Application of Galectin13 recombinant protein and mimic peptide thereof in preparation of antitumor drugs

Abstract

The invention belongs to the field of biological medicine, and discloses application of Galectin13 recombinant protein and mimic peptide thereof in preparation of antitumor drugs. The inventor finds that the Gal13 recombinant protein and the mimic peptide HYNEYEIKVNGIRIYGFV of the Gal13 recombinant protein can be combined with CD44, so that the combination of the CD44 and SLC7A11 is blocked, and the ferroptosis sensitivity of tumor cells is enhanced. The Gal13 recombinant protein and the mimic peptide thereof are combined with Erastin to strongly induce ferroptosis. Animal experiment data show that the Gal13 recombinant protein and the mimic peptide thereof significantly promote tumor cell ferroptosis mediated by radiotherapy or immunotherapy, and greatly enhance the curative effect of radiotherapy or immunotherapy. The Gal13 recombinant protein and the mimic peptide thereof have good patent medicine prospects, and can be clinically combined with radiotherapy or immunotherapy in the future.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of Galectin13 recombinant protein and its mimetic peptides in the preparation of anti-tumor drugs. Background Art

[0002] Ferroptosis is a new type of programmed cell death different from apoptosis and necrosis, and its core feature is the abnormal accumulation of iron ion-dependent lipid peroxidation. This process is precisely regulated by the system Xc- / glutathione / GPX4 antioxidant axis: the system Xc- is responsible for transporting cystine into the cell and converting it into cysteine, and then synthesizing glutathione (GSH); glutathione peroxidase 4 (GPX4) then uses GSH to reduce toxic lipid peroxides to harmless lipid alcohols. When this pathway is inhibited, lipid peroxidation products accumulate excessively on the cell membrane, ultimately leading to cell membrane rupture and the occurrence of ferroptosis.

[0003] Recent studies have found that tumor cells often exhibit an "iron addiction" phenotype due to abnormal metabolism: on the one hand, they enhance iron uptake by upregulating transferrin receptor (TfR1), and on the other hand, they reduce the expression of ferroportin, resulting in a continuous expansion of the intracellular free iron pool. This iron metabolism imbalance makes tumor cells more sensitive to ferroptosis, providing an important target for selectively killing tumors. Inhibiting ferroptosis significantly weakens the efficacy of tumor radiotherapy and immunotherapy. Therefore, effectively inducing ferroptosis in tumor cells using compounds or drugs or polypeptides can significantly enhance the efficacy of tumor radiotherapy and immunotherapy.

[0004] Galectin-13 (Galactoside-binding soluble lectin 13), abbreviated as Gal13, belongs to the galectin family. Its structure contains a typical galectin domain, indicating that it may participate in cell-cell or cell-matrix interactions by recognizing β-galactoside glycosylation modifications. Such interactions usually involve immune regulation, cell adhesion, and signal transduction. Gal13 shows a highly specific distribution in human tissues, mainly expressed in placental tissue, spleen, kidney, and bladder. Existing studies have shown that Gal13 can bind to β-galactoside and lactose (PubMed: 10527825), is a strong inducer of T cell apoptosis (PubMed: 19497882), and has hemagglutination activity against chicken red blood cells (PubMed: 29343868). Gal13 is mainly expressed by syncytiotrophoblasts and released from the placenta into the maternal circulation, playing an important role in regulating the relationship between maternal and fetal tissues. Low expression or mutation of Gal13 can lead to preeclampsia (PubMed: 25191322). However, there are few reports on the role of Gal13 in tumors. In particular, no studies have shown that Gal13 is related to ferroptosis, nor have any studies suggested the role of Gal13 in tumor treatment. Summary of the Invention

[0005] An object of the present invention is to overcome at least one deficiency of the prior art and provide a Gal13 mimetic peptide and its application in enhancing the efficacy of tumor radiotherapy and immunotherapy.

[0006] The technical solution adopted by the present invention is as follows: In the first aspect of the present invention, provided is: A Gal13 mimetic peptide with the amino acid sequence HYNEYEIKVNGIRIYGFV.

[0007] In the second aspect of the present invention, provided is: A nucleotide sequence encoding the Gal13 mimetic peptide described in the first aspect of the present invention.

[0008] In the third aspect of the present invention, provided is: The application of Galectin-13 recombinant protein or the Gal13 mimetic peptide described in the first aspect of the present invention in the preparation of anti-tumor drugs.

[0009] In some examples, the tumor is selected from breast cancer and melanoma.

[0010] In the fourth aspect of the present invention, provided is: The application of Galectin-13 recombinant protein or the Gal13 mimetic peptide described in the first aspect of the present invention in the preparation of a synergist for ferroptosis inducer.

