RESUMEN
The development of multitargeted drugs represents an innovative approach to cancer treatment, aiming to enhance drug effectiveness while minimizing side effects. Herein, we sought to elucidate the inhibitory effect of selagibenzophenone B derivatives on the survival of cancer cells and dual topoisomerase I/II enzyme activity. Results demonstrated that among the compounds, SelB-1 selectively inhibited the proliferation and migration of prostate cancer cells while exhibiting minimal effects on healthy cells. Furthermore, SelB-1 showed a dual inhibitory effect on topoisomerases. Computational analyses mirrored the results from enzyme inhibition assays, demonstrating the compound's strong binding affinity to the catalytic sites of the topoisomerases. To our surprise, SelB-1 did not induce apoptosis in prostate cancer cells; instead, it induced autophagic gene expression and lipid peroxidation while reducing GSH levels, which might be associated with ferroptotic death mechanisms. To summarize, the findings suggest that SelB-1 possesses the potential to serve as a dual topoisomerase inhibitor and can be further developed as a promising candidate for prostate cancer treatment.
RESUMEN
The human kallikrein-related peptidase (KLK) family which consists of 15 members is associated with prostate cancer and other cancers. It has been reported that overexpression of KLK4 in prostate cancer correlates with bone metastasis or advanced stage. Hypoxia occurs in the early stages of prostate cancer due to the accumulation of acidic metabolites or reactive oxygen species (ROS). In our study, KLK4 gene expression in hypoxic conditions in PC-3 and LNCaP cells which are treated with TGF-ß was evaluated with mRNA, protein, and promoter activity levels. A chemical hypoxia model was created and confirmed at mRNA and protein level. No statistically significant cytotoxic effect of CoCl2 and TGF-ß was observed in PC-3 and LNCaP cells with the MTT test. Four different truncated KLK4 gene promoter constructs were cloned in pmetLuc expression vector and basal activities of all promoter fragments were analyzed. The activities of P1 (-447/ + 657), P2 (-103/ + 657), and P3 (-267/ + 657) promoter fragments increased in hypoxic conditions except P4 (+555/ + 657), which does not contain the SMAD and HRE region. KLK4 mRNA levels in both PC-3 and LNCaP cells increased in the hypoxia and hypoxia/TGF groups compared to the non-treated groups. The stimulating effect of TGF-ß is correlated with the increase in SMAD2/3 mRNA levels. KLK4 expression is up-regulated by TGF-ß, especially under hypoxic conditions, and its interaction with the SMAD pathway is determined with different inhibitor experiments. HIF-1α and SMAD transcription factors bind to the KLK4 promoter showing the direct interaction of HIF-1α (-80/-52) and SMAD (+163/+194) regions with EMSA.
RESUMEN
BACKGROUND: The most challenging and mortal complication of gastric sleeve surgery (SG) is staple line leakage. Although many agents have been used for increasing tissue healing on the stapler line, there is still no consensus on its effectiveness and efficacy. The aim of study is to determine the effect of phenytoin on the healing process of gastric sleeve surgery in rats. METHODS: On the 10th post-operative day, the effects of phenytoin on bursting pressure in the stapler line were evaluated along-side pathohistological examinations. To investigate the molecular impact of phenytoin on the expression of TGF-ß, VEGF, FGF2, and p53 genes, quantitative real-time polymerase chain reaction was utilized. In addition, gene expressions at the protein level were deter-mined by immunohistochemical analysis. RESULTS: No signs of intra-abdominal leakage were observed in the resected samples. A statistically essential extend in stable line bursting pressure measure was observed between the control group and the group treated with phenytoin application. Pathohisto-logical results indicate that the mean score of collagens of the study group (3.2±0.42) was significantly higher than the control group (2.3±0.48) (P=0.003). In addition, the mean epithelization score of the study group (3.4±0.52) was significantly higher than the control group (2.1±0.57) (P=0.001). mRNA of TGFß, FGF2, VEGF, and p53 genes drastically increased phenytoin treated group. High FGF2 protein expression levels were determined from phenytoin use compared to the control group. CONCLUSION: Molecular studies suggest that phenytoin may increase the healing process of Gastric sleeve following SG in rats and may become a new agent for the prevention of human gastric leaks.
