Use of cathepsin e in the preparation of a medicament for treating or preventing lung cancer

Abstract

The application discloses application of cathepsin E in preparation of a drug for treating or preventing lung cancer, relates to the technical field of biological medicine, and first proves that CTSE is highly expressed in lung adenocarcinoma tissues and has the biological function of inhibiting lung cancer cell proliferation through clinical sample analysis, cell function experiment and animal experiment. Mechanism research shows that CTSE plays the anticancer role by down-regulating the expression of cathepsin B (CTSB) and promoting the degradation of STING protein through a lysosome pathway and inhibiting the cGAS-STING natural immune pathway. The application clarifies the application value of CTSE as a new target for treating lung adenocarcinoma, provides a theoretical basis and experimental basis for developing a novel lung cancer treatment drug, and has an important clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of cathepsin E in the preparation of drugs for the treatment or prevention of lung cancer. Background Technology

[0002] Lung cancer is one of the malignant tumors with the highest incidence and mortality rates in my country and even globally. Lung adenocarcinoma accounts for more than 40% of the pathological types of lung cancer. Its pathogenesis is complex, its prognosis is poor, and its 5-year survival rate is less than 20%. Currently, lung cancer treatment methods include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy, but problems such as drug resistance, serious adverse reactions, and low treatment efficacy still exist. There is an urgent need to explore new therapeutic targets and intervention strategies.

[0003] The cathepsin family is a class of proteases primarily located in lysosomes, participating in various physiological and pathological processes. Cathepsin E (CTSE), a member of the aspartic protease family, has its progenitor mainly located in the endoplasmic reticulum and Golgi apparatus, while its mature enzyme is primarily located in endosomes. It has been reported to participate in antigen processing and the maturation of secretory proteins. However, the function and mechanism of CTSE in the development and progression of lung cancer remain unclear, and its application as a therapeutic target for lung cancer has not been reported.

[0004] Therefore, in-depth research on the expression characteristics and biological functions of CTSE in lung adenocarcinoma, and the development of novel drugs for the treatment or prevention of lung cancer based on this, provides an important theoretical foundation and experimental basis for improving the precision treatment system for lung cancer. Summary of the Invention

[0005] This invention discloses the application of cathepsin E in the preparation of drugs for the treatment or prevention of lung cancer. The aim is to inhibit the proliferation of lung cancer cells and improve the tumor immune microenvironment by upregulating the expression or activity of CTSE and utilizing its endogenous regulatory mechanism on cathepsin B and the cGAS STING pathway.

[0006] The technical solution provided by this invention is as follows: In a first aspect, the present invention discloses the application of cathepsin E as a target in the preparation of drugs for the treatment or prevention of lung cancer.

[0007] Furthermore, the lung cancer mentioned is lung adenocarcinoma.

[0008] Furthermore, the drug inhibits lung cancer cell proliferation by upregulating the expression or activity of CTSE.

[0009] Furthermore, the drug also contains a pharmaceutically acceptable carrier or excipient.

[0010] Furthermore, the dosage forms of the drug include injections, tablets, capsules, and aerosols.

[0011] In summary, compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention clarifies the application value of CTSE as a new target for the treatment of lung adenocarcinoma, and provides a theoretical basis for the development of new lung cancer treatment drugs; (2) This invention reveals the molecular mechanism by which CTSE exerts its anti-cancer effect by regulating CTSB expression and the cGAS-STING pathway, providing a clear pathway for drug development; (3) This invention confirms that targeted upregulation of CTSE expression or activity can effectively inhibit the proliferation of lung cancer cells and regulate the tumor immune microenvironment, providing a new strategy for targeted therapy and immunotherapy of lung cancer. Attached Figure Description

