Application of IL-1beta receptor inhibitor analhysteresis in preparation of sensitizer for improving radiotherapy effect of cervical cancer

Abstract

The invention provides an application of an IL-1beta receptor inhibitor analhysteresis in preparation of a sensitizer for improving the radiotherapy effect of cervical cancer. Furthermore, the pharmaceutical composition also contains a pharmaceutically acceptable carrier or excipient. The invention also provides a sensitizer for improving the radiotherapy effect of cervical cancer, and the sensitizer contains an effective dose of analectin as an IL-1beta receptor inhibitor. Under the condition of combined use of the IL-1beta receptor inhibitor analectin and radiotherapy, the capability of reducing migration and invasion of cervical cancer tumor cells is remarkably enhanced, and the sensibilization effect is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a sensitizer, specifically the use of anakinra, an IL-1β receptor inhibitor, in the preparation of a sensitizer for improving the radiotherapy effect of cervical cancer. Background Art

[0002] Cervical cancer is a common malignant tumor in women, and its disease-related mortality rate ranks third. The main treatment for locally advanced cervical cancer is synchronous chemoradiotherapy. Although the treatment efficacy of patients with locally advanced cervical cancer has been significantly improved with the application of technical means such as image-guided brachytherapy and immunotherapy, 30-50% of patients still face disease recurrence; the recurrence includes local regional recurrence and distant metastasis. How to improve the treatment efficacy has always been the focus of clinical attention.

[0003] More and more evidence shows that the efficacy of radiotherapy, in addition to the killing effect of radiation on tumor cells, depends largely on the impact of changes in the immune microenvironment caused by radiotherapy on the fate of tumor cells. The tumor microenvironment is composed of various malignant cells, fibroblasts, vascular endothelial cells, immune cells, etc. In tumor progression and treatment response, various cells interact and influence each other. Previous studies have shown that chemotherapy drugs can ultimately cause the activation of NLRP3-inflammasomes, increase the release of IL-1β, and IL-1β has a strong immune regulatory effect. Its high expression is associated with treatment resistance to chemotherapy drugs. Inhibiting the IL-1β signaling pathway can improve the efficacy of PD-1 inhibition. However, little is known about the relationship between IL-1β and radiotherapy sensitivity.

[0004] Based on this background, we explored the relationship between IL-1β and radiosensitivity of cervical cancer using tumor specimens from patients with cervical cancer as well as in vivo and in vitro experiments. Summary of the invention

[0005] In view of the above technical problems in the prior art, the present invention provides the use of anakinra, an IL-1β receptor inhibitor, in the preparation of a sensitizer for improving the radiotherapy effect of cervical cancer. The use is intended to solve the technical problem in the prior art that radiotherapy for cervical cancer has a poor therapeutic effect.

[0006] The present invention provides the use of anakinra, an IL-1β receptor inhibitor, in preparing a sensitizer for improving the radiotherapy effect of cervical cancer.

[0007] Furthermore, it also contains a pharmaceutically acceptable carrier or excipient.

[0008] Furthermore, the dosage form of the sensitizer is tablets, capsules, granules, oral liquids, sustained-release preparations, controlled-release preparations, nano preparations or injections.

[0009] The present invention also provides a sensitizer for improving the radiotherapy effect of cervical cancer, which contains an effective dose of IL-1β receptor inhibitor anakinra.

[0010] Specifically, the anakinra is a commercially available product, and its structural formula is:

[0011]

[0012] Furthermore, it also contains a pharmaceutically acceptable carrier or excipient.

[0013] Furthermore, the dosage form of the sensitizer is tablets, capsules, granules, oral liquids, sustained-release preparations, controlled-release preparations, nano preparations or injections.

[0014] Compared with the prior art, the technical effect of the present invention is positive and obvious. When used in combination with radiotherapy, the IL-1β receptor inhibitor anakinra significantly enhances the ability to reduce the migration and invasion of cervical cancer tumor cells and plays a sensitizing role. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The relationship between IL-1R1 expression in tumor specimens of cervical cancer patients and patient prognosis was shown.

[0016] Figure 2 The killing effect of IL-1β pathway inhibitors on tumor cells was verified in vitro.

[0017] Figure 3 It was shown that anakinra significantly reduced the migration and invasion ability of tumor cells, and when used in combination with radiotherapy, it significantly enhanced the ability to reduce the migration and invasion ability of tumor cells.

[0018] Figure 4 The radiosensitizing effect of IL-1β signaling pathway inhibitors was verified in vivo. DETAILED DESCRIPTION

[0019] Example 1 Relationship between IL-1R1 (interleukin 1β receptor 1) expression in tumor specimens of cervical cancer patients and patient prognosis

[0020] We collected tumor tissue biopsy specimens before and after 10 rounds of radiotherapy from 15 patients with locally advanced cervical cancer who received radical chemoradiotherapy. Among them, 5 patients had no recurrence, 5 patients had distant metastasis, and 5 patients had local recurrence. Tumor tissue specimens were subjected to immunofluorescence staining to analyze the correlation between the expression of IL1R1, the main receptor of the proinflammatory cytokine IL-1β, and the outcome (recurrence or not). Multiple immunofluorescence staining was performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissue sections, mainly using antibodies against IL-1R1 (abcam, ab106278). Tissue sections were dewaxed, rehydrated, and antigen retrieval was performed at high temperature using citrate buffer (pH 6.0). To block nonspecific binding, sections were treated with 5% bovine serum albumin (BSA) for 1 hour and then incubated with primary antibodies against the protein of interest at 4°C overnight. The next day, sections were washed and incubated with fluorescein-labeled secondary antibodies at room temperature for 1 hour. Nuclei were counterstained with DAPI, and slides were mounted using antifade medium before capturing fluorescent signals.

