RESUMO
The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway mediates many cytokine and growth factor signals. Tyrosine kinase 2 (TYK2), one of the members of this pathway and the first described member of the JAK family. TYK2 associates with inflammatory and autoimmune diseases, cancer and diabetes. Here, we present novel compounds as selective inhibitors of the canonical kinase domain of TYK2 enzyme. These compounds were rationally designed and synthesized with appropriate reactions. Molecular modeling techniques were used to design and optimize the candidates for TYK2 inhibition and to determine the estimated binding orientations of them inside JAKs. Designed compounds potently inhibited TYK2 with good selectivity against other JAKs as determined by in vitro assays. In order to verify its selectivity properties, compound A8 was tested against 58 human kinases (KinaseProfiler™ assay). The effects of the selected seven compounds on the protein levels of members of the JAK/STAT family were also detected in THP-1 monocytes although the basal level of these proteins is poorly detectable. Therefore, their expression was induced by lipopolysaccharide treatment and compounds A8, A15, A18, and A19 were found to be potent inhibitors of the TYK2 enzyme, (9.7 nM, 6.0 nM, 5.0 nM and 10.3 nM, respectively), and have high selectivity index for the JAK1, JAK2, and JAK3 enzymes. These findings suggest that triazolo[1,5-a]pyrimidinone derivatives may be lead compounds for developing potent TYK2-selective inhibitors targeting enzymes' active site.
Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases , TYK2 Quinase , Humanos , Relação Dose-Resposta a Droga , Descoberta de Drogas , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirimidinonas/farmacologia , Pirimidinonas/síntese química , Pirimidinonas/química , Relação Estrutura-Atividade , Triazóis/farmacologia , Triazóis/química , Triazóis/síntese química , TYK2 Quinase/antagonistas & inibidores , TYK2 Quinase/metabolismo , Janus Quinases/química , Janus Quinases/metabolismoRESUMO
Background: Androgen deprivation therapy remains the first-line therapy option for prostate cancer, mostly resulting in the transition of the disease to a castration-resistant state. The lack of androgen signaling during therapy affects various cellular processes, which sometimes paradoxically contributes to cancer progression. As androgen receptor (AR) signaling is known to contribute to oxidative stress regulation, loss of AR may also affect DNA damage level and the response mechanism in oxidant and inflammatory conditions of the prostate tumor microenvironment. Therefore, this study aimed to investigate the role of AR and AR-regulated tumor suppressor NKX3.1 upon oxidative stress-induced DNA damage response (DDR) in the inflammatory tumor microenvironment of the prostate. Materials and methods: Intracellular reactive oxygen species (ROS) level was induced by either inflammatory conditioned media obtained from lipopolysaccharide-induced macrophages or oxidants and measured by dichlorodihydrofluorescein diacetate. In addition to this, DNA damage was subsequently quantified by counting gH2AX foci using an immunofluorescence-based Aklides platform. Altered expression of proteins function in DDR detected by western blotting. Results: Cellular levels of ROS and ROS-induced DNA double-strand break damage were analyzed in the absence and presence of AR signaling upon treatment of prostate cancer cells by either oxidants or inflammatory microenvironment exposure. The results showed that AR suppresses intracellular ROS and contributes to DNA damage recognition under oxidant conditions. Besides, increased DNA damage due to loss of NKX3.1 under inflammatory conditions was alleviated by its overexpression. Moreover, the activation of the DDR mediators caused by AR and NKX3.1 activation in androgen-responsive and castration-resistant prostate cancer cells indicated that the androgen receptor function is essential both in controlling oxidative stress and in activating the ROS-induced DDR. Conclusion: Taken together, it is concluded that the regulatory function of androgen receptor signaling has a vital function in the balance between antioxidant response and DDR activation.
