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Steroid sulfatase (STS) deficiency is responsible for X-linked ichthyosis (XLI), a genetic disorder characterized by rough and dry skin caused by excessive keratinization. The impaired keratinization process leads to reduced cell mobility and increased apoptosis, which can cause an excessive buildup of the stratum corneum. In this study, we investigated the mechanisms underlying XLI and found that STS deficiency reduces cell mobility and increases apoptosis in human keratinocyte HaCaT cells. To explore these mechanisms further, RNA-sequencing was conducted on skin tissues from STS transgenic and knockout mice. Our RNA-seq results revealed that STS deficiency plays a critical role in regulating multiple signaling pathways associated with cell mobility and apoptosis, such as Wnt/ß signaling and the Hippo signaling pathway. Knockdown of the STS gene using shRNA in HaCaT cells led to an upregulation of E-cadherin expression and suppression of key factors involved in epithelial-mesenchymal transition (EMT), such as N-cadherin and vimentin. Inhibition of EMT involved the Hippo signaling pathway and reduction of HIF-1α. Interestingly, inhibiting STS with shRNA increased mitochondrial respiration levels, as demonstrated by the extracellular flux oxygen consumption rate. Additionally, we observed a significant increase in ROS production in partial STS knockout cells compared to control cells. Our study demonstrated that the excessive generation of ROS caused by STS deficiency induces the expression of Bax and Bak, leading to the release of cytochrome c and subsequent cell death. Consequently, STS deficiency impairs cell mobility and promotes apoptosis, offering insights into the pathophysiological processes and potential therapeutic targets for XLI.
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Ictiose Ligada ao Cromossomo X , Ictiose , Animais , Camundongos , Humanos , Ictiose Ligada ao Cromossomo X/genética , Esteril-Sulfatase/genética , Espécies Reativas de Oxigênio , Ictiose/genética , Apoptose , RNA Interferente PequenoRESUMO
The Hippo pathway is a signaling pathway that controls organ size in animals by regulating cell proliferation and apoptosis. Yes-associated protein 1 (YAP1), an oncogene associated with the development and progression of breast cancer, is downregulated by the Hippo pathway and is associated with the development and progression of breast cancer. Yippee-like 3 (YPEL3) is a target gene of the tumor suppressor protein p53, and its activation has been shown to inhibit cell growth, induce cellular senescence, and suppress tumor cell metastasis. In this study, we found that YAP1 inhibits the expression of YPEL3 expression in breast cancer cells. Furthermore, a decrease in lamin B1, a marker protein of cellular senescence, coupled with the activation of senescence-associated ß-galactosidase indicated that upregulating YPEL3 levels through YAP1 downregulation can induce cellular senescence. Additionally, elevated YPEL3 levels resulted in higher levels of oxygen consumption rate in mitochondria, thus promoting apoptosis. This suggests that YPEL3 plays a crucial role in regulating oxidative stress and cell apoptosis in breast cancer cells. Therefore, the interaction between YAP1 and YPEL3 represents a novel mechanism of cellular senescence mediated by the Hippo signaling pathway. Collectively, our findings suggest that the Hippo signaling pathway plays an important role in regulating cellular senescence, which could have implications for the development of new therapeutic strategies for diseases such as cancer.
