CN115414465A - Application of JWA polypeptide in preparation of antitumor drug synergist - Google Patents
Application of JWA polypeptide in preparation of antitumor drug synergist Download PDFInfo
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- CN115414465A CN115414465A CN202211269118.5A CN202211269118A CN115414465A CN 115414465 A CN115414465 A CN 115414465A CN 202211269118 A CN202211269118 A CN 202211269118A CN 115414465 A CN115414465 A CN 115414465A
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Abstract
The invention relates to an application of JWA polypeptide in preparing antineoplastic drug synergist, wherein the amino acid sequence of the polypeptide is shown as I or II: i: FPGSDRF-Z; II: X-FPGSDRF-Z; wherein, the amino acid S is modified by phosphorylation, and X and Z are respectively amino acid or amino acid sequence; x is selected from F, (R) 9 、(R) 9 -F, 6-aminocaproic acid-F, 6-aminocaproic acid- (R) 9 6-aminocaproic acid- (R) 9 -one of F; z is selected from (G) n ‑RGD、A‑(G) n -one of 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 tumor microenvironment anoxia, and promote tumor blood vessel normalization to inhibit tumorThe initiation and occurrence of tumor metastasis can increase the effective injection amount of the medicine in the tumor, and the medicine can be used as an antitumor synergistic agent to provide new clinical medication possibility for treating the tumor.
Description
Technical Field
The invention relates to an application of JWA polypeptide in preparing an antitumor drug synergist, belonging to the technical field of antitumor auxiliary drugs.
Background
In recent years, emerging tumor diagnosis and treatment techniques have significantly improved the cure rate of primary tumors, and some completely inhibit tumor growth even at some stages [1,2]. However, tumor metastasis is the leading cause of death in patients with tumors [3] and once metastasis occurs, existing treatments are difficult to benefit patients. Prevention of metastasis initiation may be an unmet significant clinical need in tumor therapy due to limited therapeutic strategies for tumor metastasis [4,5]. The pre-colonization phase of tumor cell metastasis is a complex multi-step process involving shedding of tumor cells from the primary tumor, penetration of basement membrane, and entry into blood vessels [6], and the break-through of tumor cells into the vasculature by the basement membrane is the core of initiation of tumor metastasis [7]. The immaturity and instability of the tumor microenvironment vasculature provides a prerequisite for the migration of primary tumor cells into the vasculature to distant organs [8,9]. Therefore, the disordered blood vessel is reconstructed to become a mature and stable blood vessel, and the occurrence of tumor metastasis can be effectively inhibited. Tumor angiogenesis is a regenerative process highly influenced by tumor cells, promoting tumor growth and metastasis [10]. Cytokines secreted by tumor cells interact with perivascular cells and convert them into fibroblasts, thereby depriving these cells of the vascular protective effect [11,12]. In addition, tumor cells release pseudo-endothelial signals that stimulate endothelial cell migration, leading to discontinuous endothelial lining and basement membrane defects [13]. All these features are indicative of vascular immaturity and dysfunction. Therefore, the high permeability of the blood vessel provides convenience for the tumor cells to invade the blood vessel. Normalization of tumor blood vessels places endothelial cells surrounded by mature pericytes in a quiescent state. Endothelial cells and pericytes are separated by a basement membrane and filled with various adhesion proteins to maintain vascular stability [14]. Tumor vascularization inhibits metastasis by reducing the invasion of blood vessels by tumor cells [15,16]. In addition, regular mature vascularity in the tumor microenvironment promotes blood perfusion, relieving hypoxia and acidosis [17]. IL8 is a known chemokine cytokine, which has been reported to be highly expressed in a variety of tumors [18,19]; IL8 promotes the proliferation and metastasis of malignant tumors and plays a key regulatory role in the tumor microenvironment [20,21]. IL8 also exacerbates tumorigenicity by mediating crosstalk between ECFC and TNBC [22]. Hepatitis B virus coding gene (HBX) induces high IL8 production by activating MEK-ERK signal, leading to enhanced endothelial cell permeability and promoting tumor vascular invasion [23]. More importantly, IL8 mediated blood vessel immaturity induces tumor microenvironment hypoxia, and further stimulates tumor cells to secrete IL8[24], thereby causing malignancy feedback of tumor microenvironment hypoxia, IL8 secretion and blood vessel disorder. Therefore, inhibition of IL8 may be an effective therapeutic approach to correct vascular disorders in the tumor microenvironment.
JWA is a tumor suppressor gene that inhibits malignant phenotypes such as melanoma, gastric cancer, breast cancer growth, metastasis [25-27]. JWA promotes mitochondrial metabolism reprogramming and improves hypoxia of a tumor microenvironment by regulating AMPK/FOXO3a/UQCRC2 signals, so that tumor metastasis [28] is inhibited; anti-tumor peptide JP1 designed based on JWA functional sequence can inhibit melanoma metastasis effectively [29]; however, the mechanism of the antitumor peptide JP1 for inhibiting tumor metastasis is not clear at present, and further research and study are urgently needed if the antitumor peptide JP1 can be used as a synergistic agent of the existing antitumor drugs. The inventors have made recent studies and applied for the present invention.
Reference to the literature
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7.Klein CA.Cancer progression and the invisible phase of metastatic colonization.Nature reviews Cancer.2020；20(11):681-94.
8.Yip RKH,Rimes JS,Capaldo BD,Vaillant F,Mouchemore KA,Pal B,et al.Mammary tumour cells remodel the bone marrow vascular microenvironment to support metastasis.Nature communications.2021；12(1):6920.
9.Tavora B,Mederer T,Wessel KJ,Ruffing S,Sadjadi M,Missmahl M,et al.Tumoural activation of TLR3-SLIT2 axis in endothelium drives metastasis.Nature.2020；586(7828):299-304.
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11.Bakir B,Chiarella AM,Pitarresi JR,and Rustgi AK.EMT,MET,Plasticity,and Tumor Metastasis.Trends in cell biology.2020；30(10):764-76.
12.Vieugue P,and Blanpain C.Recording EMT Activity by Lineage Tracing during Metastasis.Developmental cell.2020；54(5):567-9.
13.Genova T,Grolez GP,Camillo C,Bernardini M,Bokhobza A,Richard E,et al.TRPM8 inhibits endothelial cell migration via a non-channel function by trapping the small GTPase Rap1.The Journal of cell biology.2017；216(7):2107-30.
14.Xu Z,Guo C,Ye Q,Shi Y,Sun Y,Zhang J,et al.Endothelial deletion of SHP2 suppresses tumor angiogenesis and promotes vascular normalization.Nature communications.2021；12(1):6310.
