Use of Kaempferoside in the Preparation of Anti-non-small Cell Lung Cancer Drugs
A technology for non-small cell lung cancer and kaempferol, which is used in antitumor drugs, drug combinations, pharmaceutical formulations, etc.
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Embodiment 1
[0056] Example 1 Determined that AurkC is highly expressed in NSCLC drug-resistant cell lines by Western Blot experiment
[0057] experiment method:
[0058] HCC827 and HCC827GR cells in the logarithmic growth phase were digested with 0.25% trypsin to form a cell suspension, counted, and seeded 3×10 in a 10 cm culture dish. 6 cells, 37°C, 5% CO 2 Cultivate in the incubator for 24h. When the cell density reaches 80%, discard the clean medium, add 300μL RIPA to lyse the cells, collect the lysed cells into a 1.5mL EP tube, sonicate three times, 15 seconds each time, centrifuge at 12000rpm for 10 minutes, transfer the supernatant to a new tube In EP tube. Western Blot detection of related molecules.
[0059] results and analysis:
[0060] Depend on figure 1 It can be seen that MET, Aurk and p-H3(Ser10) are all highly expressed in HCC827GR cells. Among the three Aurk proteins, the expression of AurkC changes most significantly, which is consistent with the change trend of p-H...
Embodiment 2
[0061] Example 2 Determination of AurkC as the target of kaempferol by Kinase Profiler experiment
[0062] experiment method:
[0063] (1) Add 10 mM kaempferin, 47 kinds of kinases and kinase substrates respectively with Mosquito protein crystallization screening workstation and mix well. After adding 10 mM ATP to each well with an automatic dispenser, incubate at room temperature for 2 hours. (2) After the reaction is finished, filter the reaction solution with a filter membrane, and the rest is a solution containing unconsumed ATP. (3) Use a Biomek instrument to read the value of the remaining ATP. (4) The off-board data was analyzed with IDBS Activity Base software.
[0064] test results:
[0065] target Activity(%) target Activity(%) Abl(h) 79 ICK(h) 96 ALK(h) 90 IGF-1R(h) 114 A-Raf(h) 98 IKKα(h) 97 ATM(h) 99 JAK2(h) 90 ATR / ATRIP(h) 105 JNK2α2(h) 101 Aurora-A(h) 72 Lck(h) 82 Aurora-B(h) 56 LIMK...
Embodiment 3
[0067] Embodiment 3 Kaempferoside can combine with AurkC kinase and inhibit its enzymatic activity
[0068] 1. Through drug binding experiments, it was determined that kaempferrin can bind to AurkC
[0069] experiment method:
[0070] (1) Accurately weigh 0.1 g of CNBr Beads into a cryovial, add 1 mL of Activating Buffer, 800 μL of Coupling Buffer and kaempferol at a final concentration of 10 mM, and invert overnight at 4°C. (2) The next day, take out the kaempferiside-Beads conjugate that was turned overnight at 4°C, add 1mL of Coupling Buffer for centrifugation, add 1mL of Blocking Buffer, and turn over overnight at 4°C. (3) On the third day, take out the kaempferiside-Beads conjugate that was turned overnight at 4°C, add 500 μL 1×PBS to resuspend the kaempferiside-Beads conjugate, and store at 4°C for use. (4) Add the harvested HCC827GR cell protein to the Kaempferoside-Beads conjugate, add 1mL Reaction Buffer 1, and invert overnight at 4°C. (5) On the fourth day, take o...
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