RESUMO
Neuroendocrine (NE) differentiation is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. NE tumor cells are nonproliferating and escape apoptotic cell death; therefore, an understanding of the apoptotic status of the NE phenotype is imperative for the development of new therapies for prostate cancer. Here, we report for the first time on alterations in intracellular Ca(2+) homeostasis, which is a key factor in apoptosis, caused by NE differentiation of androgen-dependent prostate cancer epithelial cells. NE-differentiating regimens, either cAMP elevation or androgen deprivation, resulted in a reduced endoplasmic reticulum Ca(2+)-store content due to both SERCA 2b Ca(2+) ATPase and luminal Ca(2+) binding/storage chaperone calreticulin underexpression, and to a downregulated store-operated Ca(2+) current. NE-differentiated cells showed enhanced resistance to thapsigargin- and TNF-alpha-induced apoptosis, unrelated to antiapoptotic Bcl-2 protein overexpression. Our results suggest that targeting the key players determining Ca(2+) homeostasis in an attempt to enhance the proapoptotic potential of malignant cells may prove to be a useful strategy in the treatment of advanced prostate cancer.
Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Diferenciação Celular , Homeostase , Sistemas Neurossecretores , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/fisiopatologia , Western Blotting , Canais de Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Calreticulina/metabolismo , Linhagem Celular Tumoral , Capacitância Elétrica , Impedância Elétrica , Eletrofisiologia , Retículo Endoplasmático/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Corantes Fluorescentes , Fura-2 , Humanos , Cinética , Masculino , Modelos Biológicos , Técnicas de Patch-Clamp , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tapsigargina/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
This study investigates the calcium mechanisms involved in growth arrest induced by extracellular ATP in DU-145 androgen-independent human prostate cancer cells. Exposure of DU-145 cells to 100 microM ATP produced an increase in cytoplasmic calcium concentration ([Ca(2+)](i)), due to a mobilization of calcium from the endoplasmic reticulum stores and to subsequent capacitative calcium entry (CCE). We have shown that this [Ca(2+)](i) increase occurs after stimulation by ATP of the phospholipase C (PLC) pathway. For the first time, we have identified the inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms expressed in this cell line and have demonstrated a participation of protein kinase C in CCE. Using fluorescence imaging, we have shown that a long-term treatment with ATP leads to a decrease in the intraluminal endoplasmic reticulum calcium concentration as well as in the amount of releasable Ca(2+). Modulating extracellular free calcium concentrations indicated that variations in [Ca(2+)](i) did not affect the ATP-induced growth arrest of DU-145 cells. However, treating cells with 1 nM thapsigargin (TG) to deplete intracellular calcium pools prevented the growth arrest induced by ATP. Altogether, these results indicate that growth arrest induced in DU-145 cells by extracellular ATP is not correlated with an increase in [Ca(2+)](i) but rather with a decrease in intracellular calcium pool content.
Assuntos
Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Carcinoma/metabolismo , Neoplasias da Próstata/metabolismo , Trifosfato de Adenosina/farmacologia , Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carcinoma/fisiopatologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Masculino , Neoplasias da Próstata/fisiopatologia , Proteína Quinase C/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Tapsigargina/farmacologia , Fosfolipases Tipo C/efeitos dos fármacos , Fosfolipases Tipo C/metabolismoRESUMO
Ca2+ homeostasis mechanisms, in which the Ca2+ entry pathways play a key role, are critically involved in both normal function and cancerous transformation of prostate epithelial cells. Here, using the lymph node carcinoma of the prostate (LNCaP) cell line as a major experimental model, we characterize prostate-specific store-operated Ca2+ channels (SOCs)--a primary Ca2+ entry pathway for non-excitable cells--for the first time. We show that prostate-specific SOCs share major store-dependent, kinetic, permeation, inwardly rectifying, and pharmacological (including dual, potentiation/inhibition concentration-dependent sensitivity to 2-APB) properties with "classical" Ca2+ release-activated Ca2+ channels (CRAC), but have a higher single channel conductance (3.2 and 12pS in Ca2+- and Na+-permeable modes, respectively). They are subject to feedback inhibition via Ca2+-dependent PKC, CaMK-II and CaM regulatory pathways and are functionally dependent on caveolae integrity. Caveolae also provide a scaffold for spatial co-localization of SOCs with volume-regulated anion channels (VRAC) and their Ca2+-mediated interaction. The TRPC1 and TRPV6 members of the transient receptor potential (TRP) channel family are the most likely molecular candidates for the formation of prostate-specific endogenous SOCs. Differentiation of LNCaP cells to an androgen-insensitive, apoptotic-resistant neuroendocrine phenotype downregulates SOC current. We conclude that prostate-specific SOCs are important determinants in the transition to androgen-independent prostate cancer.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Células Epiteliais/metabolismo , Neoplasias da Próstata/metabolismo , Biomarcadores , Canais de Cálcio/genética , Eletrofisiologia , Retículo Endoplasmático/metabolismo , Células Epiteliais/patologia , Humanos , Cinética , Masculino , Oligonucleotídeos Antissenso/farmacologia , Neoplasias da Próstata/patologia , RNA Mensageiro/efeitos dos fármacos , Canais de Cátion TRPC , Canais de Cátion TRPV , Células Tumorais CultivadasRESUMO
Ca(2+) chelating agents are widely used in biological research for Ca(2+) buffering. Here we report that BAPTA, EDTA and HEDTA produce fast, reversible, voltage-dependent inhibition of swelling-activated Cl(-) current (I(Cl,swell)) in LNCaP prostate cancer epithelial cells that is unrelated to their Ca(2+) binding. BAPTA was the most effective (maximal blockade 67%, IC(50)=70 microM, at +100 mV) followed by EDTA and HEDTA. I(Cl,swell) blockade by EDTA was pH-dependent. BAPTA blocked I(Cl,swell) also in other cell types. We conclude that Ca(2+) chelating agents block I(Cl,swell) by acting directly on the underlying channel, and that the negative charge of the free chelator form is critical for the blockade.