15.La Porta S,Roth L,Singhal M,Mogler C,Spegg C,Schieb B,et al.Endothelial Tie1-mediated angiogenesis and vascular abnormalization promote tumor progression and metastasis.The Journal of clinical investigation.2018；128(2):834-45.
16.Cartier A,Leigh T,Liu CH,and Hla T.Endothelial sphingosine 1-phosphate receptors promote vascular normalization and antitumor therapy.Proceedings of the National Academy of Sciences of the United States of America.2020；117(6):3157-66.
17.Park JS,Kim IK,Han S,Park I,Kim C,Bae J,et al.Normalization of Tumor Vessels by Tie2 Activation and Ang2 Inhibition Enhances Drug Delivery and Produces a Favorable Tumor Microenvironment.Cancer cell.2016；30(6):953-67.
18.Yuen KC,Liu LF,Gupta V,Madireddi S,Keerthivasan S,Li C,et al.High systemic and tumor-associated IL-8correlates with reduced clinical benefit of PD-L1 blockade.Nature medicine.2020；26(5):693-8.
19.Gao S,Jiang J,Jin C,Gao J,Xiong D,Yang P,et al.Interleukin-8 as a candidate for thymoma identification and recurrence surveillance.Nature communications.2020；11(1):4881.
20.Xu Y,Ren W,Li Q,Duan C,Lin X,Bi Z,et al.LncRNA Uc003xsl.1-mediated activation of the NF-kappaB/IL8 axis promotes progression of triple-negative breast cancer.Cancer research.2021.
21.Han ZJ,Li YB,Yang LX,Cheng HJ,Liu X,and Chen H.Roles of the CXCL8-CXCR1/2Axis in the Tumor Microenvironment and Immunotherapy.Molecules.2021；27(1).
22.Kim ES,Nam SM,Song HK,Lee S,Kim K,Lim HK,et al.CCL8 mediates crosstalk between endothelial colony forming cells and triple-negative breast cancer cells through IL-8,aggravating invasion and tumorigenicity.Oncogene.2021；40(18):3245-59.
23.Zhang C,Gao Y,Du C,Markowitz GJ,Fu J,Zhang Z,et al.Hepatitis B-Induced IL8 Promotes Hepatocellular Carcinoma Venous Metastasis and Intrahepatic Treg Accumulation.Cancer research.2021；81(9):2386-98.
24.Watanabe K,Shiga K,Maeda A,Harata S,Yanagita T,Suzuki T,et al.Chitinase 3-like 1secreted from cancer-associated fibroblasts promotes tumor angiogenesis via interleukin-8secretion in colorectal cancer.International journal of oncology.2022；60(1).
25.Lu J,Tang Y,Farshidpour M,Cheng Y,Zhang G,Jafarnejad SM,et al.JWA inhibits melanoma angiogenesis by suppressing ILK signaling and is an independent prognostic biomarker for melanoma.Carcinogenesis.2013；34(12):2778-88.
26.Wang S,Wu X,Chen Y,Zhang J,Ding J,Zhou Y,et al.Prognostic and predictive role of JWA and XRCC1 expressions in gastric cancer.Clinical cancer research:an official journal of the American Association for Cancer Research.2012；18(10):2987-96.
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Disclosure of Invention
The main purposes of the invention are: aiming at the problems in the prior art, the application of the JWA polypeptide in the aspect of preparing the antitumor drug synergist is provided, the hypoxic state of a tumor microenvironment can be improved, tumor blood vessels can be promoted to be normalized so as to inhibit the initiation and the occurrence of tumor metastasis, the effective perfusion amount of the drug in the tumor can be increased by combining with a chemotherapeutic drug, the synergistic effect is exerted, and a new clinical medication possibility is provided for treating the tumor.
The technical scheme for solving the technical problems of the invention is as follows:
the use of a polypeptide for the preparation of a synergistic agent for antitumor drugs;
the amino acid sequence of the polypeptide is shown as I or II:
I：FPGSDRF-Z；
II：X-FPGSDRF-Z；
wherein, the amino acid S is modified by phosphorylation, and X and Z are respectively amino acid or amino acid sequence;
x is selected from F, (R) 9 、(R) 9 -F, 6-aminocaproic acid-F, 6-aminocaproic acid- (R) 9 6-aminocaproic acid- (R) 9 One of-F;
z is selected from (G) n -RGD、A-(G) n -one of RGD, n being an integer greater than or equal to 0, and n having a value ranging from 0 to 10.
Preferably, the antitumor drug synergist has the functions of: the hypoxia of the tumor microenvironment is improved by promoting the oxidative phosphorylation of the tumor cells so as to reduce the secretion of IL8, and the mitochondrial metabolic reprogramming of the tumor cells is regulated through an AMPK/FOXO3a/UQCRC2 signal channel so as to improve the hypoxia state of the tumor microenvironment.
Preferably, the antitumor drug synergist has the functions of: the IL8 is inhibited to promote the normalization of tumor blood vessels and reduce the opportunity of tumor cells to enter the blood vessels, thereby inhibiting the initiation and occurrence of tumor metastasis.
Preferably, the antitumor drug synergist has the functions of: increasing the effective perfusion amount of the medicine in the tumor.
Preferably, the antitumor drug aimed by the antitumor drug synergistic agent is a chemical drug, an antibody drug or a cell drug for tumor treatment.
More preferably, the antineoplastic agent targeted by the antineoplastic agent synergist is paclitaxel.
Preferably, the tumor targeted by the antineoplastic drug synergist is a pan-solid tumor having at least one of the following characteristics:
the method is characterized in that: hypoxia of microenvironment in tumor, disturbance of blood vessel distribution and low drug perfusion in tumor;
the second characteristic: the energy metabolism of tumor cell mitochondria is disturbed, the oxidative phosphorylation level is reduced, and the glycolytic metabolism is enhanced;
the characteristics are three: IL8 secretion is abnormally increased and the integrity of the intratumoral vessel wall is destroyed.
More preferably, the tumor targeted by the antineoplastic drug synergist is melanoma, lung cancer, or a human melanocyte nevus.
Preferably, the N end of the polypeptide is modified by acetylation, and the C end of the polypeptide is modified by amidation; the amino acid of the polypeptide is L-type natural amino acid or D-type unnatural amino acid.
Preferably, the amino acid sequence of the polypeptide is FPGSDRF-RGD, wherein the amino acid S is modified by phosphorylation.
