CN114432442A - Epirubicin as125I sensitizers - Google Patents

Abstract

The invention belongs to the field of biomedicine, and provides epirubicin as a new compound¹²⁵I use of a sensitizer for radiotherapy of malignant tumors. The malignant tumor is selected from pancreatic cancer, lung cancer or liver cancer. The present invention provides epirubicin as¹²⁵The application of the I radiotherapy sensitizer is verified by experiments compared with the I radiotherapy sensitizer¹²⁵I Radioactive particle Single action, epirubicin and¹²⁵the combined action of the I radioactive particles can effectively enhance the proliferation multiple of target cells, enhance the capacity of resisting invasion and migration, and also enhance the capacity of promoting apoptosis. In clinical applications, for the presence¹²⁵Patients with radiation resistance, the combined use of epirubicin can enhance¹²⁵The therapeutic effect of I; and is¹²⁵The use of the I radiotherapy sensitizer can be reduced¹²⁵Use of IAnd clinical treatment dose, thereby reducing the incidence of radiation therapy related complications. Thereby improving the treatment effect and the life quality of the patients.

Description

Epirubicin as a sensitizer for 125I

technical field

The invention belongs to the field of biomedicine, and particularly relates to epirubicin enhancing the radiation sensitivity of hepatocellular carcinoma to ¹²⁵ I radioactive particles.

Background technique

Hepatocellular carcinoma is the third most common malignancy in the digestive system and one of the leading causes of cancer-related deaths. The prognosis of hepatocellular carcinoma is generally poor, the 5-year survival rate is lower than that of prostate cancer, breast cancer and lung cancer, so it has a serious impact on the quality of life of patients. The incidence of other cancers has declined over the past decade, but the incidence of hepatocellular carcinoma has continued to rise, especially among women, increasing by 2.1% per year. The use and promotion of ^125I radioactive seed implantation technology has improved the safety and efficacy of clinical treatment of advanced hepatocellular carcinoma, especially when combined with other chemotherapeutic drugs (such as lobaplatin) and traditional Chinese medicine preparations, the prognosis has been significantly improved. However, the specific mechanism of the effect of ¹²⁵ I radioactive particles on hepatocellular carcinoma cells remains to be studied. In addition, whether chemotherapeutic drugs can enhance the radiosensitivity of hepatocellular carcinoma to ¹²⁵ I radioactive particles is still unclear. Exploring the mechanism of action and new targets of ¹²⁵ I radioactive particles will help the better clinical application of ¹²⁵ I radioactive particles. , and provide new treatment ideas for hepatocellular carcinoma.

It is well known that any abnormality or alteration of signaling pathways may lead to tumor formation, and STAT1, as a signal transducer and activator of transcription, can play an important role in many cytokine-induced signal transduction. STAT1 is mainly involved in antiviral and antibacterial responses, inhibits tumor growth, and induces apoptosis by regulating anti-apoptotic genes such as Bcl-XL, caspases, and Bax. Studies have shown that there are 12 abnormal signaling pathways that are mainly involved in the occurrence and development of cancer, and the JAK/STAT1 signaling pathway is one of them. The JAK/STAT1 signaling pathway is closely related to the occurrence of tumors and plays a regulatory role in the metastasis of solid tumors.

Epirubicin (EPI) is a traditional anthracycline chemotherapy drug with few side effects, so it is widely used in the clinical treatment of breast cancer, liver cancer, gastric cancer and non-small cell lung cancer. Epirubicin inhibits the proliferation of tumor cells by interfering with the DNA transcription process and inhibiting the synthesis of DNA and messenger RNA. In clinical practice, epirubicin is often used in combination with other drugs or carriers, such as trastuzumab, paclitaxel, polymer micelles, and hyaluronic acid, to enhance the antitumor effect. However, the effect of epirubicin in combination with ¹²⁵ I radioactive seeds in the treatment of hepatocellular carcinoma is still unknown.

