Use of an nr2f2 agonist in the manufacture of a medicament for treating renal clear cell carcinoma in a female

Abstract

The application provides a use of an NR2F2 agonist levomilnacipran in preparation of a drug for treating female renal clear cell carcinoma (ccRCC). In an estrogen environment, the levomilnacipran can significantly promote expression of NR2F2, and significantly inhibit proliferation, invasion and migration of ccRCC cells. The levomilnacipran can be used as a novel therapeutic drug for female ccRCC, and can specifically inhibit progression of the female ccRCC, and has important significance for disease prognosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of an NR2F2 agonist in the preparation of a drug for treating clear cell renal cell carcinoma in women. Background Technology

[0002] Kidney cancer is one of the most common malignant tumors. Clear cell renal cell carcinoma (ccRCC) is the most common pathological subtype of kidney cancer, accounting for about 70%-80% of kidney cancers.

[0003] NR2F2 (nuclear receptor subfamily 2 group F member 2) belongs to the orphan nuclear receptor family. Furthermore, NR2F2 is one of the most evolutionarily conserved nuclear receptors across all species, exhibiting extremely high homology between its DNA-binding and ligand-binding domains. The ligand-binding domain of NR2F2 is essentially identical across different species, including humans, indicating its importance to life processes. Our preliminary studies revealed that the function of NR2F2 in ccRCCs is related to estrogen and androgen environments and its receptor: in estrogen environments, overexpression of NR2F2 significantly inhibits ccRCC cell proliferation, invasion, and migration, while in androgen environments, overexpression of NR2F2 significantly promotes ccRCC cell proliferation, invasion, and migration. In other words, NR2F2 acts as a double-edged sword in different hormonal environments.

[0004] Therefore, there is an urgent need to develop a drug that can effectively alter NR2F2 abnormalities for the treatment of clear cell renal cell carcinoma in women. Summary of the Invention

[0005] To overcome the problems existing in the prior art, the present invention provides the following technical solution:

[0006] Use of an NR2F2 agonist in the preparation of a drug for treating clear cell renal cell carcinoma in women;

[0007] Furthermore, the therapeutic effect is to inhibit the proliferation, invasion, or migration ability of renal cell carcinoma cells;

[0008] Furthermore, the NR2F2 agonist is levomirnacitabine;

[0009] Furthermore, the levamisole is an oral formulation.

[0010] Levomilnacipran, CAS number 96847-55-1, molecular formula C 15 H 22 N₂O, with a molecular weight of 246.35, has the structural formula shown in Formula I:

[0011]

[0012] Levomilnacipran is the fourth 5-HT / NE reuptake inhibitor (SNRI) approved by the FDA. It was approved in 2009 for the treatment of fibromyalgia. On July 26, 2013, the FDA approved the novel antidepressant levomilnacipran for the treatment of major-depressive disorder (MDD) in adults, marketed under the brand name Fetzima. It is available as extended-release capsules in four strengths (20, 40, 80, and 120 mg). Attached Figure Description

[0013] Figure 1 The expression levels of NR2F2 in ccRCC in the TCGA and CPTAC databases are shown in Figure 1A. NR2F2 expression in unpaired samples in the TCGA database is shown in Figure 1B. NR2F2 expression in the CPTAC database is shown in Figure 1C. NR2F2 expression levels and OS in ccRCC patients of different genders are shown in Figures 1C and 1D.

[0014] Figure 2 The results of the subcutaneous tumor formation experiment in male and female nude mice are shown in Figure 2A, which shows the tumor growth between different groups observed by fluorescence in vivo imaging; Figure 2B shows the weight of subcutaneous tumor samples in the female group of model mice at the study endpoint; and Figure 2C shows the weight of subcutaneous tumor samples in the male group of model mice at the study endpoint.

[0015] Figure 3 For differential gene heatmap;

[0016] Figure 4 The diagram shows the drug screening process. 4A is a simplified drug screening procedure diagram, 4B is a molecular docking diagram, and 4C is a verification diagram of Western blot (WB) experiment.

[0017] Figure 5 The results of the experiment using the Biacore biomolecular interaction analysis system coupled with fluorescence resonance technology are shown in Figure 5A, which is the SPR curve and Figure 5B, which is the affinity curve.

[0018] Figure 6 To validate levomirnacitabine at the cellular and animal levels. 6A represents the CCK-8 cell proliferation assay. Figure 6 B represents the Transwell cell migration assay. Figure 6 C represents the Transwell cell invasion assay. Figure 6 D represents the subcutaneous tumor formation experiment in nude mice. Figure 6E is a graph showing the volume and statistics of subcutaneous tumors in nude mice. Figure 6 F represents the lung transfer experiment via tail vein injection in nude mice;

[0019] Figure 7 This is a schematic diagram illustrating the inhibition of female ccRCC by levomirnacitabine.

[0020] Beneficial effects

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] In an estrogen-rich environment, levamisole can significantly promote NR2F2 expression and significantly inhibit ccRCC cell proliferation, invasion, and migration. It could serve as a novel therapeutic agent for female ccRCC, specifically inhibiting its progression and holding significant importance for disease prognosis. Detailed Implementation

[0023] The present application will now be described in further detail with reference to the embodiments. It is to be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0024] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0025] Example 1

[0026] Through TCGA transcriptomics sequencing database ( Figure 1 A) CPTAC Proteomics Database ( Figure 1 B) Analysis revealed that NR2F2 mRNA and protein expression levels in ccRCC tissues were lower than those in adjacent normal tissues. However, survival analysis showed that in male patients, NR2F2 expression levels were not significantly correlated with overall survival (OS), while in female patients, high NR2F2 expression was significantly associated with better OS. Figure 1 CD), ***P value < 0.001.

