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1.
Int J Mol Sci ; 24(17)2023 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-37686085

RESUMO

The protein disulfide isomerase A3 (PDIA3) is directly or indirectly involved in various physiopathological processes and participates in cancer initiation, progression and chemosensitivity. However, little is known about its involvement in glioblastoma. To obtain specific information, we performed cellular experiments in the T98G and U-87 MG glioblastoma cell lines to evaluate the role of PDIA3. The loss of PDIA3 functions, either through inhibition or silencing, reduced glioblastoma cells spreading by triggering cytotoxic phenomena. PDIA3 inhibition led to a redistribution of PDIA3, resulting in the formation of protein aggregates visualized through immunofluorescence staining. Concurrently, cell cycle progression underwent arrest at the G1/S checkpoint. After PDIA3 inhibition, ROS-independent DNA damage and the activation of the repair system occurred, as evidenced by the phosphorylation of H2A.X and the overexpression of the Ku70 protein. We also demonstrated through a clonogenic assay that PDIA3 inhibition could increase the chemosensitivity of T98G and U-87 MG cells to the approved glioblastoma drug temozolomide (TMZ). Overall, PDIA3 inhibition induced cytotoxic effects in the analyzed glioblastoma cell lines. Although further in vivo studies are needed, the results suggested PDIA3 as a novel therapeutic target that could also be included in already approved therapies.


Assuntos
Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Isomerases de Dissulfetos de Proteínas/genética , Temozolomida/farmacologia , Fosforilação , Bioensaio
2.
Int J Mol Sci ; 24(3)2023 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-36769334

RESUMO

In the present study, we used a mouse model of Alzheimer's disease (AD) (3×Tg-AD mice) to longitudinally analyse the expression level of PDIA3, a protein disulfide isomerase and endoplasmic reticulum (ER) chaperone, in selected brain limbic areas strongly affected by AD-pathology (amygdala, entorhinal cortex, dorsal and ventral hippocampus). Our results suggest that, while in Non-Tg mice PDIA3 levels gradually reduce with aging in all brain regions analyzed, 3×Tg-AD mice showed an age-dependent increase in PDIA3 levels in the amygdala, entorhinal cortex, and ventral hippocampus. A significant reduction of PDIA3 was observed in 3×Tg-AD mice already at 6 months of age, as compared to age-matched Non-Tg mice. A comparative immunohistochemistry analysis performed on 3×Tg-AD mice at 6 (mild AD-like pathology) and 18 (severe AD-like pathology) months of age showed a direct correlation between the cellular level of Aß and PDIA3 proteins in all the brain regions analysed, even if with different magnitudes. Additionally, an immunohistochemistry analysis showed the presence of PDIA3 in all post-mitotic neurons and astrocytes. Overall, altered PDIA3 levels appear to be age- and/or pathology-dependent, corroborating the ER chaperone's involvement in AD pathology, and supporting the PDIA3 protein as a potential novel therapeutic target for the treatment of AD.


Assuntos
Doença de Alzheimer , Camundongos , Animais , Doença de Alzheimer/metabolismo , Camundongos Transgênicos , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Encéfalo/metabolismo , Camundongos Endogâmicos , Modelos Animais de Doenças , Peptídeos beta-Amiloides/metabolismo , Proteínas tau/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo
3.
Cell Mol Biol Lett ; 27(1): 12, 2022 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-35109791

RESUMO

The ERp57/PDIA3 protein is a pleiotropic member of the PDIs family and, although predominantly located in the endoplasmic reticulum (ER), has indeed been found in other cellular compartments, such as the nucleus or the cell membrane. ERp57/PDIA3 is an important research target considering it can be found in various subcellular locations. This protein is involved in many different physiological and pathological processes, and our review describes new data on its functions and summarizes some ligands identified as PDIA3-specific inhibitors.


Assuntos
Retículo Endoplasmático , Isomerases de Dissulfetos de Proteínas , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo
4.
Molecules ; 26(23)2021 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-34885717

RESUMO

The ß-isomer of hexachlorocyclohexane (ß-HCH) is a globally widespread pollutant that embodies all the physicochemical characteristics of organochlorine pesticides, constituting an environmental risk factor for a wide range of noncommunicable diseases. Previous in vitro studies from our group disclosed the carcinogenic potential of ß-HCH, which contributes to neoplastic transformation by means of multifaceted intracellular mechanisms. Considering the positive evidence regarding the protective role of natural bioactive compounds against pollution-induced toxicity, micronutrients from olive and tomato endowed with the capability of modulating ß-HCH cellular targets were tested. For this purpose, the solution obtained from a patented food supplement (No. EP2851080A1), referred to as Tomato and Olive Bioactive Compounds (TOBC), was administered to the androgen-sensitive prostate cancer cells LNCaP and different biochemical and cellular assays were performed to evaluate its efficiency. TOBC shows a dose-dependent significant chemoprotection by contrasting ß-HCH-induced intracellular responses such as STAT3 and AhR activation, disruption of AR signaling, antiapoptotic and proliferative activity, and increase in ROS production and DNA damage. These experimental outcomes identified TOBC as a suitable functional food to be included in a diet regimen aimed at defending cells from ß-HCH negative effects, recommending the development of tailored enriched formulations for exposed individuals.


