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1.
J Cell Biochem ; 121(12): 4974-4990, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32692912

RESUMO

In preclinical cancer studies, three-dimensional (3D) cell spheroids and aggregates are preferred over monolayer cell cultures due to their architectural and functional similarity to solid tumors. We performed a proof-of-concept study to generate physiologically relevant and predictive preclinical models using non-small cell lung adenocarcinoma, and colon and colorectal adenocarcinoma cell line-derived 3D spheroids and aggregates. Distinct panels were designed to determine the expression profiles of frequently studied biomarkers of the two cancer subtypes. The lung adenocarcinoma panel included ALK, EGFR, TTF-1, and CK7 biomarkers, and the colon and colorectal adenocarcinoma panel included BRAF V600E, MSH2, MSH6, and CK20. Recent advances in immunofluorescence (IF) multiplexing and imaging technology enable simultaneous detection and quantification of multiple biomarkers on a single slide. In this study, we performed IF staining of multiple biomarkers per section on formalin-fixed paraffin-embedded 3D spheroids and aggregates. We optimized protocol parameters for automated IF and demonstrated staining concordance with automated chromogenic immunohistochemistry performed with validated protocols. Next, post-acquisition spectral unmixing of the captured fluorescent signals were utilized to delineate four differently stained biomarkers within a single multiplex IF image, followed by automated quantification of the expressed markers. This workflow has the potential to be adapted to preclinical high-throughput screening and drug efficacy studies utilizing 3D spheroids from cancer cell lines and patient-derived organoids. The process allows for cost, time, and resource savings through concurrent staining of several biomarkers on a single slide, the ability to study the interactions of multiple expressed proteins within a single region of interest, and enable quantitative assessment of biomarkers in cancer cells.

2.
PLoS Pathog ; 10(4): e1004018, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24763432

RESUMO

The role of calcium (Ca2+) and its dependent protease calpain in Aeromonas hydrophila-induced head kidney macrophage (HKM) apoptosis has been reported. Here, we report the pro-apoptotic involvement of calmodulin (CaM) and calmodulin kinase II gamma (CaMKIIg) in the process. We observed significant increase in CaM levels in A. hydrophila-infected HKM and the inhibitory role of BAPTA/AM, EGTA, nifedipine and verapamil suggested CaM elevation to be Ca2+-dependent. Our studies with CaM-specific siRNA and the CaM inhibitor calmidazolium chloride demonstrated CaM to be pro-apoptotic that initiated the downstream expression of CaMKIIg. Using the CaMKIIg-targeted siRNA, specific inhibitor KN-93 and its inactive structural analogue KN-92 we report CaM-CaMKIIg signalling to be critical for apoptosis of A. hydrophila-infected HKM. Inhibitor studies further suggested the role of calpain-2 in CaMKIIg expression. CaMK Kinase (CaMKK), the other CaM dependent kinase exhibited no role in A. hydrophila-induced HKM apoptosis. We report increased production of intracellular cAMP in infected HKM and our results with KN-93 or KN-92 implicate the role of CaMKIIg in cAMP production. Using siRNA to PKACA, the catalytic subunit of PKA, anti-PKACA antibody and H-89, the specific inhibitor for PKA we prove the pro-apoptotic involvement of cAMP/PKA pathway in the pathogenicity of A. hydrophila. Our inhibitor studies coupled with siRNA approach further implicated the role of cAMP/PKA in activation of extracellular signal-regulated kinase 1 and 2 (ERK 1/2). We conclude that the alteration in intracellular Ca2+ levels initiated by A. hydrophila activates CaM and calpain-2; both pathways converge on CaMKIIg which in turn induces cAMP/PKA mediated ERK 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected HKM.


Assuntos
Aeromonas hydrophila/metabolismo , Apoptose , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Doenças dos Peixes , Proteínas de Peixes/metabolismo , Infecções por Bactérias Gram-Negativas , Rim/metabolismo , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Animais , Peixes-Gato , Doenças dos Peixes/metabolismo , Doenças dos Peixes/microbiologia , Doenças dos Peixes/patologia , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/patologia , Infecções por Bactérias Gram-Negativas/veterinária , Rim/patologia , Macrófagos/patologia
3.
Chemosphere ; 332: 138848, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37156291

