WO2024082408A1 - Use of jwa polypeptide in preparation of anti-tumor drug co-potentiator - Google Patents

Use of jwa polypeptide in preparation of anti-tumor drug co-potentiator Download PDF

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WO2024082408A1
WO2024082408A1 PCT/CN2022/138739 CN2022138739W WO2024082408A1 WO 2024082408 A1 WO2024082408 A1 WO 2024082408A1 CN 2022138739 W CN2022138739 W CN 2022138739W WO 2024082408 A1 WO2024082408 A1 WO 2024082408A1
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tumor
drug
cells
polypeptide
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周建伟
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苏州明人医药生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to application of JWA polypeptide in preparing anti-tumor drug synergistic enhancer, belonging to the technical field of anti-tumor auxiliary drugs.
  • tumor metastasis is the main cause of death in cancer patients [3]. Once metastasis occurs, existing treatments are unlikely to benefit patients. Due to the limited treatment strategies for tumor metastasis, preventing the initiation of metastasis may be a major unmet clinical need in tumor treatment [4,5].
  • the pre-colonization stage of tumor cell metastasis is a complex multi-step process, including tumor cells detaching from the primary tumor, penetrating the basement membrane, and entering the blood vessels [6]. Breaking through the basement membrane and entering the vascular system is the core of initiating tumor metastasis [7].
  • Tumor angiogenesis is a regenerative process that is highly influenced by tumor cells and promotes tumor growth and metastasis [10]. Cytokines secreted by tumor cells interact with perivascular cells and transform them into fibroblasts, thereby causing these cells to lose their ability to protect blood vessels [11,12].
  • tumor cells release pseudo-endothelial signals to stimulate endothelial cell migration, resulting in discontinuity of endothelial lining and defects in basement membrane [13]. All of these characteristics are manifestations of vascular immaturity and dysfunction. Therefore, the high permeability of blood vessels facilitates the invasion of tumor cells into blood vessels. Normalization of tumor blood vessels can keep endothelial cells surrounded by mature pericytes in a quiescent state. Endothelial cells and pericytes are separated by basement membranes and filled with various adhesion proteins to maintain vascular stability [14]. Normalization of tumor blood vessels inhibits metastasis by reducing tumor cell invasion of blood vessels [15,16].
  • IL8 is a known chemotactic cytokine that has been reported to be highly expressed in a variety of tumors [18,19]; IL8 can promote the proliferation and metastasis of malignant tumors and play a key regulatory role in the tumor microenvironment [20,21]. IL8 also aggravates tumorigenicity by mediating crosstalk between ECFC and TNBC [22].
  • the hepatitis B virus-encoded gene (HBX) induces high IL8 production by activating MEK-ERK signaling, leading to increased endothelial cell permeability and promoting tumor vascular invasion[23].
  • IL8-mediated vascular immaturity induces hypoxia in the tumor microenvironment, which in turn stimulates tumor cells to secrete IL8[24], leading to a vicious feedback of hypoxia in the tumor microenvironment, IL8 secretion, and vascular disorder. Therefore, inhibiting IL8 may be an effective treatment for correcting vascular disorder in the tumor microenvironment.
  • JWA is a tumor suppressor gene that can inhibit the growth and metastasis of melanoma, gastric cancer, breast cancer and other malignant phenotypes[25-27]. JWA regulates AMPK/FOXO3a/UQCRC2 signals, promotes mitochondrial metabolic reprogramming, improves hypoxia in the tumor microenvironment, and thus inhibits tumor metastasis[28].
  • the anti-tumor peptide JP1 designed based on the JWA functional sequence can effectively inhibit melanoma metastasis[29].
  • the mechanism by which the anti-tumor peptide JP1 inhibits tumor metastasis is still unclear, and whether the anti-tumor peptide JP1 can be used as a synergist of existing anti-tumor drugs also needs further exploration and research.
  • the inventor's research group has the latest research results and applied for the patent of this invention based on them.
  • the main purpose of the present invention is to propose an application of JWA polypeptide in the preparation of anti-tumor drug synergistic enhancers in view of the problems existing in the prior art, which can improve the hypoxic state of the tumor microenvironment, promote the normalization of tumor blood vessels to inhibit the initiation and occurrence of tumor metastasis, and can be used in combination with chemotherapy drugs to increase the effective perfusion amount of drugs in the tumor, exert a synergistic effect, and provide new clinical drug possibilities for the treatment of tumors.
  • a use of a polypeptide characterized in that the use is used for preparing an anti-tumor drug synergist;
  • amino acid sequence of the polypeptide is shown in I or II:
  • amino acid S is phosphorylated
  • X and Z are amino acids or amino acid sequences, respectively;
  • X is selected from one of F, (R) 9 , (R) 9 -F, 6-aminocaproic acid, 6-aminocaproic acid-F, 6-aminocaproic acid-(R) 9 , and 6-aminocaproic acid-(R) 9 -F;
  • Z is selected from (G) n -RGD and A-(G) n -RGD, n is an integer greater than or equal to 0, and the value range of n is 0-10.
  • the function of the anti-tumor drug synergist is to improve the hypoxia of the tumor microenvironment by promoting oxidative phosphorylation of tumor cells and thus reducing the secretion of IL8, and to improve the hypoxic state of the tumor microenvironment by regulating the mitochondrial metabolic reprogramming of tumor cells through the AMPK/FOXO3a/UQCRC2 signaling pathway.
  • the function of the anti-tumor drug synergist is to promote the normalization of tumor blood vessels by inhibiting IL8, reduce the chance of tumor cells entering blood vessels, and thus inhibit the initiation and occurrence of tumor metastasis.
  • the function of the anti-tumor drug synergist is to increase the effective perfusion amount of the drug in the tumor.
  • the anti-tumor drug targeted by the anti-tumor drug synergist is a chemical drug, antibody drug, or cell drug used for tumor treatment.
  • the anti-tumor drug targeted by the anti-tumor drug synergist is paclitaxel.
  • the tumor targeted by the anti-tumor drug synergist is a pan-solid tumor having at least one of the following characteristics:
  • Feature 1 hypoxia in the tumor microenvironment, disordered vascular distribution, and low intratumoral drug perfusion;
  • the tumor targeted by the anti-tumor drug synergist is melanoma, lung cancer, or human melanocytic nevus.
  • the N-terminus of the polypeptide is modified by acetylation, and the C-terminus is modified by amidation;
  • the amino acids of the polypeptide are L-type natural amino acids or D-type non-natural amino acids.
  • amino acid sequence of the polypeptide is FPGSDRF-RGD, wherein the amino acid S is phosphorylated.
  • the polypeptide involved in the present invention is a part of the series of polypeptides recorded in the Chinese invention patent with patent number CN201310178099X and authorization announcement number CN103239710B.
  • the inventor has found through practical research that the above polypeptide can regulate mitochondrial metabolic reprogramming through the AMPK/FOXO3a/UQCRC2 signaling pathway, and reduce the secretion of interleukin-8 (IL8) by promoting oxidative phosphorylation of tumor cells, thereby improving the hypoxia of the tumor microenvironment; the oxygen-rich tumor microenvironment inhibits the secretion of IL8 by tumor cells and promotes the normalization of tumor blood vessels; blood vessel normalization can make blood vessels mature and distribute normally, so that the tumor microenvironment forms a benign feedback of blood vessel regularization, full perfusion, and oxygen-rich microenvironment, preventing tumor cells from entering blood vessels and inhibiting the initiation of metastasis.
  • IL8 interleukin-8
  • the above polypeptide combined with paclitaxel (PTX) can maintain a certain blood vessel density in the tumor, promote the normalization of tumor blood vessel growth, increase the delivery of oxygen and drugs, and enhance the anti-tumor effect. Therefore, the above-mentioned polypeptides can be used as synergistic enhancers of anti-tumor drugs, providing new technical means to overcome the bottleneck of poor efficacy caused by reduced intratumor drug infusion due to hypoxia in the tumor microenvironment, abnormal vascular structure and distribution, increased IL8 expression, etc. in the treatment of tumors, especially solid tumors, and have good application prospects.
  • FIG1 is a diagram of the implementation process and results of Example 1 of the present invention.
  • the main points of each figure are as follows:
  • (BC) Quantitative results (n 6) of mice with lung metastasis after surgical resection of primary B16F10 (B) or LLC tumors (C) after designated treatment.
  • (F) Flow cytometric detection of GFP+B16F10 cells transfected with GFP-specific plasmid.
  • G Schematic diagram of CTC clone counting and flow cytometric analysis.
  • JK CTCs from 6 mice were subjected to migration and invasion assays, and the quantitative results of migrating cells (J) and invasive cells (K) per 30,000 cells were plotted (*P ⁇ 0.05;**P ⁇ 0.01; ns: the difference was not statistically significant).
  • Figure 2 is a graph showing the results of Example 1 of the present invention.
  • the key points of each figure are as follows:
  • C Representative lung HE staining images of each group, used to detect the starting point of B16F10 tumor metastasis, scale: 500 ⁇ m.
  • F Representative lung HE staining images of each group, used to detect the starting point of LLC tumor metastasis, scale: 500 ⁇ m.
  • FIG3 is a graph showing the results of Example 1 of the present invention.
  • the key points of each graph are as follows: (A) Representative images of CTC clone formation after Ctrl-R or JP1 treatment. (B) Representative images of CTC quantification per 1.5 ⁇ 107 blood cells by FACS analysis after Ctrl-R or JP1 treatment. (C) Representative images of CTC migration after Ctrl-R or JP1 treatment. (D) Representative images of CTC invasion after Ctrl-R or JP1 treatment.
  • Figure 4 is a result diagram of Example 2 of the present invention.
  • the key points of each figure are as follows:
  • F-H Result diagrams of ⁇ -SMA coverage (F), claudin5 coverage (G), and desmin coverage (H) of LLC tumor nodules, scale bar: 100 ⁇ m.
  • I-J Schematic diagram of vascular permeability analysis (I) and quantitative diagram of vascular permeability (J). (*P ⁇ 0.05; **P ⁇ 0.01).
  • Figure 5 is a graph of the results of Example 3 of the present invention.
  • the key points of each figure are as follows:
  • F Representative images of human melanocytic nevus and melanoma stained with immunofluorescence for ⁇ -SMA (green) and IL8 (red), scale bar: 200 ⁇ m.
  • GH Quantification of IL8 + area (G) and ⁇ -SMA + area (H) between human melanocytic nevus and melanoma.
  • JK Tumor growth curves and tumor/body weight (%) results of each group are shown.
  • LM Representative fluorescence images of ⁇ -SMA (green), CD31 (red), and DAPI nuclear staining in each group (L), and the quantitative analysis of ⁇ -SMA coverage is shown in the figure (M), with a scale bar of 100 ⁇ m.
  • Figure 6 is a graph showing the results of Example 3 of the present invention.
  • the key points of each figure are as follows:
  • A-B Representative Western Blot analysis of whole cell lysates from B16F10 (A) and LLC (B) cells after treatment with specified concentrations of JP1 to evaluate the expression of IL8.
  • C-D Representative Western Blot analysis results of IL8 protein extracted from B16F10 (C) and LLC (D) tumor nodules.
  • E-F Quantitative results of IL8 mRNA after treatment of B16F10 (E) and LLC (F) cells with specified concentrations of JP1.
  • FIG. 7 is a result diagram of Example 4 of the present invention.
  • the key points of each figure are as follows:
  • A-B After 48 hours of treatment with JP1 at a specified concentration, the oxidative phosphorylation levels of B16F10 (A) and LLC (B) cells were analyzed by OCR.
  • C-D Representative immunohistochemical staining of HIF1 ⁇ in B16F10 tumor nodules (C), HIF1 ⁇ intensity quantification diagram (D), scale bar: 100 ⁇ m.
  • E-F Representative immunohistochemical staining of HIF1 ⁇ in LLC tumor nodules (E), HIF1 ⁇ intensity quantification diagram (F), scale bar: 100 ⁇ m.
  • HIF1 ⁇ was detected by Western Blot after being treated with JP1 at a specified concentration for 48 hours.
  • I-J HIF1 ⁇ protein was extracted from B16F10 (I) and LLC (J) tumor nodules for Western Blot detection; quantitative results of HIF1 ⁇ protein concentration. (**P ⁇ 0.01; ***P ⁇ 0.001; ns: the difference was not statistically significant).
  • Figure 8 is a result graph of Example 4 of the present invention.
  • the key points of each figure are as follows:
  • A-B Representative Western Blot analysis of whole cell lysates from B16F10 (A) and LLC (B) cells after treatment with specified concentrations of JP1 to evaluate the expression of HIF1 ⁇ .
  • C-D Representative Western Blot analysis of HIF1 ⁇ protein extracted from B16F10 (C) and LLC (D) tumor nodules.
  • E-F Quantification of HIF1 ⁇ mRNA in B16F10 (E) and LLC (F) cells after treatment with specified concentrations of JP1.
  • Figure 9 is a result diagram of Example 5 of the present invention.
  • the key points of each figure are as follows:
  • A-B Western Blot (A) and quantitative RT-PCR (B) analysis of IL8 levels in B16F10 and LLC cells.
  • C-D Western Blot (C) and quantitative RT-PCR (D) analysis of IL8 levels in B16F10 and LLC cells after 24 hours of Ctrl-R or JP1 treatment under normoxic or hypoxic conditions.
  • F-G Tumor growth curves and tumor/body weight (%) result diagrams of each group are shown respectively.
  • H-I Representative immunohistochemical staining of HIF1 ⁇ in B16F10 tumor nodules of each group (H), HIF1 ⁇ intensity quantification diagram (I), scale bar is 50 ⁇ m.
  • J-K Western Blot detection of HIF1 ⁇ (J) and IL8 (K) protein expression in B16F10 tumor nodules in each group.
  • L-N Representative fluorescence images of ⁇ -SMA (green), desmin (green), CD31 (red), and DAPI nuclear staining in each group (L); Quantitative graph of ⁇ -SMA (M) and desmin (N) coverage, scale: 50 ⁇ m. (*P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ns: no statistically significant difference).
  • Figure 10 is a graph showing the results of Example 5 of the present invention.
  • the key points of each figure are as follows:
  • Figure 11 is a result diagram of Example 6 of the present invention.
  • the main points of each figure are as follows:
  • H Representative HIF1 ⁇ immunohistochemical staining images in B16F10 tumor nodules in each group, scale bar: 50 ⁇ m.
  • I Quantitative results of HIF1 ⁇ intensity.
  • J Quantitative results of vascular permeability after treatment with solvent, JP1, PTX, and JP1 combined with PTX.
  • K According to high-performance liquid chromatography analysis, the PTX standard solution has a peak at the retention time.
  • L Quantitative results of PTX concentration in tumors of each group.
  • M Illustration of the tumor microenvironment status after treatment with solvent, PTX, and JP1.
  • N The working mode of JP1 promoting tumor vascular normalization and thus inhibiting metastasis initiation. (*P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ns: the difference was not statistically significant).