Assuntos
Compostos Fitoquímicos/farmacologia , Neoplasias da Próstata/dietoterapia , Receptores Androgênicos/genética , Fator de Transcrição STAT3/genética , Androgênios/metabolismo , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hexaclorocicloexano/toxicidade , Humanos , Solanum lycopersicum/química , Masculino , Micronutrientes/química , Micronutrientes/farmacologia , Olea/química , Compostos Fitoquímicos/química , Neoplasias da Próstata/induzido quimicamente , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Espécies Reativas de Oxigênio/química , Fatores de Risco , Transdução de Sinais/efeitos dos fármacos
5.
Biomedicines ; 9(11)2021 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-34829762

RESUMO

In a previous work, it was shown that punicalagin, an active ingredient of pomegranate, is able to bind to PDIA3 and inhibit its disulfide reductase activity. Here we provide evidence that punicalagin can also bind to PDIA1, the main expressed form of protein disulfide isomerase (PDI). In this comparative study, the affinity and the effect of punicalagin binding on each protein were evaluated, and a computational approach was used to identify putative binding sites. Punicalagin binds to either PDIA1 or PDIA3 with a similar affinity, but the inhibition efficacy on protein reductase activity is higher for PDIA3. Additionally, punicalagin differently affects the thermal denaturation profile of both proteins. Molecular docking and molecular dynamics simulations led to propose a punicalagin binding mode on PDIA1 and PDIA3, identifying the binding sites at the redox domains a' in two different pockets, suggesting different effects of punicalagin on proteins' structure. This study provides insights to develop punicalagin-based ligands, to set up a rational design for PDIA3 selective inhibitors, and to dissect the molecular determinant to modulate the protein activity.

6.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072471

RESUMO

Organochlorine pesticides constitute the majority of the total environmental pollutants, and a wide range of compounds have been found to be carcinogenic to humans. Among all, growing interest has been focused on ß-hexachlorocyclohexane (ß-HCH), virtually the most hazardous and, at the same time, the most poorly investigated member of the hexachlorocyclohexane family. Considering the multifaceted biochemical activities of ß-HCH, already established in our previous studies, the aim of this work is to assess whether ß-HCH could also trigger cellular malignant transformation toward cancer development. For this purpose, experiments were performed on the human normal bronchial epithelium cell line BEAS-2B exposed to 10 µM ß-HCH. The obtained results strongly support the carcinogenic potential of ß-HCH, which is achieved through both non-genotoxic (activation of oncogenic signaling pathways and proliferative activity) and indirect genotoxic (ROS production and DNA damage) mechanisms that significantly affect cellular macroscopic characteristics and functions such as cell morphology, cell cycle profile, and apoptosis. Taking all these elements into account, the presented study provides important elements to further characterize ß-HCH, which appears to be a full-fledged carcinogenic agent.


Assuntos
Carcinógenos/farmacologia , Transformação Celular Neoplásica/induzido quimicamente , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Hexaclorocicloexano/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/patologia , Apoptose/efeitos dos fármacos , Biomarcadores , Biomarcadores Tumorais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células , Células Cultivadas , Células Epiteliais/metabolismo , Expressão Gênica , Hexaclorocicloexano/efeitos adversos , Humanos , Espécies Reativas de Oxigênio/metabolismo , Mucosa Respiratória/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
Biomedicines ; 8(11)2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33207735

RESUMO

Organochlorine pesticides (OCPs) belong to a heterogeneous class of organic compounds blacklisted by the Stockholm Convention in 2009 due to their harmful impact on human health. Among OCPs, ß-hexachlorocyclohexane (ß-HCH) is one of the most widespread and, at the same time, poorly studied environmental contaminant. Due to its physicochemical properties, ß-HCH is the most hazardous of all HCH isomers; therefore, clarifying the mechanisms underlying its molecular action could provide further elements to draw the biochemical profile of this OCP. For this purpose, LNCaP and HepG2 cell lines were used as models and were subjected to immunoblot, immunofluorescence, and RT-qPCR analysis to follow the expression and mRNA levels, together with the distribution, of key biomolecules involved in the intracellular responses to ß-HCH. In parallel, variations in redox homeostasis and cellular bioenergetic profile were monitored to have a complete overview of ß-HCH effects. Obtained results strongly support the hypothesis that ß-HCH could be an endocrine disrupting chemical as well as an activator of AhR signaling, promoting the establishment of an oxidative stress condition and a cellular metabolic shift toward aerobic glycolysis. In this altered context, ß-HCH can also induce DNA damage through H2AX phosphorylation, demonstrating its multifaceted mechanisms of action.