[0011] In some examples, the ferroptosis inducer is selected from Erastin, RSL3, ML210, FIN56, FINO2.

[0012] The fifth aspect of the present invention provides: The application of the Gal13 mimetic peptide described in the first aspect of the present invention in the preparation of a synergist for tumor radiotherapy or immunotherapy.

[0013] In some examples, the tumor is selected from breast cancer, melanoma.

[0014] In some examples, the immunotherapy is selected from PD-1, PD-L1 immunotherapy.

[0015] The sixth aspect of the present invention provides: A composition for treating tumors, comprising: At least one ferroptosis inducer; At least one of the Gal13 mimetic peptides of the Galectin-13 recombinant protein described in the first aspect of the present invention.

[0016] In some examples, the tumor is selected from breast cancer, melanoma.

[0017] In some examples, the ferroptosis inducer is selected from Erastin, RSL3, ML210, FIN56, FINO2.

[0018] The beneficial effects of the present invention are: The Gal13 recombinant protein or the Gal13 mimetic peptide HYNEYEIKVNGIRIYGFV (SEQ ID NO.1) of the present invention can bind to CD44, thereby blocking the binding of CD44 to SLC7A11 and enhancing the ferroptosis sensitivity of tumor cells. The combination of the Gal13 recombinant protein or the Gal13 mimetic peptide with Erastin can strongly induce ferroptosis. The data of animal experiments show that the Gal13 recombinant protein or the Gal13 mimetic peptide significantly promotes ferroptosis of tumor cells mediated by radiotherapy or immunotherapy, and greatly enhances the efficacy of radiotherapy or immunotherapy. This suggests that the Gal13 recombinant protein or the Gal13 mimetic peptide has good prospects for drug development and can be combined with radiotherapy or immunotherapy in the future clinic. Description of the Drawings

[0019] Figure 1: The 96 - 113 peptide segment in the Gal13 protein sequence mediates the binding of Gal13 to CD44. (a) The Gal13 protein was split into two segments, namely the N - terminal and the C - terminal. The full - length or truncated Gal13 and CD44 were co - transfected into 293T cells. Co - immunoprecipitation was used to analyze the interaction between CD44 and the full - length Gal13 and truncated Gal13. (b) CD44 and each truncated Gal13 were co - transfected into 293T cells, and then co - immunoprecipitation experiments were performed to analyze the binding of each truncated protein to CD44. (c) Endogenous CD44 was immunoprecipitated from MDA - MB - 231 cells treated with Gal13 recombinant protein, and then immunoblotting was performed using an antibody against SLC7A11. (d) The Gal13 mimetic peptide or Gal13 mimetic peptide - Mut was incubated with the cell lysates of MDA - MB - 231, pulled down with streptavidin beads, and the eluate was analyzed by western blotting. (e) Endogenous CD44 was immunoprecipitated from MDA - MB - 231 cells treated with Gal13 mimetic peptide or Gal13 mimetic peptide - Mut, and then immunoblotting was performed using an antibody against SLC7A11.

[0020] Figure 2 : Gal13 recombinant protein or Gal13 mimetic peptide enhances ferroptosis induced by ferroptosis inducers Erastin, RSL3, ML210, FIN56, FINO2 in tumor cells. (a, b) MDA - MB - 231 cells were treated with DMSO or 3 μM Erastin for 8 hours (with or without treatment with Gal13 recombinant protein or Gal13 mutant recombinant protein), and then cell death and lipid peroxidation of each group of cells were detected. (c, d) MDA - MB - 231 cells were treated with DMSO or 1 μM RSL3, 0.5 μM ML210, 2 μM FIN56, 1 μM FINO2 for 8 hours (with or without treatment with Gal13 recombinant protein), and then cell death and lipid peroxidation of each group of cells were detected. (e, f) MDA - MB - 231 cells were treated with DMSO or 3 μM Erastin for 8 hours (with or without treatment with Gal13 mimetic peptide or Gal13 mimetic peptide - Mut or Lipro - 1), and then cell death and lipid peroxidation of each group of cells were detected. (g, h) MDA - MB - 231 cells were treated with DMSO or 1 μM RSL3, 0.5 μM ML210, 2 μM FIN56, 1 μM FINO2 for 8 hours (with or without treatment with Gal13 mimetic peptide), and then cell death and lipid peroxidation of each group of cells were detected.

