RESUMO
INTRODUCTION: The breast microenvironment can either retard or accelerate the events associated with progression of latent cancers. However, the actions of local physiological mediators in the context of breast cancers are poorly understood. Serotonin (5-HT) is a critical local regulator of epithelial homeostasis in the breast and other organs. Herein, we report complex alterations in the intrinsic mammary gland serotonin system of human breast cancers. METHODS: Serotonin biosynthetic capacity was analyzed in human breast tumor tissue microarrays using immunohistochemistry for tryptophan hydroxylase 1 (TPH1). Serotonin receptors (5-HT1-7) were analyzed in human breast tumors using the Oncomine database. Serotonin receptor expression, signal transduction, and 5-HT effects on breast cancer cell phenotype were compared in non-transformed and transformed human breast cells. RESULTS: In the context of the normal mammary gland, 5-HT acts as a physiological regulator of lactation and involution, in part by favoring growth arrest and cell death. This tightly regulated 5-HT system is subverted in multiple ways in human breast cancers. Specifically, TPH1 expression undergoes a non-linear change during progression, with increased expression during malignant progression. Correspondingly, the tightly regulated pattern of 5-HT receptors becomes dysregulated in human breast cancer cells, resulting in both ectopic expression of some isoforms and suppression of others. The receptor expression change is accompanied by altered downstream signaling of 5-HT receptors in human breast cancer cells, resulting in resistance to 5-HT-induced apoptosis, and stimulated proliferation. CONCLUSIONS: Our data constitutes the first report of direct involvement of 5-HT in human breast cancer. Increased 5-HT biosynthetic capacity accompanied by multiple changes in 5-HT receptor expression and signaling favor malignant progression of human breast cancer cells (for example, stimulated proliferation, inappropriate cell survival). This occurs through uncoupling of serotonin from the homeostatic regulatory mechanisms of the normal mammary epithelium. The findings open a new avenue for identification of diagnostic and prognostic markers, and valuable new therapeutic targets for managing breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Serotonina/metabolismo , Processos de Crescimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Epiteliais/patologia , Feminino , Humanos , Imuno-Histoquímica , Receptores de Serotonina/biossíntese , Receptores de Serotonina/metabolismo , Serotonina/biossíntese , Transdução de Sinais , Triptofano Hidroxilase/metabolismoRESUMO
Previously we observed that the antiestrogens tamoxifen and 4-hydroxytamoxifen (4OHT) induce CYP3A4 in primary human hepatocytes and activate human pregnane X receptor (PXR) in cell-based reporter assays. Given the complex cross-talk between nuclear receptors, tissue-specific expression of CYP3A4, and the potential for tamoxifen and 4OHT to interact with a myriad of receptors, this study was undertaken to gain mechanistic insights into the inductive effects of tamoxifen and 4OHT. First, we observed that transfection of the primary cultures of human hepatocytes with PXR-specific small interfering RNA reduced the PXR mRNA expression and the extent of CYP3A4 induction by tamoxifen and 4OHT by 50%. Second, in LS174T colon carcinoma cells, which were observed to have significantly lower PXR expression relative to human hepatocytes, neither tamoxifen nor 4OHT induced CYP3A4. Third, N-desmethyltamoxifen, which did not induce CYP3A4 in human hepatocytes, also did not activate PXR in LS174T cells. We then used cell-based reporter assay to evaluate the effects of other receptors such as glucocorticoid receptor GR alpha and estrogen receptor ER alpha on the transcriptional activation of PXR. The cotransfection of GR alpha in LS174T cells augmented PXR activation by tamoxifen and 4OHT. On the other hand, the presence of ER alpha inhibited PXR-mediated basal activation of CYP3A4 promoter, possibly via competing for common cofactors such as steroid receptor coactivator 1 and glucocorticoid receptor interacting protein 1. Collectively, our findings suggest that the CYP3A4 induction by tamoxifen and 4OHT is primarily mediated by PXR but the overall stoichiometry of other nuclear receptors and transcription cofactors also contributes to the extent of the inductive effect.