  • Figure 12 is a result diagram of Example 6 of the present invention. The key points of each figure are as follows: (A) Representative HPLC analysis image of PTX at a specified concentration. (B) Linear standard curve of PTX concentration and peak area. (C-F) Representative HPLC analysis images of PTX in a specific group.
  • Example 1 JP1 inhibits metastasis by reducing tumor cell entry into blood vessels
  • an active metastasis model was established by surgical resection of the primary tumor, a melanoma model was established with B16F10 cells, and a lung cancer model was established with LLC cells.
  • group a is the Ctrl-R group, which was not intervened by JP1 from beginning to end; group b was intervened by JP1 from the beginning, but JP1 treatment was stopped after the primary tumor was resected; group c was not intervened by JP1 at the beginning, but JP1 treatment was given after the primary tumor was resected; group d was the JP1 intervention group from the beginning of tumor bearing, and JP1 treatment was given from beginning to end.
  • the dosage of JP1 was: 50 mg/kg, intraperitoneal injection, once a day. After the primary tumor was resected, mice developed multi-organ metastasis, the most common of which was lung metastasis.
  • a melanoma-bearing model was constructed using GFP-labeled B16F10 cells, and CTCs were analyzed by cell cloning and FACS sorting ( Figure 1, Figure F and Figure G).
  • the Ctrl-R group did not have JP1 intervention from beginning to end, and the JP1 group was treated with JP1 from beginning to end, and the dosage of JP1 was the same as above.
  • Cell cloning analysis showed that 4 out of 6 mice in the CTCs extracted from the Ctrl-R group formed clonal clusters in vitro, while only 1 out of 6 mice in the JP1 group formed clonal clusters ( Figure 1, Figure H, Figure 3, Figure A).
  • JP1 inhibits metastasis initiation by reducing tumor cell entry into the vasculature, but has a weaker inhibitory effect on the invasive ability of tumor cells.
  • JP1 The sequence of JP1 is FPGSDRF-GGGG-RGD, in which the amino acid S is phosphorylated.
  • this example evaluates the morphology of tumor blood vessels extracted from melanoma and lung cancer samples, wherein the Ctrl-R group was not intervened by JP1 from beginning to end, and the JP1 group was treated with JP1 from beginning to end, and the dosage of JP1 was the same as that in Example 1.
  • Immunofluorescence staining showed that in melanoma (B16F10), the coverage of ⁇ -SMA (mural cell marker), Claudin 5 (endothelial cell marker), and Desmin (pericyte marker) increased after JP1 treatment ( Figures A-D in Figure 4).
  • JP1 can reduce the chance of tumor cells entering blood vessels by promoting the normalization of tumor blood vessels, thereby inhibiting the occurrence of tumor metastasis.
  • Example 3 JP1 promotes normalization of tumor blood vessels by inhibiting IL8
  • JP1 promoted tumor vascular normalization by increasing pericyte and endothelial cell coverage in IL8WT B16F10; however, knockout of IL8 significantly improved tumor vascular normalization, while JP1 treatment failed to enhance the vascular normalization index (Figure 5, L-O panels).
  • JP1 promotes tumor vascular normalization by inhibiting IL8.
  • Example 4 JP1 promotes tumor oxidative phosphorylation and improves tumor microenvironment hypoxia
  • hypoxia is an inevitable result of tumor development, and it has been reported that hypoxia stimulates IL8 secretion.
  • the JWA gene can inhibit pancreatic cancer metastasis by improving tumor microenvironment hypoxia by promoting aerobic respiration.
  • OCR oxygen consumption rate
  • JP1 can effectively promote oxidative phosphorylation of B16F10 and LLC cells.
  • HIF1 ⁇ staining of melanoma and lung cancer model tumor samples showed that JP1 significantly improved the hypoxia of the tumor microenvironment ( Figures C-F of Figure 7).
  • JP1 can effectively promote tumor oxidative phosphorylation and improve tumor microenvironment hypoxia.
  • Example 5 Hypoxia induces tumor cells to secrete IL8
  • this example uses B16F10 and LLC cells to establish normoxic (oxygen content in air 21%) and hypoxic (oxygen content in air 8%) models, and found that IL8 expression increased under hypoxic conditions ( Figure 9 A and Figure B).
  • JP1 significantly inhibited the expression of HIF1 ⁇ under normoxic conditions; while under hypoxic conditions, the regulatory effect of JP1 on IL8 was weakened ( Figure 9 C and Figure D).
  • this example established a melanoma tumor-bearing model under normoxic and hypoxic conditions (Figure 9 E).
  • Vascular morphological evaluation showed that under hypoxic conditions, regardless of whether JP1 was treated or not, the vascular normalization index was lower than that under normoxic conditions ( Figure 9 L and Figure 9 M).
  • the Ctrl-R group had no JP1 intervention from beginning to end, the JP1 group was treated with JP1 from beginning to end, and the dosage of JP1 was the same as that in Example 1; Normoxia was under normoxic conditions, and Hypoxia was under hypoxic conditions.
  • JP1 improves tumor microenvironment hypoxia by promoting oxidative phosphorylation in tumor cells and thereby reducing the secretion of IL8.
  • this example verifies the expression of related molecular proteins in B16F10 and LLC cells.
  • JP1 regulates mitochondrial metabolic reprogramming of B16F10 and LLC cells through the AMPK/FOXO3a/UQCRC2 signaling pathway, thereby improving the hypoxic state of the tumor microenvironment (Figure 10, Figure C).
  • Example 6 JP1 promotes intratumoral delivery of paclitaxel (PTX) and enhances anti-tumor effects
  • Normalization of tumor vascular structure reduces its permeability, can reduce tumor cell invasion of blood vessels, and inhibit tumor metastasis; in addition, normalization of tumor vascular distribution also promotes the entry of drugs into the tumor and enhances the anti-tumor effect.
  • this example completes the combined treatment of melanoma model with JP1 and paclitaxel (PTX) ( Figure A of Figure 11).
  • this example performs hypoxia analysis on tumor tissues and finds that although PTX can promote normalization of tumor blood vessels, its inhibitory effect on blood vessels cannot improve the hypoxic state, while the combined treatment of JP1 and PTX significantly improves the hypoxic state of the tumor microenvironment ( Figure H and Figure I of Figure 11), and the vascular permeability experiment further verifies this observation (Figure J of Figure 11).
  • this example detects the content of PTX in the tumor by HPLC, and finds that compared with the combined treatment of JP1, after 11 days of PTX treatment, the delivery of PTX into the tumor is significantly reduced, while the combined treatment of JP1 and PTX significantly increases the delivery of PTX in the tumor (Figure K-N of Figure 11, Figure 12).
  • the Vehicle group was the control group without JP1 and PTX intervention; the JP1 group was treated with JP1 only, with a dose of 50 mg/kg, intraperitoneal injection, once a day; the PTX group was treated with PTX only, with a dose of 10 mg/kg, intraperitoneal injection, twice a week (Monday and Thursday); the JP1+PTX group was a group treated with JP1 and PTX together, with the dose of JP1 being the same as that of the JP1 group, and the dose of PTX being the same as that of the PTX group.
  • PTX inhibits metastasis initiation by promoting tumor vascular normalization, but its inhibitory effect on blood vessels reduces the delivery of drugs into tumors; the combination of JP1 and PTX can not only promote tumor vascular normalization to inhibit metastasis initiation, but also increase the delivery of PTX into tumors.
  • each JWA polypeptide shown in the table below was used for detection according to Examples 1 to 6, and the amino acid S of each JWA polypeptide was phosphorylated.
  • the present invention confirms that the series of JWA polypeptides represented by JP1: (1) improve the hypoxia of the tumor microenvironment by promoting oxidative phosphorylation of tumor cells and thus reducing the secretion of IL8, and regulate the mitochondrial metabolic reprogramming of tumor cells through the AMPK/FOXO3a/UQCRC2 signaling pathway to improve the hypoxic state of the tumor microenvironment; (2) promote the normalization of tumor vascular structure and intratumoral distribution by inhibiting IL8, reduce the chance of tumor cells entering blood vessels, thereby inhibiting the initiation and occurrence of tumor metastasis; (3) can increase the effective perfusion amount of drugs in the tumor.
  • JWA targeting peptides not only exhibit effective anti-tumor activity when used alone, but can also be combined with other therapies such as chemical drugs, antibody drugs, and cell drugs to exert synergistic tumor inhibition. These unique biological characteristics of JWA polypeptides will provide new clinical drug possibilities for the treatment of tumors, especially solid tumors, and have good application prospects.
  • Mouse melanoma B16F10 and lung cancer LLC cells were purchased from ATCC (MD, USA).
  • IL8KO (IL8 knockout) B16F10 cells were generated using IL8-specific CRISPR/Cas9 plasmids (synthesized by Corues Biotechnology). Sequencing analysis and western blot experiments confirmed that the gene knockout was complete.
  • GFP-B16F10 cells were generated by GFP-specific plasmids (synthesized by Shanghai Genechem Co., Ltd.). All cell lines were maintained in DMEM medium supplemented with streptomycin 100 ⁇ g/ml, penicillin 100 U/ml and 10% fetal bovine serum and stored in a 37°C, 5% CO 2 incubator.
  • mice All care and treatment of experimental mice were approved by the Guidelines of the Animal Care and Use Committee of Nanjing Medical University (IACUC: 1811067-1). Experimental mice were purchased from the Shanghai SLAC Laboratory Animal Center and stored in the Animal Core Facility of Nanjing Medical University.
  • mice were humanely killed, and the tumors were measured and recorded.
  • the oxygen concentration controller purchased from Shanghai Yuyan Instrument Co., Ltd.
  • the melanoma/lung cancer active metastasis model was performed using a general protocol described previously.
  • CTC (circulating tumor cell) analysis and sorting A melanoma model was constructed using GFP-labeled B16F10 cells. When the tumor volume reached 2000 mm 3 , 500 ⁇ l of blood was taken from each mouse to lyse red blood cells (red blood cell lysis buffer was purchased from Fcmacs Biotech Co., Ltd.). FACS analysis was performed using an LSRII flow cytometer (BD Biosciences) to detect CTCs in every 1.5 million cells, and the data were analyzed using FlowJo software (Tree Star Inc.). FACS sorting was performed using an Aria III instrument (BD Biosciences).
  • the mouse CD31 (Servicebio, GB12063) antibody solution was incubated with rabbit ⁇ -SMA (Servicebio, GB111364), Claudin 5 (Servicebio, GB11290), and Desmin (Servicebio, GB11081) antibody solutions at 4°C with gentle shaking for 24 hours.
  • the images were scanned after incubation with mouse (red) and rabbit (green) fluorescent secondary antibodies.
  • the staining intensity was analyzed with J-graphs.
  • Vascular permeability determination 50 mg/kg Evans Blue solution was injected into mice carrying melanoma through the tail vein. Blood was collected 1 hour later, the supernatant was centrifuged and diluted with formamide at 1:100. Evans blue was dissolved in formamide (0ng/ml, 125ng/ml, 250ng/ml, 500ng/ml, 1000ng/ml, 5000ng/ml, 10000ng/ml, 25000ng/ml, 50000ng/ml) to make a standard curve. The absorbance of the supernatant was measured at 620nm to calculate the concentration of Evans blue in the blood.
  • vascular permeability [ ⁇ l/(g ⁇ h)] [Evans blue ( ⁇ g)/tumor dry weight (g)]/[Evans blue concentration in blood ( ⁇ g/ ⁇ l) ⁇ circulation time (h)].
  • Angiogenesis array analysis Angiogenesis array GS1000 (GSH-ANG-1000-1, Ray Biotech Inc.) was used according to the manufacturer's instructions. Briefly, slides were removed from the box and equilibrated to room temperature in a sealed plastic bag for 20-30 min. Slides were removed from the plastic bag, the cover film was peeled off, and air-dried for 1-2 h. Standards or samples were collected and incubated with the array at 4 °C for 24 h with gentle shaking. Chemiluminescent signals were captured by InnoScan 300 microarray scanner (Innopsys). Data were extracted using the microarray analysis software ScanArray Express.
  • RNA from cells and tissues was extracted using TRIzon (Thermo Fisher Scientific, 10296010) and reverse transcribed using a reverse transcription kit (Vazyme, R323-01).
  • AB RT-PCR Q5 was performed using SYBR (Vazyme, TSE202). Finally, the difference value was calculated by GAPDH standardization.
  • the primers used in RT-PCR were: HIF1 ⁇ -F (ACGTTCCTTCGATCAGTTGTCACC), HIF1 ⁇ -R (GGCAGTGGTAGTGGTGGCATTAG), IL8-F (tcctgctttcctc), IL8-R (GGGTGGAAAGGTGTGGAATG), GAPDH-F (gctctctgctcctcctgtttttc), GAPDH-R (ACGACCAAATCCGTTGACTC).
  • Western Blot analysis was performed as previously reported. Briefly, cells and tissue samples were lysed with cell lysis buffer and tissue protein extraction reagent (purchased from Thermo Fisher Scientific, 78510), respectively.
  • Hif1 ⁇ CST, 36169T
  • IL8 Abcam, ab106350
  • p-AMPK CST, 2535T
  • AMPK Abcam, 32047
  • FOXO3A Abcam, 53287)
  • UQCRC2 Abcam, ab203832
  • Tubulin Beyotime, AF0001.
  • OCR analysis of B16F10 and LLC cells was performed using Seahorse XFp Analyzer (Seahorse XF96). B16F10 cells (6000) and LLC cells (10000) were seeded onto hippocampal plates and placed in growth medium for 24 h. Oligomycin (1 ⁇ M) and FCCP (B16F10: 1.5 ⁇ M, LLC: 1 ⁇ M) were added sequentially to XF matrix containing 1 mM sodium pyruvate, 2 mM l-glutamine, and 10 mM glucose to measure mitochondrial oxygen consumption.
  • PTX Paclitaxel
  • NSC 125973 Selleck
  • the peak time of PTX was determined by HPLC (chromatographic column: Agilent Zorbax Exlipse Plus C18, 100 ⁇ 4.6 mm, 3.5 ⁇ M particle size; flow rate: 1 ml/min; wavelength: UV 254 nm; mobile phase: CH 3 OH 0.1% TFA/H 2 O 0.1% TFA, 0 min: 10:90, 0-15 min: 10:90-100:0, 15-20 min: 100:0).
  • Tumor-bearing mice were injected with 20 mg/kg PTX solution through the tail vein. After 5 min, 200 mg of the tumor was taken and dissolved in 1 ml of methanol solution.
  • PTX was dissolved in methanol solution (200 ⁇ g/ml, 100 ⁇ g/ml, 50 ⁇ g/ml, 25 ⁇ g/ml, 12.5 ⁇ g/ml, 6.25 ⁇ g/ml, 3.125 ⁇ g/ml, 1.56 ⁇ g/ml) to prepare a standard curve.
  • the standard or sample was measured by high performance liquid chromatography to calculate the concentration of paclitaxel in the tumor.