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The leishmaniasis are a group of vector-borne diseases caused by a protozoan parasite from the genus Leishmania. In this study, a series of thiazolopyrimidine derivatives were designed and synthesized as novel antileishmanial agents with LmPTR1 inhibitory activity. The final compounds were evaluated for their in vitro antipromastigote activity, LmPTR1 and hDHFR enzyme inhibitory activities, and cytotoxicity on RAW264.7 and L929 cell lines. Based on the bioactivity results, three compounds, namely L24f, L24h and L25c, were selected for evaluation of their in vivo efficacy on CL and VL models in BALB/c mice. Among them, two promising compounds, L24h and L25c, showed in vitro antipromastigote activity against L. tropica with the IC50 values of 0.04 µg/ml and 6.68 µg/ml; against L. infantum with the IC50 values of 0.042 µg/ml and 6.77 µg/ml, respectively. Moreover, the title compounds were found to have low in vitro cytotoxicity on L929 and RAW264.7 cell lines with the IC50 14.08 µg/ml and 21.03 µg/ml, and IC50 15.02 µg/ml and 8.75 µg/ml, respectively. LmPTR1 enzyme inhibitory activity of these compounds was determined as 257.40 µg/ml and 59.12 µg/ml and their selectivity index (SI) over hDHFR was reported as 42.62 and 7.02, respectively. In vivo studies presented that L24h and L25c have a significant antileishmanial activity against footpad lesion development of CL and at weight measurement of VL group in comparison to the reference compound, Glucantime®. Also, docking studies were carried out with selected compounds and other potential Leishmania targets to detect the putative targets of the title compounds. Taken together, all these findings provide an important novel lead structure for the antileishmanial drug development.
Assuntos
Antiprotozoários , Leishmania , Leishmaniose , Animais , Camundongos , Leishmaniose/tratamento farmacológico , Camundongos Endogâmicos BALB CRESUMO
Despite the availability of an effective prophylactic vaccine, 820,000 people die annually of hepatitis B virus (HBV)-related liver disease according to WHO. Since current antiviral therapies do not provide a curative treatment for the 296 million HBV carriers around the globe, novel strategies to cure HBV are urgently needed. A promising approach is the redirection of T cells towards HBV-infected hepatocytes employing chimeric antigen receptors or T-cell engager antibodies. We recently described the effective redirection of T cells employing a second-generation chimeric antigen receptor directed against the envelope protein of hepatitis B virus on the surface of infected cells (S-CAR) as well as bispecific antibodies that engage CD3 or CD28 on T cells employing the identical HBV envelope protein (HBVenv) binder. In this study, we added a trispecific antibody comprising all three moieties to the tool-box. Cytotoxic and non-cytolytic antiviral activities of these bi- and trispecific T-cell engager antibodies were assessed in co-cultures of human PBMC with HBV-positive hepatoma cells, and compared to that of S-CAR-grafted T cells. Activation of T cells via the S-CAR or by either a combination of the CD3- and CD28-targeting bispecific antibodies or the trispecific antibody allowed for specific elimination of HBV-positive target cells. While S-CAR-grafted effector T cells displayed faster killing kinetics, combinatory treatment with the bispecific antibodies or single treatment with the trispecific antibody was associated with a more pronounced cytokine release. Clearance of viral antigens and elimination of the HBV persistence form, the covalently closed circular (ccc) DNA, through cytolytic as well as cytokine-mediated activity was observed in all three settings with the combination of bispecific antibodies showing the strongest non-cytolytic, cytokine-mediated antiviral effect. Taken together, we demonstrate that bi- and trispecific T-cell engager antibodies can serve as a potent, off-the-shelf alternative to S-CAR-grafted T cells to cure HBV.
