Oligopeptide, lentivirus and cell for expressing oligopeptide and application of oligopeptide

Abstract

The invention provides an oligopeptide, and a lentiviral expression vector and a recombinant cell for expressing the oligopeptide. The invention further provides application of the compound in tumor treatment. After the oligopeptide is delivered to HT1080 cells through a lentivirus system, the intracellular SLC7A11 protein level is reduced, the intracellular Fe < 2 + > and lipid peroxidation level is remarkably increased, and the oligopeptide has a remarkable inhibition effect on cell proliferation and has the tumor treatment potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of an SLC7A11-derived peptide FP-01 as a ferroptosis inducer in tumor treatment. Background Technology

[0002] Ferroptosis is an iron-dependent programmed cell death characterized by lipid peroxidation and glutathione depletion. Morphologically, ferroptosis is characterized by mitochondrial atrophy, increased membrane density, and reduced mitochondrial cristae. Mechanistically, it is primarily regulated by multiple pathways, including the cysteine-glutamate antitransporter system (System XC-), the mevalonate pathway, and the mitochondrial DHODH pathway. As a novel form of cell death, the biological effects of ferroptosis activation or inhibition have attracted widespread attention and research. Ferroptosis inhibitors can protect normal tissues by inhibiting ferroptosis and are potential or adjuvant therapies for age-related injuries such as acute kidney injury, acute liver injury, radiation injury, and neurodegenerative diseases. Ferroptosis activators can directly induce ferroptosis in some tumor cells or, in combination with clinical oncology drugs, achieve synergistic therapeutic effects on tumors. However, the toxicity and unclear mechanisms of action of both ferroptosis inhibitors and ferroptosis activators are significant obstacles to their clinical translation.

[0003] The cysteine-glutamate antitransporter system (System XC-) is a cell membrane-embedded dimer composed of SLC3A2 and SLC7A11, and is one of the main mechanisms regulating ferroptosis. SLC7A11 is the main subunit of System XC- that functions, and it is highly expressed in various cancer cells. Patients with high SLC7A11 expression have significantly lower survival than those with low expression, indicating that high SLC7A11 expression is a poor prognostic factor for tumors. Mechanistically, overexpression of SLC7A11 leads to increased biosynthesis of reduced glutathione (GSH), thereby inhibiting lipid peroxidation, suppressing ferroptosis, and promoting tumor growth. Therefore, targeting and regulating SLC7A11 or the System XC- system is a potential therapeutic approach for cancer.

[0004] SLC7A11 is a multi-transmembrane protein composed of an N-terminus (1-43), a C-terminus (471-501), and a core transmembrane region (44-470) located in the cytoplasm. Summary of the Invention

[0005] The purpose of this invention is to provide a short peptide drug (FP-01) with ferroptosis activity and its application in tumor treatment. FP-01, as a ferroptosis-promoting agent, can promote ferroptosis in tumor cells by downregulating the protein level of the core subunit SLC7A11 in the System XC- pathway, thereby alleviating tumor-bearing stress in mice and demonstrating potential for tumor treatment.

[0006] The present invention first provides a short peptide, the amino acid sequence of which is SEQ ID NO:1.

[0007] The present invention further provides the encoding gene of the above-mentioned short peptide, the nucleotide sequence of which is SEQ ID NO:2.

[0008] The present invention provides a recombinant lentiviral expression vector containing the above-mentioned coding gene.

[0009] In one embodiment of the invention, it comprises a CMV promoter; preferably a pLVX lentiviral vector.

[0010] The present invention also provides a recombinant cell comprising the encoding gene of the recombinant lentiviral expression vector described above;

[0011] In one embodiment of the present invention, the recombinant cells are prepared by a method comprising the following steps:

[0012] The recombinant lentiviral expression vector described above was co-transfected with packaging plasmids psPAX2 and pMD2G into HEK293T cells. After 48-72 hours of transfection, the culture medium containing viral particles was collected. The culture medium containing viral particles was then co-cultured with the target cells. After 48 hours, cell clones that stably expressed the virus were screened using puromycin.

[0013] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned short peptide, recombinant lentiviral expression vector, or recombinant cell.