SUMMARY OF THE INVENTION

In view of the problems in the prior art, the present invention provides a new use of epirubicin, which can enhance the radiation sensitivity of hepatocellular carcinoma to ¹²⁵ I radioactive particles, enhance the anti-proliferation of ¹²⁵ I, inhibit cell migration and invasion, and can promote apoptosis.

In order to achieve the above objects, the present invention adopts the following technical solutions.

Use of epirubicin as a sensitizer for ^125I radiotherapy for malignant tumors.

The malignant tumor is selected from pancreatic cancer, lung cancer or liver cancer.

The present invention has the following advantages:

The invention provides the use of epirubicin as a sensitizer for ¹²⁵ I radiotherapy. It is verified through experiments that compared with the single action of ¹²⁵ I radioactive particles, the combined action of epirubicin and ¹²⁵ I radioactive particles can effectively enhance the target cells. Proliferation times, enhance the ability of anti-invasion and migration, but also enhance its ability to promote apoptosis. In clinical application, for patients with ¹²⁵ I radiation resistance, the combined use of epirubicin can enhance the therapeutic effect of ¹²⁵ I; and the use of ¹²⁵ I radiotherapy sensitizers can reduce the activity of ¹²⁵ I and clinical treatment dose, thereby reducing the incidence of radiation therapy-related complications. Thereby improving the treatment effect and quality of life of patients.

Description of drawings

Figure 1 is the logarithm of epirubicin concentration-HCC cell inhibition rate curve;

Figure 2 shows the effects of different treatments on cell proliferation;

Figure 3 shows the effects of different treatments on the cell cycle;

Figure 4 is the pictures of cell migration (A) and invasion (B) under different treatments;

Figure 5 shows the effects of different treatments on apoptosis;

Figure 6 is an analysis of differentially expressed proteins in cells treated with ¹²⁵ I;

Figure 7 is the Western Blot diagram and relative content of differentially expressed proteins and corresponding phosphorylated proteins in cells treated with ¹²⁵ I;

Figure 8 is the Western Blot diagram and relative content of JAK and STAT1 proteins and their phosphorylated proteins in SMMC-7721 cells treated with different treatments;

Figure 9 is a Western Blot of STAT1 protein of NC-RNAi SMMC-7721 and STAT1-RNAi SMMC-7721;

Figure 10 is a comparison of the volume (A) and weight (B) of xenografted tumor tissues with different treatments;

Among them, * means p<0.05, ** means p<0.01, and *** means p<0.001.

Detailed ways

The present invention will be further described below with reference to the embodiments and the accompanying drawings, but the present invention is not limited by the following embodiments.

Example 1 In vitro sensitization test of epirubicin to ¹²⁵ I

Hepatocellular carcinoma cell lines SMMC7721 and HepG2 were purchased from Zhongqiao Xinzhou Biotechnology Company. SMMC7721 cells were cultured in RPMI 1640 (Corning, Inc.) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. HepG2 cells were cultured in Dulbecco's modified Eagle's medium (Corning, Inc.) supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C and 5% carbon dioxide.

1. Screening of epirubicin sensitizing concentration

Sensitivity of SMMC-7721 and HepG2 cells to epirubicin (Zhejiang Hisun Pharmaceutical Co., Ltd.) drug cytotoxicity was assessed using the CCK-8 kit (Dojindo, Kumamoto): cells cultured in 96-well plates at concentrations Epirubicin was treated with 0-5 μg/mL for 72 hours. The median inhibitory concentration ( _IC50 ) was calculated using GraphPad Prism 9.0, and 10% of the _IC50 was chosen as the epirubicin sensitizing concentration. The logarithmic-inhibitory rate curves of different epirubicin concentrations on two types of HCC cells are shown in Figure 1. After calculation, the sensitizing concentration for SMMC-7721 cells is 0.023 μg/mL, and the sensitizing concentration for HepG2 cells is 0.02 μg/mL.

