RESUMEN
This study investigated the nature and mechanism of juglone-induced apoptosis in the human breast cancer cell line MCF-7. The inhibitory effect of juglone on MCF-7 cell growth was evaluated by the dimethylthiazol tetrazolium assay. Morphological apoptotic changes were characterized using an inverted microscope, Hoechst 33258 fluorescence staining, and Giemsa staining. The rate of cell apoptosis, intracellular levels of reactive oxygen species (ROS), and mitochondrial membrane potential were detected using flow cytometry. Intracellular Ca(2+) concentrations were detected using laser scanning confocal fluorescence microscopy. Expression of the proteins Bcl-2, Bax, and cytochrome C was assessed by western blotting. Caspase-3 activity was quantified using a caspase-3 activity kit. Juglone inhibited the growth of MCF-7 cell line with an IC50 of 11.99 µM. The rates of MCF-7 cell apoptosis at 24 h after exposure to 5, 10, and 20 µM juglone were 9.29, 20.67, and 28.39%, respectively; compared to unexposed cells, juglone-exposed cells exhibited significant elevation in intracellular ROS level, decrease in mitochondrial membrane potential, and increase in intracellular Ca(2+) concentration. Juglone upregulated the expression of Bax, and downregulated the expression of Bcl-2, promoting the release of cytochrome C, thereby upregulating the activity of caspase-3. The results suggest that the mechanism of juglone-induced apoptosis in MCF-7 cells is characterized by elevated ROS levels, reduced Bcl-2 expression, increased Bax expression, decreased mitochondrial membrane potential, increased intracellular Ca(2+) concentration, outer mitochondrial-membrane rupture, cytochrome C release, and caspase-3 activation.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Naftoquinonas/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Caspasa 3/metabolismo , Línea Celular Tumoral , Citocromos c/metabolismo , Femenino , Humanos , Células MCF-7 , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
BACKGROUND The purpose of this study was to evaluate telomerase activity in peripheral whole blood from head and neck squamous cell carcinoma (HNSCC) patients as a biomarker for diagnosis of HNSCC or detection of recurrence during follow-up. MATERIALS AND METHODS Telomerase activity was measured from peripheral whole blood extracts by telomerase repeat amplification protocol (TRAP) in HNSCC patients before and after surgery and in a control group. Sixty-two HNSCC patients and 42 control subjects were included. RESULTS Telomerase activity was found in 41 out of 62 (66.1%) HNSCC patients before surgery and in 8 out of 42 (19.0%) controls (p<0.001). Among 41 HNSCC patients who showed positive telomerase activity before surgery, 32 (78.1%) showed a conversion of telomerase activity to negative after surgery. In follow-up, 6 out of 8 (75%) showed conversion of telomerase activity from negative to positive after recurrence. Telomerase activity was changed to negative in 4 out of 6 (66%) recurred patients with positive telomerase activity after second surgery. CONCLUSION The telomerase activity in peripheral whole blood extracts of HNSCC patients might be a useful biomarker for detecting recurrence after treatment. Further study with larger sample size using a more sensitive detection method of telomerase activity is necessary to verify these results.