  • the present invention may also have other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

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Abstract

The present invention relates to a use of a JWA polypeptide in the preparation of an anti-tumor drug co-potentiator. The amino acid sequence of the polypeptide is as shown in I or II: I: FPGSDRF-Z, and II: X-FPGSDRF-Z, the amino acid S being subject to phosphorylation modification, and X and Z each being amino acids or amino acid sequences; X is selected from one of F, (R)9, (R)9-F, 6-aminocaproic acid, 6-aminocaproic acid-F, 6-aminocaproic acid-(R)9, 6-aminocaproic acid-(R)9-F; Z is selected from one of (G)n-RGD and A-(G)n-RGD, n being an integer greater than or equal to 0, and n having a value ranging from 0 to 10. The polypeptide can improve a hypoxic state of a tumor microenvironment, can promote tumor blood vessel normalization so as to inhibit initiation and occurrence of tumor metastasis, can increase the effective perfusion amount of drugs in tumors, and may be used as an anti-tumor drug co-potentiator, possibly providing a new clinical medication for treating tumors.

Description

JWA多肽在制备抗肿瘤药协同增效剂方面的应用Application of JWA polypeptide in the preparation of anti-tumor drug synergist 技术领域Technical Field
本发明涉及JWA多肽在制备抗肿瘤药协同增效剂方面的应用，属于抗肿瘤辅助药物技术领域。The present invention relates to application of JWA polypeptide in preparing anti-tumor drug synergistic enhancer, belonging to the technical field of anti-tumor auxiliary drugs.
背景技术Background technique
近年来，新兴的肿瘤诊断和治疗技术显著提高了原发肿瘤的治愈率，有些甚至在某些阶段完全抑制了肿瘤的生长[1,2]。然而，肿瘤转移是肿瘤患者死亡的主要原因[3]，一旦发生转移，现有的治疗方法难以使患者获益。由于肿瘤转移的治疗策略有限，防止转移起始可能是肿瘤治疗中未满足的重大临床需求[4,5]。肿瘤细胞转移的定植前阶段是一个复杂的多步骤过程，包括肿瘤细胞从原发肿瘤脱落，穿透基底膜，进入血管[6]，肿瘤细胞突破基底膜进入脉管系统是启动肿瘤转移的核心[7]。肿瘤微环境脉管系统的不成熟和不稳定为原发肿瘤细胞进入脉管系统转移到远处器官提供了前提条件[8,9]。因此，重构紊乱的血管，使其成为成熟稳定的血管，可有效抑制肿瘤转移的发生。肿瘤血管生成是一个受肿瘤细胞高度影响、促进肿瘤生长和转移的再生过程[10]。肿瘤细胞分泌的细胞因子与血管周围细胞相互作用并将其转化为成纤维细胞，从而使这些细胞失去保护血管的作用[11,12]。此外，肿瘤细胞释放伪内皮信号刺激内皮细胞迁移，导致内皮内衬不连续和基底膜缺陷[13]。所有这些特征都是血管不成熟和功能失调的表现。因此，血管的高通透性，为肿瘤细胞侵入血管提供了便利。肿瘤血管正常化可使成熟周细胞包围的内皮细胞处于静息状态。内皮细胞和周细胞被基膜分隔，并充满各种黏附蛋白以维持血管稳定[14]。肿瘤血管正常化通过减少肿瘤细胞对血管的侵袭来抑制转移[15,16]。此外，肿瘤微环境中规则成熟的血管分布促进血液灌注，缓解缺氧和酸中毒[17]。IL8是已知的趋化细胞因子，已被报道在多种肿瘤中高表达[18,19]；IL8可促进恶性肿瘤增殖和转移，在肿瘤微环境中起关键调控作用[20,21]。IL8还通过介导ECFC和TNBC之间的串扰而加重致瘤性[22]。乙肝病毒编码基因(HBX)通过激活MEK-ERK信号诱导高IL8产生，导致内皮细胞通透性增强，促进肿瘤血管侵袭[23]。更重要的是，IL8介导的血管不成熟诱导肿瘤微环境缺氧，进而刺激肿瘤细胞分泌IL8[24]，从而引起肿瘤微环境缺氧、IL8分泌及血管紊乱的恶性反馈。因此，抑制IL8可能是纠正肿瘤微环境中血管紊乱的有效治疗方法。In recent years, emerging tumor diagnostic and therapeutic technologies have significantly improved the cure rate of primary tumors, and some have even completely inhibited tumor growth at certain stages [1,2]. However, tumor metastasis is the main cause of death in cancer patients [3]. Once metastasis occurs, existing treatments are unlikely to benefit patients. Due to the limited treatment strategies for tumor metastasis, preventing the initiation of metastasis may be a major unmet clinical need in tumor treatment [4,5]. The pre-colonization stage of tumor cell metastasis is a complex multi-step process, including tumor cells detaching from the primary tumor, penetrating the basement membrane, and entering the blood vessels [6]. Breaking through the basement membrane and entering the vascular system is the core of initiating tumor metastasis [7]. The immaturity and instability of the tumor microenvironment vasculature provides the prerequisite for primary tumor cells to enter the vasculature and metastasize to distant organs [8,9]. Therefore, remodeling disordered blood vessels into mature and stable blood vessels can effectively inhibit the occurrence of tumor metastasis. Tumor angiogenesis is a regenerative process that is highly influenced by tumor cells and promotes tumor growth and metastasis [10]. Cytokines secreted by tumor cells interact with perivascular cells and transform them into fibroblasts, thereby causing these cells to lose their ability to protect blood vessels [11,12]. In addition, tumor cells release pseudo-endothelial signals to stimulate endothelial cell migration, resulting in discontinuity of endothelial lining and defects in basement membrane [13]. All of these characteristics are manifestations of vascular immaturity and dysfunction. Therefore, the high permeability of blood vessels facilitates the invasion of tumor cells into blood vessels. Normalization of tumor blood vessels can keep endothelial cells surrounded by mature pericytes in a quiescent state. Endothelial cells and pericytes are separated by basement membranes and filled with various adhesion proteins to maintain vascular stability [14]. Normalization of tumor blood vessels inhibits metastasis by reducing tumor cell invasion of blood vessels [15,16]. In addition, regular and mature blood vessel distribution in the tumor microenvironment promotes blood perfusion and relieves hypoxia and acidosis [17]. IL8 is a known chemotactic cytokine that has been reported to be highly expressed in a variety of tumors [18,19]; IL8 can promote the proliferation and metastasis of malignant tumors and play a key regulatory role in the tumor microenvironment [20,21]. IL8 also aggravates tumorigenicity by mediating crosstalk between ECFC and TNBC [22]. The hepatitis B virus-encoded gene (HBX) induces high IL8 production by activating MEK-ERK signaling, leading to increased endothelial cell permeability and promoting tumor vascular invasion[23]. More importantly, IL8-mediated vascular immaturity induces hypoxia in the tumor microenvironment, which in turn stimulates tumor cells to secrete IL8[24], leading to a vicious feedback of hypoxia in the tumor microenvironment, IL8 secretion, and vascular disorder. Therefore, inhibiting IL8 may be an effective treatment for correcting vascular disorder in the tumor microenvironment.
JWA是一种肿瘤抑制基因，可抑制黑色素瘤、胃癌、乳腺癌生长、转移等恶性表型[25-27]。JWA通过调控AMPK/FOXO3a/UQCRC2信号，促进线粒体代谢重编程，改善肿瘤微环境缺氧，从而抑制肿瘤转移[28]；基于JWA功能序列设计的抗肿瘤肽JP1能有效抑制黑色素瘤转移[29]；然而，抗肿瘤肽JP1抑制肿瘤转移的机理目前尚不清楚，抗肿瘤肽JP1是否能作为现有抗肿瘤药物的协同增效剂，也亟待进一步探索和研究。对此，发明人课题组已有最新的研究成果，并以此来申请本发明专利。JWA is a tumor suppressor gene that can inhibit the growth and metastasis of melanoma, gastric cancer, breast cancer and other malignant phenotypes[25-27]. JWA regulates AMPK/FOXO3a/UQCRC2 signals, promotes mitochondrial metabolic reprogramming, improves hypoxia in the tumor microenvironment, and thus inhibits tumor metastasis[28]. The anti-tumor peptide JP1 designed based on the JWA functional sequence can effectively inhibit melanoma metastasis[29]. However, the mechanism by which the anti-tumor peptide JP1 inhibits tumor metastasis is still unclear, and whether the anti-tumor peptide JP1 can be used as a synergist of existing anti-tumor drugs also needs further exploration and research. In this regard, the inventor's research group has the latest research results and applied for the patent of this invention based on them.
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发明内容Summary of the invention
本发明的主要目的是：针对现有技术存在的问题，提出一种JWA多肽在制备抗肿瘤药协同增效剂方面的应用，能改善肿瘤微环境的缺氧状态，能促进肿瘤血管正常化以抑制肿瘤转移的起始和发生，与化疗药物联合使用能增加肿瘤内药物的有效灌注量，发挥协同增效作用，为治疗肿瘤提供新的临床用药可能。The main purpose of the present invention is to propose an application of JWA polypeptide in the preparation of anti-tumor drug synergistic enhancers in view of the problems existing in the prior art, which can improve the hypoxic state of the tumor microenvironment, promote the normalization of tumor blood vessels to inhibit the initiation and occurrence of tumor metastasis, and can be used in combination with chemotherapy drugs to increase the effective perfusion amount of drugs in the tumor, exert a synergistic effect, and provide new clinical drug possibilities for the treatment of tumors.
本发明解决其技术问题的技术方案如下：The technical solution of the present invention to solve the technical problem is as follows:
一种多肽的用途，其特征是，所述用途为用于制备抗肿瘤药协同增效剂；A use of a polypeptide, characterized in that the use is used for preparing an anti-tumor drug synergist;
所述多肽的氨基酸序列如I或II所示：The amino acid sequence of the polypeptide is shown in I or II:
I：FPGSDRF-Z；I: FPGSDRF-Z;
II：X-FPGSDRF-Z；II: X-FPGSDRF-Z;
其中，氨基酸S经磷酸化修饰，X、Z分别为氨基酸或氨基酸序列；Wherein, the amino acid S is phosphorylated, and X and Z are amino acids or amino acid sequences, respectively;
X选自F、(R) 9、(R) 9-F、6-氨基己酸、6-氨基己酸-F、6-氨基己酸-(R) 9、6-氨基己酸-(R) 9-F之一； X is selected from one of F, (R) 9 , (R) 9 -F, 6-aminocaproic acid, 6-aminocaproic acid-F, 6-aminocaproic acid-(R) 9 , and 6-aminocaproic acid-(R) 9 -F;
Z选自(G) n-RGD、A-(G) n-RGD之一，n为大于或等于0的整数，且n的取值范围为0-10。 Z is selected from (G) n -RGD and A-(G) n -RGD, n is an integer greater than or equal to 0, and the value range of n is 0-10.
优选地，所述抗肿瘤药协同增效剂的功能为：通过促进肿瘤细胞氧化磷酸化从而减少IL8的分泌来改善肿瘤微环境缺氧，并且通过AMPK/FOXO3a/UQCRC2信号通路调控肿瘤细胞的线粒体代谢重编程以改善肿瘤微环境的缺氧状态。Preferably, the function of the anti-tumor drug synergist is to improve the hypoxia of the tumor microenvironment by promoting oxidative phosphorylation of tumor cells and thus reducing the secretion of IL8, and to improve the hypoxic state of the tumor microenvironment by regulating the mitochondrial metabolic reprogramming of tumor cells through the AMPK/FOXO3a/UQCRC2 signaling pathway.
优选地，所述抗肿瘤药协同增效剂的功能为：通过抑制IL8促进肿瘤血管正常化，减少肿瘤细胞进入血管的机会，从而抑制肿瘤转移的起始和发生。Preferably, the function of the anti-tumor drug synergist is to promote the normalization of tumor blood vessels by inhibiting IL8, reduce the chance of tumor cells entering blood vessels, and thus inhibit the initiation and occurrence of tumor metastasis.
优选地，所述抗肿瘤药协同增效剂的功能为：增加肿瘤内药物的有效灌注量。Preferably, the function of the anti-tumor drug synergist is to increase the effective perfusion amount of the drug in the tumor.
优选地，所述抗肿瘤药协同增效剂针对的抗肿瘤药为用于肿瘤治疗的化学药、抗体药、细胞类药物。Preferably, the anti-tumor drug targeted by the anti-tumor drug synergist is a chemical drug, antibody drug, or cell drug used for tumor treatment.
更优选地，所述抗肿瘤药协同增效剂针对的抗肿瘤药为紫杉醇。More preferably, the anti-tumor drug targeted by the anti-tumor drug synergist is paclitaxel.
优选地，所述抗肿瘤药协同增效剂针对的肿瘤为至少具有以下特征之一的泛实体瘤：Preferably, the tumor targeted by the anti-tumor drug synergist is a pan-solid tumor having at least one of the following characteristics:
特征一：瘤内微环境缺氧、血管分布紊乱、瘤内药物灌注量低；Feature 1: hypoxia in the tumor microenvironment, disordered vascular distribution, and low intratumoral drug perfusion;
特征二：肿瘤细胞线粒体能量代谢紊乱，氧化磷酸化水平降低、糖酵解代谢增强；Feature 2: Mitochondrial energy metabolism in tumor cells is disrupted, with decreased oxidative phosphorylation and enhanced glycolysis metabolism;
特征三：IL8分泌量异常增加、瘤内血管壁完整性被破坏。Feature three: Abnormal increase in IL8 secretion and destruction of the integrity of the vascular wall within the tumor.
更优选地，所述抗肿瘤药协同增效剂针对的肿瘤为黑色素瘤、肺癌、或人类黑色素细胞痣。More preferably, the tumor targeted by the anti-tumor drug synergist is melanoma, lung cancer, or human melanocytic nevus.
优选地，所述多肽的N端经乙酰化修饰、C端经酰胺化修饰；所述多肽的氨基酸为L型天然氨基酸或D型非天然氨基酸。Preferably, the N-terminus of the polypeptide is modified by acetylation, and the C-terminus is modified by amidation; the amino acids of the polypeptide are L-type natural amino acids or D-type non-natural amino acids.
优选地，所述多肽的氨基酸序列为FPGSDRF-RGD，其中，氨基酸S经磷酸化修饰。Preferably, the amino acid sequence of the polypeptide is FPGSDRF-RGD, wherein the amino acid S is phosphorylated.
