Application of TSP-1-sourced polypeptide PKHB1 in preparation of medicine for treating NSCLC

Abstract

The invention discloses application of TSP-1-sourced polypeptide PKHB1 in preparation of a medicine for treating NSCLC, and belongs to the field of biomedicine.TSP-1-sourced CD47 targeted activation polypeptide PKHB1 and CD47-sourced TSP-1 targeted blocking polypeptide TAX2 are used for targeted simulation or blocking of a TSP-1 / CD47 signal axis, and research results of the invention are beneficial to improvement of clinical treatment of middle and advanced NSCLC: on one hand, the TSP-1-sourced polypeptide PKHB1 and the TSP-1-sourced TSP-1 targeted blocking polypeptide TAX2 can be used for preparing a TSP-1 / CD47 targeted activation polypeptide PKHB1 and the TSP-1-sourced TSP-1 targeted blocking polypeptide TAX2; a brand new potential technical means is provided for treatment and prevention of middle and advanced NSCLC, namely the PKHB1 polypeptide which induces immunogenic death of NSCLC cancer cells, is derived from TSP-1 and is subjected to CD47 targeted activation; on the other hand, a theoretical basis is provided for inducing middle and advanced NSCLC radiotherapy from the perspective of active oxygen dependent endoplasmic reticulum stress induced cancer cell immunogenic death.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of a polypeptide PKHB1 derived from TSP-1 in the preparation of a drug for treating NSCLC. Background Art

[0002] Lung cancer is one of the most common malignant tumors in the world and has currently become the leading cause of death from malignant tumors among urban populations in China. Compared with small cell lung cancer, non-small cell lung cancer (NSCLC), which accounts for about 80% of the total lung cancer, has slower cancer cell growth and division and relatively later spread and metastasis. However, about 75% of NSCLC patients are found to be in the middle and advanced stages at the time of diagnosis. Despite the great progress made in the surgery, radiotherapy, chemotherapy, molecular targeted therapy, and immunotherapy of NSCLC, the overall cure rate and survival rate of NSCLC patients are still very low. Therefore, the clinical treatment of middle and advanced NSCLC is still very difficult and urgently needs to be solved.

[0003] As a classical tumor suppressor, thrombospondin-1 (TSP-1) is an important glycoprotein complex in the extracellular matrix with a complex molecular structure. It can bind to various molecules in the cell membrane and extracellular matrix through its different domains and perform complex and diverse biological functions.

[0004] At present, it has been reported that the TSP-1 protein has the potential to inhibit the progression of NSCLC, but its specific cellular and molecular mechanisms are not clear. At present, the research on TSP-1 against NSCLC focuses on its inhibition of tumor angiogenesis, thus unable to provide effective targeted agents for the effective targeted treatment of NSCLC, which is a technical problem urgently to be solved in this field. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of a polypeptide PKHB1 derived from TSP-1 in the preparation of a drug for treating NSCLC to solve the above problems.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: the application of a polypeptide PKHB1 derived from TSP-1 in the preparation of a drug for treating NSCLC, and the amino acid sequence of the polypeptide PKHB1 is: kRFYVVMWKK.

[0007] As a preferred technical solution, the polypeptide PKHB1 is also used in combination with a TSP-1 targeting blocking polypeptide TAX2 derived from CD47.

[0008] As a further preferred technical solution, the amino acid sequence of the polypeptide TAX2 is CEVSQLLKGDAC.

