Anti-tumor short peptide, pharmaceutical composition and use thereof

Abstract

Disclosed is an anti-tumor short peptide. The short peptide comprises an amino acid sequence shown as Seq ID No: 1; and the N-terminus of the amino acid sequence shown as Seq ID No: 1 is further linked to a cell-penetrating peptide. The short peptide inhibits tumor growth by inducing tumor cell differentiation, the tumor being glioma, and the short peptide being a short peptide derived from LGP2. Also disclosed is an anti-tumor pharmaceutical composition comprising a PD-1 antibody and the short peptide. Also disclosed is the use of the short peptide or the anti-tumor pharmaceutical composition in the preparation of an anti-tumor drug. A novel differentiation therapy route is provided for tumors, especially gliomas.

Description

A short antitumor peptide, pharmaceutical composition and its application Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an anti-tumor short peptide, a pharmaceutical composition, and its application. Background Technology

[0002] Differentiation therapy is an innovative treatment strategy that inhibits malignant proliferation by reactivating the differentiation potential of cancer cells, promoting their development towards a mature state. Unlike traditional cytotoxic therapy, this approach does not directly kill cancer cells but reduces their tumorigenicity by altering their differentiation state. Due to its fewer side effects, limited impact on normal cells, and long-lasting efficacy, it has attracted considerable attention in recent years. The earliest successful case came from the application of all-trans retinoic acid (ATRA) in acute promyelocytic leukemia (APL). This therapy, by degrading the PML-RARα fusion protein, relieved the differentiation arrest of leukemia cells, significantly improving patient prognosis. Currently, with the development of molecular biology techniques, differentiation therapy is gradually revealing its potential new targets in the treatment of malignant tumors and is increasingly being applied to solid tumors such as neuroblastoma and hepatocellular carcinoma. However, the therapeutic effects on solid tumors and the specific mechanisms of differentiation induction remain unclear.

[0003] The RIG-I-like receptor family (RLRs) includes retinoic acid-induced gene-I (RIG-I), melanoma differentiation-associated receptor 5 (MDA5), and Laboratory Genetics and Physiology 2 (LGP2). They interact with exogenous RNA, causing conformational changes. MDA5 and RIG-I, upon sensing non-self RNA, expose their CARD domains. The exposed CARD interacts with the CARD domain of the mitochondrial antiviral signaling protein (MAVS), promoting the activation of transcriptional regulators including IRF-3 and NFκB, leading to the transcription of type I interferon (IFN) and IFN-inducible genes (ISGs), which participate in the immune response to viral infection. IFN and ISGs not only affect viral replication but also have a direct tumor-killing effect. Although LGP2 can bind to RNA and its binding is stronger than that of MDA5 and RIG-I, it lacks a CARD domain or other signal transduction domains and cannot transmit signals, thus failing to directly interact with the adaptor protein MAVS to induce the production of IFN and inflammatory factors. The function of LGP2 in immune responses remains controversial. Numerous studies have shown that LGP2 promotes MDA5 antiviral signaling. LGP2 is primarily involved in innate and antiviral immunity, but its role in tumors has not yet been reported. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an anti-tumor short peptide, a pharmaceutical composition and its application.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] On one hand, the present invention provides an anti-tumor short peptide comprising an amino acid sequence as shown in Seq ID No: 1.

[0007] As a further improvement of the present invention, the N-terminus of the amino acid sequence shown in Seq ID No:1 is also connected to a membrane-penetrating peptide.

[0008] Furthermore, the amino acid sequence of the transmembrane peptide is shown in Seq ID No: 2.

[0009] Furthermore, the short peptide inhibits tumor growth by inducing tumor cell differentiation.

[0010] Furthermore, the tumor is a glioma.

[0011] Furthermore, the short peptide is a short peptide derived from LGP2.

[0012] On the other hand, the present invention also provides an antitumor pharmaceutical composition comprising a PD-1 antibody and the aforementioned short peptide.

