Application of hesperetin and/or derivative thereof in preparation or serving as medicine for preventing and/or treating renal cell carcinoma

[0071] Example 1 Analysis of Cx43 expression, correlation and its relationship with the prognosis of renal cell carcinoma

[0072] Except for a few cells (such as sperm, platelets, etc.), almost all cells in mammals are connected to each other by gap junctions (GJs). Small molecules below 1-1.5kDa, such as calcium ions, IP3, cAMP, and free amino acids, can pass between cells via GJ, allowing cells to share some small molecule metabolites and regulate signal transmission between cells. Some small-molecule ribonucleic acids (miRNAs) can also be transmitted from cells to neighboring cells via GJs. Cx43 is one of the most abundantly expressed connexins in GJs formed between cells, and is related to the occurrence and development of various diseases such as ischemia-reperfusion injury, inflammation, diabetes, and tumors. However, the expression of Cx43 in renal cell carcinoma is rarely reported. On this basis, we further explored the relationship between Cx43 and renal cell carcinoma progression.

[0073] The expression and correlation of GJA1 (Cx43) and other receptor tyrosine kinases (RTKs, which are key targets for renal cancer therapy), and the relationship between Cx43 and RTKs and the prognosis of different tumors can be determined by TIMER2 (Tumor immune estimation resource, version 2) Obtained after analysis in the "Gene_DE", "Gene_Corr" or "Gene_Outcome" modules in the database (http://timer.cistrome.org/). Correlation analysis showed that GJA1 and RTKs were significantly correlated in multiple different tumors ( image 3 A), the relationship between the expression of GJA1 (Cx43) and RTKs (FLT1, KDR, FLT4, EFNB2, etc.) and the prognosis of clear cell renal cell carcinoma (KIRC) showed the same trend ( image 3 B).

[0074] The expression analysis of Cx43 in different renal clear cell carcinoma subtypes and grades was carried out by the "TCGA analysis" module of the UALCAN (http://ualcan.path.uab.edu/analysis-prot.html) database. Compared with normal tissues, Cx43 was significantly increased in KIRC, and its expression in different subtypes and tumor grades was higher than that in normal tissues ( Figure 4 A).

[0075] The protein expression analysis was performed by the CPTAC (Clinical proteomic tumor analysis consortium) unit in the UALCAN website. The results showed that the expression of Cx43 protein and phosphorylated Cx43 protein was significantly up-regulated compared with normal tissues ( Figure 4 B. Figure 4 C), the expression of Cx43 is associated with the prognosis of KIRC ( Figure 4 D).

[0076] The boxplot of the expression difference between the carcinoma and adjacent tissues of renal cancer can be found in the "Expression analysis- Select GTEx (Genotype-tissue expression) data in the "Box Plots" module, set P-value cutoff=0.01, log2FC (Fold change) cutoff=1, check "Match TCGA normal and GTEx data" to obtain, for Cx43 in KIRC The expressions of , renal papillary cell carcinoma (KIRP) and renal chromophobe cell carcinoma (KICH) and their relationship with the prognosis of KIRP and KICH were also analyzed ( Figure 4 E. Figure 4 F), the results showed that there was no significant correlation between Cx43 and KIRP and KICH.

[0077] The "Survival Analysis" module of GEPIA2 can also analyze the relationship between different RTKs (FLT1, FLT4, KDR, EFNB2, PDGFD) and KIRC's overall survival and disease progression-free survival ( Figure 4 G~ Figure 4 K), Cutoff-high (50%) and cutoff-low (50%) can be set to distinguish high expression group and low expression group; and through UALCAN's TCGAanalysis" module to analyze the relationship between Cx43 and FLT1, KDR, FLT4, EFNB2 The results showed that Cx43 was highly correlated with FLT1, KDR, FLT4, EFNB2 in KIRC ( Figure 4 L~ Figure 4 O).

[0078] Example 2 Inhibition of hesperidin on Cx43 expression and renal cancer cells

[0079] 1. Experimental method

[0080] (1) According to the method in the aforementioned "Western blot detection of protein expression", after collecting clinical tissue samples and different renal cell samples from renal cancer patients, protein extraction and sample preparation were performed, and the expression of proteins such as Cx43 and EGFR was detected by western blotting ; and use the methods in the aforementioned "clone formation assay", "detection of cell migration ability by cell scratch method", and "detection of cell invasion ability by Transwell chamber culture method" to detect the ability of cell clone formation, migration and invasion.

[0081] (2) The cells were seeded into a 96-well plate at a certain density (about 3000-5000 cells per well). After 24 hours of cell adhesion, the medium was changed and 11 kinds of Chinese medicine monomers (1-Cyclocytidine HCl, 2 -Apigenin, 3-Berberine HCl, 4-Cytisine, 5-Glycyrrhizin, 6-Icariin, 7-(+)-Matrine, 8-Neohesperidin, 9-(-)-Parthenolide, 10-Salicin, 11-Tangeretin) treatment 24 After hours, protein extraction and sample preparation were performed, and the expression of Cx43 and other proteins was detected by western blotting.