[0021] The results of the analysis based on the intensity of expression showed that the expression of IL-1R1 in patients with local or distant metastasis recurrence was significantly higher than that in the non-recurrence group (see Figure 1 A), suggesting that IL-1R1 may be associated with radiosensitivity.

[0022] In addition, we also conducted a correlation analysis between IL-1β expression and overall survival in patients with cervical cancer (TCGA-CESC) in the TCGA database ( Figure 1 B), the results showed that the expression level of IL-1β was negatively correlated with the overall survival of patients, and the prognosis of patients with high IL-1β expression was relatively worse.

[0023] Example 2 Anakinra enhances the killing effect of radiotherapy on cervical cancer cells in vitro

[0024] We tested the killing effect of IL1R1 inhibitor anakinra (ChemEgen, C109584) on cervical cancer cells with or without radiation. To this end, we conducted a plate clone formation experiment.

[0025] 1. Cell preparation: Cervical cancer U14 cells (purchased from icell) in the logarithmic growth phase were digested with trypsin, resuspended in complete culture medium into a cell suspension, and accurately counted.

[0026] 2. Inoculation of cells: 700 cells / well were inoculated in each experimental group in a 6-well plate.

[0027] 3. Drug treatment: After cells adhered, 10 μg / mL of anakinra was added to the anakinra and anakinra+radiotherapy groups.

[0028] 4. Radiation treatment: The radiotherapy group and the anakinra + radiotherapy group were given radiotherapy, and the culture dishes were placed in Synregy TM A 1-cm-thick glass plate was placed under the culture dish on the accelerator treatment bed, and a single X-ray irradiation of 6 Gy was given.

[0029] 5. Cultivate cells: The treated cells are cultured continuously for 14 days or until the number of cells in most individual clones is greater than 50. Replace the culture medium and drugs every 3 days and observe the cell status.

[0030] 6. Fixation and staining: Fix with 4% paraformaldehyde for 30 minutes, then wash with PBS. Then stain with crystal violet stain for 20 minutes, then wash several times with PBS.

[0031] 7. Count clones: Count the number of clones containing more than 50 cells under a microscope, and take the average value of clones from three replicate wells for calculation. The clone formation rate = (average number of clones / number of single cells inoculated) × 100%.

[0032] The results showed that after treatment with anakinra and radiotherapy alone, the long-term survival of cervical cancer cells was significantly inhibited and the clone formation was significantly reduced. The inhibitory effect was further enhanced when anakinra was combined with radiotherapy ( Figure 2 A), suggesting the radiosensitizing effect of anakinra.

[0033] Example 3 Anakinra enhances the growth inhibition effect of radiotherapy on cervical cancer cells

[0034] We investigated the inhibitory effect of IL1R1 inhibitor anakinra (ChemEgen, C109584) on cervical cancer cells in vitro with and without irradiation and its radiosensitizing effect. To this end, we performed CCK-8 to evaluate the effect of anakinra on the proliferation of irradiated SiHa cells.

[0035] 1. Cell inoculation: 5000 U14 cervical cancer cell lines were inoculated into a 96-well plate, 100 μL of cell suspension was added to each well, and then pre-cultured in an incubator at 37° C. and 5% CO 2 for 24 hours.

[0036] 2. Drug treatment: According to the experimental design, anakinra (10 μg / mL) was added to the anakinra and anakinra + radiotherapy groups, and incubation continued under the same conditions for 24 hours.

[0037] 3. Radiotherapy: The anakinra + radiotherapy group and the radiotherapy group were irradiated with 6 Gy; the culture dish was placed in Synregy TM On the accelerator treatment bed, a 1cm thick glass plate was placed under the culture dish, and a single 6Gy X-ray was given. After the irradiation, the culture was continued for 24 hours.

[0038] 3.CCK-8 solution: After the incubation, add 10 μL of CCK-8 solution (abcam, UK, ab65475) to each well, mix gently, and continue to incubate in the incubator for 1-4 hours to allow WST-8 to be fully reduced by living cells.

[0039] 4. Detect absorbance: Use an enzyme-labeled instrument to measure the absorbance of each well at a wavelength of 450 nm, and set a reference wavelength (such as 630 nm) to reduce background interference.

[0040] 5. Data analysis: Calculate the average OD value of each well, analyze the changes in cell proliferation or toxicity by comparing with the control group, draw a dose-response curve, and calculate indicators such as the half-maximal inhibitory concentration (IC50) or cell proliferation rate.

