Application of shizukaol D in preparing anti-tumor target medicines
An anti-tumor and drug technology, applied in the fields of medicine and genetic engineering, can solve the problem that there is no report of shizukaolD targeting and inhibiting cell signaling pathway.
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Embodiment 1
[0093] Example 1 The signal of Wnt pathway is inhibited
[0094] Take 293T cells and plant a 24-well plate (4~6×10 per well 4 Cells). After adherence, it was treated with 50mmol / L LiCl for 12 hours, and then transferred to Topflash, Fopflash and internal reference RL plasmids. Shizukaol D and DMSO control were added the next day (that is, 0.1% DMSO was added as a control) for 24 hours, and 3 replicates were made for each sample. Receive samples and detect luciferase activity. Take XAV939 as a positive control. XAV939 is a tankryase enzyme inhibitor that can indirectly inhibit the Wnt pathway (Huang SM, Mishina YM, Liu S, et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling[J].Nature,2009,461(7264):614-20. ). XAV-939 selectively inhibits Wnt / β-catenin-mediated transcription by inhibiting tankyrase1 / 2, IC 50 It is 11nM / 4nM, which regulates the level of axin, but has no effect on CRE, NF-κB and TGF-β.
[0095] Such as figure 1 As shown, shizukaol D at 6.25 a...
Embodiment 2
[0096] Example 2 Verification of differential genes in Wnt pathway
[0097] After SMMC-7721 cells adhere to the wall, shizukaol D is added, and samples are collected at 0, 6 and 12 hours respectively. RNA is extracted by Trizol method, and genomic RNA is removed. After reverse transcription, real-time PCR is used to detect gene expression in the samples. Use GAPDH as an internal reference. Real-time Quantity PCR (Q-PCR) was used to detect the difference in the number of amplification cycles (-ΔΔCt) of related genes in the Wnt pathway to determine the change in the expression of the gene.
[0098] Such as figure 2 As shown, the expression levels of c-Myc, cyclin D, Tcf-1, LEF1, wnt3a and FGF18 were all decreased in the samples with shizukaol D added for 12 hours compared with the samples without shizukaol D. QPCR results showed that shizukaol D down-regulated the expression of c-myc and other genes. This may be because shizukaol D can inhibit the expression of β-catenin, thereby ...
Embodiment 3
[0100] Example 3 The addition of wnt3a partially offsets the inhibitory effect of shizukaol D on the growth of liver cancer cells
[0101] Adding wnt3a is a classic way to activate the Wnt pathway. The medium of L-wnt3a cells contains sufficient wnt3a, which usually activates the Wnt pathway of the cells. Take SMMC-7721 cells, add medium containing wnt3a after adherence, and incubate overnight. After adding 6.25 and 12.50μmol / L shizukaol D, after 48 hours of treatment, use CCK-8 kit to detect cell viability. Take 0.1% DMSO as control.
[0102] The result is image 3 As shown, adding medium containing wnt3a can significantly improve the survival rate of SMMC-7721 cells under the action of shizukaol D. Regardless of whether 6.25 or 12.50μmol / L shizukaol D is added, once wnt3a is added to the medium, the viability of SMMC-7721 cells can be restored to the same level, which shows that adding wnt3a to the medium can offset the difference in compound concentration between the two grou...
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