[0009] The present invention discloses the induction of ICD in NSCLC cancer cells by the TSP-1 / CD47 signaling axis and its molecular mechanism, thereby laying a foundation for subsequent translational medicine research on NSCLC treatment. On the one hand, the present invention discovers that the PKHB1 polypeptide derived from TSP-1 can exert an anti-NSCLC effect by inducing ICD in NSCLC cancer cells. This research result will lay a foundation for the translational medicine application of the PKHB1 polypeptide derived from TSP-1 against NSCLC, and contribute to the further preclinical animal experiments and clinical trials of the PKHB1 polypeptide derived from TSP-1 against NSCLC. This will provide a brand-new potential technical means for the treatment of advanced NSCLC. On the other hand, the present invention discovers that reactive oxygen species (ROS)-dependent endoplasmic reticulum stress is a key link in the induction of ICD in NSCLC cancer cells by the TSP-1 / CD47 signaling axis. This suggests that ROS-dependent endoplasmic reticulum stress can serve as a key target for NSCLC treatment centered on ICD in NSCLC cancer cells. Radiotherapy has been considered in recent years to cause ROS-dependent endoplasmic reticulum stress and induce ICD in cancer cells, thereby exerting its anti-cancer effect. However, currently, the ICD induced by NSCLC radiotherapy is not sufficient to bring a significant anti-cancer effect. The present invention provides a theoretical basis for optimizing NSCLC radiotherapy.

[0010] Compared with the prior art, the advantages of the present invention are as follows: The research results of the present invention contribute to improving the clinical treatment of advanced NSCLC: On the one hand, it will provide a brand-new potential technical means for the treatment and prevention of advanced NSCLC, namely the PKHB1 polypeptide derived from TSP-1 that induces immunogenic death of NSCLC cancer cells and targets and activates CD47; on the other hand, it will provide a theoretical basis for inducing radiotherapy for advanced NSCLC from the perspective of ROS-dependent endoplasmic reticulum stress-induced immunogenic death of cancer cells. The present invention helps to provide a brand-new polypeptide drug and an optimized radiotherapy plan for the prevention and treatment of advanced NSCLC. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 are the MTT assay results of different experimental groups;

[0012] Figure 2 are the cell migration results graphs of different experimental groups;

[0013] Figure 3 are the statistical analysis results of cell migration of different experimental groups;

[0014] Figure 4 are the photos of scratch assays of different experimental groups;

[0015] Figure 5 are the statistical results of scratch assays of different experimental groups;

[0016] Figure 6It is immunogenic cell death of mouse LLC1 cell line;

[0017] Figure 7 It is the immunodependence of the anti-NSCLC effect of TSP-1. Specific implementation manners

[0018] The present invention will be further described below in conjunction with embodiments.

[0019] Some experimental drugs used in the following embodiments:

[0020] According to the polypeptide sequences and techniques described in the existing literature, with solid-phase polypeptide synthesis technology as the core, PKHB1 (k-R-F-Y-V-V-M-W-K-k) polypeptide and its control TAX2 (C-E-V-S-Q-L-L-K-G-D-A-C) polypeptide are artificially synthesized in vitro.

[0021] Establishment of syngeneic orthotopic NSCLC mouse model:

[0022] In the preliminary experiment, a syngeneic orthotopic NSCLC mouse model has been established according to the method described in the literature (Liu P, et al. Nat Commun. 2019) to accurately and reliably simulate NSCLC. Specifically, C57BL / 6 mice are anesthetized with 3 vol.% isoflurane and placed in the supine position. The surgical area is depilated and disinfected and sterilized with alcohol / iodophor, and a lateral chest wall incision is made. 2×10 5 mouse lung adenocarcinoma cells LLC1 (suspended in 50 μl PBS containing 20 vol.% Matrigel) are injected transcutaneously with a 0.3 ml insulin syringe loaded with a 30G subcutaneous injection needle. The skin incision is sutured, and the success of model establishment is observed 5 days after the operation.

[0023] Example 1:

[0024] MTT experiment

[0025] Add 100 μL cell suspension to each well in a 96-well plate so that the number of cells in each well is 5×10³ (6 parallel sample wells in each group, and 2 culture plates are seeded), and culture overnight in an incubator at 37 °C and 5 vol.% CO2. As Figure 1 shown, the concentration of TSP-1 recombinant protein added in the experimental group is 5 μg / mL (TSP1 group), and the concentrations of PKHB-1 (PKHB1 group) or TAX-2 (TAX2 group) polypeptides are both 125 ng / mL, and only PBS is added to the control group.