Assuntos
Anticorpos Biespecíficos , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/genética , Vírus da Hepatite B , Antivirais , Proteínas do Envelope Viral/genética , Linfócitos T , Antígenos CD28/genética , Leucócitos Mononucleares , DNA Circular , Citocinas/genéticaRESUMO
BACKGROUND: siRNAs hold a great potential for cancer therapy, however, poor stability in body fluids and low cellular uptake limit their use in the clinic. To enhance the bioavailability of siRNAs in tumors, novel, safe, and effective carriers are needed. RESULTS: Here, we developed cationic solid lipid nanoparticles (cSLNs) to carry siRNAs targeting EphA2 receptor tyrosine kinase (siEphA2), which is overexpressed in many solid tumors including prostate cancer. Using DDAB cationic lipid instead of DOTMA reduced nanoparticle size and enhanced both cellular uptake and gene silencing in prostate cancer cells. DDAB-cSLN showed better cellular uptake efficiency with similar silencing compared to commercial transfection reagent (Dharmafect 2). After verifying the efficacy of siEphA2-loaded nanoparticles, we further evaluated a potential combination with a histone lysine demethylase inhibitor, JIB-04. Silencing EphA2 by siEphA2-loaded DDAB-cSLN did not affect the viability (2D or 3D culture), migration, nor clonogenicity of PC-3 cells alone. However, upon co-administration with JIB-04, there was a decrease in cellular responses. Furthermore, JIB-04 decreased EphA2 expression, and thus, silencing by siEphA2-loaded nanoparticles was further increased with co-treatment. CONCLUSIONS: We have successfully developed a novel siRNA-loaded lipid nanoparticle for targeting EphA2. Moreover, preliminary results of the effects of JIB-04, alone and in combination with siEphA2, on prostate cancer cells and prostate cancer tumor spheroids were presented for the first time. Our delivery system provides high transfection efficiency and shows great promise for targeting other genes and cancer types in further in vitro and in vivo studies.
Assuntos
Histona Desmetilases/efeitos dos fármacos , Lipídeos/química , Nanopartículas/química , Neoplasias da Próstata/tratamento farmacológico , RNA Interferente Pequeno/farmacologia , Receptor EphA2/metabolismo , Aminopiridinas/farmacologia , Cátions , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Hidrazonas/farmacologia , Masculino , Nanopartículas/uso terapêutico , Tamanho da Partícula , Próstata , RNA Interferente Pequeno/genética , Receptor EphA2/genética , TransfecçãoRESUMO
A series of thiazolopyrimidine derivatives was designed and synthesized as a Leishmania major pteridine reductase 1 (LmPTR1) enzyme inhibitor. Their LmPTR1 inhibitor activities were evaluated using the enzyme produced by Escherichia coli in a recombinant way. The antileishmanial activity of the selected compounds was tested in vitro against Leishmania sp. Additionally, the compounds were evaluated for cytotoxic activity against the murine macrophage cell line RAW 264.7. According to the results, four compounds displayed not only a potent in vitro antileishmanial activity against promastigote forms but also low cytotoxicity. Among them, compound L16 exhibited an antileishmanial activity for both the promastigote and amastigote forms of L. tropica, with IC50 values of 7.5 and 2.69 µM, respectively. In addition, molecular docking studies and molecular dynamics simulations were also carried out in this study. In light of these findings, the compounds provide a new potential scaffold for antileishmanial drug discovery.
Assuntos
Antiprotozoários/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Leishmania major/efeitos dos fármacos , Oxirredutases/antagonistas & inibidores , Pirimidinas/farmacologia , Tiazóis/farmacologia , Animais , Antiprotozoários/síntese química , Antiprotozoários/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Leishmania major/enzimologia , Macrófagos/efeitos dos fármacos , Camundongos , Modelos Moleculares , Estrutura Molecular , Oxirredutases/metabolismo , Testes de Sensibilidade Parasitária , Pirimidinas/síntese química , Pirimidinas/química , Células RAW 264.7 , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/químicaRESUMO
Escape from apoptosis, one of the characteristic features of cancer cells, is a case that reduces the therapeutic efficacy of apoptosis-inducing molecules used in the cancer treatment. Stabilization of the P53 protein, which is responsible for the regulation of apoptosis mechanism in the cell, is therefore an important therapeutic goal. Nutlin3a inhibits the degradation of the P53 protein, triggers P53-mediated apoptosis in cancer cells and enhances the effectiveness of chemotherapeutics. However, its low aqueous solubility is the major disadvantage when it comes to in vivo administration. In order to facilitate an aqueous formulation of Nutlin3a and to enhance its apoptotic activity on cancer cells, Nutlin3a was encapsulated in solid lipid nanoparticles (SLNs) prepared by Ouzo method. Physicochemical characterization was performed and activity of apoptosis induction on wild-type P53 expressing LNCaP prostate cancer cell line was evaluated. Nutlin3a-loaded cationic solid lipid nanoparticles were found to stabilize functional P53 at protein level. In addition, induction rate of apoptosis by nanoparticles was higher than Nutlin3a solution in DMSO, proving this nanoparticle formulation is a promising candidate for increasing the efficiency of Nutlin3a for P53(+) cancer cases. Thus, it is anticipated that the results will contribute to evaluate the use of lipid-based nanocarriers to enhance the therapeutic potential of small molecules that are important in cancer cure.