本发明涉及的多肽是专利号CN201310178099X、授权公告号CN103239710B的中国发明专利中记载的系列多肽中的一部分。发明人经实践研究发现，上述多肽能通过AMPK/FOXO3a/UQCRC2信号通路调控线粒体代谢重编程，并通过促进肿瘤细胞氧化磷酸化从而减少白细胞介素-8(IL8)的分泌，以此来改善肿瘤微环境缺氧；富氧的肿瘤微环境抑制肿瘤细胞分泌IL8，促进肿瘤血管正常化；血管正常化可使血管成熟、分布正常，使肿瘤微环境形成血管规正化、充分灌注、富氧微环境的良性反馈，阻止肿瘤细胞进入血管，抑制转移起始。此外，上述多肽联合紫杉醇(PTX)可维持肿瘤内一定的血管密度，促进肿瘤血管生长正常化，增加氧和药物的输送，增强抗肿瘤作用。因此，上述多肽可作为抗肿瘤药协同增效剂，为攻克肿瘤尤其是泛实体瘤治疗中因肿瘤微环境缺氧、血管结构和分布异常、IL8表达增加等导致的瘤内药物灌注量减少而疗效差的瓶颈提供新的技术手段，具有良好的应用前景。The polypeptide involved in the present invention is a part of the series of polypeptides recorded in the Chinese invention patent with patent number CN201310178099X and authorization announcement number CN103239710B. The inventor has found through practical research that the above polypeptide can regulate mitochondrial metabolic reprogramming through the AMPK/FOXO3a/UQCRC2 signaling pathway, and reduce the secretion of interleukin-8 (IL8) by promoting oxidative phosphorylation of tumor cells, thereby improving the hypoxia of the tumor microenvironment; the oxygen-rich tumor microenvironment inhibits the secretion of IL8 by tumor cells and promotes the normalization of tumor blood vessels; blood vessel normalization can make blood vessels mature and distribute normally, so that the tumor microenvironment forms a benign feedback of blood vessel regularization, full perfusion, and oxygen-rich microenvironment, preventing tumor cells from entering blood vessels and inhibiting the initiation of metastasis. In addition, the above polypeptide combined with paclitaxel (PTX) can maintain a certain blood vessel density in the tumor, promote the normalization of tumor blood vessel growth, increase the delivery of oxygen and drugs, and enhance the anti-tumor effect. Therefore, the above-mentioned polypeptides can be used as synergistic enhancers of anti-tumor drugs, providing new technical means to overcome the bottleneck of poor efficacy caused by reduced intratumor drug infusion due to hypoxia in the tumor microenvironment, abnormal vascular structure and distribution, increased IL8 expression, etc. in the treatment of tumors, especially solid tumors, and have good application prospects.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为本发明实施例1实施过程及结果图。各图要点如下：(A)B16F10(黑色素瘤细胞)/LLC(肺腺癌细胞)同种异体移植示意图，Ctrl-R或JP1治疗后的活性转移两阶段模型。(B-C)经指定治疗手术切除原发性B16F10(B)或LLC肿瘤(C)后发生肺转移的小鼠的定量(n＝6)结果图。(D-E)B16F10(D)和LLC(E)肿瘤小鼠的Kaplan-Meier生存分析(n＝10)结果图。(F)流式细胞仪检测GFP+B16F10细胞转染GFP特异性质粒后的结果图。(G)CTC克隆计数和流式细胞术分析示意图。(H)Ctrl-R或JP1处理后CTC集落形成的定量(n＝6)结果图。(I)Ctrl-R或JP1处理后每个1.5×10 7血细胞的CTC定量(n＝6)结果图。(J-K)来自6只小鼠的CTC进行迁移和侵袭试验，每30000个细胞中迁移细胞(J)和侵袭细胞(K)的定量结果图。(*P<0.05；**P<0.01；ns：差异没有统计学意义)。 FIG1 is a diagram of the implementation process and results of Example 1 of the present invention. The main points of each figure are as follows: (A) Schematic diagram of B16F10 (melanoma cells)/LLC (lung adenocarcinoma cells) allogeneic transplantation, two-stage model of active metastasis after Ctrl-R or JP1 treatment. (BC) Quantitative results (n=6) of mice with lung metastasis after surgical resection of primary B16F10 (B) or LLC tumors (C) after designated treatment. (DE) Kaplan-Meier survival analysis (n=10) of B16F10 (D) and LLC (E) tumor mice. (F) Flow cytometric detection of GFP+B16F10 cells transfected with GFP-specific plasmid. (G) Schematic diagram of CTC clone counting and flow cytometric analysis. (H) Quantitative results (n=6) of CTC colony formation after Ctrl-R or JP1 treatment. (I) Quantitative results (n=6) of CTCs per 1.5×10 7 blood cells after Ctrl-R or JP1 treatment. (JK) CTCs from 6 mice were subjected to migration and invasion assays, and the quantitative results of migrating cells (J) and invasive cells (K) per 30,000 cells were plotted (*P<0.05;**P<0.01; ns: the difference was not statistically significant).
图2为本发明实施例1的结果图。各图要点如下：(A-B)B16F10肿瘤经Ctrl-R或JP1治疗后体重及肿瘤体积测量结果图。(C)各分组具有代表性的肺HE染色图，用于检测B16F10肿瘤转移起始点，比例尺:500μm。(D-E)LLC肿瘤经Ctrl-R或JP1治疗后的体重和肿瘤体积测量结果图。(F)各分组具有代表性的肺HE染色图，用于检测LLC肿瘤转移起始，比例尺:500μm。Figure 2 is a graph showing the results of Example 1 of the present invention. The key points of each figure are as follows: (A-B) B16F10 tumor body weight and tumor volume measurement results after treatment with Ctrl-R or JP1. (C) Representative lung HE staining images of each group, used to detect the starting point of B16F10 tumor metastasis, scale: 500μm. (D-E) LLC tumor body weight and tumor volume measurement results after treatment with Ctrl-R or JP1. (F) Representative lung HE staining images of each group, used to detect the starting point of LLC tumor metastasis, scale: 500μm.
图3为本发明实施例1的结果图。各图要点如下：(A)Ctrl-R或JP1处理后CTC克隆形成的代表性图像。(B)Ctrl-R或JP1处理后，通过FACS分析每个1.5×107血细胞的CTCs定量的代表性图像。(C)CTCs在Ctrl-R或JP1处理后迁移的代表性图像。(D)Ctrl-R或JP1处理后CTC侵袭的代表性图像。FIG3 is a graph showing the results of Example 1 of the present invention. The key points of each graph are as follows: (A) Representative images of CTC clone formation after Ctrl-R or JP1 treatment. (B) Representative images of CTC quantification per 1.5×107 blood cells by FACS analysis after Ctrl-R or JP1 treatment. (C) Representative images of CTC migration after Ctrl-R or JP1 treatment. (D) Representative images of CTC invasion after Ctrl-R or JP1 treatment.
图4为本发明实施例2的结果图。各图要点如下：(A)Ctrl-R或JP1处理B16F10肿瘤结节α-SMA(绿色)、claudin5(绿色)、desmin(绿色)、CD31(红色)和DAPI核染色代表性荧光图像。(B-D)B16F10肿瘤结节间质微环境α-SMA覆盖率(B)、claudin5覆盖率(C)、desmin覆盖率(D)结果图，比例尺:100μm。 (E)Ctrl处理LLC肿瘤结节α-SMA(绿色)、claudin5(绿色)、desmin(绿色)、CD31(红色)和DAPI核染色的代表性荧光图像Ctrl-R或JP1。(F-H)LLC肿瘤结节间质微环境α-SMA覆盖率(F)、claudin5覆盖率(G)、desmin覆盖率(H)结果图，比例尺:100μm。(I-J)血管通透性分析示意图(I)和血管通透性定量图(J)。(*P<0.05；**P<0.01)。Figure 4 is a result diagram of Example 2 of the present invention. The key points of each figure are as follows: (A) Representative fluorescence images of α-SMA (green), claudin5 (green), desmin (green), CD31 (red) and DAPI nuclear staining of B16F10 tumor nodules treated with Ctrl-R or JP1. (B-D) Result diagrams of α-SMA coverage (B), claudin5 coverage (C), and desmin coverage (D) of the stromal microenvironment of B16F10 tumor nodules, scale bar: 100μm. (E) Representative fluorescence images of α-SMA (green), claudin5 (green), desmin (green), CD31 (red) and DAPI nuclear staining of LLC tumor nodules treated with Ctrl-R or JP1. (F-H) Result diagrams of α-SMA coverage (F), claudin5 coverage (G), and desmin coverage (H) of LLC tumor nodules, scale bar: 100μm. (I-J) Schematic diagram of vascular permeability analysis (I) and quantitative diagram of vascular permeability (J). (*P<0.05; **P<0.01).
图5为本发明实施例3的结果图。各图要点如下：(A)Ctrl-R或JP1处理B16F10细胞中涉及肿瘤血管正常化的60个基因的热图。(B-C)在B16F10(B)和LLC(C)细胞中，以指定浓度的JP1处理48h后，IL8蛋白的定量图。(D-E)从B16F10(D)和LLC(E)肿瘤结节中提取的IL8蛋白进行Western Blot检测，IL8蛋白浓度的定量结果图。(F)α-SMA(绿色)和IL8(红色)的免疫荧光染色人类黑色素细胞痣和黑色素瘤的代表性图像，比例尺:200μm。(G-H)定量测定人类黑色素细胞痣与黑色素瘤之间的IL8 +面积(G)和α-SMA +面积(H)。(I)B16F10(IL8WT)和B16F10(IL8KO)细胞异体移植物按Ctrl-R或JP1处理示意图(n＝6)。(J-K)分别显示各组的肿瘤生长曲线和肿瘤/体重(％)结果图。(L-M)各组α-SMA(绿色)、CD31(红色)、DAPI核染色代表性荧光图(L)，α-SMA覆盖率的定量如图(M)所示，比例尺为100μm。(N-O)各组desmin(绿色)、CD31(红色)和DAPI核染色的代表性荧光图像(N)；图(O)显示了desmin覆盖率的定量，比例尺为100μm。(*P<0.05；**P<0.01；***P<0.001；ns:差异没有统计学意义)。 Figure 5 is a graph of the results of Example 3 of the present invention. The key points of each figure are as follows: (A) Heat map of 60 genes involved in tumor vascular normalization in B16F10 cells treated with Ctrl-R or JP1. (BC) Quantification of IL8 protein in B16F10 (B) and LLC (C) cells after 48h treatment with the specified concentration of JP1. (DE) Western Blot detection of IL8 protein extracted from B16F10 (D) and LLC (E) tumor nodules, and quantitative results of IL8 protein concentration. (F) Representative images of human melanocytic nevus and melanoma stained with immunofluorescence for α-SMA (green) and IL8 (red), scale bar: 200μm. (GH) Quantification of IL8 + area (G) and α-SMA + area (H) between human melanocytic nevus and melanoma. (I) Schematic diagram of B16F10 (IL8WT) and B16F10 (IL8KO) cell allografts treated with Ctrl-R or JP1 (n=6). (JK) Tumor growth curves and tumor/body weight (%) results of each group are shown. (LM) Representative fluorescence images of α-SMA (green), CD31 (red), and DAPI nuclear staining in each group (L), and the quantitative analysis of α-SMA coverage is shown in the figure (M), with a scale bar of 100 μm. (NO) Representative fluorescence images of desmin (green), CD31 (red), and DAPI nuclear staining in each group (N); the figure (O) shows the quantitative analysis of desmin coverage, with a scale bar of 100 μm. (*P<0.05;**P<0.01;***P<0.001; ns: no statistically significant difference).
图6为本发明实施例3的结果图。各图要点如下：(A-B)在指定浓度的JP1处理后，对来自B16F10(A)和LLC(B)细胞的全细胞裂解液进行代表性的Western Blot分析，以评估IL8的表达。(C-D)提取自B16F10(C)和LLC(D)肿瘤结节的IL8蛋白的代表性Western Blot分析结果图。(E-F)以指定浓度的JP1处理B16F10(E)和LLC(F)细胞后，IL8 mRNA的定量结果图。(G-H)采用qPCR方法检测B16F10(G)和LLC(H)肿瘤组织中IL8 mRNA的表达，定量检测IL-8 mRNA表达，代表性Western Blot分析表明，IL8敲除有效。(J)各组小鼠体重图。(K)Ctrl-R或JP1处理后，从IL8WT和IL8KO B16F10肿瘤中提取的代表性IL8蛋白Western Blot分析图。(*P<0.05；**P<0.01；***P<0.001；ns：差异没有统计学意义)。Figure 6 is a graph showing the results of Example 3 of the present invention. The key points of each figure are as follows: (A-B) Representative Western Blot analysis of whole cell lysates from B16F10 (A) and LLC (B) cells after treatment with specified concentrations of JP1 to evaluate the expression of IL8. (C-D) Representative Western Blot analysis results of IL8 protein extracted from B16F10 (C) and LLC (D) tumor nodules. (E-F) Quantitative results of IL8 mRNA after treatment of B16F10 (E) and LLC (F) cells with specified concentrations of JP1. (G-H) qPCR method was used to detect the expression of IL8 mRNA in B16F10 (G) and LLC (H) tumor tissues, and IL-8 mRNA expression was quantitatively detected. Representative Western Blot analysis showed that IL8 knockout was effective. (J) Body weight of mice in each group. (K) Representative Western Blot analysis of IL8 protein extracted from IL8WT and IL8KO B16F10 tumors after Ctrl-R or JP1 treatment. (*P<0.05; **P<0.01; ***P<0.001; ns: the difference was not statistically significant).
图7为本发明实施例4的结果图。各图要点如下：(A-B)在指定浓度的JP1作用48h后，通过OCR分析B16F10(A)和LLC(B)细胞的氧化磷酸化水平。(C-D)B16F10肿瘤结节中HIF1α代表性免疫组化染色图(C)，HIF1α强度定量图(D)，比例尺:100μm。(E-F)LLC肿瘤结节中HIF1α代表性免疫组化染色图(E)，HIF1α强度定量图(F)，比例尺:100μm。(G-H)在B16F10(G)和LLC(H)细胞中，用指定浓度的JP1处理48h后，通过Western Blot检测HIF1α。(I-J)从B16F10(I)和LLC(J)肿瘤结节中提取HIF1α蛋白进行Western Blot检测；HIF1α蛋白浓度的定量结果图。(**P<0.01；***P<0.001；ns:差异没有统计学意义)。Figure 7 is a result diagram of Example 4 of the present invention. The key points of each figure are as follows: (A-B) After 48 hours of treatment with JP1 at a specified concentration, the oxidative phosphorylation levels of B16F10 (A) and LLC (B) cells were analyzed by OCR. (C-D) Representative immunohistochemical staining of HIF1α in B16F10 tumor nodules (C), HIF1α intensity quantification diagram (D), scale bar: 100μm. (E-F) Representative immunohistochemical staining of HIF1α in LLC tumor nodules (E), HIF1α intensity quantification diagram (F), scale bar: 100μm. (G-H) In B16F10 (G) and LLC (H) cells, HIF1α was detected by Western Blot after being treated with JP1 at a specified concentration for 48 hours. (I-J) HIF1α protein was extracted from B16F10 (I) and LLC (J) tumor nodules for Western Blot detection; quantitative results of HIF1α protein concentration. (**P<0.01; ***P<0.001; ns: the difference was not statistically significant).
