Application of AHNAK in inhibiting generation of drug resistance of EGFR-TKI drug in HCC

A technology of EGFR-TKI and drug resistance, which is applied in the field of hepatocellular carcinoma, can solve the problems of EGFR-TKI not fully clarified, curative effect deterioration, tumor cell secondary mutation, etc., achieve good liver cancer treatment effect and reduce drug resistance effect

Pending Publication Date: 2022-07-22
BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] EGFR-TKI drug resistance is divided into primary drug resistance and acquired drug resistance. Primary drug resistance refers to the lack of benefit after EGFR-TKI treatment, and secondary drug resistance refers to the efficacy of EGFR-TKI treatment. (tumor remission, delayed progression, improvement of symptoms, etc.) and then worsened, the mechanism of acquired resistance to EGFR TKI is still not fully clear
Existing studies have shown that acquired drug resistance in about 50% of patients after effective EGFR TKI treatment may be due to secondary mutations in tumor cells

Method used

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  • Application of AHNAK in inhibiting generation of drug resistance of EGFR-TKI drug in HCC
  • Application of AHNAK in inhibiting generation of drug resistance of EGFR-TKI drug in HCC
  • Application of AHNAK in inhibiting generation of drug resistance of EGFR-TKI drug in HCC

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Expression analysis of AHNAK protein in the progression from liver disease to liver cancer and liver cancer tissue

[0024] Liver tissues, paracancerous tissues and cancer tissues of healthy people, chronic hepatitis, and early stage of liver cancer were obtained from samples stored in our laboratory.

[0025] Western blot (WB): cells or tissues were treated with lysate (RIPA) to extract total protein, then electrophoresed on 10% SDS-PAGE, transferred to PVDF membrane (Amersham Biosciences), and blocked with 5% (w / v) BSA at room temperature After 1 hour, the membranes were incubated with primary antibodies against various proteins at 4°C overnight. After washing with PBST, the membranes were incubated with HRP-conjugated secondary antibodies at 37°C for 1 hour. Chemiluminescence reagent ECL (GE) was used for detection.

[0026] Immunohistochemical detection: Tissue specimens from patients with liver cancer were sectioned at 4um thickness and embedded in paraf...

Embodiment 2

[0028] Example 2 Specific knockout of AHNAK protein can inhibit the proliferation of HepG2 cells

[0029] The AHNAK gene was specifically knocked out by lentiviral vector, and the stable replication HepG2 cell line (HepG2 AHNAK-KO cells), detect the activity of CCK8 cells for 5 days and draw a growth curve ( figure 2 ), it was found that the proliferation ability of HepG2 cells with knockout of AHNKA protein was reduced, which directly proved that AHNAK protein has the function of promoting the occurrence and development of HCC, and laid a foundation for further research on its role and mechanism in the progression of HCC.

Embodiment 3

[0030] Example 3 Discovery of AHNAK-related signaling pathways

[0031] Co-immunoprecipitation combined with protein spectroscopy (coIP-MS): The paracancerous tissues and liver cancer tissues (n=6) of 3 patients were collected, an appropriate amount of reducing Co-IP RIPA lysate was added according to the mass, the samples were ground, and the total tissue protein was extracted. The AHNAK antibody and protienA / G beats were specifically bound to the AHNAK protein complex. After boiling and denaturation, they were separated by 10% SDS-PAGE coagulation electrophoresis. After completion, Coomassie brilliant blue and silver nitrate reagents were used for coagulation and staining. The clear protein bands were cut from the gel according to the protein size, placed in a 1.5mL centrifuge tube, and subjected to mass spectrometry analysis after decolorization, reduction, alkylation, enzymatic hydrolysis, extraction and desalting.

[0032] Chromatographic separation before mass spectromet...

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Abstract

The invention discloses an application of AHNAK in inhibiting generation of drug resistance of EGFR-TKI drugs in HCC. It is found that the AHNAK protein is a key substance for activating the drug resistance of HCC in EGFR-TKI drugs, and the drug resistance of HCC can be reduced by blocking the expression of the AHNAK protein in vivo, so that a better liver cancer treatment effect is achieved. The invention further researches the biochemical mechanism of the AHNAK protein participating in the activation of the IGF-1R pathway, and provides a theoretical basis for further screening of drugs for resisting HCC drug resistance.

Description

technical field [0001] The invention belongs to the technical field of hepatocellular carcinoma, and in particular relates to the application of AHNAK to inhibit the drug resistance of EGFR-TKI drugs in HCC. Background technique [0002] Hepatocellular carcinoma (HCC), a primary liver cancer with high mortality, is the third most common cause of cancer-related mortality worldwide, with genetic, epigenetic alterations, chronic hepatitis B, hepatitis C virus infection, Aflatoxin exposure, smoking, obesity, and diabetes are major risk factors for liver cancer. Cancer cells lined up to form solid clumps surrounded by dilated sinusoids. Cancer cells have unclear boundaries, uniform size, and wide pulp. The nucleus is single, round, and the atypia is not obvious. [0003] EGFR-TKI resistance can be divided into primary resistance and acquired resistance. Primary resistance refers to no benefit after EGFR-TKI treatment, and secondary resistance refers to EGFR-TKI treatment with ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/82C12N15/12C12N15/867C12N5/10
CPCC07K14/82C12N15/86C12N5/067C12N5/0693C12N2740/15043C12N2510/00C12N2800/107
Inventor 李康张永宏
Owner BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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