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Application of isoliensinine in the preparation of drugs targeted to inhibit akt activation

A technology of isoliensinine and medicine, applied in the application field of medicine, can solve problems such as pharmacokinetics, cell tolerance and limited curative effect, and achieve the effect of broadening medical use

Active Publication Date: 2021-06-29
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 the preparation of drugs targeted to inhibit akt activation
  • Application of isoliensinine in the preparation of drugs targeted to inhibit akt activation
  • Application of isoliensinine in the preparation of drugs targeted to inhibit akt activation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0034] Cervical cancer SiHa, C33A, CaSki and HeLa cells were taken for routine culture respectively. When the cells grew to the logarithmic phase, the cells were collected, digested with trypsin, neutralized with complete medium and adjusted the concentration of the cell suspension to 1×10 5 cells / mL, and then the cell suspension was planted into 96-well culture plates, and 180 μL was added to each well, and then the concentration of 0 μM, 5 μM, 10 μM, 20 μM, 30 μM and 40 μM was added to SiHa, C33A, CaSki and HeLa cells respectively. Liensinine (ISO) was treated for 24 hours. The Western blot method was used to detect the expression of pAKT (Ser 473 phosphorylated) protein in cervical cancer cells. The specific method is as follows:

[0035] (1) Assemble the electrophoresis tank, check for leaks, configure different separating gels according to needs, add stacking gel after soli...

Embodiment 2

[0041] Embodiment 2, the inhibitory effect of isoliensinine on the viability of cervical cancer cells

[0042] According to the method of embodiment 1, process cervical cancer SiHa, C33A, CaSki and HeLa cells with isoliensinine (ISO), use CCK8 method to measure cervical cancer cell viability, specific method is as follows:

[0043] (1) Take 5,000-10,000 cells and inoculate them in a 96-well plate at 37°C, 5% CO 2 to cultivate;

[0044] (2) Add 0 μM, 5 μM, 10 μM, 15 μM, 20 μM and 25 μM isoliensinine after the cells adhere to the wall, and add an equal volume of DMSO to the blank as a control, and culture for 24 hours and 48 hours.

[0045] (3) Abandon the medium, add premixed CCK8 (10%) medium at 37°C, 5% CO 2 Incubate for 1-2 hours.

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

[0047] Test results such as image 3 As shown, with the increase of isoliensinine concentration or treatment time, the viability of cervical cancer SiHa, C33A, CaSki and HeLa cells gradual...

Embodiment 3

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

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

[0050]According to the above method, cervical cancer SiHa, C33A, CaSki and HeLa cells were treated with isoliensinine (ISO) at concentrations of 0 μM, 5 μM, 10 μM, 20 μM, 30 μM and 40 μM, and the cell cycle was measured by flow cytometry. The specific steps were as follows:

[0051] a. Take 1×10 cells 6 Cells / mL were seeded in a 6-well plate, and 0 μM, 5 μM, 10 μM, 20 μM, 30 μM and 40 μM of isoliensinine were added after the wall adhered, and an equal volume of DMSO was added to the blank as a control, 37 ° C, 5% CO 2 Cultivate for 24h.

[0052] b. Centrifuge at 2000 rpm for 5 minutes, collect the cells in EP tubes, add 300 μL of pre-cooled PBS to resuspend the cells and add 700 μL of absolute ethanol to mix, and fix overnight at 4°C. After ethanol fix...

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Abstract

The invention discloses the application of isoliensinine in the preparation of drugs for targeting and inhibiting AKT activation. It is found through research that after using isoliensinine to treat cervical cancer cells, AKT activation is significantly inhibited, and isoliensinine inhibits AKT activation pathway and up-regulates the expression of p21. , while down-regulating the expression of Cyclin E1 and CDK2, the cell cycle of cervical cancer cells is arrested in the G1 phase, and finally the proliferation and migration of cervical cancer cells are inhibited, and apoptosis is induced; Liensinine provides a new medical application, and also provides a new drug candidate for the treatment of cervical cancer, which has important clinical significance.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of isoliensinine in the preparation of drugs targeting to inhibit AKT activation. Background technique [0002] AKT, also known as protein kinase B (protein kinase B, PKB), its excessive activation in cells will lead to continuous flow of cell matrix through the glycolysis 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] As a target of oncogenic activation in various tumors, AKT plays a key role in tumor cell proliferation, migration, autophagy and apoptosis. Therefore, effective control of AKT activation is crucial for drug therapy and prognosis of cancer patients. [0004] At present, a variety of anti-tumor AKT inhibitors have been successfully developed in the market, but these inhibitors have problems of pharmacokine...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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