图8为本发明实施例4的结果图。各图要点如下：(A-B)在指定浓度的JP1处理后，对来自B16F10(A)和LLC(B)细胞的全细胞裂解液进行代表性的Western Blot分析，以评估HIF1α的表达。(C-D)提取自B16F10(C)和LLC(D)肿瘤结节的HIF1α蛋白的代表性Western Blot分析图。(E-F)在B16F10(E)和LLC(F)细胞中，以指定浓度的JP1处理后的HIF1αmRNA的定量图。(G-H)采用qPCR方法检测B16F10(G)和LLC(H)瘤体中HIF1α的mRNA表达，定量检测HIF1αmRNA表达结果图。(*P<0.05；**P<0.01；***P<0.001；ns：差异没有统计学意义)。Figure 8 is a result graph of Example 4 of the present invention. The key points of each figure are as follows: (A-B) Representative Western Blot analysis of whole cell lysates from B16F10 (A) and LLC (B) cells after treatment with specified concentrations of JP1 to evaluate the expression of HIF1α. (C-D) Representative Western Blot analysis of HIF1α protein extracted from B16F10 (C) and LLC (D) tumor nodules. (E-F) Quantification of HIF1α mRNA in B16F10 (E) and LLC (F) cells after treatment with specified concentrations of JP1. (G-H) qPCR method was used to detect the mRNA expression of HIF1α in B16F10 (G) and LLC (H) tumors, and the results of quantitative detection of HIF1α mRNA expression were shown. (*P<0.05; **P<0.01; ***P<0.001; ns: the difference was not statistically significant).
图9为本发明实施例5的结果图。各图要点如下：(A-B)Western Blot(A)和定量RT-PCR(B)分析B16F10和LLC细胞的IL8水平。(C-D)Western Blot(C)和定量RT-PCR(D)分析在常氧或缺氧条件下，Ctrl-R或JP1处理24h后B16F10和LLC细胞的IL8水平。(E)常氧或缺氧条件下B16F10细胞异体移植Ctrl-R或JP1处理示意图(n＝6)。(F-G)分别显示各组的肿瘤生长曲线和肿瘤/体重(％)结果图。(H-I)各组B16F10肿瘤结节中HIF1α代表性免疫组化染色图(H)，HIF1α强度定量图(I)，比例尺为50μm。(J-K)Western Blot检测各组B16F10肿瘤结节中HIF1α(J)和IL8(K)蛋白的表达结果图。(L-N)各组α-SMA(绿色)、desmin(绿色)、CD31(红色)、DAPI核染色的代表性荧光图像(L)；α-SMA(M)和desmin(N)覆盖率的定量图，比例尺:50μm。(*P<0.05；**P<0.01；***P<0.001；ns:差异没有统计学意义)。Figure 9 is a result diagram of Example 5 of the present invention. The key points of each figure are as follows: (A-B) Western Blot (A) and quantitative RT-PCR (B) analysis of IL8 levels in B16F10 and LLC cells. (C-D) Western Blot (C) and quantitative RT-PCR (D) analysis of IL8 levels in B16F10 and LLC cells after 24 hours of Ctrl-R or JP1 treatment under normoxic or hypoxic conditions. (E) Schematic diagram of B16F10 cell allogeneic transplantation Ctrl-R or JP1 treatment under normoxic or hypoxic conditions (n=6). (F-G) Tumor growth curves and tumor/body weight (%) result diagrams of each group are shown respectively. (H-I) Representative immunohistochemical staining of HIF1α in B16F10 tumor nodules of each group (H), HIF1α intensity quantification diagram (I), scale bar is 50μm. (J-K) Western Blot detection of HIF1α (J) and IL8 (K) protein expression in B16F10 tumor nodules in each group. (L-N) Representative fluorescence images of α-SMA (green), desmin (green), CD31 (red), and DAPI nuclear staining in each group (L); Quantitative graph of α-SMA (M) and desmin (N) coverage, scale: 50μm. (*P<0.05; **P<0.01; ***P<0.001; ns: no statistically significant difference).
图10为本发明实施例5的结果图。各图要点如下：(A)各组小鼠体重图。(B)常氧和缺氧状态下，Ctrl-R或JP1处理B16F10肿瘤结节提取的HIF-1α和IL8蛋白的代表性Western Blot分析结果图。(C)在指定浓度的JP1处理后，对来自B16F10和LLC细胞的全细胞裂解液进行代表性的Western Blot分析，以评估p-AMPK、AMPK、FOXO3a、UQCRC2、HIF1α和IL8的表达。Figure 10 is a graph showing the results of Example 5 of the present invention. The key points of each figure are as follows: (A) Body weight of mice in each group. (B) Representative Western Blot analysis results of HIF-1α and IL8 proteins extracted from B16F10 tumor nodules treated with Ctrl-R or JP1 under normoxic and hypoxic conditions. (C) Representative Western Blot analysis of whole cell lysates from B16F10 and LLC cells after treatment with the specified concentrations of JP1 to evaluate the expression of p-AMPK, AMPK, FOXO3a, UQCRC2, HIF1α and IL8.
图11为本发明实施例6的结果图。各图要点如下：(A)B16F10细胞异体移植模型示意图。小鼠腹腔注射JP1和PTX单独或联合治疗及相关溶剂。(B-C)各组的肿瘤生长曲线及肿瘤/体重(％)结果图。(D-G)各组α-SMA(绿色)、desmin(绿色)、CD31(红色)、DAPI核染色代表性荧光图像；α-SMA、desmin和CD31覆盖率的定量图，比例尺:50μm。(H)各组B16F10肿瘤结节中具有代表性的HIF1α免疫组化染色图，比例尺:50μm。(I)HIF1α强度的定量结果图。(J)溶剂、JP1、PTX及JP1联合PTX处理后血管通透性的定量结果图。(K)通过高效液相色谱分析，PTX标准溶液在保留时间上有一个峰。(L)各组肿瘤内PTX浓度的定量结果图。(M)溶剂、PTX、JP1治疗后肿瘤微环境状态的图解。(N)JP1促进肿瘤血管正常化从而抑制转移起始的工作模式。(*P<0.05；**P<0.01；***P<0.001；ns:差异没有统计学意义)。Figure 11 is a result diagram of Example 6 of the present invention. The main points of each figure are as follows: (A) Schematic diagram of the B16F10 cell xenograft model. Mice were intraperitoneally injected with JP1 and PTX alone or in combination and related solvents. (B-C) Tumor growth curves and tumor/body weight (%) results of each group. (D-G) Representative fluorescent images of α-SMA (green), desmin (green), CD31 (red), and DAPI nuclear staining in each group; quantitative graphs of α-SMA, desmin, and CD31 coverage, scale bar: 50μm. (H) Representative HIF1α immunohistochemical staining images in B16F10 tumor nodules in each group, scale bar: 50μm. (I) Quantitative results of HIF1α intensity. (J) Quantitative results of vascular permeability after treatment with solvent, JP1, PTX, and JP1 combined with PTX. (K) According to high-performance liquid chromatography analysis, the PTX standard solution has a peak at the retention time. (L) Quantitative results of PTX concentration in tumors of each group. (M) Illustration of the tumor microenvironment status after treatment with solvent, PTX, and JP1. (N) The working mode of JP1 promoting tumor vascular normalization and thus inhibiting metastasis initiation. (*P<0.05; **P<0.01; ***P<0.001; ns: the difference was not statistically significant).
图12为本发明实施例6的结果图。各图要点如下：(A)指定浓度的PTX代表性高效液相色谱分析图像。(B)PTX浓度与峰面积的线性标准曲线。(C-F)特定组中PTX的代表性高效液相色谱分析图像。Figure 12 is a result diagram of Example 6 of the present invention. The key points of each figure are as follows: (A) Representative HPLC analysis image of PTX at a specified concentration. (B) Linear standard curve of PTX concentration and peak area. (C-F) Representative HPLC analysis images of PTX in a specific group.
具体实施方式Detailed ways
下面参照附图并结合实施例对本发明作进一步详细描述。但是本发明不限于所给出的例子。各实施例所用的材料、方法、实验模型条件等内容附在各实施例之后，除此之外，如无特殊说明，所用材料、实验方法均为常规材料、常规实验方法。The present invention is further described in detail below with reference to the accompanying drawings and in combination with the examples. However, the present invention is not limited to the examples given. The materials, methods, experimental model conditions and other contents used in each embodiment are attached after each embodiment. In addition, unless otherwise specified, the materials and experimental methods used are conventional materials and conventional experimental methods.
实施例1、JP1通过减少肿瘤细胞进入血管来抑制转移Example 1: JP1 inhibits metastasis by reducing tumor cell entry into blood vessels
为确定JP1在转移起始中的作用，通过手术切除原发肿瘤建立了活性转移模型，以B16F10细胞建立黑色素瘤模型，以LLC细胞建立肺癌模型。如图1的A图所示，a组为Ctrl-R组，自始至终没有JP1干预；b组为JP1自始干预，但在原发肿瘤切除后停止JP1治疗；c组为开始没有JP1干预，但在原发肿瘤切除后给予JP1治疗；d组为自荷瘤起全程JP1干预组，自始至终给予JP1治疗。其中，JP1的给药剂量为：50mg/kg，腹腔注射，每天1次。原发性肿瘤切除后，小鼠发生多器官转移，最常见的是肺转移。随后进行肺组织染色获得肺转移的发生率，发现a组(Ctrl-R组)6只小鼠中有5只出现肺转 移，而d组(自荷瘤起全程JP1干预组)在黑色素瘤模型中只有1只小鼠出现肺转移。重要的是，在原发肿瘤切除后停止JP1治疗的b组中，6只小鼠中也只有1只发生肺转移，而在原发肿瘤切除后给予JP1治疗的c组中，6只小鼠中有4只出现肺转移(图1的B图，以及图2的A-C图)。这一观察结果在肺癌模型中得到了进一步验证(图1的C图，以及图2的D-F图)。此外，还建立了平行生存模型，研究发现，与对照组相比，在黑色素瘤和肺癌模型中，JP1干预延长了小鼠生存时间，其中JP1全程干预组效果最好，其次为荷瘤期间JP1干预组，这两组小鼠生存时间均显著长于对照组小鼠；切除荷瘤后JP1干预组对小鼠生存也有一定改善作用，但效果不如早期干预和全程干预组(图1的D图、E图)。这些数据表明JP1能抑制肿瘤转移的发生。To determine the role of JP1 in the initiation of metastasis, an active metastasis model was established by surgical resection of the primary tumor, a melanoma model was established with B16F10 cells, and a lung cancer model was established with LLC cells. As shown in Figure 1A, group a is the Ctrl-R group, which was not intervened by JP1 from beginning to end; group b was intervened by JP1 from the beginning, but JP1 treatment was stopped after the primary tumor was resected; group c was not intervened by JP1 at the beginning, but JP1 treatment was given after the primary tumor was resected; group d was the JP1 intervention group from the beginning of tumor bearing, and JP1 treatment was given from beginning to end. Among them, the dosage of JP1 was: 50 mg/kg, intraperitoneal injection, once a day. After the primary tumor was resected, mice developed multi-organ metastasis, the most common of which was lung metastasis. Subsequently, lung tissue staining was performed to obtain the incidence of lung metastasis. It was found that 5 out of 6 mice in group a (Ctrl-R group) had lung metastasis, while only 1 mouse in group d (JP1 intervention group from the beginning of tumor bearing) had lung metastasis in the melanoma model. Importantly, in group b where JP1 treatment was stopped after primary tumor resection, only 1 of 6 mice developed lung metastasis, while in group c where JP1 treatment was given after primary tumor resection, 4 of 6 mice developed lung metastasis (Figure 1, Panel B, and Figure 2, Panels A-C). This observation was further verified in the lung cancer model (Figure 1, Panel C, and Figure 2, Panels D-F). In addition, a parallel survival model was established, and the study found that compared with the control group, JP1 intervention prolonged the survival time of mice in melanoma and lung cancer models, among which the JP1 full-course intervention group had the best effect, followed by the JP1 intervention group during tumor bearing, and the survival time of these two groups of mice was significantly longer than that of the control group mice; the JP1 intervention group after tumor resection also had a certain improvement on the survival of mice, but the effect was not as good as the early intervention and full-course intervention groups (Figure 1, Panel D, Panel E). These data indicate that JP1 can inhibit the occurrence of tumor metastasis.
为了进一步验证该假设，用GFP标记的B16F10细胞构建了携带黑色素瘤的模型，并通过细胞克隆和FACS分选分析了CTCs(图1的F图、G图)。其中，Ctrl-R组为自始至终没有JP1干预，JP1组为自始至终给予JP1治疗，且JP1的给药剂量与前文相同。细胞克隆分析显示，Ctrl-R组小鼠提取的CTCs中6只小鼠有4只在体外形成克隆簇，而JP1组6只小鼠只有1只形成克隆簇(图1的H图，图3的A图)。此外，FACS分析显示，原发肿瘤进入循环系统的肿瘤细胞在JP1治疗后明显减少(图1的I图，图3的B图)。之后，对CTC进行迁移和侵袭实验，发现经过JP1处理的CTC迁移能力减弱(图1的J图，图3的C图)，但侵袭能力保持不变(图1的K图，图3的D图)。To further verify this hypothesis, a melanoma-bearing model was constructed using GFP-labeled B16F10 cells, and CTCs were analyzed by cell cloning and FACS sorting (Figure 1, Figure F and Figure G). Among them, the Ctrl-R group did not have JP1 intervention from beginning to end, and the JP1 group was treated with JP1 from beginning to end, and the dosage of JP1 was the same as above. Cell cloning analysis showed that 4 out of 6 mice in the CTCs extracted from the Ctrl-R group formed clonal clusters in vitro, while only 1 out of 6 mice in the JP1 group formed clonal clusters (Figure 1, Figure H, Figure 3, Figure A). In addition, FACS analysis showed that the number of tumor cells entering the circulation system from the primary tumor was significantly reduced after JP1 treatment (Figure 1, Figure I, Figure 3, Figure B). Afterwards, migration and invasion experiments were performed on CTCs, and it was found that the migration ability of CTCs treated with JP1 was weakened (Figure 1, Figure J, Figure 3, Figure C), but the invasion ability remained unchanged (Figure 1, Figure K, Figure 3, Figure D).
这些结果表明，JP1通过减少肿瘤细胞进入血管系统来抑制转移起始，但对肿瘤细胞的侵袭能力的抑制作用较弱。These results suggest that JP1 inhibits metastasis initiation by reducing tumor cell entry into the vasculature, but has a weaker inhibitory effect on the invasive ability of tumor cells.
注：JP1的序列为FPGSDRF-GGGG-RGD，其中，氨基酸S经磷酸化修饰。Note: The sequence of JP1 is FPGSDRF-GGGG-RGD, in which the amino acid S is phosphorylated.