[0014] In another aspect, the present invention provides the use of the above-mentioned short peptides, recombinant lentiviral expression vectors, or recombinant cells in the preparation of medicaments for treating tumors.

[0015] In one embodiment of the invention, the tumor is selected from one or more of fibrosarcoma (FS), sarcoma (SARC), breast cancer (BRCA), hepatocellular carcinoma (LIHC), or pancreatic cancer (PAAD).

[0016] In one embodiment of the invention, the tumor is a fibrosarcoma.

[0017] The beneficial effects of the above-described technical solution of the present invention are as follows:

[0018] The short peptide FP-01 provided by this invention is an active peptide that can induce ferroptosis in cells. It can promote ferroptosis in tumor cells by downregulating the protein level of the core subunit SLC7A11 in the cystine-glutamate antitransporter system (System XC-), thereby alleviating tumor-bearing pressure in mice and showing potential for tumor therapy. Attached Figure Description

[0019] Figure 1 High expression of SLC7A11 is a poor prognostic factor for tumors. (A) Kaplan-Meier survival curve analysis of SARC (sarcoma) patients in the TCGA database; (B) Kaplan-Meier survival curve analysis of BRCA (breast cancer) patients in the TCGA database; (C) Kaplan-Meier survival curve analysis of LIHC (hepatocellular carcinoma) patients in the TCGA database; (D) Kaplan-Meier survival curve analysis of PAAD (pancreatic cancer) patients in the TCGA database;

[0020] (E) Western blot analysis of SLC7A11 protein levels in liver cancer patients and corresponding adjacent normal samples;

[0021] Figure 2 FP-01 downregulates intracellular protein expression levels. (A) Cartoon diagram of SLC7A11 protein structure, with yellow marking at the N-terminus, magenta marking at the C-terminus, and gray marking at the transmembrane region; (B) Schematic diagram of truncated expression region of SLC7A11 protein, with yellow marking at the N-terminus, magenta marking at the C-terminus, and gray marking at the transmembrane region; (C) Western blot detection of different SLC7A11 protein truncated expression levels; (D) Multiple sequence alignment and conservation analysis of the C-terminus of SLC7A11 proteins in human (Q9UPY5_HUM), mouse (Q9WTR9_MOU), and bovine (G3N179_BOV) species, with the relevant secondary structure of human SLC7A11 protein shown as a schematic diagram above the multiple sequence alignment results; (E) Western blot detection of the effect of FP-01 on cellular SLC7A11 protein levels; (F) Western blot detection of the recovery of SLC7A11 protein level decline caused by FP-01 by proteasome inhibitors (MG132) and autophagy inhibitors (CQ and Baf1); (G) Western blot detection of the effect of FP-01 on SLC7A11 protein level decline; blot analysis of the effect of FP-01 on the ubiquitination level of SLC7A11 protein;

[0022] Figure 3FP-01 promotes ferroptosis in HT1080 cells in vitro; (A) The left figure shows the effect of crystal violet assay on cell proliferation and the recovery of FP-01 proliferation inhibition by the ferroptosis inhibitor (Lip1). The right figure shows the statistical results of cell clone numbers in the relevant experimental groups; (B) The left figure shows the effect of FerroOrange staining on intracellular Fe in HT1080 cells by FP-01. 2+ The effect of FP-01 on intracellular Fe in HT1080 cells is shown in the right figure, which is a statistical representation of fluorescence signal intensity. (C) The left figure shows the effect of FP-01 on intracellular Fe in HT1080 cells by flow cytometry. 2+ (D) Effect of FP-01 on GSH levels in HT1080 cells; (E) Effect of C11-BODIPY staining on intracellular lipid peroxidation levels in HT1080 cells, with the right figure showing the relative signal intensity statistics; (F) Effect of FP-01 on intracellular lipid peroxidation levels in HT1080 cells, with the left figure showing the effect of C11-BODIPY staining on GSH levels in HT1080 cells, and the right figure showing the relative signal intensity statistics.

