Use of disulfiram in the manufacture of a radiosensitizer

Abstract

This invention discloses the application of disulfiram or its pharmaceutical salt in the preparation of radiosensitizers, wherein the radiosensitizer refers to a radiosensitizer for malignant tumors, specifically osteosarcoma. The radiation involved in the radiosensitizer is selected from alpha, beta, gamma, or X-ray radiation. This invention is the first to discover and confirm a novel use of disulfiram in enhancing the radiosensitivity of malignant tumors, significantly inhibiting the proliferation of osteosarcoma cells after radiotherapy and promoting cell death in vitro. It also demonstrates outstanding efficacy in inhibiting the growth of subcutaneously implanted osteosarcoma in mice after radiotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to the application of disulfiram in the preparation of radiosensitizers. Background Technology

[0002] Osteosarcoma is a highly malignant tumor derived from mesenchymal stem cells and is the most common primary malignant bone tumor, predominantly affecting adolescents. Since the 1980s, when neoadjuvant chemotherapy, surgery, and postoperative chemotherapy became the preferred treatment for osteosarcoma, the prognosis of osteosarcoma has significantly improved. However, in the last 20 years, the prognosis of osteosarcoma has plateaued again, without any major breakthroughs. The lack of sensitivity to radiotherapy and chemotherapy, and the absence of effective targeted therapies, are important reasons for this phenomenon. Radiotherapy, as an important local treatment for malignant tumors, plays a crucial role in various radiosensitive tumors, including head and neck malignancies and cervical cancer. However, due to the low sensitivity of osteosarcoma cells to radiotherapy, and the severe side effects associated with high-dose radiation, its application in refractory osteosarcoma is limited. Therefore, after improving the radiosensitivity of osteosarcoma through various adjuvant methods, preoperative adjuvant radiotherapy can be used to reduce surgical margins, and it can also be used for further treatment of patients with positive surgical margins after osteosarcoma surgery. Furthermore, for advanced osteosarcoma cases where surgery is not feasible, chemotherapy and targeted therapy can be used in conjunction to increase the effectiveness of palliative treatment. Overall, increasing the sensitivity of osteosarcoma to radiotherapy has extremely high clinical value.

[0003] Current research on the radiosensitization mechanism involves multiple pathways, including promoting reactive oxygen species (ROS) generation, inhibiting DNA damage repair, promoting apoptosis, and regulating the cell cycle. Interestingly, although some studies have shown that cell cycle inhibition provides a time window for DNA damage repair after radiotherapy, sustained cell cycle arrest can also induce apoptosis and senescence, thereby promoting cell death after radiotherapy. CDK4 / 6 proteins mainly control the activation of the G1 / S phase checkpoint in the cell cycle and have been found to be highly expressed in various cancers. Studies have shown that inhibiting CDK4 / 6 proteins can not only lead to G1 cycle arrest but also reduce radiotherapy resistance in esophageal cancer and head and neck squamous cell carcinoma. This suggests that targeting CDK4 / 6 to inhibit cell cycle progression could be an effective radiosensitization method. The p53 / p21 pathway and activation of CDKN2C can both exert inhibitory effects on CDK4 / 6 proteins.

[0004] Disulfiram (DSF) is a clinically approved drug for alcohol withdrawal by the U.S. Food and Drug Administration (FDA) and has a good biocompatibility. Recent studies suggest that DSF has a positive effect on reducing acute lung and intestinal injury caused by radiation (IR), with specific mechanisms including inhibiting pyroptosis and promoting DNA damage repair. Furthermore, some researchers have confirmed that therapeutic doses of DSF have no significant toxic effects on bone marrow in vitro and in vivo. Although DSF exhibits radiation-protective effects in benign tissues, some studies have revealed that DSF may act as a radiosensitizer in some malignant tumors.

[0005] An SCI article with PMID 30121967 indicates that disulfiram can reduce the radioresistance of glioblastoma cells by inhibiting the repair of DNA damage after X-ray irradiation and inducing apoptosis (in vitro effects only).

[0006] An SCI article with PMID 33116640 points out that disulfiram can promote the death rate of glioblastoma cells after X-ray irradiation, thereby reducing the radioresistance of the tumor, but does not further explore its mechanism (only the in vitro effects were studied).