[0026] Take the time of adding the treatment drug as 0 hour, and continue culturing for 24 hours after adding the treatment drug. At 0 hour, take any culture plate, add MTT reagent (final concentration 0.5 mg / mL) to each well, incubate for 4 hours, aspirate the culture medium, add 150 μL DMSO to each well, shake at room temperature for 10 minutes, and after complete dissolution, measure the absorbance value (OD value) at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Another culture plate is detected in the same manner after culturing for 24 hours.

[0027] The proliferation index calculation formula is: (OD value at 24 hours - OD value at 0 hour) / OD value at 0 hour × 100%.

[0028] The experimental results are as Figure 1 shown, Figure 1 in which, A is the cell proliferation index of different experimental groups, and B is the OD value (B) detected by MTT. It can be seen from Figure 1 that: the cell proliferation ability of the TSP1 group, PKHB1 group, and TAX2 break group is significantly lower than that of the control group. Among them, the effect of PKHB-1 on alleviating the inhibitory effect of TSP-1 is significantly higher than that of the TAX2 group. There are significant differences in pairwise comparisons between groups, and * indicates P < 0.05.

[0029] Example 2:

[0030] Cell migration experiment

[0031] The cells are cultured to the logarithmic growth phase, digested and resuspended in serum-free medium, and a Transwell culture plate pre-coated with Matrigel is used. Add 600 μL of medium containing 10 vol.% FBS to the lower chamber (bottom of the 24-well plate). Dissolve each group of drugs in serum-free medium, mix with 150 μL of cell suspension (serum-free), and add them together to the upper chamber of the Transwell, and continue culturing for 72 hours. Each group has 3 parallel sample wells, and the drug concentration is Figure 1 the same. Figure 2 In Figure 1 A, B, C, and D groups, the drugs added correspond to

[0032] the control group, TSP1 group, PKHB1 group, and TAX2 group of

[0033] respectively. Gently wipe the inner Matrigel and non-penetrating cells of the upper chamber with a cotton swab, immerse the Transwell insert in 4 vol.% paraformaldehyde, fix at room temperature for 15 minutes, and gently rinse once with PBS. Then immerse the insert in 0.1 vol.% crystal violet staining solution, stain at room temperature for 20 minutes, gently rinse 3 times with PBS to remove excess dye, and air dry. Carefully remove the insert membrane, place it on a glass slide (membrane facing up), and take 5 random fields of view under a 10× objective of an inverted microscope to calculate the number of migrating cells. Figure 3As shown, through small chamber cell counting, it was found that in the TSP1 group, the number of cells passing through the membrane significantly decreased (P<0.05); after being blocked by PKHB-1, the number of cells passing through the membrane approached the control group level, while after being blocked by TAX-2, the change in the number of cells passing through the membrane was not significantly different compared with the TSP-1 group (P>0.05).

[0034] Example 3:

[0035] Wound healing assay

[0036] Cells were cultured to the logarithmic growth phase and seeded at a density of 5×10 5 cells / well in a six-well plate, incubated statically for 24 hours until the confluence reached about 90%, and then switched to serum-free medium and continued to culture for 6 hours. A 200 μL sterile pipette tip was used to draw a straight line perpendicular to the bottom of the plate along a ruler, and two parallel lines were drawn in each well. The cells detached due to scratching were removed by rinsing twice with PBS, and then switched to normal medium containing the treatment drug, with the concentrations of each group the same as in Example 1. Photos were taken immediately after scratching, the positions for taking photos were marked and recorded as 0 hour, and then photos were taken at the same positions again after 3 days and 5 days. An inverted microscope with a 4× objective lens was used, and the ImageJ software was used to calculate the number of cells migrating into the scratched area.