[0041] Results: CCK8 cell proliferation experiments showed that radiotherapy and anakinra alone could inhibit the proliferation of tumor cells, and anakinra combined with radiotherapy further enhanced the ability of radiation to stimulate tumor cell proliferation ( Figure 2 B).

[0042] Example 4 Anakinra improves the inhibitory effect of radiotherapy on the migration and invasion of cervical cancer cells

[0043] To verify the effect of anakinra on the migration and invasion of cervical cancer cells, we performed a transwell experiment.

[0044] 1. Prepare cells: Take U14 cells with good growth status and starve them for 24 hours to synchronize the cell cycle and reduce the influence of proliferation on the experimental results;

[0045] 2. Prepare Transwell chamber (Costar, USA, #3422): According to the experimental design, choose whether to add Matrigel (Becton Dickinson Compary). The preparation and coating of Matrigel are carried out according to the instructions;

[0046] 3. Preparation of cell suspension: Digest and resuspend the prepared cells and adjust the cell concentration to an appropriate density using serum-free culture medium.

[0047] 4. Add induction factors: Add an appropriate amount of inducer, such as complete culture medium, to the lower chamber;

[0048] 5. Inoculate cells: Add the cell suspension into the upper chamber of the Transwell.

[0049] 6. Culture: Place the Transwell chamber back into the incubator and culture for 48 hours.

[0050] 7. Fixation and staining: After the experiment, fix the cells with 4% paraformaldehyde for 30 minutes, discard the fixative and wash the chamber once with PBS; then stain with crystal violet for 10 minutes, and then wash three times with PBS to facilitate observation under a microscope.

[0051] 8. Migration / invasion cell counting: Randomly select 5 fields of view under the microscope and count the number of cells that migrate to the underside of the membrane.

[0052] 9. Data analysis: Based on the counting results, calculate the percentage of cell migration or invasion or related statistical data.

[0053] Results: It was found that both radiotherapy and anakinra significantly reduced the migration and invasion ability of tumor cells, and this effect was further enhanced after anakinra was combined with radiotherapy ( Figure 3 ).

[0054] Example 5: Radiosensitization effect of anakinra on cervical cancer tumors (in vivo)

[0055] In order to verify the radiosensitizing effect of anakinra in vivo, we used a subcutaneous transplant tumor in vivo irradiation model. In the BALB / c nude mouse model, SiHA cells formed subcutaneous tumors and were given radiotherapy or anakinra to observe their tumor-killing effects in vivo.

[0056] Subcutaneous tumor formation in mice with cervical cancer cell lines:

[0057] Subcutaneous tumors were grown in the inguinal region of 5-6 week-old female BALB / c nude mice, and 1×10 6 Tumor cells SiHA were suspended in Matrigel and injected subcutaneously in the groin area until the tumor grew to 75-100 mm 3 After treatment (including intraperitoneal injection of anakinra (ChemEgen, C109584) 1.5 mg / kg / day for 5 consecutive days, local irradiation 12 Gy, and intraperitoneal injection of anakinra + 12 Gy irradiation), the patients were observed for 1 month after treatment or until the tumor volume was >2 cm 3 The mice were killed, the tumors were removed, and the tumor diameter was measured and recorded every 3 days after the tumor grew. The mice were killed and the tumors were removed and the tumor volume and weight were recorded for subsequent analysis. There were 5 mice in each of the 4 experimental groups. All animal experimental procedures were approved by the Ethics Committee of Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine.

[0058] Irradiation of subcutaneous tumors:

[0059] After anesthesia, tumor-bearing BALB / c nude mice were placed in a supine position to expose the tumor site (inguinal region) and fixed on a fixing board. TMThe accelerator uses 6MV X-rays and designs the anterior and posterior irradiation fields according to the tumor volume to give conformal irradiation to the tumor area with a dose of 8Gy in a single irradiation.

[0060] The results showed that after radiotherapy or anakinra treatment alone, the growth rate of the tumor slowed down, and the combination of radiotherapy and anakinra had a stronger ability to kill tumors (see Figure 4 ).

Claims

1. Use of anakinra, an inhibitor of IL-1β receptor, in the preparation of a sensitizer for improving the radiotherapy effect of cervical cancer.

2. The use according to claim 1, characterized in that It also contains a pharmaceutically acceptable carrier or excipient.

3. The use according to claim 1, characterized in that The dosage form of the sensitizer is tablet, capsule, granule, oral solution, sustained-release preparation, controlled-release preparation, nano preparation or injection.

4. A sensitizer for improving the radiotherapy effect of cervical cancer, characterized in that: Contains an effective dose of anakinra, an inhibitor of IL-1β receptor.

5. A sensitizer for improving the radiotherapy effect of cervical cancer according to claim 4, characterized in that: It also contains a pharmaceutically acceptable carrier or excipient.

6. A sensitizer for improving the radiotherapy effect of cervical cancer according to claim 4, characterized in that: The dosage form of the sensitizer is tablet, capsule, granule, oral solution, sustained-release preparation, controlled-release preparation, nano preparation or injection.

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