A polypeptide tat-gper1c and application thereof

Abstract

The application discloses a polypeptide TAT-GPER1C and an application thereof, and the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1; wherein, positions 1-11 are a cell-penetrating peptide TAT, and positions 12-60 are GPER1C. The polypeptide TAT-GPER1C provided by the application is derived from GPER, the polypeptide TAT-GPER1C and GPER competitively combine with beta-arrestin 2, interfere with the interaction between GPER and beta-arrestin 2, and thus inhibit symptoms such as tumor cell proliferation and chemotherapy drug tolerance mediated by beta-arrestin 2. The polypeptide TAT-GPER1C of the application has application potential in preparation of a medicine for treating cancers in which beta-arrestin 2 plays a role, such as breast cancer and ovarian cancer.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a polypeptide TAT-GPER1C and its applications. Background Technology

[0002] Estrogen plays a significant role in both physiological and pathological changes in women. As a seven-transmembrane G protein-coupled estrogen receptor (GPER), it can specifically bind to estrogen and its derivatives, mediating their biological effects. GPER is widely involved in the occurrence and development of various hormone-responsive cancers such as breast cancer, ovarian cancer, endometrial cancer, and cervical cancer. Studies show that GPER function is mainly regulated by the multifunctional scaffold protein β-arrestin 2, and their interaction plays a crucial role in cancer cell proliferation and chemotherapy resistance.

[0003] β-arrestin 2 is highly expressed in related cancer tissues and cells and is closely associated with poor prognosis. Knockdown of β-arrestin 2 expression can effectively inhibit cancer cell proliferation and improve their chemosensitivity, and may become an effective therapeutic target. Drug research targeting β-arrestin 2 has great potential for clinical treatment.

[0004] Estrogen or its derivatives activate GPER, causing a conformational change that activates the G protein signaling pathway, subsequently attracting β-arrestin 2 to accumulate on the cell membrane. β-arrestin 2 competitively binds to the cytoplasmic region of GPER, releasing it from G protein coupling and preventing further G protein binding because β-arrestin 2 covers the functional cytoplasmic region of GPER. The β-arrestin 2-mediated signaling pathway then begins to play a role in tumor proliferation and chemotherapy resistance. GPER affects cancer cell proliferation and chemotherapy resistance through its interaction with β-arrestin 2; therefore, intervening in its signaling pathway may be an effective clinical treatment strategy.

[0005] GPER belongs to the GPCR family and is a seven-transmembrane receptor. Its seven α-helical structures are connected by three extracellular loops (ECLs 1-3) and three intracellular loops (ICLs 1-3). Studies have shown that the interaction between GPER and β-arrestin 2 may be mainly mediated by C-terminal recognition.

[0006] Peptides designed based on the intracellular domain of GPER can selectively interfere with the interaction between β-arrestin 2 and GPER, potentially blocking the function of β-arrestin 2. Peptide drugs that block the interaction between β-arrestin 2 and GPER may have promising anti-cancer effects, playing a role in the treatment of breast cancer, cervical cancer, endometrial cancer, and ovarian cancer, providing new insights into the treatment of female-related malignancies. However, current research in this area is still lacking. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a polypeptide TAT-GPER1C and its applications, which have potential applications in the preparation of drugs for treating breast cancer and ovarian cancer.

[0008] This invention is achieved through the following technical solution:

[0009] A polypeptide TAT-GPER1C, the amino acid sequence of which is shown in SEQ ID NO.1; wherein, positions 1-11 are the transmembrane peptide TAT, and positions 12-60 are GPER1C.

[0010] The gene encoding the above-mentioned polypeptide TAT-GPER1C has the nucleotide sequence shown in SEQ ID NO.2; wherein, positions 1-33 from the 5' end are the nucleotide sequence encoding the membrane-penetrating peptide TAT, and positions 34-180 are the nucleotide sequence encoding GPER1C.

[0011] Expression cassettes, recombinant vectors, or recombinant bacteria containing the above-mentioned genes.

[0012] The application of the above-mentioned polypeptide TAT-GPER1C, the above-mentioned gene or the above-mentioned expression cassette, recombinant vector or recombinant bacteria in the preparation of antitumor drugs.

[0013] Preferably, the tumor is tumor tissue or cells in which β-arrestin 2 plays a role.

[0014] Preferably, the tumor is breast cancer or ovarian cancer.

[0015] Preferably, the drug is in any pharmacologically acceptable dosage form.

[0016] Preferably, the drug further includes a pharmaceutically acceptable drug delivery carrier.