The polypeptide related to the invention is a part of the series of polypeptides described in Chinese invention patent with patent number CN201310178099X and issued publication number CN 103239710B. The inventor finds out through practical research that the polypeptide can regulate and control mitochondrial metabolism reprogramming through an AMPK/FOXO3a/UQCRC2 signal path, and reduce the secretion of interleukin-8 (IL 8) by promoting the oxidative phosphorylation of tumor cells so as to improve the hypoxia of the tumor microenvironment; the oxygen-rich tumor microenvironment inhibits the tumor cells from secreting IL8 and promotes the normalization of tumor blood vessels; the normalization of blood vessels can ensure that the blood vessels are mature and normally distributed, so that a tumor microenvironment forms benign feedback of blood vessel normalization, full perfusion and oxygen-rich microenvironment, tumor cells are prevented from entering the blood vessels, and the initiation of metastasis is inhibited. In addition, the above polypeptide in combination with Paclitaxel (PTX) can maintain a certain blood vessel density in tumor, promote tumor blood vessel growth normalization, increase oxygen and drug delivery, and enhance antitumor effect. Therefore, the polypeptide can be used as an antitumor drug synergistic agent, provides a new technical means for overcoming the bottleneck of poor curative effect caused by reduction of intratumoral drug perfusion amount due to tumor microenvironment hypoxia, abnormal vascular structure and distribution, increase of IL8 expression and the like in treatment of tumors, particularly pan-solid tumors, and has good application prospect.
Drawings
FIG. 1 is a graph showing the operation and results of example 1 of the present invention. The points of the drawings are as follows: (A) Schematic B16F10 (melanoma cells)/LLC (lung adenocarcinoma cells) allograft, two-stage model of active metastasis after Ctrl-R or JP1 treatment. (B-C) quantitative (n = 6) results plot of mice developing lung metastases after surgical resection of primary B16F10 (B) or LLC tumor (C) with indicated treatment. (D-E) B16F10 (D) and LLC (E) tumor mice Kaplan-Meier survival analysis (n = 10) results. (F) And detecting the result of GFP + B16F10 cell transfection with GFP specific plasmid by flow cytometry. (G) schematic representation of CTC clone enumeration and flow cytometry analysis. (H) Quantitative (n = 6) result plots of CTC colony formation after Ctrl-R or JP1 treatment. (I) Ctrl-R or JP1 treatment followed by 1.5X 10 each 7 CTC quantification (n = 6) of blood cells. (J-K) CTCs from 6 mice were subjected to migration and invasion assays, and the results of quantification of migrated (J) and invaded (K) cells were plotted for each 30000 cells. (. P)<0.05；**P<0.01; ns: the difference is not statistically significant).
FIG. 2 is a graph showing the results of example 1 of the present invention. The points of the drawings are as follows: (A-B) B16F10 tumors body weight and tumor volume measurements after Ctrl-R or JP1 treatment. (C) Each group had a representative lung HE staining pattern for detection of B16F10 tumor metastasis initiation sites at a scale of 500 μm. (D-E) graph of body weight and tumor volume measurements of LLC tumors after treatment with Ctrl-R or JP1. (F) Each group had a representative lung HE staining pattern for LLC tumor metastasis initiation at a scale of 500 μm.
FIG. 3 is a graph showing the results of example 1 of the present invention. The points of the drawings are as follows: (A) Representative images of CTC clones formed after Ctrl-R or JP1 treatment. (B) Representative images of the quantification of CTCs per 1.5X 107 blood cells after Ctrl-R or JP1 treatment were analyzed by FACS. (C) Representative images of CTCs migrating after Ctrl-R or JP1 treatment. (D) Ctrl-R or representative images of CTC invasion after JP1 treatment.
FIG. 4 is a graph showing the results of example 2 of the present invention. The points of the drawings are as follows: (A) Ctrl-R or JP1 treated B16F10 tumor nodules α -SMA (green), claudin5 (green), desmin (green), CD31 (red) and DAPI nuclear staining representative fluorescence images. (B-D) B16F10 tumor nodule interstitial microenvironment alpha-SMA coverage (B), claudin5 coverage (C), desmin coverage (D) results, scale bar: 100 μm. (E) Ctrl treatment LLC tumor nodules alpha SMA (green), claudin5 (green), desmin (green), CD31 (red) and DAPI nuclear staining representative fluorescence images Ctrl-R or JP1. (F-H) LLC tumor nodule interstitial microenvironment alpha-SMA coverage (F), claudin5 coverage (G) and desmin coverage (H) result chart, scale bar: 100 μm. (I-J) schematic diagram of vascular permeability analysis (I) and quantitative map of vascular permeability (J). (. P < 0.05;. P < 0.01).
FIG. 5 is a graph showing the results of example 3 of the present invention. The points of the drawings are as follows: (A) Thermography of 60 genes involved in tumor vessel normalization in Ctrl-R or JP 1-treated B16F10 cells. (B-C) quantitative profile of IL8 protein in B16F10 (B) and LLC (C) cells after 48h treatment with JP1 at the indicated concentrations. (D-E) Western Blot analysis of IL8 protein from tumor nodules of B16F10 (D) and LLC (E), and quantification of IL8 protein concentration. (F) Representative images of immunofluorescence staining of α -SMA (green) and IL8 (red) human melanocyte nevi and melanoma, scale bar: 200 μm. (G-H) quantitative determination of IL8 between human melanocytic nevus and melanoma + Area (G) and alpha-SMA + Area (H). (I) B16F10 (IL 8 WT) and B16F10 (IL 8 KO) cellular allografts are schematically treated with Ctrl-R or JP1 (n = 6). (J-K) shows the tumor growth curves and the tumor/body weight (%) results of the respective groups, respectively. (L-M) representative fluorescence plots (L) for each group of α -SMA (green), CD31 (red), DAPI nuclear staining, quantitation of α -SMA coverage is shown in plot (M) on a scale bar of 100 μ M. (N-O) representative fluorescence images of sets of desmin (green), CD31 (red) and DAPI nuclear staining (N); panel (O) shows quantification of desmin coverage, at a scale bar of 100 μm. (. P)<0.05；**P<0.01；***P<0.001; ns: differences have no statistical significance).
FIG. 6 is a graph showing the results of example 3 of the present invention. The points of the drawings are as follows: (A-B) representative Western Blot analysis of whole cell lysates from B16F10 (A) and LLC (B) cells after JP1 treatment at the indicated concentrations was performed to assess IL8 expression. (C-D) representative Western Blot analysis results of IL8 protein extracted from B16F10 (C) and LLC (D) tumor nodules. (E-F) quantification of IL 8mRNA after treatment of B16F10 (E) and LLC (F) cells with JP1 at the indicated concentrations. (G-H) the expression of IL 8mRNA in B16F10 (G) and LLC (H) tumor tissues is detected by a qPCR method, the expression of IL-8mRNA is quantitatively detected, and a representative Western Blot analysis shows that the IL8 knockout is effective. (J) body weight plots of mice in each group. (K) Western Blot analysis of representative IL8 proteins 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 is not statistically significant).