[0013] Furthermore, the present invention also provides the application of the above-mentioned short peptide in the preparation of antitumor drugs.

[0014] In another aspect, the present invention also provides the use of the above-mentioned antitumor pharmaceutical composition in the preparation of antitumor drugs.

[0015] This invention investigates the antitumor activity of the aforementioned short peptide (H1), examining its effects on the survival of glioma cell lines U87MG and T98G, its influence on glioma organoid growth and clonogenic capacity, and its effect on inducing the expression of differentiation markers. Subsequently, tumor growth was observed in an experiment involving intracranial implantation of gliomas in BALB / c nude mice. The results indicate that H1 can be used as a short peptide to inhibit tumor growth and induce differentiation. Further experiments show that the H1 short peptide can enhance the efficacy of PD-1 antibody therapy and can be used in combination for antitumor treatment. This invention provides a novel differentiation therapy approach for tumors, especially gliomas. Attached Figure Description

[0016] The above is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0017] Figure 1 shows the expression of markers indicating that the H1 short peptide inhibits glioma cell growth and induces differentiation.

[0018] Figure A shows the effect of H1 on the survival of U87MG and T98G cells; Figures B and C show the effect of H1 on the colony formation of U87MG and T98G cells; Figure D shows the effect of H1 on the growth of glioma organoids; Figures E and F show the effect of H1 on the protein levels of organoid differentiation markers.

[0019] Figure 2 shows that the H1 short peptide significantly inhibits glioma growth in tumor-bearing mice and induces glioma cell differentiation; where:

[0020] A is a schematic diagram of the U87MG-Luc tumor xenograft; B and C are bioluminescent images (n=3) of the orthotopic glioma in mice on day 14 after tumor implantation and the corresponding statistical analysis of fluorescence values; D is a graph showing the size of the orthotopic glioma after HE staining (within the blue dashed box); E is a graph showing the protein expression levels of differentiation markers TUBB3 and GFAP in four groups (H1, Hnc, PBS, TMZ) of mouse gliomas after H1 treatment using immunohistochemistry; F is a graph showing the survival curve analysis of mice.

[0021] Figure 3 shows the effect of H1 short peptide significantly enhancing PD-1 antibody treatment, where:

[0022] A is a schematic diagram of Gl261-Luc tumor xenografts; B and C are bioluminescent images (n=3) of orthotopic gliomas in mice on day 14 after tumor implantation, along with corresponding statistical analysis of fluorescence values; D is a mouse survival curve analysis diagram; E is a diagram showing the size of orthotopic gliomas (within the blue dashed box) after HE staining; F and H are statistical analysis of CD8 and TNFα levels and fluorescence values ​​of CD8 and TNFα detected by immunohistofluorescence in six groups (PBS, Hnc, H1, aPD-1, aPD-1+Hnc, aPD-1+H1) of mouse gliomas after treatment with H1 and PD-1 antibodies.

[0023] Detailed Implementation

[0024] This invention discovers that LGP2 interacts with MCCC1 (methylcrotonyl-CoA carboxylase 1), upregulates the protein levels of MCCC1 and MAVS, and promotes the interaction between MCCC1 and MAVS, thereby activating the production of downstream type I IFN and interferon-stimulated genes and inhibiting glioma growth.

[0025] This invention experimentally revealed that LGP2 interacts with MCCA, thereby inhibiting the enzymatic activity of MCC. Ultimately, this upregulation of intracellular crotonylation modification levels is mediated by ECHS1. Histone H3K18cr crotonylation modification enhances the transcriptional level of PPARGC1A. PGC1a can influence mitochondrial division by promoting DRP1 phosphorylation, producing an anti-Warburg effect. Ultimately, this drives glioma cell differentiation.

[0026] The H1 short peptide derived from LGP2 can mimic the function of LGP2. On the one hand, it interacts with MCCA and MCCB to inhibit the enzymatic activity of MCC and induce differentiation. On the other hand, it stabilizes the protein of MCCA, thereby activating MAVS and its downstream pathways.