[0017] The beneficial effects of this invention are as follows:

[0018] The polypeptide TAT-GPER1C provided by this invention is derived from GPER. TAT-GPER1C competitively binds to β-arrestin 2, interfering with the interaction between GPER and β-arrestin 2, thereby inhibiting β-arrestin 2-mediated tumor cell proliferation and chemotherapy drug resistance. The polypeptide TAT-GPER1C of this invention has potential application in the preparation of drugs for treating cancers such as breast cancer and ovarian cancer, where β-arrestin 2 plays a role. Attached Figure Description

[0019] Figure 1 The effect of peptide TAT-GPER1C on the proliferation of breast cancer cells MCF-7 (A) or ovarian cancer cells SKOV3 (B) in Example 2;

[0020] Figure 2 This study examines the effect of the peptide TAT-GPER1C in Example 2 on the sensitivity of breast cancer cells MCF-7 / ADM (A) or ovarian cancer cells SKOV3 / cDDP (B) to chemotherapeutic drugs. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0022] Unless otherwise specified, the quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional molecular biology methods.

[0024] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0025] In the following examples, MCF-7 cells, MCF-7 / ADM cells, SKOV3 cells, and SKOV3 / cDDP cells were all derived from ATCC (American Type Culture Collection).

[0026] Example 1: Obtaining the peptide TAT-GPER1C

[0027] This embodiment illustrates the process of obtaining the peptide TAT-GPER1C with penetrating activity. The specific steps are as follows:

[0028] (1) Total RNA was extracted from SKOV3 cells and cDNA was obtained by reverse transcription. The GPER C-terminal polypeptide sequence was obtained by PCR using cDNA as a template. The upstream primer is shown in SEQ ID NO.3 and the downstream primer is shown in SEQ ID NO.4. The TAT coding sequence was inserted into the upstream primer (as shown in SEQ ID NO.5). XhoI and BamHI restriction sites were added to the upstream and downstream primers, respectively. The pWaldo-TAT-GPER1C Escherichia coli expression vector was constructed by restriction enzyme ligation and transformed into BL21(DE3) expression strain to obtain recombinants.

[0029] pWaldo is a vector with GFP-8His inserted into pET28(a+). Its synthesis can be found in the following reference: Drew, DE, von Heijne, G., Nordlund, P. & de Gier, JW. Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichiacoli. FEBS Lett 507, 220-4 (2001).

[0030] (2) Inoculate the BL21(DE3) recombinant strain into 200 mL of LB medium and incubate overnight at 37°C. Transfer 50 mL of the overnight culture to 1 L of LB medium and incubate at 37°C until OD500. 600 Add IPTG to a final concentration of 0.1 mM at a concentration between approximately 0.5 and 0.6, incubate at 22°C for about 18 hours, and collect cells by centrifugation at 6000g for 15 minutes (approximately 22g of cells can be collected from 5L of culture medium).

[0031] (3) Resuspend cells in 100 mL of lysis buffer (20 mM Tris-HCl, pH 8.5, 300 mM NaCl and 10% glycerol), add 200 U Dnase I, 50 μg / mL lysozyme and 1 tablet of protease inhibitor (cocktail), homogenize twice at 1200 bar and 4°C, centrifuge at 18,000 rpm and 4°C for 30 min to remove cell debris; pass the supernatant through a Ni-NTA column; wash 100 mL with washing buffer (20 mM Tris-HCl, pH 8.5, 300 mM NaCl, 10% glycerol and 30 mM imidazole), elute with elution buffer (20 mM Tris-HCl, pH 8.5, 300 mM NaCl, 10% glycerol and 300 mM imidazole), collect the 5 mL sample with the highest protein content, and pass through a molecular sieve (buffer: 20 mM Tris-HCl, pH 8.5, 300 mM NaCl, 300 mM glycerol and 10% glycerol). Purification was performed using NaCl (10% glycerol). This step yielded a high-purity TAT-GPER1C fusion protein, which can be used for related studies in subsequent examples.

[0032] The purified polypeptide TAT-GPER1C amino acid sequence is shown in SEQ ID NO.1, consisting of 60 amino acid residues. Among them, amino acid residues 1-11 form the region with cell membrane penetration function, namely the transmembrane peptide TAT. The positions of amino acid residues 12-60 can be interchanged and can be replaced by other sequences with the same function. Amino acid residues 12-60 constitute the region that can interfere with the binding of GPER and β-arrestin 2 proteins, namely GPER1C.

