PI3K and MTH1 targeting drug composition and applications thereof
A composition, the technology of BKM120, applied in the field of medicine, can solve the problem that cell proliferation is not inhibited, and achieve the effects of improving drug sensitivity, enhancing therapeutic effect, and broad application prospects
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Embodiment 1
[0043] Western blot was used to detect the changes in the signaling pathways of U251, T98G, and U87 glioma cell lines under different concentrations of BKM120 monotherapy. The specific implementation plan is as follows:
[0044] 1. Press 6ⅹ10 for U251, T98G, and U87 cell lines one day in advance 5 / Dish spread in 6cm dishes, 7 dishes for each kind of cells.
[0045] 2. Prepare single-drug BKM120 at concentrations of 0.1 μM, 1 μM, 2 μM, 4 μM, 8 μM, and 10 μM.
[0046] 3. Add 5mL of the drug in step 2 to each plate 24 hours after the cells are plated, and add DMSO with a concentration of 2‰ to the control group for 24 hours.
[0047] 4. After 24 hours of drug treatment, wash the cells twice with PBS, drain, add 200 μL of cell lysate to each dish, collect the lysed cells into a 1.5mL EP tube with a scraper, and then lyse at 4°C for half an hour, 13000rpm Centrifuge for 15 min to take the supernatant and store at -80°C.
[0048] 5. Use the BCA method to measure the protein cont...
Embodiment 2
[0052] The colony formation experiment was used to detect the long-term growth of various glioma cell lines under the single drug action of BKM120. The specific implementation plan is as follows:
[0053] 1. Eight glioma cell lines U251, LN18, SNB19, U118, LN229, U87, A172, T98G were planted in a 12-well plate at an amount of 800 cells per well.
[0054] 2. Prepare BKM120 at concentrations of 500nM, 750nM and 1μM, and set up a group of blank controls with DMSO at a concentration of 2‰.
[0055] 3. After 48 hours, wait for the cells in step 1 to adhere to the wall, add 2 mL of the drug prepared in step 2 to each well, change the dressing every 3 days, and withdraw the drug for 3 days after continuous culture for 14 days.
[0056] 4. Remove the medium 3 days after drug withdrawal, wash twice with PBS, wash once with deionized water, fix with 4% paraformaldehyde for 15 minutes, wash once with deionized water, stain with 1× Giemsa stain in the dark for 30 minutes, wash with deioni...
Embodiment 3
[0059] Alamar blue was used to detect the cell proliferation of BKM120, TH588 single drug and two drugs in combination, and the SI value was calculated. The specific implementation plan is as follows:
[0060] 1. The day before, U251 and SNB19 were seeded in 96-well plates at 1000 cells per well; LN229, A72, LN18, U118MG, and T98G were planted in 96-well plates at 2000 cells per well. 3 replicate wells, and set up a control group.
[0061] 2. Prepare single-drug BKM120 100nM, BKM120 1μM, TH588 10μM, TH588 2μM; double-drug BKM120 100nM+TH588 10μM, BKM120 100nM+TH588 2μM, BKM120 1μM+TH588 10μM, 1KM28+TH.
[0062] 3. After the cells adhere to the wall in step 1, the old medium is discarded, and then 100 μL of the mixture of complete medium and 10 μL of Lamar Blue is added to each well. After incubation in a cell culture incubator with 5% carbon dioxide at 37°C for 4 hours, use a chemiluminescence instrument to Under the conditions of excitation light wavelength 534nm and emissio...
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