ABSTRACT
In this study, we demonstrate that apoptosis and G2/M cell cycle arrest were easily induced by treatment with the oral-antifungal agent, griseofulvin (GF). The mechanisms of GF-induced G2/M arrest were characterized as (a) induction of abnormal mitotic spindle formation, (b) elevation of cyclin B1/cdc2 kinase activity and (c) down-regulation of myt-1 protein expression. On the other hand, caspase 3 activation, Bcl-2 hyperphosphorylation and inhibition of the normal function of Bcl-2 associated with Bax were demonstrated to be the mechanisms of GF-induced apoptosis. DNA fragmentation and flow cytometry analyses demonstrated that combined treatment of GF with the cancer chemotherapeutic agent, nocodazole (ND), strongly potentiates the apoptotic effect and arrest of the G2/M cell cycle in 5 types of human cancer cells, but not in normal human keratinocytes (#76 KhGH). The combined treatment of GF and ND triggered the polymerization of purified tubulin in HT 29 but not in #76 KhGH cells. To further confirm these observations, the therapeutic efficacy was further examined in vivo by treating athymic mice bearing COLO 205 tumor xenografts, with GF (50 mg/kg), ND (5 mg/kg) or GF + ND. Combined treatment of GF and ND significantly enhanced the effect of ND, and led to cessation of tumor growth. These results suggest that chemotherapeutic agents (such as ND) administered in the presence of GF might provide a novel therapy for colorectal cancer.
Subject(s)
Antifungal Agents/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , G2 Phase/drug effects , Griseofulvin/pharmacology , Nocodazole/pharmacology , Animals , Antifungal Agents/administration & dosage , Apoptosis/physiology , Caspase 3 , Caspases/metabolism , Colorectal Neoplasms/drug therapy , Cyclin B/metabolism , Cyclin B1 , DNA Fragmentation , Down-Regulation , Drug Synergism , Drug Therapy, Combination , Enzyme Activation , Griseofulvin/administration & dosage , HT29 Cells/drug effects , Humans , Keratinocytes/drug effects , Membrane Proteins , Mice , Mice, Nude , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Tubulin/drug effects , Tubulin/metabolismABSTRACT
In this study, the amount of S-nitrosoglutathione (GSNO) was measured spectrophotometrically at 334 nm. A spontaneous decrease in absorbency at 334 nm was detected when GSNO was exposed to 37 degrees C and a high pH (pH 8.0). We investigated the catalytic roles of various metal ions on the decomposition of GSNO. The degradation of GSNO (0.5 mM) was enhanced by the presence of Cu(2+) and Ni(2+) ions. The amount of nitric oxide (NO) released from GSNO degradation was estimated by the Griess reaction based on nitrite accumulation. The results indicated that nitrite production was elevated at least twofold in the presence of Cu(2+). Our study further indicated that Cu(2+) enhanced GSNO-induced apoptosis in human colon adenocarcinoma HT 29 cells. We also found that copper ions modulated the expression of bad, bax, and bcl-2 in GSNO-treated HT 29 cells. The levels of bax and bad proteins in treated cells were significantly elevated about fourfold to sixfold when compared with mock-treated cells 24 h after combined treatment with GSNO plus Cu(2+) or Ni(2+). On the other hand, significant inhibition of bcl-2 occurred in HT 29 cells with simultaneous treatment of GSNO with Cu(2+) (or Ni(2+)). It seemed that Cu(2+) and Ni(2+) can enhance the decomposition of GSNO, which liberates NO to activate the pathways. Our results demonstrated that the apoptotic effects induced by GSNO were promoted by Ni(2+) and Cu(2+) through two different mechanisms: depletion of intracellular glutathione and triggering of NO release from GSNO, which then promotes NO-induced apoptosis in human cells.