FIG. 7 is a graph showing the results of example 4 of the present invention. The points of the drawings are as follows: (A-B) the oxidative phosphorylation levels of B16F10 (A) and LLC (B) cells were analyzed by OCR 48h after JP1 at the indicated concentrations. (C-D) representative immunohistochemical staining pattern (C) for HIF1 α in B16F10 tumor nodules, intensity quantification (D) for HIF1 α, scale bar: 100 μm. (E-F) representative immunohistochemical staining pattern (E) for HIF 1. Alpha. In LLC tumor nodules, intensity quantitation pattern (F) for HIF 1. Alpha. On a scale of 100. Mu.m. (G-H) HIF 1. Alpha. Was detected by Western Blot after 48H of treatment with JP1 at the indicated concentrations in B16F10 (G) and LLC (H) cells. (I-J) extracting HIF1 alpha protein from B16F10 (I) and LLC (J) tumor nodules for Western Blot detection; graph showing the results of quantification of HIF 1. Alpha. Protein concentration. (P < 0.01;. P <0.001 ns: the difference is not statistically significant).
FIG. 8 is a graph showing the results of example 4 of the present invention. The points of the drawings are as follows: (A-B) representative Western Blot analyses of whole cell lysates from B16F10 (A) and LLC (B) cells following JP1 treatment at the indicated concentrations were performed to assess HIF1 α expression. (C-D) representative Western Blot analysis of HIF 1. Alpha. Proteins extracted from B16F10 (C) and LLC (D) tumor nodules. (E-F) quantitative mapping of HIF 1. Alpha. MRNA after JP1 treatment at the indicated concentrations in B16F10 (E) and LLC (F) cells. (G-H) detecting HIF1 alpha mRNA expression in B16F10 (G) and LLC (H) tumor bodies by adopting a qPCR method, and quantitatively detecting an HIF1 alpha mRNA expression result chart. (P < 0.05;. P < 0.01;. P <0.001 ns: the difference is not statistically significant).
FIG. 9 is a graph showing the results of example 5 of the present invention. The points of the drawings 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 Ctrl-R or JP1 treatment for 24h under normoxic or hypoxic conditions. (E) Schematic representation of treatment of B16F10 cells xenografted with Ctrl-R or JP1 under normoxic or hypoxic conditions (n = 6). (F-G) shows the tumor growth curves and the tumor/body weight (%) results of the respective groups. (H-I) representative immunohistochemical staining pattern (H) for HIF1 α in B16F10 tumor nodules in each group, and intensity quantitation pattern (I) for HIF1 α at a scale bar of 50 μm. (J-K) Western Blot analysis results of HIF1 alpha (J) and IL8 (K) protein expression in B16F10 tumor nodules of each group. (L-N) representative fluorescence images (L) of each set of α -SMA (green), desmin (green), CD31 (red), DAPI nuclear staining; quantitative plots of α -SMA (M) and desmin (N) coverage, scale bar: 50 μ M. (P < 0.05;. P < 0.01;. P <0.001 ns: the difference is not statistically significant).
FIG. 10 is a graph showing the results of example 5 of the present invention. The points of the drawings are as follows: and (A) the body weight of each group of mice. (B) Representative Western Blot analysis results of HIF-1. Alpha. And IL8 proteins from B16F10 tumor nodules extracted by Ctrl-R or JP1 treatment under normoxic and hypoxic conditions are shown. (C) Representative Western Blot analyses of whole cell lysates from B16F10 and LLC cells were performed following JP1 treatment at the indicated concentrations to assess the expression of p-AMPK, FOXO3a, UQCRC2, HIF1 α and IL 8.
FIG. 11 is a graph showing the results of example 6 of the present invention. The points of the drawings are as follows: (A) B16F10 cell allotransplantation model. Mice were injected intraperitoneally with JP1 and PTX alone or in combination with the treatment and associated solvents. (B-C) tumor growth curves and tumor/body weight (%) results of the respective groups. (D-G) representative fluorescence images of each group of α -SMA (Green), desmin (Green), CD31 (Red), DAPI nuclear staining; quantitative plots of α -SMA, desmin and CD31 coverage at scale bar 50 μm. (H) Representative HIF 1. Alpha. Immunohistochemical staining patterns were shown in B16F10 tumor nodules in each group, scale bar: 50 μm. (I) HIF 1. Alpha. Intensity quantification results. (J) Results of quantification of vascular permeability after solvent, JP1, PTX and JP1 in combination with PTX treatment. (K) The PTX standard solution had a peak in retention time as analyzed by high performance liquid chromatography. (L) quantification of PTX concentration in each group of tumors. (M) illustration of tumor microenvironment status after solvent, PTX, JP1 treatment. (N) the mode of operation of JP1 in promoting normalization of tumor vessels and thereby inhibiting the initiation of metastasis. (P < 0.05;. P < 0.01;. P <0.001 ns: the difference is not statistically significant).
FIG. 12 is a graph showing the results of example 6 of the present invention. The points of the drawings are as follows: (A) PTX representative high performance liquid chromatography images at the indicated concentrations. (B) Linear standard curve of PTX concentration versus peak area. (C-F) representative high performance liquid chromatography images of PTX in the specified group.
Detailed Description
The invention is described in further detail below with reference to embodiments and with reference to the drawings. The invention is not limited to the examples given. Materials, methods, experimental model conditions, and the like used in the examples are given in the following description, and unless otherwise specified, the materials and experimental methods used are conventional materials and conventional experimental methods.
