Application of Leflunomide in Inhibiting Prolactinoma

Abstract

The invention discloses the application of leflunomide in inhibiting prolactinoma, and belongs to the technical field of medical application. The leflunomide is used for treating prolactinoma. The research results of the invention show that leflunomide has a good inhibitory effect on prolactinoma through programmed cell death mediation; in addition, the combined use of cabergoline and leflunomide effectively enhances the inhibitory effect of cabergoline on prolactinoma cells, which once again proves that leflunomide has a potential therapeutic effect in drug-resistant prolactinoma.

Description

Technical Field

[0001] The invention belongs to the technical field of medical application, and particularly relates to the application of leflunomide in inhibiting prolactinoma. Background Art

[0002] Prolactinoma is the most common functional pituitary adenoma in clinical practice, accounting for 40-45% of functional pituitary adenomas in adults. In severe cases, it can cause headaches, visual dysfunction, pituitary dysfunction and hyperprolactinemia. In clinical treatment, dopamine agonists are a commonly used drug for the treatment of prolactinoma, among which cabergoline (CAB) has a better therapeutic effect and lower side effects than other products. Currently, among prolactinoma patients undergoing surgery, the overall epidemiology of bromocriptine (BRC) resistance is 20-30%, and cabergoline resistance is about 10%.

[0003] Leflunomide is a dihydrogenase (DHODH) inhibitor, which is routinely used in the clinical treatment of rheumatoid arthritis. In recent years, studies have found that leflunomide and its active metabolites have anti-tumor effects by inhibiting the proliferation of neuroblastoma and multiple myeloma cells and inducing cell apoptosis. Therefore, studying new uses of leflunomide is of great clinical significance. Summary of the invention

[0004] In view of this, the object of the present invention is to provide the use of leflunomide in inhibiting prolactinoma, so as to provide a new clinical use of leflunomide.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides application of leflunomide in inhibiting prolactinoma. The leflunomide is used for inhibiting the growth of prolactinoma.

[0007] Furthermore, the combined administration of leflunomide and cabergoline synergistically enhances the inhibitory effect of cabergoline on prolactinoma growth.

[0008] The beneficial effects of the present invention are:

[0009] 1. The present invention uses pituitary tumor cell lines to observe the tumor inhibitory effects of cabergoline, leflunomide and drug combinations in CCK-8 inhibition experiments and xenograft experiments, explores differentially expressed genes through functional and pathway analysis, analyzes PPI and TF gene networks to study gene interactions, and the results show that leflunomide has a good inhibitory effect on prolactinoma mediated by programmed cell death.

[0010] 2. The research results of the present invention show that the combined use of cabergoline and leflunomide can further effectively enhance the inhibitory effect of cabergoline on prolactinoma cells, indicating that leflunomide has a potential therapeutic effect in inhibiting prolactinoma.

[0011] Other advantages, objectives and features of the present invention will be described in the following description and will be apparent to those skilled in the art to some extent, or those skilled in the art may be taught from the practice of the present invention. The objectives and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to make the purpose, technical solution and beneficial effects of the invention clearer, the present invention provides the following drawings for illustration:

[0013] Figure 1 A is a graph showing the results of the inhibition of proliferation of MMQ cells by the experimental groups of different concentrations of the present invention;

[0014] Figure 1 B is a graph showing the results of the inhibition of proliferation of GH3 cells by the experimental groups of different concentrations of the present invention;

[0015] Figure 2 This is a graph showing the proliferation inhibition results of the combined group of the present invention on MMQ;

[0016] Figure 3 The diagram shows the tumors removed in different treatment groups of the present invention;

[0017] Figure 4 It is a growth curve diagram of the change of tumor volume in mice after different treatments of the present invention;

[0018] Figure 5 This is a graph showing the enrichment of DEGs in leflunomide GO of the present invention;

[0019] Figure 6 It is the KEGG pathway enrichment map of leflunomide DEGs of the present invention;

[0020] Figure 7 Heat map of DEGs related to leflunomide death in the present invention;

[0021] Figure 8 It is the PPI development diagram of leflunomide death-related DEGs of the present invention;

[0022] Fig. 9 This is a module analysis diagram of leflunomide of the present invention;

[0023] Fig.10 is a heat map of DEGs in cabergoline of the present invention;

[0024] Fig.11 GO and pathway enrichment diagram of DEGs in cabergoline of the present invention;

[0025] Fig.12 This is a module analysis diagram of the PPI network of the present invention;

[0026] Fig.13 It is a module analysis diagram of the PPI network of the present invention;

[0027] Fig.14 DEGs between the combined use of cabergoline and leflunomide of the present invention;

[0028] Fig.15 GO and pathway enrichment diagram of DEGs between the combination of cabergoline and leflunomide of the present invention. DETAILED DESCRIPTION

[0029] like Figure 1-15 As shown, the present invention provides the use of leflunomide in inhibiting prolactinoma.