Assuntos
Citocromo P-450 CYP3A/metabolismo , Receptores de Esteroides/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Receptor alfa de Estrogênio/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Receptor de Pregnano X , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Esteroides/genética , TransfecçãoRESUMO
BACKGROUND: We previously showed that the anti-inflammatory drug, sulfasalazine (salicylazosulfapyridine, SASP), can arrest proliferation of MCF-7 and MDA-MB-231 mammary cancer cells by inhibiting uptake of cystine via the x(c-) cystine/glutamate antiporter. Here we examined SASP with regard to reduction of cellular glutathione (GSH) levels and drug efficacy-enhancing ability. METHODS: GSH levels were measured spectrophotometrically. Cellular drug retention was determined with 3H-labeled methotrexate, and drug efficacy with a colony formation assay. RESULTS: Incubation of the mammary cancer cells with SASP (0.3-0.5 mM) led to reduction of their GSH content in a time- and concentration-dependent manner. Similar to MK-571, a multidrug resistance-associated protein inhibitor, SASP increased intracellular accumulation of methotrexate. Preincubation of cells with SASP (0.3 mM) significantly enhanced the potency of the anticancer agent doxorubicin (2.5 nM). CONCLUSIONS: SASP-induced reduction of cellular GSH levels can lead to growth arrest of mammary cancer cells and enhancement of anticancer drug efficacy.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/farmacologia , Sulfassalazina/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Antimetabólitos Antineoplásicos/metabolismo , Carcinoma/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Glutationa/análise , Glutationa/metabolismo , Humanos , Metotrexato/metabolismo , Oxirredução , Propionatos/farmacologia , Quinolinas/farmacologia , Ensaio Tumoral de Célula-Tronco , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
The prolactin (PRL)-dependent rat Nb2 T lymphoma is a valuable model for investigation of molecular mechanisms that underlie tumor progression in hormone-dependent cancers. mRNA differential display was used to screen for novel gene products expressed in hormone-stimulated or differentiating agent-treated Nb2 sublines. From numerous transcripts identified, DNA sequencing and GenBank analysis revealed a novel 289-bp fragment. Using 5'-rapid amplification of complementary ends-PCR, this fragment was used to clone a unique 2117-bp cDNA, designated HRPAP20 (hormone-regulated proliferation-associated protein), in rat lymphoma cells. Computer-assisted sequence analysis revealed a single open reading frame that encoded a putative 20.2-kDa protein. The effect of hormone stimulation to alter expression of HRPAP20 was evaluated by Northern blot analysis of total RNA obtained from PRL-stimulated, lactogen-dependent Nb2-11 cells. Quiescent cells, synchronized in the G(0)-G(1) phase of cell cycle, exhibited reduced HRPAP20 expression compared with exponentially proliferating cultures. The addition of mitogenic concentrations of PRL to stationary cells increased HRPAP20 mRNA accumulation within 4-6 h, corresponding to G(1) cell cycle progression. Immunoblot analysis showed that PRL also increased HRPAP20 protein levels within 4 h. In addition, PRL stimulated serine phosphorylation of the HRPAP20 protein with a similar kinetic pattern. Stable transfection of the HRPAP20 cDNA into Nb2-11 cells significantly (P < 0.01) increased proliferation in the absence of hormonal stimulation and inhibited apoptosis induced by lactogen deprivation (P < 0.001). In the hormone-independent and highly malignant Nb2-SFJCD1 subline, the constitutive expression of HRPAP20 was markedly reduced by exposure of the cells to dietary differentiating agents (butyrate, retinoic acid, and vitamin D(3)). After removal of these substances, PRL stimulated its expression in a manner similar to that observed in PRL-dependent Nb2-11 cells. HRPAP20 expression was also evaluated in MCF-7 cells. Its expression was detectable in quiescent cultures; addition of PRL significantly (P < 0.05) increased HRPAP20 during G(1) cell cycle progression. Exposure of the cells to butyrate or retinoic acid reduced HRPAP20 expression, similar to the effects of these substances in the malignant rat lymphoma. Stable transfection of HRPAP20 into MCF-7 cells significantly (P < 0.006) increased proliferation in the absence of hormone stimulation and augmented survival in the absence of serum (P < 0.05). We conclude that HRPAP20 is a phosphoprotein that is required for proliferation and survival of hormone-dependent tumor cells.
Assuntos
Proteínas de Neoplasias/fisiologia , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/patologia , Fosfoproteínas/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Galinhas , Clonagem Molecular , Perfilação da Expressão Gênica , Humanos , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Linfoma de Células T/patologia , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Hormônio-Dependentes/metabolismo , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Fosforilação , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Homologia de Sequência de AminoácidosRESUMO
The role of cannabinoid receptor I (CBR-1) in the induction of decidualization was examined using decidual fibroblasts and human endometrial stromal cells as model systems. Decidual fibroblasts decidualized in vitro for 3 and 6 days in the presence of the CBR-1 agonist R(+)-WIN 55,212-2 mesylate (WIN, 0.1-10 microM) expressed less of the decidualization-specific markers prolactin, CBR-1, forkhead (FKHR), TIMP-3, laminin, endometrial bleeding associated factor (EBAF), decorin and insulin-like growth factor binding protein-1 (IGFBP-1) mRNA levels compared to control cells. The maximal decrease for each transcript was in the range of 50-99%. In contrast, cells exposed to the CBR-1 inhibitor AM-251 (1 microM) expressed about two-fold higher levels of the decidualization-specific marker gene mRNAs. The WIN-exposed cells showed a marked decrease in intracellular cAMP levels and a progressive, concentration-dependent increase in DNA fragmentation (TUNEL assay) and caspase 3 levels during decidualization compared to control cells. These studies strongly suggest that activation of CBR-1 inhibits human decidualization and stimulates apoptosis by a cAMP-dependent mechanism.