[0021] Figure 3: Gal13 recombinant protein or Gal13 mimetic peptide enhances radiotherapy- or immunotherapy-mediated tumor suppression. (a, c) Tumors were irradiated with a dose of 10 Gy using a JL Shepherd Mark I-68A irradiator. Gal13 recombinant protein or Gal13 mimetic peptide or Gal13 mimetic peptide-Mut was dissolved in a saline solution, and then the Gal13 recombinant protein was administered to mice via the tail vein every other day at a dose of 30 mg / kg, and the Gal13 mimetic peptide or Gal13 mimetic peptide-Mut was administered at a dose of 10 mg / kg. Lipro-1 diluted with PBS was intraperitoneally injected into mice at a dose of 15 mg / kg per day. Mice were treated with Lipro-1 3 times before irradiation and then continuously administered until the end point. (b, d) 1.5×10 5 B16 was subcutaneously inoculated into C57 mice. On the 3rd day, mice were treated in the following groups: control, anti-PD1 antibody, Gal13 recombinant protein or Gal13 mimetic peptide (or Gal13 mimetic peptide-Mut), anti-PD1 antibody and Gal13 recombinant protein or Gal13 mimetic peptide (or Gal13 mimetic peptide-Mut), Lipro-1, anti-PD1 antibody and Gal13 recombinant protein or Gal13 mimetic peptide and Lipro-1. The anti-PD1 antibody was intraperitoneally injected once every 3 days at a dose of 100 μg / mouse. The Gal13 recombinant protein was injected into the tail vein at a dose of 30 mg / kg every other day, or the Gal13 mimetic peptide or Gal13 mimetic peptide-Mut was injected at a dose of 10 mg / kg. Lipro-1 (15 mg / kg) was administered 3 times before anti-PD1 antibody treatment and then continued to be administered daily until the end point. Detailed implementation mode

[0022] The technical solution of the present invention will be further described below in conjunction with experiments.

[0023] First, the specific segment of Gal13 binding to CD44 was analyzed. Using molecular cloning technology, truncated plasmids of the Gal13 gene fragment were constructed, and each truncated plasmid was transfected into tumor cells to express truncated Gal13 protein. Using this strategy, the Gal13 protein was first split into two segments, namely the N-terminal (amino acids 1-68) and the C-terminal (amino acids 69-139). The full-length and truncated Gal13 plasmids and the CD44 plasmid were co-transfected into 293T cells. The above cells were lysed, and the technique of immunoprecipitation combined with immunoblotting was used to analyze which truncated proteins CD44 bound to. The specific process: Incubate the CD44 antibody with cell lysate, then enrich CD44 using microbeads, and analyze the proteins bound to CD44 by immunoblotting. The results of co-immunoprecipitation showed that CD44 could bind to full-length Gal13 and Gal13 containing the C-terminal sequence, that is, CD44 could bind to the 69-139 amino acid sequence of Gal13 (Figure 1a).

[0024] Further truncate the 69-139 amino acid sequence of Gal13 protein into 7 segments (69-78, 79-88, 89-95, 96-113, 114-120, 121-127, 128-139). Co-transfect these truncated plasmids with full-length CD44 into 293T cells, and then perform co-immunoprecipitation experiments to analyze the binding of each truncated protein to CD44. The results showed that the 96-113 peptide segment in the Gal13 protein sequence mediated the binding of Gal13 to CD44 (Figure 1b).

[0025] Based on the above research, we purified the recombinant Gal13 protein in vitro. And by designing and synthesizing the 96-113 amino acid fragment of Gal13 (small molecule polypeptide) to mimic the binding of Gal13 to CD44, thereby inhibiting the binding of CD44 to SLC7A11, and finally playing a role in promoting ferroptosis of tumor cells. This polypeptide was named Gal13 mimetic peptide, and the anti-tumor effect of Gal13 mimetic peptide was evaluated. Gal13 mimetic peptide (HYNEYEIKVNGIRIYGFV, SEQ ID NO.1) was synthesized in vitro. In addition, the key amino acids in the Gal13 mimetic peptide were mutated to synthesize Gal13 mimetic peptide-Mut (HYNEPEPKPNGIPIPGPV, SEQ ID NO.2), and Gal13 mimetic peptide-Mut was used as a negative control. First, incubate the Gal13 mimetic peptide or Gal13 mimetic peptide-Mut with the lysate of breast cancer MDA-MB-231 cells, and then it was found by immunoblotting experiment that the Gal13 mimetic peptide could bind to CD44, while the Gal13 mimetic peptide-Mut could not bind to CD44 (Figure 1d).

[0026] Subsequently, tumor cells were treated with Gal13 recombinant protein or Gal13 mimetic peptide or Gal13 mimetic peptide-Mut in combination with ferroptosis inducers. The results of immunoblotting experiments showed that the Gal13 mimetic peptide significantly inhibited the binding of CD44 to SLC7A11 by binding to CD44, and reduced the formation of the CD44 / SLC7A11 protein complex (Figure 1c, e). The CD44 / SLC7A11 protein complex plays a role in inhibiting ferroptosis. This suggests that Gal13 may promote ferroptosis by reducing the CD44 / SLC7A11 protein complex. The results of cell death detection showed that Gal13 recombinant protein or Gal13 mimetic peptide greatly enhanced the ferroptosis of tumor cells mediated by ferroptosis inducers Erastin, RSL3, ML210, FIN56, and FINO2 (Figure 2a-h).