Asunto(s)
Laparoscopía , Obesidad Mórbida , Ratas , Humanos , Animales , Gastrectomía/efectos adversos , Fenitoína/farmacología , Fuga Anastomótica/etiología , Factor 2 de Crecimiento de Fibroblastos/genética , Factor A de Crecimiento Endotelial Vascular/genética , Laparoscopía/efectos adversos , Obesidad Mórbida/complicacionesRESUMEN
Although it is known that organophosphate insecticides are harmfull to aquatic ecosystems, oxidative damages caused by Dimethoate and Chlorpyrifos are not studied on Arthrospira platensis Gomont. In this study, various Chlorpyrifos (0-150 µg mL-1) and Dimethoate (0-250 µg mL-1) concentrations were added to the culture medium in laboratory to evaulate growth rate, chlorophyll-a content and antioxidant parameters of A. platensis. Optical Density (OD560) and chlorophyll-a decreased compared to the control for seven days in both pesticide applications. Superoxide dismutase (SOD) activity increased at 50 µg mL-1 Chlorpyrifos concentration but it decreased at all concentrations. Although Ascorbate peroxidase (APX) and glutathione reductase (GR) activities increased with Chlorpyrifos application, they did not change with Dimethoate application. Malondialdehyde (MDA) amount decreased at 150 µg mL-1 Chlorpyrifos concentration but it increased in Dimethoate application. The H2O2 content were increased in both applications. Proline decreased in 50 and 75 µg mL-1 Chlorpyrifos concentrations and increased at 150 µg mL-1 concentration, while it increased at 25 µg mL-1 Dimethoate concentration. The results were tested at 0.05 significance level. These pesticides inhibit A. platensis growth and chlorophyll-a production and cause oxidative stress. The excessive use may affect the phytoplankton and have negative consequences in the aquatic ecosystem.
Asunto(s)
Cloropirifos , Insecticidas , Plaguicidas , Insecticidas/toxicidad , Cloropirifos/toxicidad , Dimetoato/toxicidad , Ecosistema , Peróxido de Hidrógeno , Estrés Oxidativo , Plaguicidas/toxicidad , Antioxidantes , Clorofila , Clorofila A , Compuestos OrganofosforadosRESUMEN
ADAMTS-2 and ADAMTS-3, known as procollagen amino proteases (PNP), are primarily responsible for processing the amino ends of the fibrillar collagen precursors. ADAMTS-2 is a highly expressed gene in type I collagen-rich tissues, such as skin, bones, tendons, and aorta. ADAMTS-3 is mainly expressed in cartilage, where it colocalizes with type II procollagen and in the nervous system. Studies about ADAMTS-2 and ADAMTS-3 enzymes primarily focused on their collagen processing activity. Knowledge about the transcriptional regulations of these genes is rather limited. Here we analyzed the transcriptional regulations of ADAMTS-2 and ADAMTS-3 genes under chemically induced hypoxic conditions in endothelial cell model, HUVECs. We elucidated that hypoxia is the potent positive regulator of ADAMTS-2 and ADAMTS-3 genes. qRT-PCR and western blotting studies revealed that ADAMTS-2 and ADAMTS-3 expressions were increased at mRNA and protein levels under chemically induced hypoxic conditions in HUVECs. In addition, Transient transfection experiments of ADAMTS-2 and ADAMTS-3 promoter-reporter constructs indicated that low oxygen conditions increased ADAMTS-2 and ADAMTS-3 promoter activities. Furthermore, the DNA-protein interaction assay provided evidence of the functional binding of HIF-1α on bioinformatically determined HRE regions on the ADAMTS-2 and ADAMTS-3 promoters.
