Molecular chaperone function regulator
A technology of molecular chaperones and regulators, which can be used in organic active ingredients, endocrine system diseases, medical preparations containing active ingredients, etc.
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Embodiment 1
[0223] Example 1 Relationship between induction of HSP70 and time when using A-431 cells derived from human epithelial carcinoma
[0224] (Methods) A-431 cells were cultured on a 12-well plate, and when the cell proliferation reached about 70%-80%, compound A and the control drug gefitinib were added to 10 μM, respectively.
[0225] DMSO (0.1%) was added to the control group. After addition, cells were harvested 6, 9, 12 and 24 hours later, and cell extracts were prepared. The expression of HSP70 protein in the cell extract was detected by using HSP70 specific antibody by Western blot method. T / C (%) was calculated by taking the amount of chemiluminescence of the HRP-labeled secondary antibody on the PVDF membrane as the expression level of HSP70, and taking the expression level of HSP70 in the cells in the control well as 100.
[0226] The result is shown in Figure 1.
[0227] (Results) In A-431 cells treated with compound A (-○-), the expression of HSP70 increased over ti...
Embodiment 2
[0229] Example 2 Induction of HSP70 in various human tumor-derived cells
[0230] (Method) Regarding induction of HSP70 by compound A, induction in various human tumor cells was examined.
[0231] TE-8 cells, NCI-H520 cells, HPAC cells, DU145 cells, KPL-4 cells, MKN-45 cells, HL-60 cells and DLD-1 cells were respectively cultured in 12-well plates.
[0232] When the cell proliferation reached about 70-80%, the compound A and the control drug gefitinib were added to 10 μM respectively. DMSO was added to the control group (0.1%). After the addition, the cells were collected 9 hours later, and a cell extract was prepared. The expression of HSP70 protein in each tumor cell extract was detected by Western blot method using HSP70 specific antibody.
[0233] The result is shown in Figure 2.
[0234] (Results) The expression of HSP70 was increased in any of the human tumor-derived cells treated with Compound A for 9 hours, compared to the DMSO-treated control group. In contrast, ...
Embodiment 3
[0236] Example 3 Inhibition of HSP90 Guest Protein Expression
[0237] (Methods) The effect of compound A on the expression of HSP90 chaperone guest proteins EGFR, HER2, ERα, AR, AKT and CDK4 was detected.
[0238] MCF-7 cells were cultured in a culture dish (10 cm in diameter), and when the cell proliferation reached about 70-80%, compound A and the control drug gefitinib were added to 10 μM respectively. Geldanamycin (1 μM) was added to the positive control group, and DMSO (0.1%) was added to the negative control group.
[0239] Cells were harvested 24 hours after addition of the compounds and divided into two. Cell extracts were prepared from half of the cells, and cytoplasmic and nuclear fractions were prepared from the remaining half of the cells. Cytoplasmic fractions and nuclear fractions were prepared using NER-PER (trademark) nuclear and cytoplasmic extractant (manufactured by PIERCE, #78833). Using specific antibodies to each object, the expression of the object i...
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