Polypeptide composition DFBP capable of inhibiting ovarian cancer and protecting ovary and application of polypeptide composition DFBP

Abstract

The invention discloses a polypeptide composition DFBP for inhibiting ovarian cancer and protecting ovary and application thereof. The polypeptide composition comprises oDFBP polypeptide as shown in SEQ ID NO. 1 and nDFBP polypeptide as shown in SEQ ID NO. 2. As the DFBP is used for specifically inhibiting the BIN2-T423Aamp, the DFBP is used for inhibiting the Phosphorylation of S424 is carried out, so that the CIBB does not influence phosphorylation of other sites of BIN2, other post-translational modifications and related physiological functions; as a polypeptide drug, compared with an inhibitor, DFBP has the remarkable advantages of high specificity, high efficiency, high safety, low immunogenicity, high membrane permeability, low cost and the like; the DFBP can be combined with various tumor treatment schemes, so that the dual purposes of inhibiting the ovarian cancer process and protecting the ovarian function are achieved.

Description

Technical Field

[0001] The present invention belongs to the field of ovarian medical treatment, and specifically relates to a polypeptide composition capable of inhibiting the progression of ovarian cancer and protecting the ovary and an application thereof. Background Art

[0002] Malignant tumors or cancer are increasingly threatening people's health, and the radiotherapy and chemotherapy commonly used in clinical tumor treatment can significantly damage female ovarian function and even lead to complete infertility. For young female patients with ovarian cancer (OC), the ideal treatment should achieve a dual purpose: inhibit tumor progression and protect ovarian function, because young female patients with ovarian cancer still have fertility needs. We previously found that activated (phosphorylated) BIN2 in mouse ovaries regulates primordial follicle activation and oocyte quality through p-RPS6. In this study, we found that inhibiting BIN2 phosphorylation by knocking out Bin2 or using DFBP can inhibit the occurrence and development of OC. However, in human female OC tissues, although we observed a significant increase in p-BIN2, we did not see an increase in p-RPS6. Therefore, based on this difference between normal ovarian and OC tissues, we speculate that p-BIN2 has other more important targets in OC tissues. Through mass spectrometry identification, we found that only the constitutively activated forms of BIN2 (T423D and S424D) hooked HDAC1, indicating that HDAC1 is a more critical target of BIN2 in OC tissues. Next, we did see that Bin2 knockout or inhibition significantly reduced p-HDAC1 (S421) while increasing H3K27ac. In addition, chip-seq showed that BIN2 inhibition significantly increased the binding of H3K27ac to multiple tumor suppressor genes. In addition, in mice with in situ OC or ectopic OC invasion induced by chemical carcinogens, BIN2 knockout or inhibition can protect ovarian function. This study shows that inhibiting BIN2 can both inhibit the occurrence and development of OC and protect ovarian function. Therefore, for women with OC, it can achieve the dual purpose of inhibiting the progression of ovarian cancer and protecting the ovaries, thus providing a more reasonable and ideal innovative solution for female patients with OC relative to radiotherapy and chemotherapy. Summary of the invention

[0003] Technical problem to be solved: The present invention provides a polypeptide composition DFBP that can inhibit ovarian cancer and protect ovaries and its application. The polypeptide composition can inhibit the progression of ovarian cancer and protect ovarian function. This "dual function" is very important for women of childbearing age with ovarian cancer to maintain their fertility.

[0004] Technical solution: A polypeptide composition DFBP, comprising an oDFBP polypeptide shown in SEQ ID NO.1 and an nDFBP polypeptide shown in SEQ ID NO.2.

[0005] Preferably, the molar ratio of the above-mentioned oDFBP polypeptide to the nDFBP polypeptide is 1:1 to 10:1.

[0006] A gene encoding the oDFBP polypeptide.

[0007] A gene encoding the nDFBP polypeptide.

[0008] The polypeptide composition DFBP is used in the preparation of a drug for inhibiting the progression of ovarian cancer while protecting ovarian function.

[0009] A drug for inhibiting the progression of ovarian cancer and protecting ovarian function, comprising the above-mentioned polypeptide composition DFBP.

[0010] The method for preparing the drug for inhibiting the progression of ovarian cancer and protecting ovarian function is prepared by mixing the polypeptide composition DFBP with a pharmaceutically acceptable carrier or excipient.

