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Application of isoliensinine in preparation of drugs for targeted inhibition of AKT activation

A technology of isoliensinine and medicine, which can be applied in the directions of drug combination, anti-tumor drug, pharmaceutical formula, etc., can solve the problems of pharmacokinetics, cell tolerance and limited curative effect, etc., and achieve the effect of broadening the use of medicine

Active Publication Date: 2019-06-21
长沙市中心医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, a variety of anti-tumor AKT inhibitors have been successfully developed in the market, but these inhibitors have problems of pharmacokinetics, cell tolerance and limited efficacy

Method used

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  • Application of isoliensinine in preparation of drugs for targeted inhibition of AKT activation
  • Application of isoliensinine in preparation of drugs for targeted inhibition of AKT activation
  • Application of isoliensinine in preparation of drugs for targeted inhibition of AKT activation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. The inhibitory effect of isoliensinine on the activation of cervical cancer AKT (pAKT)

[0034] Take cervical cancer SiHa, C33A, CaSki and HeLa cells for routine culture. When the cells grow to the logarithmic phase, collect the cells, digest with trypsin, neutralize the reaction with complete medium and adjust the cell suspension concentration to 1×10 5 Cells / mL, and then plant the cell suspension into a 96-well culture plate, add 180μL to each well, and then add different concentrations of 0μM, 5μM, 10μM, 20μM, 30μM and 40μM to SiHa, C33A, CaSki and HeLa cells. Liensinine (ISO) treatment for 24h. Westernblot method is used to detect the expression of pAKT (Ser 473 phosphorylation) protein in cervical cancer cells. The specific method is as follows:

[0035] (1) Assemble the electrophoresis tank, check for leaks, configure different separation gels as needed, add concentrated gels after solidification, and solidify for 30 minutes.

[0036] (2) Add the electrophore...

Embodiment 2

[0041] Example 2. The inhibitory effect of isoliensinine on the vitality of cervical cancer cells

[0042] According to the method in Example 1, isoliensinine (ISO) was used to treat cervical cancer SiHa, C33A, CaSki and HeLa cells, and the CCK8 method was used to measure the viability of cervical cancer cells. The specific method is as follows:

[0043] (1) Inoculate 5000~10000 cells in a 96-well plate, 37℃, 5% CO 2 to cultivate;

[0044] (2) Add 0μM, 5μM, 10μM, 15μM, 20μM and 25μM isoliensinine and blank plus equal volume of DMSO as controls after cell attachment, and culture for 24h and 48h.

[0045] (3) Discard the medium and add pre-mixed CCK8 (10%) medium at 37°C, 5% CO 2 Incubate for 1 to 2h.

[0046] (4) OD value at 450nm of the microplate reader.

[0047] Test results such as image 3 As shown, as the treatment concentration of isoliensinine increases or the treatment time is prolonged, the viability of cervical cancer SiHa, C33A, CaSki and HeLa cells gradually decreases.

Embodiment 3

[0048] Example 3. The inhibitory effect of isoliensinine on the proliferation of cervical cancer cells

[0049] (1) The inhibitory effect of isoliensinine on the proliferation of cervical cancer SiHa, C33A, CaSki and HeLa cells

[0050] Use isoliensinine (ISO) with concentrations of 0 μM, 5 μM, 10 μM, 20 μM, 30 μM and 40 μM to treat cervical cancer SiHa, C33A, CaSki and HeLa cells according to the above method, and flow cytometry to measure the cell cycle. The specific steps are as follows:

[0051] a. Take cell 1×10 6 Cells / mL were seeded in a 6-well plate. After attachment, 0μM, 5μM, 10μM, 20μM, 30μM and 40μM isoliensinine and blank plus equal volume of DMSO were added as a control, 37℃, 5% CO 2 Cultivate for 24h.

[0052] b. Centrifuge at 2000 rpm for 5 min, collect the cells in an EP tube, add 300 μL of pre-cooled PBS to resuspend the cells, then add 700 μL of absolute ethanol to mix, and fix overnight at 4°C. After ethanol fixation, centrifuge at 2000 rpm for 5 min, remove ethano...

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Abstract

The invention discloses application of isoliensinine in preparation of drugs for targeted inhibition of AKT activation. Studies show that the AKT activation is obviously inhibited after cervical cancer cells are treated by the isoliensinine; the isoliensinine inhibits AKT activation passages, up-regulates p21 expression, and down-regulates expression of cyclin E1 and cyclin-dependent kinase CDK2,so that the cervical cancer cells are arrested in the G1 phase, the proliferation and migration of the cervical cancer cells are inhibited, and apoptosis is induced. A new medial use of the isoliensinine is provided, a new alternate drug is provided for treatment of cervical cancer, and the application is of great clinical significance.

Description

Technical field [0001] The present invention relates to the field of biomedicine, in particular to the application of isoliensinine in preparing drugs for targeted inhibition of AKT activation. Background technique [0002] AKT is also known as protein kinase B (PKB), and its over-activation in the cell will cause the cell matrix to flow continuously through the glycolytic pathway, thereby causing changes in cell biological behavior. There are two pathways for its activation, one is the phosphorylation pathway (pAKT), and the other is the methylation pathway. [0003] AKT, as a target of oncogenic activation of many tumors, has a key impact on tumor cell proliferation, migration, autophagy and apoptosis. Therefore, the effective control of AKT activation is very important for the drug treatment and prognosis of cancer patients. [0004] At present, a variety of anti-tumor AKT inhibitors have been successfully developed on the market, but these inhibitors have problems with pharmaco...

Claims

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Application Information

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IPC IPC(8): A61K31/4725A61P35/00A61P35/04
Inventor 高利臣李红丽文丹丹程艳胡锦跃李杰平
Owner 长沙市中心医院
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