Assuntos
Apoptose/efeitos dos fármacos , AMP Cíclico/metabolismo , Decídua/fisiologia , Endométrio/fisiologia , Fibroblastos/fisiologia , Receptor CB1 de Canabinoide/metabolismo , Células Estromais/fisiologia , Benzoxazinas , Bloqueadores dos Canais de Cálcio/farmacologia , Canabinoides/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endométrio/citologia , Endométrio/efeitos dos fármacos , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead , Humanos , Marcação In Situ das Extremidades Cortadas , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Laminina/genética , Laminina/metabolismo , Fatores de Determinação Direita-Esquerda , Morfolinas/farmacologia , Naftalenos/farmacologia , Piperidinas/farmacologia , Prolactina/genética , Prolactina/metabolismo , Pirazóis/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/genética , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
The hypothesis that prolactin (PRL) functions as an immunomodulator was based on studies showing lymphocyte PRL receptors, and its effects on growth, differentiation, and apoptosis in lymphoid cells. However, studies of PRL (PRL-/-) and PRL receptor knockout mice indicated that PRL was not required for immune system development or function under basal conditions. Because PRL maintains survival in glucocorticoid (GC)-treated Nb2-T lymphocytes in vitro, and PRL and GCs are elevated during stress, we investigated whether PRL protected T cells in vivo from GC-induced apoptosis. Adrenalectomized mice [PRL -/-, undetectable PRL; pituitary grafted PRL-/- (PRL-/-Graft), elevated PRL; and PRL+/-, normal PRL] were treated with dexamethasone (DEX) or PBS. Thymocytes and splenocytes were isolated and annexin V labeling of phosphatidylserine, DNA fragmentation, and caspase-3 activation were assessed as indices of apoptosis. Total thymocytes and CD4+ and CD8+ T cells obtained from DEX-treated PRL-/- mice exhibited significantly increased annexin V binding. In contrast, binding was not altered by DEX in PRL-/-Graft thymocytes. In addition, DEX induced classic DNA fragmentation in PRL-/- thymocytes. Elevated serum PRL reduced this effect. Thymocytes from DEX-treated PRL-/- mice exhibited increased caspase-3 activation, which was inhibited in cells from PRL-/-Graft mice. Finally, elevated expression of X-linked inhibitor of apoptosis, XIAP, was observed in thymi from DEX-treated PRL -/-Graft mice. This is the first demonstration that elevated PRL antagonizes apoptosis in thymocytes exposed to GCs in vivo. These observations suggest that, under conditions of increased GCs, such as during stress, elevated PRL functions physiologically to maintain survival and function of T-lymphocytes.
Assuntos
Apoptose/fisiologia , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Prolactina/fisiologia , Timo/efeitos dos fármacos , Timo/fisiologia , Animais , Sobrevivência Celular/genética , Corticosterona/sangue , Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout/genética , Prolactina/genética , Baço/citologia , Baço/efeitos dos fármacos , Baço/fisiologia , Timo/citologiaRESUMO
The importance of prolactin (PRL) in mammopoiesis and milk production is undisputed. However, previous studies investigating the role of PRL in immune function have yielded inconsistencies. These inconsistencies have led to our hypothesis that the immunomodulatory effects of PRL are only manifest under conditions in which the organism is subjected to stress. Thermal injury is a well-known stressor. The goal of this study was to determine whether the lack of PRL enhanced the negative effects of thermal injury-induced immune alterations utilizing a mouse model in which the PRL gene had been disrupted. Mice received either sham or burn treatment, and were sacrificed 4 days later. The immune parameters studied were the capacity of bone marrow cells to form granulocyte-macrophage colony forming units (GM-CFU) in the presence of granulocyte-macrophage colony stimulating factor, and the ability of the splenic T lymphocytes to proliferate in response to phytohemagglutin (PHA). As shown by others, our results reveal that burn increased the number of GM-CFU compared to sham controls; however, this elevation was only significant in the PRL-/- mice. Thermal injury increased PHA-stimulated proliferation of splenic T lymphocytes, however this increase was only significant in the PRL+/- group. We conclude that under conditions of a controlled stress event (thermal injury) [a] the increase in the GM-CFU is exaggerated in the absence of PRL, and [b] the enhancement of PHA-induced proliferation of splenic lymphocytes required PRL. This study supports the hypothesis that the immunomodulatory effects of PRL are manifest when the organism is subjected to stress.