Asunto(s)
Desintegrinas , Procolágeno , Humanos , Proteínas ADAM/genética , Proteína ADAMTS4 , Células Endoteliales/metabolismo , Hipoxia , Metaloproteinasas de la Matriz , Procolágeno N-Endopeptidasa/genética , Procolágeno N-Endopeptidasa/metabolismo , Trombospondinas , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismoRESUMEN
BACKGROUND: Anastomotic leakage is the most feared complication after colonic anastomosis. The purpose of the study is to determine the effects of phenytoin applied by different application routes, on the healing process of colorectal anastomoses. METHODS: Wistar Albino rats were divided into Intraperitoneal Phenytoin Group, Oral Phenytoin Group (OAP), Rectal Phenytoin Group (RAP), and control groups. The molecular effect of phenytoin on the expression of vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-ß), fibroblast growth factor 2 (FGF2), and p53 genes was evaluated at mRNA and protein level. The effects of phenytoin on anastomotic bursting pressure analysis measured as well as pathohistological examinations. RESULTS: There are statistically significant increase in anastomotic bursting pressure values between control and application groups. Inflammatory cell infiltration of all groups increased in the intestinal anastomosis region compared to control. Collagen scores were found to be significantly higher in the OAP and RAP groups compared to the control group. mRNA of TGF-ß and FGF2 expression increased in all routes of phenytoin applications. CONCLUSION: Three different administration routes show considerably increase on the bursting pressure. Regarding the results of the expression of FGF2, TGF-ß, p53, and VEGF genes, there is a significant increase FGF2 and TGF-ß at mRNA and protein level in most administration routes.
Asunto(s)
Neoplasias Colorrectales , Fenitoína , Anastomosis Quirúrgica/efectos adversos , Animales , Colon/cirugía , Neoplasias Colorrectales/patología , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fenitoína/metabolismo , Fenitoína/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Recto/cirugía , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Strigolactones (SLs), carotenoid-derived phytohormones, control the plant response and signaling pathways for stressful conditions. In addition, they impact numerous cellular processes in mammalians and present new scaffolds for various biomedical applications. Recent studies demonstrated that SLs possess potent antitumor activity against several cancer cells. Herein, we sought to elucidate the inhibitory effects of SL analogs on the growth and survival of human brain tumor cell lines. Among four tested SLs, we showed for the first time that two lead bioactiphores, indanone-derived SL and EGO10, can inhibit cancer cell proliferation, induce apoptosis, and induce G1 cell cycle arrest at low concentrations. SL analogs were marked by increased expression of Bax/Caspase-3 genes and downregulation of Bcl-2. In silico studies were conducted to identify drug-likeness, blood-brain barrier penetrating properties, and molecular docking with Bcl-2 protein. Taken together, this study indicates that SLs may be promising antiglioma agents, presenting novel pharmacophores for further preclinical and clinical assessment.
Asunto(s)
Glioblastoma , Animales , Glioblastoma/tratamiento farmacológico , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Lactonas/farmacología , Simulación del Acoplamiento MolecularRESUMEN
In this study, the chemical composition, antimicrobial, antioxidant, and anticancer effects of Thymus convolutus Klokov oil and its main compound camphor were investigated. The oil was isolated from T. convolutus using hydrodistillation method, analyzed by gas chromatography/mass spectrometry (GC-MS), and 66 compounds were identified. The main component was determined as camphor at 16.6%. The antioxidant properties were identified with the DPPH (2,2'-diphenyl-1-picrylhydrazyl) radical-scavenging method and, 33.39 ± 0.25% DPPH was scavenging in 1000 µg/mL of essential oil. The strong antimicrobial activity was observed against Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, and Pseudomonas aeruginosa with MIC values of 125 µg/mL. Aspergillus flavus was more sensitive (28%) against T. convolutus essential oil than other fungi. The cytotoxic effect of oil was analyzed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method. Camphor was effective on human hepatoma cells (Hep3B) at concentrations of 1 mg/mL, 500, 250, and 125 µg/mL, while essential oil of T. convolutus was found to be effective at concentrations of 250 and 125 µg/mL. A reduction in cell proliferation was observed in colon carcinoma cells (HT-29) treated with 500 µg/mL camphor for 48 h. No statistically significant effect was found in Umbilical Vein Endothelial Cells (HUVEC) treated with essential oil and camphor.