[0027] Both radiotherapy and immunotherapy can play an anti-tumor role by inducing ferroptosis of tumor cells. Effectively inducing ferroptosis of tumor cells with compounds or drugs can significantly enhance the efficacy of tumor radiotherapy and immunotherapy. Therefore, on the basis of the above research, it was proposed to further explore the enhancing effect of Gal13 recombinant protein or Gal13 mimetic peptide on the efficacy of tumor radiotherapy and immunotherapy by inducing ferroptosis. A mouse subcutaneous tumor model was constructed by subcutaneously injecting breast cancer MDA-MB-231 cells into nude mice. When the tumor volume reached 50-100 mm 3 After that, the mice were treated with radiotherapy, Gal13 recombinant protein or Gal13 mimetic peptide, radiotherapy combined with Gal13 recombinant protein or Gal13 mimetic peptide, radiotherapy combined with Gal13 recombinant protein or Gal13 mimetic peptide and ferroptosis inhibitor Lipro-1, ferroptosis inhibitor Lipro-1, etc. It was found that neither radiotherapy alone nor Gal13 recombinant protein or Gal13 mimetic peptide alone could play a good anti-tumor effect. The combination of Gal13 recombinant protein or Gal13 mimetic peptide and radiotherapy significantly enhanced the anti-tumor effect of radiotherapy, and this enhanced effect could be reversed by the ferroptosis inducer Lipro-1, indicating that the Gal13 mimetic peptide significantly enhanced the efficacy of tumor radiotherapy by promoting radiotherapy-mediated ferroptosis of tumor cells (Figure 3a, c). In the immunotherapy mouse model, a mouse subcutaneous tumor model was constructed by subcutaneously injecting melanoma B16 cells into C57 mice. When the tumor volume reached 50-100 mm 3After the mice were treated with anti-PD1 antibody, Gal13 recombinant protein or Gal13 mimetic peptide, anti-PD1 antibody combined with Gal13 recombinant protein or Gal13 mimetic peptide, anti-PD1 antibody combined with Gal13 recombinant protein or Gal13 mimetic peptide and ferroptosis inhibitor Lipro-1, ferroptosis inhibitor Lipro-1 alone, etc., it was found that neither the administration of anti-PD1 antibody alone nor the administration of Gal13 recombinant protein or Gal13 mimetic peptide alone could achieve a good tumor inhibitory effect. However, the combination of Gal13 recombinant protein or Gal13 mimetic peptide and anti-PD1 antibody significantly enhanced the tumor inhibitory effect of anti-PD1 antibody, and this enhanced effect could be reversed by the ferroptosis inducer Lipro-1, indicating that Gal13 recombinant protein or Gal13 mimetic peptide significantly enhanced the efficacy of tumor immunotherapy by promoting immunotherapy-mediated ferroptosis of tumor cells (Figure 3 b, d).

[0028] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For those of ordinary skill in the technical field to which the present invention pertains, simple deductions or substitutions without departing from the concept of the present invention are within the protection scope of the present invention.

Claims

1. A Gal13 mimetic peptide with the amino acid sequence HYNEYEIKVNGIRIYGFV.

2. A nucleotide sequence encoding the Gal13 mimetic peptide according to claim 1.

3. Use of the Galectin-13 recombinant protein or the Gal13 mimetic peptide according to claim 1 in the preparation of an anti-tumor drug or a synergist for tumor radiotherapy or immunotherapy.

4. The application according to claim 3, characterized in that The tumor is selected from breast cancer and melanoma.

5. The application according to claim 3, wherein The immunotherapy is selected from PD-1 and PD-L1 immunotherapy.

6. Use of the Galectin-13 recombinant protein or the Gal13 mimetic peptide according to claim 1 in the preparation of a synergist for ferroptosis inducers.

7. The application according to claim 6, characterized in that, The ferroptosis inducer is selected from at least one of Erastin, RSL3, ML210, FIN56, and FINO2.

8. A composition for treating tumors, comprising: At least one ferroptosis inducer; At least one of the Gal13 mimetic peptide and the Galectin-13 recombinant protein according to claim 1.

9. The composition according to claim 8, characterized in that, The tumor is selected from breast cancer and melanoma.

10. The composition according to claim 8, characterized in that, The death inducer is selected from at least one of Erastin, RSL3, ML210, FIN56, and FINO2.

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