Assuntos
Queimaduras/patologia , Queimaduras/fisiopatologia , Leucopoese/fisiologia , Prolactina/fisiologia , Baço/patologia , Linfócitos T/patologia , Animais , Medula Óssea/patologia , Queimaduras/metabolismo , Divisão Celular/fisiologia , Corticosterona/sangue , Granulócitos/patologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Prolactina/deficiência , Células-Tronco/patologia , Fatores de TempoRESUMO
In this study, we sought to determine if prolactin (PRL) had any influence on burn-induced alterations in myelopoiesis and serum IL-6, IL-10, IL-12, IFN-gamma, TNF-alpha, and MCP-1 levels. To do this, we used mice that were PRL normal, PRL deficient, or hyperprolactinemic and had received a 15% total body surface area burn, sham treatment, or no treatment. We performed clonogenic assays of bone marrow cells, and we found that sham treatment significantly decreased monocyte/macrophage (M) colony formation relative to the control group in the PRL-deficient and PRL-normal mice (P < 0.01). Hyperprolactinemia attenuated the sham-induced decrease in M colony formation. Burn injury significantly increased M colony formation relative to the sham group with an equal significance in the PRL-deficient and PRL-normal mice (P < 0.05). We also showed that burn led to a significant increase in GM colony formation relative to the sham group. This burn-induced increase was significant in the PRL-normal (P < 0.05) and the PRL-deficient (P < 0.01) mice. In the PRL-normal mice, burn injury caused a 2.1-fold increase in the GM colony number, whereas in the PRL-deficient mice burn led to a 2.6-fold increase in GM colony number. When comparing the effects of burn injury on colony formation to the control groups, there were no significant differences seen, irrespective of the PRL level. We observed that all of the cytokines studied, with the exception of IL-10, were influenced by either sham treatment, burn injury, or both forms of stress. This stress-induced response occurred most often in animals that were either hypo- or hyperprolactinemic. We conclude that the PRL level was able to influence the sham-induced and burn-induced alterations in GM and M colony formation. Under euprolactinemic conditions, mice exhibited less often with stress-induced serum cytokine level alterations. We did not find any significant correlations with any of the serum cytokine levels and the ability to form colonies. Importantly, the sham treatment led to immune alterations independent of, and sometimes opposite of burn-induced effects.
Assuntos
Medula Óssea/patologia , Queimaduras , Citocinas/biossíntese , Macrófagos/metabolismo , Monócitos/metabolismo , Prolactina/biossíntese , Animais , Células da Medula Óssea/metabolismo , Quimiocina CCL2/biossíntese , Citocinas/metabolismo , Citometria de Fluxo , Glucocorticoides/metabolismo , Temperatura Alta , Interleucina-10/biossíntese , Interleucina-12/biossíntese , Interleucina-6/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Prolactina/sangueRESUMO
The antiretroviral agent efavirenz enhances the systemic clearance of coadministered drugs that are cytochrome P450 (CYP) 3A4 substrates. The mechanism of the apparent increase in CYP3A4 activity by efavirenz and the magnitude of change relative to other known inducers are not known. The authors tested the hypothesis that increased enzymatic activity by efavirenz entails CYP3A4 induction and activation of the human pregnane X receptor (hPXR), a key transcriptional regulator of CYP3A4. Employing primary cultures of human hepatocytes, they compared the CYP3A4 inductive effects of efavirenz (1-10 microM) to rifampin (10 microM) and phenobarbital (2 mM). A cell-based reporter assay was employed to assess hPXR activation. The authors observed that efavirenz caused a concentration-dependent CYP3A4 induction and hPXR activation. Based on the CYP3A4 activity assay, the average magnitude of induction by efavirenz (5-10 microM) was approximately 3- to 4-fold. In comparison, phenobarbital (2 mM) and rifampin (10 microM) caused a 5- and 6-fold induction, respectively.
Assuntos
Fármacos Anti-HIV/farmacologia , Sistema Enzimático do Citocromo P-450/biossíntese , Hepatócitos/efeitos dos fármacos , Oxazinas/farmacologia , Fenobarbital/farmacologia , Rifampina/farmacologia , Alcinos , Benzoxazinas , Células Cultivadas , Ciclopropanos , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Relação Dose-Resposta a Droga , Indução Enzimática , Hepatócitos/enzimologia , Humanos , Receptor de Pregnano X , RNA Mensageiro/biossíntese , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores de Esteroides/agonistasRESUMO
