Application of therapeutic target to preparation of medicine for resisting cancer cell cis-platinum resistance

Abstract

The invention discloses application of a therapeutic target to preparation of a drug for resisting cancer cell cis-platinum resistance. The therapeutic target is NPR1; the NPR1 is used for inhibiting mitochondrial autophagy-dependent ferroptosis by inhibiting the ubiquitination degradation level of PARL. The invention discloses a therapeutic target NPR1 and an NPR1-MARCH8-PARL axis, expression of PARL can be down-regulated by blocking or inhibiting the NPR1 or NPR1-MARCH8-PARL axis, and a mitochondrial autophagy-dependent ferroptosis process can be recovered, so that drug resistance of gastric cancer cells is inhibited, and a new method is further provided for preparing drugs for resisting cisplatin drug resistance of the gastric cancer cells.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a therapeutic target in preparing a cisplatin-resistant drug for cancer cells. Background Art

[0002] Currently, non-surgical treatments for gastric cancer mainly focus on radiotherapy and chemotherapy, and cisplatin-based chemotherapy is the recommended treatment for patients with advanced gastric cancer. Although chemotherapy is effective, resistance to chemotherapy is a key factor affecting the prognosis of gastric cancer. For example, with the continuous use of cisplatin, cancer cells gradually develop resistance to cisplatin, which greatly reduces its anti-cancer effect and requires the use of other drugs.

[0003] However, constantly changing drugs will lead to obvious drug reactions and increased side effects in patients, and the cost of treatment will increase significantly in the later stages. Therefore, how to alleviate the resistance of cancer cells to cisplatin or inhibit the resistance of cancer cells to cisplatin has become a problem that needs to be solved by existing technologies. Therefore, exploring the potential mechanism of chemotherapy resistance is of great significance for finding new treatment strategies for adjuvant chemotherapy. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a therapeutic target in the preparation of an anti-cancer cell cisplatin resistance drug, so as to solve the technical problem that the cisplatin drug resistance of cancer cells is difficult to inhibit in the prior art.

[0005] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

[0006] The present invention provides an application of a therapeutic target in the preparation of a cisplatin-resistant drug for cancer cells, wherein the therapeutic target is NPR1;

[0007] NPR1 inhibits mitophagy-dependent ferroptosis by suppressing the ubiquitination and degradation of PARL.

[0008] As a preferred embodiment of the present invention, the NPR1 competitively binds to MARCH8 to affect the ubiquitin degradation process of PARL, thereby upregulating the expression of PARL.

[0009] As a preferred embodiment of the present invention, the cancer cells are gastric cancer cells.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The present invention discloses a therapeutic target NPR1, which is highly expressed in drug-resistant gastric cancer cells. It inhibits the ubiquitination degradation level of PARL to inhibit mitochondrial autophagy-dependent ferroptosis, thereby leading to chemotherapy resistance of gastric cancer cells. The present invention provides a solution and new ideas for preparing drugs against cisplatin resistance of gastric cancer cells by inhibiting NPR1 or inhibiting the NPR1-PARL pathway.

[0012] The present invention further discloses the NPR1-MARCH8-PARL axis, which can downregulate the expression of PARL and restore the mitochondrial autophagy-dependent ferroptosis process by blocking or inhibiting the NPR1-MARCH8-PARL axis, thereby inhibiting the drug resistance of gastric cancer cells, and further providing a new method for preparing drugs against cisplatin resistance of gastric cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0014] Figure 1 A schematic diagram of Western Blot detection of differential expression of NPR1 in cisplatin-resistant and sensitive gastric cancer cell lines is provided for Example 1 of the present invention;

[0015] Figure 2 A dendrogram of the tolerance of AGS / DDP and HGC27 / DDP cell lines to cisplatin after knocking down NPR1 is provided for Example 1 of the present invention by using CCK8 cytotoxicity assay;

[0016] Figure 3 Provided for Example 1 of the present invention, a diagram showing the tolerance of AGS / DDP and HGC27 / DDP cell lines to cisplatin after NPR1 knockdown in a clone formation experiment;

[0017] Figure 4 Provided for the present invention is a graph showing the tolerance of AGS / DDP and HGC27 / DDP cells to cisplatin after knocking down NPR1 in a mouse subcutaneous tumor formation experiment;

[0018] Figure 5 Provided for Example 2 of the present invention is a graph showing the tolerance of AGS / DDP and HGC27 / DDP cell lines to cisplatin when NPR1 is knocked down and Fer-1 is added simultaneously by a CCK8 cytotoxicity experiment;