2. The effect of epirubicin on the anti-proliferation of ¹²⁵ I

The cells were treated with epirubicin at the sensitizing concentration of each cell line, combined with ¹²⁵ I particle in vitro irradiation model. The cells were divided into: control group, single EPI treatment group, single ¹²⁵ I treatment group, and EPI combined with ¹²⁵ I treatment group. The sensitizing concentration of EPI is prepared from the cell culture medium. When the normal medium of the cells is replaced with the medium containing EPI, the cells are then placed in the ¹²⁵ I particle in vitro irradiation to start the irradiation. The irradiation time is 72 hours. The activity level was 3.0 mCi and the dose rate was 3.412 cGy/h.

Cells with different treatments were prepared into a cell suspension with a concentration of 3×10 ³ cells/mL, and put into a 96-well plate, with 200 μL per well. Add CCK-8 reagent at 0h, 24h, 48h and 72h, incubate at 37°C for 2h, then use a microplate reader to measure the absorbance value at 450nm, and calculate the cell proliferation fold according to the following formula: Proliferation fold = absorbance value at different treatment times /0 hour absorbance value. The results are shown in Figure 2: Compared with the treatment group treated with ¹²⁵ I radioactive particles alone, the combined application of epirubicin and ¹²⁵ I radioactive particles can significantly reduce HepG2 (**, p<0.01) and SMMC7721 cells (** *, p<0.001), which indicated that the combined application of epirubicin and ¹²⁵ I radioactive particles could better inhibit the proliferation of HepG2 and SMMC7721 cells.

Cells were cultured in 6-well plates (2×10 ⁵ cells/well), and cells were treated with sensitizing concentrations of EPI, ¹²⁵ I particles, and a combination of the two, respectively, and then collected and fixed with pre-chilled 70% ethanol for 1 hour. , then stained with PI and RNase A (BDBiosciences) and then detected using flow cytometry (Beckman). The results are shown in Figures 3A-B: the degree of cell cycle arrest in the G2/M phase in the combined treatment group of EPI and ¹²⁵ I radioactive particles was greater than that of the group treated with either ¹²⁵ I radioactive particles or EPI alone.

3. The effect of epirubicin on the anti-invasion and migration of ¹²⁵ I

The invasive and migratory abilities of cells were assessed by Transwell assay. For invasion assays, Matrigel (BD Biosciences) and Opti-MEM (Gibco) were usually mixed at a ratio of 1:5, and 50 μL of the diluted Matrigel was then spread into Transwell chambers (BD Biosciences) and incubated at 37°C Incubate for 1h. Then, 200 μL of cell suspension (2×10 ⁵ cells/mL) was added to the Transwell chamber and incubated at 37° C. for 24 h. Finally, cells were fixed with 4% paraformaldehyde, stained with crystal violet for 30 min, and then slowly washed twice with water. After washing, the cells were photographed with an inverted microscope (Olympus CKX53, Tokyo, Japan) and counted with Image J software. The migration assay is roughly the same as the invasion assay, but does not require matrigel to be applied to the Transwell chamber. The results are shown in Figure 4: the number of cells in the migration and invasion stages in the combined treatment group was significantly lower than that in the epirubicin and ¹²⁵ I radioactive seed monotherapy group.

4. The effect of epirubicin on ¹²⁵ I-promoted apoptosis

Cells (2×10 ⁵ cells/well) were cultured in 6-well plates, and the cells were collected after treatment with sensitizing concentrations of EPI, ¹²⁵ I particles, and a combination of the two. Cells were resuspended in binding buffer, and then 5 μL propidium iodide (PI) and Annexin V-APC (Elabscience) or Annexin V-FITC (BD Biosciences) were added for staining, and flow cytometry (Beckman) was used for detection in the dark for 20 min. Annexin V-FITC/PI assay was used to evaluate the effect of epirubicin on the apoptosis of HCC cells induced by ¹²⁵ I radioactive particles. The results are shown in Figure 5: the apoptosis rate of the combined treatment group was significantly higher than that of epirubicin or ¹²⁵ I radioactive seed monotherapy group. This indicated that epirubicin promoted the apoptosis induced by ¹²⁵ I radioactive particles.