RESUMO

Bifenthrin (BF), a synthetic pyrethroid is used worldwide for both agricultural and non-agricultural purposes due to its high insecticidal activity and low toxicity in mammals. However, its improper usage implies a possible risk to aquatic life. The study was aimed to correlate the association of BF toxicity with mitochondrial DNA copy number variation in edible fish Punitus sophore. The 96-h LC50 of BF in P. sophore was 3.4 µg/L, fish was treated with sub-lethal doses ((⅒ and ⅕ of LC50;0.34 µg/L, 0.68 µg/L) of BF for 15 days. The activity and expression level of cytochrome c oxidase (Mt-COI) were measured to assess mitochondrial dysfunction caused by BF. Results showed BF reduced the level of Mt-COI mRNA in treated groups, hindered complex IV activity and increased ROS generation leading to oxidative damage. mtDNAcn was decreased in the muscle, brain and liver after BF treatment. Furthermore, BF induced neurotoxicity in brain and muscle cells through the inhibition of AchE activity. The treated groups showed elevated level of malondialdehyde (MDA) and an imbalance of antioxidant enzymes activity. Molecular docking and simulation analysis also predicted that BF binds to the active sites of the enzyme and restricts the fluctuation of its residues. Hence, outcome of the study suggests reduction of mtDNAcn could be a potential biomarker to assess Bifenthrin induced toxicity in aquatic ecosystem.


Assuntos
Cyprinidae , Piretrinas , Animais , Complexo IV da Cadeia de Transporte de Elétrons/genética , Variações do Número de Cópias de DNA , DNA Mitocondrial/genética , Ecossistema , Simulação de Acoplamento Molecular , Piretrinas/toxicidade , Piretrinas/química , Estresse Oxidativo , Antioxidantes , Mitocôndrias , Mamíferos
4.
Toxicol Appl Pharmacol ; 256(1): 44-51, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21798276

RESUMO

We had earlier shown that exposure to arsenic (0.50 µM) caused caspase-3 mediated head kidney macrophage (HKM) apoptosis involving the p38-JNK pathway in Clarias batrachus. Here we examined the roles of calcium (Ca(2+)) and extra-cellular signal-regulated protein kinase (ERK), the other member of MAPK-pathway on arsenic-induced HKM apoptosis. Arsenic-induced HKM apoptosis involved increased expression of ERK and calpain-2. Nifedipine, verapamil and EGTA pre-treatment inhibited the activation of calpain-2, ERK and reduced arsenic-induced HKM apoptosis as evidenced from reduced caspase-3 activity, Annexin V-FITC-propidium iodide and Hoechst 33342 staining. Pre-incubation with ERK inhibitor U 0126 inhibited the activation of calpain-2 and interfered with arsenic-induced HKM apoptosis. Additionally, pre-incubation with calpain-2 inhibitor also interfered with the activation of ERK and inhibited arsenic-induced HKM apoptosis. The NADPH oxidase inhibitor apocynin and diphenyleneiodonium chloride also inhibited ERK activation indicating activation of ERK in arsenic-exposed HKM also depends on signals from NADPH oxidase pathway. Our study demonstrates the critical role of Ca(2+) homeostasis on arsenic-induced HKM apoptosis. We suggest that arsenic-induced alteration in intracellular Ca(2+) levels initiates pro-apoptotic ERK and calpain-2; the two pathways influence each other positively and induce caspase-3 mediated HKM apoptosis. Besides, our study also indicates the role of ROS in the activation of ERK pathway in arsenic-induced HKM apoptosis in C. batrachus.


Assuntos
Apoptose/efeitos dos fármacos , Arsênio/toxicidade , Cálcio/fisiologia , Calpaína/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Homeostase/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Animais , Apoptose/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Peixes-Gato , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Homeostase/fisiologia , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/enzimologia , Isoenzimas/metabolismo , Rim/efeitos dos fármacos , Rim/enzimologia , Macrófagos/citologia
5.
Dev Comp Immunol ; 76: 392-402, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28713009

RESUMO

Alterations in intracellular-calcium (Ca2+)i homeostasis is critical to Aeromonas hydrophila-induced headkidney macrophages (HKM) apoptosis of Clarias gariepinus, though the implications are poorly understood. Here, we describe the role of intermediate molecules of Ca2+-signaling pathway that are involved in HKM apoptosis. We observed phosphoinositide-3-kinase/phospholipase C is critical for (Ca2+)i release in infected HKM. Heightened protein kinase-C (PKC) activity and phosphorylation of MEK1/2-ERK1/2 was noted which declined in presence of 2-APB, Go6976 and PD98059, inhibitors to IP3-receptor, conventional PKC isoforms (cPKC) and MEK1/2 respectively implicating Ca2+/cPKC/MEK-ERK1/2 axis imperative in A. hydrophila-induced HKM apoptosis. Significant tumor necrosis factor-α (TNFα) production and its subsequent reduction in presence of MEK-ERK1/2 inhibitor U0126 suggested TNFα production downstream to cPKC-mediated signaling via MEK1/2-ERK1/2 pathway. RNAi and inhibitor studies established the role of TNFα in inducing caspase-8-mediated apoptosis of infected HKM. We conclude, alterations in A. hydrophila-induced (Ca2+)i alterations activate cPKC-MEK1/2-ERK1/2-TNFα signaling cascade triggering HKM apoptosis.