[0019] Figure 6Provided in Example 2 of the present invention, malondialdehyde (MDA), glutathione (GSH) and Fe are detected and analyzed by an ELISA instrument 2+ Schematic diagram of ferroptosis levels in AGS / DDP and HGC27 / DDP cell lines with NPR1 knockdown;

[0020] Figure 7 Provided for Example 2 of the present invention is a graph of the expression levels of ferroptosis-related proteins in AGS / DDP and HGC27 / DDP cell lines detected by Western Blot after NPR1 knockdown;

[0021] Figure 8 Provided for Example 2 of the present invention is an immunofluorescence detection of the fluorescence signal intensity of LC3B after NPR1 knockdown in AGS / DDP and HGC27 / DDP and a schematic diagram of the co-localization of LC3B and Mitotrack;

[0022] Fig. 9 Provided for Example 2 of the present invention is a co-localization diagram of mitochondria and lysosomes after knocking down NPR1 by immunofluorescence detection;

[0023] Fig.10 Provides a statistical graph of mitochondrial membrane potential level detected by JC-1 experiment after NPR1 knockdown for Example 2 of the present invention;

[0024] Fig.11 Provide a Western Blot to detect the expression level of mitochondrial autophagy-related proteins for Example 2 of the present invention;

[0025] Fig.12 Provides a schematic diagram of the change in ROS level caused by NPR1 knockdown detected by flow cytometry for Example 2 of the present invention;

[0026] Fig.13 The present invention provides Example 2 that knocks down NPR1 and adds Mdivi-1 at the same time, and the intracellular MDA, GSH and Fe 2+ Horizontal analysis chart;

[0027] Fig.14 Provides a Western Blot diagram for detecting ferroptosis-related protein expression levels for Example 2 of the present invention;

[0028] Fig.15 Provided for Example 3 of the present invention, IP / MS was performed by overexpressing NPR1 in the cisplatin-sensitive cell line AGS, identifying proteins that interact with NPR1, and jointly analyzing the graph with the mitochondrial autophagy and ferroptosis databases in NCBI and GENECARDS;

[0029] Fig.16 Provides a Western Blot result diagram for Example 3 of the present invention;

[0030] Fig.17 Provides a CCK8 experimental result diagram for Example 3 of the present invention;

[0031] Fig.18 Provides a result diagram of clone formation experiment for Example 3 of the present invention;

[0032] Fig.19 Provides a graph of the results of a mouse subcutaneous tumor formation experiment for Example 3 of the present invention;

[0033] Fig. 20 Provided for Example 3 of the present invention is an immunofluorescence detection of LC3B fluorescence signal intensity and LC3B and Mitotrack co-localization diagram after NPR1 knockdown and PARL overexpression in AGS / DDP and HGC27 / DDP;

[0034] Fig.21 Provides a co-localization diagram of mitochondria and lysosomes after NPR1 knockdown and PARL overexpression detected by immunofluorescence for Example 3 of the present invention;

[0035] Fig. 22 Provides a diagram of mitochondrial membrane potential detected by JC-1 after NPR1 knockdown and PARL overexpression for Example 3 of the present invention;

[0036] Fig.23 Provides a Western Blot diagram of the expression level of mitochondrial autophagy-related proteins for Example 3 of the present invention;

[0037] Fig.24 Provide a flow cytometry statistical graph of intracellular ROS levels after NPR1 knockdown and PARL overexpression for Example 3 of the present invention;

[0038] Fig.25 Provide MDA, GSH and Fe for Example 3 of the present invention 2+ Level detection shows the statistical graph of ferroptosis levels after knocking down NPR1 and overexpressing PARL;

[0039] Fig.26 Provide a statistical graph of the expression level of ferroptosis-related proteins detected by Western Blot for Example 3 of the present invention;

[0040] Fig. 27 Provided for the present invention embodiment, PARL mRNA level knockdown NPR1 after RT-qPCR detection;

[0041] Fig.28 Provide a statistical graph of PARL half-life detected by Western Blot for Example 4 of the present invention;

[0042] Fig.29Provides a statistical diagram of Western Blot detection of two protein degradation pathways of CQ and MG132 for Example 4 of the present invention;

[0043] Fig.30 Provides a statistical graph of the ubiquitin signal of PARL detected in NPR1 knockdown cells after immunoprecipitation of endogenous PARL in cells transfected with HA-Ub for Example 4 of the present invention;

[0044] Fig.31 Provide a statistical graph of the prediction of the E3 ubiquitin ligase of PARL through the website for Example 5 of the present invention;

[0045] Fig.32 Provides a statistical graph of the effect of knocking down MARCH8 and NEDD4L on PARL expression detected by Western Blot for Example 5 of the present invention;

[0046] Fig.33 Provides a statistical graph of the correlation between MARCH8, NEDD4L and PARL detected by co-IP for Example 5 of the present invention;

[0047] Fig.34 Provides a statistical graph for co-IP detection of transfected NPR1 and PARL overexpression transcription for Example 5 of the present invention. DETAILED DESCRIPTION

[0048] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] The present invention provides a therapeutic target NPR1, which plays an important role in cisplatin resistance of gastric cancer, and proposes the application of the therapeutic target in preparing cisplatin resistance drugs for anticancer cells.

