Effect Of Inhibition Of Extracellular-Signal Regulated Protein Kinase On Arsenic Trioxide-induced Apoptosis In MGC-803 Cells

IntroductionArsenic trioxide (As₂O₃) is an ancient reagent for about 2400 years. It was used to treat syphilis, psoriasis, rheumatism, and so on. Recently arsenic trioxide has shown effective on inducing complete remission in acute promyelocytic leukemia (APL). Its principal mechanism elucidated is to induce APL cells apopto-sis.Gastric cancer is the most common malignant tumor in our country , less sensitive to both chemotherapy and radiation. A lot of reports showed that arsenic trioxide can inhibit tumor cell growth and this effect is mainly dependent on apoptosis and cell cycle arrest. But the specific mechanism has not been identified.Apoptosis and cell cycle arrest are regulated by a variety of genes. Extracellular - Signal regulated protein kinase (ERK) is a member of the mitogen -activated protein kinase( MAPK) familys. It is an important mediator of signal transduction and is activated by a variety of stimuli such as growth factors. The ERK pathway and its upstream kinases are activated by growth factor stimuli and have the effect of transducing the signal to the nucleus and of increasing the level of expression of genes that are related to cellular proliferation and differentiation. The ERK pathway is also associated with cellular apoptosis. Recently it was suggested that inhibition of ERK activities causes a downregulation of antiapop-totic homlogs and that activation of the pathway functions to protect cells fromapoptosis.The objective of the present study is to explore the effect of inhibition of ERK on arsenic trioxide - induced apoptosis in gastric cancer cell line.Methods and Materials1. Materials0Arsenic trioxide was purchased from Harbin Yierda pharmaceutic corporation. RPMI1640 was purchased from GIBCO BRL USA. Fetal bovine serum was purchased from Institue of Hematology CAMS. The selective inhibitor of ERK (PD98059) was purchased from Promega corporation USA.2. Cell cultureThe gastric cancer cell line MGC - 803 was cultured in RPMI1640 medium, supplemented with 10% fetal bovine serum with addition of 12U/ml genta-mycin and was incubated at 37℃ in 5% CO₂ atomosphere. In all experiments, cells were used in logarithmic growth phase.3. Assay for growth inhibition of arsenic trioxide to gastric cancer cell line MGC-803Growth inhibition of tumor cells was determined by MTT.4. Assay for apoptosis and cell cycle arrestThe cells treated with arsenic trioxide were dyed by Wright — Giemsa staining and observed with microscope.The ratio of apoptosis and cell cycle arrest were determined by flow cyto-metric DNA analysis.5. Assay for the effect of inhibition of ERK on arsenic trioxide - induced apoptosisCells in suspension were planted to culture plate and incubated at 37℃ in a humidified atomosphere of 0.5% CO₂ in air. The selective inhibitor of ERK was added 1 hour prior to arsenic trioxide. The cultures were again incubated for 24 hours ,48 hours respectively. The ratio of apoptosis was determined by Wright -Giemsa staining and flow cytometric DNA analysis.