实施例2、JP1促进肿瘤血管正常化Example 2: JP1 promotes normalization of tumor blood vessels
为研究JP1减少肿瘤细胞进入血管的功能，本实施例评估从黑色素瘤和肺癌样本中提取的肿瘤血管的形态，其中，Ctrl-R组为自始至终没有JP1干预，JP1组为自始至终给予JP1治疗，且JP1的给药剂量与实施例1相同。免疫荧光染色显示，在黑色素瘤(B16F10)中，JP1处理后α-SMA(壁细胞标记物)、Claudin 5(内皮细胞标记物)、Desmin(周细胞标记物)的覆盖量增加(图4的A-D图)。这一观察结果在肺癌(LLC)中得到了进一步证实(图4的E-H图)。这些结果表明，与对照组相比，肿瘤在JP1治疗后表现出接近正常血管结构表型。进一步进行了血管通透性实验，发现与Ctrl-R处理相比，JP1降低了血管通透性(图4的I图、J图)。To study the function of JP1 in reducing the entry of tumor cells into blood vessels, this example evaluates the morphology of tumor blood vessels extracted from melanoma and lung cancer samples, wherein the Ctrl-R group was not intervened by JP1 from beginning to end, and the JP1 group was treated with JP1 from beginning to end, and the dosage of JP1 was the same as that in Example 1. Immunofluorescence staining showed that in melanoma (B16F10), the coverage of α-SMA (mural cell marker), Claudin 5 (endothelial cell marker), and Desmin (pericyte marker) increased after JP1 treatment (Figures A-D in Figure 4). This observation was further confirmed in lung cancer (LLC) (Figures E-H in Figure 4). These results indicate that compared with the control group, the tumor showed a phenotype close to the normal vascular structure after JP1 treatment. Further vascular permeability experiments were performed, and it was found that JP1 reduced vascular permeability compared with Ctrl-R treatment (Figures I and J in Figure 4).
综合来看，JP1通过促进肿瘤血管正常化能够减少肿瘤细胞进入血管的机会，从而抑制了肿瘤转移的发生。In summary, JP1 can reduce the chance of tumor cells entering blood vessels by promoting the normalization of tumor blood vessels, thereby inhibiting the occurrence of tumor metastasis.
实施例3、JP1通过抑制IL8促进肿瘤血管正常化Example 3: JP1 promotes normalization of tumor blood vessels by inhibiting IL8
为获得JP1作用的下游靶分子，对JP1处理后的差异调控基因进行血管生成阵列分析，其中，Ctrl-R组为自始至终没有JP1干预，JP1组为自始至终给予JP1治疗，且JP1的给药剂量与实施例1相同。结果显示，IL8是下调最多的基因(图5的A图)。Western blot分析发现，随着JP1浓度的增加，IL8显著降低(图5的B图和C图，图6的A图和B图)；随后在小鼠肿瘤样本中证实了这一发现(图5的D图和E图，图6的C图和D图)。RT-PCR定量分析也验证了体外和体内实验的结果(图6的E-H图)。进一步检测了IL8在人类黑色素细胞痣和黑色素瘤组织芯片中的表达，正如预测的那样，IL8在 黑色素瘤中的表达明显高于黑色素细胞痣(图5的F图和G图)。相比之下，黑色素瘤组织中的壁细胞覆盖率较低(图5的H图)。这些结果表明，IL8在JP1的抑制下与血管正常化呈负相关。In order to obtain the downstream target molecules of JP1, the differentially regulated genes after JP1 treatment were analyzed by angiogenesis array, wherein the Ctrl-R group was not intervened by JP1 from beginning to end, and the JP1 group was treated with JP1 from beginning to end, and the dosage of JP1 was the same as that in Example 1. The results showed that IL8 was the most downregulated gene (Figure A of Figure 5). Western blot analysis found that IL8 was significantly reduced with the increase of JP1 concentration (Figures B and C of Figure 5, Figures A and B of Figure 6); this finding was subsequently confirmed in mouse tumor samples (Figures D and E of Figure 5, Figures C and D of Figure 6). RT-PCR quantitative analysis also verified the results of in vitro and in vivo experiments (Figures E-H of Figure 6). The expression of IL8 in human melanocytic nevus and melanoma tissue chips was further detected. As expected, the expression of IL8 in melanoma was significantly higher than that in melanocytic nevus (Figures F and G of Figure 5). In contrast, the coverage of parietal cells in melanoma tissue was lower (Figure H of Figure 5). These results suggest that IL8 is inversely correlated with vascular normalization upon inhibition of JP1.
为进一步探索IL8是如何被JP1调控的，用Crispr-Cas9敲除B16F10细胞中的IL8基因(图6的I图)，并观察JP1是否在IL8缺失的情况下仍可促进肿瘤血管正常化。其中，Ctrl-R组为自始至终没有JP1干预，JP1组为自始至终给予JP1治疗，且JP1的给药剂量与实施例1相同。然后用含有IL8WT和IL8KO的B16F10细胞构建了黑色素瘤的荷瘤模型(图5的I图)。结果显示，IL8KO B16F10细胞的生长速度明显低于IL8WT B16F10细胞。此外，与IL8WT B16F10细胞相比，JP1显著抑制黑色素瘤的生长，但对IL8KO B16F10细胞无明显抑制作用(图5的J图和K图，图6的J图和K图)。从小鼠肿瘤标本中提取的血管的形态学评价也获得相同的结果。JP1通过增加周细胞和内皮细胞在IL8WT B16F10中的覆盖率促进肿瘤血管正常化；然而，敲除IL8可显著改善肿瘤血管正常化，而JP1治疗不能增强血管正常化指数(图5的L-O图)。To further explore how IL8 is regulated by JP1, Crispr-Cas9 was used to knock out the IL8 gene in B16F10 cells (Figure 6, Figure I), and to observe whether JP1 can still promote tumor vascular normalization in the absence of IL8. Among them, the Ctrl-R group was not intervened by JP1 from beginning to end, and the JP1 group was treated with JP1 from beginning to end, and the dosage of JP1 was the same as that in Example 1. Then, a melanoma tumor-bearing model was constructed using B16F10 cells containing IL8WT and IL8KO (Figure 5, Figure I). The results showed that the growth rate of IL8KO B16F10 cells was significantly lower than that of IL8WT B16F10 cells. In addition, compared with IL8WT B16F10 cells, JP1 significantly inhibited the growth of melanoma, but had no significant inhibitory effect on IL8KO B16F10 cells (Figures J and K of Figure 5, Figures J and K of Figure 6). The same results were obtained in the morphological evaluation of blood vessels extracted from mouse tumor specimens. JP1 promoted tumor vascular normalization by increasing pericyte and endothelial cell coverage in IL8WT B16F10; however, knockout of IL8 significantly improved tumor vascular normalization, while JP1 treatment failed to enhance the vascular normalization index (Figure 5, L-O panels).
综上所述，这些数据表明，JP1通过抑制IL8促进肿瘤血管正常化。Taken together, these data suggest that JP1 promotes tumor vascular normalization by inhibiting IL8.
实施例4、JP1促进肿瘤氧化磷酸化，改善肿瘤微环境缺氧Example 4: JP1 promotes tumor oxidative phosphorylation and improves tumor microenvironment hypoxia
缺氧是肿瘤发展的必然结果，并已报道缺氧刺激IL8分泌。已知JWA基因通过促进有氧呼吸改善肿瘤微环境缺氧，可抑制胰腺癌转移。为了验证JP1是否能起到与JWA基因类似的促进肿瘤细胞氧化磷酸化的作用，本实施例进行了耗氧率(Oxygen consumption rate,OCR)分析。如图7的A图、B图所示，JP1能有效促进B16F10和LLC细胞的氧化磷酸化。黑色素瘤和肺癌模型肿瘤样本的HIF1α染色显示，JP1显著改善了肿瘤内微环境缺氧(图7的C-F图)。免疫印迹分析显示，在B16F10和LLC细胞中，随着JP1浓度的增加，HIF1α显著降低(图7的G图和H图，图8的A图和B图)。此外，这一发现随后在小鼠肿瘤样本中得到了证实(图7的I图和J图，图8的C图和D图)。RT-PCR定量分析进一步验证了JP1在体外和体内实验中对HIF1α的调控作用(图8的E-H图)。注：本实施例中涉及Ctrl-R组和JP1组对照实验时，Ctrl-R组为自始至终没有JP1干预，JP1组为自始至终给予JP1治疗，且JP1的给药剂量与实施例1相同。Hypoxia is an inevitable result of tumor development, and it has been reported that hypoxia stimulates IL8 secretion. It is known that the JWA gene can inhibit pancreatic cancer metastasis by improving tumor microenvironment hypoxia by promoting aerobic respiration. In order to verify whether JP1 can play a role in promoting oxidative phosphorylation of tumor cells similar to the JWA gene, oxygen consumption rate (OCR) analysis was performed in this example. As shown in Figures A and B of Figure 7, JP1 can effectively promote oxidative phosphorylation of B16F10 and LLC cells. HIF1α staining of melanoma and lung cancer model tumor samples showed that JP1 significantly improved the hypoxia of the tumor microenvironment (Figures C-F of Figure 7). Immunoblotting analysis showed that in B16F10 and LLC cells, HIF1α was significantly reduced with increasing JP1 concentration (Figures G and H of Figure 7, Figures A and B of Figure 8). In addition, this finding was subsequently confirmed in mouse tumor samples (Figures I and J of Figure 7, Figures C and D of Figure 8). RT-PCR quantitative analysis further verified the regulatory effect of JP1 on HIF1α in vitro and in vivo experiments (Figure 8 E-H). Note: In this example, when the control experiment involving the Ctrl-R group and the JP1 group was performed, the Ctrl-R group was not intervened by JP1 from beginning to end, and the JP1 group was treated with JP1 from beginning to end, and the dosage of JP1 was the same as that in Example 1.
这些结果表明，JP1能有效促进肿瘤氧化磷酸化，改善肿瘤微环境缺氧。These results indicate that JP1 can effectively promote tumor oxidative phosphorylation and improve tumor microenvironment hypoxia.
实施例5、缺氧诱导肿瘤细胞分泌IL8Example 5: Hypoxia induces tumor cells to secrete IL8
为确定肿瘤微环境缺氧对IL8表达的影响，本实施例采用B16F10和LLC细胞建立常氧(空气中含氧量21％)和缺氧(空气中含氧量8％)模型，发现缺氧条件下IL8表达增加(图9的A图、B图)。此外，JP1在常氧条件下显著抑制HIF1α的表达；而在缺氧条件下，JP1对IL8的调控作用减弱(图9的C图、D图)。为了进一步证明这一假设，本实施例建立了常氧和缺氧条件下的黑色素瘤荷瘤模型(图9的E图)。结果表明，与在常氧条件下JP1对肿瘤生长有显著抑制作用相比，在缺氧条件下JP1对肿瘤生长无显著抑制作用(图9的F图和G图，图10的A图)。小鼠肿瘤HIF1α染色显示，在常氧条件下，JP1显著改善肿瘤内微环境缺氧；而在缺氧条件下，瘤内缺氧严重，JP1无法逆转瘤内微环境的缺氧状态(图9的H图、I图)，这一结果通过免疫印迹分析得到验证(图9的J图，图10的B图)。此外，在缺氧条件下，肿瘤样品的IL8表达明显升高(图9的K图，图10的B图)。血管形态学评价显示，在缺氧条件下，无论有无JP1处理，血管正常化指数均低于常氧条件下(图9的L图、M图)。注： 本实施例中，Ctrl-R组为自始至终没有JP1干预，JP1组为自始至终给予JP1治疗，且JP1的给药剂量与实施例1相同；Normoxia为常氧条件下，Hypoxia为缺氧条件下。To determine the effect of hypoxia in the tumor microenvironment on IL8 expression, this example uses B16F10 and LLC cells to establish normoxic (oxygen content in air 21%) and hypoxic (oxygen content in air 8%) models, and found that IL8 expression increased under hypoxic conditions (Figure 9 A and Figure B). In addition, JP1 significantly inhibited the expression of HIF1α under normoxic conditions; while under hypoxic conditions, the regulatory effect of JP1 on IL8 was weakened (Figure 9 C and Figure D). To further prove this hypothesis, this example established a melanoma tumor-bearing model under normoxic and hypoxic conditions (Figure 9 E). The results showed that compared with the significant inhibitory effect of JP1 on tumor growth under normoxic conditions, JP1 had no significant inhibitory effect on tumor growth under hypoxic conditions (Figures F and G of Figure 9, Figure 10 A). HIF1α staining of mouse tumors showed that under normoxic conditions, JP1 significantly improved the hypoxia of the tumor microenvironment; under hypoxic conditions, the hypoxia in the tumor was severe, and JP1 could not reverse the hypoxic state of the tumor microenvironment (Figure 9 H and Figure 10), and this result was verified by immunoblotting analysis (Figure 9 J, Figure 10 B). In addition, under hypoxic conditions, the expression of IL8 in tumor samples was significantly increased (Figure 9 K, Figure 10 B). Vascular morphological evaluation showed that under hypoxic conditions, regardless of whether JP1 was treated or not, the vascular normalization index was lower than that under normoxic conditions (Figure 9 L and Figure 9 M). Note: In this example, the Ctrl-R group had no JP1 intervention from beginning to end, the JP1 group was treated with JP1 from beginning to end, and the dosage of JP1 was the same as that in Example 1; Normoxia was under normoxic conditions, and Hypoxia was under hypoxic conditions.
这些结果提示JP1通过促进肿瘤细胞氧化磷酸化从而减少IL8的分泌来改善肿瘤微环境缺氧。These results suggest that JP1 improves tumor microenvironment hypoxia by promoting oxidative phosphorylation in tumor cells and thereby reducing the secretion of IL8.
此外，在机制方面，本实施例验证了相关分子蛋白在B16F10和LLC细胞中的表达。正如我们所预期的那样，JP1通过AMPK/FOXO3a/UQCRC2信号通路调控B16F10和LLC细胞的线粒体代谢重编程，从而改善肿瘤微环境的缺氧状态(图10的C图)。In addition, in terms of mechanism, this example verifies the expression of related molecular proteins in B16F10 and LLC cells. As expected, JP1 regulates mitochondrial metabolic reprogramming of B16F10 and LLC cells through the AMPK/FOXO3a/UQCRC2 signaling pathway, thereby improving the hypoxic state of the tumor microenvironment (Figure 10, Figure C).