[0027] The H1 short peptide derived from LGP2 has the function of inhibiting glioma growth and inducing glioma cell differentiation in gliomas. This will be illustrated through experiments below:

[0028] I. Experimental Methods:

[0029] (1) Cell culture

[0030] Cell lines U87MG, T98G, and glioma organoids were cultured in Dulbecco-modified Eagle medium (DMEM; Meilunbio: MA0212). The medium consisted of 10% fetal bovine serum (FBS) and antibiotics (penicillin (100 U / ml) / streptomycin (0.1 mg / ml)). Cell culture conditions were 5% CO2 and 37°C, with the medium changed daily.

[0031] (2) The H1 short peptide derived from LGP2 was synthesized by Nanjing Leon Biotechnology, with Hnc serving as a negative control. The amino acid sequences and N-terminal transmembrane peptide (TAT) sequences of H1 and Hnc are as follows:

[0032] H1: YGRKKRRQRRR-KDTVYNVIMSQYLELKL (Seq ID No: 2- Seq ID No: 1)

[0033] Hnc: YGRKKRRQRRR-KTRAAAYVAKRHLET (Seq ID No: 2- Seq ID No: 3)

[0034] It should be noted that H1 and Hnc used in the following experiments contain sequences of the transmembrane peptide (TAT). However, the transmembrane peptide YGRKKRRQRRR (Seq ID No.: 2) is only used to carry the corresponding sequence (Seq ID No.: 1: KDTVYNVIMSQYLELKL) into the cell; the actual sequence that exerts the antitumor effect should be the one it carries. In addition to the aforementioned transmembrane peptide, other types of transmembrane peptides can also be used. This embodiment only uses the transmembrane peptide (Seq ID No.: 2) as an example for illustration.

[0035] (3) Western blot assay

[0036] To detect the induced expression of differentiation marker proteins by the H1 short peptide, Western blot assays were used for protein validation. Western blotting and IP cell lysis buffer (Beyotime, product number: P0013) were used to lyse glioma organoids and collect proteins. 20 μg of total protein from each group was used for protein electrophoresis. After transfer, the membrane was incubated with 5% skim milk at 22°C for 2 hours, and then incubated overnight with primary antibody at 4°C. The major antibody and its dilutions are as follows:

[0037] Beta-Actin Mouse mAb (Proteintech #23660-1-AP): Dilution-1:5000;

[0038] GFAP Mouse mAb (Proteintech #60190-1-Ig): Dilution-1:5000;

[0039] MAP2 Rabbit mAb (Proteintech #17490-1-AP): Dilution-1:5000;

[0040] TUBB3 Mouse mAb (Proteintech #66375-1-Ig;): Dilution-1:5000;

[0041] Subsequently, anti-rabbit secondary antibody (HRP-linked antibody (#7074, CST, USA): 1:5000) and anti-mouse secondary antibody (HRP-linked antibody (#7076, CST, USA): 1:5000) were used at room temperature for 1 hour. Protein bands were visualized using a chemiluminescent ECL kit (Tanon, Shanghai, China).

[0042] (4) CCK-8 experiment

[0043] U87MG and T98G cells in logarithmic growth phase were centrifuged at 300g for 5 min, and the cell pellet was collected. The cells were resuspended in fresh culture medium, diluted 100-fold, and cell counts were performed. Five experimental groups were set up: 0 μM, 1 μM, 5 μM, 10 μM, and 15 μM, with five replicates per group. 100 μL of cell suspension was added to each well of a 96-well plate at a density of 1 x 10³ cells / well. H1 short peptides of corresponding concentrations were added to the experimental groups, with Hnc short peptides serving as controls. After incubation at 37℃ and 5% CO2 for 72 h, 10 μL of CCK8 solution (yeasen, #40203ES76) was added to each well. The 96-well plates were placed in a cell culture incubator and incubated for another 2 h. The absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability was obtained by calculating ODexperimental group / OD0μM.