Example 2 The sensitization mechanism of epirubicin to ¹²⁵ I

1. Analysis of differentially expressed proteins in cells treated with ¹²⁵ I

SMMC-7721 cells were irradiated with ¹²⁵ I radioactive particles at a dose of 4Gy, and no irradiation was used as a negative control. Total protein was extracted with RIPA lysis buffer (TIANGEN), and then sent to Jiyun (Shanghai) Biotechnology Co., Ltd. for isotope labeling quantification. Signal pathway and biological function analysis were performed with IPA software. Significant up-regulation was determined based on a fold change >1.2 and a P value <0.05 in the mean value of the labeling between the ^125I radioactive particle-treated group and the control group. The results are shown in Figure 6: Figure 6A shows a heat map of iTRAQ labeling, dividing HCC cells into ¹²⁵ I radioactive particle-treated and control groups to quantify the difference in protein expression after ¹²⁵ I radioactive particle treatment. Figure 6B is a volcano plot, there are a total of 207 differentially expressed proteins in HCC cells treated with ¹²⁵ I radioactive particles, including 119 up-regulated proteins and 88 down-regulated proteins, which are significantly different from those in the control group. As shown in Figure 6C, the expression level of STAT1 was significantly different between the ¹²⁵ I radioactive particle-treated group and the control group (***, p<0.001). This indicates that ¹²⁵ I radioactive particles have effects on cell apoptosis, survival and protein synthesis, as well as on the disease and functional status of HCC cells. These findings suggest that ¹²⁵ I radioactive particles have a specific role in the upregulation of the JAK/STAT1 signaling pathway, which is involved in the regulation of cellular states, including survival and intracellular protein synthesis (Fig. 6D).

2. Western blot of differentially expressed proteins and corresponding phosphorylated proteins in cells treated with ¹²⁵ I

SMMC-7721 cells were irradiated with ¹²⁵ I radioactive particles for 48h and 72h, and no irradiation was used as a negative control. Cells were washed twice with 1x PBS. Then, cells were lysed with RIPA lysis buffer for 20 min and centrifuged (12,000 rpm, 10 min, 4°C) to obtain the supernatant, protein loading buffer was added to the supernatant, and the protein was denatured in a metal bath at 95°C, and then 10 wt. % SDS-PAGE for electrophoretic analysis of protein samples (10 μL/well). Then, the proteins were transferred to PVDF membranes (Millipore). Subsequently, the blots were blocked with 5% nonfat dry milk for 1 h and incubated at 4°C with primary antibodies (JAK primary antibody (ab133666), p-JAK primary antibody (ab138005), STAT1 primary antibody (ab109320) or p-STAT1 primary antibody (ab109461)) overnight. Then, Western blots were washed with 1×TBST buffer and incubated with the corresponding secondary antibodies (goat anti-rabbit IgG (GenScript) or goat anti-mouse IgG (GenScript)) for 1 h at room temperature, with GAPDH protein as a reference. The results are shown in Figure 7: The expression of p-JAK and p-STAT1 proteins in the ¹²⁵ I radioactive particle-treated group increased in a dose-dependent manner.

3. The effect of epirubicin and ¹²⁵ I combined treatment on the protein content of JAK and STAT1

By treating SMMC-7721 cells with ¹²⁵ I radioactive particles alone, epirubicin, or a combination of the two, we investigated whether epirubicin affects cell proliferation and induced apoptosis via the JAK-STAT1 pathway ^. , the method of each test is carried out according to Example 1.

The relative contents of JAK and STAT1 proteins and their phosphorylated proteins in SMMC-7721 cells after different treatments were detected by Western blot. The results are shown in Figure 8: Although the total amount of JAK and STAT1 proteins did not change significantly compared with the monotherapy group, the phosphorylated forms of these two proteins were significantly increased when combined treatment.