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Genomic analysis indicated that the genome of Drosophila melanogaster contains more than 80 cytochrome P450 genes. To date, the enzymatic activity of these P450s has not been extensively studied. Here, the biochemical properties of CYP6A8 were characterized. CYP6A8 was cloned into the pCW vector, and its recombinant enzyme was expressed in Escherichia coli and purified using Ni2+-nitrilotriacetate affinity chromatography. Its expression level was approximately 130 nmol per liter of culture. Purified CYP6A8 exhibited a low-spin state in the absolute spectra of the ferric forms. Binding titration analysis indicated that lauric acid and capric acid produced type Ð spectral changes, with Kd values 28 ± 4 and 144 ± 20 µM, respectively. Ultra-performance liquid chromatography-mass spectrometry analysis showed that the oxidation reaction of lauric acid produced (ω-1)-hydroxylated lauric acid as a major product and ω-hydroxy-lauric acid as a minor product. Steady-state kinetic analysis of lauric acid hydroxylation yielded a kcat value of 0.038 ± 0.002 min-1 and a Km value of 10 ± 2 µM. In addition, capric acid hydroxylation of CYP6A8 yielded kinetic parameters with a kcat value of 0.135 ± 0.007 min-1 and a Km value of 21 ± 4 µM. Because of the importance of various lipids as carbon sources, the metabolic analysis of fatty acids using CYP6A8 in this study can provide an understanding of the biochemical roles of P450 enzymes in many insects, including Drosophila melanogaster.
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2,4,3',5'-Tetramethoxystilbene (TMS) is a selective inhibitor of cytochrome P450 1B1 to block the conversion from estradiol to 4-OH-estradiol. Several studies suggested that TMS may act as a potent anti-cancer agent for hormone-related cancer including cervical cancer. Nutlin-3a is a cis-imidazoline analog that interferes with the interaction between mouse double minute 2 homolog (MDM2) and the tumor suppressor p53. The purpose of the study was to compare the cytotoxic effect of TMS and nutlin-3a treatment individually and in combination in HeLa cells. To assess the potential synergistic effects between TMS and nutlin-3a, low concentrations of TMS and nutlin-3a were simultaneously treated in HeLa cells. Based on cell viability, apoptosis assays, and the increase in cleaved caspase-3 and poly (ADP-ribose) polymerase cleavage, it was demonstrated that the combination with TMS and nutlin-3a exerts a synergistic effect on cancer cell death. Isobologram analysis of HeLa cells noted synergism between TMS and nutlin-3a. The combined treatment increased the expression of mitochondrial pro-apoptotic factors such as Bax and Bak, and decreased the expression of the XIAP. In addition, combination treatment significantly enhanced the translocation of AIF to the nucleus in HeLa cells. In conclusion, the results demonstrate that the combination of TMS and nutlin-3a induces synergistic apoptosis in HeLa cells, suggesting the possibility that this combination can be applied as a novel therapeutic strategy for cervical cancer.
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Lung cancer has the highest incidence and mortality rates among all types of cancer worldwide, and 80%-85% of patients with lung cancer are diagnosed with non-small cell lung cancer (NSCLC), which has 5-year survival rate of only 5% at advanced stages. Development of new therapeutic agents and strategies is required to enhance the treatment efficiency in patients with NSCLC. Metabolic alterations and anticancer effects of plant hormones and their derivatives have not been investigated in NSCLC in vitro and in vivo. The present study investigated the cytotoxic effects of 11 plant hormones and their derivatives against NSCLC cell lines; ortho-topolin riboside (oTR) showed the highest cytotoxicity among all tested compounds against NSCLC cells. Alteration of metabolites and lipids was investigated using gas chromatography-mass spectrometry and nano electrospray ionization-mass spectrometry in oTR-treated NSCLC cells and a xenograft mouse model. oTR reduced amino acid and pyrimidine synthesis in NSCLC cells and xenograft tumors. Moreover, oTR reduced glycolytic function and decreased mitochondrial respiration function by inhibiting glutamine and fatty acid oxidation. Increased levels of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine species suggested that oTR might act as a fatty acid oxidation inhibitor. In addition, the increased level of phosphatidylserine species implied that phosphatidylserine-mediated apoptosis occurred in oTR-treated NSCLC cells and xenograft tumor. The antiproliferative and apoptotic effects of oTR were mediated by the reduced p-ERK and p-AKT levels and increased cleaved Caspase-3 levels, respectively. This is the first study to investigate the metabolic alterations and anticancer activity of oTR in in vitro and in vivo models of NSCLC. Our results provide basis for the development of oTR-based therapeutic agent for patients with NSCLC.