[0030] In the present invention, in order to verify the therapeutic effect of leflunomide, the following experiments are included:

[0031] ①CCK-8 experiment: MMQ and GH3 cells were evenly distributed in a 96-well plate, with 5×10 3 or 1×10 4 cells, and different concentrations of reagents; Cabergoline and leflunomide were purchased from MedChemExpress and dissolved in DMSO; Cell viability in each well was assessed by measuring the absorbance at 450 nm after 0, 24, 48, and 72 h of culture; Each experiment was performed three times.

[0032] ② Cell line experiment: MMQ (CRL-10609, ATCC) and GH3 (CCL-82.1, ATCC) rat prolactinoma cell lines were cultured in complete medium based on Gibco's Ham's F-12K medium, containing 2.5% fetal bovine serum, 15% horse serum and 1% penicillin / streptomycin; the cells were cultured at a temperature of 37°C and an environment containing 5% CO2.

[0033] ③ Xenotransplantation experiment: BALB / cA-nu mice were purchased from HFK Bioscience Co., Ltd., Beijing, China, and were raised under specific pathogen-free (SPF) conditions. Several MMQ cells (4×10 6 ) was suspended in a solution containing 50% Matrigel, and then 100 μl of the solution was injected into the flank of the mouse. Then, the tumor-bearing animals were randomly divided into 4 groups (9 mice / group), and cabergoline or leflunomide was taken 9 days after inoculation and continued for 3 weeks until the mice were killed. The mice were weighed and their tumor volumes were measured, and the tumor volume was calculated as V (mm 3 )=[AB 2] / 2 (A represents the length of the tumor and B represents the width of the tumor). All animal experiments were performed in accordance with the ARRIVE guidelines and in compliance with protocols approved by the Institutional Animal Care and Ethics Committee. This study was approved by the Ethics Committee of the Army Medical University (AMUWEC20210074).

[0034] ④ Transcriptome experiment: Total RNA was extracted, and the ND-1000 Nanodrop spectrophotometer was used to evaluate the quantity and quality of total RNA; the integrity of RNA was evaluated using Agilent 2200 TapeStation, and the RINe value of each sample was higher than 7.0; rRNA was then extracted and then decomposed into fragments of approximately 200 base pairs in length. RNA was first converted into cDNA through the first and second strand synthesis process, and then the adapters were connected and the RNA was analyzed according to Illumina's Instructions for RNA LT / HT sample preparation kit, enrichment using the low cycle method, evaluation of library projects, and subsequent sequencing using HiSeq 2500 (2 x 100 bp).

[0035] ⑤ Enrichment map and protein interaction experiment: Gene ontology, pathway enrichment and death-related DEGs identification were performed using ToppGene and Metascape websites, which were statistically significant with p values ​​lower than 0.05.

[0036] To determine the status of GO terms and pathways under specific conditions, the Z score was calculated according to the following formula, N up represents the number of overexpressed DEGs, while N down Indicates the number of under-expressed DEGs:

[0037]

[0038] To investigate drug-related PPIs in MMQ, differentially expressed genes (DEGs) were linked to the STRING online database, which has NetworkAnalyst and Metascape built in. Modules within the PPI network were identified using InfoMap and MCODE algorithms, and the results were displayed using Cytoscape.

[0039] Statistical analysis: Data are expressed as mean ± SEM, and two-tailed Student's t test was used to determine the statistical significance between the two groups. The above analysis was performed using SPSS for Windows version 13.0.

[0040] ⑥ Differentially expressed gene selection experiment: Cufflinks (v1.0.3) was used to evaluate the transcription level of each gene. The genetic data used in this experimental analysis were from the UCSC database for reference genome annotation. The data were normalized to exon fragments per kilobase per million mapped reads by adjusting for changes in sequencing depth. Next, genes with low or no expression were filtered out, and DEGs were identified between the experimental and control groups using the edgeR R package. Genes with fold change (FC) > 2 and p value < 0.05 between the experimental and control groups were classified as DEGs.