Assuntos
Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Imidazóis/administração & dosagem , Nanopartículas , Piperazinas/administração & dosagem , Linhagem Celular Tumoral , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
The 8-hydroxy-2'-deoxyguanosine (8-OHdG) damages are base damages induced by reactive oxygen species. We aimed to investigate the role of Androgen Receptor and NKX3.1 in 8-OHdG formation and repair activation by quantitating the DNA damage using Aklides.NUK system. The data demonstrated that the loss of NKX3.1 resulted in increased oxidative DNA damage and its overexpression contributes to the removal of menadione-induced 8-OHdG damage even under oxidative stress conditions. Moreover, 8-oxoguanine DNA glycosylase-1 (OGG1) expression level positively correlates to NKX3.1 expression. Also in this study, first time a reliable cell-based quantitation method for 8-OHdG damages is reported and used for data collection.
Assuntos
Dano ao DNA/genética , Proteínas de Homeodomínio/genética , Estresse Oxidativo/genética , Neoplasias da Próstata/genética , Fatores de Transcrição/genética , Linhagem Celular Tumoral , Desoxiguanosina/genética , Genômica/métodos , Humanos , Masculino , Células PC-3 , Receptores Androgênicos/genéticaRESUMO
OBJECTIVES: This was a phytochemical study of endemic Ferula tenuissima roots and determined the cytotoxic activity of pure compounds on PC-3. MATERIALS AND METHODS: Air-dried and powdered roots of F. tenuissima (1 kg) were extracted consecutively with n-hexane, chloroform (CHCl3), and methanol (MeOH) (3×2 L, each) by sonication at 30°C for 24 h. The extracts were then filtered. The solvents were separately evaporated under reduced pressure to dryness. The compounds were isolated by chromatographic methods and their structures were determined by spectral methods (1D and 2D NMR and LC-MS). The compounds were tested for their cytotoxic activities versus the PC-3 cell line by WST assay. RESULTS: A phytochemical investigation of the dried roots of endemic F. tenuissima was performed and three sesquiterpene esters were isolated. The daucane-type sesquiterpenes teferidin, ferutinin, and elaeochytrin-A were identified. In the bioactivity study, ferutinin exhibited the highest cytotoxic activity, with an IC50 value of 19.7 µM. CONCLUSION: The results indicate that the main compounds of F. tenuissima roots are daucane sesquiterpenes and ferutinin has a potential effect on PC-3 cells.
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Chronic inflammation is associated to 25% of cancer cases according to epidemiological data. Therefore, inhibition of inflammation-induced carcinogenesis can be an efficient therapeutic approach for cancer chemoprevention in drug development studies. It is also determined that anti-inflammatory drugs reduce cancer incidence. Cell culture-based in vitro screening methods are used as a fast and efficient method to investigate the biological activities of the biomolecules. In addition, saponins are molecules that are isolated from natural sources and are known to have potential for tumor inhibition. Studies on the preparation of analogues of cycloartane-type sapogenols (9,19-cyclolanostanes) have so far been limited. Therefore we have decided to direct our efforts toward the exploration of new anti-tumor agents prepared from cycloastragenol and its production artifact astragenol. The semi-synthetic derivatives were prepared mainly by oxidation, condensation, alkylation, acylation, and elimination reactions. After preliminary studies, five sapogenol analogues, two of which were new compounds (2 and 3), were selected and screened for their inhibitory activity on cell viability and NFκB signaling pathway activity in LNCaP prostate cancer cells. We found that the astragenol derivatives 1 and 2 as well as cycloastragenol derivatives 3, 4, and 5 exhibited strong inhibitory activity on NFκB signaling leading the repression of NFκB transcriptional activation and suppressed cell proliferation. The results suggested that these molecules might have significant potential for chemoprevention of prostate carcinogenesis induced by inflammatory NFκB signaling pathway.