[0007] An SCI article with PMID 33671083 indicates that disulfiram can promote the death of head and neck squamous cell carcinoma cells by reducing G2 / M phase arrest after X-ray irradiation, thereby reducing the radioresistance of this tumor (in vitro effects only).

[0008] An SCI article with PMID 34631519 points out that disulfiram can reduce the radioresistance of pancreatic cancer in vitro by inducing DNA double-strand breaks, apoptosis, and cell cycle arrest in pancreatic cancer cells after X-ray irradiation, and this effect has been verified in mouse tumor implantation.

[0009] An SCI article with PMID 28340172 points out that disulfiram can reduce the radioresistance of atypical teratogenic / rhabdomyosarcoma tumors in vitro by inducing DNA double-strand breaks, apoptosis, autophagy, and cell cycle arrest in pancreatic cancer cells after X-ray irradiation, and this effect has been verified in mouse tumor implantation.

[0010] Patent application CN115227688A discloses the application of disulfiram in enhancing the efficacy of paclitaxel in the treatment of lung cancer. Specifically, it describes the use of a disulfiram-paclitaxel combination to enhance the therapeutic effect of paclitaxel in lung cancer, providing a novel use for disulfiram. Paclitaxel, a chemotherapy drug, exerts its anti-tumor effect by inhibiting tumor cell mitosis and slowing tumor cell proliferation. The combined use of disulfiram enhances the efficacy of paclitaxel in treating lung cancer. Disulfiram is an inhibitor of acetaldehyde dehydrogenase 1A1; the combination of disulfiram and paclitaxel can increase the sensitivity of lung cancer cells to paclitaxel treatment. This improves the efficacy of paclitaxel chemotherapy and provides a highly effective chemotherapy sensitizer for the clinical treatment of lung cancer.

[0011] Patent application CN114146073A discloses the application of disulfiram in enhancing immune responses, particularly the application of DSF in enhancing anti-tumor immune responses. Through extensive experimental research, the inventors discovered that DSF can promote CD8... + T cell receptor activation in T cells binds to and activates LCK, directly activating T cells and metabolic reprogramming, significantly promoting the production of IFN-γ and TNF-α, and enhancing CD8. + T-cell immune response. Animal experiments showed that tumor-infiltrating CD8+ cells were more pronounced in the DSF treatment group. + The proportion and number of T cells are both increased, which can significantly inhibit tumor growth (such as colon cancer, melanoma, breast cancer, etc.) and prolong survival. Summary of the Invention

[0012] The purpose of this invention is to provide an application of disulfiram in the preparation of radiosensitizers.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] In a first aspect, the present invention provides the use of disulfiram or a pharmaceutical salt thereof in the preparation of a radiosensitizer.

[0015] The structure of the disulfiram is shown below:

[0016]

[0017] The radiosensitizer mentioned refers to a radiosensitizer for malignant tumors.

[0018] The malignant tumor referred to is osteosarcoma.

[0019] The radiation involved in the radiosensitizer is selected from alpha, beta, gamma, or X-ray radiation.

[0020] The pharmaceutical salt is an acid addition salt formed by disulfiram with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0021] The present invention relates to the application of disulfiram or its pharmaceutical salt in the preparation of radiosensitizers, wherein disulfiram or its pharmaceutical salt is used as a single active ingredient.

[0022] The present invention also provides a pharmaceutical preparation made from disulfiram or a pharmaceutical salt thereof, and medically acceptable excipients or other pharmaceutically acceptable ingredients.

[0023] The dosage forms of the pharmaceutical preparations include injections, capsules, tablets, granules, pills, microcapsule preparations, microsphere preparations, and nano-preparations.

[0024] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0025] This invention is the first to discover and demonstrate the novel use of disulfiram in enhancing the radiosensitivity of malignant tumors, significantly inhibiting the proliferation of osteosarcoma cells after radiotherapy and promoting cell death in vitro. It also shows outstanding efficacy in inhibiting the growth of subcutaneously implanted osteosarcoma in mice after radiotherapy.