Asunto(s)
Antiinfecciosos/química , Antineoplásicos Fitogénicos/química , Antioxidantes/química , Aceites Volátiles/química , Thymus (Planta)/química , Antiinfecciosos/farmacología , Antineoplásicos Fitogénicos/farmacología , Candida/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Cromatografía de Gases y Espectrometría de Masas , Humanos , Pruebas de Sensibilidad Microbiana , Extractos Vegetales/química , Pseudomonas aeruginosa/efectos de los fármacos , Thymus (Planta)/metabolismo , TurquíaRESUMEN
URG-4/URGCP is a gene that may be associated with the onset of tumorigenesis and cell cycle regulation. In the literature, there is no study about inflammatory cytokine-mediated URG-4/URGCP regulation. In this study, the effect of TNF-α cytokine was investigated on URG-4/URGCP expression in serum-starved and serum-cultured hepatoma cells. The effect of TNF-α on hepatoma cells was shown using MTT and Annexin-V/PI staining with flow cytometer analyses. As a result, TNF-α leads to the cytotoxicity of hepatoma cells in serum-starved condition whereas no decrease was detected from serum-cultured condition. TNF-α-mediated URG-4/URGCP expression was determined at mRNA and protein level with qRT-PCR analyses and Western blotting method. URG-4URGCP mRNA expression was upregulated in both serum-starved and serum-cultured hepatoma cells. The transfection studies were carried out with URG-4/URGCP promoter constructs for determining the transcriptional activity. TNF-α caused to the upregulation of the activities of URG/URGCP promoter constructs. The basal activities of the URG-4/URGCP promoter conditions are differential according to serum conditions. In addition, some pathway inhibitors were added into hepatoma cells for blocking specific pathways to find out TNF-α-mediated URG-4/URGCP upregulation at mRNA and protein level. TNF-α used JNK and PI3K pathways for regulating URG-4/URGCP gene at serum-starved Hep3B cells. In serum-cultured condition, wortmannin (PI3K inhibitor), MEK-1 (MAPK inhibitor), and SP600125 (JNK inhibitor) did not inhibit the activation response of TNF-α on URGCP.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Citocinas/metabolismo , Humanos , Inflamación , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Transcripción GenéticaRESUMEN
In this study, various N-heterocyclic nitro prodrugs (NHN1-16) containing pyrimidine, triazine and piperazine rings were designed and synthesized. The final compounds were identified using FT-IR, 1H NMR, 13C NMR as well as elemental analyses. Enzymatic activities of compounds were conducted by using HPLC analysis to investigate the interaction of substrates with Ssap-NtrB nitroreductase enzyme. MTT assay was performed to evaluate the toxic effect of compounds against Hep3B and PC3 cancer cell lines and healthy HUVEC cell. It was observed that synthesized compounds NHN1-16 exhibited different cytotoxic profiles. Pyrimidine derivative NHN3 and triazine derivative NHN5 can be good drug candidates for prostate cancer with IC50 values of 54.75 µM and 48.9 µM, respectively. Compounds NHN6, NHN10, NHN12, NHN14 and NHN16 were selected as prodrug candidates because of non-toxic properties against three different cell models. The NHN prodrugs and Ssap-NtrB combinations were applied to SRB assay to reveal the prodrug capabilities of these selected compounds. SRB screening results showed that the metabolites of all selected non-toxic compounds showed remarkable cytotoxicity with IC50 values in the range of 1.71-4.72 nM on prostate cancer. Among the tested compounds, especially piperazine derivatives NHN12 and NHN14 showed significant toxic effect with IC50 values of 1.75 nM and 1.79 nM against PC3 cell compared with standart prodrug CB1954 (IC50: 1.71 nM). Novel compounds NHN12 and NHN14 can be considered as promising prodrug candidates for nitroreductase-prodrug based prostate cancer therapy.
Asunto(s)
Antineoplásicos/química , Colletotrichum/química , Compuestos de Anillos Fusionados/química , Compuestos Heterocíclicos/química , Nitrocompuestos/química , Nitrorreductasas/antagonistas & inhibidores , Profármacos/química , Neoplasias de la Próstata/tratamiento farmacológico , Antineoplásicos/farmacología , Aziridinas/farmacología , Aziridinas/normas , Mezclas Complejas/química , Mezclas Complejas/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fermentación , Compuestos de Anillos Fusionados/farmacología , Compuestos Heterocíclicos/farmacología , Humanos , Masculino , Policétidos/química , Profármacos/farmacología , Relación Estructura-ActividadRESUMEN
Ubiquitin-proteasome pathways have a crucial role in tumor progression. PSMD4 (Rpn10, 26S proteasome non-ATPase subunit 4), which is a subunit of the regulatory particle, is a major ubiquitin (Ub) receptor of 26S proteasome. PSMD4 overexpression has been observed in colon carcinoma, hepatocellular carcinoma, and breast cancer. In this work, we elucidated the effect of hypoxia on PSMD4 gene expression in prostate cancer cells (PC3). Chemically mimicked hypoxia drastically upregulated PSMD4 gene expression at both mRNA and protein levels. Transient transfection experiments indicated that all promoter fragments were active in PC3 cells. Hypoxia increased transcriptional activity of all PSMD4 promoter constructs. EMSA analysis shows that HIF-1a transcription factor binds to the hypoxia response element (HRE) present within the -98/+52 region of PSMD4 promoter. We also used human umbilical vein endothelial cell (HUVEC) as a different cell model, in which increased PSMD4 expression was seen only at 24 h. The increased expression of the PSMD4 level in the PC3 cell line was not parallel to the expression in hypoxic HUVEC.