Assuntos
Carcinogênese/efeitos dos fármacos , NF-kappa B/metabolismo , Neoplasias da Próstata/patologia , Sapogeninas/química , Sapogeninas/farmacologia , Triterpenos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimioprevenção , Dinoprostona/metabolismo , Humanos , Inflamação/patologia , Masculino , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.
Assuntos
Cooperação Internacional , Espécies Reativas de Oxigênio/metabolismo , Animais , União Europeia , Humanos , Biologia Molecular/organização & administração , Biologia Molecular/tendências , Oxirredução , Espécies Reativas de Oxigênio/química , Transdução de Sinais , Sociedades CientíficasRESUMO
OBJECTIVES: Chronic inflammation has been known as one of the major causes of cancer progression and 25% of cancer cases initiate due to chronic inflammation according to epidemiologic data. It has been determined that chronic inflammation induces carcinogenesis through the abrogation of cell proliferation, apoptosis, and angiogenesis mechanisms. Therefore, it is believed that inhibition of inflammation-induced carcinogenic mechanisms is an efficient therapeutic strategy in drug development studies of cancer chemoprevention. It has also been observed that use of anti-inflammatory drugs reduces the incidence of cancer, and the risk of developing prostate cancer decreases 15-20% with regular use of aspirin and non-steroidal anti-inflammatory drugs (NSAID). MATERIALS AND METHODS: In this study, we investigated the effects of some clinically used NSAIDs on cellular mechanisms that play a role in inflammation-induced prostate carcinogenesis. Inhibition activities on the nuclear factor kappa-B signaling pathway, which activates tumorigenic mechanisms, as well as alterations on androgen receptor signaling, which regulates the proliferation of prostate cells, were investigated. In addition, protein kinase B (Akt) activation, which is stimulated a the inflammatory microenvironment, was examined. RESULTS: The results showed that anti-inflammatory agents alter the protein levels of androgen receptors as well as tumor suppressor NKX3.1, and might trigger an unexpected increase in Akt(S473) level, which induces tumorigenesis. CONCLUSION: It is suggested that inflammatory pathways and prostate carcinogenesis-specific mechanisms should be taken into account for the use of anti-inflammatory drugs for chemoprevention of inflammation-induced prostate cancer.
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Detection of 8-OHdG-base damage has been a big challenge for decades, though different analytical methods are developed. The recent approaches that are used for quantitating either the total amount of base damage or the amount of base damage per cell from different sources of samples are not automated. We have developed a method for automated damage detection from a single cell and applied it to 8-OHdG quantitation.