[0012] Figure 1 The images show CTSE immunohistochemical staining, with cancer cells on the left and adjacent normal cells on the right. Figure 2 Western blot analysis was performed to detect CTSE protein gel images in different cell lines. Figure 3 A bar chart showing the statistical analysis of CTSE expression levels in different cell lines using qPCR. Figure 4 A bar chart showing the statistical analysis of CTSE and CTSB expression levels in the H1299 knockdown cell line using qPCR validation. Figure 5 Western blot analysis of protein gel images to verify CTSE in H1299 knockdown cell lines; Figure 6 A bar chart showing the statistical analysis of CTSE and CTSB expression levels in the H1299 overexpressing cell line using qPCR validation. Figure 7 Western blot image of CTSE in H1299 overexpressing cell line for validation. Figure 8 A bar chart showing the statistical analysis of CTSE and CTSB expression levels in the A549 knockdown cell line using qPCR validation. Figure 9 Western blot validation of CTSE in A549 knockdown cell lines; Figure 10 A bar chart showing the statistical analysis of CTSE and CTSB expression levels in A549 overexpressing cell lines using qPCR validation. Figure 11 Western blot validation of CTSE in A549 overexpressing cell lines; Figure 12 Line graph showing the proliferation of H1299 knockdown cell lines detected by CCK8 assay; Figure 13A diagram illustrating the clonogenic assay for detecting H1299 knockdown cell lines using CCK8. Figure 14 Line graph showing the proliferation of H1299 overexpressing cell lines detected by CCK8 assay; Figure 15 A diagram of a CCK8 assay for detecting colony formation in H1299-overexpressing cell lines; Figure 16 Line graph showing the proliferation of A549 knockdown cell lines detected by CCK8; Figure 17 A diagram of a CCK8 assay for detecting clonogenesis in A549 knockdown cell lines; Figure 18 Line graph showing the proliferation of A549 overexpressing cell lines detected by CCK8; Figure 19 A diagram of a CCK8 assay for detecting colony formation in A549-overexpressing cell lines; Figure 20 Animal experiments involving the seeding of CTSE-knockdown H1299 cells onto nude mouse skin, and statistical diagrams of tumor volume and mass. Figure 21 The image shows a gel image of STING protein expression at different CHX addition time points, comparing CTSE-overexpressing A549 and H1299 overexpressing cell lines with the corresponding control group. Figure 22 This is a WB gel image to verify the expression of STING protein in A549-overexpressing cell lines and H1299-overexpressing cell lines that overexpress CTSE and their corresponding control groups after the addition of different drugs. Figure 23 This is a diagram showing the colocalization of STING and LC3 in the A549 CTSE overexpression group and the control group; Figure 24 This is a diagram showing the colocalization of STING and LC3 in the H1299 CTSE overexpression group and the control group; Figure 25 This is a diagram showing the colocalization of STING and P62 in the A549 CTSE overexpression group and the control group; Figure 26 This is a diagram showing the colocalization of STING and P62 in the H1299 CTSE overexpression group and the control group. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and various embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0014] Unless otherwise stated, the terms or phrases used herein should not be considered uncertain or unclear unless specifically defined, and should be understood in their ordinary sense. When trade names appear in this document, they are intended to refer to the corresponding product or its active ingredient.

[0015] Unless otherwise stated, all reagents and raw materials used in this invention are commercially available.

[0016] Example 1 This embodiment detects the expression of CTSE in lung adenocarcinoma tissues and cell lines.

[0017] 1. Clinical sample collection: Tumor tissue and paired adjacent normal tissue were collected from 89 patients with pathologically confirmed lung adenocarcinoma. None of the patients had received radiotherapy or chemotherapy before surgery.

[0018] 2. Immunohistochemical detection: After fixation, dehydration, embedding, and sectioning, tissue specimens undergo CTSE immunohistochemical staining, such as... Figure 1 The results showed that CTSE expression in tumor tissues was significantly higher than that in adjacent normal tissues (P < 0.0001), and analysis revealed that high expression was positively correlated with tumor pathological grade but not with prognosis.

[0019] 3. Cell line detection: Normal lung epithelial cells (HBE) and lung adenocarcinoma cell lines (H1299, A549, PC9, H1650, H1975) were cultured. Total protein and RNA were extracted and analyzed by Western blotting and qPCR, respectively. Figure 2 and Figure 3 The results showed that the protein and mRNA expression levels of CTSE in lung adenocarcinoma cell lines were significantly higher than those in normal lung epithelial cells.

[0020] Example 2 This embodiment experimentally investigated the regulatory function of CTSE in lung cancer cell proliferation.

