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1.
J Enzyme Inhib Med Chem ; 34(1): 1298-1306, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31307242

RESUMO

10H-1,9-diazaphenothiazine was obtained in the sulphurisation reaction of diphenylamine with elemental sulphur and transformed into new 10-substituted derivatives, containing alkyl and dialkylaminoalkyl groups at the thiazine nitrogen atom. The 1,9-diazaphenothiazine ring system was identified with advanced 1H and 13C NMR techniques (COSY, NOESY, HSQC and HMBC) and confirmed by X-ray diffraction analysis of the methyl derivative. The compounds exhibited significant anticancer activities against the human glioblastoma SNB-19, melanoma C-32 and breast cancer MDA-MB-231 cell lines. The most active 1,9-diazaphenothiazines were the derivatives with the propynyl and N, N-diethylaminoethyl groups being more potent than cisplatin. For those two compounds, the expression of H3, TP53, CDKN1A, BCL-2 and BAX genes was detected by the RT-QPCR method. The proteome profiling study showed the most probable compound action on SNB-19 cells through the intrinsic mitochondrial pathway of apoptosis. The 1,9-diazaphenotiazine system seems to be more potent than known isomeric ones (1,6-diaza-, 1,8-diaza-, 2,7-diaza- and 3,6-diazaphenothiazine).


Assuntos
Antineoplásicos/farmacologia , Fenotiazinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Fenotiazinas/síntese química , Fenotiazinas/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas
2.
Eur J Med Chem ; 177: 302-315, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31158746

RESUMO

Betulin-1,4-quinone hybrids were obtain by connecting two active structures with a linker. This strategy allows for obtaining compounds showing a high biological activity and better bioavailability. In this research, synthesis, anticancer activity and molecular docking study of betulin-1,4-quinone hybrids are presented. Newly synthesized compounds were characterized by 1H, 13C NMR, IR and HR-MS. Hybrids were tested in vitro against a panel of human cell lines including glioblastoma, melanoma, breast and lung cancer. They showed a high cytotoxic activity depending on the type of 1,4-quinone moiety and the applied tumor cell lines. It was found that cytotoxic activities of the studied hybrids were increasing against the cell line with higher NQO1 protein level, like melanoma (C-32), breast (MCF-7) and lung (A-549) cancer. Selected hybrids were tested on the transcriptional activity of the gene encoding a proliferation marker (H3 histone), a cell cycle regulators (p53 and p21) and an apoptosis pathway (BCL-2 and BAX). The obtained results suggested that the tested compounds caused a mitochondrial apoptosis pathway in A549 and MCF-7 cell lines. The molecular docking was used to examine the probable interaction between the hybrids and human NAD[P]H-quinone oxidoreductase (NQO1) protein. The computational studies showed that the type of the 1,4-quinone moiety affected the location of the compound in the active site of the enzyme. Moreover, it was shown that an interaction of 1,4-quinone fragment with the hydrophobic matrix of the active site near Tyr128, Phe178, Trp105 and FAD cofactor could explain the observed increase of TP53 gene expression.


Assuntos
Antineoplásicos/farmacologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , Quinonas/farmacologia , Triterpenos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Betula/química , Domínio Catalítico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/genética , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , NAD(P)H Desidrogenase (Quinona)/química , Ligação Proteica , Quinonas/síntese química , Quinonas/química , Quinonas/metabolismo , Relação Estrutura-Atividade , Triterpenos/síntese química , Triterpenos/química , Triterpenos/isolamento & purificação , Triterpenos/metabolismo , Proteína Supressora de Tumor p53/genética
3.
Bioorg Chem ; 87: 810-820, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30981160

