A polypeptide composition DFBP with inhibitory and protective effects on ovarian cancer and its application.

Abstract

A polypeptide composition DFBP with the functions of inhibiting ovarian cancer and protecting the ovary, and its application, comprising the oDFBP polypeptide shown in SEQ ID NO.1 and the nDFBP polypeptide shown in SEQ ID NO.2. Since DFBP specifically inhibits the phosphorylation of BIN2-T423A&S424, CIBB does not affect the phosphorylation of other sites of BIN2 or other post-translational modifications and related physiological functions. As a polypeptide drug, DFBP has significant advantages over inhibitors, including high specificity, high efficiency, high safety, low immunogenicity, high membrane permeability, and low cost. DFBP can be used in combination with various tumor treatment regimens to achieve the "dual purpose" of inhibiting ovarian cancer progression and protecting ovarian function.

Description

Technical Field

[0001] This invention belongs to the field of ovarian medicine, specifically relating to a polypeptide composition that can inhibit the progression of ovarian cancer and protect the ovaries, and its application. Background Technology

[0002] Malignant tumors or cancers are increasingly threatening people's health, and radiotherapy and chemotherapy, commonly used in clinical cancer treatment, can significantly damage women's ovarian function and even lead to complete infertility. For young women with ovarian cancer (OC), ideal treatment should achieve a dual purpose: inhibiting tumor progression and protecting ovarian function, as young women with OC 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 knocking out Bin2 or inhibiting BIN2 phosphorylation with DFBP can inhibit the occurrence and development of OC. However, in human female OC tissue, 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 tissue, we speculated that p-BIN2 has other more important targets in OC tissue. Through mass spectrometry identification, we found that only the persistently activated forms of BIN2 (T423D and S424D) hooked HDAC1, indicating that HDAC1 is a more critical target of BIN2 in OC tissue. Next, we observed that Bin2 knockout or inhibition significantly reduced p-HDAC1 (S421) while increasing H3K27ac. Furthermore, chip-seq showed that Bin2 inhibition significantly increased the binding of H3K27ac to multiple tumor suppressor genes. Additionally, in mice with chemically induced in situ or ectopic ovarian cancer (OC), Bin2 knockout or inhibition protected ovarian function. This study demonstrates that inhibiting Bin2 can both suppress the development and progression of OC and protect ovarian function. Therefore, for women with OC, this approach can achieve the dual goals of inhibiting ovarian cancer progression and protecting the ovaries, thus providing a more rational and ideal innovative treatment option compared to radiotherapy and chemotherapy. Summary of the Invention

[0003] Technical problem solved: This invention provides a polypeptide composition DFBP that inhibits ovarian cancer and protects the ovaries, and its application. This polypeptide composition can inhibit the progression of ovarian cancer while protecting ovarian function. This "dual function" is crucial for women of childbearing age with ovarian cancer to maintain their fertility.

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

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

[0006] A gene that encodes the oDFBP polypeptide.

[0007] A gene that encodes the nDFBP polypeptide.

[0008] The above-mentioned polypeptide composition DFBP is used in the preparation of drugs that inhibit the progression of ovarian cancer while protecting ovarian function.

[0009] A drug that inhibits the progression of ovarian cancer while protecting ovarian function, comprising the above-mentioned polypeptide composition DFBP.

[0010] The preparation method of the above-mentioned drug that inhibits the progression of ovarian cancer and protects ovarian function involves mixing the above-mentioned polypeptide composition DFBP with a pharmaceutically acceptable carrier or excipient.

[0011] Women with ovarian cancer were given oral DFBP, 2 mg / kg, once daily for four weeks. Serum samples were then collected to measure tumor markers (CRP, HE4, CD31, RB1, etc.) to determine if ovarian cancer progression was suppressed, and six hormones (FSH, LH, E2, P, T, PRL) and NNT (nicotinamide nucleotide transhydrogenase) were measured to assess 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 peptide drug, DFBP has significant advantages over inhibitors, including high specificity, high efficiency, high safety, low immunogenicity, high membrane permeability, and low cost.

[0014] 3. DFBP can be used in combination with various tumor treatment regimens to achieve the "dual purpose" of inhibiting the progression of ovarian cancer and protecting ovarian function. Attached Figure Description

[0015] Figure 1 Bin2 knockout inhibits the development of chemically carcinogenic ovarian tumors;