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Antineoplásicos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Citocininas/metabolismo , Neoplasias Pulmonares/metabolismo , Metaboloma/fisiologia , Células A549 , Animais , Apoptose/fisiologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/metabolismoRESUMO
Animal models are used for preclinical toxicity studies, and the need for in vitro alternative methods has been strongly raised. Our study aims to elucidate the potential mechanism of change in EGR1 expression under situations of toxic injury and to develop an Egr1 promoter-luciferase gene reporter assay for an in vitro alternative method for toxicity prediction in drug discovery. We first found an increase in early growth response-1 (EGR1) mRNA/protein expressions in the liver and kidney of cisplatin-treated injured rats. Additionally, the EGR1 protein level was also elevated under situations of ocular injury after sodium lauryl sulfate (SLS) eye drops. These in vivo observations on injury-related EGR1 induction were confirmed by in vitro studies, where human corneal epithelial cells were treated with representative irritants (SLS and benzalkonium chloride) and 17 chemicals having different UN GHS irritant categories. Additionally, our results suggest the involvement of ERK, JNK, p38 MAPK pathways in EGR1 elevation in response to gamma-butyrolactone-induced injury. As EGR1 is considered to be a pivotal factor in proliferation and regeneration, siRNA-mediated knockdown of Egr1 promoted cytotoxic potential through a delay of injury-related recovery. More importantly, the elevation of promoter activities was observed by various irritants in cells transfected with Egr1 promoter-reporter vector. In conclusion, Egr1 can be a potential biomarker in a promoter-reporter system to improve the accuracy of in vitro predictions for ocular irritation.
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Human steroid sulfatase (STS) is an enzyme that catalyzes the hydrolysis of dehydroepiandrosterone sulfate (DHEAS), estrone sulfate (E1S), and cholesterol sulfate. Abnormal expression of STS causes several diseases including colorectal, breast, and prostate cancer and refractory skin disease. In particular, accumulation of intracellular cholesterol sulfate by STS deficiency leads to a skin disorder with abnormal keratinization called X-linked ichthyosis (XLI). To determine the detailed mechanisms of XLI, we performed RNA sequencing (RNA-seq) analysis using human keratinocyte HaCaT cells treated with cholesterol and cholesterol sulfate. Of the genes with expression changes greater than 1.5-fold, Yippee-like 3 (YPEL3), a factor expected to affect cell differentiation, was found. Induction of YPEL3 causes permanent growth arrest, cellular senescence, and inhibition of metastasis in normal and tumor cells. In this study, we demonstrate that YPEL3 expression was induced by STS deficiency and, using the CRISPR/Cas9 system, a partial knock-out (STS+/-) cell line was constructed to establish a disease model for XLI studies. Furthermore, we show that increased expression of YPEL3 in STS-deficient cell lines promoted cellular senescence and expression of keratinization-related proteins such as involucrin and loricrin. Our results suggest that upregulation of YPEL3 expression by STS deficiency may play a crucial role in inducing cellular senescence and abnormal differentiation in human keratinocytes.