PURPOSE: The induction of cytochrome P450 (CYP) 3A4 by drugs and other xenobiotics is a common cause of serious drug interactions. The aim of this study was to comparatively examine the effects of paclitaxel and docetaxel, two structurally related taxane anticancer agents, on the activity and expression of hepatic CYP3A4. METHODS: Employing primary cultures of human hepatocytes from multiple donors, we investigated the differences in the magnitude of CYP3A4 induction and relative accumulation of paclitaxel and docetaxel. The CYP3A4 activity of intact hepatocytes was measured as the rate of testosterone 6beta-hydroxylation. The CYP3A4-specific immunoreactive protein and mRNA levels were measured employing Western blot and Northern blot analysis, respectively. Furthermore, employing cell-based reporter gene assay in CV-1 cells, we evaluated the capacity of paclitaxel and docetaxel to activate human pregnane X receptor (hPXR), an orphan nuclear receptor that plays a key role in the transcriptional regulation of CYP3A4. RESULTS: In concurrence with previous reports, we observed that paclitaxel potently induced CYP3A4 activity and expression in hepatocytes treated for 48-96 h. However, docetaxel did not increase the activity or the CYP3A4 immunoreactive protein levels for treatment periods up to 96 h. A marginal increase in the CYP3A4 mRNA levels was observed in cells treated with higher levels (5 and 10 microM) of docetaxel. Furthermore, while paclitaxel effectively activated hPXR (the half-maximal effective concentration, EC50, being about 5.2 microM), docetaxel weakly activated hPXR, and moreover the activation occurred only at high concentrations relative to paclitaxel. A comparison of the cellular concentrations of paclitaxel and docetaxel, in the cell culture models employed for evaluating CYP3A4 induction and hPXR activation, revealed that the intracellular paclitaxel levels were three-fold higher than that of docetaxel. Thus, it appears that both pharmacokinetic (drug concentration) and pharmacodynamic differences (hPXR activation) may account for the observed differences in CYP3A induction by paclitaxel and docetaxel. CONCLUSION: Our studies suggest that docetaxel has markedly reduced propensity to cause drug interactions that may entail hepatic CYP3A4 induction.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Sistema Enzimático do Citocromo P-450/biossíntese , Hepatócitos/patologia , Paclitaxel/farmacologia , Taxoides/farmacologia , Northern Blotting , Western Blotting , Técnicas de Cultura de Células , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Docetaxel , Interações Medicamentosas , Indução Enzimática , Fígado/efeitos dos fármacos , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides/efeitos dos fármacos , Receptores de Esteroides/fisiologiaRESUMO
BACKGROUND: Malignant progression of lymphoma cells is associated with acquisition of the cystine/glutamate antiporter, xc-, enhancing cystine uptake. Recently, we showed that sulfasalazine (SASP) is a specific xc- inhibitor. Here, we investigated xc- in mammary cancer cell lines. MATERIALS AND METHODS: Expression and function of xc- were evaluated by RT-PCR and 35S-cystine uptake analysis. RESULTS: Xc- expression was elevated 4-fold (p < 0.001) in cells of the most malignant line, MDA-MB-231, associated with increased 35S-cystine uptake (p < 0.001). Proliferation was inhibited by 0.2-0.5 mM SASP. 2-Mercaptoethanol (60 microM), a cystine uptake enhancer, completely prevented SASP-mediated growth inhibition in MDA-MB-231 cultures, but only partially in 184A1 and MCF-7 cultures. SASP-induced growth arrest was reversible and not cell cycle-specific. CONCLUSION: The results suggest: (i) malignant progression of human mammary cancer may be associated with acquisition of xc- expression potentially leading to increased growth autonomy and drug resistance, (ii) xc- may act as a therapeutic target.
Assuntos
Transporte Biológico/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Cistina/farmacocinética , Sulfassalazina/farmacologia , Mama/citologia , Mama/efeitos dos fármacos , Neoplasias da Mama/patologia , Linhagem Celular , Cistina/antagonistas & inibidores , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Cinética , Radioisótopos de Enxofre , Células Tumorais CultivadasRESUMO
The objective of this study was to determine the effect of different cell culture media glucose concentrations on the functional activity of PepT-1 in Caco-2 cells. Uptake kinetics of Gly-Sar into Caco-2 cells that were maintained in iso-osmotic media containing 25 or 5.5 mM glucose were determined in the presence and absence of amino acid-selective chemical modifiers and dithiothreitol. Inhibition of Gly-Sar uptake into Caco-2 cells was measured in the presence of dipeptides and xenobiotics exhibiting various binding affinities for the PepT-1. The effect of extracellular glucose on PepT-1 gene expression was assessed using comparative RT-PCR. Long-term exposure of Caco-2 cells to 25 mM glucose reduced maximum transport capacity for Gly-Sar uptake without altering PepT-1 gene expression. In contrast, binding affinity of Gly-Sar and other dipeptides or xenobiotics was not significantly changed. Chemical modification of Lys and Tyr residues decreased V(max), while Cys modification increased the maximum transport capacity of the carrier. Preincubation of Caco-2 cells with dithiothreitol restored PepT-1 activity in cells maintained at 25 mM glucose. In conclusion, cell culture media containing 25 mM glucose decreases maximum transport capacity of PepT-1 in Caco-2 cells without affecting substrate recognition, at least in part, mediated via an oxidative pathway.