[0016] A and B. We induced tumorigenesis in situ within the ovary using the chemical inducers DMBA and NMU. White light imaging and quantification showed that, under chemical carcinogen treatment, the area of ​​white ovarian tissue transforming into cancer in 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 its ovarian structural characteristics. C and D. Immunofluorescence and quantification of the vascular marker CD31 showed that, under chemical carcinogen treatment, the intensity of the vascular marker CD31 in the WT group ovary was significantly increased, while the intensity of CD31 in the Bin2-KO group was similar to that in the untreated control. E and F. Immunofluorescence and quantification of the proliferation marker Ki67 showed that, under chemical carcinogen treatment, the intensity of the proliferation marker Ki67 in the WT group ovary was significantly increased, while the intensity of Ki67 in the Bin2-KO group was similar to that in 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 WT group ovaries, while the levels in the Bin2-KO ovaries were similar to those in the untreated ovaries. Scale bar: A is 2 mm at the top and 100 μm at the bottom; C and E are 100 μm. *: 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 in normal ovaries. The OC group had n=426 patients, and normal ovarian tissue had n=88 patients. B. Kaplan-Meier correlation analysis showed that OC patients with high BIN2 levels had significantly lower progression-free survival (PFS) in grade I and II OC (GSE14764, n=293). C. We collected data from 32 OC patients, and Western blot and quantification showed that BIN2 and p-BIN2 were significantly upregulated in 60% of the OC tissues from these patients. D. Western blot and quantification showed that BIN2 (F and G) and p-BIN2 (H and I) levels in OC cells (A2780 cells) were significantly higher than in normal ovarian epithelial cells (Moody cells). Immunohistochemistry on human OC microarrays by J and K showed that p-BIN2 levels were significantly upregulated in high-grade serous ovarian cancer (HGSOC), ovarian endometrioid carcinoma (OEC), and clear cell ovarian 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 enhance 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 apoptosis levels in A2780 OC cells. J and K. DFBP treatment significantly reduced mitochondrial membrane potential in A2780 OC cells. L and M. DFBP treatment significantly increased ROS levels in A2780 OC cells. NQ. In immunodeficient mice injected with A2780, DFBP treatment significantly reduced the percentage of tumor formation (N and O) and tumor volume (P and Q). Scale bar, 20 μm. **: P < 0.01; ***: P < 0.001; ***: P < 0.0001.

[0021] Figure 4 BIN2 directly binds to 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 and LC-MS of A2780 cells transfected with Strep II antibody in BIN2-WT, S423A&T424A inactive, and S423D&T424D persistently activating mutants showed that only the activating mutants induced HDAC1; however, none of the BIN2 plasmids pulled down RPS6. C. Co-transfection of A2780 cells with the Bin2-DD-Strep II and HDAC1-FLAG constructs (in pcDNA3.1+) showed that BIN2-DD interacted with HDAC1. D and E. Western blotting and quantification showed that p-HDAC1 levels in OCs were significantly higher than in ovaries, while p-RPS6 levels in tumors were significantly lower than in ovaries. Therefore, the ratio of p-HDAC1 to p-RPS6 in OC was more than six times higher than in ovaries. FI. Blot and quantification showed that DFBP significantly reduced p-HDAC1 (S421) (FH) in A2780 cells by inhibiting BIN2, 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 mutants (inactive forms, S423A and T424A) purified from Sf9 cells. KM. In vitro phosphorylation experiments and quantification using proteins from J showed that BIN2-WT increased p-HDAC1 in a dose-dependent manner, while the BIN2-AA mutant had much lower activity for HDAC1 phosphorylation. N. SDS-PAGE and Coomassie staining showed good protein purity of BIN2-DUF (the region containing the phosphorylation active sites of S423 & T424) and BIN2-BAR purified from Sf9 cells. OQ. In vitro phosphorylation assays using proteins derived from N showed that BIN2-DUF increased p-HDAC1 in a dose-dependent manner, while BIN2-BAR showed much lower activity in phosphorylating HDAC1. R and S. Immunohistochemistry and quantification of p-BIN2 or p-HDAC1 in the same region of the same tissue (but in different Z sections) of human ovarian tissue microarrays showed that both p-BIN2 and p-HDAC1 were higher in OC tissue than in normal ovaries, and p-BIN2 levels were positively correlated with p-HDAC1 levels. Scale bar of R, 100 μm. *: P < 0.05; **: P < 0.01.

[0023] Figure 5 Inhibit BIN2 upregulation of H3K27ac;

[0024] AC. Blot and quantification showed that under in situ chemical carcinogen treatment, the level of H3K27ac in Bin2-KO ovaries was significantly higher than that in the WT group, but the level of H3K27me3 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 the binding of H3K27ac to the 5kb upstream and downstream regions of transcription start sites (TSS) of many tumor suppressor genes (G), and KEGG analysis showed that many of these genes are involved in cancer-related signaling pathways (H). I and J. BIN2 inhibition significantly increased the binding intensity of H3K27ac to eight representative tumor suppressor genes. K. RNA sequencing heatmap showed that at a threshold of |log2(DFBP / CTR)|≥1.2, there were 578 differentially expressed genes (DEGs) between CTR- and DFBP-treated A2780 OC cells. 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 (I and J) mentioned above. *: 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 the ovaries of Bin2-KO mice retained 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 and growing follicles at each stage of the ovary in Bin2-KO mice was significantly higher than that in the WT group. Black arrows indicate primordial follicles. Scale bar, 400 μm above D, 100 μm below D. *: P<0.05; **: P<0.01; ***: P<0.001; ***: P<0.0001.