Example 1 JP1 inhibits metastasis by reducing tumor cell entry into blood vessels
To determine the role of JP1 in metastasis initiation, an active metastatic 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 FIG. 1, panel A is Ctrl-R panel, with no JP1 intervention all the time; group b was JP1 naive, but JP1 treatment was discontinued after primary tumor resection; group c had no JP1 intervention at the beginning, but was given JP1 treatment after primary tumor resection; group d is the JP1 intervention group all the way from tumor bearing, with JP1 treatment given all the way through. Wherein, the administration dosage of JP1 is as follows: 50mg/kg, i.e. intraperitoneal injection, 1 time per day. After primary tumor resection, mice develop multiple organ metastases, most commonly lung metastases. Subsequent lung tissue staining was performed to obtain the incidence of lung metastases, and it was found that 5 of 6 mice in group a (Ctrl-R group) had lung metastases, while only 1 mouse in group d (whole course JP1 intervention group from tumor-bearing) had lung metastases in the melanoma model. Importantly, lung metastasis also occurred in only 1 of 6 mice in group B where JP1 treatment was discontinued after primary tumor resection, while lung metastasis occurred in 4 of 6 mice in group C where JP1 treatment was given after primary tumor resection (panel B of fig. 1, and panels a-C of fig. 2). This observation was further validated in the lung cancer model (panel C of figure 1, and panel D-F of figure 2). In addition, a parallel survival model is established, and researches show that compared with a control group, JP1 intervention prolongs the survival time of mice in melanoma and lung cancer models, wherein the JP1 whole-course intervention group has the best effect, and the JP1 intervention group is the JP1 intervention group in a tumor-bearing period, and the survival time of the two groups of mice is obviously longer than that of the control group of mice; the JP1 intervention group also had some improvement in mouse survival after tumor resection, but the effect was inferior to the early intervention and global intervention groups (fig. 1, panels D, E). These data indicate that JP1 is able to inhibit the development of tumor metastasis.
To further validate this hypothesis, a melanoma-bearing model was constructed with GFP-labeled B16F10 cells, and CTCs were analyzed by cell cloning and FACS sorting (fig. 1, panels F, G). Wherein Ctrl-R group was given with no JP1 intervention throughout, JP1 group was given JP1 treatment throughout, and JP1 was administered at the same dose as before. Cell clone analysis showed that 4 of 6 mice in the CTCs extracted from Ctrl-R group mice formed clonal clusters in vitro, while 1 of 6 mice in JP1 group formed clonal clusters (FIG. 1, H, A of FIG. 3). In addition, FACS analysis showed a significant reduction in tumor cells from the primary tumor entering the circulation after JP1 treatment (fig. 1, panel I, fig. 3, panel B). Thereafter, the migration and invasion experiments of CTCs were carried out, and it was found that the migration ability of CTCs subjected to JP1 treatment was reduced (fig. 1, J panel, fig. 3, C panel), but the invasion ability remained unchanged (fig. 1, K panel, fig. 3, D panel).
These results indicate that JP1 inhibits the initiation of metastasis by reducing the entry of tumor cells into the vascular system, but has a weak inhibitory effect on the invasive capacity of tumor cells.
Note: the sequence of JP1 is FPGSDRF-GGGG-RGD, wherein amino acid S is modified by phosphorylation.
Example 2 JP1 promotion of tumor vascular normalization
To investigate the function of JP1 to reduce the entry of tumor cells into blood vessels, this example evaluated the morphology of tumor blood vessels extracted from melanoma and lung cancer samples, where Ctrl-R group was without JP1 intervention all the time, JP1 group was treated with JP1 all the time, and JP1 was administered at the same dose as in example 1. Immunofluorescent staining showed an increase in coverage of α -SMA (parietal cell marker), claudin5 (endothelial cell marker), desmin (pericyte marker) after JP1 treatment in melanoma (B16F 10) (fig. 4, panels a-D). This observation was further confirmed in lung cancer (LLC) (FIG. 4, panels E-H). These results indicate that tumors exhibited a near normal vascular structure phenotype after JP1 treatment compared to the control group. Further, the vascular permeability test was conducted, and it was found that JP1 decreased the vascular permeability as compared with Ctrl-R treatment (FIG. 4, panels I, J).
Taken together, JP1 can reduce the chance of tumor cells entering blood vessels by promoting tumor vessel normalization, thereby inhibiting the occurrence of tumor metastasis.
Example 3 JP1 promotion of tumor vascular normalization by inhibition of IL8
To obtain downstream target molecules of JP1 effect, angiogenesis array analysis was performed on the differential regulatory genes after JP1 treatment, wherein Ctrl-R group was given with no JP1 intervention all the time, JP1 group was given JP1 treatment all the time, and the dose of JP1 administration was the same as in example 1. The results show that IL8 is the most downregulated gene (panel a of figure 5). Western blot analysis revealed that IL8 decreased significantly with increasing JP1 concentration (panels B and C of FIG. 5, panels A and B of FIG. 6); this finding was subsequently confirmed in mouse tumor samples (fig. 5, panels D and E, fig. 6, panels C and D). RT-PCR quantification also confirmed the results of in vitro and in vivo experiments (FIG. 6, panels E-H). IL8 expression was further examined in human melanocytic nevi and melanoma tissue chips, as predicted, IL8 expression was significantly higher in melanoma than in melanocytic nevi (fig. 5, panels F and G). In contrast, parietal cell coverage was lower in melanoma tissue (fig. 5, panel H). These results indicate that IL8 is negatively associated with vascular normalization under JP1 inhibition.
To further explore how IL8 is regulated by JP1, the IL8 gene in B16F10 cells was knocked out with criprpr-Cas 9 (fig. 6, panel I), and it was observed whether JP1 could still promote tumor vessel normalization in the absence of IL 8. Wherein Ctrl-R group was without JP1 intervention all the time, JP1 group was given JP1 treatment all the time, and JP1 was administered at the same dose as in example 1. A tumor-bearing model of melanoma was then constructed using B16F10 cells containing IL8WT and IL8KO (fig. 5, panel I). The results show that the growth rate of IL8KO B16F10 cells is significantly lower than that of IL8WT B16F10 cells. Furthermore, JP1 significantly inhibited the growth of melanoma, but did not have significant inhibitory effect on IL8KO B16F10 cells, as compared to IL8WT B16F10 cells (fig. 5, J and K, fig. 6). The same results were obtained for morphological evaluation of blood vessels extracted from mouse tumor specimens. JP1 promotes tumor vessel normalization by increasing pericyte and endothelial cell coverage in IL8WT B16F 10; however, knock-out of IL8 significantly improved tumor normalization, whereas JP1 treatment did not enhance the vascular normalization index (L-O plot of fig. 5).
Taken together, these data suggest that JP1 promotes tumor vascular normalization by inhibiting IL 8.