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Ictiose Ligada ao Cromossomo X/genética , Queratinócitos/patologia , Esteril-Sulfatase/genética , Proteínas Supressoras de Tumor/genética , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Senescência Celular , Humanos , Ictiose Ligada ao Cromossomo X/patologia , Queratinócitos/metabolismo , Regulação para CimaRESUMO
Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer with a poor prognosis for which no effective therapeutic measures are currently available. The present study aimed to investigate whether interactions with endothelial colony-forming cells (ECFCs) promote aggressive progression of TNBC cells. Herein, using an indirect co-culture system, we showed that co-culture increased the invasive and migratory phenotypes of both MDA-MB-231 TNBC cells and ECFCs. Through a cytokine antibody array and RT-PCR analysis, we revealed that co-culture markedly induced secretion of the chemokine C-C motif ligand (CCL)8 from ECFCs and that of interleukin (IL)-8 from MDA-MB-231 cells. CCL8 was crucial for ECFC-induced IL-8 secretion and invasion of MDA-MB-231 cells as well as for MDA-MB-231-enhanced MMP-2 secretion and angiogenesis of ECFCs. We suggest c-Jun as a transcription factor for CCL8-induced IL-8 expression in MDA-MB-231 cells. IL-8 was important for co-culture-induced CCL8 and MMP-2 upregulation and invasion of ECFCs. Notably, our findings reveal a positive feedback loop between CCL8 and IL-8, which contributes to the aggressive phenotypes of both ECFC and TNBC cells. Using an MDA-MB-231 cell-based xenograft model, we show that tumor growth and metastasis are increased by co-injected ECFCs in vivo. Increased expression of IL-8 was observed in tissues with bone metastases in mice injected with conditioned media from co-cultured cells. High IL-8 levels are correlated with poor recurrence-free survival in TNBC patients. Together, these results suggest that CCL8 and IL-8 mediate the crosstalk between ECFCs and TNBC, leading to aggravation of tumorigenicity in TNBC.
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Neoplasias de Mama Triplo Negativas , Animais , Linhagem Celular Tumoral , Movimento Celular , Células Endoteliais , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-8/metabolismo , CamundongosRESUMO
Human cytochrome P450 enzymes (CYPs) play a critical role in various biological processes and human diseases. CYP1 family members, including CYP1A1, CYP1A2, and CYP1B1, are induced by aryl hydrocarbon receptors (AhRs). The binding of ligands such as polycyclic aromatic hydrocarbons activates the AhRs, which are involved in the metabolism (including oxidation) of various endogenous or exogenous substrates. The ligands that induce CYP1 expression are reported to be carcinogenic xenobiotics. Hence, CYP1 enzymes are correlated with the pathogenesis of cancers. Various endogenous substrates are involved in the metabolism of steroid hormones, eicosanoids, and other biological molecules that mediate the pathogenesis of several human diseases. Additionally, CYP1s metabolize and activate/inactivate therapeutic drugs, especially, anti-cancer agents. As the metabolism of drugs determines their therapeutic efficacy, CYP1s can determine the susceptibility of patients to some drugs. Thus, understanding the role of CYP1s in diseases and establishing novel and efficient therapeutic strategies based on CYP1s have piqued the interest of the scientific community.
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Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Xenobióticos/farmacocinética , Animais , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1B1/genética , Modelos Animais de Doenças , Predisposição Genética para Doença , Humanos , Ligantes , Receptores de Hidrocarboneto Arílico/metabolismoRESUMO
Auranofin is a gold complex used as an anti-rheumatic agent and may act as a potent anticancer drug against breast tumors. Trametinib is a specific mitogen-activated protein kinase inhibitor, approved for the treatment of metastatic melanoma. The aim of this study was to examine the synergistic effects of auranofin and trametinib on apoptosis in MCF-7 human breast cancer cells. The combination treatment inhibited cancer cell proliferation and induced cell cycle arrest at the sub-G1 phase and apoptosis via poly (ADP-ribose) polymerase cleavage and caspase-3/7 activation. It is noteworthy that this treatment significantly increased p38 mitogen-activated protein kinase (MAPK) phosphorylation to induce mitochondrial stress, subsequently promoting cancer cell apoptosis through release of apoptosis-inducing factor. Further data demonstrated that combined treatment significantly induced increase in nuclear translocation of AIF. These results indicated that activation of the p38 MAPK signaling pathway and mitochondrial apoptosis may contribute to the synergistic consequences in MCF-7 cells. Collectively, our data demonstrated that combined treatment with auranofin and trametinib exhibited synergistic breast cancer cell death and this combination might be utilized as a novel therapeutic strategy for breast cancer.
