitro ; 58: 118-125, 2019 Aug.
Article in English | MEDLINE | ID: mdl-30905860

ABSTRACT

As knowledge regarding mechanisms of pentachlorophenol (PCP) toxicity in neuronal cell lines is limited, the aim of the study was to evaluate the effects of PCP and its active metabolites, tetrachloro-1,4-benzoquinone (TCBQ) and tetrachlorohydroquinone (TCHQ) in human neuroblastoma SH-SY5Y cells. All compounds induced cytotoxic effects in time- and dose-dependent manners, and resulted in differential modes of cell death. Reduced mitochondrial membrane potential (Δá´ªM) and oxidative damage lead to apoptosis and necrosis following TCBQ and PCP exposure, respectively. Time-dependent investigations revealed transient Δá´ªM recovery in TCHQ exposed cells, and redox stress. Sufficient Δá´ªM recovery allowed apoptosis completion in TCHQ exposed cells, whereas overwhelming metabolic and oxidative stress saw a conversion from apoptotic to necrotic-like cell death. The onset of mitochondrial dysfunction preceded that of redox damage for all compounds, indicating that oxidative damage is secondary to Δá´ªM insult. Cytotoxic events were further linked to cell cycle. S phase and G2/M blocks were observed after 12 h exposure to TCBQ and TCHQ, respectively, while a G1 block occurred after 24 h exposure to PCP. This study provides new insight regarding time-dependant toxic effects of PCP and its metabolites in human neuronal cells.


Subject(s)
Pentachlorophenol/toxicity , Pesticides/toxicity , Acetylcholinesterase/metabolism , Apoptosis/drug effects , Caspase 3/metabolism , Cell Cycle/drug effects , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Glutathione/metabolism , Humans , Membrane Potential, Mitochondrial/drug effects , Reactive Oxygen Species/metabolism
2.
PLoS One ; 13(7): e0198248, 2018.
Article in English | MEDLINE | ID: mdl-29969443

ABSTRACT

The field of dermal fillers is evolving rapidly and numerous products are currently on the market. Biodegradable polymers such as polycaprolactone (PCL) have been found to be compatible with several body tissues, and this makes them an ideal material for dermal filling purposes. Hollow PCL spheres were developed by the Council for Scientific and Industrial Research (CSIR) to serve both as an anchor point and a "tissue harbour" for cells. Particles were tested for cytotoxicity and cell adherence using mouse embryo fibroblasts (MEF). MEFs adhered to the particles and no significant toxic effects were observed based on morphology, cell growth, cell viability and cell cycle analysis, suggesting that the particles are suitable candidates for cell delivery systems in an in vivo setting. The objective of providing a "tissue harbour" was however not realized, as cells did not preferentially migrate into the ported particles. In vivo studies were conducted in BALB/c mice into whom particles were introduced at the level of the hypodermis. Mice injected with PCL particles (ported and non-ported; with or without MEFs) showed evidence of local inflammation and increased adipogenesis at the site of injection, as well as a systemic inflammatory response. These effects were also observed in mice that received apparently inert (polystyrene) particles. Ported PCL particles can therefore act as a cell delivery system and through their ability to induce adipogenesis, may also serve as a dermal bulking agent.


Subject(s)
Dermal Fillers/pharmacology , Fibroblasts/transplantation , Polyesters/pharmacology , Tissue Engineering/methods , Tissue Scaffolds , Adipogenesis/drug effects , Animals , Cell Adhesion/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Embryo, Mammalian , Female , Fibroblasts/cytology , Fibroblasts/physiology , Mice , Mice, Inbred BALB C , Models, Animal , Polyesters/chemistry , Polystyrenes/chemistry , Polystyrenes/pharmacology , Primary Cell Culture
3.
Cell Physiol Biochem ; 35(4): 1499-526, 2015.
Article in English | MEDLINE | ID: mdl-25791820

ABSTRACT

BACKGROUND: C9, a newly in silico-designed inhibitor of microtubule dynamics induces G2/M arrest culminating in apoptosis. Dichloroacetate (DCA) inhibits pyruvate dehydrogenase kinase, an enzyme that promotes pyruvate entry into mitochondria. The use of antitumor drugs targeting different cancer features can be a more effective way to overcome drug resistance. METHODS: The influence of C9 (130 nM) + DCA (7.5 mM) on MCF-7 and MCF-12 cells was assessed via microscopy spectrophotometry global gene expression and flow cytometry assays. RESULTS: An LDH assay showed that C9+DCA treatment decreased cell viability to 83.5% in MCF-7 cells when compared to the non-tumorigenic MCF-12A cells 92.4% (P < 0.05). C9- and C9+DCA treatment induced mitochondrial membrane potential depolarization in MCF-7 cells but not in MCF-12A cells (P < 0.05). The occurrence of apoptosis was associated with increased hypo- and hyper-phosphorylation of Bcl-2 Ser(70) and caspase 7 activation. Kinase inhibition revealed sustained activation of the JNK pathway caused increased Bcl-2 protein Ser(70) hypo-and hyper-phosphorylation. Elevated levels of DCF fluorescence was observed in DCA-, C9- and C9+DCA-exposed MCF-7 cells, but not in MCF-12A cells, indicating cytosolic H2O2/Fe(2+) formation in treated tumorigenic cells. LC3-II expression was elevated in C9+DCA-treated cells in both cell lines, indicating that autophagy was also induced. CONCLUSIONS: Synergistic effects of C9+DCA were demonstrated on breast carcinoma and non-tumorigenic cells with selectivity towards the MCF-7 cells. Antimitotic compound C9 in combination with a glycolytic inhibitor dichloroacetate eradicates breast cancer cells through ROS-JNK-Bcl-2-mediated signalling pathways in vitro and it is argued that autophagy acts as protective mechanism in the treated cells before apoptosis occurs.