RESUMO

We present efficient synthesis of isomeric types of angularly fused diquinothiazines in the reactions of 2,2'-dichloro-3,3'-diquinolinyl disulfide and diquinodithiin with 3-, 5-, 6- and 8-aminoquinolines. The pentacyclic diquinothiazine ring systems were identified as diquino[3,2-b;3',4'-e][1,4]thiazine, diquino[3,2-b;5',6'-e][1,4]thiazine, diquino[3,2-b;6',5'-e][1,4]thiazine and diquino[3,2-b;8',7'-e][1,4]thiazine with advanced two-dimensional 1H and 13C NMR techniques (COSY, ROESY, HSQC and HMBC) of N-methyl derivatives. The identification of pentacyclic ring system was confirmed by X-ray diffraction analysis of selected N-alkyl derivatives. The X-ray analysis revealed different spatial structures of the ring system (planar and folded). NH-diquinothiazines were further transformed into N-alkyl and N-dialkylaminoalkyl derivatives. Most of diquinothiazines exhibited significant cancer cell growth inhibition against the human glioblastoma SNB-19, colorectal carcinoma Caco-2, breast cancer MDA-MB-231 and lung cancer A549 cell lines with the IC50 values < 3 µM. This anti-proliferative activity was found to be more than for cisplatin. The most promising compound, 7-dimethylaminopropyldiquino[3,2-b;6',5'-e]thiazine, was used for gene expression analysis by reverse transcription-quantitative real-time PCR (RT-QPCR) method. The expression of H3, TP53, CDKN1A, BCL-2 and BAX genes revealed that this compound inhibited the proliferation in all cells (H3) and activated mitochondrial events of apoptosis (BAX/BCL-2) in two cancer cell lines (SNB-19 and Caco-2).

4.
Molecules ; 24(2)2019 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-30642021

RESUMO

New 10-substituted derivatives of 3,6-diazaphenothiazine, containing the triple bond linker terminated with tertiary cyclic and acyclic amine groups, were synthesized and screened for their anticancer action. The compounds exhibited varied anticancer activities against human glioblastoma SNB-19, melanoma C-32, and breast cancer MDA-MB231 cell lines, depending on the nature of the substituents. The most active 3,6-diazaphenothiazine, 4, was the derivative with the N,N-diethylamino-2-butynyl substituent against glioblastoma SNB-19, and was ten times more potent than cisplatin. For this compound, the expression of H3, TP53, CDKN1A, BCL-2, and BAX genes was detected by the RT-qPCR method. The gene expression ratio BAX/BCL-2 indicated the induction of mitochondrial apoptosis in cancer cell lines. The transformation of the propynyl substituent into amino-2-butynyl can be a method applicable to the search for more anticancer-active azaphenothiazines.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Fenotiazinas/síntese química , Fenotiazinas/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Fenotiazinas/química
5.
Molecules ; 22(10)2017 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-28972559

RESUMO

Abstract: AKT, a serine/threonine protein kinase and mammalian target of rapamycin (mTOR) plays a critical role in the proliferation and resistance to apoptosis that are essential to the development and progression of colon cancer. Therefore, AKT/mTOR signaling pathway has been recognized as an attractive target for anticancer therapy. Inositol hexaphosphate (InsP6), a natural occurring phytochemical, has been shown to have both preventive and therapeutic effects against various cancers, however, its exact molecular mechanisms of action are not fully understood. The aim of the in vitro study was to investigate the anticancer activity of InsP6 on colon cancer with the focus on inhibiting the AKT1 kinase and p70S6K1 as mTOR effector, in relation to proliferation and apoptosis of cells. The colon cancer Caco-2 cells were cultured using standard techniques and exposed to InsP6 at different concentrations (1 mM, 2.5 mM and 5 mM). Cellular proliferative activity was monitored by 5-bromo-2'-deoxyuridine (BrdU) incorporation into cellular DNA. Flow cytometric analysis was performed for cell cycle progression and apoptosis studies. Real-time RT-qPCR was used to validate mRNA levels of CDNK1A, CDNK1B, CASP3, CASP9, AKT1 and S6K1 genes. The concentration of p21 protein as well as the activities of caspase 3, AKT1 and p70S6K1 were determined by the ELISA method. The results revealed that IP6 inhibited proliferation and stimulated apoptosis of colon cancer cells. This effect was mediated by an increase in the expression of genes encoding p21, p27, caspase 3, caspase 9 as well a decrease in transcription of AKT1 and S6K1. InsP6 suppressed phosphorylation of AKT1 and p70S6K1, downstream effector of mTOR. Based on these studies it may be concluded that InsP6 can reduce proliferation and induce apoptosis through inhibition of the AKT/mTOR pathway and mTOR effector followed by modulation of the expression and activity of several key components of these pathways in colon cancer cells.