实施例6、JP1促进紫杉醇(PTX)的瘤内传送，增强抗肿瘤作用Example 6: JP1 promotes intratumoral delivery of paclitaxel (PTX) and enhances anti-tumor effects
肿瘤血管结构正常化减少了其通透性，能够减少肿瘤细胞对血管的侵袭，抑制肿瘤转移；此外，肿瘤血管的分布正常化也促进了药物进入瘤内，增强了抗肿瘤作用。为评估JP1促进肿瘤血管正常化的效果，以便药物进入瘤内，本实施例完成了JP1和紫杉醇(PTX)的联合治疗黑色素瘤模型(图11的A图)。结果显示，JP1和PTX单独治疗的抗肿瘤作用分别为37％和49.7％，联合治疗的抑制作用为65.4％，提示JP1联合PTX具有更好的抗肿瘤作用(图11的B图、C图)，即在抗肿瘤作用方面JP1能对PTX产生协同增效作用。肿瘤血管形态验证，与JP1促进肿瘤血管正常化的作用相比，PTX在抑制血管总数的范围内促进了血管正常化(图11的D-G图)。此外，本实施例对肿瘤组织进行缺氧分析，发现PTX虽然能促进肿瘤血管正常化，但其对血管的抑制作用并不能改善缺氧状态，而JP1和PTX联合治疗明显改善了肿瘤微环境的缺氧状态(图11的H图、I图)，血管通透性实验进一步验证了这一观察结果(图11的J图)。为了进一步证明JP1促进药物向瘤内传递，本实施例通过HPLC检测了肿瘤中PTX的含量，发现与JP1联合治疗相比，PTX治疗11天后，PTX向瘤内传递明显降低，而JP1与PTX联合治疗显著增加了肿瘤中PTX的传递(图11的K-N，图12)。注：本实施例中，Vehicle组为对照组，没有JP1和PTX干预；JP1组为仅给予JP1治疗，JP1的给药剂量为50mg/kg，腹腔注射，每天1次；PTX组为仅给予PTX治疗，PTX给药剂量为10mg/kg，腹腔注射，每周2次(周一、四)；JP1+PTX组为JP1和PTX联合给药组，JP1的给药剂量同JP1组，PTX的给药剂量同PTX组。Normalization of tumor vascular structure reduces its permeability, can reduce tumor cell invasion of blood vessels, and inhibit tumor metastasis; in addition, normalization of tumor vascular distribution also promotes the entry of drugs into the tumor and enhances the anti-tumor effect. In order to evaluate the effect of JP1 in promoting the normalization of tumor blood vessels so that drugs can enter the tumor, this example completes the combined treatment of melanoma model with JP1 and paclitaxel (PTX) (Figure A of Figure 11). The results showed that the anti-tumor effects of JP1 and PTX alone were 37% and 49.7%, respectively, and the inhibitory effect of combined treatment was 65.4%, indicating that JP1 combined with PTX has a better anti-tumor effect (Figure B and Figure C of Figure 11), that is, JP1 can produce a synergistic effect on PTX in terms of anti-tumor effect. Tumor vascular morphology verified that, compared with the effect of JP1 in promoting the normalization of tumor blood vessels, PTX promoted vascular normalization within the range of inhibiting the total number of blood vessels (Figure D-G of Figure 11). In addition, this example performs hypoxia analysis on tumor tissues and finds that although PTX can promote normalization of tumor blood vessels, its inhibitory effect on blood vessels cannot improve the hypoxic state, while the combined treatment of JP1 and PTX significantly improves the hypoxic state of the tumor microenvironment (Figure H and Figure I of Figure 11), and the vascular permeability experiment further verifies this observation (Figure J of Figure 11). In order to further prove that JP1 promotes drug delivery into the tumor, this example detects the content of PTX in the tumor by HPLC, and finds that compared with the combined treatment of JP1, after 11 days of PTX treatment, the delivery of PTX into the tumor is significantly reduced, while the combined treatment of JP1 and PTX significantly increases the delivery of PTX in the tumor (Figure K-N of Figure 11, Figure 12). Note: In this example, the Vehicle group was the control group without JP1 and PTX intervention; the JP1 group was treated with JP1 only, with a dose of 50 mg/kg, intraperitoneal injection, once a day; the PTX group was treated with PTX only, with a dose of 10 mg/kg, intraperitoneal injection, twice a week (Monday and Thursday); the JP1+PTX group was a group treated with JP1 and PTX together, with the dose of JP1 being the same as that of the JP1 group, and the dose of PTX being the same as that of the PTX group.
这些结果表明，PTX通过促进肿瘤血管正常化来抑制转移起始，但其对血管的抑制作用减少了药物向肿瘤内的输送；采用JP1与PTX联用后则既能促进肿瘤血管正常化来抑制转移起始，又能增加PTX向肿瘤内的输送。These results indicate that PTX inhibits metastasis initiation by promoting tumor vascular normalization, but its inhibitory effect on blood vessels reduces the delivery of drugs into tumors; the combination of JP1 and PTX can not only promote tumor vascular normalization to inhibit metastasis initiation, but also increase the delivery of PTX into tumors.
实施例7Example 7
本实施例采用下表所示的各JWA多肽按实施例1至6进行检测，各JWA多肽的氨基酸S经磷酸化修饰。In this example, each JWA polypeptide shown in the table below was used for detection according to Examples 1 to 6, and the amino acid S of each JWA polypeptide was phosphorylated.
Figure PCTCN2022138739-appb-000001
Figure PCTCN2022138739-appb-000001
Figure PCTCN2022138739-appb-000002
Figure PCTCN2022138739-appb-000002
受篇幅所限，本实施例未列出具体实验数据。所得实验数据表明，以上各JWA多肽的各项检测结果均与JP1基本一致。Due to space limitations, this example does not list specific experimental data. The experimental data obtained show that the test results of the above JWA polypeptides are basically consistent with those of JP1.
结论in conclusion
由以上各实施例可知，本发明证实了以JP1为代表的系列JWA多肽：(1)通过促进肿瘤细胞氧化磷酸化从而减少IL8的分泌来改善肿瘤微环境缺氧，并通过AMPK/FOXO3a/UQCRC2信号通路调控肿瘤细胞的线粒体代谢重编程以改善肿瘤微环境的缺氧状态；(2)通过抑制IL8促进肿瘤血管结构和瘤内分布正常化，减少肿瘤细胞进入血管的机会，从而抑制肿瘤转移的起始和发生；(3)能够增加肿瘤内药物的有效灌注量。因此，JWA靶向肽不仅单独用药体现有效的抗肿瘤活性，还可与其它疗法如化学药、抗体药、细胞类药联合发挥协同抑瘤作用。JWA多肽的这些独特生物学特性，将为治疗肿瘤尤其是实体瘤提供新的临床用药可能，具有良好的应用前景。As can be seen from the above embodiments, the present invention confirms that the series of JWA polypeptides represented by JP1: (1) improve the hypoxia of the tumor microenvironment by promoting oxidative phosphorylation of tumor cells and thus reducing the secretion of IL8, and regulate the mitochondrial metabolic reprogramming of tumor cells through the AMPK/FOXO3a/UQCRC2 signaling pathway to improve the hypoxic state of the tumor microenvironment; (2) promote the normalization of tumor vascular structure and intratumoral distribution by inhibiting IL8, reduce the chance of tumor cells entering blood vessels, thereby inhibiting the initiation and occurrence of tumor metastasis; (3) can increase the effective perfusion amount of drugs in the tumor. Therefore, JWA targeting peptides not only exhibit effective anti-tumor activity when used alone, but can also be combined with other therapies such as chemical drugs, antibody drugs, and cell drugs to exert synergistic tumor inhibition. These unique biological characteristics of JWA polypeptides will provide new clinical drug possibilities for the treatment of tumors, especially solid tumors, and have good application prospects.
以上各实施例所用的材料、方法、实验模型条件等如下文所示。The materials, methods, experimental model conditions, etc. used in the above examples are shown below.
1.细胞系和细胞培养：小鼠黑色素瘤B16F10和肺癌LLC细胞均购自ATCC(MD,USA)。IL8KO(IL8基因敲除)B16F10细胞使用IL8特异性CRISPR/Cas9质粒(由Corues Biotechnology合成)生成。测序分析和western blot实验证实基因敲除完成。GFP-B16F10细胞由GFP特异性质粒(上海Genechem有限公司合成)生成。所有细胞系在添加链霉素100μg/ml、青霉素100U/ml和10％胎牛血清的DMEM培养基中保存，在37℃、5％CO 2培养箱中保存。 1. Cell lines and cell culture: Mouse melanoma B16F10 and lung cancer LLC cells were purchased from ATCC (MD, USA). IL8KO (IL8 knockout) B16F10 cells were generated using IL8-specific CRISPR/Cas9 plasmids (synthesized by Corues Biotechnology). Sequencing analysis and western blot experiments confirmed that the gene knockout was complete. GFP-B16F10 cells were generated by GFP-specific plasmids (synthesized by Shanghai Genechem Co., Ltd.). All cell lines were maintained in DMEM medium supplemented with streptomycin 100 μg/ml, penicillin 100 U/ml and 10% fetal bovine serum and stored in a 37°C, 5% CO 2 incubator.
2.小鼠同种异体移植瘤模型：实验小鼠的所有护理和治疗均经南京医科大学动物护理和使用委员会指南(IACUC:1811067-1)批准。实验小鼠购自上海SLAC实验动物中心，保存于南京医科大学动物核心设施。黑色素瘤生长模型：将B16F10、IL8WT B16F10、IL8KO B16F10和GFP-B16F10细胞(5×10 5)皮下注射至C57BL/6雄性小鼠：肺癌生长模型：LLC细胞(5×10 6)皮下注射至C57BL/6雄性小鼠。当肿瘤体积达到100mm 3时，将小鼠随机分组并进行干预。每隔一天测量体重和肿瘤大小。实验结束后，人道地处死小鼠，测量并记录肿瘤。氧浓度控制器(购自上海宇燕仪器有限公司)创建含氧量8％的环境，小鼠停留6h/d建立缺氧模型。采用先前描述的通用方案进行了黑色素瘤/肺癌活性转移模型。 2. Mouse allograft tumor model: All care and treatment of experimental mice were approved by the Guidelines of the Animal Care and Use Committee of Nanjing Medical University (IACUC: 1811067-1). Experimental mice were purchased from the Shanghai SLAC Laboratory Animal Center and stored in the Animal Core Facility of Nanjing Medical University. Melanoma growth model: B16F10, IL8WT B16F10, IL8KO B16F10 and GFP-B16F10 cells (5×10 5 ) were subcutaneously injected into C57BL/6 male mice: Lung cancer growth model: LLC cells (5×10 6 ) were subcutaneously injected into C57BL/6 male mice. When the tumor volume reached 100mm 3 , the mice were randomly divided into groups and intervened. Body weight and tumor size were measured every other day. After the experiment, the mice were humanely killed, and the tumors were measured and recorded. The oxygen concentration controller (purchased from Shanghai Yuyan Instrument Co., Ltd.) created an environment with an oxygen content of 8%, and the mice stayed for 6h/d to establish a hypoxia model. The melanoma/lung cancer active metastasis model was performed using a general protocol described previously.
3.CTC(循环肿瘤细胞)分析与分选：采用GFP标记的B16F10细胞构建黑色素瘤模型。当肿瘤体积 达到2000mm 3时，每只小鼠取血液500μl裂解红细胞(红细胞裂解缓冲液购自Fcmacs Biotech Co.，Ltd.)。使用LSRII流式细胞仪(BD Biosciences)进行FACS分析，检测每150万个细胞中的CTC，并使用FlowJo软件(Tree Star Inc.)分析数据。FACS分选使用Aria III仪器(BD Biosciences)。 3. CTC (circulating tumor cell) analysis and sorting: A melanoma model was constructed using GFP-labeled B16F10 cells. When the tumor volume reached 2000 mm 3 , 500 μl of blood was taken from each mouse to lyse red blood cells (red blood cell lysis buffer was purchased from Fcmacs Biotech Co., Ltd.). FACS analysis was performed using an LSRII flow cytometer (BD Biosciences) to detect CTCs in every 1.5 million cells, and the data were analyzed using FlowJo software (Tree Star Inc.). FACS sorting was performed using an Aria III instrument (BD Biosciences).
4.细胞增殖，迁移和侵袭分析：取携带黑色素瘤小鼠血液，裂解红细胞，直接加入DMEM培养基培养21天进行增殖实验。使用FACS排序的CTC进行迁移和入侵实验。在迁移试验中，CTC接种在Transwell TM过滤器(购自美国康宁公司)上腔的无血清培养基中，并在下腔中加入含10％FBS的培养基。12h后，用甲醇固定1h，用结晶紫(Beyotime,Shanghai,China)染色30min，计数。对于侵袭试验，在下腔膜上放置一层基质(从BD生物科学公司购买)。 4. Cell proliferation, migration and invasion analysis: Blood from mice carrying melanoma was taken, red blood cells were lysed, and DMEM medium was directly added for 21 days of culture for proliferation experiments. Migration and invasion experiments were performed using CTCs sorted by FACS. In the migration experiment, CTCs were inoculated in serum-free medium in the upper chamber of the Transwell TM filter (purchased from Corning, USA), and medium containing 10% FBS was added to the lower chamber. After 12 hours, they were fixed with methanol for 1 hour, stained with crystal violet (Beyotime, Shanghai, China) for 30 minutes, and counted. For the invasion experiment, a layer of matrix (purchased from BD Biosciences) was placed on the membrane of the lower chamber.
5.免疫组织化学和免疫荧光染色：组织标本在福尔马林浸泡24h后，石蜡包埋，切片(石蜡切片机，Thermo HM340E)。脱蜡和抗原回收后，用10％正常山羊血清在室温下阻断30min,4℃下用HIF1α(CST,36169T)抗体溶液轻轻摇匀孵育24h。DAB染色后用病理切片扫描仪(Pannoramic MIDI)扫描。双免疫荧光染色，将小鼠源CD31(Servicebio,GB12063)抗体溶液分别与兔源α-SMA(Servicebio,GB111364)、Claudin 5(Servicebio,GB11290)、Desmin(Servicebio,GB11081)抗体溶液在4℃温和摇晃下孵育24h。用鼠源(红色)和兔源(绿色)荧光二抗孵育后扫描图像。用J图分析染色强度。5. Immunohistochemistry and immunofluorescence staining: After soaking in formalin for 24 hours, tissue specimens were embedded in paraffin and sliced (paraffin slicer, Thermo HM340E). After dewaxing and antigen retrieval, the specimens were blocked with 10% normal goat serum at room temperature for 30 minutes and incubated with HIF1α (CST, 36169T) antibody solution at 4°C with gentle shaking for 24 hours. After DAB staining, the specimens were scanned with a pathological section scanner (Pannoramic MIDI). For double immunofluorescence staining, the mouse CD31 (Servicebio, GB12063) antibody solution was incubated with rabbit α-SMA (Servicebio, GB111364), Claudin 5 (Servicebio, GB11290), and Desmin (Servicebio, GB11081) antibody solutions at 4°C with gentle shaking for 24 hours. The images were scanned after incubation with mouse (red) and rabbit (green) fluorescent secondary antibodies. The staining intensity was analyzed with J-graphs.
