RESUMO
Mesenchymal stem cells (MSC) as vehicles of therapeutic genes represent a unique tool to activate drugs within a neoplastic mass due to their property to home and engraft into tumours. In particular, MSC expressing the cytosine deaminase::uracil phosphoribosyltransferase (CD-MSC) have been previously demonstrated to inhibit growth of subcutaneous prostate cancer xenografts thanks to their ability to convert the non-toxic 5-fluorocytosine into the antineoplastic 5-fluorouracil. Since both the immune system and the tumour microenvironment play a crucial role in directing cancer progression, in order to advance towards clinical applications, we tested the therapeutic potential of this approach on animal models that develop autochthonous prostate cancer and preserve an intact immune system. As cell vectors, we employed adipose-tissue and bone-marrow MSC. CD-MSC toxicity on murine prostate cancer cells and tumour tropism were verified in vitro and ex-vivo before starting the preclinical studies. Magnetic Resonance Imaging was utilised to follow orthotopic tumour progression. We demonstrated that intravenous injections of CD-MSC cells, followed by intraperitoneal administration of 5-fluorocytosine, caused tumour regression in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, which develops aggressive and spontaneous prostate cancer. These results add new insights to the therapeutic potential of specifically engineered MSC in prostate cancer disease.
Assuntos
Adenocarcinoma/terapia , Terapia Genética/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Neoplasias da Próstata/terapia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Citosina Desaminase/sangue , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Modelos Animais de Doenças , Flucitosina/farmacologia , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pentosiltransferases/biossíntese , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Oligomeric guanidines are highly efficient biocides against a broad spectrum of microorganisms. However, their antitumor effects have not been studied so far. We investigated an antiproliferative effect of Akacid-medical-formulation (AMF), a member of the oligoguanidine family of biocides, against solid cancer cell lines and primary cells by measuring [3H]-thymidine incorporation. Additionally, we examined cell cycle distribution in two AMF-sensitive prostate cancer cell lines (DU-145, LNCaP) using flow cytometry. Finally, the influence of AMF on cell cycle regulatory molecules and intracellular kinase cascade-related signaling molecules was assessed. We found that AMF has variable antiproliferative effects on all tested cells. In DU-145 and LNCaP cells, flow cytometric studies showed a reduction of S-phase with a maximum extent of 24 and 58%, respectively. This was associated with a decrease in expression of cyclin D1, cyclin-dependent kinases 2 and 4, while having varying effects on expression of cyclin E and p27. Additionally, reduced phosphorylation of Erk1 and Erk2 was found, whereas expression of phospho-Akt1 remained unchanged. Herein we report for the first time that AMF exerts potent antiproliferative activity against various malignant cell lines, including those of prostate. We therefore recommend further investigation of the anticancer activity of this biocidal oliguanidine.
Assuntos
Antineoplásicos/farmacologia , Guanidinas/química , Guanidinas/uso terapêutico , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias da Próstata/metabolismo , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Ativação Enzimática , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Fase SRESUMO
Peripheral nervous system possesses both classical and non-classical steroid receptors and consequently may represent a target for the action of neuroactive steroids. The present review summarizes the state of art of this intriguing field of research reporting data which indicate that neuroactive steroids, like for instance progesterone, dihydroprogesterone, tetrahydroprogesterone, dihydrotestosterone and 3alpha-diol, stimulate the expression of two important proteins of the myelin of peripheral nerves, the glycoprotein P0 (P0) and the peripheral myelin protein 22 (PMP22). Interestingly, the mechanisms by which neuroactive steroids exert their effects involve classical steroid receptors, like for instance progesterone and androgen receptors, in case of P0 and non-classical steroid receptors, like GABA(A) receptor, in case of PMP22. Moreover, neuroactive steroids not only control the expression of these specific myelin proteins, but also influence the morphology of myelin sheaths and axons suggesting that these molecules may represent an interesting new therapeutic approach to maintain peripheral nerve integrity during neurodegenerative events.