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Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Auranofina/farmacologia , Neoplasias da Mama/tratamento farmacológico , Piridonas/farmacologia , Pirimidinonas/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Axl receptor tyrosine kinase has been implicated in cancer progression, invasion, and metastasis in various cancer types. Axl overexpression has been observed in many cancers, and selective inhibitors of Axl, including R428, may be promising therapeutic agents for several human cancers, such as breast, lung, and pancreatic cancers. Here, we examined the cell growth inhibition mediated by R428 and auranofin individually as well as in combination in the human breast cancer cell lines MCF-7 and MDAMB- 231 to identify new advanced combination treatments for human breast cancer. Our data showed that combination therapy with R428 and auranofin markedly inhibited cancer cell proliferation. Isobologram analyses of these cells indicated a clear synergism between R428 and auranofin with a combination index value of 0.73. The combination treatment promoted apoptosis as indicated by caspase 3 activation and poly (ADP-ribose) polymerase cleavage. Cancer cell migration was also significantly inhibited by this combination treatment. Moreover, we found that combination therapy significantly increased the expression level of Bax, a mitochondrial proapoptotic factor, but decreased that of the X-linked inhibitor of apoptosis protein. Furthermore, the suppression of cell viability and induction of Bax expression by the combination treatment were recovered by treatment with N-acetylcysteine. In conclusion, our data demonstrated that combined treatment with R428 and auranofin synergistically induced apoptosis in human breast cancer cells and may thus serve as a novel and valuable approach for cancer therapy.
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BACKGROUND: Gut microbiota are known to be closely related to irritable bowel syndrome (IBS). However, not much is known about characteristic fecal metabolic profiles of IBS. We aimed to characterize fecal metabolites in patients with IBS with predominant diarrhea (IBS-D) using 1 H-nuclear magnetic resonance (1 H-NMR) spectroscopy. METHODS: In this study, we enrolled 29 patients diagnosed with IBS-D according to the Rome IV criteria, 22 healthy controls (HC) and 11 HC administered laxatives (HC-L) in the age group of 20-69 year. The usual diet of the patients and HC was maintained, their fecal samples were collected and investigated by NMR-based global metabolic profiling coupled with multivariate statistical analysis. RESULTS: We detected 55 metabolites in 1 H-NMR spectra of fecal samples: four amines, 16 amino acids, six fatty acids, eight organic acids, three sugars, and 18 other compounds. Orthogonal partial least square-discriminant analysis derived score plots showed clear separation between the IBS-D group and the HC and HC-L groups. Among the 55 metabolites identified, we found five disease-relevant potential biomarkers distinguishing the IBS-D from the HC, namely, cadaverine, putrescine, threonine, tryptophan, and phenylalanine. CONCLUSIONS: The patients with IBS-D were clearly differentiated from the HC and HC-L by fecal metabolite analysis using 1 H-NMR spectroscopy, and five fecal metabolites characteristic of IBS-D were found. The findings of this study could be used to develop alternative and complementary diagnostic methods and as a source of fundamental information for developing novel therapies for IBS-D.