[0011] Women with ovarian cancer were given DFBP orally, 2 mg / kg, once a day, for four weeks. Then serum was taken to measure tumor markers (CRP, HE4, CD31, RB1, etc.) to determine whether the progression of ovarian cancer was suppressed, and six hormones (FSH, LH, E2, P, T, PRL) and NNT (nicotinamide nucleotide transhydrogenase) were measured to determine ovarian function.

[0012] Beneficial effects: 1. Since DFBP specifically inhibits the phosphorylation of BIN2-T423A&S424, CIBB does not affect the phosphorylation of other sites of BIN2 and other post-translational modifications and related physiological functions;

[0013] 2. As a polypeptide drug, DFBP has significant advantages over inhibitors, such as high specificity, high efficiency, high safety, low immunogenicity, high membrane permeability and low cost;

[0014] 3. DFBP can be used in combination with a variety of tumor treatment regimens, thereby achieving the "dual purpose" of inhibiting the progression of ovarian cancer and protecting ovarian function. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 .Bin2 knockout inhibits chemical carcinogen-induced ovarian tumorigenesis;

[0016] A and B. We used the chemical inducer DMBA plus NMU to induce tumorigenesis in situ in the ovary. White light imaging and quantification showed that under chemical carcinogen treatment, the area of ​​white ovarian tissue transformed into cancer in the ovaries of the WT group was significantly larger than that in Bin2-KO female mice; HE staining of paraffin sections showed that the white tumor-like tissue had lost the ovarian structural characteristics. C and D. Immunofluorescence and quantification of vascular marker CD31 showed that under chemical carcinogen treatment, the intensity of vascular marker CD31 in the ovaries of the WT group increased significantly, while the intensity of CD31 in the Bin2-KO group was similar to that of the untreated control. E and F. Immunofluorescence and quantification of proliferation marker Ki67 showed that under chemical carcinogen treatment, the intensity of proliferation marker Ki67 in the ovaries of the WT group increased significantly, while the intensity of Ki67 in the Bin2-KO group was similar to that of the untreated group. GJ.Western blot and quantification showed that under chemical carcinogen treatment, common tumor markers including CD31, RB1 and HE4 were significantly increased (HE4, CD31) or decreased (RB1) in the ovaries of the WT group, while the levels in the Bin2-KO ovaries were similar to those of the untreated ovaries. Scale bars: 2 mm in the upper row of A and 100 μm in the lower row; 100 μm in C and E. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ***: P < 0.0001.

[0017] Figure 2 .Both BIN2 and p-BIN2 are upregulated in ovarian tumors;

[0018] A. Analysis of BIN2 expression data from the GEPIA database showed that BIN2 mRNA levels in ovarian cancer (OC) tissues were higher than those in normal ovaries. OC group n = 426, normal ovarian tissue n = 88. B. Kaplan-Meier correlation analysis showed that OC patients with high levels of BIN2 had a much lower progression-free survival (PFS) in grade I and II OC (GSE14764, n = 293). CE. We collected data from 32 OC patients, and Western blot and quantification showed that BIN2 and p-BIN2 were significantly upregulated in OC tissues in 60% of these patients. FI. Western blot and quantification showed that the levels of BIN2 (F and G) and p-BIN2 (H and I) in OC cells (A2780 cells) were significantly higher than those in normal ovarian epithelial cells (Moody cells). J and K. Immunohistochemistry on human OC microarray showed that p-BIN2 levels were significantly upregulated in high-grade serous ovarian carcinoma (HGSOC), ovarian endometrioid carcinoma (OEC), and ovarian clear cell carcinoma (CCOC) compared with normal ovaries. Scale bar, J, 200 μm. *: P < 0.05; **: P < 0.01; ***: P < 0.0001.

[0019] Figure 3 .Inhibiting BIN2 phosphorylation prevents OC progression;

[0020] A. DFBP is a mixture of omnipresent BPP (oBPP) and nucleus-targeting BPP (nBPP) designed to improve its potency in inhibiting BIN2 phosphorylation. BF. DFBP treatment significantly reduced colony formation (B and C), cell proliferation (D), and cell migration (E and F) in A2780 OC cells. GI. PI staining showed that iBPP treatment significantly increased the apoptosis level of A2780 OC cells. J and K. DFBP treatment significantly reduced the mitochondrial membrane potential of A2780 OC cells. L and M. DFBP treatment significantly increased the ROS level of A2780 OC cells. NQ. DFBP treatment significantly reduced the percentage of tumor formation (N and O) and tumor volume (P and Q) in A2780-injected immunodeficient mice. Scale bar, 20 μm. **: P < 0.01; ***: P < 0.001; ***: P < 0.0001.