[0026] In this invention, DSF's different effects in regulating the radiosensitivity of benign and malignant tissues allow it to minimize radiation damage to adjacent benign tissues while increasing tumor killing, making it particularly suitable as a radiosensitizing drug for malignant tumor radiotherapy.

[0027] This invention reveals that the combined application of DSF and IR in vitro, compared to IR alone, increases cell viability inhibition by over 28.7%, apoptosis inhibition by over 72%, and proliferation inhibition by over 43.3% (based on colony formation). In vivo, the combined application of DSF and IR, compared to IR alone, increases tumor growth inhibition by 84%, confirming that DSF has a radiosensitizing effect on osteosarcoma in both in vitro and in vivo. Furthermore, this effect is confirmed to be based on DSF enhancing the activation of apoptosis pathways after radiotherapy and inhibiting cell cycle progression after radiotherapy. Attached Figure Description

[0028] Figure 1 This is a schematic diagram showing the growth curve and specimen photograph of a mouse after subcutaneous implantation of osteosarcoma following radiotherapy.

[0029] Figure 2 This is a schematic diagram of Ki67 immunohistochemical staining after radiotherapy for subcutaneous osteosarcoma implantation in mice.

[0030] Figure 3 This is a schematic diagram of Tunel staining after radiotherapy for subcutaneous implantation of osteosarcoma in mice.

[0031] Figure 4 IC50 of DSF in different osteosarcoma cell lines 50 Schematic diagram of the curve.

[0032] Figure 5 A statistical diagram illustrating the detection of cell viability of different osteosarcoma cell lines using CCK8.

[0033] Figure 6 This diagram illustrates the clonogenic patterns and statistical representations of different osteosarcoma cell lines.

[0034] Figure 7 Scratch assay diagrams and statistical schematics of different osteosarcoma cell lines.

[0035] Figure 8 The image shows the results of apoptosis detection by Annexin V / PI flow cytometry for different osteosarcoma cell lines and a statistical diagram.

[0036] Figure 9 This is a Western blotting image of apoptosis-related proteins BAX, BCL2, Caspase-3, and Cleaved Caspase-3, along with a statistical diagram of their relative protein expression levels.

[0037] Figure 10 A bar chart showing the cell cycle detection results for different osteosarcoma cell lines.

[0038] Figure 11 Western blotting plot showing the changing trends of cycle-related proteins P53, P21, CDKN2C, CDK4, CDK6, and Cyclin D1 within 24 hours after IR. Detailed Implementation

[0039] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0040] In this embodiment of the invention, male wild-type BALB / c mice (6 weeks old) were purchased from Jihui Biotechnology (Shanghai). All mice were housed in the Specific Pathogen-Free (SPF) Laboratory of the Animal Experiment Center of Naval Medical University (Shanghai) of the Chinese People's Liberation Army.

[0041] The irradiation (IR) used in this invention is all gamma rays, developed by the Naval Medical University. 60 Co radiation source implementation.

[0042] Experimental Example 1

[0043] Disulfiram's radiosensitizing effect on subcutaneously implanted osteosarcoma in mice

[0044] Twenty-four 6-week-old male BALB / c mice were randomly divided into four groups: DMSO group (intraperitoneal injection of DMSO, no irradiation), DSF group (intraperitoneal injection of disulfiram, no irradiation), DMSO+IR group (intraperitoneal injection of DMSO, irradiation), and DSF+IR group (intraperitoneal injection of DSF, irradiation). Note: DMSO is an abbreviation for dimethyl sulfoxide, the solvent for DSF.

[0045] Steps for modeling osteosarcoma subcutaneously in mice: 1. Digest and count HOS osteosarcoma cells, take a sufficient amount of HOS cells, centrifuge to obtain the precipitate, and discard the supernatant (calculate 2 million HOS osteosarcoma cells per mouse, and prepare 120% of the calculated amount); 2. Resuspend the cells in PBS at a ratio of 2 million cells / 100ul PBS; 3. Inject 100ul of cell suspension subcutaneously into the left inguinal region of the aforementioned mice; 4. Observe and measure the growth of subcutaneous tumors in vitro.