6.
Molecules ; 21(11)2016 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-27827930

RESUMO

A novel series of tetracyclic quinobenzothiazine derivatives was synthetized. Compounds containing a substituent (hydroxyl, methyl, phenyl, piperidyl, or piperazinyl) in positions 9 and 11 were obtained by cyclization of suitable 4-aminoquinolinium-3-thiolates. Quinobenzothiazine 10-O-substituted derivatives were obtained by alkylating the hydroxyl group in position 10 of the parent (quinobenzothiazine) system. Antiproliferative activity of the synthesized compounds was studied using cultured neoplastic cells (MDA-MB-231, SNB-19, and C-32 cell lines). Four selected compounds were investigated in more detail for cytotoxicity and antiproliferative effect. Transcriptional activity of genes regulating cell cycle (TP53), apoptosis (BAX, BCL-2), as well as proliferation (H3) were assessed. Finally, the ability of the selected compounds to bind DNA was checked in the presence of ethidium bromide.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Tiazinas/síntese química , Tiazinas/farmacologia , Antineoplásicos/química , Apoptose , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Tiazinas/química , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/genética
7.
J Enzyme Inhib Med Chem ; 31(6): 1132-8, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27677322

RESUMO

New derivatives of two isomeric types of azaphenothiazines, 1,8- and 2,7-diazaphenothiazine, containing the triple bond substituents and additionally tertiary cyclic and acyclic amine groups, were synthesized and tested for their anticancer activity. The compounds exhibited differential inhibitory activities. Better results were obtained when the acetylenic group was transformed via the Mannich reaction to the dialkylaminobutynyl groups. The most active was 2,7-diazaphenothiazine with the N-methylpiperazine-2-butynyl substituent against the human ductal breast epithelial tumor cell line T47D, more potent than cisplatin. The 2,7-diazaphenothiazine system turned out to be more active than isomeric 1,8-diaza one. For the most active compound, the expression of TP53, CDKN1A, BCL-2 and BAX genes was detected by the RT-QPCR method. The gene expression ratio BACL-2/BAX suggests the mitochondrial apoptosis in T47D cells. The synthesis makes possible to obtain many new bioactive phenothiazines with the dialkylaminoalkynyl substituents inserting various tertiary cyclic and acyclic amine moieties to the substituents.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Fenotiazinas/síntese química , Fenotiazinas/farmacologia , Linhagem Celular Tumoral , Humanos , Fenotiazinas/química , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas de Bombardeamento Rápido de Átomos
8.
J Enzyme Inhib Med Chem ; 31(6): 1512-9, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26950280

RESUMO

3,6-Diazaphenothiazines were obtained in cyclization of 3-amino-3'-nitro-2,4'-dipyridinyl sulfide and the reaction of sodium 3-amino-2-pyridinethiolate with 4-chloro-3-nitropyridine followed by alkylation and heteroarylation. The thiazine ring formation ran via the Smiles rearrangement. The structure elucidation was based on 2D NMR and X-ray analysis of N-methylated product. 3,6-Diazaphenothiazines were investigated for antitumor activity using glioblastoma SNB-19, melanoma C-32 and breast cancer MCF-7 cells. 10H-3,6-diazaphenothiazine was 10 times more active (IC50 < 0.72 µg/mL) than cisplatin. Two diazaphenothiazines with the 2-pyrimidinyl and dimethylaminopropyl substituents were selectively active against MCF-7 and C-32 cells. The expressions of H3 (proliferation marker), TP53, CDKN1A (cell cycle regulators), BAX and BCL-2 (proapoptopic and antiapoptopic genes) were detected by RT-QPCR method. The expression analysis suggests the cell cycle arrest and the mitochondrial apoptosis pathway activation in MCF-7 and SNB-19 cells.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Fenotiazinas/síntese química , Fenotiazinas/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Fenotiazinas/química , Espectroscopia de Prótons por Ressonância Magnética
9.
Acta Pol Pharm ; 72(4): 719-25, 2015 Jul-Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26647629