[0021] Figure 4 .BIN2 directly binds and phosphorylates HDAC1;

[0022] A. Blot and quantification showed no clear correlation between p-BIN2 and p-RPS6 in clinical OC samples (n=31). B. Immunoprecipitation with Strep II antibody in A2780 cells transfected with BIN2-WT, S423A&T424A inactive, and S423D&T424D constitutively active mutants and LC-MS showed that only the active mutants induced HDAC1; but none of the BIN2 plasmids pulled down RPS6. C. Bin2-DD-Strep II and HDAC1-FLAG constructs (in pcDNA3.1+) were co-transfected into A2780 cells. Co-IP with Strep II (lower panel) and FLAG (upper panel) showed that BIN2-DD interacted with HDAC1. D and E. Blot and quantification showed that p-HDAC1 levels in OC were much higher than in ovaries, while p-RPS6 levels in tumors were much lower than in ovaries. Thus, the ratio of p-HDAC1 to p-RPS6 in OC was more than sixfold higher than that in ovary.FI.Blot and quantification showed that DFBP inhibition of BIN2 in A2780 cells significantly reduced p-HDAC1(S421)(FH), but did not affect the level of p-HDAC2(S424)(F and I).J.SDS-PAGE and Coomassie staining showed good protein purity of BIN2-WT and -AA mutant (inactive forms, S423A and T424A) purified from Sf9 cells.KM. In vitro phosphorylation assay and quantification with proteins from J showed that BIN2-WT increased p-HDAC1 in a dose-dependent manner, while BIN2-AA mutant was much less active for HDAC1 phosphorylation.N.SDS-PAGE and Coomassie staining showed good protein purity of BIN2-DUF (S423 & T424 phosphorylation active site region) and BIN2-BAR purified from Sf9 cells. OQ. In vitro phosphorylation assays with proteins from N showed that BIN2-DUF increased p-HDAC1 in a dose-dependent manner, whereas BIN2-BAR was much less active on HDAC1 phosphorylation. R and S. Immunohistochemistry and quantification of p-BIN2 or p-HDAC1 in the same area of ​​the same tissue (but in different Z sections) of human ovarian tissue microarrays showed that both p-BIN2 and p-HDACl were higher in OC tissue than in normal ovaries, and that p-BIN2 levels were positively correlated with p-HDACI levels. Scale bar in R, 100 μm. *: P < 0.05; **: P < 0.01.

[0023] Figure 5 .Inhibit BIN2 upregulation of H3K27ac;

[0024] AC. Blot and quantification showed that H3K27ac levels were significantly higher in Bin2-KO ovaries than in WT groups under in situ chemical carcinogen treatment conditions, but H3K27me3 levels remained unchanged. DF. Blot and quantification showed that inhibition of BIN2 with DFBP significantly upregulated H3K27ac in A2780 OC cells, but did not affect H3K27me3. G and H. H3K27ac ChIP-seq data showed that BIN2 inhibition significantly increased H3K27ac binding to the 5kb regions upstream and downstream of the transcription start site (TSS) of many tumor suppressor genes (G), and KEGG analysis showed that many of these genes were associated with cancer-related signaling pathways (H). I and J. BIN2 inhibition significantly increased the intensity of H3K27ac binding to eight representative tumor suppressor genes. K. RNA sequencing heat map showed that there were 578 differentially expressed genes (DEGs) between CTR and DFBP-treated A2780 OC cells at a threshold of |log2(DFBP / CTR)|≥1.2. L and M. Analysis of representative RPKM values ​​from RNA sequencing (K) showed that DFBP treatment significantly upregulated the expression levels of the eight tumor suppressor genes mentioned above (I and J). *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0025] Figure 6 .Bin2 knockout protects ovarian function under OC invasion;

[0026] A. Analysis of cumulative litter size (A, n = 5) showed that under in situ chemical carcinogen treatment, the ovaries of WT mice almost completely lost their fertility, while Bin2-KO ovaries maintained partial fertility. B and C. Under ectopic (subcutaneous) invasion of mouse ID8 OC cells, the tumor size and weight of Bin2-KO mice were significantly smaller than those of WT mice. DH. HE staining and follicle counting of ovarian sections (n ​​= 4) showed that under ectopic invasion of mouse ID8 OC cells, the number of primordial follicles and growing follicles at each stage in the ovaries of Bin2-KO mice was significantly higher than that of the WT group. Black arrows indicate primordial follicles. Scale bars, 400 μm in D upper and 100 μm in D lower. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ***: P < 0.0001.

