An anti-tumor short peptide, pharmaceutical composition and application thereof

Abstract

The present invention discloses an anti-tumor short peptide, comprising an amino acid sequence as shown in Seq ID No: 1; the amino acid sequence as shown in Seq ID No: 1 is further connected to a cell-penetrating peptide at its N-terminus; the short peptide inhibits tumor growth by inducing tumor cell differentiation; the tumor is a glioma; and the short peptide is derived from LGP2. The present invention also discloses an anti-tumor pharmaceutical composition comprising a PD-1 antibody and the short peptide. The present invention also discloses a use of the short peptide or anti-tumor pharmaceutical composition in the preparation of an anti-tumor drug. The present invention provides a new differentiation therapy approach for tumors, particularly gliomas.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-tumor short peptide, a pharmaceutical composition and applications thereof. Background Art

[0002] Differentiation therapy is an innovative therapeutic strategy that reactivates the differentiation potential of cancer cells, promoting their maturation and thereby inhibiting malignant proliferation. Unlike traditional cytotoxic therapy, this therapy does not directly kill cancer cells, but rather reduces their tumorigenicity by altering their differentiation state. It has attracted considerable attention in recent years due to its minimal side effects, limited impact on normal cells, and long-lasting efficacy. One of the earliest successful cases came from the use of all-trans retinoic acid (ATRA) in acute promyelocytic leukemia (APL). This therapy degrades the PML-RARα fusion protein, relieving the differentiation block of leukemia cells and significantly improving patient prognosis. Currently, with the advancement of molecular biology techniques, differentiation therapy is gradually revealing potential new targets for the treatment of malignant tumors and is gradually being applied to solid tumors such as neuroblastoma and hepatocellular carcinoma. However, the therapeutic effect on solid tumors and the specific mechanisms of differentiation induction remain unclear.

[0003] The RIG-I-like receptor family (RLRs) includes retinoic acid-inducible gene-I (RIG-I), melanoma differentiation-associated receptor 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). They can interact with exogenous RNA, inducing conformational changes. MDA5 and RIG-I, upon sensing non-self RNA, expose their CARD domains. The exposed CARD domain 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 interferons (IFNs) and IFN-inducible genes (ISGs), which participate in the immune response to viral infection. IFNs and ISGs not only affect viral replication but also have direct anti-tumor effects. Although LGP2 can bind to RNA more strongly than MDA5 and RIG-I, it lacks the CARD domain or other signaling domains, preventing it from transmitting signals and thus being unable to directly interact with the adaptor protein MAVS to induce the production of IFNs and inflammatory factors. The function of LGP2 in immune responses remains controversial. Numerous studies have shown that LGP2 promotes antiviral signaling in MDA5. While LGP2 is primarily involved in innate and antiviral immunity, its role in tumorigenesis remains unknown. 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 applications thereof.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] In one aspect, the present invention provides an anti-tumor short peptide comprising the amino acid sequence 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 further connected to a cell-penetrating peptide.

[0008] Furthermore, the amino acid sequence of the cell-penetrating 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 anti-tumor pharmaceutical composition comprising a PD-1 antibody and the above-mentioned short peptide.

[0013] In another aspect, the present invention also provides a use of the above-mentioned short peptide in the preparation of anti-tumor drugs.

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

[0015] The present invention studies the anti-tumor activity of the above-mentioned short peptide (H1), detects its effect on the survival of glioma cell lines U87MG and T98G, and detects its effect on the growth and clone formation ability of glioma organoids and the effect of inducing the expression of differentiation markers. Subsequently, in an experiment in which gliomas were implanted in the skull of BALB / c nude mice, the growth of the tumor was observed. The results showed that H1 can be used as a short peptide that inhibits tumor growth and induces differentiation. Additional experiments showed that the H1 short peptide can enhance the effect of PD-1 antibody treatment and can be used in combination for anti-tumor treatment. The present invention provides a new differentiation therapy approach for tumors, especially gliomas. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The above is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0017] Figure 1 The figure shows that H1 short peptide inhibits the growth of glioma cells and induces the expression of differentiation markers, where:

[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 levels of organoid differentiation marker proteins;

[0019] Figure 2 The H1 short peptide significantly inhibited the growth of glioma in tumor-bearing mice and induced glioma cell differentiation;

[0020] A is a schematic diagram of the U87MG-Luc tumor transplant; B and C are bioluminescent images of mouse orthotopic gliomas on the 14th day after tumor implantation (n=3) and the corresponding fluorescence numerical statistical analysis graphs; D is a HE staining diagram showing the size of the orthotopic glioma (in the blue dotted box); E is an immunohistochemical method for detecting the protein expression levels of differentiation markers TUBB3 and GFAP in four groups of mouse brain gliomas after H1 treatment (H1, Hnc, PBS, TMZ); F is a mouse survival curve analysis graph.

[0021] Figure 3 This is a diagram showing that H1 short peptide significantly enhances the effect of PD-1 antibody treatment, where:

[0022] A is a schematic diagram of the Gl261-Luc tumor transplant; B and C are bioluminescence images of mouse orthotopic gliomas on the 14th day after tumor implantation (n=3) and the corresponding fluorescence numerical statistical analysis graphs; D is a mouse survival curve analysis graph; E is a HE staining diagram showing the size of the orthotopic glioma (in the blue dotted box); FH are immunohistofluorescence methods used to detect the levels of CD8 and TNFa in six groups of mouse brain gliomas after treatment with H1 and PD-1 antibodies, as well as fluorescence numerical statistical analysis graphs of CD8 and TNFa. DETAILED DESCRIPTION

[0023] The present invention found 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, inhibiting glioma growth.

[0024] Experiments in this study revealed that LGP2 interacts with MCCA, inhibiting its enzymatic activity. This interaction ultimately upregulates intracellular crotonylation through ECHS1. Crotonylation of histone H3K18cr enhances PPARGC1A transcription. PGC1a can influence mitochondrial fission by promoting DRP1 phosphorylation, resulting in an anti-Warburg effect, ultimately driving glioma cell differentiation.

[0025] 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 MCCA protein, thereby activating MAVS and its downstream pathways.

[0026] The H1 short peptide derived from LGP2 has the function of inhibiting glioma growth and inducing glioma cell differentiation in glioma. The following experiments are used to illustrate the effect:

[0027] 1. Experimental methods:

[0028] (1) Cell culture

[0029] Cell lines U87MG, T98G, and glioma organoids were cultured in Dulbecco's modified Eagle's 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 medium changes daily.

[0030] (2) The H1 short peptide derived from LGP2 was synthesized by Nanjing Leon Biotechnology Co., Ltd., and Hnc was used as a negative control. The amino acid sequences of H1 and Hnc and the N-terminal transmembrane peptide (TAT) sequence are as follows:

[0031] H1:YGRKKRRQRRR-KDTVYNVIMSQYLELKL(Seq ID No: 2-Seq ID No: 1)

[0032] Hnc:YGRKKRRQRRR-KTRAAAYVAKRHLET(Seq ID No: 2-Seq ID No: 3)

[0033] It should be noted that the H1 and Hnc used in the following experiments contain a transmembrane peptide (TAT).

[0034] However, the cell-penetrating peptide YGRKKRRQRRR (Seq ID No: 2) is only used to carry the corresponding sequence (Seq ID No: 1: KDTVYNVIMSQYLELKL) into cells. The sequence that actually has an anti-tumor effect should be the sequence it carries. In addition to the aforementioned cell-penetrating peptides, other types of cell-penetrating peptides can also be used. This example only uses the cell-penetrating peptide (Seq ID No: 2) as an example for illustration.