8.
Cells ; 8(9)2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500219

RESUMO

Prostate cancer (PCa) is a multifactorial disease characterized by the aberrant activity of different regulatory pathways. STAT3 protein mediates some of these pathways and its activation is implicated in the modulation of several metabolic enzymes. A bioinformatic analysis indicated a STAT3 binding site in the upstream region of SHMT2 gene. We demonstrated that in LNCaP, PCa cells' SHMT2 expression is upregulated by the JAK2/STAT3 canonical pathway upon IL-6 stimulation. Activation of SHTM2 leads to a decrease in serine levels, pushing PKM2 towards the nuclear compartment where it can activate STAT3 in a non-canonical fashion that in turn promotes a transient shift toward anaerobic metabolism. These results were also confirmed on FFPE prostate tissue sections at different Gleason scores. STAT3/SHMT2/PKM2 loop in LNCaP cells can modulate a metabolic shift in response to inflammation at early stages of cancer progression, whereas a non-canonical STAT3 activation involving the STAT3/HIF-1α/PKM2 loop is responsible for the maintenance of Warburg effect distinctive of more aggressive PCa cells. Chronic inflammation might thus prime the transition of PCa cells towards more advanced stages, and SHMT2 could represent a missing factor to further understand the molecular mechanisms responsible for the transition of prostate cancer towards a more aggressive phenotype.


Assuntos
Glicina Hidroximetiltransferase/metabolismo , Neoplasias da Próstata/metabolismo , Fator de Transcrição STAT3/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Metabolismo Energético , Glicina Hidroximetiltransferase/genética , Humanos , Masculino , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais , Ativação Transcricional
9.
Int J Mol Sci ; 20(8)2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-31013746

RESUMO

STAT3 is an oncoprotein overexpressed in different types of tumors, including prostate cancer (PCa), and its activity is modulated by a variety of post-translational modifications (PTMs). Prostate cancer represents the most common cancer diagnosed in men, and each phase of tumor progression displays specific cellular conditions: inflammation is predominant in tumor's early stage, whereas oxidative stress is typical of clinically advanced PCa. The aim of this research is to assess the correspondence between the stimulus-specificity of STAT3 PTMs and definite STAT3-mediated transcriptional programs, in order to identify new suitable pharmacological targets for PCa treatment. Experiments were performed on less-aggressive LNCaP and more aggressive DU-145 cell lines, simulating inflammatory and oxidative-stress conditions. Cellular studies confirmed pY705-STAT3 as common denominator of all STAT3-mediated signaling. In addition, acK685-STAT3 was found in response to IL-6, whereas glutC328/542-STAT3 and pS727-STAT3 occurred upon tert-butyl hydroperoxyde (tBHP) treatment. Obtained results also provided evidence of an interplay between STAT3 PTMs and specific protein interactors such as P300 and APE1/Ref-1. In accordance with these outcomes, mRNA levels of STAT3-target genes seemed to follow the differing STAT3 PTMs. These results highlighted the role of STAT3 and its PTMs as drivers in the progression of PCa.


Assuntos
Neoplasias da Próstata/metabolismo , Processamento de Proteína Pós-Traducional , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Biomarcadores , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neoplasias da Próstata/genética , Transcrição Gênica
10.
Int J Mol Sci ; 19(7)2018 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-30036966

RESUMO

BACKGROUND: Organochlorine pesticides (OCPs) are widely distributed in the environment and their toxicity is mostly associated with the molecular mechanisms of endocrine disruption. Among OCPs, particular attention was focused on the effects of ß-hexaclorocyclohexane (ß-HCH), a widely common pollutant. A detailed epidemiological study carried out on exposed population in the "Valle del Sacco" found correlations between the incidence of a wide range of diseases and the occurrence of ß-HCH contamination. Taking into account the pleiotropic role of the protein signal transducer and activator of transcription 3 (STAT3), its function as a hub protein in cellular signaling pathways triggered by ß-HCH was investigated in different cell lines corresponding to tissues that are especially vulnerable to damage by environmental pollutants. MATERIALS AND METHODS: Human prostate cancer (LNCaP), human breast cancer (MCF-7 and MDA-MB 468), and human hepatoma (HepG2) cell lines were treated with 10 µM ß-HCH in the presence or absence of specific inhibitors for different receptors. All samples were subjected to analysis by immunoblotting and RT-qPCR. RESULTS AND CONCLUSIONS: The preliminary results allow us to hypothesize the involvement of STAT3, through both its canonical and non-canonical pathways, in response to ß-HCH. Moreover, we ascertained the role of STAT3 as a master regulator of energy metabolism via the altered expression and localization of HIF-1α and PKM2, respectively, resulting in a Warburg-like effect.


Assuntos
Hexaclorocicloexano/farmacologia , Fator de Transcrição STAT3/metabolismo , Metabolismo Energético/efeitos dos fármacos , Feminino , Células Hep G2 , Humanos , Células MCF-7 , Masculino , Transdução de Sinais/efeitos dos fármacos
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