RESUMEN
Carbonic Anhydrase III (CAIII) belongs to a member of the alpha Carbonic Anhydrase (CA) family. Although some CA members are strongly up-regulated by HIF1-α, it is not known about the transcriptional regulation of CAIII in prostate cancer cells, PCa. Therefore, we aimed to identify regulatory regions important for the regulation of CAIII gene under hypoxic conditions in human prostate cancer cells (PC3). The present study, for the first time, demonstrated that the chemically mimicked hypoxic condition led to the induced CAIII mRNA and protein expression in prostate cancer cells. Transcriptional regulation of CAIII was investigated by transient transfection assay that indicates that the most active promoter activity was in the region of P2 -699/+86. Hypoxic condition also upregulates the basal activity of for P1;-941/+86 and P2;-699/+86 constructs containing putative Hypoxia Response Element (HRE) region located in -268/-252. EMSA analysis of HRE located in -268/-252 bases, showed one DNA-protein binding complexes. Competition assays indicated this complex is resulted from HIF1α interactions. In addition, site-directed mutagenesis of potential HIF1α binding sites diminished a DNA-protein complex. These findings suggest that CAIII is a hypoxia-regulated gene and valuable for targeting of prostate cancer tumors in hypoxic condition.
Asunto(s)
Anhidrasa Carbónica III/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias de la Próstata/metabolismo , Anhidrasa Carbónica III/metabolismo , Hipoxia de la Célula , Regulación Neoplásica de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Masculino , Células PC-3 , Regiones Promotoras Genéticas , Regulación hacia ArribaRESUMEN
Prodrugs for targeted tumor therapies have been extensively studied in recent years due to not only maximising therapeutic effects on tumor cells but also reducing or eliminating serious side effects on healthy cells. This strategy uses prodrugs which are safe for normal cells and form toxic metabolites (drugs) after selective reduction by enzymes in tumor tissues. In this study, prodrug candidates (1-36) containing nitro were designed, synthesized and characterized within the scope of chemical experiments. Drug-likeness properties of prodrug candidates were analyzed using DS 2018 to investigate undesired toxicity effects. In vitro cytotoxic effects of prodrug canditates were performed with MTT assay for human hepatoma cells (Hep3B) and prostate cancer cells (PC3) and human umbilical vein endothelial cells (HUVEC) as healthy control. Non-toxic compounds (3, 5, 7, 10, 12, 15, 17, 19 and 21-23), and also compounds (1, 2, 5, 6, 9, 11, 14, 16, 20 and 24) which had low toxic effects, were selected to examine their suitability as prodrug canditates. The reduction profiles and kinetic studies of prodrug/Ssap-NtrB combinations were performed with biochemical analyses. Then, selected prodrug/Ssap-NtrB combinations were applied to prostate cancer cells to determine toxicity. The results of theoretical, in vitro cytotoxic and biochemical studies suggest 14/Ssap-NtrB, 22/Ssap-NtrB and 24/Ssap-NtrB may be potential prodrug/enzyme combinations for nitroreductase (Ntr)-based prostate cancer therapy.