[0027] Figure 7 In mice with ectopic OC invasion, BIN2 inhibitors protected ovarian function, whereas HDAC1 inhibitors did not.

[0028] AC. Blot and quantification showed that both DFBP and HDAC1 inhibitor treatment significantly reduced p-HDAC1 in ectopic ID8-OC tissues, while significantly increasing H3K27ac. DF. Blot and quantification showed that both DFBP and HDAC1 inhibitor treatment significantly reduced tumor markers CD31 and HE4 in ectopic ID8-OC tissues. G and H. Tumor size measurements showed that both DFBP and HDAC1 inhibitor treatment significantly inhibited ID8 OC growth. IK. Blot and quantification showed that both DFBP and HDAC1 treatment significantly reduced p-HDAC1 and increased H3K27ac in ovaries under ectopic ID8-OC invasion. LP. HE staining of ovarian sections showed that under ectopic ID8-OC invasion, inhibition of BIN2 with DFBP significantly restored the number of follicles at each stage that was significantly reduced by ectopic ID8-OC invasion; however, HDAC1 inhibitor treatment had no rescue effect at all. QS.Blot and quantification showed that ectopic ID8 OC invasion significantly reduced NNT levels, and DFBP treatment but not HDAC1 treatment significantly upregulated ovarian NNT levels. Scale bar, L left 400μm, L right 100μm. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ***: P < 0.0001.

[0029] Figure 8 .Model: BIN2 inhibition suppresses tumor progression by downregulating p-HDAC1 in OC, while protecting normal ovarian function by downregulating p-RPS6.

[0030] In OC tissues, p-HDAC1 is more dominant than p-RPS6 in binding to p-BIN2, and p-HDAC1 negatively regulates H3K27ac. Therefore, DFBP inhibition of BIN2 upregulates tumor suppressor genes by downregulating p-HDAC1 and upregulating H3K27ac, thereby upregulating the expression level of tumor suppressor proteins. In normal ovaries, p-RPS6 is more dominant than p-HDAC1 in binding to p-BIN2. Therefore, DFBP inhibition of BIN2 significantly increases the number of follicles per stage and improves oocyte quality by downregulating p-RPS6 and upregulating NNT.

[0031] Fig. 9 Mass spectrometry identification of oDFBP.

[0032] Fig.10 It is the mass spectrometry identification of nDFBP. DETAILED DESCRIPTION

[0033] Example 1

[0034] Currently, oDFBP (CYGRKKRRQRRRQRASAPPHRPPP) and nDFBP (CYGRKKRRQRRRPKKKRKVAAQRASAPPHRPPP) are synthesized by the company. The product was identified by mass spectrometry ( Fig. 9 , Fig.10 ).