RESUMO

In developed countries, chronic rhinosinusitis with nasal polyps is one of the diseases that diminish patients' quality of life most significantly. Treatment of that often incurable disease is based on the steroids and surgery in patients who had failed thorough conservative management. It appears that the introduction of new treatment agents suppressing inflammation process and inhibiting cells' proliferation would be a valuable therapeutic option. The aim of the present study was to evaluate the in vitro effect of genistein and phytic acid on the viability and growth rate of fibroblasts derived from nasal polyps. Cells were incubated with various concentrations of genistein (5-500 µM) and phytic acid (100-20,000 µM). After 72 h incubation, cells survivability and cells' growth rate were estimated by combination of WST-1 and LDH methods. QRT-PCR technique was used to determine the expression of histone H3, BCL-2, BAX and P53 genes. Caspase-8 and -9 expressions were evaluated by ELISA assay. Genistein and phytic acid significantly and in dose-specific manner decreased nasal polyps fibroblasts survivability and growth rate. Both agents in similar way decreased cell proliferation as measured by the expression of histone H3. They induce apoptotic machinery by modulating the expression of BCL-2, BAX and caspase-8 activity. Genistein and phytic acid have significant potential for a therapeutic role in the treatment of chronic rhinosinusitis.


Assuntos
Genisteína/farmacologia , Pólipos Nasais/tratamento farmacológico , Ácido Fítico/farmacologia , Apoptose/efeitos dos fármacos , Caspases/análise , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Genes p53 , Humanos , L-Lactato Desidrogenase/metabolismo , Pólipos Nasais/patologia , Proteína X Associada a bcl-2/genética
10.
Acta Pol Pharm ; 72(5): 909-15, 2015 Sep-Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26665397

RESUMO

Cancer therapy is challenging for scientists because of low effectiveness of so far existing therapies (especially in case of great invasiveness and advanced tumor stage). Such need for new drug development and search for more efficient new findings in therapeutical applications is therefore still valid. There are also conducted studies on modifying so far existing drugs and their new methods of usage in oncology practice. One of them is phenothiazine and its derivatives which are used in psychiatric treatment for years. They also exhibit antiprion, antiviral, antibacterial and antiprotozoal properties. Cytotoxic activity, influence on proliferation, ability to induce apoptosis suggest also a possibility of phenothiazine derivatives usage in cancer cells termination. The aim of our the study was to evaluate the influence of two amine derivatives of phenothiazine on cancer cells in vitro. Amelanotic melanoma C-32 cell line (ATCC) and glioma SNB-19 cells (DSMZ) were used in this study and two derivatives were analyzed. In view of examined substances tumor potential toxicity cells proliferation and viability exposed to phenothiazine derivatives were established. Cell cycle regulatory genes expression (TP53 and CDKN1A), S-phase marker--H3 gene and intracellular apoptosis pathway genes (BAX, BCL-2) were analyzed using RT-QPCR method. The influence of examined derivatives on total cell oxidative status (TOS), total antioxidative status (TAS), malondialdehyde concentration (MDA) and superoxide dismutase activity (SOD) were analyzed. As a result, examined phenothiazine derivatives cytotoxic action on C-32 and SNB-19 and also cells proliferation inhibition were determined. Cell cycle regulatory genes (TP53, CDKN1A) expression and protein products of genes involved in mitochondial apoptosis pathway (BAX, BCL-2) expression are changed by the presence of phenothiazine derivatives during culturing. There were also noted small changes in redox potential in cells exposed to two mentioned phenothiazine derivatives.