[0046] The drug administration time points for mice described below are 24 hours and 2 hours before the planned irradiation time (a total of 2 administrations were given before irradiation to maintain an effective drug concentration).

[0047] DSF group: Disulfiram was administered intraperitoneally at a dose of 25 mg / kg;

[0048] DMSO group: Intraperitoneal injection of the same volume of DMSO as the DSF group;

[0049] DMSO+IR group: The same volume of DMSO as the DSF group was injected intraperitoneally, and the subcutaneous tumor area in the left groin was treated with a local dose of 20 Gy of gamma rays.

[0050] DSF+IR group: Disulfiram was injected intraperitoneally at a dose of 25 mg / kg, followed by local 20 Gy gamma irradiation of the subcutaneous tumor area in the left groin.

[0051] Tumor growth in all groups of mice was continuously monitored after irradiation, and samples were collected on day 6: mice were sacrificed by cervical dislocation, subcutaneous tumors were collected, photographed according to group, and fixed with 4% paraformaldehyde.

[0052] Test separately:

[0053] (1) Proliferative capacity of subcutaneous tumors in mice (Ki67 staining);

[0054] Ki67 immunohistochemical detection steps: 1. After dissecting the mouse subcutaneous tumor, embed it in paraffin and section it; 2. Dewax the paraffin sections to water; 3. Antigen retrieval; 4. Block endogenous peroxidase; 5. Serum blocking; 6. Add primary antibody; 7. Add secondary antibody; 8. DAB staining; 9. Counterstain cell nuclei; 10. Dehydrate, mount, and examine under a microscope for photography; 11. Assess proliferation capacity based on DAB positivity (displayed by brown-yellow staining). The stronger the signal (the more obvious the brown-yellow staining), the higher the proliferation capacity.

[0055] (2) Apoptosis level of subcutaneous tumors in mice (Tunel staining).

[0056] Tunel staining detection steps: 1. After dissecting the mouse subcutaneous tumor, embed it in paraffin and section it; 2. Dewax the paraffin sections to water; 3. Proteinase K repair; 4. Perforate the membrane; 5. Equilibrate to room temperature; 6. Add reaction solution; 7. Counterstain the cell nuclei with DAPI; 8. After mounting, observe and photograph under a microscope; 9. Assess the apoptosis level based on the strength of the FITC signal (displayed as green fluorescence). The stronger the signal (the higher the proportion of green fluorescence), the higher the apoptosis level.

[0057] The results are as follows Figure 1 As shown, Figure 1 This image shows the growth curve and specimen photographs of osteosarcoma after subcutaneous implantation in mice following radiotherapy. Figure A shows the tumor growth curve, and Figure B shows a specimen photograph. As shown in Figure A, the tumor growth curves indicate that DMSO (n=5) or DSF (n=5) alone did not significantly inhibit tumor growth. IR (n=6) alone showed some inhibition. However, the combined application of DSF and IR (n=6) significantly inhibited subcutaneous tumor growth in mice. Furthermore, compared to IR alone, the combined application of DSF and IR resulted in an additional 84% inhibition of subcutaneous tumor growth.

[0058] Additional inhibition of subcutaneous tumor growth in mice (%) = (1 - mean subcutaneous tumor volume in the combined treatment group / mean subcutaneous tumor volume in the DMSO group) / (1 - mean subcutaneous tumor volume in the irradiation-only group / mean subcutaneous tumor volume in the DMSO group) * 100% - 100%.

[0059] ns indicates that there is no statistically significant difference between the two groups. *, **, and *** represent P < 0.05, 0.01, and 0.001, respectively, indicating a statistically significant difference between the groups; the values ​​are expressed as mean ± standard deviation.

[0060] As can be seen from the photos in Figure B, the subcutaneous tumor photos of each group after sampling also show that DSF combined with IR can significantly inhibit tumor growth (to comply with ethical requirements, one mouse in each of the DMSO and DSF groups was sacrificed prematurely due to severe emaciation and could not be sampled).