Assuntos
Aeromonas hydrophila/imunologia , Cálcio/metabolismo , Peixes-Gato/imunologia , Citosol/metabolismo , Doenças dos Peixes/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Rim Cefálico/patologia , Macrófagos/imunologia , Animais , Apoptose , Caspase 8/metabolismo , MAP Quinase Quinase 1/metabolismo , Macrófagos/microbiologia , Proteína Quinase C/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
6.
Toxicol Res (Camb) ; 5(5): 1477-1489, 2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30090451

RESUMO

Fluoride is known to induce apoptosis though the mechanisms remain obscure. The aim of the present study was to understand the underlying molecular mechanisms of fluoride-induced apoptosis using fish headkidney macrophages (HKMs). Exposure to fluoride triggered HKM cell apoptosis as evidenced by Hoechst 333432 and AnnexinV-propidium iodide staining, the presence of an internucleosomal DNA ladder and the comet assay. Our results suggest the influx of extra-cellular Ca2+ to be an initial event in fluoride-induced HKM cell apoptosis. We observed persistently elevated levels of superoxide anions and our inhibitor studies with EGTA suggested the primal role of the Ca2+ flux in triggering superoxide production in fluoride-exposed HKM cells. Fluoride exposure led to elevated levels of Ca2+/CaM dependent protein kinase II gamma (CaMKIIg) and pre-treatment with the inhibitor KN-93 but not its inactive structural analogue KN-92 reduced the number of apoptotic cells establishing the pro-apoptotic role of CaMKIIg in fluoride-induced HKM cell apoptosis. We report that the sustained superoxide generation is primarily responsible for the increased CaMKIIg levels observed in fluoride-exposed HKM cells. Our inhibitor studies further implicated CaMKIIg in the activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) culminating in caspase-8/caspase-3 mediated apoptosis of HKM cells. We conclude that fluoride-induced apoptosis is largely dependent on Ca2+ induced superoxide generation leading to elevation in CaMKIIg which in turn induces the phosphorylation of ERK 1/2 and downstream activation of extrinsic caspase cascade in HKM cells.

7.
PLoS One ; 11(1): e0146554, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26752289

RESUMO

Mycobacterium fortuitum causes 'mycobacteriosis' in wide range of hosts although the mechanisms remain largely unknown. Here we demonstrate the role of calcium (Ca+2)-signalling cascade on M. fortuitum-induced apoptosis in headkidney macrophages (HKM) of Clarias sp. M. fortuitum could trigger intracellular-Ca+2 influx leading to the activation of calmodulin (CaM), protein kinase C alpha (PKCα) and Calmodulin kinase II gamma (CaMKIIg). Gene silencing and inhibitor studies established the role of CaM in M. fortuitum pathogenesis. We noted that CaMKIIg activation is regulated by CaM as well as PKCα-dependent superoxide anions. This is altogether first report of oxidised CaMKIIg in mycobacterial infections. Our studies with targeted-siRNA and pharmacological inhibitors implicate CaMKIIg to be pro-apoptotic and critical for the activation of extra-cellular signal regulated kinase 1/2 (ERK1/2). Inhibiting the ERK1/2 pathway attenuated nitric oxide synthase 2 (NOS2)-induced nitric oxide (NO) production. Conversely, inhibiting the NOS2-NO axis by specific-siRNA and inhibitors down-regulated ERK1/2 activation suggesting the crosstalk between ERK1/2 and NO is essential for pathogenesis induced by the bacterium. Silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase-8 mediated activation of caspase-3 in the infected HKM. Our findings unveil hitherto unknown mechanism of M. fortuitum pathogenesis. We propose that M. fortuitum triggers intracellular Ca+2 elevations resulting in CaM activation and PKCα-mediated superoxide generation. The cascade converges in common pathway mediated by CaMKIIg resulting in the activation of ERK1/2-NOS2 axis. The crosstalk between ERK1/2 and NO shifts the balance in favour of caspase dependent apoptosis of M. fortuitum-infected HKM.