Assuntos
Aminas/farmacologia , Glioma/tratamento farmacológico , Melanoma Amelanótico/tratamento farmacológico , Fenotiazinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/genética , Genes p53 , Glioma/genética , Glioma/patologia , Humanos , Melanoma Amelanótico/genética , Melanoma Amelanótico/patologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia
11.
Acta Pol Pharm ; 72(5): 923-9, 2015 Sep-Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26665399

RESUMO

Besides well-known effect on bone and mineral metabolism vitamin D is involved in essential non-calcemic regulatory mechanisms, such as cellular proliferation, differentiation and apoptosis in various cell types. Major limitation for therapeutic use of calcitriol, a hormonally active form of vitamin D, is its calcemic and phosphatemic action. Recently, more selective vitamin D analogs which retain clinically useful activities with reduced toxicity have been designed. The aim of the present study was to evaluate the in vitro effect of vitamin D analogs on proliferation rate and survivability of cells with increased proliferative activity. The effect of calcitriol, PRI-2191, PRI-1890, PRI-1906 and PRI-2205 was examined. The experiments were performed on cultures derived from nasal polyps and cancer cells lines (SNB-19, C32 and SH-4). Cultures were incubated 72 h with tested compounds, each at the concentration of 0.025, 0.25, 2.5 and 25 µg/mL. The cytotoxic effect of vitamin D analogs and their influence on growth rate were determined using WST-1 assay. RT-QPCR technique was used to evaluate the expression of anti-apoptotic BCL-2 and pro-apoptotic BAX gene. Each of the tested compounds presented significant effect at the concentrations above 0.25 µg/mL. The strongest inhibition of the growth rate and decrease in cell survivability was observed after treatment with PRI-1890 and PRI-2191. Stimulation with calcitriol and other vitamin D analogs led to decrease BCL-2/BAX mRNA ratio in each cell lines. The apparent pro-apoptotic action revealed PRI-2191 followed by PRI-1890. It might be hypothesized that vitamin D analogs supplementation may provide therapeutic benefits not only in oncological patients but also in chronic rhinosinusitis.


Assuntos
Pólipos Nasais/tratamento farmacológico , Vitamina D/análogos & derivados , Calcitriol/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Genes bcl-2 , Humanos , Pólipos Nasais/patologia , Vitamina D/farmacologia , Proteína X Associada a bcl-2/genética
12.
Adv Clin Exp Med ; 24(1): 37-46, 2015 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25923085

RESUMO

BACKGROUND: Breast cancer is the most common malignant tumour in women in the whole world. Despite significant developments in the early diagnosis of breast cancer, there is no effective method which would assure total recovery of the patient. Currently available clinical data and laboratory tests indicate a possibility to introduce photodynamic therapy (PDT) to the supplementary treatment of breast cancer. OBJECTIVES: The aim of this study was to assess the influence of PDT with Photolon as a photosensibilizator on the expression of apoptosis associated genes (BCL-2, BAX, TP53) in human breast cancer cell lines, preceded by assessment of survivorship and proliferative activity in the tested cells after PDT. MATERIAL AND METHODS: In the present study human breast cancer cell lines MCF-7 and T-47D were used. Photolon (chlorin e6 complex: PVP 1:1) was used as a photosensitizer. Assessments of survivorship and proliferative activity of cells under the influence of PDT (WST-1 test) were conducted along with the expression of selected genes involved in the process of apoptosis: BCL-2, BAX, TP53 (RT-QPCR). RESULTS: PDT limited both survivorship and proliferative activity of breast cancer cells in the two tested lines. In case of T-47D cell line was found increase of BAX and BCL-2 genes expression after PDT and sustained activity of TP53 gene. Conversely, in MCF-7 cell line a decrease in expression was found for both BAX and TP53 genes, but also an increase of BCL-2 gene expression. CONCLUSIONS: A progressing decrease (24, 48 and 72 h after PDT) in the count of culture cells, which suggests the occurrence of apoptosis initiated by a photodynamic reaction with simultaneous increase of BCL-2/BAX index, indicates activation of a different endogenous apoptosis pathway than the one examined, namely pointing to suicidal death of cells after PDT.