Assuntos
Dano ao DNA/efeitos dos fármacos , Desoxiguanosina/análogos & derivados , Análise de Célula Única/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , 8-Hidroxi-2'-Desoxiguanosina , Dano ao DNA/genética , Reparo do DNA/genética , Desoxiguanosina/química , Desoxiguanosina/isolamento & purificação , Humanos , Estresse Oxidativo/genéticaRESUMO
The review pinpoints operational concepts related to the redox biology network applied to the pathophysiology and therapeutics of solid tumors. A sophisticated network of intrinsic and extrinsic cues, integrated in the tumor niche, drives tumorigenesis and tumor progression. Critical mutations and distorted redox signaling pathways orchestrate pathologic events inside cancer cells, resulting in resistance to stress and death signals, aberrant proliferation and efficient repair mechanisms. Additionally, the complex inter-cellular crosstalk within the tumor niche, mediated by cytokines, redox-sensitive danger signals (HMGB1) and exosomes, under the pressure of multiple stresses (oxidative, inflammatory, metabolic), greatly contributes to the malignant phenotype. The tumor-associated inflammatory stress and its suppressive action on the anti-tumor immune response are highlighted. We further emphasize that ROS may act either as supporter or enemy of cancer cells, depending on the context. Oxidative stress-based therapies, such as radiotherapy and photodynamic therapy, take advantage of the cytotoxic face of ROS for killing tumor cells by a non-physiologically sudden, localized and intense oxidative burst. The type of tumor cell death elicited by these therapies is discussed. Therapy outcome depends on the differential sensitivity to oxidative stress of particular tumor cells, such as cancer stem cells, and therefore co-therapies that transiently down-regulate their intrinsic antioxidant system hold great promise. We draw attention on the consequences of the damage signals delivered by oxidative stress-injured cells to neighboring and distant cells, and emphasize the benefits of therapeutically triggered immunologic cell death in metastatic cancer. An integrative approach should be applied when designing therapeutic strategies in cancer, taking into consideration the mutational, metabolic, inflammatory and oxidative status of tumor cells, cellular heterogeneity and the hypoxia map in the tumor niche, along with the adjoining and systemic effects of oxidative stress-based therapies.
Assuntos
Neoplasias/patologia , Estresse Oxidativo , Antioxidantes/uso terapêutico , Fatores de Transcrição Forkhead/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fármacos Fotossensibilizantes/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
The oxidative stress response is a cellular defense mechanism that protects cells from oxidative damage and cancer development. The exact molecular mechanism by which reactive oxygen species (ROS) contribute to DNA damage and increase genome instability in prostate cancer merits further investigation. Here, we aimed to determine the effects of NKX3.1 loss on antioxidant defense in response to acute and chronic inflammation in an in vitro model. Oxidative stress-induced DNA damage resulted in increased H2AX((S139)) phosphorylation (a hallmark of DNA damage), along with the degradation of the androgen receptor (AR), p53 and NKX3.1, upon treatment with conditioned medium (CM) obtained from activated macrophages or H2O2. Furthermore, the expression and stability of SIRT1 were increased by CM treatment but not by H2O2 treatment, although the level of ATM((S1981)) phosphorylation was not changed compared with controls. Moreover, the deregulated antioxidant response resulted in upregulation of the pro-oxidant QSCN6 and the antioxidant GPX2 and downregulation of the antioxidant GPX3 after CM treatment. Consistently, the intracellular ROS level increased after chronic treatment, leading to a dose-dependent increase in the ability of LNCaP cells to tolerate oxidative damage. These data suggest that the inflammatory microenvironment is a major factor contributing to DNA damage and the deregulation of the oxidative stress response, which may be the underlying cause of the increased genetic heterogeneity during prostate tumor progression.
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Inflammation-induced carcinogenesis is associated with increased proliferation and migration/invasion of various types of tumor cells. In this study, altered ß-catenin signaling upon TNFα exposure, and relation to loss of function of the tumor suppressor NKX3.1 was examined in prostate cancer cells. We used an in vitro prostate inflammation model to demonstrate altered sub-cellular localization of ß-catenin following increased phosphorylation of Akt(S473) and GSK3ß(S9). Consistently, we observed that subsequent increase in ß-catenin transactivation enhanced c-myc, cyclin D1 and MMP2 expressions. Consequently, it was also observed that the ß-catenin-E-cadherin association at the plasma membrane was disrupted during acute cytokine exposure. Additionally, it was demonstrated that disrupting cell-cell interactions led to increased migration of LNCaP cells in real-time migration assay. Nevertheless, ectopic expression of NKX3.1, which is degraded upon proinflammatory cytokine exposure in inflammation, was found to induce the degradation of ß-catenin by inhibiting Akt(S473) phosphorylation, therefore, partially rescued the disrupted ß-catenin-E-cadherin interaction as well as the cell migration in LNCaP cells upon cytokine exposure. As, the disrupted localization of ß-catenin at the cell membrane as well as increased Akt(S308) priming phosphorylation was observed in human prostate tissues with prostatic inflammatory atrophy (PIA), high-grade prostatic intraepithelial neoplasia (H-PIN) and carcinoma lesions correlated with loss of NKX3.1 expression. Thus, the data indicate that the ß-catenin signaling; consequently sub-cellular localization is deregulated in inflammation, associates with prostatic atrophy and PIN pathology.
