RESUMO
Disruption of intracellular calcium initiates multiple cell-damaging processes, such as apoptosis. In normal cells, the levels of Ca(2+) are low in the mitochondria, whereas in apoptotic cells, Ca(2+) increases. Mitochondria uptake Ca(2+) via an inner membrane channel called the uniporter and extrude it into the cytoplasm through a Na(+)/Ca(2+) exchanger. Overload of Ca(2+) in the mitochondria in CGP-treated cells leads to its damage, thus affecting cellular function and survival. The goal of these experiments was to determine the importance of mitochondrial calcium ([Ca(2+)](m)) in apoptosis of prostate cancer cells. Furthermore, we have examined the advantages of increasing the [Ca(2+)](m) and treating the cells with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a potent apoptotic agent. Our results show that, under these treatment conditions, inhibiting the Na(+)/Ca(2+) exchanger using benzothiazepin CGP-37157 (CGP) did not induce apoptosis. However, combination of CGP and TRAIL increased the apoptotic response approximately 25-fold compared with control. Increase in apoptosis followed enhanced levels of [Ca(2+)](m) and was accompanied by pronounced mitochondrial changes characteristic of mitochondria-mediated apoptosis. Experiments with calcium ionophores showed that mere increase in cytosolic and/or mitochondrial Ca(2+) was not sufficient to induce apoptosis. These results have therapeutic implications as inhibitors of Na(+)/Ca(2+) exchanger are being used for treating some neurologic and cardiologic ailments, and TRAIL induces apoptosis preferentially in cancer cells. Furthermore, this system provides an excellent model to investigate the role of [Ca(2+)](m) in apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Androgênios/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/administração & dosagem , Clonazepam/análogos & derivados , Clonazepam/farmacologia , Sinergismo Farmacológico , Humanos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/metabolismo , Tiazepinas/farmacologia , Células Tumorais CultivadasRESUMO
BACKGROUND: The goal of this study was to compare the antagonism of elevated dietary Cu (250 mg/kg) from CuSO4 on three different Zn sources (ZnSO4 · H2O; [Zn bis(-2-hydroxy-4-(methylthio)butanoic acid)], Zn(HMTBa)2, a chelated Zn methionine hydroxy analogue; and Zn-Methionine), as measured using multiple indices of animal performance in ROSS 308 broilers. METHODS: Three experiments were conducted in broiler chicks fed a semi-purified diet. All birds were fed a Zn-deficient diet (8.5 mg/kg diet) for 1 wk, and then provided with the experimental diets for 2 wks. RESULTS: Experiment 1 was a 2 × 2 factorial design with two levels of Cu (8 vs. 250 mg/kg diet from CuSO4) and two Zn sources at 30 mg/kg [ZnSO4 · H2O vs. Zn(HMTBa)2]. Elevated Cu impaired growth performance only in birds fed ZnSO4. Compared to ZnSO4 · H2O, Zn(HMTBa)2 improved feed intake (12 %; P < 0.001) and weight gain (12 %, P < 0.001) and the benefits were more pronounced in the presence of 250 mg/kg diet Cu. Experiment 2 was a dose titration of ZnSO4 · H2O and Zn(HMTBa)2 at 30, 45, 60, and 75 mg/kg diet in the presence of 250 mg/kg CuSO4. Feed:gain was decreased and tibia Zn was increased with increasing Zn levels from 30 to 75 mg/kg. Birds fed Zn(HMTBa)2 consumed more food and gained more weight compared to birds fed ZnSO4, especially at lower supplementation levels (30 and 45 mg/kg; interaction P < 0,05). Experiment 3 compared two organic Zn sources (Zn(HMTBa)2 vs. Zn-Methionine) at 30 mg/kg with or without 250 mg/kg CuSO4. No interactions were observed between Zn sources and Cu levels on performance or tissue mineral concentrations. High dietary Cu decreased weight gain (P < 0.01). Tibia Cu and liver Cu were significantly increased with 250 mg/kg dietary Cu supplementation (P < 0.01). No difference was observed between the two Zn sources. CONCLUSIONS: Dietary 250 mg/kg Cu significantly impaired feed intake and weight gain in birds fed ZnSO4 · H2O, but had less impact in birds fed Zn(HMTBa)2. No difference was observed between the two organic zinc sources. These results are consistent with the hypothesis that chelated organic Zn is better utilized than inorganic zinc in the presence of elevated Cu.
RESUMO
The proteasome inhibitor Velcade (bortezomib/PS-341) has been shown to block the targeted proteolytic degradation of short-lived proteins that are involved in cell maintenance, growth, division, and death, advocating the use of proteasomal inhibitors as therapeutic agents. Although many studies focused on the use of one proteasomal inhibitor for therapy, we hypothesized that the combination of proteasome inhibitors Lactacystin (AG Scientific, Inc., San Diego CA) and MG132 (Biomol International, Plymouth Meeting, PA) may be more effective in inducing apoptosis. Additionally, this regimen would enable the use of sublethal doses of individual drugs, thus reducing adverse effects. Results indicate a significant increase in apoptosis when LNCaP prostate cancer cells were treated with increasing levels of Lactacystin, MG132, or a combination of sublethal doses of these two inhibitors. Furthermore, induction in apoptosis coincided with a significant loss of IKKalpha, IKKbeta, and IKKgamma proteins and NFkappaB activity. In addition to describing effective therapeutic agents, we provide a model system to facilitate the investigation of the mechanism of action of these drugs and their effects on the IKK-NFkappaB axis.
Assuntos
Acetilcisteína/análogos & derivados , Apoptose/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Leupeptinas/farmacologia , Neoplasias da Próstata/patologia , Acetilcisteína/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica , Sinergismo Farmacológico , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/metabolismo , Masculino , NF-kappa B/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Inibidores de Proteassoma , Células Tumorais CultivadasRESUMO
Treatment options for androgen-independent prostate cancer cells are limited. Therefore, it is critical to identify agents that induce death of both androgen-responsive and androgen-insensitive cells. Here we demonstrate that a product of plant cell walls, pectin, is capable of inducing apoptosis in androgen-responsive (LNCaP) and androgen-independent (LNCaP C4-2) human prostate cancer cells. Commercially available fractionated pectin powder (FPP) induced apoptosis (approximately 40-fold above non-treated cells) in both cell lines as determined by the Apoptosense assay and activation of caspase-3 and its substrate, poly(ADP-ribose) polymerase. Conversely, citrus pectin (CP) and the pH-modified CP, PectaSol, had little or no apoptotic activity. Glycosyl residue composition and linkage analyses revealed no significant differences among the pectins. Mild base treatment to remove ester linkages destroyed FPP's apoptotic activity and yielded homogalacturonan (HG) oligosaccharides. The treatment of FPP with pectinmethylesterase to remove galacturonosyl carboxymethylesters and/or with endopolygalacturonase to cleave nonmethylesterified HG caused no major reduction in apoptotic activity, implicating the requirement for a base-sensitive linkage other than the carboxymethylester. Heat treatment of CP (HTCP) led to the induction of significant levels of apoptosis comparable to FPP, suggesting a means for generating apoptotic pectic structures. These results indicate that specific structural elements within pectin are responsible for the apoptotic activity, and that this structure can be generated, or enriched for, by heat treatment of CP. These findings provide the foundation for mechanistic studies of pectin apoptotic activity and a basis for the development of pectin-based pharmaceuticals, nutraceuticals, or recommended diet changes aimed at combating prostate cancer occurrence and progression.