[0061] The results are as follows Figure 2 As shown, Figure 2 This is a schematic diagram of Ki67 immunohistochemical staining after radiotherapy for subcutaneous osteosarcoma implantation in mice. According to... Figure 2 It is evident that there are significant differences in Ki67 staining levels (the more pronounced the brownish-yellow staining, the stronger the corresponding proliferative capacity) among different treatment groups. The DSF+IR group showed the fewest brownish-yellow staining spots, suggesting that DSF combined with irradiation can effectively reduce the growth and proliferation capacity of osteosarcoma.

[0062] The results are as follows Figure 3 As shown, Figure 3 This is a schematic diagram of Tunel staining after radiotherapy for subcutaneous implantation of osteosarcoma in mice. According to... Figure 3 It was observed that there were significant differences in TUNEL staining levels (a higher proportion of FITC-positive cells corresponded to a higher level of apoptosis) among different treatment groups. The DSF+IR group had the highest proportion of FITC-positive cells, suggesting that DSF combined with irradiation treatment can effectively increase the apoptosis level of osteosarcoma.

[0063] Experiment Example 2

[0064] IC50 of disulfiram in different osteosarcoma cell lines 50 Value detection

[0065] Grouping: Both HOS and K7M2 cell lines were divided into a DMSO negative control group and experimental groups with different final DSF concentrations for DSF IC50 analysis. 50 The detection methods used include the following final DSF concentrations: 1.25 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 15 μM, and 20 μM.

[0066] IC 50 Detection steps: 1. Evenly seed HOS and K7M2 cells into 96-well plates; 2. Add DMSO and different concentrations of DSF to each well; 3. Detect cell viability 24 hours after drug treatment using a CCK8 assay kit and a multi-functional microplate reader; 4. Perform IC50 analysis using Graphpad Prism 9 software. 50 Curve fitting was used to obtain the IC50 values ​​for the HOS and K7M2 cell lines. 50 value.

[0067] The results are as follows Figure 4 As shown, Figure 4 IC50 of DSF in different osteosarcoma cell lines 50 A schematic diagram of the curve. Based on... Figure 4As shown, different gradient concentrations of DSF were applied to two different osteosarcoma cell lines, HOS (human osteosarcoma cells) and K7M2 (mouse osteosarcoma cells). Cell viability was detected using a Cell-Counting Kit 8 (CCK8) kit in conjunction with a multi-functional microplate reader, and finally, IC50 analysis was performed using Graphpad Prism 9 software. 50 Curve fitting was used to obtain the IC50 values ​​for the HOS and K7M2 cell lines. 50 The values ​​were 13.41 μM and 7.44 μM, respectively. Furthermore, it was determined that the critical DSF concentration that did not produce viability inhibition in the above cell lines was 5 μM. Using this concentration can reduce the interference of DSF toxicity effects on their radiosensitization effects. A DSF concentration of 5 μM was used for subsequent cellular effect studies of the two cell lines.

[0068] Experimental Example 3

[0069] Verification and investigation of the radiosensitizing effect of disulfiram in different osteosarcoma cell lines

[0070] DSF group: DSF was added alone (DSF was added to the cell culture medium 2 hours before the planned irradiation time to a final concentration of 5 uM, but no irradiation was performed);

[0071] DMSO group: DMSO was added alone (DMSO was added to the cell culture medium 2 hours before irradiation treatment, the volume of which was the same as that of the DSF group, but no irradiation was performed);

[0072] DMSO+IR group: DMSO was added and irradiated (DMSO was added to the cell culture medium 2 hours before irradiation treatment, the volume of which was the same as that of the DSF group, and 8 Gy or 16 Gy of γ-ray irradiation was performed).

[0073] DSF+IR group: DSF was added and irradiated (DSF was added to the cell culture medium 2 hours before the planned irradiation time to make a final concentration of 5 uM, and 8 Gy or 16 Gy of γ-ray irradiation was performed).

[0074] Cell viability CCK8 assay procedure: 1. Evenly seed HOS and K7M2 cells onto 96-well plates; 2. Add DMSO or DSF to each well 2 hours before the planned irradiation time, and irradiate with 8 Gy and 16 Gy gamma rays respectively; 3. 24 hours after irradiation, detect cell viability using a CCK8 assay kit and a multi-functional microplate reader; 4. Additional inhibition of cell viability by combined treatment (%) = (100% - mean percentage of cell viability in the combined treatment group) / (100% - mean percentage of cell viability in the irradiation-only group) * 100% - 100%.