Assuntos
Células CACO-2/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Espaço Extracelular/efeitos dos fármacos , Glucose/farmacologia , Simportadores , Células CACO-2/metabolismo , Proteínas de Transporte/genética , Espaço Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Transportador 1 de Peptídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
PURPOSE: To determine whether the xc- cystine transporter could be a useful therapeutic target for small-cell lung cancer (SCLC). METHODS: Human SCLC cell cultures were examined for growth dependence on extracellular cystine, xc- expression, glutathione levels and response to highly specific xc- inhibitors, i.e., monosodium glutamate (MSG) and the anti-inflammatory drug, sulfasalazine (SASP). In studying tumor growth inhibition by SASP, use was also made of a novel SCLC tissue xenograft model, LU6-SCLC, derived from a chemoresistant patient's SCLC specimen. RESULTS: Growth of NCI-H69 and NCI-H82 SCLC cells greatly depended on xc- -mediated uptake of cystine. SASP substantially reduced their glutathione levels (>70%; 0.3 mM SASP; 24 h) and growth (72 h) with IC(50)s of 0.21 and 0.13 mM, respectively; MSG also inhibited growth markedly. Both SASP- and MSG-induced growth arrests were largely prevented by cystine uptake-enhancing 2-mercaptoethanol (66 approximately microM) indicating they were primarily due to cystine starvation. Without major side-effects, SASP (i.p.) restrained growth of NCI-H69 cell xenografts (approximately 50%) and, importantly, substantially inhibited growth of the clinically more relevant LU6-SCLC tissue xenografts (approximately 70% by stereological analysis), reducing tumor glutathione contents. CONCLUSIONS: The xc- cystine/glutamate antiporter is potentially useful as a target for therapy of SCLC based on glutathione depletion. Sulfasalazine may be readily used for this approach, especially in combination chemotherapy.
Assuntos
Sistema y+ de Transporte de Aminoácidos/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/farmacologia , Carcinoma de Células Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Sulfassalazina/farmacologia , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/efeitos adversos , Transporte Biológico , Carcinoma de Células Pequenas/patologia , Linhagem Celular Tumoral , Cistina/metabolismo , Sistemas de Liberação de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutationa/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Glutamato de Sódio/farmacologia , Sulfassalazina/administração & dosagem , Sulfassalazina/efeitos adversos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Certain cancers depend for growth on uptake of cystine/cysteine from their environment. Here we examined advanced human prostate cancer cell lines, DU-145 and PC-3, for dependence on extracellular cystine and sensitivity to sulfasalazine (SASP), a potent inhibitor of the x(c)(-) cystine transporter. METHODS: Cultures were evaluated for growth dependence on exogenous cystine, x(c)(-) transporter expression, response to SASP (growth and glutathione content). In vivo, effect of SASP was determined on subrenal capsule xenograft growth. RESULTS: Cystine omission from culture medium arrested DU-145 and PC-3 cell proliferation; both cell lines expressed the x(c)(-) transporter and were growth inhibited by SASP (IC(50)s: 0.20 and 0.28 mM, respectively). SASP-induced growth inhibition was associated with vast reductions in cellular glutathione content - both effects based on cystine starvation. SASP (i.p.) markedly inhibited growth of DU-145 and PC-3 xenografts without major toxicity to hosts. CONCLUSIONS: SASP-induced cystine/cysteine starvation leading to glutathione depletion may be useful for therapy of prostate cancers dependent on extracellular cystine.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Cistina/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Sulfassalazina/farmacologia , Sistema ASC de Transporte de Aminoácidos/metabolismo , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cistina/deficiência , Cistina/metabolismo , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfassalazina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Patients with refractory inflammatory bowel disease (IBD) exhibit increased expression of intestinal P-glycoprotein (P-gp) as well as elevated luminal IFN-gamma and nitric oxide (NO) levels. Using the in vitro Caco-2 cell culture model, we investigated whether these pathological mediators associated with the etiology of IBD affect functional activity of intestinal efflux systems. IFN-gamma reduced cellular uptake of cyclosporin A (CysA) but not methotrexate (MTX) in a time- and concentration-dependent manner. Simultaneously, P-gp expression increased by approximately twofold. Coincubation with the inducible NO synthase inhibitor l-N(6)-(1-iminoethyl)lysine (l-NIL) dramatically reduced production of intracellular NO in response to IFN-gamma stimulus. The presence of l-NIL also abrogated the cytokine-mediated increase in P-gp expression and function suggesting that NO is required for IFN-gamma-mediated activation of this efflux system. Exposure of Caco-2 cells to the chemical NO donor S-nitroso-N-acetylpenicillamine (SNAP) produced a concentration-dependent decrease in intracellular CysA accumulation that was paralleled by an increase in P-gp expression. Both IFN-gamma and SNAP enhanced DNA binding of NF-kappaB, whereas inclusion of l-NIL dramatically decreased this cytokine-induced effect on NF-kappaB binding. These results suggest that NO mediates IFN-gamma-induced increase in expression and function of intestinal P-gp in the human Caco-2 cell culture model by altering DNA binding of NF-kappaB, which may enhance transcription of the ABCB1 gene encoding for this efflux system.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Interferon gama/farmacologia , Mucosa Intestinal/metabolismo , Óxido Nítrico/fisiologia , Células CACO-2 , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Immunoblotting , Doenças Inflamatórias Intestinais/patologia , Intestinos/citologia , Intestinos/efeitos dos fármacos , NF-kappa B/biossíntese , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , S-Nitroso-N-Acetilpenicilamina/farmacologiaRESUMO