[0035] The corresponding DNA sequences of oDFBP and nDFBP were cloned into pGEX-6P2 and transformed into BL21-DE-Plys Escherichia coli for prokaryotic protein expression. A small amount of bacteria was picked and added to 100 mL LB culture medium to proliferate. The bacterial solution was added to 4 L LB+ culture medium at a volume ratio of 1:50 and cultured in a shaker at 200 rpm and 37°C until the OD 600 =0.6, 0.1 mmol / L IPTG was added, and induced overnight at 16°C, 200 rpm for 16 hours. The cells were collected by centrifugation at 3500 rpm for 10 minutes at 4°C, washed with ice-cold PBS, pH 7.3, and incubated in 25 mL lysis buffer (PBS, pH 7.3, 1 mM DTT, 1 mM PMSF, 1% Triton X-100, 1:100 InStab TM Protease inhibitor cocktail (Yeasen), 1:100 InStab TM Lyse in a phosphatase inhibitor cocktail (Yeasen). After standing on ice for 15 minutes, the cells were broken with a high-pressure crusher and the supernatant (containing the fusion protein) was collected by centrifugation at 16,000 rpm at 4°C. The fusion protein was captured with 1 mL of glutathione agarose resin at 4°C for 30 minutes. Next, the resin bound to the GST fusion polypeptide was transferred to a 10 mL chromatographic column and the resin was washed three times with 20 mL of washing buffer. Finally, the GST fusion polypeptide was eluted with elution buffer (1x PBS, 1 mM MgSO4, 0.5 mM DTT, 10 mM Glutathione, pH 7.4). To cut off the GST tag, the GST fusion polypeptide was transferred into the PreScission column by centrifugation using a size-exclusion spin column. TM Protease digestion buffer (50mM Tris, pH 8.0, 0.5mM EDTA, 1mM DTT), PreScission TM The ratio of protease enzyme to GST fusion polypeptide was 1:25-1:100 U / μg fusion polypeptide, and the enzyme digestion was carried out overnight at 4°C. Finally, the oDFBP and nDFBP polypeptides were recovered, and the concentration of the polypeptides was determined by comparing the intensity of the polypeptides with the standard BSA on SDS-PAGE gel.

[0036] Example 2

[0037] DFBP (i.e. Figure 7 iBPP) is a mixture of equal amounts of nDFBP (i.e., nBPP in the figure) and oDFBP (oBPP in the figure). DFBP was dissolved in 10% DMSO (Sigma) diluted with sterile ultrapure water. The concentration of the concentrate is 5 mg / mL. The concentrate was diluted with 0.9% sodium chloride solution to a final concentration of 0.5 mg / mL and injected into the peritoneal cavity of experimental mice at a dose of 6 mg / kg. The TAT sequence CYGRKKRRQRRR was used as a control peptide and injected intraperitoneally into control mice at a dose of 6 mg / kg. An ectopic ovarian cancer model was constructed by transplanting ID8 mouse ovarian cancer cells into normal B6 female mice subcutaneously, and tumor-bearing female mice were treated with DFBP, and HDAC1 inhibitors were used as controls. Blot and quantification showed that both DFBP and HDAC1 inhibitors significantly reduced the tumor markers CD31 and HE4 (in ectopic ovarian cancer tissues. Figure 7 DF) and significantly inhibited the growth of ID8 OC ( Figure 7 G and H). Both DFBP and HDAC1 inhibitor treatment significantly reduced p-HDAC1 and increased H3K27ac in ovarian cancer tissues ( Figure 7 AC, IK). Meanwhile, HE staining of ovarian sections showed that under ectopic ID8-OC invasion, inhibition of BIN2 with DFBP significantly restored the number of follicles at each stage that was significantly reduced by ectopic ovarian cancer invasion; however, HDAC1 inhibitor treatment had no rescue effect at all ( Figure 7 LP). Blot and quantification showed that DFBP still protected ovarian reserve and improved oocyte quality by reducing p-RPS6 and increasing NNT, but HDAC1 inhibitor could not reduce p-RPS6 and increase NNT ( Figure 7 QS). Therefore, DFBP shows a good "dual function" of inhibiting ovarian cancer and protecting ovarian function.

Claims

1. A polypeptide composition DFBP, characterized in that: It comprises the oDFBP polypeptide shown in SEQ ID NO.1 and the nDFBP polypeptide shown in SEQ ID NO.

2.

2. The polypeptide composition DFBP according to claim 1, characterized in that: The molar ratio of the oDFBP polypeptide to the nDFBP polypeptide is 1:1 to 10:

1.

3. A gene, characterized in that The gene encodes the oDFBP polypeptide according to claim 1.

4. A gene, characterized in that The gene encodes the nDFBP polypeptide described in claim 1.

5. Use of the polypeptide composition DFBP according to claim 1 or 2 in the preparation of a drug for inhibiting the progression of ovarian cancer while protecting ovarian function.

6. A drug for inhibiting the progression of ovarian cancer while protecting ovarian function, characterized in that: Comprising the polypeptide composition DFBP according to claim 1 or 2.

7. The method for preparing the drug for inhibiting the progression of ovarian cancer and protecting ovarian function according to claim 6, characterized in that: The polypeptide composition DFBP according to claim 1 or 2 is prepared by mixing with a pharmaceutically acceptable carrier or excipient.

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