Abstract:

Objective To explore the molecular mechanism by which the mitogen-activated extracellular signal-regulated kinase （MEK） inhibitor PD98059 regulates the MEK/extracellular signal-regulated kinase （ERK） signaling pathway and reverses the multiple drug resistance （MDR） of human glioma stem cells （GSCs）. Methods According to the instructions of CD133 immunomagnetic isolation kit， human glioma SHG44 cells with CD133 positive were isolated， and they were cultured with serum-free stem cell culture media. Then， GSCs were divided into control group （normal culture）， inhibitor group （200 μmol/L PD98059）， activation group （25 μmol/L erucin） and MDR1 knockout group （200 μmol/L PD98059+MDR1 gene knockout）. The proliferation of GSCs after treatment with PD98059 （0， 25， 50， 100， 200， 300， 400 μmol/L） was detected by CCK-8. The apoptosis of GSCs after being treated with doxorubicin or vincristine respectively was detected by TUNEL staining. Levels of MEK， ERK and MDR1 mRNA in GSCs were quantitatively analyzed by real-time PCR， expressions of p-MEK/MEK， p-ERK/ERK and MDR1 proteins were detected by Western blotting， and sensitivity of cells to chemotherapy drugs was analyzed by CCK-8. Results In GSCs after treatment with PD98059 （0， 25， 50， 100， 200， 300， 400 μmol/L）， there were statistically significant differences in absorbance （A）₄₅₀ values at different time points （24， 48， 72， 96 h） （F=56.22， P<0.001； F=42.69， P<0.001； F=34.19， P<0.001； F=60.28， P<0.001）， there were statistically significant differences in the A₄₅₀ values at concentrations of 25， 50， 100， 200， 300， and 400 μmol/L compared with 0 μmol/L （all P<0.05）， while there were no statistically significant differences when comparing 200 μmol/L with 300 and 400 μmol/L concentrations （both P>0.05）. After treatment with doxorubicin， apoptosis rates of GSCs in control group， inhibitor group， activation group and MDR1 knockout group were （18.21±0.33）%， （27.73±1.86）%， （20.11±2.06）% and （25.77±2.61）%， while which were （22.07±1.51）%， （33.89±3.12）%， （25.41±2.65）% and （30.19±3.08）% after treatment with vincristine， with statistically significant differences （F=36.46， P<0.001； F=40.14， P<0.001）. The above indexes in inhibitor group， activation group and MDR1 knockout group were significantly higher than those in control group （all P<0.05）， these indexes in activation group were significantly lower than those in inhibitor group （both P<0.05）， and which in MDR1 knockout group were significantly higher than those in activation group （both P<0.05）. The expressions of MEK mRNA in GSCs in the four groups were 1.00±0.00， 0.29±0.05， 0.68±0.07， 0.33±0.03， the expressions of ERK mRNA were 1.00±0.00， 0.35±0.06， 0.74±0.07， 0.38±0.04， the expressions of MDR1 mRNA were 1.00±0.00， 0.51±0.08， 0.89±0.09， 0.56±0.06， respectively， with statistically significant differences （F=30.26， P<0.001； F=22.59， P<0.001； F=18.75， P<0.001）. The above indexes in inhibitor group， activation group and MDR1 knockout group were significantly lower than those in control group （all P<0.05）， these indexes in activation group were significantly higher than those in inhibitor group （all P<0.05）， and which in MDR1 knockout group were lower than those in activation group （all P<0.05）. The levels of p-MEK/MEK proteins in GSCs in the four groups were 0.90±0.09， 0.29±0.05， 0.47±0.05， 0.32±0.04， the levels of p-ERK/ERK proteins were 1.19±0.13， 0.37±0.06， 0.55±0.06， 0.40±0.04， the levels of MDR1 proteins were 1.08±0.12， 0.62±0.07， 0.73±0.07， 0.65±0.06， respectively， with statistically significant differences （F=51.74， P<0.001； F=42.30， P<0.001； F=36.58， P<0.001）. The above indexes in inhibitor group， activation group and MDR1 knockout group were significantly lower than those in control group （all P<0.05）， these indexes in activation group were significantly higher than those in inhibitor group （all P<0.05）， and which in MDR1 knockout group were significantly lower than those in activation group （all P<0.05）. The half maximal inhibitory concentration （IC₅₀） values of GSCs to doxorubicin in the four groups were 0.88， 0.23， 0.79， 0.56 mg/L， the IC₅₀ values to vincristine were 0.84， 0.18， 0.75， 0.51 mg/L， with statistically significant differences （H=17.84， P<0.001； H=15.43， P<0.001）. The above indexes in inhibitor group were lower than those in control group （both P<0.05）， these indexes in activation group were higher than those in inhibitor group （both P<0.05）， and which in MDR1 knockout group were lower than those in activation group （both P<0.05）. Conclusions The MEK inhibitor PD98059 can reduce the MDR1 mRNA level in GSCs by inhibiting the MEK/ERK signaling pathway， thereby enhancing the sensitivity to chemotherapeutic drugs such as doxorubicin and vincristine.

Key words: Glioma, Neoplastic stem cells, Drug resistance， neoplasm, MAP kinase signaling system