Asunto(s)
Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Nitrorreductasas/antagonistas & inhibidores , Profármacos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Compuestos de Anilina/síntesis química , Compuestos de Anilina/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Terapia Genética , Humanos , Masculino , Modelos Moleculares , Estructura Molecular , Nitrorreductasas/genética , Nitrorreductasas/metabolismo , Profármacos/síntesis química , Profármacos/química , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Relación Estructura-ActividadRESUMEN
The use of nitroreductases (NTR) that catalyze the reduction of nitro compounds by using NAD(P)H in GDEPT (Gene-directed enzyme prodrug therapy) studies which minimize toxicity at healthy cells and increases concentration of drugs at cancer cells is remarkable. Discovery of new prodrug/NTR combinations is necessary to be an alternative to known prodrug candidates such as CB1954, SN23862, PR-104A. For this aim, nitro containing aromatic amides (A1-A23)2 were designed, synthesized, performed in silico ADMET and molecular docking techniques in this study. Prodrug candidates were studied on reduction potentials with Ssap-NtrB by HPLC system. Also, cyototoxic properties and prodrug ability of these amides were investigated using different cancer cell lines such as Hep3B and PC3. As a result of theoretical and biological studies, combinations of A5, A6 and A20 with Ssap-NtrB can be suggested as potential prodrugs/enzyme combinations at NTR based cancer therapy compared with CB1954/NfsB.
Asunto(s)
Amidas/farmacología , Antineoplásicos Fitogénicos/farmacología , Nitrorreductasas/metabolismo , Profármacos/farmacología , Amidas/síntesis química , Amidas/química , Antineoplásicos Fitogénicos/síntesis química , Antineoplásicos Fitogénicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Simulación de Dinámica Molecular , Estructura Molecular , Nitrorreductasas/química , Células PC-3 , Profármacos/síntesis química , Profármacos/química , Relación Estructura-ActividadRESUMEN
ADAMTS3 is a member of procollagen N-proteinase subfamily of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family. It has an important function in the procollagen maturation process. The removal of N-peptidases is required for the accurate processing of fibrillar collagens. Otherwise, several disorders can occur that is related with the collagenous tissues. ADAMTS3 mainly maturates type II collagen molecule which is the main component of the bone and cartilage. There are several expression studies about ADAMTS3 gene however its transcriptional regulation has not been lightened up, yet. Here we first time cloned and functionally analyzed the promoter region of ADAMTS3 gene, approximately 1380â¯bp upstream of the transcription start site. Transient transfection experiments showed that all truncated promoter constructs are active and 171â¯bp fragment is sufficient to activate gene expression in both Saos-2 and MG63 cells. In silico analysis showed that ADAMTS3 has a TATA-less promoter and contains several SP1/GC box binding motifs and a CpG island. Therefore we mainly investigated the SP1 dependent regulation of ADAMTS3 promoter. SP1 downregulated ADAMTS3 transcriptional activity. As consistent with the transcriptional activity, mRNA, and protein expression levels were also decreased by SP1. On the other hand, functional binding of the SP1 on multiple regions of ADAMTS3 promoter was confirmed by EMSA studies. As ADAMTS3 is responsible for the collagen maturation and biosynthesis, further we investigated the effect of SP1 on type I-II and III collagen gene expressions. We point out that SP1 increased type II and III collagen expression and in contrast decreased type I collagen expression levels in Saos-2 cells. mRNA expression level was decreased for all collagen types in MG63 model. Decrease in the type II collagen expression was also demonstrated at the protein level by SP1. Collectively these results provide first findings for the SP1-related transcriptional regulation of ADAMTS3 and collagen genes in osteosarcoma cell lines.
Asunto(s)
Proteínas ADAMTS/genética , Proteínas ADAMTS/metabolismo , Neoplasias Óseas/genética , Osteosarcoma/genética , Procolágeno N-Endopeptidasa/genética , Procolágeno N-Endopeptidasa/metabolismo , Factor de Transcripción Sp1/metabolismo , Proteínas ADAMTS/química , Sitios de Unión , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Clonación Molecular , Colágeno/genética , Simulación por Computador , Islas de CpG , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Modelos Biológicos , Osteosarcoma/metabolismo , Procolágeno N-Endopeptidasa/química , Regiones Promotoras GenéticasRESUMEN
