Application of 3,4 cleavage cyclic altine triterpenoids in the preparation of autophagy inhibitors and antitumor drugs
An antineoplastic drug, cycloaltin-type technology, applied in the field of medicine, to achieve the diversification of dosage forms and administration methods, wide clinical application prospects, and the effect of inhibiting the growth of tumor cells
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Embodiment 1
[0053] The 3,4-cleavage cyclic Altin-type triterpenoid black schisandrin inhibits the ubiquitination of the autophagy receptor p62.
[0054] (1) Detection of p62 ubiquitination in vitro: ATP, HA-Ub, E1 ubiquitin-activating enzyme UbE1, E2 ubiquitin-conjugating enzyme UbcH5b, E3 ubiquitin ligase HECTD3 and their substrates were sequentially added to the reaction buffer. p62 (with His tag), different concentrations (5 μM, 10 μM, 20 μM) of black schisandrin were added, DMSO was added as a positive control, and ATP was not added as a negative control, and the reaction was performed at 37°C for 40 minutes. After incubation with nickel column overnight, the ubiquitination of p62 was detected by Western Blot after sufficient elution.
[0055] The result is as Figure 1A shown, 20 μM black schisandrin significantly inhibited the ubiquitination of p62 compared to the negative and positive controls.
[0056] (2) Detection of intracellular p62 ubiquitination: p62-Flag, HECTD3 or HECTD3C...
Embodiment 2
[0059] The 3,4-cleavage cyclic altine-type triterpenoid black schisandrin inhibits lapatinib-induced autophagy.
[0060] (1) MDA-MB-468 cells were seeded at a density of 8,000 cells / well in a 24-well culture plate with glass slides in advance. After the cells were completely adherent, the GFP-LC3 plasmid was transfected, and 24 hours later, lapatinib was added respectively. Nitrile (10μM), black schisandrin (20μM) and their combination (10μM lapatinib and 20μM black schisandra) were treated for 4 hours (DMSO was also used as a control group), and fixed with 4% paraformaldehyde for 1 hour , immerse the slides in PBS for 3 times, then seal the slides with DAPI-containing blocking solution, and finally observe the GFP-LC3 fluorescent spots under a fluorescence microscope, and quantitatively calculate the number of GFP-LC3 fluorescent spots.
[0061] The experimental results see Figure 2A and 2B , it can be seen from the figure: the number of fluorescent spots in the lapatinib ...
Embodiment 3
[0065] The 3,4-fragmented cyclic Altin-type triterpenoid black schisandrin inhibits cisplatin-induced autophagy.
[0066] (1) Inoculate MDA-MB-468 cells at a density of 8,000 cells / well in a 24-well culture plate pre-placed with glass slides, and transfect GFP-LC3 plasmid after the cells are completely adherent, and add black schisandrin 16 hours later. (20μM), cisplatin (5μM) and their combination (20μM black schisandrin and 5μM cisplatin) for 4 hours (DMSO was set as a control group), fixed with 4% paraformaldehyde for 1 hour, and immersed in PBS The slides were repeated three times, and then sealed with DAPI-containing blocking solution. Finally, the GFP-LC3 fluorescent spots were observed under a fluorescence microscope, and the number of GFP-LC3 fluorescent spots was quantitatively calculated.
[0067] The experimental results see Figure 3A and 3B , the results showed that the number of fluorescent spots increased significantly in the cisplatin-treated group, indicatin...
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