[0035] (3) Western blot

[0036] To detect the induction of differentiation marker protein expression by the H1 short peptide, Western blot experiments were used for protein verification. Western and IP cell lysis buffer (Biyuntian, product number: P0013) was used to lyse glioma organoids and collect proteins. 20 μg of total protein was collected from each group for protein electrophoresis. After transfer, the membrane was incubated with 5% skim milk at 22°C for 2 hours and then incubated with the primary antibody at 4°C overnight. The primary antibodies and their 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] MAP2Rabbit mAb(Proteintech#17490-1-AP):Dilution-1:5000;

[0040] TUBB3Mouse 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] Take U87MG and T98G cells in the logarithmic growth phase, centrifuge at 300g for 5 minutes, and collect the cell pellet. Resuspend the cells in fresh culture medium, dilute 100 times, and count the cells. Set up 5 experimental groups, namely 0μM, 1μM, 5μM, 10μM, and 15μM, with 5 replicates in each group, and measure 1x10 3 At a density of 100 cells / well, 100 μL of cell suspension was added to a 96-well plate. The corresponding concentration of H1 short peptide was added to the experimental group, and the Hnc short peptide was used as a control. After incubation at 37°C 5% CO2 for 72 hours, 10 μL of CCK8 solution (yeasen, #40203ES76) was added to each well. The 96-well plate was placed in a cell culture incubator and cultured for another 2 hours. The absorbance value (OD value) at a wavelength of 450 nm was detected using a microplate reader. The cell activity was obtained by calculating the formula OD experimental group / OD0 μM.

[0044] (5) Plate cloning experiment

[0045] 5x10 cells were seeded in a six-well plate 2 For U87MG or T98G cells, add 2mL of DMEM medium, shake gently back and forth and left and right to ensure that the cells are evenly distributed, and culture in a 37°C incubator. Replace with fresh medium every 3 days. After about 14 days of culture, discard the medium and wash once with PBS to remove excess medium components. Add 3mL of 4% paraformaldehyde (Suzhou Xinsaimei Biological) and fix the cells in the dark for 20 minutes at room temperature. Wash twice with PBS, then add 3mL of 0.1% crystal violet dye (Biyuntian C0121-100ml) and continue staining in the dark for 20 minutes at room temperature. Finally, wash twice more with PBS, dry the culture dish, and use a microscope to capture images of the clones.

[0046] (6) Immunofluorescence experiment

[0047] Carefully place the 12-well plate slide in the 12-well plate and plate the glioma organoids into the wells. Wash the cells three times with PBS, making sure to remove any residual liquid each time. Subsequently, add 4% paraformaldehyde to fix the cells for 15 minutes at room temperature and then wash again three times with PBS. Permeabilize the cells with 0.1% TritonX-100 for 10 minutes. After permeabilization, wash twice with PBS. Then, block with 5% BSA (0.5g dissolved in 10mL PBS) for 1 hour at room temperature. Discard the BSA solution and add 1:500 diluted primary antibodies (150μL), including TUBB3 and GFAP antibodies. Maintain humidity and incubate overnight on a shaker at 4°C. After incubation, wash the membrane four times with PBS for 5 minutes each. Add a fluorescently labeled secondary antibody at a dilution of 1:500 and incubate in the dark for 1 hour. After incubation, wash four times with PBS. Incubate with DAPI in the dark for 5 minutes and then wash four times with PBS. Place the coverslip upside down on a glass slide with mounting medium and seal the edges with nail polish. Observe the fluorescence signal under a microscope.

[0048] (7) Intracranial tumor implantation in BALB / c nude mice and H1 short peptide treatment

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

[0050] 5×10 5A suspension of U87MG-Luc cells was injected in 10 μL of buffer at a distance of 3 mm from the brain surface for 5 minutes. After an additional 5 minutes, the syringe was removed and the injection site sealed with bone wax. Starting on day 7, each mouse received a stereotactic injection of 10 μL of H1 (2 mg / kg / 3 days) or Hnc at the same site. Intraperitoneal injection of TMZ (25 mg / kg / 3 days) or an equal volume of the solvent, PBS, served as a control. Fourteen days later, each mouse received a 100 μL intraperitoneal injection of 15 mg / ml D-luciferin, and in vivo images were acquired using an IVIS bioluminescence imaging system. Mouse survival was recorded.