BACKGROUND: Directed Enzyme Prodrugs Therapy (DEPT) as an alternative method against conventional cancer treatments, in which the non-toxic prodrugs is converted to highly cytotoxic derivative, has attracted an ample attention in recent years for cancer therapy studies. OBJECTIVE: The metabolite profile, cell cytotoxicity and molecular modeling interactions of a series of nitro benzamides with Ssap-NtrB were investigated in this study. METHOD: A series of nitro-substituted benzamide prodrugs (1-4) were synthesized and firstly investigated their enzymatic reduction by Ssap-NtrB (S. saprophyticus Nitroreductase B) using HPLC analysis. Resulting metabolites were analyzed by LC-MS/MS. Molecular docking studies were performed with the aim of investigating the relationship between nitro benzamide structures (prodrugs 1-4) and Ssap-NtrB at the molecular level. Cell viability assay was conducted on two cancer cell lines, hepatoma (Hep3B) and colon (HT-29) cancer models and healthy cell model HUVEC. Upon reduction of benzamide prodrugs by Ssap-NtrB, the corresponding amine effectors were tested in a cell line panel comprising PC-3, Hep3B and HUVEC cells and were compared with the established NTR substrates, CB1954 (an aziridinyl dinitrobenzamide). RESULTS: Cell viability assay resulted in while prodrugs 1, 2 and 3 had no remarkable cytotoxic effects, prodrug 4 showed the differential effect, showing moderate cytotoxicity with Hep3B and HUVEC. The metabolites that obtained from the reduction of nitro benzamide prodrugs (1-4) by Ssap-NtrB, showed differential cytotoxic effects, with none toxic for HUVEC cells, moderate toxic for Hep3B cells, but highly toxic for PC3 cells. CONCLUSION: Amongst all metabolites of prodrugs after Ssap-NtrB reduction, N-(2,4- dinitrophenyl)-4-nitrobenzamide (3) was efficient and toxic in PC3 cells as comparable as CB1954. Kinetic parameters, molecular docking and HPLC results also confirm that prodrug 3 is better for Ssap-NtrB than 1, 2 and 4 or known cancer prodrugs of CB1954 and SN23862, demonstrating that prodrug 3 is an efficient candidate for NTR based cancer therapy.
Asunto(s)
Antineoplásicos/farmacología , Benzamidas/farmacología , Nitrobencenos/farmacología , Nitrorreductasas/metabolismo , Profármacos/farmacología , Mostaza de Anilina/análogos & derivados , Mostaza de Anilina/farmacología , Antineoplásicos/metabolismo , Antineoplásicos/toxicidad , Aziridinas/farmacología , Benzamidas/metabolismo , Benzamidas/toxicidad , Línea Celular Tumoral , Células Endoteliales de la Vena Umbilical Humana , Humanos , Cinética , Simulación del Acoplamiento Molecular , Nitrobencenos/metabolismo , Nitrobencenos/toxicidad , Nitrorreductasas/química , Profármacos/metabolismo , Profármacos/toxicidad , Staphylococcus saprophyticus/enzimologíaRESUMEN
URG-4/URGCP gene was implicated as an oncogene that contributes hepatocarcinogenesis regulated by Hepatitis-B-virus-encoded X antigen. However, the mechanism of transcriptional regulation of this gene remains largely unknown. For this reason, we focused on the functional analyses of URG4/URGCP promoter site. First, 545 bp of URG-4/URGCP, -482/+63, and three different 5'-truncated constructs, -109/+63, -261/+63, -344/+63 were cloned by PCR-based approach into pMetLuc luciferase reporter vector. Transient transfection assay showed that, -109/+63 construct has the highest activity. The promoter of URG-4/URGCP gene contained a CpG island region spanning 400 bp from translation start site. Many SP1/GC boxes, named GC-1 to GC-10 are present in 545 bp of URG-4/URGCP promoter. Because of presence of multiple SP1/GC boxes, promoter constructs were transiently co-transfected with SP1 expression vector to determine the effect of SP1 on URG-4/URGCP promoter activity. Co-transfection analyses induced the basal activity of -268/+63, -344/+63 and -482/+63 constructs. EMSA analysis of GC-4, GC-5, GC-6 and GC-7 binding sites located in -128/-148 bases, showed two DNA-protein binding complexes. Competition assay and super-shifted complexes indicated these complexes are resulted from SP1 binding. Also, site-directed mutagenesis of potential SP1 binding sites diminished both DNA-protein complexes and SP1-mediated upregulation of URG-4 promoter activity. These findings are valuable for understanding transcriptional regulation of URG4/URGCP that has a pivotal role in cancer progression.