Recent reports have documented a functional deficit of organic cation transport in diabetic rats by an unknown mechanism. This study was designed to test the hypothesis that experimental diabetes decreases expression of organic cation transporters at the basolateral membrane. Streptozotocin-induced diabetic rats were maintained for varying durations after induction of diabetes. A second group of age-matched control rats was maintained in a parallel manner. Kinetic analysis of tetraethylammonium accumulation in freshly isolated proximal tubular cells indicated a significantly lower V(max) value for the diabetics versus controls with no statistical difference in K(m) values between the two groups. Cortex sections were processed by standard procedures for Northern and immunoblot analysis. Protein expression of the organic cation transporters rOCT1 and rOCT2 progressively decreased with increasing duration of diabetes. After 21 days of diabetes, rOCT1 and rOCT2 were maximally reduced by 50 and 70%, respectively. Quantification of mRNA expression revealed that the roct1 transcript remained unchanged, whereas the roct2 transcript was decreased by 50% after 14 days of diabetes. Treatment with insulin prevented the reductions in transporter levels. These results support the hypothesis by demonstrating that experimental diabetes decreased expression of both rOCT1 and rOCT2 protein and also of roct2 mRNA accumulation. On the other hand, roct1 mRNA levels were unaffected by the diabetic state. This suggests that differences in rOCT2 protein may result from transcriptional and/or translational changes, whereas rOCT1 deficits may be due to posttranscriptional alterations.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 1 de Cátions Orgânicos/metabolismo , Animais , Proteínas da Membrana Plasmática de Transporte de Catecolaminas , Insulina/farmacologia , Cinética , Masculino , Transportador 2 de Cátion Orgânico , Ratos , Ratos Sprague-DawleyRESUMO
Paclitaxel, a taxane anti-microtubule agent, is known to induce CYP3A in rat and human hepatocytes. Recent studies suggest that a member of the nuclear receptor family, pregnane X Receptor (PXR), is a key regulator of the expression of CYP3A in different species. We investigated the role of PXR activation, in vitro and in vivo, in mediating Cyp3a induction by paclitaxel. Pregnenolone 16 alpha-carbonitrile (PCN), an antiglucocorticoid, was employed as a positive control for mouse PXR (mPXR) activation in vitro, and Cyp3a induction in vivo. In cell based reporter gene assays paclitaxel and PCN activated mPXR with an EC(50) of 5.6 and 0.27 microM, respectively. Employing PXR wild-type and transgenic mice lacking functional PXR (-/-), we evaluated the expression and activity of CYP3A following treatment with paclitaxel and PCN. Paclitaxel significantly induced CYP3A11 mRNA and immunoreactive CYP3A protein in PXR wild-type mice. Consistent with kinetics of CYP3A induction, the V(max) of testosterone 6 beta-hydroxylation in microsomal fraction increased 15- and 30-fold in paclitaxel- and PCN-treated mice, respectively. The Cyp3a induction response was completely abolished in paclitaxel- and PCN-treated PXR-null mice. This suggests that paclitaxel-mediated CYP3A induction in vivo requires an intact PXR-signaling mechanism. Our study validates the use of PXR activation assays in screening newer taxanes for potential drug interactions that may be related to PXR-target gene induction.
Assuntos
Antineoplásicos/farmacologia , Hidrocarboneto de Aril Hidroxilases/biossíntese , Oxirredutases N-Desmetilantes/biossíntese , Paclitaxel/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Animais , Antineoplásicos/metabolismo , Citocromo P-450 CYP3A , Indução Enzimática , Técnicas In Vitro , Proteínas de Membrana , Camundongos , Camundongos Knockout , Microssomos Hepáticos/metabolismo , Paclitaxel/metabolismo , Receptor de Pregnano X , RNA Mensageiro/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genéticaRESUMO
PURPOSE: In clinical studies, topiramate (TPM) was shown to cause a dose-dependent increase in the clearance of ethinyl estradiol. We hypothesized that this interaction results from induction of hepatic cytochrome P450 (CYP) 3A4 by TPM. Accordingly, we investigated whether TPM induces CYP3A4 in primary human hepatocytes and activates the human pregnane X receptor (hPXR), a nuclear receptor that serves as a regulator of CYP3A4 transcription. METHODS: Human hepatocytes were treated for 72 h with TPM (10, 25, 50, 100, 250, and 500 microM) and known inducers, phenobarbital (PB; 2 mM), and rifampicin (10 microM). The rate of testosterone 6beta-hydroxylation by hepatocytes served as a marker for CYP3A4 activity. The CYP3A4-specific protein and mRNA levels were determined by using Western and Northern blot analyses, respectively. The hPXR activation was assessed with cell-based reporter gene assay. RESULTS: Compared with controls, TPM (50-500 microM)-treated hepatocytes exhibited a considerable increase in the CYP3A4 activity (1. 6- to 8.2-fold), protein levels (4.6- to 17.3-fold), and mRNA levels (1.9- to 13.3-fold). Comparatively, rifampicin (10 microM) effected 14.5-, 25.3-, and a 20.3-fold increase in CYP3A4 activity, immunoreactive protein levels, and mRNA levels, respectively. TPM (50-500 microM) caused 1.3- to 3-fold activation of the hPXR, whereas rifampicin (10 microM) caused a 6-fold activation. CONCLUSIONS: The observed induction of CYP3A4 by TPM, especially at the higher concentrations, provides a potential mechanistic explanation of the reported increase in the ethinyl estradiol clearance by TPM. It also is suggestive of other potential interactions when high-dose TPM therapy is used.