Assuntos
Proteínas de Homeodomínio/metabolismo , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/patologia , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Atrofia/patologia , Caderinas/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Movimento Celular , Meios de Cultivo Condicionados/farmacologia , Proteínas de Homeodomínio/genética , Humanos , Masculino , Fosforilação , Próstata/patologia , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasias da Próstata/metabolismo , Prostatite/metabolismo , Prostatite/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , beta Catenina/metabolismoRESUMO
As a link between inflammation and cancer has been reported in many studies, we established an in vitro model of prostatic inflammation to investigate the loss of androgen receptor (AR)-mediated signaling in androgen responsive prostate cell lines. First, the U937 monocyte cell line was differentiated into macrophages using phorbol acetate (PMA), and cells were induced with lipopolysaccharide (LPS) for cytokine secretion. Next, the cytokine levels (TNFα, IL-6, and IL1ß) in conditioned media (CM) were analyzed. Prostate cells were then fed with CM containing varying concentrations of TNFα, and IkB degradation, nuclear factor kappa B (NFκB) translocation and transactivation, and the expression of matrix metalloproteinase-8 (MMP8) and matrix metalloproteinase-9 (MMP9) were then assessed. As a result of CM treatment, ubiquitin-mediated AR degradation, which was restored using anti-TNFα antibody neutralization, led to both a decrease in KLK4, PSA, and NKX3.1 expression levels and the upregulation of GPX2. In addition to the loss of AR, acute and chronic CM exposure resulted in p53 degradation and consequent p21 downregulation, which was also restored by either androgen administration or ectopic NKX3.1 expression via the stabilization of MDM2 levels in LNCaP cells. Additionally, CM treatment enhanced H2AX((S139)) phosphorylation (a hallmark of DNA damage) and genetic heterogeneity in the absence of androgens in prostate cells without activating mitochondrial apoptosis. Thus, the data suggest that inflammatory cytokine exposure results in the loss of AR and p53 signaling in prostate cells and facilitates genetic heterogeneity via ROS accumulation to promote prostate carcinogenesis.
Assuntos
Androgênios/metabolismo , Inflamação/metabolismo , Prostatite/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Androgênios/genética , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Inflamação/genética , Mediadores da Inflamação/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Calicreínas/genética , Calicreínas/metabolismo , Lisina/análogos & derivados , Lisina/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Prostatite/tratamento farmacológico , Prostatite/patologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismoRESUMO
HOXB13 is a homeobox protein that is expressed in normal adult prostate and colon tissues; however, its deregulated expression was evidenced in various malignancies. To characterize the putative role of HOXB13 in cell cycle progression, we performed overexpression and siRNA-mediated knockdown studies in PC-3 and LNCaP cells. Immunohistochemistry (IHC) analyses were also performed using formalin-fixed, paraffin-embedded tissues containing normal, H-PIN and PCa sections from 20 radical prostatectomy specimens. Furthermore, when the role of HOXB13 during cell cycle progression, association with cyclins, cell growth and colony formation using real-time cell proliferation were assessed, we observed that ectopic expression of HOXB13 accumulated cells at G1 through decreasing the cyclin D1 level by promoting its ubiquitination and degradation. This loss slowed S phase entry in both cell lines examined, with an associated decrease in pRb((S780) and (S795)) phosphorylations. Contrary, siRNA-mediated depletion of HOXB13 expression noticeably increased cyclin levels, stabilized E2F1 and CDC25C, subsequent to increased pRb phosphorylations. This increase in Cyclin B1 and CDC25C both together facilitated activation of cyclin B complex via dephosphorylating CDK1((T14Y15)), and resumed the G2/M transition after nocodazole synchronization. Despite an increase in the total expression level and cytoplasmic retention of HOXB13 in H-PIN and PCa samples that were observed via IHC evaluation of prostate tissues, HOXB13 depletion facilitated to an increase in PC-3 and LNCaP cell proliferation. Thus, we suggest that HOXB13 expression is required for cell cycle regulation, and increases by an unknown mechanism consequent to its functional loss in cancer.
