Application of enantiomer-kaurenoid diterpene kamebacetal A in preparation of medicine for treating lung cancer
A kind of kaurane diterpenoid, the technology of therapeutic effect, is applied in the field of biomedicine
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Embodiment 1
[0029] Example 1: Kamebacetal A can concentration-dependently inhibit the proliferation of A549 and H1299 cells
[0030] 1.1) MTT method to detect cell proliferation rate
[0031] Take the A549 and H1299 cells in the logarithmic growth phase, and use 6×10 4 Cells / mL were inoculated in a 96-well plate, 100 μL of cell suspension was added to each well, and three parallels were set up in each group. Place at 37°C, 5% CO 2 After 24 hours of adherent culture in the cell culture incubator, add 100 μL of complete medium containing different concentrations of kamebacetal A or the positive control drug paclitaxel, continue to culture for 24 hours, add 20 μL of MTT (5 mg / mL) to each well, and discard the supernatant after incubation for 4 hours , add 150 μL of DMSO, shake for 10 min, detect the OD value of each well at 490 nm with a microplate reader and calculate the cell viability.
[0032] 1.2) Experimental results
[0033] MTT assay found that with the increase of drug concentra...
Embodiment 2
[0034] Example 2: Low concentration of kamebacetal A produces G on A549 and H1299 cells 2 / M phase arrest without causing apoptosis
[0035] 2.1) The effect of kamebacetal A on A549 and H1299 cell cycle detected by flow cytometry
[0036] Take the A549 and H1299 cells in the logarithmic growth phase, and use 6×10 4 Cells / mL were inoculated in a 60mm culture dish. After culturing for 24 hours, different concentrations of kamebacetal A were added to continue culturing for 24 hours. The original medium was discarded and washed three times with PBS (pH7.4). The cells were collected and used Cells were resuspended in pre-cooled PBS (pH 7.4), added with pre-cooled absolute ethanol (final concentration: 70%), and fixed overnight at 4°C. Cells were collected by centrifugation at 1200 rpm, washed with pre-cooled PBS, then added RNaseA (final concentration 50 μg / mL) in a water bath at 37°C for 30 minutes, then added PI (final concentration 50 μg / mL) and incubated at 4°C in the dark fo...
Embodiment 3
[0046] Example 3: Kamebacetal A promotes the assembly and stability of microtubules in A549 and H1299 cells
[0047] 3.1) The effect of kamebacetal A on the microtubule network assembly of A549 and H1299 cells was detected by indirect immunofluorescence technique
[0048] The A549 and H1299 cells in the logarithmic growth phase were taken, and the inoculation and drug addition were the same as described in "2.1)". After culturing the cells for 24 h, add complete medium containing different concentrations of drugs to continue culturing for 24 h, wash with PBS (pH7.4) three times, fix with 4% formaldehyde for 15 min, and then add PBS containing 0.3% Triton X-100 for 30 min to permeate. Add the primary antibody (α-tubulin antibody) and incubate at 37°C for 1 h in a wet box; wash 3 times with PBS (pH7.4), add the secondary antibody and incubate at 37°C in the dark for 1 h in a wet box; then wash with PBS (pH7.4) for 1 h; pH7.4) washed 3 times, mounted (glycerol: PBS = 1: 1), obse...
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