[0027] Example 2

[0028] To clarify whether there are differences in the function of NR2F2 between different sexes, we subcutaneously injected OSRC-2 cells (ccRCC cells overexpressing NR2F2 (OE-NR2F2) and normal negative control (Vector)) into nude mice of different sexes to construct a nude mouse xenograft tumor model. Figure 2 As shown, fluorescence in vivo imaging was used to observe tumor growth among different groups. Figure 2A), and at the study endpoint, subcutaneous tumor samples were collected from each group of model mice, and the tumor weight was measured. Results showed that in female nude mice, overexpression of NR2F2 significantly inhibited the proliferation of ccRCC cells. Figure 2 B); while in male nude mice, overexpression of NR2F2 significantly enhanced the proliferation of ccRCC cells (B); Figure 2 C). ***P value < 0.001. This indicates that NR2F2 agonists can significantly inhibit the proliferation of ccRCC cells in females.

[0029] Example 3

[0030] To further explore the mechanism of NR2F2 function in ccRCC and the influence of estrogen and androgen environments on its mechanism of action, we performed RNA transcriptome sequencing on OSRC-2 ccRCC cell lines overexpressing NR2F2 cultured in normal and hormone-free media. The results are as follows: Figure 3 The results showed no significant difference in downstream gene expression between the Vector control group and the OE-NR2F2 experimental group in normal culture medium. However, there was a significant difference in gene expression between Vector and OE-NR2F2 in the hormone-free culture group. Since normal cell culture environment contains both estrogen and androgen, we hypothesize that the anti-cancer effect of NR2F2 in an estrogen environment and the pro-cancer effect of NR2F2 in an androgen environment interact, causing the differences in normal culture medium to be masked. This is also consistent with... Figure 2 The results are consistent with those from animal experiments.

[0031] Example 4

[0032] The distinctly different efficacy of NR2F2 in different sexes has potential clinical value. NR2F2 inhibitors can be used in male patients, while NR2F2 agonists can be used in female patients, potentially aiding in the precise treatment of ccRCC. Given the existing literature on NR2F2 inhibitors, we conducted a high-throughput screening of NR2F2 agonists (…). Figure 4 A). High-precision molecular docking of the top 5 compounds ( Figure 4 B) and cell experiments to verify ( Figure 4C). The results indicated that amikacin, micronomicin, and levomilnacipran all significantly increased NR2F2 expression levels, with levomilnacipran showing the best NR2F2 agonist effect. However, as the drug concentration increased, the agonist effect of micronomicin on NR2F2 decreased, leading to its initial exclusion. Considering that amikacin, as an aminoglycoside, has nephrotoxicity, and our treatment target is ccRCC, we further excluded amikacin. Based on the above, we ultimately selected levomilnacipran as the effective NR2F2 agonist. Figure 4 The diagram shows the drug screening process, where 4A is a simplified drug screening procedure diagram, 4B is a molecular docking diagram, and 4C is a Western blot (WB) experimental verification diagram.

[0033] Example 5

[0034] Figure 5 The use of the Biacore biomolecular interaction analysis system coupled with fluorescence resonance (SPR) further confirmed the strong affinity between levomirnacitabine and NR2F2. Figure 5 (AB) provides direct evidence that levomirnacitabine may specifically activate NR2F2. Figure 5 A is the SPR curve. Figure 5 B represents the affinity curve.

[0035] Example 6

[0036] Figure 6 This study validated the anti-cancer function of levomirnacitabine at the cellular and animal levels. To further verify whether levomirnacitabine has anti-cancer efficacy, we cultured OSRC-2 cells with 10 μM levomirnacitabine, as shown below. Figure 6 As shown in Figure A, levamisole significantly inhibited the proliferation of renal cell carcinoma cells. Migration and invasion assays confirmed that levamisole inhibited the invasion and migration abilities of renal cell carcinoma cells. Figure 6 BC). Subsequently, we treated female nude mice subcutaneously inoculated with OSRC-2 cells with levamisole via gavage (30 mg / kg / day) and found that levamisole significantly inhibited the growth of subcutaneous tumors in female nude mice. Figure 6 DE). Tail vein metastasis experiments also found that levamisole significantly inhibited hematogenous metastasis of tumors in female nude mice. Figure 6 F).

[0037] Therefore, oral administration of levamisole can significantly promote NR2F2, inhibit the proliferation and metastasis of tumor cells, and thus inhibit the progression of ccRCC in women.

[0038] The above description is merely a preferred embodiment of this application and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the inventive concept. For example, technical solutions formed by substituting the above features with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. Use of an NR2F2 agonist, levamisole, in the preparation of a drug for treating clear cell renal cell carcinoma in women.

2. The use according to claim 1, characterized in that, The therapeutic effect is to inhibit the proliferation, invasion, or migration of renal cell carcinoma cells.

3. The use according to claim 2, characterized in that, The levamisole is an oral preparation.

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