[0027] Figure 7 In mice with ectopic ovarian invasion, BIN2 inhibitors protected ovarian function, while 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 tissue and significantly increased H3K27ac. DF. Blot and quantification showed that both DFBP and HDAC1 inhibitor treatment significantly reduced the tumor markers CD31 and HE4 in ectopic ID8-OC tissue. 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 under ectopic ID8-OC invasion, both DFBP and HDAC1 treatment significantly reduced p-HDAC1 and increased H3K27ac in the ovary. 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 had been significantly reduced due to ectopic ID8-OC invasion; however, HDAC1 inhibitor treatment had no rescue effect. QS.Blot and quantification showed that ectopic ID8 OC invasion significantly reduced NNT levels, and DFBP treatment, instead of HDAC1 treatment, significantly upregulated ovarian NNT levels. Scale bar: L left = 400 μm, L right = 100 μm. *: 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 ovarian tissue, p-HDAC1 is more dominant than p-RPS6 in binding p-BIN2, while 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 p-BIN2. Therefore, DFBP inhibition of BIN2 significantly increases the number of follicles at each stage and improves oocyte quality by downregulating p-RPS6 and upregulating NNT.

[0031] Figure 9 Identification using oDFBP mass spectrometry.

[0032] Figure 10 Identification by nDFBP mass spectrometry. Detailed Implementation

[0033] Example 1

[0034] Currently, both oDFBP (CYGRKKRRQRRRQRASAPPHRPPP) and nDFBP (CYGRKKRRQRRRPKKKRKVAAQRASAPPHRPPP) were synthesized by our company. The products were identified by mass spectrometry. Figure 9 , Figure 10 ).

[0035] The corresponding DNA sequences of oDFBP and nDFBP were cloned into pGEX-6P2, transformed into BL21-DE-Plys prokaryotic protein expression-specific *E. coli*, and a small amount of bacteria was added to 100 mL of LB medium to promote proliferation. The bacterial culture was then added to 4 L of LB+ medium at a volume ratio of 1:50 and cultured on a shaker at 200 rpm and 37 °C until OD was reached. 600 =0.6, add 0.1 mmol / L LIPTG, and induce overnight at 16°C and 200 rpm for 16 hours. Cells were collected by centrifugation at 3500 rpm for 10 minutes at 4°C, washed with ice-cold PBS (pH 7.3), and lysed in 25 mL of lysis buffer (PBS, pH 7.3, 1 mM DTT, 1 mM PMSF, 1% Triton X-100, 1:100 InStab). TM Protease inhibitor mixture (Yeasen), 1:100 InStab TM Cells were lysed in a mixture of phosphatase inhibitors (Yeasen). After standing on ice for 15 minutes, cells were lysed using a high-pressure homogenizer, 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 containing the GST fusion peptide was transferred to a 10 mL column and washed three times with 20 mL of wash buffer. Finally, the GST fusion peptide was eluted with elution buffer (1x PBS, 1 mM MgSO4, 0.5 mM DTT, 10 mM Glutathione, pH 7.4). To remove the GST tag, the GST fusion peptide was transferred to PreScission via 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 to GST fusion peptide was 1:25-1:100 U / μg fusion peptide, and digestion was performed overnight at 4°C. Finally, oDFBP and nDFBP peptides were recovered, and peptide concentrations were determined by comparing the peptide strength on an SDS-PAGE gel with standard BSA.

[0036] Example 2

[0037] DFBP (i.e.) Figure 7 iBPP was prepared by mixing 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 was 5 mg / mL. The concentrate was diluted to a final concentration of 0.5 mg / mL with 0.9% sodium chloride solution and injected intraperitoneally into 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 allogeneic ectopic ovarian cancer model was constructed by transplanting ID8 mouse ovarian cancer cells subcutaneously into normal B6 female mice. The 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 tissue. Figure 7 DF) and significantly inhibited ID8 OC growth ( Figure 7 G and H). Treatment with both DFBP and HDAC1 inhibitors 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 had been significantly reduced due to ectopic ovarian cancer invasion; however, HDAC1 inhibitor treatment had no salvage effect whatsoever. Figure 7 Blot and quantitative analysis showed that DFBP still protects ovarian reserve and improves oocyte quality by reducing p-RPS6 and increasing NNT, but HDAC1 inhibitors do not reduce p-RPS6 or increase NNT. Figure 7 Therefore, DFBP demonstrates a good "dual function" of inhibiting ovarian cancer and protecting ovarian function.

Claims

1. A polypeptide composition DFBP, characterized in that, It contains the oDFBP peptide as shown in SEQ ID NO.1 and the nDFBP peptide as shown in SEQ ID NO.

2.

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

1.

3. The use of the polypeptide composition DFBP according to claim 1 or 2 in the preparation of a drug that inhibits the progression of ovarian cancer while protecting ovarian function.

4. A drug that inhibits the progression of ovarian cancer while protecting ovarian function, characterized in that, It comprises the polypeptide composition DFBP according to claim 1 or 2.

5. The method for preparing the drug for inhibiting ovarian cancer progression and protecting ovarian function as described in claim 4, characterized in that, The polypeptide composition DFBP of claim 1 or 2 is prepared by mixing it with a pharmaceutically acceptable carrier or excipient.

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