Asunto(s)
Hepatocitos/metabolismo , Proteínas de Neoplasias/metabolismo , Elementos de Respuesta/fisiología , Factor de Transcripción Sp1/metabolismo , Transcripción Genética/fisiología , Regulación hacia Arriba/fisiología , Línea Celular Tumoral , Humanos , Proteínas de Neoplasias/genética , Factor de Transcripción Sp1/genéticaRESUMEN
Increases in the risk of infections and malignancy due to immune suppressive therapies of inflammatory bowel diseases (IBDs) have led the researchers to focus on more nontoxic and acceptable natural products like curcumin. Here we investigate whether prophylactic and therapeutic application of the curcumin alters the enzyme activities of paraoxonase (PON), carbonic anhydrase (CA), glucose-6-phosphate dehydrogenase (G6PD) and cytosolic ß-glucosidase in dextran sulphate sodium (DSS)-induced ulcerative colitis mice. Prophylactic application of curcumin resulted in higher MPO activity, less body weight loss and longer colon lengths compared to therapeutic group indicating preventive role of curcumin in IBDs. DSS-induced decrease in liver and serum PON activities were completely recovered by prophylactic administration of curcumin. DSS-induced reduction in liver cytosolic ß-glucosidase activity was not affected by curcumin neither in the prophylactic group nor in the therapeutic group. Erythrocyte CA activity was significantly increased in curcumin groups, however no remarkable change in G6PD activity was observed.
Asunto(s)
Arildialquilfosfatasa/metabolismo , Colitis Ulcerosa/tratamiento farmacológico , Curcumina/uso terapéutico , Sulfato de Dextran/toxicidad , Glucosafosfato Deshidrogenasa/metabolismo , beta-Glucosidasa/metabolismo , Animales , Colitis Ulcerosa/patología , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
The aims of this study were to examine the antiproliferation of Humulus lupulus extracts on human hepatoma carcinoma (Hep3B) and human colon carcinoma (HT-29) cell lines along with enzyme inhibitory effects of the crude extracts. Potential cell cytotoxicity of six different H. lupulus extracts were assayed on various cancer cells using MTT assay at 24, 48 and 72 h intervals. Methanol-1 extract has inhibited the cell proliferation with doses of 0.6-1 mg/mL in a time dependent (48 and 72 hours) manner in Hep3B cells with 70% inhibition, while inhibitory effect was not seen in colon cancer cells. Acetone extract has increased the cell proliferation at low doses of 0.1 mg/mL for 72 h in Hep3B cells and 0.1-0.2 mg/mL for 48 and 72 h in HT29 cells. The inhibitory effects of the extracts were compared by relative maximum activity values (V(max)) using proteases such as α-chymotrypsin, trypsin and papain, tyrosinase and ß-lactamase (penicillinase).
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Humulus/química , Monofenol Monooxigenasa/antagonistas & inhibidores , Péptido Hidrolasas/metabolismo , Extractos Vegetales/farmacología , beta-Lactamasas/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Células HT29 , Humanos , Estructura Molecular , Monofenol Monooxigenasa/metabolismo , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Relación Estructura-Actividad , Células Tumorales Cultivadas , Inhibidores de beta-Lactamasas/síntesis química , Inhibidores de beta-Lactamasas/química , Inhibidores de beta-Lactamasas/farmacologíaRESUMEN
ADAM metallopeptidase with thrombospondin type I motif, 1 (ADAMTS1) that has both antiangiogenic and aggrecanase activity was dysregulated in many pathophysiologic circumstances. However, there is limited information available on the transcriptional regulation of ADAMTS1 gene. Therefore, this study mainly aimed to identify regulatory regions important for the regulation of ADAMTS1 gene under normoxic and hypoxic conditions in human hepatoma cells (HEP3B). Cultured HEP3B cells were exposed to normal oxygen condition, and Cobalt chloride (CoCl2) induced the hypoxic condition, which is an HIF-1 inducer. The cocl2-induced hypoxic condition led to the induced ADAMTS1 mRNA and protein expression in Hepatoma cells. Differential regulation of SP1 and USF transcription factors on ADAMTS1 gene expression was determined by transcriptional activity, mRNA and protein level of ADAMTS1 gene. Ectopic expression of SP1 and USF transcription factors resulted in the decrease in ADAMTS1 transcriptional activity of all promoter constructs consistent with mRNA and protein level in normoxic condition. However, overexpression of SP1 and USF led to the increase of ADAMTS1 gene expressions at mRNA and protein level in hypoxic condition. On the other hand, C/EBPα transcription factor didn't show any statistically significant effect on ADAMTS1 gene expression at mRNA, protein and transcriptional level under normoxic and hypoxic condition.