[0033] The nucleotide sequence of the gene encoding the polypeptide TAT-GPER1C is shown in SEQ ID NO.2, and is 180 bp in length; wherein, from the 5' end, positions 1-33 are the nucleotide sequence encoding the transmembrane peptide TAT, and positions 34-180 are the nucleotide sequence encoding GPER1C.

[0034] Example 2: Application Study of Peptide TAT-GPER1C

[0035] 1. Effects on MCF-7 breast cancer cells and SKOV3 ovarian cancer cells

[0036] MCF-7 and SKOV3 cells were seeded into 96-well plates, respectively. After 12 hours of adhesion, 0.1 mg / mL of the peptide TAT-GPER1C obtained in Example 1 was added. Two hours later, 0.1 mg / mL of the peptide TAT-GPER1C was added again. Cells treated with physiological saline served as a control. After 48 hours of treatment, 10 μL of 5 mg / mL MTT solution was added to the cell wells, and the cells were incubated on a low-speed shaker for 3–4 hours. Subsequently, the mixture of MTT and culture medium was removed, and 100 μL of DMSO solution was added. Due to the presence of MTT, live cells formed crystals, which subsequently dissolved in the DMSO solution. The absorbance at 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0037] like Figure 1 As shown, the peptide TAT-GPER1C can significantly inhibit the growth of breast cancer cells MCF-7 (…). Figure 1 (A) and ovarian cancer cells SKOV3 ( Figure 1 The proliferation of B).

[0038] 2. Effects on breast cancer cells MCF-7 / ADM and ovarian cancer cells SKOV3 / cDDP

[0039] Drug-resistant breast cancer cells MCF-7 / ADM and ovarian cancer cells SKOV3 / cDDP were seeded into 96-well plates. After 12 h of adhesion, 0.2 mg / mL of the peptide TAT-GPER1C obtained in Example 1 was added. Two h later, 0.2 mg / mL of the peptide TAT-GPER1C was added again. After 30 min, 0.1–100 μg / mL of doxorubicin was added to MCF-7 / ADM cells, and 0.1–20 μg / mL of cisplatin was added to SKOV3 / cDDP cells. An equal volume of physiological saline was used as a control. After 48 h of drug treatment, 10 μL of 5 mg / mL MTT solution was added to the cell wells, and the cells were incubated on a low-speed shaker for 3–4 h. Then, the mixture of MTT and culture medium was removed, and 100 μL of DMSO solution was added. Due to the presence of MTT, live cells formed crystals, which then dissolved in the DMSO solution. The absorbance at 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0040] like Figure 2 As shown, the peptide TAT-GPER1C can significantly enhance the sensitivity of MCF-7 / ADM cells to doxorubicin chemotherapy. Figure 2 (A), and the sensitivity of SKOV3 / cDDP cells to cisplatin chemotherapy ( Figure 2 (B)

[0041] The experimental results of this embodiment show that the polypeptide TAT-GPER1C of the present invention can competitively bind to β-arrestin 2 with GPER, thereby interfering with the function of β-arrestin 2 and inhibiting the effects of β-arrestin 2-mediated cancer cell proliferation and chemotherapy drug resistance.

[0042] The foregoing has shown and described specific embodiments of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and various changes and modifications can be made without departing from the spirit and scope of the invention. All such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A polypeptide TAT-GPER1C, characterized in that, The amino acid sequence of the polypeptide TAT-GPER1C is shown in SEQ ID NO.1; wherein, positions 1-11 are the membrane-penetrating peptide TAT, and positions 12-60 are GPER1C; the polypeptide TAT-GPER1C is used to competitively bind to β-arrestin2 with GPER to interfere with the interaction between GPER and β-arrestin2.

2. The gene encoding the polypeptide TAT-GPER1C of claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.2; wherein, positions 1-33 from the 5' end are the nucleotide sequence encoding the transmembrane peptide TAT, and positions 34-180 are the nucleotide sequence encoding GPER1C.

3. An expression cassette, recombinant vector, or recombinant bacteria containing the gene as described in claim 2.

4. The use of the polypeptide TAT-GPER1C of claim 1, the gene of claim 2, or the expression cassette of claim 3, the recombinant vector, or the recombinant bacteria in the preparation of antitumor drugs, characterized in that, The tumor is either breast cancer or ovarian cancer.

5. The application according to claim 4, characterized in that, The drug is in any pharmacologically acceptable dosage form.

6. The application according to claim 4, characterized in that, The drug also includes pharmaceutically acceptable drug delivery carriers.

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