Subject(s)
Antineoplastic Agents/pharmacology , Dichloroacetic Acid/pharmacology , Estradiol/pharmacology , Signal Transduction/drug effects , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Autophagy/drug effects , Caspase 7/metabolism , Cell Line , Cell Survival/drug effects , Dichloroacetic Acid/chemistry , Drug Synergism , Estradiol/analogs & derivatives , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , MCF-7 Cells , Membrane Potential, Mitochondrial/drug effects , Microtubule-Associated Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Oxidative Stress/drug effects , Phosphorylation/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Oxygen Species/metabolism
4.
PLoS One ; 8(1): e53853, 2013.
Article in English | MEDLINE | ID: mdl-23382857

ABSTRACT

UNLABELLED: The aim of this study was to characterize the in vitro action of 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) on non-tumorigenic MCF-12A, tumorigenic MCF-7 and metastatic MDA-MB-231 breast cancer cells. ESE-16 is able to inhibit the activity of a carbonic anhydrase II and a mimic of carbonic anhydrase IX in the nanomolar range. Gene and protein expression studies using various techniques including gene and antibody microarrays and various flow cytometry assays yielded valuable information about the mechanism of action of ESE-16. The JNK pathway was identified as an important pathway mediating the effects of ESE-16 while the p38 stress-induced pathway is more important in MDA-MB-231 cells exposed to ESE-16. Lysosomal rupture and iron metabolism was identified as important mediators of mitochondrial membrane depolarization. Abrogation of Bcl-2 phosphorylation status as a result of ESE-16 also plays a role in inducing mitochondrial membrane depolarization. The study provides a basis for future research projects to develop the newly synthesized compound into a clinically usable anticancer agent either alone or in combination with other agents. KEYWORDS: Antimitotic, anticarbonic anhydrase IX, apoptosis, autophagy, cell cycle arrest, Bcl-2, JNK, p38, mitochondrial membrane depolarization, flow cytometry, gene expression and protein microarray, anticancer.


Subject(s)
Apoptosis/drug effects , Autophagy/drug effects , Estradiol/pharmacology , MAP Kinase Kinase 4/metabolism , Carbonic Anhydrase II/antagonists & inhibitors , Carbonic Anhydrase II/metabolism , Cell Cycle Checkpoints/drug effects , Estradiol/analogs & derivatives , Female , Gene Expression Regulation, Neoplastic , Humans , MCF-7 Cells , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction/drug effects , p38 Mitogen-Activated Protein Kinases/metabolism
5.
PLoS One ; 7(12): e52205, 2012.
Article in English | MEDLINE | ID: mdl-23300615

ABSTRACT

Antimitotic compounds are still one of the most widely used chemotherapeutic anticancer drugs in the clinic today. Given their effectiveness against cancer it is beneficial to continue enhancing these drugs. One way is to improve the bioavailability and efficacy by synthesizing derivatives that reversibly bind to carbonic anhydrase II (CAII) in red blood cells followed by a slow release into the blood circulation system. In the present study we describe the in vitro biological activity of a reduced derivative of 2-ethyl-3-O-sulphamoyl-estradiol (2EE), 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol). ESE-15-ol is capable of inhibiting carbonic anhydrase activity in the nanomolar range and is selective towards a mimic of carbonic anhydrase IX when compared to the CAII isoform. Docking studies using Autodock Vina suggest that the dehydration of the D-ring plays a role towards the selectivity of ESE-15-ol to CAIX and that the binding mode of ESE-15-ol is substantially different when compared to 2EE. ESE-15-ol is able to reduce cell growth to 50% after 48 h at 50-75 nM in MCF-7, MDA-MB-231, and MCF-12A cells. The compound is the least potent against the non-tumorigenic MCF-12A cells. In vitro mechanistic studies demonstrate that the newly synthesized compound induces mitochondrial membrane depolarization, abrogates the phosphorylation status of Bcl-2 and affects gene expression of genes associated with cell death and mitosis.


Subject(s)
Antimitotic Agents/pharmacology , Carbonic Anhydrase Inhibitors/pharmacology , Carbonic Anhydrases/metabolism , Estradiol/analogs & derivatives , Estradiol/pharmacology , Sulfonamides/metabolism , Sulfonamides/pharmacology , Antimitotic Agents/chemistry , Antimitotic Agents/metabolism , Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrase Inhibitors/metabolism , Carbonic Anhydrases/chemistry , Cell Cycle/drug effects , Cell Proliferation/drug effects , Estradiol/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Humans , MCF-7 Cells , Membrane Potential, Mitochondrial/drug effects , Molecular Docking Simulation , Neoplasm Metastasis , Phosphorylation/drug effects , Protein Conformation , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins c-bcl-2/metabolism , Serine/metabolism , Tubulin/chemistry , Tubulin/metabolism
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