[0023] Figure 4 The effects of FP-01 on tumors were detected in a mouse subcutaneous tumor model. (A) Tumor status 15 days after subcutaneous tumor formation in mice, n=6; (B) Tumor weight statistics 15 days after subcutaneous tumor formation in mice, n=6; (C) Overall growth trend statistics of mouse subcutaneous tumors, n=6; (D) Individual growth trend statistics of mouse subcutaneous tumors (left figure is the control group, right figure is the FP-01 treatment group); (E) Immunohistochemical analysis of the effects of FP-01 on tumor ki67 / 4-HNE and SLC7A11 protein levels; (F) Western blot detection of the effect of FP-01 on tumor SLC7A11 protein levels. Detailed Implementation

[0024] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0025] Unless otherwise specified, all reagents used in this embodiment are of analytical grade, and the progress of all chemical reactions is detected by thin-layer chromatography.

[0026] The reagent and consumable information used in the experiments of this invention is as follows:

[0027]

[0028] This invention truncates SLC7A11 (SLC7A11) 1-470 SLC7A11 44-470SLC7A11 44-501 Expression analysis showed that SLC7A11 1-501 and SLC7A11 44-501 The expression level was significantly higher than that of SLC7A11 1-470 and SLC7A11 44-470 It is speculated that the C-terminal region plays an important role in the expression level, stability, or function of the SLC7A11 protein. Based on this, overexpression of SLC7A11 in tumor cells is recommended. 470-501 Western blotting analysis results showed that SLC7A11 470-501 Overexpression of SLC7A11 significantly reduced intracellular SLC7A11 protein levels, which were significantly restored by the proteasome inhibitor MG132, indicating that SLC7A11 protein levels are significantly reduced. 470-501 The protein level of SLC7A11 can be directly regulated intracellularly via the ubiquitin-proteasome pathway. At the cellular level, SLC7A11... 470-501 It can significantly increase intracellular Fe 2+ SLC7A11 significantly inhibited cell proliferation by reducing lipid peroxidation levels, and this inhibition was significantly reversed by the ferroptosis inhibitor (Lip1). Furthermore, in a mouse tumor burden model, SLC7A11... 470 -501 It can significantly inhibit tumor size, and histochemical experiments showed that tumor proliferation (Ki67) was inhibited, while the level of tumor lipid peroxidation (4-HNE) was significantly increased. These results indicate that SLC7A11... 470-501 It is an active peptide that can induce ferroptosis in cells, named "FP-01". FP-01 can downregulate the intracellular protein level of SLC7A11, and has the ability to inhibit cell proliferation and reduce tumor-bearing stress in mice, showing potential for tumor treatment. This result has not yet been reported domestically or internationally, and has good application prospects.

[0029] Example 1: FP-01 downregulates SLC7A11 protein levels in tumor cells

[0030] 1.1 Experimental Methods

[0031] 1) Kaplan-Meier survival curve analysis: Survival prognostic data of common and frequently occurring diseases such as SARC, LIHC, PADD, and BRCA were analyzed in the TCGA database using R packages such as Survival;

[0032] 2) Detection of SLC7A11 protein level in liver cancer samples: Liver cancer tissue and corresponding adjacent normal tissue were collected from clinical liver cancer patients (6 cases). After washing with PBS, the tissue was homogenized and 200 μL of 1% SDS was added. The sample was lysed at 100 ℃ for 20 min. Then, 70 μL of LDS Sample Buffer (4X) was added and the sample was lysed at 100 ℃ for 10 min. 10 μL of the sample was taken for Western blotting experiment, and the relevant proteins were detected with SLC7A11 antibody (CST, 12691S) and GAPDH antibody (proteintech, 60004-1).