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Diarreia/metabolismo , Fezes/química , Síndrome do Intestino Irritável/metabolismo , Adulto , Idoso , Biomarcadores/metabolismo , Diarreia/complicações , Feminino , Humanos , Síndrome do Intestino Irritável/complicações , Masculino , Metaboloma , Metabolômica , Pessoa de Meia-Idade , Análise Multivariada , Espectroscopia de Prótons por Ressonância Magnética , Adulto JovemRESUMO
Cytochrome P450 1B1 (CYP1B1) is a key enzyme that catalyzes the metabolism of 17ß-estradiol (E2) into catechol estrogens, such as 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2). CYP1B1 is related to tumor formation and is over-expressed in a variety of cancer cells. In particular, CYP1B1 is highly expressed in hormone-related cancers such as breast cancer, ovarian cancer, or prostate cancer compared to other cancers. However, the detailed mechanisms involving this protein remain unclear. In this study, we demonstrate that CYP1B1 affects X-linked inhibitor of apoptosis protein (XIAP) expression. When CYP1B1 was over-expressed in cells, there was significant increase in the XIAP protein level, whereas the XIAP mRNA level was not affected by CYP1B1 expression. Treatment with 4-OHE2, mainly formed by CYP1B1 activity, also increased XIAP protein levels, whereas treatment with 2-OHE2 did not have a significant effect. Treatment with 4-OHE2 significantly prevented proteasome-mediated XIAP degradation. In addition, phosphorylation of XIAP on serine 87, which is known to stabilize XIAP, was up-regulated by 4-OHE2, indicating that 4-OHE2 affects XIAP stability through XIAP phosphorylation. We also found that phosphorylation of protein kinase C (PKC)ε, which is required for XIAP phosphorylation, increased when cells were treated with 4-OHE2. In summary, our data show that CYP1B1 may play an important role in preventing ubiquitin-proteasome-mediated XIAP degradation through the activation of PKCε signaling in cancer cells.
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Citocromo P-450 CYP1B1/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Quinase C-épsilon/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Células MCF-7 , FosforilaçãoRESUMO
Auranofin is a gold complex categorized as an anti-rheumatic agent. Recently, several investigators suggested that auranofin may act as a potent anti-cancer drug for breast tumors. Nutlin-3a is a cis-imidazoline analog which prevents interaction between mouse double minute 2 homolog (MDM2) and the tumor suppressor p53. The aim of this study was to examine cell growth inhibition mediated by auranofin or nutlin-3a individually as well as in combination with MCF-7 and MDA-MB-231 cells. To assess any potential synergistic effects between auranofin and nutlin-3a, low concentrations of auranofin and nutlin-3a were simultaneously incubated with MCF-7 and MDA-MB-231 cells. Cell viability assay, caspase-3/7 assay, and poly (ADP-ribose) polymerase cleavage revealed that auranofin and nutlin-3a exerted a synergistic effect on cancer cell apoptosis. Isobologram analysis of MCF-7 and MDA-MB-231 cells noted evident synergism between auranofin and nutlin-3a. The combined treatment increased the expression of mitochondrial pro-apoptotic factors such as Bcl-2 associated X protein and Bcl-2 homologous antagonist/killer. Further, combination treatment signiï¬cantly enhanced reactive oxygen species (ROS) generation in MCF-7 and MDA-MB-231 cells. In conclusion, data demonstrated that combined treatment with auranofin and nutlin-3a exhibited a synergistic action on breast cancer cells and this combination may be considered for use as a novel therapeutic strategy for breast cancer.
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Antineoplásicos/uso terapêutico , Auranofina/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Citotoxinas/uso terapêutico , Imidazóis/uso terapêutico , Piperazinas/uso terapêutico , Células Tumorais Cultivadas/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Modelos AnimaisRESUMO
Human breast cancer cell line, MDA-MB-231, is highly invasive and aggressive, compared to less invasive cell line, MCF-7. To explore the genes that might influence the malignancy of MDA-MB-231, DNA microarray analysis was performed. The results showed that G0/G1 switch 2 (G0S2) was one of the most highly expressed genes among the genes upregulated in MDA-MB-231. Although G0S2 acts as a direct inhibitor of adipose triglyceride lipase, action of G0S2 in cancer progression is not yet understood. To investigate whether G0S2 affects invasiveness of MDA-MB-231 cells, G0S2 expression was inhibited using siRNA, which led to decreased cell proliferation, migration, and invasion of MDA-MB-231 cells. Consequently, G0S2 inhibition inactivated integrinregulated FAK-Src signaling, which promoted Hippo signaling and inactivated ERK1/2 signaling. In addition, G0S2 downregulation decreased ß-catenin expression, while E-cadherin expression was increased. It was demonstrated for the first time that G0S2 mediates the Hippo pathway and induces epithelial to mesenchymal transition (EMT). Taken together, our results suggest that G0S2 is a major factor contributing to cell survival and metastasis of MDA-MB-231 cells.