Example 3 In vivo sensitization test of epirubicin to ¹²⁵ I

1. Construction of STAT1 knockdown SMMC-7721 cell line

Synthesize NC-siRNA (negative control) and STAT1-siRNA respectively, package lentivirus (Gene Chem), and transfect the two lentiviruses into SMMC7721 cells at MOI=10: complete medium, HiTransGA A (Gene Chem) and lentivirus Mixed, then added to a 6-well plate for transfection. After 16 hours of transfection, the old medium was replaced with a complete medium containing 2.5 μg/mL puromycin for 48 hours, and then the concentration of puromycin was gradually reduced to Complete screening. The knockout efficiency was confirmed by Western blot after 72 hours. Figure 9 shows that the STAT1 protein in the cells transfected with STAT1-siRNA was significantly reduced compared with the cells transfected with NC-siRNA. It can be seen that the SMMC-7721 cell line with knockdown of the STAT1 gene was successfully constructed and named STAT1-RNAi SMMC -7721.

2. Construction of Xenograft Tumor Model

BALB/C male nude mice were used to construct tumor models by xenografting, and all animals were fed and handled according to the standards of Shandong University Animal Care and Use Committee. SMMC-7721 cells or STAT1-RNAi SMMC-7721 cells were prepared into sterile fresh cell suspensions (1×10 ⁶ cells/mL) with 0.9% normal saline, and injected into the left hind limb of nude mice (0.2 mL/mice) . Tumor volume was measured using a vernier caliper every 3 days thereafter, and the tumor volume was calculated as follows: length (mm) × width (mm) × height (mm) × 0.5.

3. In vivo sensitization test of epirubicin to ¹²⁵ I

The nude mice successfully modeled in Example 1 were used for relevant experiments when the tumor volume reached about 400 mm ³ . The skin at the tumor site was disinfected with iodine disinfectant, anesthetized with lidocaine, and then punctured at the center of the tumor with an 18-G needle. Then, ¹²⁵ I radioactive particles (0.4 mCi) or epirubicin solution (2 mg/kg) and ¹²⁵ I radioactive particles were co-implanted into the tumor using a particle implantation device. After implantation, apply pressure with a sterile cotton swab to stop the bleeding. The mice were kept in a single cage until the end of the experiment, the tumor volume was measured with a vernier caliper, the mice were sacrificed, and the tumors were excised and weighed.

The tumor volume and weight of each treated mice are shown in Figure 10. Compared with the control group, ¹²⁵ I treatment alone or epirubicin and ¹²⁵ I combined treatment can significantly reduce the tumor volume and weight; epirubicin and The inhibitory effect of ¹²⁵ I combined treatment was significantly higher than that of ¹²⁵ I particles alone, which indicated that epirubicin had a sensitizing effect on ¹²⁵ I particles. According to the instructions of epirubicin, the dose for humans is 60-90 mg/m ² , and the dose for mice is 13.4-20.1 mg/kg after conversion based on the doses of humans and mice, and the dose used in the experiment is 2mg/kg, far less than the dose used, basically has no effect when used alone, so it is the effect of a sensitizer when used in combination with ¹²⁵ I particles.

Compared with the SMMC-7721 cell line, the inhibitory effects of ¹²⁵ I treatment or epirubicin and ¹²⁵ I combined treatment on tumor volume and weight were significantly decreased after knockdown of STAT1 gene. This indicates that knockdown of STAT1 will attenuate the anticancer effect of ¹²⁵ I radioactive particles and the sensitizing effect of epirubicin on ¹²⁵ I radioactive particles.

Claims (2)

1. Epirubicin as a new compound¹²⁵I use of a sensitizer for radiotherapy of malignant tumors.

2. Use according to claim 1, characterized in that said malignant tumor is selected from pancreatic cancer, lung cancer or liver cancer.

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