[0050] The relationship between NPR1 and cisplatin resistance in gastric cancer is verified by the following examples.

[0051] Example 1: NPR1 is highly expressed in gastric cancer cells

[0052] (1) Clinical samples were collected from January 2023 to December 2023 in the Department of Gastrointestinal Surgery, Yijishan Hospital, Wannan Medical College, including gastric cancer tissues from patients resistant to cisplatin chemotherapy and gastric cancer tissues from patients sensitive to cisplatin chemotherapy. The samples were divided into two groups, and the difference in NPR1 expression between the two groups was detected by PCR and Western Blot experiments.

[0053] (2) The results were validated in gastric cancer cell lines that were resistant or sensitive to cisplatin, including AGS and AGS / DDP, HGC27 and HGC27 / DDP, to detect the difference in NPR1 expression between the groups.

[0054] (3) The NPR1 level was knocked down in cisplatin-resistant cell lines and overexpressed in cisplatin-sensitive cell lines, and the results were analyzed by clone formation assay, CCK8 cytotoxicity assay, flow cytometry apoptosis assay, and mouse subcutaneous tumor formation assay.

[0055] The characterization results are shown in Figure 1-Figure 4 .

[0056] Example 1 shows that NPR1 is highly expressed in gastric cancer cells as they develop.

[0057] The following examples further verify the role of NPR1 in affecting cisplatin resistance in gastric cancer in functional experiments.

[0058] Example 2: NPR1 affects cisplatin resistance in gastric cancer via ferroptosis pathway in vivo and in vitro

[0059] (1) The ferroptosis inhibitor Fer-1 and the ferroptosis inducer Erastin were used to inhibit and promote the ferroptosis of gastric cancer cells, respectively. NPR1 levels were knocked down and overexpressed, respectively. The effect of NPR1 on the drug resistance of gastric cancer via ferroptosis was verified by cloning experiments, CCK8 cytotoxicity assays, and flow cytometry apoptosis assays.

[0060] (2) The mitochondrial autophagy levels of gastric cancer cells were detected under conditions of knockdown and overexpression of NPR1 by LC3B-Mito and Lyso-Mito immunofluorescence experiments, JC-1 mitochondrial membrane potential experiments, flow cytometry for ROS detection, and Western Blot for detection of mitochondrial autophagy-related proteins.

[0061] (3) The mitochondrial autophagy inhibitor Mdivi-1 and the mitochondrial autophagy inducer CCCP were used to inhibit and promote the mitochondrial autophagy level of gastric cancer cells, respectively. At the same time, the NPR1 level was knocked down and overexpressed, respectively. The GSH, MDA, and Fe2+ level experiments and the ferroptosis-related proteins were detected by Western Blot to detect the ferroptosis level of cells, thereby verifying the effect of NPR1 on the ferroptosis level of gastric cancer through mitochondrial autophagy.

[0062] The characterization results are shown in Figure 5-Figure 14 .

[0063] The results showed that NPR1 could affect cisplatin resistance of gastric cancer through the ferroptosis pathway both in vivo and in vitro, leading to drug resistance of gastric cancer cells.

[0064] Example 3: Role of NPR1 in cisplatin resistance in gastric cancer via PARL

[0065] (1) Through IP / MS experiments, we identified PARL, a downstream interacting protein of NPR1. We knocked down NPR1 and simultaneously overexpressed PARL in cisplatin-resistant strains and overexpressed NPR1 and simultaneously knocked down PARL in cisplatin-sensitive strains. We then used cloning experiments, CCK8 cytotoxicity assays, flow cytometry apoptosis assays, and mouse subcutaneous tumor formation experiments to verify the role of NPR1 in affecting cisplatin resistance in gastric cancer through PARL.

[0066] (2) Through LC3B-Mito and Lyso-Mito immunofluorescence experiments, JC-1 mitochondrial membrane potential experiments, flow cytometry experiments to detect ROS, GSH, MDA, and Fe2+ levels, and Western Blot experiments to detect mitochondrial autophagy-related proteins and ferroptosis-related proteins, it was verified that NPR1 affects cisplatin resistance in gastric cancer through PARL-mediated mitochondrial autophagy-dependent ferroptosis.