6.血管渗透性测定：将50mg/kg Evans Blue溶液通过尾静脉注射给携带黑色素瘤的小鼠。1h后采血，离心取上清，甲酰胺1:100稀释。伊文思蓝溶于甲酰胺(0ng/ml、125ng/ml、250ng/ml、500ng/ml、1000ng/ml、5000ng/ml、10000ng/ml、25000ng/ml、50000ng/ml)，制作标准曲线。620nm处测量上清吸光度，计算血液中伊文思蓝的浓度。取血后，迅速将小鼠暴露于心脏，向心脏灌注10ml 0.9％生理盐水，冲洗血管内的伊文思蓝。将肿瘤组织200mg置于60℃烘箱中5h，加入200μl甲酰胺，连续置于60℃烘箱中18h。上清离心，620nm处测定吸光度，计算肿瘤组织伊文思蓝含量。血管通透性计算公式为：血管通透性[μl/(g×h)]＝[伊文思蓝(μg)/肿瘤干重(g)]/[血液中伊文思蓝浓度(μg/μl)×循环时间(h)]。6. Vascular permeability determination: 50 mg/kg Evans Blue solution was injected into mice carrying melanoma through the tail vein. Blood was collected 1 hour later, the supernatant was centrifuged and diluted with formamide at 1:100. Evans blue was dissolved in formamide (0ng/ml, 125ng/ml, 250ng/ml, 500ng/ml, 1000ng/ml, 5000ng/ml, 10000ng/ml, 25000ng/ml, 50000ng/ml) to make a standard curve. The absorbance of the supernatant was measured at 620nm to calculate the concentration of Evans blue in the blood. After blood collection, the mouse was quickly exposed to the heart, and 10ml of 0.9% saline was perfused into the heart to flush the Evans blue in the blood vessels. 200mg of tumor tissue was placed in a 60℃ oven for 5h, 200μl of formamide was added, and it was placed in a 60℃ oven for 18h. The supernatant was centrifuged, and the absorbance was measured at 620 nm to calculate the Evans blue content in the tumor tissue. The formula for calculating vascular permeability is: vascular permeability [μl/(g×h)] = [Evans blue (μg)/tumor dry weight (g)]/[Evans blue concentration in blood (μg/μl)×circulation time (h)].
7.血管生成阵列分析：血管生成阵列GS1000(GSH-ANG-1000-1,Ray Biotech Inc.)按照厂家说明书使用。简单地说，从盒子中取出玻片，在密封的塑料袋中平衡至室温20-30min。从塑料袋中取出玻片，剥去盖膜，再风干1-2h。收集标准品或样品，与阵列在4℃下轻轻摇晃孵育24h。化学发光信号由InnoScan 300微阵列扫描仪(Innopsys)捕获。数据提取使用微阵列分析软件ScanArray Express。7. Angiogenesis array analysis: Angiogenesis array GS1000 (GSH-ANG-1000-1, Ray Biotech Inc.) was used according to the manufacturer's instructions. Briefly, slides were removed from the box and equilibrated to room temperature in a sealed plastic bag for 20-30 min. Slides were removed from the plastic bag, the cover film was peeled off, and air-dried for 1-2 h. Standards or samples were collected and incubated with the array at 4 °C for 24 h with gentle shaking. Chemiluminescent signals were captured by InnoScan 300 microarray scanner (Innopsys). Data were extracted using the microarray analysis software ScanArray Express.
8.定量RT-PCR和Western Blot分析：用TRIzon(Thermo Fisher Scientific,10296010)提取细胞和组织的总RNA，用反转录试剂盒(Vazyme,R323-01)进行反转录。RT-PCR采用SYBR(Vazyme,TSE202)进行AB RT-PCR(Q5)。最后通过GAPDH标准化计算差异值。RT-PCR使用的引物分别为:HIF1α-F(ACGTTCCTTCGATCAGTTGTCACC)、HIF1α-R(GGCAGTGGTAGTGGTGGCATTAG)、IL8-F(tcctgctttcctc)、IL8-R(GGGTGGAAAGGTGTGGAATG)、GAPDH-F(gctctctgctcctcctgttttc)、GAPDH-R(ACGACCAAATCCGTTGACTC)。按照之前的报道进行Western Blot分析。简单地说，细胞和组织样品分别用细胞裂解缓冲液和组织蛋白提取试剂(购自Thermo Fisher Scientific,78510)进行裂解。共处理40个蛋白供后续分析。使用的抗体有:Hif1α(CST,36169T)、IL8(Abcam,ab106350)、p-AMPK(CST,2535T)、AMPK(Abcam,32047)、FOXO3A(Abcam,53287)、UQCRC2(Abcam,ab203832)、 Tubulin(Beyotime,AF0001)。8. Quantitative RT-PCR and Western Blot analysis: Total RNA from cells and tissues was extracted using TRIzon (Thermo Fisher Scientific, 10296010) and reverse transcribed using a reverse transcription kit (Vazyme, R323-01). AB RT-PCR (Q5) was performed using SYBR (Vazyme, TSE202). Finally, the difference value was calculated by GAPDH standardization. The primers used in RT-PCR were: HIF1α-F (ACGTTCCTTCGATCAGTTGTCACC), HIF1α-R (GGCAGTGGTAGTGGTGGCATTAG), IL8-F (tcctgctttcctc), IL8-R (GGGTGGAAAGGTGTGGAATG), GAPDH-F (gctctctgctcctcctgttttc), GAPDH-R (ACGACCAAATCCGTTGACTC). Western Blot analysis was performed as previously reported. Briefly, cells and tissue samples were lysed with cell lysis buffer and tissue protein extraction reagent (purchased from Thermo Fisher Scientific, 78510), respectively. A total of 40 proteins were processed for subsequent analysis. The antibodies used were: Hif1α (CST, 36169T), IL8 (Abcam, ab106350), p-AMPK (CST, 2535T), AMPK (Abcam, 32047), FOXO3A (Abcam, 53287), UQCRC2 (Abcam, ab203832), Tubulin (Beyotime, AF0001).
9.线粒体耗氧率(OCR)分析：采用Seahorse XFp Analyzer(Seahorse XF96)对B16F10和LLC细胞进行OCR分析。将B16F10细胞(6000个)和LLC细胞(10000个)接种到海马板上，置于生长培养基中24h。在含1mM丙酮酸钠、2mM l-谷氨酰胺和10mM葡萄糖的XF基质中，依次添加寡霉素(1μM)和FCCP(B16F10:1.5μM,LLC:1μM)，测定线粒体耗氧量。9. Analysis of mitochondrial oxygen consumption rate (OCR): OCR analysis of B16F10 and LLC cells was performed using Seahorse XFp Analyzer (Seahorse XF96). B16F10 cells (6000) and LLC cells (10000) were seeded onto hippocampal plates and placed in growth medium for 24 h. Oligomycin (1 μM) and FCCP (B16F10: 1.5 μM, LLC: 1 μM) were added sequentially to XF matrix containing 1 mM sodium pyruvate, 2 mM l-glutamine, and 10 mM glucose to measure mitochondrial oxygen consumption.
10.高效液相色谱分析：紫杉醇(PTX)购自Selleck公司(NSC 125973)，HPLC(色谱柱:Agilent Zorbax Exlipse Plus C18,100×4.6mm,3.5μM粒径；流速:1ml/min；波长:UV 254nm；流动相:CH 3OH 0.1％TFA/H 2O 0.1％TFA,0min:10:90,0-15min:10:90～100:0,15-20min:100:0)测定PTX的峰值时间。通过尾静脉给荷瘤小鼠注射20mg/kg的PTX溶液。5min后取肿瘤200mg溶于1ml甲醇溶液中。将PTX溶于甲醇溶液(200μg/ml、100μg/ml、50μg/ml、25μg/ml、12.5μg/ml、6.25μg/ml、3.125μg/ml、1.56μg/ml)，制作标准曲线。用高效液相色谱法测定标准品或样品，计算肿瘤中紫杉醇的浓度。 10. High performance liquid chromatography analysis: Paclitaxel (PTX) was purchased from Selleck (NSC 125973). The peak time of PTX was determined by HPLC (chromatographic column: Agilent Zorbax Exlipse Plus C18, 100×4.6 mm, 3.5 μM particle size; flow rate: 1 ml/min; wavelength: UV 254 nm; mobile phase: CH 3 OH 0.1% TFA/H 2 O 0.1% TFA, 0 min: 10:90, 0-15 min: 10:90-100:0, 15-20 min: 100:0). Tumor-bearing mice were injected with 20 mg/kg PTX solution through the tail vein. After 5 min, 200 mg of the tumor was taken and dissolved in 1 ml of methanol solution. PTX was dissolved in methanol solution (200 μg/ml, 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 3.125 μg/ml, 1.56 μg/ml) to prepare a standard curve. The standard or sample was measured by high performance liquid chromatography to calculate the concentration of paclitaxel in the tumor.
11.统计分析：所有数据以个体样本平均值±SEM进行分析，使用GraphPad Prism 8和SPSS 20软件进行分析。使用不配对双尾学生t检验或单因素方差分析(ANOVA)来确定实验组之间的统计学差异。P<0.05为差异有统计学意义。(*P<0.05；**P<0.01；***P<0.001；ns：没有意义)。11. Statistical analysis: All data were analyzed as mean ± SEM of individual samples using GraphPad Prism 8 and SPSS 20 software. Unpaired two-tailed Student's t-test or one-way analysis of variance (ANOVA) was used to determine the statistical differences between experimental groups. P < 0.05 was considered statistically significant. (*P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant).
除上述实施例外，本发明还可以有其他实施方式。凡采用等同替换或等效变换形成的技术方案，均落在本发明要求的保护范围。In addition to the above embodiments, the present invention may also have other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims (10)

  1. 一种多肽的用途，其特征是，所述用途为用于制备抗肿瘤药协同增效剂；A use of a polypeptide, characterized in that the use is used for preparing an anti-tumor drug synergist;
    所述多肽的氨基酸序列如I或II所示：The amino acid sequence of the polypeptide is shown in I or II:
    I：FPGSDRF-Z；I: FPGSDRF-Z;
    II：X-FPGSDRF-Z；II: X-FPGSDRF-Z;
    其中，氨基酸S经磷酸化修饰，X、Z分别为氨基酸或氨基酸序列；Wherein, the amino acid S is phosphorylated, and X and Z are amino acids or amino acid sequences, respectively;
    X选自F、(R) 9、(R) 9-F、6-氨基己酸、6-氨基己酸-F、6-氨基己酸-(R) 9、6-氨基己酸-(R) 9-F之一； X is selected from one of F, (R) 9 , (R) 9 -F, 6-aminocaproic acid, 6-aminocaproic acid-F, 6-aminocaproic acid-(R) 9 , and 6-aminocaproic acid-(R) 9 -F;
    Z选自(G) n-RGD、A-(G) n-RGD之一，n为大于或等于0的整数，且n的取值范围为0-10。 Z is selected from (G) n -RGD and A-(G) n -RGD, n is an integer greater than or equal to 0, and the value range of n is 0-10.
  2. 根据权利要求1所述的用途，其特征是，所述抗肿瘤药协同增效剂的功能为：通过促进肿瘤细胞氧化磷酸化从而减少IL8的分泌来改善肿瘤微环境缺氧，并且通过AMPK/FOXO3a/UQCRC2信号通路调控肿瘤细胞的线粒体代谢重编程以改善肿瘤微环境的缺氧状态。The use according to claim 1 is characterized in that the function of the anti-tumor drug synergist is to improve the hypoxia of the tumor microenvironment by promoting oxidative phosphorylation of tumor cells and thus reducing the secretion of IL8, and to regulate the mitochondrial metabolic reprogramming of tumor cells through the AMPK/FOXO3a/UQCRC2 signaling pathway to improve the hypoxic state of the tumor microenvironment.
  3. 根据权利要求1所述的用途，其特征是，所述抗肿瘤药协同增效剂的功能为：通过抑制IL8促进肿瘤血管正常化，减少肿瘤细胞进入血管的机会，从而抑制肿瘤转移的起始和发生。The use according to claim 1 is characterized in that the function of the anti-tumor drug synergist is to promote the normalization of tumor blood vessels by inhibiting IL8, reduce the chance of tumor cells entering blood vessels, and thus inhibit the initiation and occurrence of tumor metastasis.
  4. 根据权利要求1所述的用途，其特征是，所述抗肿瘤药协同增效剂的功能为：增加肿瘤内药物的有效灌注量。The use according to claim 1 is characterized in that the function of the anti-tumor drug synergist is to increase the effective perfusion amount of the drug in the tumor.
  5. 根据权利要求1至4任一项所述的用途，其特征是，所述抗肿瘤药协同增效剂针对的抗肿瘤药为用于肿瘤治疗的化学药、抗体药、细胞类药物。The use according to any one of claims 1 to 4 is characterized in that the anti-tumor drug targeted by the anti-tumor drug synergist is a chemical drug, antibody drug, or cell drug used for tumor treatment.
  6. 根据权利要求5所述的用途，其特征是，所述抗肿瘤药协同增效剂针对的抗肿瘤药为紫杉醇。The use according to claim 5 is characterized in that the anti-tumor drug targeted by the anti-tumor drug synergist is paclitaxel.
  7. 根据权利要求1至4任一项所述的用途，其特征是，所述抗肿瘤药协同增效剂针对的肿瘤为至少具有以下特征之一的泛实体瘤：The use according to any one of claims 1 to 4 is characterized in that the tumor targeted by the anti-tumor drug synergist is a pan-solid tumor having at least one of the following characteristics:
    特征一：瘤内微环境缺氧、血管分布紊乱、瘤内药物灌注量低；Feature 1: hypoxia in the tumor microenvironment, disordered vascular distribution, and low intratumoral drug perfusion;
    特征二：肿瘤细胞线粒体能量代谢紊乱，氧化磷酸化水平降低、糖酵解代谢增强；Feature 2: Mitochondrial energy metabolism in tumor cells is disrupted, with decreased oxidative phosphorylation and enhanced glycolysis metabolism;
    特征三：IL8分泌量异常增加、瘤内血管壁完整性被破坏。Feature three: Abnormal increase in IL8 secretion and destruction of the integrity of the vascular wall within the tumor.
  8. 根据权利要求7所述的用途，其特征是，所述抗肿瘤药协同增效剂针对的肿瘤为黑色素瘤、肺癌、或人类黑色素细胞痣。The use according to claim 7 is characterized in that the tumor targeted by the anti-tumor drug synergist is melanoma, lung cancer, or human melanocytic nevus.
  9. 根据权利要求1至4任一项所述的用途，其特征是，所述多肽的N端经乙酰化修饰、C端经酰胺化修饰；所述多肽的氨基酸为L型天然氨基酸或D型非天然氨基酸。The use according to any one of claims 1 to 4, characterized in that the N-terminus of the polypeptide is acetylated and the C-terminus is amidated; and the amino acids of the polypeptide are L-type natural amino acids or D-type non-natural amino acids.
  10. 根据权利要求1至4任一项所述的用途，其特征是，所述多肽的氨基酸序列为FPGSDRF-RGD，其中，氨基酸S经磷酸化修饰。The use according to any one of claims 1 to 4, characterized in that the amino acid sequence of the polypeptide is FPGSDRF-RGD, wherein the amino acid S is phosphorylated.
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