[0037] The experimental results were as Figure 4 shown. In the wound healing assay, it was found that in the TSPl group, the number of cells migrating into the scratched area was significantly lower than that in the control group. After blocking with PKHB-1 and TAX-2, although the inhibitory effect of TSP-l on cell migration was alleviated to some extent, it was not significant;

[0038] The statistical results of the cell count migrating into the scratched area were as Figure 5 shown. It could be seen from Figure 5 that there was a significant difference between the TSP-l group and the control group (p<0.05). After blocking CD36 or CD47 with antibodies, the number of cells migrating to the scratched area was not different from that in the control group and the TSP-l group (P>0.05).

[0039] Example 4

[0040] Effects of TSP-1, PKHB-1 and TAX-2 polypeptides on programmed cell death of LLC1 cell line in vitro

[0041] LLC-1 cells in the logarithmic growth phase were seeded at a density of 1×10 6 in a six-well plate. After incubation overnight, the corresponding drugs were added for treatment, and 3 parallel samples were set in each group. Incubation was continued, and Annexin V-FITC / PI double staining fluorescence imaging was performed at 24 hours and 72 hours. At 72 hours, cells were also collected for Annexin V-FITC / PI apoptosis detection.

[0042] Immunofluorescence imaging: First, wash the cells twice with pre-cooled PBS to remove residual culture medium. Then, dilute 5 μL of Annexin V-FITC with 100 μL of Binding Buffer, cover the cells, and incubate for 15 minutes at room temperature in the dark. Next, add 5 μL of PI (final concentration 1.5 μg / mL), and incubate for 5 minutes in the dark. Add 1 mL of 4 vol.% paraformaldehyde to fix for 10 minutes, and then wash twice with PBS. Drop anti-fluorescence quenching mounting medium, cover with a coverslip, and use an inverted fluorescence microscope to randomly select 5 fields of view (20× objective) per well for photographing. Viable cells will not be stained and thus have no fluorescence (Annexin V − / PI − ), early apoptotic cells have only green fluorescence (Annexin V + / PI − ), late apoptotic / necrotic cells have both green + red fluorescence (Annexin V + / PI + ), and necrotic cells have only red fluorescence (Annexin V − / PI + ).

[0043] Apoptosis detection: Digest adherent cells with trypsin without EDTA, centrifuge at 1000 rpm for 5 min, collect the cells, and wash twice with pre-cooled PBS to remove residual culture medium. Resuspend the cells with 100 μL of 1× Binding Buffer, adjust the density to 1×10 6 cells / mL, add 5 μL of Annexin V-FITC, incubate for 15 minutes at room temperature in the dark, then add 5 μL of PI (final concentration 1 μg / mL), incubate for 5 minutes in the dark, supplement with 400 μL of Binding Buffer, mix well, and detect within 1 hour on a BD flow cytometer. The excitation wavelength is 488 nm, and the detection channels for Annexin V and PI signals are 530 / 30 nm and 585 / 40 nm respectively. Use FlowJo software to analyze the data. First, remove signals such as debris with an FSC VS. SCC scatter plot, and only gate the single-cell population. Then analyze the staining of Annexin V and PI in the single-cell population. In Figure B, the Q1 quadrant is pure necrotic cells (Annexin V − / PI + ), the Q2 quadrant is late apoptotic / necrotic cells (Annexin V + / PI + ), the Q3 quadrant is viable cells (Annexin V − / PI − ), and the Q4 quadrant is early apoptotic cells (Annexin V+ / PI − );

[0044] Figure C only counts the values in the Q3 quadrant;

[0045] The steps of transmission electron microscopy staining for Figure D are as follows: After washing the samples treated with TSP-1 for 48 hours 3 times with pre-cooled PBS, add 1 mL of pre-cooled 2.5% glutaraldehyde for fixation, and then send them to a third-party company for subsequent processing and electron microscopy image acquisition;