[0021] 1. Cell line construction: H1299 and A549 cell lines with stable CTSE knockdown and overexpression were constructed. The knockdown and overexpression efficiencies were verified by Western blotting and qPCR. The results are as follows: Figures 4-7As shown, in the H1299 knockdown cell line, the mRNA level of CTSE was decreased, while CTSB showed no significant change, and the protein level of CTSE was significantly decreased. In the H1299 overexpression cell lines, the H1299CTSE-OE cell line showed a significant increase in CTSE mRNA expression and a significant decrease in CTSB expression, while the H1299CTSE-OE cell line showed a significant increase in CTSE protein. Figures 8-11 As shown, the mRNA levels of both CTSE and CTSB were decreased in the A549 knockdown cell line, and the CTSE protein level in the knockdown cell line was reduced to a certain extent. In the A549 overexpression cell line, the mRNA level of CTSE was significantly increased in the A549 CTSE-OE cell line, while the expression of CTSB did not change significantly. The protein level of CTSE in the A549 CTSE-OE cell line was significantly increased.

[0022] 2. Cell proliferation assay: The CCK-8 assay was used to detect cell proliferation capacity, such as... Figures 12-19 The results showed that the cell proliferation rate in the CTSE knockdown group was significantly higher than that in the control group, while the cell proliferation rate in the CTSE overexpression group was significantly lower than that in the control group; consistent results were obtained in the clonogenic assay.

[0023] 3. Animal experiments: CTSE-knockdown H1299 cells were subcutaneously seeded into nude mice, and tumor volume was measured weekly. Figure 20 The results showed that the tumor growth rate in the CTSE knockdown group was significantly faster than that in the control group.

[0024] Example 3 Mechanism of CTSE regulation of the cGAS-STING pathway 1. To investigate the specific molecular mechanism by which CTSE affects cGAS-STING expression, STING was selected as the subject of further research. To study whether CTSE affects STING degradation, cycloheximide (CHX), an inhibitor of eukaryotic protein synthesis, was added to CTSE-overexpressing cells and control cells, respectively. The degradation of STING at different time points under the inhibited protein synthesis conditions was measured. As shown in Figure 21, STING expression was lower in A549-CTSE-OE than in A549-Vector, and lower in H1299-CTSE-OE than in H1299-Vector. In A549-CTSE-OE, STING content increased at 0 h, 3 h, and 6 h after CHX addition, and STING degradation was significant after 12 h. In contrast, in the A549-Vector group, STING concentration did not change significantly. The STING concentration in the H1299-CTSE-OE and H1299-Vector groups showed a significant gradient change over time, with the STING content gradually decreasing. Furthermore, the H1299-CTSE-OE group exhibited a faster degradation rate than the H1299-Vector group.

[0025] 2. To investigate the mechanism by which CTSE affects STING degradation, this study used ubiquitination inhibitor MG132, autophagy inhibitor Baf-A1, and chloroquine (CQ) to investigate the degradation of STING in CTSE-overexpressing tumor cells and CTSE-controlled groups. Figure 22 As shown, the expression level of STING in both A549-CTSE-OE and H1299-CTSE-OE was lower than that in the corresponding Vector group. The addition of MG132 could not inhibit the degradation of STING. The expression level of STING in the A549-CTSE-OE and H1299-CTSE-OE groups was significantly increased after the addition of Baf-A1 and CQ.

[0026] 3. To further investigate the reasons why Baf-A1 and CQ inhibit STING degradation in the CTSE overexpression group, the co-localization of STING protein with P62 and LC3 in the CTSE overexpression group and the control group was observed. The results are as follows: Figures 23-26 As shown, no obvious co-localization of STING and LC3 was observed in the A549 and H1299 cell lines before and after overexpression. However, after overexpression of CTSE in both cell lines, STING and P62 showed co-localization compared to the control group.

[0027] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. Application of cathepsin E as a target in the preparation of drugs for the treatment or prevention of lung cancer.

2. The application as described in claim 1, characterized in that, The lung cancer mentioned is lung adenocarcinoma.

3. The application as described in claim 1, characterized in that, The drug inhibits lung cancer cell proliferation by upregulating the expression or activity of CTSE.

4. The application as described in claim 1, characterized in that, The drug also contains pharmaceutically acceptable carriers or excipients.

5. The application as described in claim 1, characterized in that, The dosage forms of the drug include injections, tablets, capsules, and aerosols.

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