Assuntos
Regulação Neoplásica da Expressão Gênica , Glândulas Mamárias Humanas/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Povidona/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Protoporfirinas/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/genética , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Feminino , Humanos , Luz , Células MCF-7 , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Glândulas Mamárias Humanas/efeitos da radiação , Fotoquimioterapia , Porfirinas , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo
13.
Acta Pol Pharm ; 71(6): 972-86, 2014 Nov-Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25745770

RESUMO

Keloids are characterized by overgrowth of connective tissue in the skin that arises as a consequence of abnormal wound healing. Normal wound healing is regulated by a complex set of interactions within a network of profibrotic and antifibrotic cytokines that regulate new extracellular matrix (ECM) synthesis and remodeling. These proteins include transforming growth factor ß (TGFß) isoforms and connective tissue growth factor (CTGF). TGFß1 stimulates fibroblasts to synthesize and contract ECM and acts as a central mediator of profibrotic response. CTGF is induced by TGFß1 and is considered a downstream mediator of TGFß1action in fibroblasts. CTGF plays a crucial role in keloid pathogenesis by promoting prolonged collagen synthesis and deposition and as a consequence sustained fibrotic response. During keloids formation, besides imbalanced ECM synthesis and degradation, fibroblast proliferation and it's resistance to apoptosis is observed. Key genes that may play a role in keloid formation and growth involve: suppressor gene p53.,cyclin-depend- ent kinase inhibitor CDKN1A (p21) and BCL2 family genes: antiapoptotic BCL-2 and proapoptotic BAX. Genistein (4',5,7-trihydroxyisoflavone) exhibits multidirectional biological action. The concentration of genistein is relatively high in soybean. Genistein has been shown as effective antioxidant and chemopreventive agent. Genistein can bind to estrogen receptors (ERs) and modulate estrogen action due to its structure similarity to human estrogens. Genistein also inhibits transcription factors NFκB. Akt and AP-l signaling pathways, that are important for cytokines expression and cell proliferation, differentiation, survival and apoptosis. The aim of the study was to investigate genistein as a potential inhibitor of CTGF and TGFß1, ß2 and ß3 isoforms expression and a potential regulator of p53. CDKN1A(p21), BAX and BCL-2 expression in normal fibroblasts and fibroblasts derived from keloids cultured in vitro. Real time RT-QPCR was used to estimate transcription level of selected genes in normal and keloid fibroblasts treated with genistein. Secreted/cell-associated CTGF protein was evaluated in cell growth's medium by ELISA. Total protein quantification was evaluated by fluorimetric assay in cells llsates (Quant-iT TM Protein Assay Kit). It was found that TGFß1, ß2 and ß3 genes expression are decreased by genistein. Genistein suppresses the expression of CTGF mRNA and CTGF protein in a concentration dependent manner, p53 and p21 genes expression are modulated by genistein in concentration dependent manner. The agent also modulates BAX/BCL-2 ratio in examined cells in vitro.


Assuntos
Ciclo Celular/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Genisteína/farmacologia , Queloide/tratamento farmacológico , Fitoestrógenos/farmacologia , Fator de Crescimento Transformador beta/genética , Técnicas de Cultura de Células , Ciclo Celular/genética , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Queloide/genética , Queloide/metabolismo , Queloide/patologia , Isoformas de Proteínas , Reação em Cadeia da Polimerase em Tempo Real
15.
Acta Pol Pharm ; 65(6): 731-4, 2008 Nov-Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19172856

RESUMO

Melanin formation in pigmented melanoma cells is considered as a target for the tumor therapy. The evaluation of potential correlation between melanin structure and the tumor type could be also of diagnostic and prognostic importance. One of the major problems in structural investigations of natural melanins is the lack of appropriate methods, which allow isolation of pure intact pigment. In this study the thermochemolysis technique was used to assess the purification grade of melanin isolated from the human melanoma malignum cells by two different enzymatic methods. Melanin samples were thermally degraded in the presence of tetramethylammonium hydroxide and the thermochemolysis products were analyzed by gas chromatography/mass spectrometry (GC/MS). Compounds of lipid origin, especially fatty acid methyl esters and aliphatic and cyclic hydrocarbons, were predominant among pyrolysis products of melanin isolated from the tumor cells by method I. In contrast, during thermochemolysis of the pigment sample isolated by the method II, mainly eumelanin markers (pyrrole and its methyl derivatives, toluene, styrene, phenol, benzyl nitrile and indole) were formed. The comparison of pyrolysis profiles of the analyzed samples indicate that method II is more efficient for melanoma pigment purification.


Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Melaninas/isolamento & purificação , Melanoma/química , Sistemas de Liberação de Medicamentos , Humanos , Melaninas/química , Melanoma/tratamento farmacológico , Compostos de Amônio Quaternário , Temperatura Ambiente
16.
Autoimmunity ; 40(1): 23-30, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17364494

RESUMO

The objective of this study was to determine the expression of transforming growth factor-beta1 messenger RNA (TGF-beta1 mRNA) and the expression of mRNA for TGF-beta receptors (TGF-beta Rs mRNA) in whole peripheral blood of consecutive (treated from several months to several years) systemic lupus erythematosus (SLE) patients (21 women). A further aim of this study was to evaluate the association between expression of the above mentioned parameters in relation to the form of applied therapy (9 patients treated with quinagolide and 12 with quinagolide plus prednisone, azathioprine or cyclosporine A). The control group consisted of 15 healthy women. Most of the patients had mild SLE with SLE disease activity index (SLEDAI) score < 10 at time when blood samples were collected. Laboratory measurements included real-time polymerase chain reaction (RT-QPCR). The expression levels of TGF-beta1 mRNA and mRNA for TGF-beta RII and RIII were significantly lower in patients whereas the expression level of TGF-beta RI was statistically significantly higher in SLE patients than in the controls. A very high positive correlation between TGF-beta1 mRNA expression and expression levels of TGF-beta Rs mRNA was found. In compared subgroups selected according to the form of the applied therapy no statistically significant differences were observed. We conclude that the TGF-beta signaling pathway can be altered in circulating leukocytes derived from treated patients with SLE and that the assumed forms of the applied therapy in the group of patients under consideration are accompanied by similarity in the expression level of transcripts for TGF-beta1 and TGF-beta Rs determined in whole blood. In our investigations, we cannot exclude the influence of the disease itself on the obtained results.


Assuntos
Aminoquinolinas/uso terapêutico , Agonistas de Dopamina/uso terapêutico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , RNA Mensageiro/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/genética , Fator de Crescimento Transformador beta1/genética , Adolescente , Adulto , Azatioprina/uso terapêutico , Ciclosporina/uso terapêutico , Relação Dose-Resposta a Droga , Feminino , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Pessoa de Meia-Idade , Prednisona/uso terapêutico , RNA Mensageiro/genética , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/imunologia
17.
Otolaryngol Pol ; 61(5): 661-7, 2007.
Artigo em Polonês | MEDLINE | ID: mdl-18551997

RESUMO

INTRODUCTION: Recurrent polyposis in the same patient resulting in the necessity of repeated surgeries forced to search for new pharmacological therapeutic methods. At present, locally acting glycocorticosteroids have the greatest value in the treatment of nasal polyposis. Polyps grow is connected with inflammation process and proliferation of fibroblasts. OBJECTIVE: An evaluation of calcitriol and tacalcitol influence on proliferation of fibroblasts extracted from nasal polyps. MATERIAL: consisted of 9 tissue samples coming from nasal polyps sampled during polypectomies. The testing was performed on the polyps fibroblasts after the sixth passage after the primary culture was established. Three days after the culture was started the cells were poured with nutrient medium without serum added and after further 24 hours was replaced by nutrient medium with takalcitol and calcitriol in the defined concentrations. The expression of the genes coding histone H3 was evaluated with the use of RT-PCR technique. RESULTS: Tacalcitiol and calcitriol in vitro decrease proliferation of fibroblasts sampled from nasal polyps. Inhibition is most effective for the concentration of 10-4M. Tacalcitiol and calcitriol also inhibit level of histone H3 gene expression. CONCLUSION: Experimental data suggest tacalcitiol to be more effective in the same concentration. Present studies may indicate the direction of further investigation in the potential pharmacological treatment on nasal polyps.