[0051] (8) HE staining and immunohistochemistry

[0052] Tumor tissue was fixed and embedded in paraffin. 4-μm-thick tissue sections were obtained and stained with hematoxylin-eosin (HE) for morphological observation. After dewaxing and antigen retrieval, 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 hour, then dehydrated and sealed with neutral resin. Images were acquired using a Leica Aperio AT2 and a Leica DM IRB instrument.

[0053] 2. Experimental results:

[0054] (1) LGP2-derived H1 short peptide can inhibit glioma growth and induce glioma cell differentiation.

[0055] Figure 1 The figure shows that H1 short peptide inhibits the growth of glioma cells and induces the expression of differentiation markers. Figure 1 As shown, where:

[0056] Figure A shows the effect of H1 on the survival of U87MG and T98G cells. Human glioma U87MG and T98G cells were treated with H1 or Hnc peptide at concentrations of 0μM, 1μM, 5μM, 10μM, and 15μM, and CCK-8 assays were used to assess cell viability. The results demonstrate that H1 significantly inhibits cell viability.

[0057] Figures B and C show the effect of H1 on the colony formation of U87MG and T98G cells. Human glioma U87MG and T98G cells were treated with 5 μM H1 or Hnc for 14 days before being tested for colony formation using a plate cloning assay. The results showed that overexposure to H1 significantly inhibited the cells' colony formation.

[0058] D shows the effect of H1 on glioma organoid growth. Glioma cells were treated with 5 μM H1 or Hnc for 0 and 14 days, and images were taken using phase contrast microscopy. The organoid area was then quantitatively analyzed. The results demonstrate that overexpression of H1 significantly inhibits organoid growth.

[0059] Figures E and F show the effect of H1 on the expression of differentiation marker proteins in organoids. After 21 days of treatment with 5 μM H1 or Hnc, TUBB3 and GFAP protein expression in organoids was assessed by Western blotting and immunofluorescence. The results demonstrate that H1 can induce the expression of glioma cell differentiation markers. "NS", not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

[0060] (2) H1 short peptide can significantly inhibit tumor growth.

[0061] Figure 2 The H1 short peptide significantly inhibited the growth of glioma in tumor-bearing mice and induced glioma cell differentiation. Figure 2 As shown, where:

[0062] A is a schematic diagram of U87MG-Luc tumor transplantation. 6 U87MG-Luc glioma cells were orthotopically injected into the skull of BALB / c nude mice to establish an orthotopic mouse glioma xenograft model. Seven days after transplantation, tumor growth was monitored using an IVIS bioluminescence imaging system. H1 or Hnc short peptides were stereotactically injected into the tumor at a dose of 2 mg / kg / 3 days per mouse. TMZ (25 mg / kg / 3 days) or PBS was intraperitoneally injected as a control. Tumor growth was monitored using an IVIS bioluminescence imaging system.

[0063] Figures B and C are bioluminescent images of orthotopic gliomas in mice on day 14 after tumor implantation (n=3) and corresponding fluorescence numerical statistical analysis graphs. The results show that H1 short peptide can significantly inhibit the growth of gliomas.

[0064] D is a HE staining image showing the size of the in situ glioma (in the blue dashed box). At the end of the experiment, mice were sacrificed, and coronal sections of brain tissue were removed and stained with HE. The results show that compared with the control group, mice treated with Hnc and H1 had smaller intracranial gliomas, demonstrating an effect comparable to that of TMZ.

[0065] E is immunohistochemical analysis of the protein expression levels of differentiation markers TUBB3 and GFAP in four mouse glioma groups (H1, Hnc, PBS, and TMZ) after H1 treatment. The results showed that H1 treatment significantly induced glioma cell differentiation compared with the control group (Hnc and TMZ).

[0066] F is a mouse survival curve analysis. Survival of mice treated with H1, Hnc, PBS, and TMZ is shown. Results show that H1 treatment significantly prolonged mouse survival compared to the Hnc control group, demonstrating comparable efficacy to TMZ treatment. "NS," not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

[0067] (3) H1 short peptide can enhance the effect of PD-1 antibody treatment.