Example 4 JP1 promotes tumor oxidative phosphorylation and improves tumor microenvironment hypoxia
Hypoxia is a corollary to tumor development and has been reported to stimulate IL8 secretion. It is known that the JWA gene can inhibit pancreatic cancer metastasis by promoting aerobic respiration to improve hypoxia in the tumor microenvironment. To verify whether JP1 can promote oxidative phosphorylation of tumor cells similar to JWA gene, this example performed Oxygen Consumption Rate (OCR) analysis. As shown in FIG. 7, panels A and B, JP1 is effective in promoting oxidative phosphorylation of B16F10 and LLC cells. HIF1 α staining of melanoma and lung cancer model tumor samples showed that JP1 significantly improved microenvironment hypoxia within the tumor (C-F panel of fig. 7). Immunoblot analysis showed that HIF1 α decreased significantly with increasing JP1 concentration in B16F10 and LLC cells (fig. 7, panels G and H, fig. 8, panels a and B). Furthermore, this finding was subsequently confirmed in mouse tumor samples (panels I and J of fig. 7, panels C and D of fig. 8). RT-PCR quantitation further confirmed the regulatory role of JP1 on HIF1 α in vitro and in vivo experiments (FIG. 8, E-H panels). Note: in this example, referring to control experiments of Ctrl-R group and JP1 group, ctrl-R group was given JP1 treatment throughout without JP1 intervention, and JP1 group was given JP1 treatment throughout, and the dose of JP1 was the same as in example 1.
These results indicate that JP1 can effectively promote tumor oxidative phosphorylation and improve tumor microenvironment hypoxia.
Example 5 hypoxia-induced secretion of IL8 by tumor cells
To determine the effect of hypoxia in the tumor microenvironment on IL8 expression, this example used B16F10 and LLC cells to establish normoxic (21% oxygen in air) and hypoxic (8% oxygen in air) models, and found that IL8 expression increased under hypoxic conditions (fig. 9, panels a and B). Moreover, JP1 significantly inhibits HIF1 α expression under normoxic conditions; in contrast, under hypoxic conditions, JP1 has reduced regulatory effects on IL8 (FIG. 9, panels C and D). To further demonstrate this hypothesis, this example establishes a melanoma-bearing model under normoxic and hypoxic conditions (fig. 9, panel E). The results show that JP1 has no significant inhibition of tumor growth under hypoxic conditions, compared to JP1 under normoxic conditions (fig. 9, panels F and G, fig. 10, panel a). Mouse tumor HIF1 alpha staining shows that JP1 significantly improves microenvironment hypoxia in the tumor under normoxic conditions; under hypoxic conditions, however, hypoxia in tumors was severe, and JP1 could not reverse the hypoxic state of the microenvironment in tumors (fig. 9, H, I), and this result was confirmed by immunoblot analysis (fig. 9, J, 10, B). Furthermore, under hypoxic conditions, the tumor samples were significantly elevated in IL8 expression (K-panel of fig. 9, B-panel of fig. 10). The evaluation of blood vessel morphology showed that the blood vessel normalization index was lower under hypoxic conditions than under normoxic conditions (fig. 9, L and M panels) regardless of the presence or absence of JP1 treatment. Note: in this example, ctrl-R group was free of JP1 intervention all the time, JP1 group was given JP1 treatment all the time, and the dose of JP1 administration was the same as in example 1; normoxia is under normoxic conditions and Hypoxica is under anoxic conditions.
These results suggest that JP1 improves tumor microenvironment hypoxia by promoting oxidative phosphorylation of tumor cells, thereby reducing IL8 secretion.
Furthermore, in a lateral aspect, this example demonstrates the expression of the relevant molecular proteins in B16F10 and LLC cells. As we expected, JP1 regulates the reprogramming of mitochondrial metabolism of B16F10 and LLC cells through the AMPK/FOXO3a/UQCRC2 signaling pathway, thereby improving the hypoxic state of the tumor microenvironment (FIG. 10, panel C).
Example 6 JP1 promotes intratumoral delivery of Paclitaxel (PTX) and enhances antitumor effects
The permeability of the tumor blood vessel is reduced by normalizing the structure of the tumor blood vessel, the invasion of tumor cells to the blood vessel can be reduced, and the tumor metastasis is inhibited; in addition, the normalization of the distribution of tumor blood vessels also promotes the medicine to enter the tumor, and enhances the anti-tumor effect. To evaluate the effect of JP1 on promoting normalization of tumor blood vessels so that the drug enters the tumor, this example completed a melanoma model (fig. 11, panel a) of the combination therapy of JP1 and Paclitaxel (PTX). The results show that the antitumor effects of JP1 and PTX alone are 37% and 49.7%, respectively, and the inhibitory effect of the combination therapy is 65.4%, suggesting that JP1 has a better antitumor effect (panels B and C in fig. 11) in combination with PTX, i.e., JP1 can produce a synergistic effect on PTX in terms of antitumor effect. Tumor blood vessel morphology confirmed that PTX promoted normalization of blood vessels within the range of inhibiting the total number of blood vessels, as compared with JP 1's effect of promoting normalization of tumor blood vessels (D-G diagram of fig. 11). In addition, in this example, hypoxia analysis was performed on tumor tissues, and it was found that PTX can promote tumor vascular normalization, but its inhibitory effect on blood vessels cannot improve hypoxia status, and JP1 and PTX combined treatment significantly improve hypoxia status of tumor microenvironment (fig. 11, H and I), and the vascular permeability experiment further verifies this observation (fig. 11, J). To further demonstrate that JP1 promotes intratumoral delivery of drugs, this example measured the amount of PTX in the tumor by HPLC, and found that PTX delivery to the tumor was significantly reduced after 11 days of PTX treatment compared to JP1 combination treatment, while JP1 and PTX combination treatment significantly increased PTX delivery in the tumor (K-N of fig. 11, fig. 12). Note: in this example, the Vehicle group was a control group with no JP1 and PTX intervention; the JP1 group is only given with JP1 treatment, the administration dose of JP1 is 50mg/kg, and the injection is performed in the abdominal cavity for 1 time per day; PTX group was given PTX treatment only at a dose of 10mg/kg, i.p. 2 times per week (monday, four); the JP1+ PTX group is a JP1 and PTX combined administration group, the administration dose of JP1 is the same as that of JP1 group, and the administration dose of PTX is the same as that of PTX group.
These results indicate that PTX inhibits metastasis initiation by promoting tumor vessel normalization, but its inhibition of the vessels reduces drug delivery into the tumor; the combination of JP1 and PTX not only promotes normalization of tumor blood vessels to inhibit metastasis initiation, but also increases the delivery of PTX into tumors.
Example 7
This example used each of the JWA polypeptides shown in the table below, each of which was modified by phosphorylation of amino acid S, as tested in examples 1 to 6.
The examples do not show specific experimental data, depending on the space. The obtained experimental data show that the detection results of the JWA polypeptides are basically consistent with those of JP1.