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The pathogenic fungus Candida albicans contains genes encoding five fatty acid hydroxylases belonging to the CYP52 family in its genome. Our previous study reported that CYP52A21 demonstrated typical omega-hydroxylation of lauric acid (Kim D, Cryle MJ, De Voss JJ, Ortiz de Montellano PR (2007) Arch Biochem Biophys 464, 213-220). Functional characterization of CYP52 fatty acid hydroxylases was studied, and their selectivity for hydroxylation was analyzed. Genes for four other CYP52 members (CYP52A22, CYP52A23, CYP52A24, and CYP52C3) from C. albicans were cloned, and their recombinant enzymes were expressed in Escherichia coli. CO-binding spectral analyses showed that the functional P450 holoenzyme was obtained only in CYP52A23, while no holoenzyme peak was observed in the other three CYP52 enzymes. Spectral change of the type II binding was observed in purified CYP52A23 when titrated with fatty acids but none was observed with alkanes. The gas chromatography-mass spectrometry (GC-MS) analysis revealed that CYP52A23 predominantly exhibited omega-hydroxylation activity during the oxidation reaction of fatty acids. Interestingly, it was found that CYP52A23 preferred longer-chain fatty acids (stearic acid and arachidic acid) for its catalytic activities while CYP52A21 preferred mid-chain fatty acids (lauric acid and mystic acid). To analyze the selectivity of fatty acids, hybrid mutagenesis of genes encoding CYP52A21 and CYP52A23 by overlap extension polymerase chain reaction was conducted. Two hybrid mutants containing the N-terminal fragments of CYP52A21 and C-terminal fragments of CYP52A23 displayed higher catalytic activity in palmitic acid and arachidic acid. These results suggested that the C-terminal part of CYP52A23 may be responsible for its preference to longer-chain fatty acids.
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Candida albicans/enzimologia , Sistema Enzimático do Citocromo P-450/química , Ácidos Graxos/química , Sequência de Aminoácidos , Sequência de Bases , Candida albicans/genética , Catálise , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Escherichia coli/genética , Hidroxilação , Mutação , Engenharia de Proteínas , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Human steroid sulfatase (STS) has been linked with poor prognosis in steroid-associated tumors and represents an important clinical target in cancers, yet the mechanism of STS-induced carcinogenesis remains unclear. To correlate STS with cancer metabolism, we determined the effects of STS on aerobic glycolysis. STS overexpression increased cellular levels of lactic acid, the final product of aerobic glycolysis. Moreover, STS suppressed the oxygen consumption rate (OCR), which represents mitochondrial respiration. Inhibition of STS by the specific inhibitor STX064 recovered STS-induced OCR repression and lactic acid over-production. DHEA, but not DHEA-S, suppressed the OCR level and enhanced lactic acid production. To understand the molecular mechanism of STS-induced cancer metabolism, we measured the expression of glycolytic enzymes hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2), which was highly upregulated by STS and DHEA at both protein and mRNA levels. HIF1α is a key mediator of aerobic glycolysis, and STS enhanced HIF1α promoter activity, mRNA expression, and protein expression. Down-regulation of HIF1α by siRNA suppressed the HK2 and PKM2 expression induced by both STS and DHEA. HIF1α siRNA also recovered the OCR repression and lactic acid over-production induced by both STS and DHEA. To explore the mechanism in vivo, we produced transgenic mice overexpressing STS and found that STS expression was particularly enhanced in the lung. Consistent with our in vitro results, the expression of HIF1α, HK2, and PKM2 was also increased in mouse lung tissues. In conclusion, we suggest that STS may induce aerobic glycolysis through enhancing HIF1α expression.