[0033] 3) SLC7A11 truncated fragment expression analysis: Based on the elemental features provided by the Uniprot database (https: / / www.uniprot.org / uniprotkb / Q9UPY5 / entry), the structural information provided by the PDB database (https: / / www.rcsb.org / structure / 7EPZ), and the results of multiple sequence alignments (Q9UPY5, Q9WTR6, and G3N179), the expression of SLC7A11 truncated fragments was analyzed. 1-501 SLC7A11 1-492 SLC7A11 1-470 SLC7A11 44-470 SLC7A11 44-501Fragments were constructed into the pEGFP-Myc vector, and each fragment was fused with a Myc tag for detection at its C-terminus. The vector was transiently transfected into 293T cells, and cells were collected after 48 hours. Western blotting was used to detect the expression levels of each fragment of SLC7A11 protein, with GAPDH as an internal control protein. The amino acid sequence of SLC7A11 is SEQ ID NO:3: MVRKPVVSTISKGGYLQGNVNGRLPSLGNKEPPGQEKVQLKRKVTLLRGVSIIIGTIIGAGIFISPKGVLQNTGSVGMSLTIWTVCGVLSLFGALSYAELGTTIKKSGGHYTYILEVFGPLPAFVRVWVELLIIRPAATAVISLAFGRYILEPFFIQCEIPELAIKLITAVGITVVMVLNSMSVSWSARIQIFLTFCKLTAILIIIVPGVMQLIKGQTQNFKDAFSGRDSSITRLPLAFYYGMYAYAG WFYLNFVTEEVENPEKTIPLAICISMAIVTIGYVLTNVAYFTTINAEELLLSNAVAVTFSERLLGNFSLAVPIFVALSCFGSMNGGVFAVSRLFYVASREGHLPEILSMIHVRKHTPLPAVIVLHPL TMIMLFSGDLDSLLNFLSFARWLFIGLAVAGLIYLRYKCPDMHRPFKVPLFIPALFSFTCLFMVALSLYSDPFSTGIGFVITLTGVPAYYLFIIWDKKPRWFRIMSEKITRTLQIILEVVPEEDKL.

[0034] 4) Construction of FP-01 overexpression cell line: FP-01 (SLC7A11) 471-501 The amino acid sequence of ) is SEQ ID NO:1 (WDKKPRWFRIMSEKITRTLQIILEVVPEEDKL), and FP-01 (SLC7A11) is also present. 471-501The coding sequence SEQ ID NO:2 (tgggacaagaaacccaggtggtttagaataatgtcagagaaaataaccagaacattacaaataatactggaagttgtaccagaagaagataagtta) was cloned into the pLVX lentiviral vector (Clontech) and co-transfected with the packaging plasmid (psPAX2 / pMD2G) into HEK293T cells. After 48-72 hours of transfection, the culture medium containing the virus particles was collected. The culture medium containing the virus particles was then co-cultured with the target cells. After 48 hours, cell clones that stably expressed FP-01 were screened by puromycin.

[0035] 5) Analysis of the effect of FP-01 on SLC7A11 protein level: Using the established FP-01 stable expression cell line, after treatment with MG132 (10μM), CQ (15μM), Baf1 (5nM) and DMSO for 12 hours, samples were collected for Western Blot experiments to detect SLC7A11 protein level, with GAPDH as internal control protein.

[0036] 6) Analysis of the effect of FP-01 on the ubiquitination level of SLC7A11 protein: Using the established FP-01 stable expression cell line and related control cell lines, samples were collected after treatment with MG132 (10μM) reagent for 8 hours. IP experiment was performed using SLC7A11 antibody. Western blotting was used to detect the SLC7A11 protein level and its ubiquitination modification level, with GAPDH as the internal reference protein.

[0037] 1.2 Experimental Results

[0038] Analysis of the relationship between SLC7A11 and prognosis in patients with common cancers such as SARC (sarcoma), BRCA (breast cancer), LIHC (hepatocellular carcinoma), and PAAD (pancreatic cancer) using the TCGA database revealed that patients with high SLC7A11 expression had a poorer prognosis. Figure 1 The presence of SLC7A11 in hepatocellular carcinoma (HCC) tissues and adjacent normal tissues from clinical HCC patients (AD) indicates that high SLC7A11 expression is a poor prognostic factor for these common tumors. Western blot analysis of HCC tissues and adjacent normal tissues from clinical patients revealed that SLC7A11 expression was significantly higher in HCC tissues than in adjacent normal tissues. Figure 1 (E).