[0067] (3) CO-IP and immunofluorescence colocalization analysis were used to verify whether there is interaction and colocalization between NPR1 and PARL in GC cells.

[0068] The characterization results are shown in Figure 15-Figure 26 .

[0069] Example 3 further confirmed the interaction and co-localization between NPR1 and PARL in GC cells.

[0070] Example 4: NPR1 affects the ubiquitin degradation process of PARL

[0071] The mRNA level of PARL was measured, and RT-qPCR results showed that NPR1 had no effect on the mRNA level of PARL. Therefore, it was speculated that NPR1 regulates the protein stability of PARL. The half-life of PARL was detected by CHX, and the role of two key protein degradation pathways was evaluated by CQ and MG132: proteasome and autophagy degradation pathways. The results showed that CQ had no effect on NPR1-regulated PARL expression; after MG-132 treatment, the proteasome degradation of PARL enhanced by NPR1 siRNA was blocked by MG-132. After immunoprecipitation of endogenous PARL in cells transfected with HA-Ub, a significant increase in the ubiquitin signal of PARL was detected in cells with knockdown of NPR1.

[0072] The characterization results are shown in Figure 27-30 .

[0073] Taken together, these results suggest that NPR1 upregulates PARL via the ubiquitin-proteasome pathway.

[0074] Example 5: NPR1 affects the ubiquitin degradation process of PARL by competitively binding to MARCH8

[0075] (1) The E3 ubiquitin ligase of PARL was predicted through the website, and the top three E3 ligases with the highest scores were screened out. WB and co-IP experiments verified that MARCH8 and NEDD4L were the E3 ligases of PARL.

[0076] (2) Then co-IP detection was performed by transfecting NPR1 and overexpressing PARL.

[0077] The characterization results are shown in Figure 31-34 .

[0078] The results analysis showed that NPR1 competitively bound to MARCH8, affecting the ubiquitin degradation process of PARL and thus upregulating the expression of PARL.

[0079] Natriuretic peptide receptor A (NPR1) has been recognized as an important receptor for atrial natriuretic peptide (ANP), a widely studied hormone involved in regulating cardiovascular homeostasis. The ANP-NPR1 pathway has an impact on cell proliferation, apoptosis, inflammation, and tumorigenesis.

[0080] In the above examples, by connecting the relationship between NPR1 affecting the ROS level of cells through lipid metabolism and autophagy and ferroptosis, it was confirmed that NPR1 protein independently plays an important role in cisplatin resistance of gastric cancer cells.

[0081] Mechanistically, the present invention verifies that NPR1 is highly expressed in cisplatin-resistant gastric cancer cells, and that it can affect the level of mitochondrial autophagy and ferroptosis by upregulating the expression of PARL. More in-depth, NPR1 not only inhibits mitochondrial autophagy-dependent ferroptosis by inhibiting the ubiquitination degradation level of PARL, and the reduction of ferroptosis level is one of the key mechanisms for tumor cells to acquire chemotherapy resistance, but also NPR1 affects the ubiquitination degradation process of PARL by competitively binding to MARCH8, thereby upregulating the expression of PARL, which ultimately leads to the chemotherapy resistance of gastric cancer cells.

[0082] The present invention discloses therapeutic targets NPR1 and NPR1-MARCH8-PARL axis. By blocking or inhibiting NPR1 or NPR1-MARCH8-PARL axis, the expression of PARL can be downregulated, and the mitochondrial autophagy-dependent ferroptosis process can be restored, thereby inhibiting the drug resistance of gastric cancer cells, and further providing a new method for preparing a drug against cisplatin resistance of gastric cancer cells.

[0083] The above embodiments are only exemplary embodiments of the present application and are not intended to limit the present application. The protection scope of the present application is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present application within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present application.

Claims

1. Application of a therapeutic target in the preparation of a cisplatin-resistant drug for cancer cells, characterized in that: The therapeutic target is NPR1; NPR1 inhibits mitophagy-dependent ferroptosis by suppressing the ubiquitination and degradation of PARL.

2. The use of a therapeutic target according to claim 1 in the preparation of a cisplatin-resistant drug for anticancer cells, characterized in that: The NPR1 competitively binds to MARCH8, affecting the ubiquitin degradation process of PARL, thereby upregulating the expression of PARL.

3. The use of a therapeutic target according to claim 1 in the preparation of a cisplatin-resistant drug for anticancer cells, characterized in that: The cancer cells are gastric cancer cells.

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