Assuntos
Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Neoplasias da Próstata/genética , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Ciclina B1/genética , Ciclina B1/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Fosforilação , Próstata/metabolismo , Próstata/patologia , Prostatectomia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteólise , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Ubiquitinação , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismoRESUMO
We recently reported that hematological and neurological expressed 1 (HN1) is a ubiquitously expressed, EGF-regulated gene. Expression of HN1 in prostate cell lines down-regulates PI3K-dependent Akt activation. Here, we investigate whether HN1 is regulated by androgens through the putative androgen response elements (AREs) found in its promoter. Knockdown of HN1 expression by siRNA silencing leads to an increase in Akt((S473)) phosphorylation, resulting in the translocation of androgen receptor (AR) to the nucleus; these effects can be abrogated by the non-specific Akt inhibitor LY294002 but not by the ERK inhibitor PD98059. Furthermore, HN1 overexpression correlates with an increase in ubiquitination-mediated degradation (a consequence of the decrease in S213/210 phosphorylation of AR), ultimately resulting in the down-regulation of AR-mediated expression of the KLK3, KLK4, NKX3.1 and STAMP2 genes. We also found that HN1 overexpression suppresses colony formation as well as R1881-mediated growth in LNCaP cells, while it has the opposite effect (increasing colony formation but not proliferation) in PC-3 and DU145 cells. Therefore, we suggest that HN1 maintains a balance between the androgen-regulated nuclear translocation of AR and steady-state Akt phosphorylation, predominantly in the absence of androgens. If so, the balance between cell growth and EGF- and AR-signaling must be tightly regulated by HN1. This work has important implications for prostate cancer research, as AR, EGFR and HN1 are known to be highly expressed in prostate adenocarcinomas.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores Androgênicos/metabolismo , Ativação Transcricional , Androgênios/farmacologia , Androgênios/fisiologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dexametasona/farmacologia , Estradiol/farmacologia , Expressão Gênica , Regulação da Expressão Gênica , Glucocorticoides/farmacologia , Humanos , Calicreínas/genética , Calicreínas/metabolismo , Leupeptinas , Masculino , Metribolona/farmacologia , Proteínas Associadas aos Microtúbulos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares , Fosforilação , Neoplasias da Próstata , Inibidores de Proteassoma , Transporte Proteico , Proteólise , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
NKX3.1 is an androgen-regulated homeobox gene that encodes a tissue-restricted transcription factor, which plays an important role in the differentiation of the prostate epithelium. Thus, the role of NKX3.1 as a functional topoisomerase I activity enhancer in cell cycle regulation and the DNA damage response (DDR) was explored in prostate cancer cell lines. As an early response to DNA damage following CPT-11 treatment, we found that there was an increase in the γH2AX(S139) foci number and that total phosphorylation levels were reduced in PC-3 cells following ectopic NKX3.1 expression as well as in LNCaP cells following androgen administration. Furthermore, upon drug treatment, the increase in ATM(S1981) phosphorylation was reduced in the presence of NKX3.1 expression, whereas DNA-PKcs expression was increased. Additionally, phosphorylation of CHK2(T68) and NBS1(S343) was abrogated by ectopic NKX3.1 expression, compared with the increasing levels in control PC-3 cells in a time-course experiment. Finally, NKX3.1 expression maintained a high cyclin D1 expression level regardless of drug treatment, while total γH2AX(S139) phosphorylation remained depleted in PC-3, as well as in LNCaP, cells. Thus, we suggest that androgen regulated NKX3.1 maintains an active DDR at the intra S progression and contributes to the chemotherapeutic resistance of prostate cancer cells to DNA damaging compounds.