Assuntos
Apoptose/fisiologia , Cálcio/metabolismo , Macrófagos/metabolismo , Mycobacterium fortuitum/metabolismo , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Animais , Peixes-Gato/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo
8.
Sci Rep ; 4: 5820, 2014 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-25059203

RESUMO

Endoplasmic reticulum (ER)-stress and unfolding protein response (UPR) has not been implied in Aeromonas hydrophila-pathogenicity. We report increased expression of the ER-stress markers: CHOP, BiP and phospho-eIF2α in A. hydrophila-infected headkidney macrophages (HKM) in Clarias batrachus. Pre-treatment with ER-stress inhibitor, 4-PBA alleviated ER-stress and HKM apoptosis suggesting ER-UPR critical for the process. The ER-Ca(2+) released via inositol-triphosphate and ryanodine receptors induced calpain-2 mediated superoxide ion generation and consequent NF-κB activation. Inhibiting NF-κB activation attenuated NO production suggesting the pro-apoptotic role of NF-κB on HKM pathology. Calpain-2 activated caspase-12 to intensify the apoptotic cascade through mitochondrial-membrane potential (ψm) dissipation and caspase-9 activation. Altered mitochondrial ultra-structure consequent to ER-Ca(2+) uptake via uniporters reduced ψm and released cytochrome C. Nitric oxide induced the cGMP/PKG-dependent activation of caspase-8 and truncated-Bid formation. Both the caspases converge onto caspase-3 to execute HKM apoptosis. These findings offer a possible molecular explanation for A. hydrophila pathogenicity.


Assuntos
Aeromonas hydrophila/patogenicidade , Apoptose , Estresse do Retículo Endoplasmático , Macrófagos/metabolismo , Mitocôndrias/metabolismo , Animais , Apoptose/efeitos dos fármacos , Butilaminas/farmacologia , Cálcio/metabolismo , Calpaína/metabolismo , Caspase 12/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Peixes-Gato , Citocromos c/metabolismo , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Macrófagos/citologia , Macrófagos/microbiologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/ultraestrutura , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Fator de Transcrição CHOP/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos
9.
Dev Comp Immunol ; 37(3-4): 323-33, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22366184

RESUMO

The mechanism of macrophage cytotoxicity induced by Aeromonas hydrophila is yet unresolved. We observed A. hydrophila induces Head Kidney Macrophage (HKM) apoptosis in Clarias batrachus, as evident from Hoechst 33342 and AnnexinV-Propidium Iodide staining and presence of oligonucleosomal DNA ladder. Initiation of apoptosis required the bacteria to be alive, be actively phagocytosed into HKM and was dependent on host proteins. Elevated cytosolic calcium and consequent calpain activity that declined following pre-incubation with EGTA, verapamil and nifedipine implicates the role of calcium influx through voltage gated calcium channels and calpain in A. hydrophila-induced HKM apoptosis. Though, calpain-1 and -2 were involved, calpain-2 appeared to be more important in the process. EGTA, verapamil, nifedipine and calpain-2 inhibitor reduced caspase-3 activity and apoptosis. We conclude that A. hydrophila alters cytosolic calcium homeostasis initiating the activation of calpains, more specifically calpain-2, which leads to caspase-3 mediated HKM apoptosis in C. batrachus.


Assuntos
Aeromonas hydrophila , Calpaína/metabolismo , Caspase 3/metabolismo , Peixes-Gato , Doenças dos Peixes/imunologia , Proteínas de Peixes/metabolismo , Infecções por Bactérias Gram-Negativas/veterinária , Rim Cefálico/citologia , Macrófagos/citologia , Animais , Apoptose , Cálcio/metabolismo , Doenças dos Peixes/metabolismo , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/microbiologia , Rim Cefálico/imunologia , Macrófagos/imunologia
10.
Curr Pharm Biotechnol ; 12(4): 469-87, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21342107

RESUMO

Stem-cell technology is a major area within cell therapy that promises significant therapeutic outcome. The plasticity and self-renewal capabilities of stem cells make them valuable tools for potential application in regenerative medicine and tissue replacement following injury or disease. Here, we discuss the different types of stem cells currently used in research, preclinical and early clinical development, their potential therapeutic and diagnostic applications, and current barriers to translating basic research into clinical therapies. Biomedical imaging is increasingly being used to monitor the fate of transplanted stem cells, including their survival, proliferation, differentiation and homing to targeted organs and tissues. We discuss different imaging modalities currently utilized to track stem calls, the advantages and challenges, and future implications in clinical applications.


Assuntos
Células-Tronco Adultas , Células-Tronco Embrionárias , Imagem Molecular/métodos , Transplante de Células-Tronco , Pesquisa Translacional Biomédica/métodos , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/fisiologia , Animais , Biomarcadores/análise , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Humanos
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