[0044] (5) Plate cloning experiment

[0045] Inoculate 5x10⁻⁶ cells into a six-well plate. 2 Add 2 mL of DMEM medium to U87MG or T98G cells, gently shake to ensure even cell distribution, and incubate at 37°C. Replace the medium with fresh medium every 3 days. After approximately 14 days of culture, discard the medium and wash once with PBS to remove excess medium components. Add 3 mL of 4% paraformaldehyde (Suzhou Xinsaimei Biotechnology) and fix the cells at room temperature in the dark for 20 minutes. Wash twice with PBS, then add 3 mL of 0.1% crystal violet dye (Beyotime C0121-100ml) and continue staining at room temperature in the dark for 20 minutes. Finally, wash twice more with PBS, air dry the culture dishes, and acquire images of the clones using a microscope.

[0046] (6) Immunofluorescence experiment

[0047] Carefully place the 12-well plate into the wells and seed the glioma organoids. Wash the cells three times with PBS, ensuring that any residual liquid is aspirated after each wash. Then, fix the cells with 4% paraformaldehyde at room temperature for 15 minutes, followed by three more washes with PBS. Permeabilize the cells with 0.1% Triton X-100 for 10 minutes. After permeabilization, wash twice with PBS. Then block with 5% BSA (0.5 g dissolved in 10 mL PBS) for 1 hour at room temperature. Discard the BSA solution and add 150 μL of a 1:500 dilution of primary antibody (containing TUBB3 and GFAP antibodies). Incubate overnight at 4°C on a shaker, maintaining humidity. After incubation, wash the membrane four times with PBS for 5 minutes each time. Add a 1:500 dilution of fluorescently labeled secondary antibody and incubate in the dark for 1 hour. Wash four times with PBS after incubation. Incubate with DAPI in the dark for 5 minutes, followed by four washes with PBS. Place the coverslip upside down on a slide with a sealing agent, and seal the edges with nail polish. Observe the fluorescence signal under a microscope.

[0048] (7) BALB / c nude mouse intracranial tumor implantation experiment and H1 short peptide treatment

[0049] Six-week-old female BALB / c nude mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd. The mice were anesthetized and placed in a stereotactic head frame. A 1 ml syringe (Hamilton, Switzerland) was inserted into the coronal suture of each mouse, 2 mm to the right of the midline. The nude mice were divided into four groups.

[0050] 5×10 5 U87MG-Luc cell suspension was injected into 10 μL of buffer solution and injected 3 mm from the brain surface for 5 min. After another 5 minutes, the syringe was removed and the injection site was sealed with bone wax. Starting on day 7, each mouse was stereotactically injected with 10 μL of H1 (2 mg / kg / 3 d) or Hnc at the same site. Intraperitoneal injection of TMZ (25 mg / kg / 3 d) or an equal volume of PBS served as a control. After 14 days, each mouse was intraperitoneally injected with 100 μL of 15 mg / ml D-fluorescein, and in vivo images were acquired using an IVIS bioluminescence imaging system. The survival time of the mice was recorded.

[0051] (8) HE staining and immunohistochemistry

[0052] Tumor tissue was fixed and embedded in paraffin. 4 μm thick tissue sections were stained with hematoxylin and eosin (HE) for morphological observation. After dewaxing and antigen extraction, other similarly prepared slides were incubated overnight at 4°C with primary antibodies, including anti-TUBB3 (1:500) or anti-GFAP (1:500). After thorough washing, the slides were incubated with secondary antibodies for 1 h, then dehydrated and sealed with neutral resin. Images were acquired using a Leica Aperio AT2 and a Leica DM IRB. II. Experimental Results:

[0053] (1) The H1 short peptide derived from LGP2 can inhibit glioma growth and induce glioma cell differentiation.

[0054] Figure 1 shows the expression of markers indicating that the H1 short peptide inhibits glioma cell growth and induces differentiation. As shown in Figure 1, where:

[0055] Figure A shows the effect of H1 on the survival of U87MG and T98G cells. Human glioma U87MG and T98G cells were treated with 0 μM, 1 μM, 5 μM, 10 μM, and 15 μM H1 or Hnc peptides, and cell survival was assessed using CCK-8 assay. The results showed that H1 significantly inhibited cell viability.