Conclusion
As is clear from the above examples, the present invention confirmed a series of JWA polypeptides represented by JP 1: (1) Improving tumor microenvironment hypoxia by promoting tumor cell oxidative phosphorylation and thereby reducing IL8 secretion, and regulating and controlling tumor cell mitochondrial metabolism reprogramming through AMPK/FOXO3a/UQCRC2 signal pathway to improve tumor microenvironment hypoxia state; (2) Promoting the normalization of tumor vascular structure and intratumoral distribution by inhibiting IL8, and reducing the opportunity of tumor cells to enter blood vessels, thereby inhibiting the initiation and occurrence of tumor metastasis; and (3) the effective perfusion amount of the medicine in the tumor can be increased. Therefore, the JWA targeting peptide not only shows effective antitumor activity when being taken alone, but also can play a synergistic tumor inhibition role when being combined with other therapies such as chemical drugs, antibody drugs and cell drugs. The unique biological characteristics of the JWA polypeptide provide a new clinical medication possibility for treating tumors, particularly solid tumors, and have good application prospects.
Materials, methods, experimental model conditions, and the like used in the above examples are shown below.
1. Cell lines and cell cultures: mouse melanoma B16F10 and lung cancer LLC cells were purchased from ATCC (MD, USA). IL8KO (IL 8 knock-out) B16F10 cells were generated using IL 8-specific CRISPR/Cas9 plasmid (synthesized by coress biotechnology). Sequencing analysis and western blot experiment prove that the gene knockout is finished. GFP-B16F10 cells were generated from GFP-specific plasmids (synthesized by Shanghai Genechem, inc.). All cell lines were maintained in DMEM medium supplemented with streptomycin 100. Mu.g/ml, penicillin 100U/ml and 10% fetal bovine serum at 37 ℃ with 5% CO 2 And (5) preserving in an incubator.
2. Mouse allograft tumor model: all care and treatment for the experimental mice was approved by the Nanjing university of medical animal Care and use Committee guide (IACUC: 1811067-1). Laboratory mice were purchased from Shanghai SLAC laboratory animals center and stored in the animal core facility of Nanjing medical university. Melanoma growth model: B16F10, IL8WT B16F10, IL8KO B16F10 and GFP-B16F10 cells (5X 10) 5 ) Injected subcutaneously into C57BL/6 male mice: lung cancer growth model: LLC cells (5X 10) 6 ) C57BL/6 male mice were injected subcutaneously. When the tumor volume reaches 100mm 3 Mice were randomly grouped and intervened. Body weight and tumor size were measured every other day. After the experiment, mice were sacrificed humanely and tumors were measured and recorded. An oxygen concentration controller (purchased from shanghai yuyan instruments ltd) created an environment with an oxygen content of 8%, and the mice stayed for 6h/d to establish an anaerobic model. A melanoma/lung cancer active metastasis model was performed using the general protocol described previously.
Ctc (circulating tumor cells) analysis and sorting: a melanoma model was constructed using GFP-labeled B16F10 cells. When the tumor volume reaches 2000mm 3 In time, 500 μ l of blood was taken from each mouse to lyse erythrocytes (erythrocyte lysis buffer purchased from fclacs Biotech co., ltd.). FACS analysis was performed using a LSRII flow cytometer (BD Biosciences) to detect CTCs in every 150 million cells and the data was analyzed using FlowJo software (Tree Star inc.). FACS sorting uses an Aria III instrument (BD Biosciences).
4. Cell proliferation, migration and invasion assays: taking the blood of a mouse carrying the melanoma,the red blood cells are lysed and directly added into a DMEM medium to be cultured for 21 days for proliferation experiments. Migration and invasion experiments were performed using FACS sorted CTCs. In the migration assay, CTC was inoculated in a Transwell TM Filters (purchased from corning, usa) were added to the serum-free medium in the upper chamber and medium containing 10% FBS was added to the lower chamber. After 12h, fix with methanol for 1h, stain with crystal violet (Beyotime, shanghai, china) for 30min, and count. For the invasion test, a layer of matrix (purchased from BD biosciences) was placed on the lower cavity membrane.
5. Immunohistochemistry and immunofluorescence staining: tissue specimens were soaked in formalin for 24h, paraffin embedded, and sectioned (paraffin microtome, thermo HM 340E). After deparaffinization and antigen recovery, the cells were blocked with 10% normal goat serum 30min at room temperature and incubated with HIF1 α (CST, 36169T) antibody solution gently shaking at 4 ℃ for 24h. DAB staining was followed by scanning with a pathological section scanner (Pannoramic MIDI). Double immunofluorescent staining, the mouse source CD31 (ServiceBio, GB 12063) antibody solution and rabbit source alpha-SMA (ServiceBio, GB 111364), claudin5 (ServiceBio, GB 11290), desmin (ServiceBio, GB 11081) antibody solution were incubated for 24h at 4 ℃ with gentle shaking. Images were scanned after incubation with fluorescent secondary antibodies of murine (red) and rabbit (green) origin. The staining intensity was analyzed by J-plot.
6. And (3) measuring vascular permeability: 50mg/kg Evans Blue solution was injected via tail vein into melanoma-bearing mice. Blood was collected after 1h, and the supernatant was centrifuged and diluted with formamide 1. Evans blue was dissolved in formamide (0 ng/ml, 125ng/ml, 250ng/ml, 500ng/ml, 1000ng/ml, 5000ng/ml, 10000ng/ml, 25000ng/ml, 50000 ng/ml) to prepare a standard curve. The absorbance of the supernatant was measured at 620nm, and the concentration of evans blue in the blood was calculated. After blood sampling, mice were quickly exposed to the heart, and then 10ml of 0.9% physiological saline was perfused into the heart to wash evans blue in the blood vessels. 200mg of tumor tissue was placed in a 60 ℃ oven for 5h, 200. Mu.l formamide was added, and the mixture was placed in succession in a 60 ℃ oven for 18h. Centrifuging the supernatant, measuring the absorbance at 620nm, and calculating the content of evans blue in the tumor tissue. The vascular permeability is calculated as: vascular permeability [ μ l/(g × h) ] = [ evans blue (μ g)/tumor dry weight (g) ]/[ evans blue concentration in blood (μ g/μ l) × circulation time (h) ].
7. Angiogenesis array analysis: the angiogenesis array GS1000 (GSH-ANG-1000-1, ray Biotech Inc.) was used according to the manufacturer's instructions. Briefly, the slides were removed from the cassette and equilibrated to room temperature in a sealed plastic bag for 20-30min. And taking out the slide from the plastic bag, stripping the cover film, and air-drying for 1-2h. Standards or samples were collected and incubated with the array at 4 ℃ for 24h with gentle shaking. Chemiluminescent signals were captured by an InnoScan 300 microarray scanner (Innopsys). Data extraction was performed using microarray analysis software ScanArray Express.