[0046] Figure E is the fluorescence imaging detection of calreticulin (CRT) membrane translocation. Select the six-well plate samples treated with TSP-1 for 24 hours, wash them 2 times with pre-cooled PBS, add 4% paraformaldehyde, fix them at room temperature for 15 minutes, and wash them 3 times with PBS again. Block them with 1% BSA at room temperature for 30 minutes, add rabbit anti-mouse CRT primary antibody diluted 1:100, and incubate at room temperature for 1 h. Wash 3 times with PBS, add AlexaFluor-labeled goat anti-rabbit fluorescent secondary antibody, and incubate at room temperature for 1 hour in the dark. Wash 3 times with PBS, add 1 μg / mL DAPI to stain the nuclei for 5 minutes, and wash 2 times with PBS. Drop anti-quenching mounting medium, cover with a coverslip, and take pictures with a laser confocal microscope (60× oil immersion lens). For cells positive for CRT, their cell membranes are red.

[0047] The results are as Figure 6 shown, Figure 6 in:

[0048] A represents: Annexin V-FITC / PI double staining and fluorescence microscopy imaging, N = 3 / group;

[0049] B-C represent: Annexin V-FITC / PI double staining and flow cytometry analysis (72 hours), N = 5 / group. ***: P < 0.001, compared with the PBS solvent control; : P < 0.001, compared with the simple administration of TSP-1 recombinant human protein. Isotype IgG control is only shown in the statistical chart;

[0050] D represents: Transmission electron microscopy morphology of programmed cell death of the in vitro LLC1 cell line induced by TSP-1 (5 μg / ml, 48 hours). N = 3 / group. (E) TSP-1 (5 μg / ml, 24 hours) confers immunogenic core molecular characteristics (calreticulin membrane translocation) on the in vitro LLC1 cell line; N = 3 / group.

[0051] Example 5

[0052] Immunodependence of the anti-NSCLC effect of TSP-1

[0053] Figure 7A is a schematic diagram for constructing a lung cancer model. The immunodeficient nude mouse strain is BALB / C nude, and the immunocompetent mouse strain is C57BL / 6. The LLC-1 cell line labeled with FITC was used, and the inoculation dose was 1×10 6 cells / 50 μL / mouse, with 6 mice in each experimental group. The in-situ inoculation procedure was as follows: The mice were anesthetized by inhaled isoflurane, a left intercostal incision was made to expose the left lung, 50 μL of cells were slowly injected into the lung parenchyma, and the muscle and skin were sutured layer by layer. After the operation, the mice were kept warm and recovered.

[0054] At the 4th - 5th week, the mice were treated with TSP-1 or PBS three times a week, and the dosage of TSP-1 was 1.25 μg per time. The tumor volume of the mice was monitored weekly using a small animal in vivo imaging system, and the experimental endpoint was the 8th week. The experimental data were analyzed using GraphPad Prism software, with two-way ANOVA. The significant differences between the TSP-1 group vs. the PBS group and the wild type vs. the nude mouse group were marked with * and # respectively.

[0055] The results are as Figure 7 shown in Figure 7 as follows:

[0056] A represents: A schematic diagram for constructing a syngeneic in-situ NSCLC mouse model based on immunocompetent or immunodeficient mice;

[0057] B represents: The tumor growth of syngeneic in-situ NSCLC in immunocompetent or immunodeficient mice and the anti-cancer effect of recombinant human TSP-1 protein. N = 6 / group. ***: P < 0.001, comparing the recombinant human TSP-1 protein (1.25 μg per time, three times a week) with the PBS solvent control in the syngeneic in-situ NSCLC mouse model of immunocompetent or immunodeficient mice; : P < 0.001, the difference in the anti-NSCLC effect of recombinant human TSP-1 protein between immunocompetent and immunodeficient mice.

[0058] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of the polypeptide PKHB1 derived from TSP-1 in the preparation of a medicament for treating NSCLC, characterized in that, The amino acid sequence of the polypeptide PKHB1 is: kRFYVVMWKK.

2. The application according to claim 1, wherein The polypeptide PKHB1 is also used in combination with the CD47-derived TSP-1 targeting blocking polypeptide TAX2.

3. The application according to claim 2, wherein The amino acid sequence of the polypeptide TAX2 is CEVSQLLKGDAC.

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