Assuntos
Quelantes/farmacologia , Canais de Cloreto/antagonistas & inibidores , Ácido Edético/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Cálcio , Canais de Cloreto/fisiologia , Ácido Edético/análogos & derivados , Humanos , Concentração de Íons de Hidrogênio , Células Tumorais CultivadasRESUMO
The present study demonstrates for the first time that intracellular calcium-ATPases and calcium pool content are closely associated with prostate cancer LNCaP cell growth. Cell growth was modulated by changing the amount of epidermal growth factor, serum, and androgene in culture media. Using the microspectrofluorimetric method with Fura-2 and Mag Fura-2 as probes, we show that in these cells, the growth rate is correlated with intracellular calcium pool content. Indeed, an increased growth rate is correlated with an increase in the calcium pool filling state, whereas growth-inhibited cells show a reduced calcium pool load. Using Western blotting and immunocytochemistry, we show that endoplasmic reticulum calcium pump expression is closely linked to LNCaP cell growth, and are a common target of physiological stimuli that control cell growth. Moreover, we clearly demonstrate that inhibition of these pumps, using thapsigargin, inhibits LNCaP cell growth and prevents growth factor from stimulating cell proliferation. Our results thus provide evidence for the essential role of functional endoplasmic reticulum calcium pumps and calcium pool in control of prostate cancer LNCaP cell growth, raising the prospect of new targets for the treatment of prostate cancer.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/enzimologia , Neoplasias da Próstata/metabolismo , Retículo Sarcoplasmático/enzimologia , Western Blotting , ATPases Transportadoras de Cálcio/biossíntese , Divisão Celular , Quelantes/farmacologia , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/antagonistas & inibidores , Corantes Fluorescentes/farmacologia , Fura-2/farmacologia , Humanos , Imuno-Histoquímica , Membranas Intracelulares/metabolismo , Masculino , Microscopia de Fluorescência , Microssomos/metabolismo , Ligação Proteica , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Espectrofotometria , Tapsigargina/farmacologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
1. In the present study, we investigated the mechanisms involved in the induction of apoptosis by the Ca2+-ATPase inhibitor thapsigargin (TG), in androgen-sensitive human prostate cancer LNCaP cells. 2. Exposure of fura-2-loaded LNCaP cells to TG in the presence of extracellular calcium produced an increase in intracellular Ca2+, the first phase of which was associated with depletion of intracellular stores and the second one with consecutive extracellular Ca2+ entry through plasma membrane, store-operated Ca2+ channels (SOCs). 3. For the first time we have identified and characterized the SOC-mediated membrane current (Istore) in prostate cells using whole-cell, cell-attached, and perforated patch-clamp techniques, combined with fura-2 microspectrofluorimetric and Ca2+-imaging measurements. 4. Istore in LNCaP cells lacked voltage-dependent gating and displayed an inwardly rectifying current-voltage relationship. The unitary conductance of SOCs with 80 mM Ca2+ as a charge carrier was estimated at 3.2 +/- 0.4 pS. The channel has a high selectivity for Ca2+ over monovalent cations and is inhibited by Ni2+ (0.5-3 mM) and La3+ (1 microM). 5. Treatment of LNCaP cells with TG (0.1 microM) induced apoptosis as judged from morphological changes. Decreasing extracellular free Ca2+ to 200 nM or adding 0.5 mM Ni2+ enhanced TG-induced apoptosis. 6. The ability of TG to induce apoptosis was not reduced by loading the cells with intracellular Ca2+ chelator (BAPTA-AM). 7. These results indicate that in androgen-sensitive prostate cancer cells the depletion of intracellular Ca2+ stores may trigger apoptosis but that there is no requirement for the activation of store-activated Ca2+ current and sustained Ca2+ entry in induction and development of programmed cell death.