Assuntos
Anticonvulsivantes/farmacologia , Sistema Enzimático do Citocromo P-450/biossíntese , Frutose/análogos & derivados , Frutose/farmacologia , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores de Esteroides/efeitos dos fármacos , Animais , Linhagem Celular , Células Cultivadas , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Humanos , Fenobarbital/farmacologia , Receptor de Pregnano X , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides/fisiologia , Rifampina/farmacologia , Testosterona/metabolismo , Topiramato , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
Tamoxifen is a widely utilized antiestrogen in the treatment and chemoprevention of breast cancer. Clinical studies document that tamoxifen administration markedly enhances the systemic elimination of other drugs. Additionally, tamoxifen enhances its own clearance following repeated dosing. The mechanisms that underlie these clinically important events remain unresolved. Here, we report that tamoxifen and its metabolite 4-hydroxytamoxifen markedly induce cytochrome P450 3A4, a drug-metabolizing enzyme of central importance, in primary cultures of human hepatocytes. Tamoxifen and 4-hydroxytamoxifen (1-10 microM) significantly increased the CYP3A4 expression and activity (measured as the rate of testosterone 6beta-hydroxylation). Maximal induction was achieved at the 5 microM level. At this level, tamoxifen and 4-hydroxytamoxifen caused a 1.5- to 3.3-fold (mean, 2.1-fold) and 3.4- to 17-fold (mean, 7.5-fold) increase in the CYP3A4 activity, respectively. In comparison, rifampicin treatment resulted in a 6- to 16-fold (mean, 10.5-fold) increase. We also observed corresponding increase in the CYP3A4 immunoreactive protein and mRNA levels. Furthermore, tamoxifen and 4-hydroxytamoxifen efficaciously activated the human pregnane X receptor (hPXR; also known as the steroid xenobiotic receptor), a key regulator of CYP3A4 expression. The efficacy of tamoxifen and 4-hydroxytamoxifen relative to rifampicin for hPXR activation was approximately 30 and 60%, respectively. Our results indicate that the mechanism of tamoxifen-mediated alteration in drug clearance pathways in humans may involve CYP3A4 induction by the parent drug and/or its metabolite. Furthermore, the CYP3A4 induction may be a result of hPXR activation. These findings have important implications for optimizing the use of tamoxifen and in the development of newer antiestrogens.
Assuntos
Antineoplásicos/farmacologia , Sistema Enzimático do Citocromo P-450/biossíntese , Hepatócitos/metabolismo , Oxigenases de Função Mista/biossíntese , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores de Esteroides/agonistas , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Células Cultivadas , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Hepatócitos/enzimologia , Humanos , Oxigenases de Função Mista/genética , Receptor de Pregnano X , RNA Mensageiro/análiseRESUMO
Using morphological and molecular approaches, we characterized cisplatin-induced cell necrosis and apoptosis in rat kidney. Male Sprague-Dawley rats ( n=5 per group) received a single intraperitoneal injection of either cisplatin (5 mg/kg) or saline, and were killed on day 5. Functionally, cisplatin-treated rats developed polyuric acute renal failure. Morphologically, kidneys of cisplatin-treated rats showed overt tubular necrosis associated with apoptosis in the corticomedullary junction. Cell necrosis was segment-specific and was distributed in radial fashion at the corticomedullary junction. The apoptosis was limited to discrete cells in apparently intact tubules in the vicinity of the necrosed tubules. The apoptotic changes were confirmed by TUNEL (TdT-mediated deoxyuridine triphosphate nick-end labeling) and staining for cleaved caspase-3. Analysis of outer medullary tissue for apoptosis-related molecules by RNase protection assay revealed a significant increase in the expression of pro-apoptotic mRNAs (caspases 1, 2, and 8, and Bax) in cisplatin-treated rats. On the other hand, the expression of mRNA for the anti-apoptotic Bcl-2 did not change, resulting in a decrease in relative ratio of Bcl-2/Bax, and thus favoring apoptosis. The above changes were paralleled by a marked increase in caspase-3 precursor, the executioner protease. Furthermore, these pro-apoptotic molecular changes were associated with a 3-fold increase in the activity of JNK1 in the outer medulla, but not in the cortex, of cisplatin-treated rat kidneys, localizing to the site of maximal apoptosis. Upregulation of JNK1 activity in the outer medulla was not accompanied by changes in the activities of ERK or p38 kinase. In conclusion, these data suggest that cisplatin-induced apoptotic cell death in native kidney may be mediated by cooperative activation of the JNK1 pathway and Bax in the outer medulla.