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). RT-PCR AB RT-PCR (Q5) was performed using SYBR (Vazyme, TSE 202). Finally, the difference was calculated by GAPDH normalization. The primers used in RT-PCR are HIF1 alpha-F (ACGTTCCTTCGATCAGTTGTCACC), HIF1 alpha-R (GGCAGTGGTAGTGGTGGCATTAG), IL8-F (tcctgcttcctc), IL8-R (GGGTGGAAAGGTGTGTGGAATG), GAPDH-F (gcctctgctccttctttc), and GAPDH-R (ACGACCAAATCCGTTGATCC), respectively. Western Blot analysis was performed as reported previously. Briefly, cell and tissue samples were lysed with cell lysis buffer and tissue protein extraction reagent (available from Thermo FisherScientific, 78510), respectively. A total of 40 proteins were processed for subsequent analysis. The antibodies used were Hif1 α (CST, 36169T), IL8 (Abcam, ab 106350), p-AMPK (CST, 2535T), AMPK (Abcam, 32047), FOXO3A (Abcam, 53287), UQCRC2 (Abcam, ab 203832), tubulin (Beyotime, AF 0001).
9. Mitochondrial Oxygen Consumption (OCR) analysis: OCR analysis was performed on B16F10 and LLC cells using a Seahorse XFP Analyzer (Seahorse XF 96). B16F10 cells (6000) and LLC cells (10000) were seeded onto hippocampal plates and placed in growth medium for 24h. To XF substrate containing 1mM sodium pyruvate, 2mM l-glutamine and 10mM glucose, oligomycin (1. Mu.M) and FCCP (B16F 10: 1.5. Mu.M, LLC: 1. Mu.M) were added in this order to measure the mitochondrial oxygen consumption.
10. High performance liquid chromatography analysis: paclitaxel (PTX) was purchased from Selleck (NSC 125973), HPLC (column: agilent Zorbax Exlipse Plus C18, 100X 4.6mm, 3.5. Mu.M particle size; flow;)The speed is 1ml/min; the wavelength is UV 254nm; mobile phase CH 3 OH 0.1％ TFA/H 2 O0.1% TFA,0min 10. Tumor-bearing mice were injected with 20mg/kg of PTX solution via the tail vein. After 5min, 200mg of tumor was dissolved in 1ml of methanol solution. PTX was dissolved in a methanol solution (200. Mu.g/ml, 100. Mu.g/ml, 50. Mu.g/ml, 25. Mu.g/ml, 12.5. Mu.g/ml, 6.25. Mu.g/ml, 3.125. Mu.g/ml, 1.56. Mu.g/ml) to prepare a standard curve. Measuring standard substance or sample by high performance liquid chromatography, and calculating paclitaxel concentration in tumor.
11. Statistical analysis: all data were analyzed as individual sample mean ± SEM, using GraphPad Prism 8 and SPSS 20 software. Statistical differences between experimental groups were determined using unpaired two-tailed student's t-test or one-way analysis of variance (ANOVA). P <0.05 is statistically significant for the differences. (P < 0.05;. P < 0.01;. P <0.001 ns: meaningless).
In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the claims of the present invention.
Claims (10)
1. The use of a polypeptide for the preparation of a synergistic agent for antitumor drugs;
the amino acid sequence of the polypeptide is shown as I or II:
I：FPGSDRF-Z；
II：X-FPGSDRF-Z；
wherein, the amino acid S is modified by phosphorylation, and X and Z are respectively amino acid or amino acid sequence;
x is selected from F, (R) 9 、(R) 9 -F, 6-aminocaproic acid-F, 6-aminocaproic acid- (R) 9 6-aminocaproic acid- (R) 9 -one of F;
z is selected from (G) n -RGD、A-(G) n -one of RGD, n being an integer greater than or equal to 0, and n having a value ranging from 0 to 10.
2. The use as claimed in claim 1, wherein the antineoplastic drug synergist functions as: the hypoxia of the tumor microenvironment is improved by promoting the oxidative phosphorylation of the tumor cells so as to reduce the secretion of IL8, and the mitochondrial metabolic reprogramming of the tumor cells is regulated through an AMPK/FOXO3a/UQCRC2 signal channel so as to improve the hypoxia state of the tumor microenvironment.
3. Use according to claim 1, characterized in that the antineoplastic drug synergist has the function of: the IL8 is inhibited to promote the normalization of tumor blood vessels and reduce the opportunity of tumor cells to enter the blood vessels, thereby inhibiting the initiation and occurrence of tumor metastasis.
4. The use as claimed in claim 1, wherein the antineoplastic drug synergist functions as: increasing the effective perfusion amount of the medicine in the tumor.
5. Use according to any one of claims 1 to 4, wherein the antineoplastic drug to which the antineoplastic drug synergist is directed is a chemical drug, an antibody drug or a cell-based drug for tumor therapy.
6. Use according to claim 5, wherein the antineoplastic drug co-potentiator is paclitaxel.
7. Use according to any one of claims 1 to 4, characterized in that the antineoplastic drug co-potentiator is to a tumor is pan-solid tumor having at least one of the following characteristics:
the method is characterized in that: hypoxia of microenvironment in tumor, disturbance of blood vessel distribution and low drug perfusion in tumor;
the second characteristic: the energy metabolism of tumor cell mitochondria is disturbed, the oxidative phosphorylation level is reduced, and the glycolytic metabolism is enhanced;
the characteristics are as follows: the IL8 secretion is abnormally increased and the integrity of the blood vessel wall in the tumor is destroyed.
8. The use as claimed in claim 7, wherein the antineoplastic drug synergist is directed against melanoma, lung cancer, or nevus of human melanocytes.
9. The use according to any one of claims 1 to 4, wherein the polypeptide is modified by acetylation at the N-terminus and amidation at the C-terminus; the amino acid of the polypeptide is L-type natural amino acid or D-type unnatural amino acid.
10. The use according to any one of claims 1 to 4, wherein the amino acid sequence of the polypeptide is FPGSDRF-RGD, wherein amino acid S is modified by phosphorylation.
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| CN115414465A (en) * | 2022-10-17 | 2022-12-02 | 苏州明人医药生物科技有限公司 | Application of JWA polypeptide in preparation of antitumor drug synergist |
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| WO2024082408A1 (en) * | 2022-10-17 | 2024-04-25 | 苏州明人医药生物科技有限公司 | Use of jwa polypeptide in preparation of anti-tumor drug co-potentiator |
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