[0041] In the present invention, different sample groups are set, and the specific group information is as follows:

[0042] ① Experimental groups with different concentrations: 2 μmol / L leflunomide, 4 μmol / L leflunomide and 10 μmol / L leflunomide;

[0043] ②Control group: set 0 μmol / L leflunomide.

[0044] ③Combination group: 2μmol / L leflunomide and 25μmol / L cabergoline were administered together.

[0045] ④ Cabergoline group: Cabergoline was administered alone.

[0046] verify:

[0047] 1. Inhibitory effect of leflunomide on lactoma cell lines:

[0048] To study the effect of leflunomide on prolactinoma cell lines, CCK-8 experiments were performed to demonstrate that leflunomide inhibited the proliferation of GH3 (rat pituitary tumor cells that secrete growth hormone and a small amount of prolactin) and MMQ (rat pituitary tumor cells that secrete prolactin) cells, and its inhibitory effect was concentration-dependent.

[0049] The experimental group and the control group were applied to mice for treatment, and the results were as follows Figure 1 As shown in A, after treatment with different concentrations of leflunomide, compared with the control group, 2μmol / L leflunomide, 4μmol / L leflunomide and 10μmol / L leflunomide in the experimental group significantly inhibited the growth of pituitary tumor MMQ cells to varying degrees (p<0.05). This result proves that leflunomide can inhibit the growth of pituitary tumor MMQ cells.

[0050] Similarly, if Figure 1 As shown in B, 4 μmol / L leflunomide and 10 μmol / L leflunomide in the experimental group also had a time- and dose-dependent inhibitory effect on the growth of pituitary tumor GH3 cells (p<0.05). This result also proves that leflunomide can inhibit the growth of pituitary tumor GH3 cells.

[0051] 2. Study on the enhancement of the inhibitory effect of cabergoline by leflunomide

[0052] The combination group was applied to mice for treatment, according to Figure 2 The results showed that the inhibitory effect of the combination group on MMQ cells in the cell line experiment was significantly higher than that of cabergoline alone (p<0.05). This result proves that the combined use of leflunomide and cabergoline can improve the therapeutic effect of cabergoline.

[0053] Through in vivo experiments (xenograft experiments), according to Figure 3 and Figure 4 The results showed that leflunomide could inhibit the growth of MMQ cells, that is, leflunomide could effectively enhance the efficacy of cabergoline.

[0054] 3. Significantly changed DEGs in MMQ assay with leflunomide

[0055] In order to comprehensively analyze the mechanism of leflunomide inhibiting MMQ cells at the transcriptome level, the differentially expressed genes (DEGs) associated with MMQ in leflunomide were identified by transcriptome experiments. A total of 101 up-regulated genes and 17 down-regulated genes were identified with |FC|>2 and p-value<0.05, respectively, as shown in Table 1:

[0056] Table 1

[0057]

[0058]

[0059]

[0060]

[0061] Among them, cell death regulatory molecules (Mff, Txnip and Phlda3), membrane molecule Ptprv and immune-related gene Cd80 were significantly upregulated, as shown in Table 2:

[0062] Table 2

[0063] Gene Log(FC) p-value Gene Log(FC) p-value Mf 2.627 0.002 Nhp2 -3.056 0.007 Txnip 2.323 0.002 7x -2.425 0.042 Ptprv 2.224 <![CDATA[1.824×10 -17 ]]> Rmrp -1.723 <![CDATA[5.2×10 -5 ]]> Phlda3 2.081 <![CDATA[3.622×10 -29 ]]> Rplp2 -1.231 0.0003 Cd80 2.015 <![CDATA[9.068×10 -6 ]]> Akap5 -1.070 0.001

[0064] At the same time, the expression levels of the immune-related gene Nhp2, the intracellular transport-related gene Stx7, and tumor-related genes (Rmrp and Rplp2), as well as the neuronal activity associated with Akap5, were reduced in cells treated with leflunomide.

[0065] according to Figure 5It was shown that based on the gene ontology (GO) enrichment analysis of biological processes, the top ten functional items were significantly enriched in the cell death process, and the regulation of the apoptotic process and programmed cell death both had the highest z-score (z-score = 4.58, as shown in Table 3); negative regulation of cell death, apoptotic process, and programmed cell death also had high z-scores (4.24, 4, and 4); in addition, other processes also had positive trends, such as response to abiotic stimuli (z-score = 2.98), central nervous system development (z-scores = 3.58), and response to organic nitrogen compounds (z-score = 2.9).