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Proteínas de Transporte/metabolismo , Glicólise , Hexoquinase/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Membrana/metabolismo , Esteril-Sulfatase/metabolismo , Hormônios Tireóideos/metabolismo , Animais , Proteínas de Transporte/genética , Desidroepiandrosterona/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Glicólise/efeitos dos fármacos , Células HeLa , Hexoquinase/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Pulmão/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Consumo de Oxigênio/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Esteril-Sulfatase/antagonistas & inibidores , Hormônios Tireóideos/genética , Proteínas de Ligação a Hormônio da TireoideRESUMO
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Numerous studies have attempted to develop a new in vitro eye irritation test (EIT). To obtain more reliable results from EIT, potential new biomarkers that reflect eye irritation by chemicals must be identified. We investigated candidate biomarkers for eye irritation, using a proteomics approach. Sodium lauryl sulfate (SLS) or benzalkonium chloride (BAC) was applied on a reconstructed human cornea-like epithelium model, MCTT HCE, and corneal protein expression was examined by two-dimensional gel electrophoresis. We found that ezrin (EZR) was significantly upregulated by SLS or BAC. In addition, upregulation of EZR in immortalized human corneal cells treated with SLS or BAC was confirmed by quantitative reverse transcription-PCR and western blot analysis. Furthermore, other well-known eye irritants such as cetylpyridinium bromide, Triton X-100, cyclohexanol, ethanol, 2-methyl-1-pentanol, and sodium hydroxide significantly increased EZR expression in immortalized human corneal cells. Induction of EZR promoter activity in irritant-treated human corneal cells was confirmed by a luciferase gene reporter assay. In conclusion, EZR expression may be a potential biomarker for detecting eye irritation, which may substantially improve the performance of in vitro EIT.
Assuntos
Proteínas do Citoesqueleto/biossíntese , Células Epiteliais/efeitos dos fármacos , Epitélio Corneano/efeitos dos fármacos , Irritantes/toxicidade , Modelos Biológicos , Testes de Toxicidade/métodos , Compostos de Benzalcônio/farmacologia , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/genética , Células Epiteliais/metabolismo , Epitélio Corneano/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Dodecilsulfato de Sódio/farmacologiaRESUMO
7,12-Dimethylbenz[α]anthracene (DMBA) is a hazardous component present in polluted environments. DMBA has been used as an experimental tool for in vivo tumor formation owing to its carcinogenic effects, but the detailed molecular mechanism of DMBA has not been fully established. To comprehend the carcinogenic mechanism of DMBA, we explored its effects in the breast cancer cell lines, MCF-7 and MDA-MB-231, and the cervical cancer cell line, HeLa. Cell viability assay and measurement of a proliferation marker showed that DMBA markedly increased cancer cell proliferation. Furthermore, morphological observations and wound healing assays in nontumorigenic MCF-10A cells and trans-well invasion assays in cancer cells following DMBA treatment revealed that DMBA induced cell migration and invasion. To reveal the molecular mechanism of DMBA, we investigated the effects of DMBA on the epithelial-mesenchymal transition (EMT) process and Wnt/ß-catenin signaling, a critical pathway for cell proliferation that was reported to correlate with the EMT process, by using quantitative RT-PCR (qPCR), western blot analysis, and confocal microscopy. Consequently, we found that DMBA increased cancer cell proliferation and invasion through the promotion of EMT-inducing factors and ß-catenin. Especially, it was revealed in promoter activity assay using mutated luciferase vectors on transcription factor-binding sites that TWIST1 is promoted by DMBA through induction of STAT3-mediated promoter activation. To further elucidate the detailed mechanism of DMBA, we aimed to identify the key regulator of its carcinogenic action. DMBA was shown to significantly upregulate the expression of specificity protein 1 (Sp1), a transcription factor, and the carcinogenic effects of DMBA were blocked via the suppression or interruption of Sp1 activity. In conclusion, our data suggested that DMBA induced carcinogenic effects through activation of Wnt/ß-catenin signaling and the EMT process by upregulating Sp1 activity.