Assuntos
Calcitriol/farmacologia , Di-Hidroxicolecalciferóis/farmacologia , Fibroblastos/efeitos dos fármacos , Histonas/metabolismo , Vitaminas/farmacologia , Proliferação de Células/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Técnicas In Vitro , Pólipos Nasais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vitamina D/análogos & derivados , Vitamina D/farmacologia
18.
Autoimmunity ; 38(7): 487-91, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16373253

RESUMO

The objective of this study was to determine the frequencies of human cytomegalovirus (HCMV) infection and HCMV genome copy number in blood of consecutive (treated from several months to several years) systemic lupus erythematosus (SLE) patients (22 women). The obtained results were compared to the healthy controls (15 women). All patients fulfilled at least four of the 1982 revised American rheumatism association (ARA) classification criteria for SLE. Our patients demonstrated three or four of the nine possible organ systems involved and most of them had mild SLE with SLE disease activity index (SLEDAI) score < 10 at time when blood samples were collected to detect HCMV. Quantitative analysis of HCMV genome was performed with aid of sequence analyzer ABI PRISM 7,700 Perkin Elmer. Primers and probe were constructed on the basis of IE4 region of HCMV genome. The viral load was expressed as log(10) of calculated HCMV genome copy number. Qualitative analysis revealed that 100% of our SLE patients were infected with HCMV, whereas in the control group only 73% of persons were HCMV positive. Statistically significant difference was demonstrated when the strength of the association between SLE or controls and infection of HCMV was calculated (estimated by Fisher's exact test, P value=0.02). Higher viral DNA copy number was observed in whole blood of SLE patients than in the control group (338.45+/- 221.76 and 229.00+/- 405.61 copies/ml respectively) but did not reach statistical significance level (95% confidence interval from 170.41 to 249.32, P=0.71). Furthermore percentage of patients with HCMV-DNA copy number >2.0 x 10(2) copies/ml was statistically significantly higher than this one in controls. The data show association between HCMV infection and SLE, which should be taken into account during the course of SLE.


Assuntos
Citomegalovirus/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Adolescente , Adulto , Citomegalovirus/genética , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , DNA Viral/sangue , Feminino , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/virologia , Pessoa de Meia-Idade
19.
Ginekol Pol ; 73(11): 897-903, 2002 Nov.
Artigo em Polonês | MEDLINE | ID: mdl-12722370

RESUMO

OBJECTIVES: Histones are involved in process of proliferation and differentiation of the cells. In many lesions the expression of mRNA of histones is a marker of proliferation. The expression of kinin receptors B1 and B2 coexist with inflammatory processes, but in some studies the expression of these receptors was found to influence the growth and differentiation of different cells. DESIGN: In search of proliferation markers in squamous cell vulvar cancer we have analysed the correlation between the expression of the mRNA of histone H2B and H4 and mRNA of kinin receptors B1 and B2. MATERIALS AND METHODS: The specimen obtained from 46 women operated for squamous cell vulvar cancer stages I to IV according to FIGO were analysed. RNA was extracted and the number of mRNA copies were assessed by QRT-PCR using ABI PRISM 7700 (TaqMan). RESULTS: The results obtained in this study indicate a statistically significant correlation between mRNA B1 and mRNA H4 (p < 0.01), B2 and histone H4 (p < 0.01) B1 and histone H2B (p < 0.01). CONCLUSIONS: The expression of mRNA of histones and kinins receptors may reflects the dynamics of neoplastic growth in vulvar cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Histonas/metabolismo , RNA Mensageiro/metabolismo , Receptores da Bradicinina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Vulvares/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Humanos , Receptor B1 da Bradicinina , Receptor B2 da Bradicinina , Receptores da Bradicinina/genética
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