[0039] SLC7A11 is a major subunit of the cystine-glutamate antitransporter system (System XC-), and is one of the main mechanisms regulating ferroptosis. Based on structural-functional domain analysis, different truncated expression fragments of SLC7A11 were constructed. Figure 2 China A, Figure 2 (B) The expression levels of different segments of SLC7A11 were analyzed by Western blotting, and the results showed that SLC7A11... 1-470 and SLC7A11 44-470 The protein expression level of SLC7A11 was significantly lower than that of SLC7A11 containing the C-terminal region (471-401). 1-501 and SLC7A11 44-501 Related fragments ( Figure 2 Secondary structure analysis shows that the C-terminal region (493-501) of human SLC7A11 is a disordered structure. Figure 2 (D), SLC7A11 1-492 Expression levels and SLC7A11 1-501 The expression levels are comparable ( Figure 2 The presence of C-terminal regions (471-491) in SLC7A11 indicates that these regions play a crucial role in the protein's expression level, stability, and function. Combined with multiple sequence alignment, although the C-terminal region (493-501) of SLC7A11 is a disordered structure, its sequence is relatively conserved. Figure 2 Based on this, only its C-terminal region (SLC7A11) is overexpressed in tumor cells. 471-501 Western blot analysis showed that SLC7A11 470-501 Overexpression significantly reduced intracellular SLC7A11 protein levels. Figure 2 (E), and can be significantly reversible by the proteasome inhibitor MG132 ( Figure 2 In the middle F), the ubiquitination level of SLC7A11 protein increased ( Figure 2 (G) indicates that SLC7A11 470-501 It can directly regulate the intracellular protein level of SLC7A11 through the ubiquitin-proteasome pathway.

[0040] Example 2: FP-01 induces ferroptosis in tumor cells

[0041] 2.1 Experimental Methods

[0042] 1) Plate colony formation assay (crystal violet): ① Cell preparation: Collect cells or pre-treated cells after normal digestion with trypsin, resuspend them in complete culture medium to form a single-cell suspension, and count them; ② Cell seeding: Adjust the cell suspension concentration to 1000 cells / mL, and seed 1000 cells in each well of a 6-well plate; ③ Cell colony culture: Continue to culture in a cell culture incubator for about 1-3 weeks, changing the medium every 3 days and observing the cell status; ④ Fixation and staining: After culture, wash the cells twice with PBS, add 1 mL of 4% paraformaldehyde to each well to fix the cells for 30-60 min, wash twice more with PBS, and add 1 mL of 0.1% crystal violet staining solution to each well for 10-20 min; ⑤ Washing and drying: Wash 3 times with ddH2O, dry and photograph; ⑥ Counting: Count the number of colonies formed under a microscope, or photograph the entire 6-well plate and each well.

[0043] 2) Flow cytometry detection of Fe 2+ (FerroOrange): ① Cell preparation: 12-well plate, 1x10 cells per well 5 ① Cell seeding and overnight culture; ② Dye incubation: First, wash cells three times with serum-free medium, then incubate cells for 30 min with serum-free medium containing 0.2 μM Ferro Orange dye; ③ Cell collection: After normal digestion with trypsin, collect the pretreated cells, wash three times with PBS, and then resuspend them in a single-cell suspension; ④ Flow cytometry detection: PE, SSC

[0044] 3) Fluorescence detection of Fe 2+ (FerroOrange): ① Cell slide preparation: Place appropriately sized coverslips in a 12-well plate, 1x10 cells per well. 5 ① Cell seeding and overnight culture; ② Dye incubation: First, wash the cells three times with serum-free medium, then incubate the cells for 30 min with serum-free medium containing 0.2 μM FerroOrange dye; ③ Sample preparation: Wash the coverslips three times with PBS, take out the prepared coverslips, add an appropriate amount of anti-quenching agent containing DAPI, and load them onto a suitable-sized slide; ④ Fluorescence microscopy photography and statistics: Take pictures using the red and blue light channels respectively, and count the fluorescence intensity using Fiji software.

