Exploration Of The Mechanism Of Growth Inhibition And Apoptosis Induction By Combined Therapy With RhIL-24and5-FU In Breast Cancer Cells

Objectives:To compare the inhibitory effects of the combined therapy with recombinant human interleukin-24(rhIL-24) and5-Fluorouracil (5-FU) and single therapies with either rhIL-24or5-FU on the MDA-MB-157breast cancer cells. and to explore the anti-tumor mechanism of combined therapy via the observation of the differential influences of each therapy strategy on both cell cycle and apoptosis so as to provide theoretical instructions for the combined therapy with rhIL-24and5-FU both in vitro and in the clinic.Methods:(1) MDA-MB-157cells were cultured in96-well plates at a density of104/ml at37℃in a humidified atmosphere with no CO2. and WI-38cells were cultured in96-well plates at a density of2×104/ml at37℃in a humidified atmosphere of5%CO2. The culture supernatant was discarded at24h and rhIL-24was added at a concentration of80ng/ml.100ng/ml,120ng/ml,150ng/ml and200ng/ml respectively.5-Fu was added at a concentration of5μg/ml,10μg/ml,25μg/ml,50μg/ml and75μg/ml. After cells were cultured for another24.48and72hours, the CCK-8assay was employed to detect cellular proliferation, and growth curves were drafted and the inhibitory rates were calculated.(2) The MDA-MB-157and the WI-38cells were cultured and treated as mentioned above, and120ng/ml rhIL-24and10μg/ml5-Fu were added. The CCK-8assay was employed to detect cellular proliferation, and growth curves were drafted and the inhibitory rates were calculated at24h.48h and72h. respectively.(3) MDA-MB-157cells were cultured in6-well plates at a density of104/ml at37℃in a humidified atmosphere with no CCK and WI-38cells were cultured in6-well plates at a density of2×104/ml at37℃in a humidified atmosphere of5%CO2The culture supernatant was discarded at24h and cells were treated with120ng/ml rhIL-24,10μg/ml5-Fu or120ng/ml rhIL-24and10μg/m15-FU for24hours. after which apoptosis were detected by flow cytometry.(4) AnnexinV-FITC staining was performed at24h. the cell apoptosis in both the MDA-MB-157and WI-38cells were observed under the fluorescence microscope for the above three groups. Results:(1) The optimal inhibitory concentration of rhIL-24is120ng/ml for the MDA-MB-157cells, while the optimal inhibitory concentration of5-Fu for the MDA-MB-157cells is lOug/ml; rhIL-24showed no significant inhibitory effect on WI-38cells. The optimal inhibitory concentration of5-Fu for the WI-38cells is10μg/ml.(2) Combined inhibitory effects:While the inhibitory rates of the combined group for the MDA-MB-157cells are respectively28.2%.36.5%and44.7%at24h.48h and72h. its inhibitory rates for the WI-38cells are respectively5.4%.25%and68.7%at24h.48h and72h.(3) Influences on cell apoptosis:While the apoptosis rates of the MDA-MB-157cells are respectively14.9%.20.2%and26.4%for the rhIL-24group, the5-Fu group and the combined group, the apoptosis rates of the WI-38cells are respectively6.8%.28.1%and24.1%for the above three groups.(4) Fluorescence detection of cell apoptosis:Cellular pyknosis and apoptotic bodies were observed for MDA-MB-157cells and WI-38cells at24h after the addition of rhIL-24or/and5-FU. and more cells were shown to present with cellular pyknosis and apoptotic bodies in the combined group.Conclusions:1. The optimal inhibitory concentration of rhIL-24is120ng/ml for the MDA-MB-157cells, and rhIL-24showed no significant inhibitory effect on WI-38cells.2.5-Fu inhibited the growth of both the MDA-MB-157and WI-38cells in a dose-and time-dependent manner, indicating an inhibitory effect of5-Fu on normal cells.3. rhIL-24and5-Fu showed a synergistic and time-dependent inhibitory effect on cell growth.4. The anti-tumor mechanism by combined therapy with rhIL-24and5-Fu which is apoptosis induction.