Inhibiting Pi3K/AKT Pathway Increases Effects Of Chemotherapy In The Human Glioma U251Cells In Vitro

Recent studies showed that phosphatidylinositol3-kinase(PI3K) plays an importantrole in the regulation of tumor signal transduction. Cells in effects of a series of internaland external factors were started by the PI3K/Akt signaling pathway to induce cellproliferation、differentiation、anti-apoptosis. In the process of tumor cells to escapeanti-cancer drugs, the PI3K/Akt signaling pathway may play an extremely important role.Therefore, PI3K/Akt signaling pathway is expected to become a new target for tumortreatment. There have more articles reported that lung cancer、stomach cancer、colorectalcancer and other malignancies be used of PI3K inhibitor LY294002/Wortmannin to inhibitthe PI3K/Akt pathway in domestic and foreign. Thereby enhance the effects ofchemotherapy and reduce malignance of cancer cells.This study by using chemotherapeutics cisplatin and temozolomide combined withPI3K inhibitor LY294002to treat human glioma U251cells to study chemotherapy role ofinhibiting PI3K/Akt signaling pathway in human glioma U251cells in vitro.This study from two aspects to study the PI3K/Akt pathway in human glioma U251:(1) By inhibiting of the PI3K/Akt signaling pathway,we found effects of cell proliferationand cell migration in human glioma U251.(2) By inhibiting of the PI3K/Akt signalingpathway,we found effects of cell apoptosis in human glioma U251.Part I：Inhibiting PI3K/AKT pathway in migration and proliferationof human glioma U251Objective By inhibiting of the PI3K/Akt signaling pathway, we studied effects of cellmigration and cell proliferation in human glioma U251and different concentrationsinhibitors for cell and cell migration. Methods We cultured human glioma U251cells in logarithmic phase and seededthem to6-well plates and96-well plates. This study were divided into control andtreatment groups. There were no drug in the control group. The treatment group weredivided into simply chemotherapeutics cisplatin or temozolomide group andchemotherapeutics cisplatin or temozolomide in combination with PI3K inhibitorLY294002group to inhibit the PI3K/Akt pathway. The concentration of chemotherapeuticswas remained in constant (final concentrations of DDP was2.5ug/ml. final concentrationsof TMZ was8.5ug/ml). The concentrations of LY294002was20umol/L、40umol/L inscratch test. The concentrations of LY294002was5umol/L、10umol/L、20umol/L、40umol/L in MTT assay. Scratch test was detected for changing of cell migration ability.MTT assay was measured for changing of cell proliferation.Results The scratch test results showed that cell migration ability was weakened byinhibiting of the PI3K/Akt signaling pathway. Compared with the control group, Simplychemotherapeutics group cell migration ability was weaker. Compared with simplychemotherapeutics group, Cell migration ability of chemotherapeutics combined withLY294002group was weaker. In a certain concentrations, cell migration ability wasweaker along with increase of LY294002concentrations. MTT assay results showed thatsimply chemotherapeutics group of cell proliferation of inhibition ratio better than thecontrol group. Cell proliferation of inhibition rate of chemotherapeutics combined withLY294002group better than simply chemotherapeutics group. In a certain concentrations,cell proliferation inhibition rate was increased along with increase of LY294002concentrations. As time increases, cell inhibition rate gradually increased in24h、48h、72h。Conclusion1.By inhibiting the PI3K/Akt pathway can effectively inhibit cellmigration ability of U251cells and U251cells migration.2.By inhibiting the PI3K/Aktpathway can effectively inhibit cell proliferation of U251and U251cell proliferation.3.Byinhibiting the PI3K/Akt pathway can effectively reduced migration capacity of U251cellsand inhibited proliferation of U251cells. So that by inhibiting the PI3K/Akt pathway caneffectively improve the effects of chemotherapy in the human glioma U251cells in vitro. Part II Inhibiting PI3K/AKT pathway in apoptosis of human gliomaU251Objective By inhibiting of the PI3K/Akt signaling pathway, we studied influences ofcell apoptosis in human glioma U251and different concentrations inhibitors for cellapoptosis.Methods We cultured human glioma U251cells in logarithmic phase and seededthem to6-well plates. This study were divided into control and treatment groups. Therewere no drug in the control group. The treatment group were divided into simplychemotherapeutics cisplatin or temozolomide group and chemotherapeutics cisplatin ortemozolomide in combination with PI3K inhibitor LY294002group to inhibit thePI3K/Akt pathway. The concentration of chemotherapeutics was remained in constant(final concentrations of DDP for2.5ug/ml, final concentrations of TMZ for8.5ug/ml). Theconcentrations of LY294002was20umol/L、40umol/L in flow cytometry、 situfluorescence microscopy、RT-PCR. We Used flow cytometry to detect early apoptosis rateand Situ fluorescence microscopy to observe apoptosis and RT-PCR to detect caspase-3,caspase-9mRNA expression.Results Flow cytometry results showed that early apoptosis of U251cells in controlgroup was2.1%. The early apoptosis rate of U251cells in simply chemotherapeutics groupwere15.7%;14.5%. The early apoptosis rate of U251cells in chemotherapeutics combinedwith LY294002group were18.5%,29.7%;19.6%,35.6%. Compared with the control group,the early apoptosis rate of U251cells in simply chemotherapeutics group was higher.Compared with simply chemotherapeutics group, the early apoptosis rate of U251cells inchemotherapeutics combined with LY294002group was higher. In a certain concentrations,early apoptosis rate was more higher along with increase of LY294002concentrations. Situfluorescence microscopy results showed that U251cells in early apoptotic cells were6andin necrosis cells were2in the control group. U251cells in early apoptotic cells were9and in necrosis cell was1in the simply chemotherapeutics group. U251cells in early apoptoticcells were12、16and in necrosis cells were3in the chemotherapeutics combined withLY294002group. Compared with the control group, the early apoptosis rate of U251cellsin simply chemotherapeutics group was higher. Compared with simply chemotherapeuticsgroup, the early apoptosis rate of U251cells in chemotherapeutics combined withLY294002group was higher. In a certain concentrations, U251cells in early apoptotic washigher along with increase of LY294002concentrations. RT-PCR results showed that byinhibiting the PI3K/Akt pathway, caspase-3and caspase-9mRNA expression wasincreased.Conclusion1.By inhibiting the PI3K/Akt pathway can effectively increase apoptosisrate of human glioma cells U251.2.By inhibiting the PI3K/Akt pathway can effectivelyincrease apoptosis of U251cells. So that by inhibiting the PI3K/Akt pathway caneffectively improve the effects of chemotherapy in the human glioma U251cells in vitro.