[0082] (3) According to the method in "Detecting cell viability by MTT or MTS method", 24 hours after the cells were seeded into the 96-well plate, set different groups, change the medium and add Cx43 siRNA or control to be treated differently, The absorbance of each group was measured after adding the corresponding MTS after 72 hours, and the survival rate was calculated by setting the corresponding blank control group.

[0083] (4) According to the methods in the aforementioned "Clone Formation Experiment" and "Detection of Cell Migration Ability by Cell Scratch Method", the cloning and migration ability of renal cancer cells was detected.

[0084] 2. Experimental results

[0085] (1) A comparative study on the expression of Cx43 in human kidney cells (normal cells HK-2, renal cancer cells 769-P, ACHN, Caki-1, OS-RC-2, 786-O) showed that it was significantly higher than that of human normal cells. The expression of Cx43 was higher in renal cancer cells ACHN, Caki-1, OS-RC-2 compared with renal HK-2 cells ( Figure 5 A); Inhibition of Cx43 expression in ACHN cells using siRNA ( Figure 5 B) After the proliferation of renal cancer ACHN cells ( Figure 5 C), migration ( Figure 5 D) and invasion ( Figure 5 E) The ability was significantly decreased, indicating that Cx43 expression was closely related to the proliferation, migration and invasion of renal cancer ACHN cells.

[0086] (2) In the effects of 11 kinds of Chinese medicine monomers on the expression of Cx43 in renal cancer OS-RC-2 cells ( Figure 5 F), 11-Ceretin is one of the compounds that effectively inhibit the expression of Cx43. The inhibitory effects of different concentrations (3, 10, and 30 μmol/L) of orange peretin on the expression of Cx43 in OS-RC-2 are as follows: Figure 5 As shown in G, from Figure 5 G, it can be seen that orange peretin can significantly inhibit the expression of Cx43 in renal cancer cells, and since the expression of Cx43 is closely related to the prognosis of renal cancer cells, it can be proved that orange peretin can significantly treat/prevent renal cancer.

[0087] (3) MTS detection after 24 hours of treatment with orange peretin in each cell under serum-free conditions, the results show that orange peretin can significantly inhibit OS-RC-2 ( Figure 5 H), ACHN ( Figure 5 J) and 786-O cells ( Figure 5 K) vitality. Cisperetin also significantly inhibited the migration of Caki-1 cells ( Figure 5 i).

[0088] (4) The results of clone formation ability and scratch test showed that citrus can inhibit the clone and migration ability of renal cancer cells. Orange peretin (10μmol/L, 30μmol/L) inhibited the colony formation of 786-O cells ( Image 6 A); orange peretin (10μmol/L, 30μmol/L) can inhibit the colony formation of 769P cells ( Image 6 B); orange peretin (10μmol/L, 30μmol/L) can inhibit the colony formation of ACHN cells ( Image 6 C); orange peretin (10μmol/L, 30μmol/L) can inhibit the migration of 786-O cells ( Image 6 D); orange peretin (10μmol/L, 30μmol/L) can inhibit the migration of 769-P cells ( Image 6 E).

[0089] Example 3 Inhibition of cellulite on the expression of proliferation, cloning and migration-related molecules in renal cancer cells

[0090] 1. Experimental method

[0091] According to the method in the aforementioned "Western blot detection of protein expression", different renal cancer cells were treated and administered with small interfering RNA of Cx43 and orange peretin, and the expression of orange peretin and Cx43 was examined to determine the effects of renal cancer cell proliferation, Effects of cloning, expression of molecules closely related to migration (phosphorylated EGFR, AR, MMP-9, SOD-1, CD44, phosphorylated ERK, phosphorylated Stat3, BCL-2, P65, phosphorylated AKT and Survivin).

[0092] 2. Experimental results

[0093] The expression of phosphorylated EGFR, AR, MMP-9 and SOD-1 was down-regulated after inhibition of Cx43 expression in ACHN cells with hesperidin ( Figure 7 A), and inhibited the expression of CD44 in ACHN cells ( Figure 7 B); Orange peretin can inhibit the expression of phosphorylated ERK in OS-RC-2 cells ( Figure 7 C), and after inhibiting the expression of Cx43 with hesperidin, the expression of phosphorylated EGFR, phosphorylated ERK, P65 and Survivin in renal cancer OS-RC-2 cells was reduced ( Figure 7D); Orange peretin can inhibit the expression of phosphorylated Stat3, Survivin, BCL-2 and phosphorylated AKT in 786-O cells ( Figure 7 E).

[0094] To sum up the experimental results, it can be concluded that orange peretin can inhibit the expression of Cx43 in renal cancer cells, and inhibit the proliferation, cloning, cloning, and proliferation of renal cancer cells (OS-RC-2, 769-P, 786-O, Caki-1 or ACHN). Migration can achieve a good effect on the prevention and treatment of renal cell carcinoma, providing a new option for the targeted therapy of renal cell carcinoma.