[0045] 4) GSH detection: The preparation of standard samples was carried out in accordance with Elabscience. ® Reduced glutathione (GSH) colorimetric assay kit (E-BC-K030-M) instruction manual; The general procedure for GSH detection of the target sample is as follows: ① Preparation of cell homogenate supernatant: Take 10 6Cells were homogenized with 500 μL PBS; after homogenization, the cells were centrifuged at 10000 × g for 10 min at 4℃, and the supernatant was collected and placed on ice for testing; a portion of the supernatant was reserved for protein concentration determination; ② Preparation of test supernatant: 0.1 mL of the test sample was taken, 0.1 mL of reagent I was added, mixed, centrifuged at 4500 × g for 10 min, and the supernatant was collected for testing; ③ 25 μL of reagent III was added to the standard wells (100 μL of GSH standard solutions of different concentrations of 0 / 10 / 20 / 40 / 50 / 60 / 80 / 100 μmol / L), the test wells (100 μL of test supernatant), and the test blank wells (100 μL of reagent I); ④ 100 μL of reagent II was added to each well in step ③; ⑤ The plate was shaken for 1 min with an ELISA reader, allowed to stand for 5 min, and the OD value was measured at 405 nm with an ELISA reader.

[0046] 5) Flow cytometry detection of ROS (BODIPY-C11): ① Cell preparation: 12-well plate, 1x10 cells per well 5 ① Seed cells and culture overnight; ② Dye incubation: Add 1 μM BODIPY-C11 directly to the culture medium and incubate for 30 min in a cell culture incubator; ③ Cell collection: Collect the pretreated cells after normal digestion with trypsin, wash three times with PBS and then resuspend them as a single-cell suspension; ④ Flow cytometry detection: Detect FITC and SSC by flow cytometry.

[0047] 6) Fluorescence detection of ROS (BODIPY-C11): ① Cell slide preparation: Place appropriately sized coverslips in a 12-well plate, 1x10 cells per well. 5 ① Cells were seeded and cultured overnight; ② Dye incubation: 1 μM BODIPY-C11 was added directly to the culture medium and incubated in a cell culture incubator for 30 min; ③ Sample preparation: The samples were washed three times with PBS, and the prepared coverslips were removed. An appropriate amount of anti-quenching agent containing DAPI was added and the coverslips were loaded onto appropriately sized slides; ④ Fluorescence microscopy photography and statistics: The samples were photographed using the red, green, and blue light channels, and the fluorescence intensity was statistically analyzed using Fiji software.

[0048] 2.2 Experimental Results

[0049] A stable FP-01-expressing HT1080 cell line and an empty vector control cell line were constructed using a lentiviral system. At the cellular level, crystal violet cloning assays showed that FP-01 significantly inhibited the number of colonies forming in HT1080 cells, but this inhibition was significantly reversed by the ferroptosis inhibitor (Lip1). Figure 3 (A) suggests that FP-01 may inhibit its in vitro proliferation by promoting ferroptosis. Further analysis using flow cytometry and fluorescence imaging techniques was conducted to detect the effect of FP-01 on cellular Fe...2+ The study investigated the effects of FP-01 on common ferroptosis markers such as GSH and ROS, and found that FP-01 significantly increased cellular Fe. 2+ and ROS levels, while intracellular GSH levels decreased significantly ( Figure 3 The study further confirmed that FP-01 inhibits the in vitro proliferation and expansion of cells through ferroptosis (BF).

[0050] Example 3: FP-01 reduces tumor-bearing stress in mice

[0051] 3.1 Experimental Methods

[0052] 1) Subcutaneous tumorigenesis experiment in nude mice: ① Cell preparation: FP-01 stably expressing cells and control cells were collected after normal digestion with trypsin and washed three times with PBS; the cells were then resuspended in PBS to form a single-cell suspension with a cell concentration of 1x10⁻¹. 7 Cells / mL; then mix with matrix gel 1:1 as needed to achieve a cell concentration of 5 x 10⁻⁶ cells / mL. 6 ① Cells / mL. ② Subcutaneous cell inoculation: 5-week-old BALB / c nude mice were randomly divided into 2 groups of 6 mice each, and 100 μL of relevant cells were subcutaneously inoculated.