Assuntos
Nervos Periféricos/efeitos dos fármacos , Esteroides/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Modelos Biológicos , Proteínas da Mielina/metabolismo , Nervos Periféricos/metabolismo , Receptores de Esteroides/classificação , Receptores de Esteroides/metabolismo , Células de Schwann/efeitos dos fármacosRESUMO
INTRODUCTION: Interleukin-6 (IL-6) is a pleiotropic regulator of prostate cancer cell growth. Oncostatin M (OSM), an IL-6-type cytokine, affects the growth of prostate cancers in a paracrine and autocrine manner. In order to understand better the mechanisms controlling proliferation and intracellular signaling by these cytokines in advanced prostate carcinoma, we performed studies in 22Rv1 cells derived from the relapsed xenograft CWR22R. METHODS: Expression of IL-6 and OSM receptors (OSMR-beta) and elements of signal transduction pathways in 22Rv1 cells were investigated by RT-PCR. Proliferation was assessed by cell counting after treatment with either IL-6 or OSM. IL-6 secretion was measured in conditioned medium from 22Rv1 cells by ELISA. Expression and phosphorylation status of signal transducers and activators of transcription factor (STAT) 3, mitogen-activated protein kinases (MAPK) p44/p42 and p38, and protein kinase B (Akt) was investigated by Western blot. RESULTS: 22Rv1 cells express both subunits of the IL-6 receptor (gp80 and gp130) and leukemia inhibitory factor receptor-beta (LIFR-beta) but not OSMR-beta. Their proliferation was stimulated by IL-6 or OSM and the maximal effect was observed at a concentration of 10 ng/ml of either cytokine. Interestingly, neither IL-6 nor OSM induced phosphorylation of STAT3. OSM modestly increased the phosphorylation of p38 and both cytokines exerted an effect on Akt phosphorylation. CONCLUSIONS: IL-6 and OSM stimulate proliferation of 22Rv1 cells, at least in part through activation of the phosphatidylinositol 3-kinase (PI 3-K) signaling pathway. Our data provide additional evidence for the growth-stimulatory role of IL-6 and related cytokines in advanced prostate cancer and may serve as a basis for the development of novel experimental therapies.
Assuntos
Interleucina-6/fisiologia , Peptídeos/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Neoplasias da Próstata/fisiopatologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Masculino , Oncostatina M , Transdução de Sinais/fisiologiaRESUMO
The location and the role of gamma-aminobutyric acid type B (GABA(B)) receptors in the central nervous system have recently received considerable attention, whilst relatively little is known regarding the peripheral nervous system. In this regard, here we demonstrate for the first time that GABA(B) receptor isoforms [i.e. GABA(B(1)) and GABA(B(2))] are specifically localized in the rat Schwann cell population of the sciatic nerve. Using the selective GABA(B) agonist [i.e. (-)-baclofen] and the antagonists (i.e. CGP 62349, CGP 56999 A, CGP 55845 A), such receptors are shown to be functionally active and negatively coupled to the adenylate cyclase system. Furthermore, exposure of cultured Schwann cells to (-)-baclofen inhibits their proliferation and reduces the synthesis of specific myelin proteins (i.e. glycoprotein Po, peripheral myelin protein 22, myelin-associated glycoprotein, connexin 32), providing evidence for a physiological role of GABA(B) receptors in the glial cells of the peripheral nervous system.
Assuntos
Proteínas da Mielina/metabolismo , Receptores de GABA-B/fisiologia , Células de Schwann/metabolismo , Animais , Baclofeno/farmacologia , Northern Blotting/métodos , Western Blotting/métodos , Bromodesoxiuridina/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Agonistas GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Hipoxantina Fosforribosiltransferase/metabolismo , Imuno-Histoquímica/métodos , Indóis/metabolismo , Proteínas de Membrana/metabolismo , Proteínas da Mielina/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células de Schwann/efeitos dos fármacos , Nervo Isquiático/citologia , Nervo Isquiático/metabolismo , Sais de Tetrazólio/metabolismo , Fatores de TempoRESUMO
Steroid receptor RNA activator (SRA) is a novel coactivator for steroid receptors that acts as an RNA molecule, whereas steroid receptor coactivator (SRC) family members, such as steroid receptor coactivator-1 (SRC-1) and transcriptional intermediary factor 2 (TIF2) exert their biological effects as proteins. Individual overexpression of each of these coactivators, which can form multimeric complexes in vivo, results in stimulated ERalpha transcriptional activity in transient transfection assays. However there is no information on the consequences of reducing SRC-1, TIF2, or SRA expression, singly or in combination, on ERalpha transcriptional activity. We therefore developed antisense oligodeoxynucleotides (asODNs) to SRA, SRC-1, and TIF2 mRNAs, which rapidly and specifically reduced the expression of each of these coactivators. ERalpha-dependent gene expression was reduced in a dose-dependent fashion by up to 80% in cells transfected with these oligonucleotides. Furthermore, treatment of cells with combinations of SRA, SRC-1, and TIF2 asODNs reduced ERalpha transcriptional activity to an extent greater than individual asODN treatment alone, suggesting that these coactivators cooperate, in at least an additive fashion, to activate ERalpha-dependent target gene expression. Finally, treatment of MCF-7 cells with asODN against SRC-1 and TIF2 revealed a requirement of these coactivators, but not SRA, for hormone-dependent DNA synthesis and induction of estrogen-dependent pS2 gene expression, indicating that SRA and SRC family coactivators can fulfill specific functional roles. Taken together, we have developed a rapid method to reduce endogenous coactivator expression that enables an assessment of the in vivo role of specific coactivators on ERalpha biological action and avoids potential artifacts arising from overexpression of coactivators in transient transfection assays.