[0068] Figure 3 This is a diagram showing that H1 short peptide significantly enhances the effect of PD-1 antibody treatment. Figure 3 As shown, where:

[0069] A is a schematic diagram of Gl261-Luc tumor transplantation. 6 GL261-Luc cells were injected orthotopically into the skull of C57 mice to establish an orthotopic mouse glioma xenograft model. Seven days after transplantation, tumor growth was monitored using an IVIS bioluminescence imaging system. H1 or Hnc short peptides were stereotactically injected into the tumor at a dose of 2 mg / kg / 3d per mouse. PD-1 antibodies (10 mg / kg / 3d) or PBS were injected intraperitoneally as controls. Tumor growth was monitored using an IVIS bioluminescence imaging system.

[0070] B and C are bioluminescent images of orthotopic gliomas in mice 14 days after tumor implantation (n=3) and corresponding fluorescence numerical statistical analysis graphs. The results show that H1 short peptide can significantly inhibit glioma growth and significantly enhance the therapeutic effect of PD-1 antibody.

[0071] D is a mouse survival curve analysis. It shows the survival of mice treated with H1, Hnc, PBS, and PD-1 antibodies. The results show that compared with the control group, mice treated with PD-1 and H1 had significantly prolonged survival, and the combination of PD-1 and H1 was more effective than single-agent therapy.

[0072] Figure E shows the size of the in situ gliomas (inside the blue dashed box) as determined by HE staining. At the end of the experiment, mice were sacrificed, and coronal sections of brain tissue were removed and stained with HE staining. This shows the size of gliomas after treatment with H1, Hnc, PBS, and PD-1. The results indicate that compared to the control group, mice treated with the PD-1 antibody and H1 developed smaller intracranial gliomas, demonstrating that the combination of PD-1 and H1 is more effective than monotherapy.

[0073] FH shows immunohistofluorescence analysis of CD8 and TNFα levels in six mouse glioma groups (PBS, Hnc, H1, aPD-1, aPD-1 + Hnc, and aPD-1 + H1) after treatment with H1 and PD-1 antibodies. The results show that compared to the control group, treatment with PD-1 antibodies and H1 significantly increased CD8 and TNFα levels in the tumor microenvironment. The combination of H1 and PD-1 antibodies significantly increased CD8 and TNFα levels in the tumor microenvironment compared to monotherapy.

[0074] "NS", not significant, *p<0.05, **p<0.01, ***p<0.001.

[0075] In summary, the present invention studied the anti-tumor activity of the H1 short peptide, detected its effect on the survival of glioma cell lines U87MG and T98G, detected its effect on the growth and clone formation ability of glioma organoids, and detected its effect on the expression of differentiation markers. Subsequently, in an experiment in which gliomas were implanted intracranially in BALB / c nude mice, the growth of the tumor was observed. The results showed that H1 can be used as a short peptide to inhibit glioma growth and induce differentiation. Additional experiments showed that the H1 short peptide can enhance the effect of PD-1 antibody treatment and can be used in combination for anti-tumor treatment.

[0076] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Those skilled in the art can make some simple modifications, equivalent changes or modifications based on the technical content disclosed above, which all fall within the scope of protection of the present invention.

Claims

1. An anti-tumor short peptide, characterized in that: The short peptide is the amino acid sequence shown in Seq ID No:

1.

2. An anti-tumor short peptide, characterized in that: The short peptide is a cell-penetrating peptide connected to the N-terminus of the amino acid sequence shown in Seq ID No:

1.

3. The anti-tumor short peptide according to claim 2, characterized in that: The amino acid sequence of the cell-penetrating peptide is shown in SeqID No:

2.

4. An anti-tumor pharmaceutical composition, characterized in that: Comprising a PD-1 antibody and the short peptide according to any one of claims 1 to 3.

5. Use of the short peptide according to any one of claims 1 to 3 in the preparation of an anti-tumor drug, characterized in that: The tumor is a glioma.

6. Use of the antitumor pharmaceutical composition according to claim 4 in the preparation of antitumor drugs, characterized in that: The tumor is a glioma.

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