[0056] Figures B and C show the effect of H1 on clonogenicity in U87MG and T98G cells. Human glioma U87MG and T98G cells were treated with 5 μM H1 or Hnc for 14 days, and the clonogenic ability of the cells was assessed using a plate colony assay. The results showed that H1 significantly inhibited the clonogenic ability of the cells.

[0057] Figure D shows the effect of H1 on the growth of glioma organoids. Phase-contrast microscopy images were taken after 0 and 14 days of treatment with 5 μM H1 or Hnc on gliomas, and the area of ​​the organoids was quantitatively analyzed. The results indicate that overexpression of H1 significantly inhibits the growth ability of organoids.

[0058] E and F show the effect of H1 on the protein levels of organoid differentiation markers. After 21 days of treatment with 5 μM H1 or Hnc in glioma organoids, the protein expression levels of TUBB3 and GFAP were detected by Western blotting and immunofluorescence assays. The results showed that H1 can induce the expression of glioma cell differentiation markers. "NS", not significant, * p <0.05, ** p <0.01, *** p <0.001.

[0059] (2) H1 short peptide can significantly inhibit tumor growth.

[0060] Figure 2 shows that the H1 short peptide significantly inhibits glioma growth and induces glioma cell differentiation in tumor-bearing mice. As shown in Figure 2, in which:

[0061] A is a schematic diagram of a U87MG-Luc tumor xenograft. (The diagram shows a 1x10...) 6 A mouse glioma orthotopic xenograft model was established by orthotopic injection of U87MG-Luc glioma cells into the cranium of BALB / c nude mice. Tumor growth was monitored 7 days post-transplantation using an IVIS bioluminescence imaging system. H1 or Hnc peptides were stereotactically injected into the tumor at a dose of 2 mg / kg / 3 days per mouse, with intraperitoneal injection of TMZ (25 mg / kg / 3 days) or PBS serving as controls. Tumor growth was monitored using an IVIS bioluminescence imaging system.

[0062] Figures B and C show bioluminescent images (n=3) of orthotopic gliomas in mice on day 14 after tumor implantation, along with corresponding statistical analysis of fluorescence values. The results indicate that the H1 short peptide significantly inhibits glioma growth.

[0063] Image D shows the size of the in situ glioma as indicated by HE staining (within the blue dashed box). Mice were sacrificed after the experiment reached its endpoint, and coronal sections of brain tissue were extracted and stained with HE to show the size of the glioma after treatment with H1, Hnc, PBS, and TMZ. The results showed that, compared to the control group, Hnc and H1 treatment resulted in smaller intracranial gliomas in mice, with effects comparable to TMZ treatment.

[0064] E represents the immunohistochemical detection of the protein expression levels of differentiation markers TUBB3 and GFAP in glioma cells from four groups (H1, Hnc, PBS, TMZ) after H1 treatment. The results showed that H1 treatment significantly induced glioma cell differentiation compared to the control groups (Hnc and TMZ).

[0065] F is the mouse survival curve analysis. It shows the survival time of mice treated with H1, Hnc, PBS, and TMZ. The results indicate that compared to the control group (Hnc), H1 treatment significantly prolonged the survival time of mice, with effects comparable to TMZ treatment. "NS", not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0066] (3) H1 short peptide can enhance the effect of PD-1 antibody therapy.

[0067] Figure 3 shows the effect of H1 short peptide significantly enhancing PD-1 antibody treatment. As shown in Figure 3, in which:

[0068] A is a schematic diagram of a Glu261-Luc tumor xenograft. (The diagram shows a 3 x 10...) 6Glioma GL261-Luc cells were injected orally into the cranium of C57 mice to establish a mouse orthotopic glioma xenograft model. Tumor growth was monitored 7 days post-transplantation using an IVIS bioluminescence imaging system. H1 or Hnc peptides were stereotactically injected into the tumor at a dose of 2 mg / kg / 3 days per mouse, with intraperitoneal injection of PD-1 antibody (10 mg / kg / 3 days) or PBS serving as controls. Tumor growth was monitored using an IVIS bioluminescence imaging system.