[0053] 2) Tumor size detection: Starting from the fifth day after subcutaneous inoculation of tumor cells, the tumor volume (0.5 × major axis × minor axis) was measured daily until the 15th day. GraphPad Prism software was used to analyze and statistically analyze the overall tumor growth trend of each group and the tumor growth trend of relevant individuals.

[0054] 3) Tumor sample-related experiments: Fifteen days after subcutaneous inoculation of tumor cells into mice, subcutaneous tumor tissue was harvested. After washing with PBS, the tumors were first photographed and weighed. The weight of each group of tumors was analyzed and counted using GraphPad Prism software. Then, the tumor tissue was cut, and a portion was used for immunohistochemical experiments to detect indicators such as ki67 / 4-HNE / SLC7A11. The other portion was used to extract proteins, and the SLC7A11 protein level was detected by Western Blot.

[0055] 3.2 Experimental Results

[0056] Results from a mouse subcutaneous tumor model showed that FP-01 significantly inhibited the size of HT1080 tumors. Figure 4 Immunohistochemical experiments on tumor tissue confirmed that FP-01 inhibited tumor proliferation (ki67), increased tumor lipid peroxidation (4-HNE), and reduced the protein level of SLC7A11 in tumor cells. Figure 4 Meanwhile, Western blot experiments also confirmed that FP-01 can downregulate the protein level of SLC7A11 in tumor cells (E); Figure 4 (Middle F).

[0057] Conclusion and Advantages of the Invention

[0058] The short peptide FP-01 described in this invention exhibits the ability to promote tumor ferroptosis and inhibit tumor growth at the cellular and mouse tumor model levels. Mechanistically, FP-01 increases the ubiquitination level of SLC7A11, thereby downregulating SLC7A11 protein levels, disrupting the function of the cysteine-glutamate antitransporter system (System XC-), reducing intracellular GSH levels, and increasing intracellular Fe. 2+ The levels of ions and ROS ultimately induce ferroptosis in tumor cells, thereby inhibiting tumor proliferation.

[0059] Compared to small molecule drugs, peptide drugs like FP-01 have a large surface area, resulting in a larger binding interface with the target protein. This leads to higher affinity and specificity for the target, resulting in greater efficacy while effectively reducing related treatment side effects. Furthermore, because FP-01 peptide inhibitors have multiple interaction sites on their surface, the loss of individual binding sites does not lead to a complete loss of binding ability. This characteristic makes them more tolerant of subtle changes in the target protein structure. Therefore, the short peptide drug FP-01 described in this invention not only possesses good anti-tumor effects but also demonstrates significant innovation and potential application value.

[0060] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A short peptide having the amino acid sequence SEQ ID NO:

1.

2. The gene encoding the short peptide as described in claim 1, wherein the nucleotide sequence is SEQ ID NO:

2.

3. A recombinant lentiviral expression vector comprising the coding gene as described in claim 2.

4. The recombinant lentiviral expression vector as described in claim 3, comprising a CMV promoter; preferably a pLVX lentiviral vector.

5. A recombinant cell comprising the encoding gene of the recombinant lentiviral expression vector as described in claim 3 or 4; Preferably, the recombinant cells are prepared by a method comprising the following steps: The recombinant lentiviral expression vector described in claim 3 or 4 was co-transfected with packaging plasmids psPAX2 and pMD2G into HEK293T cells. After 48-72 hours of transfection, the culture medium containing viral particles was collected. The culture medium containing viral particles was then co-cultured with the target cells. After 48 hours, cell clones that stably expressed the virus were screened using puromycin.

6. A pharmaceutical composition comprising the short peptide of claim 1, the recombinant lentiviral expression vector of claim 3 or 4, or the recombinant cell of claim 5.

7. The use of the short peptide of claim 1, the recombinant lentiviral expression vector of claim 3 or 4, or the recombinant cell of claim 5 in the preparation of a medicament for treating tumors.

8. The application as described in claim 7, wherein, The tumor is selected from one or more of fibrosarcoma (FS), sarcoma (SARC), breast cancer (BRCA), hepatocellular carcinoma (LIHC), or pancreatic cancer (PAAD).

9. The application as described in claim 8, wherein the tumor is a fibrosarcoma.

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