[0066] Table 3

[0067]

[0068] like Figure 6 As shown, regarding pathway enrichment, the p53 signaling pathway with the highest z-score (z-score = 2.45) was significantly activated, and viral carcinogenesis with the second highest z-score (2.23) showed an active trend. In addition, the PI3K-Akt signaling pathway, platinum resistance, lifespan regulation pathway, endocrine resistance, glucagon signaling pathway, FoxO signaling pathway and cell adhesion molecules also showed active trends (z-score>1).

[0069] like Figure 7 As shown, to investigate cell death caused by related biological processes in leflunomide RPL, 26 cell death-related genes were identified by GO enrichment between the experimental and control groups. After leflunomide treatment, only Hk2 decreased in MMQ, while the remaining 25 cell death-related genes all showed an increasing trend.

[0070] 4. Hub genes based on PPI network construction

[0071] To demonstrate how leflunomide controls cell death regulation, e.g. Figure 8 As shown, DEGs were used to construct protein-protein interaction (PPI) networks and modules using the STRING database. Community members were determined based on adjusted p-value < 0.05 and modularity > 20, and according to the degree of each node, the most upregulated genes Mdm2 regulated 115 genes, Cdkn1a regulated 74 genes, Plk2 regulated 53 genes, and Ccng1 regulated 27 genes.

[0072] The InfoMap algorithm of the network module recognition module was used to study the functional relationships between differentially expressed genes (DEGs). Fig. 9 As shown, the first two modules that can be identified have more than 20 community nodes each, and the adjusted statistical p-value is lower than 0.05.

[0073] The results showed that there was an interaction relationship between the modules in MMQ and leflunomide, 8 nodes were regulated by the hub genes Cdkn1a and Ccng1, and in a module with the largest number of nodes, cell senescence and ubiquitin-mediated proteolysis were regulated by the upregulated hub gene Mdm2. In addition, the results also showed that upregulated Cdkn1a and Ccng1 regulated the cell cycle and cell senescence.

[0074] 5. Combination therapy of leflunomide and cabergoline significantly changes MMQ

[0075] To investigate the possibility of PRL tumor inhibition in the cabergoline group and the combination group, as well as the mechanistic effect of cabergoline, DEGs (differentially expressed genes) were identified between the cabergoline group and the control group and the combination group.

[0076] Fig.10 As shown in Figure 2, 268 (202 up-regulated and 66 down-regulated) DEGs were identified in the cabergoline group, and as Fig.11 As shown, these genes are involved in the positive regulation of cell death and apoptosis signaling pathways.

[0077] Furthermore, DEGs were crucial in the dysfunction of metabolic processes, including steroid biosynthesis and monocarboxylic acid metabolism. Fig.12 As shown, module analysis of the PPI network using the Metascape website revealed two major modules in the PPI network, in which MCODE 1 was related to the amino acids regulating mTORC1, and MCODE 2 was related to the assembly of collagen fiber structure.

[0078] like Fig.13 As shown, the transcription factor Sp1 was predicted to regulate 15 DEGs in cabergoline, and most of these genes played key roles in controlling apoptosis and cell cycle function.

[0079] The basic mechanism by which leflunomide enhances the anticancer properties of cabergoline was then investigated, such as Fig.13 As shown, 13 DEGs were detected between the combination group and the cabergoline group, including Rplp2, Tprkb, and hnrnpab.

[0080] Compared with the cabergoline group, eight genes were downregulated in the combination group (including Mrpl53, Cttn, Snrpg, Iars1, Mir1956, Mir25, Hnrnpc, and U5), and five genes were upregulated (LOC688583, Rplp2, Tprkb, hnrnpab, and Vps50).

[0081] Fig.15As shown, GO and pathway enrichment analysis indicated that the combined drug-specific DEGs were mainly involved in translation inhibition processes but were active in gene expression processes, such as ribonucleoprotein complex, protein-containing complex binding, and translation, especially Rplp2 and hnrnpab played an important role in translation inhibition.

[0082] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. An application of leflunomide against prolactinoma, characterized in that: The leflunomide is used to inhibit the growth of prolactinoma.

2. The use of leflunomide against prolactinoma according to claim 1, characterized in that: The combined administration of leflunomide and cabergoline synergistically enhances the inhibition of prolactinoma growth by cabergoline.

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