[0069] B and C are bioluminescent images (n=3) of orthotopic gliomas in mice on day 14 after tumor implantation, along with corresponding statistical analysis of fluorescence values. The results indicate that the H1 short peptide significantly inhibits glioma growth and significantly enhances the therapeutic effect of PD-1 antibody.

[0070] D is the mouse survival curve analysis graph. It shows the survival time of mice treated with H1, Hnc, PBS, and PD-1 antibody. The results indicate that compared to the control group, PD-1 and H1 treatment significantly prolonged the survival time of mice, and the combination of PD-1 and H1 was more effective than single-agent treatment.

[0071] Image E shows the size of the in situ glioma as indicated by HE staining (within the blue dashed box). Mice were sacrificed after the experiment reached its endpoint, and coronal sections of brain tissue were extracted and stained with HE to show the size of the glioma after treatment with H1, Hnc, PBS, and PD-1. The results showed that, compared to the control group, the intracranial gliomas in mice treated with PD-1 antibody and H1 were smaller, and the combination of PD-1 and H1 was more effective than single-agent treatment.

[0072] FH is a statistical analysis of the levels of CD8 and TNFα in six groups (PBS, Hnc, H1, aPD-1, aPD-1+Hnc, aPD-1+H1) of mouse gliomas after treatment with H1 and PD-1 antibodies, using immunohistofluorescence. The results showed that, compared to the control group, treatment with PD-1 antibody and H1 significantly increased the levels of CD8 and TNFα in the tumor microenvironment. The combination of H1 and PD-1 antibody significantly increased the levels of CD8 and TNFα in the tumor microenvironment compared to monotherapy. "NS", not significant, * p <0.05, ** p <0.01, *** p <0.001.

[0073] In summary, this invention investigated the antitumor activity of the H1 short peptide, examining its effects on the survival of glioma cell lines U87MG and T98G, its influence on glioma organoid growth and clonogenic capacity, and its effect on the expression of differentiation markers. Subsequently, tumor growth was observed in an experiment involving intracranial implantation of gliomas in BALB / c nude mice. The results indicate that H1 can be used as a short peptide to inhibit glioma growth and induce differentiation. Further experiments showed that the H1 short peptide can enhance the efficacy of PD-1 antibody therapy and can be used in combination for antitumor purposes.

[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes or alterations made by those skilled in the art using the disclosed technical content shall fall within the protection scope of the present invention.

Claims

1. A short peptide for antitumor activity, characterized in that, The short peptide contains an amino acid sequence as shown in Seq ID No:

1.

2. The antitumor short peptide according to claim 1, characterized in that, The N-terminus of the amino acid sequence shown in Seq ID No:1 is also linked to a membrane-penetrating peptide.

3. The antitumor short peptide according to claim 2, characterized in that, The amino acid sequence of the membrane-penetrating peptide is shown in Seq ID No:

2.

4. The antitumor short peptide according to any one of claims 1-3, characterized in that, The short peptide inhibits tumor growth by inducing tumor cell differentiation.

5. The antitumor short peptide according to any one of claims 1-3, characterized in that, The tumor is a glioma.

6. The antitumor short peptide according to any one of claims 1-3, characterized in that, The short peptide is a short peptide derived from LGP2.

7. An antitumor pharmaceutical composition, characterized in that, It comprises a PD-1 antibody and the short peptide described in any one of claims 1-6.

8. The use of the short peptide according to any one of claims 1-6 in the preparation of an antitumor drug or in the treatment of tumors.

9. Use of the antitumor pharmaceutical composition of claim 7 in the preparation of an antitumor drug or in the treatment of tumors.

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