[0075] The results are as follows Figure 5 As shown, Figure 5 This is a statistical diagram illustrating the cell viability of different osteosarcoma cell lines detected by CCK8. Based on... Figure 5 The CCK8 assay results show that, without irradiation, DSF alone did not significantly affect the viability of HOS and K7M2 cell lines; however, irradiation alone weakened cell viability. When combined with 8 Gy and 16 Gy irradiation, DSF exhibited a further radiosensitizing effect, significantly reducing cell viability. Compared to the irradiation-only group, combined DSF application in HOS cells increased viability inhibition by an additional 51.7% (at 8 Gy) and 28.7% (at 16 Gy), and in K7M2 cells, it increased viability inhibition by an additional 75.9% (at 8 Gy) and 144% (at 16 Gy). *, **, and *** represent P < 0.05, 0.01, and 0.001, respectively, indicating statistically significant differences between groups; values ​​are expressed as mean ± standard deviation.

[0076] Colony formation detection procedure: 1. HOS and K7M2 cells were evenly seeded into 6-well plates; 2. Two hours before the planned irradiation time, DMSO or DSF was added to each well, and the cells were irradiated with 8 Gy of gamma rays; 3. After 7 days of continuous culture following irradiation treatment, the cells were fixed with 4% paraformaldehyde and stained with crystal violet; 4. The results after staining were obtained by scanning, and the number of colonies formed in each group was counted to assess the effect of intervention factors on colony formation. Additional inhibition of cell colony formation by combined treatment (%) = (1 - mean number of colonies in the combined treatment group / mean number of colonies in the DMSO group) / (1 - mean number of colonies in the irradiation-only group / mean number of colonies in the DMSO group) * 100% - 100%.

[0077] The results are as follows Figure 6 As shown, Figure 6 This image shows a clonogenic diagram and statistical illustration of different osteosarcoma cell lines. Based on... Figure 6 As shown in Figure A, without irradiation, DSF alone exhibited a mild inhibitory effect on clonogenesis in HOS and K7M2 cell lines, while irradiation alone produced a stronger inhibitory effect on clonogenesis. However, when combined with 8 Gy irradiation, DSF demonstrated a further significant inhibitory effect on cell clonogenesis, i.e., a radiosensitizing effect that inhibits cell proliferation. Figure 6 Figure B presents the statistical analysis results of colony formation number. The combined application of DSF and irradiation significantly reduced colony formation number compared to irradiation alone, with statistically significant differences. Furthermore, the combined application of DSF and irradiation increased the additional inhibition of colony formation by 43.3% (HOS cells) and 46.2% (K7M2 cells). *, **, and *** represent P < 0.05, 0.01, and 0.001, respectively, indicating statistically significant differences between groups. Values ​​are expressed as mean ± standard deviation.

[0078] Scratch assay procedure: 1. HOS and K7M2 cells were evenly seeded into 6-well plates, ensuring they were confluent before irradiation; 2. Two hours before the scheduled irradiation time, DMSO or DSF was added to each well, followed by irradiation with 8 Gy of gamma rays; 3. At 0 h post-irradiation, a straight line was drawn in each well, and images were captured using a microscope; 4. After culturing the cells for 24 hours, images were captured again at the same sampling points using a microscope; 5. The scratch area of ​​each well at the two time points was calculated using ImageJ software. Cell migration ability was assessed as the percentage reduction in scratch area over 24 hours; a higher percentage indicated stronger cell migration ability.

[0079] The percentage reduction in scratch area within 24 hours = (scratch area in 1-24h / scratch area in 0h) * 100%.

[0080] The results are as follows Figure 7 As shown, Figure 7 This image shows scratch assay results and statistical diagrams for different osteosarcoma cell lines. Based on... Figure 7 As shown in the scratch assay diagram, in the absence of irradiation, DSF alone had no significant inhibitory effect on the migration ability of HOS cell lines, but had a slight inhibitory effect on the migration ability of K7M2 cell lines. When combined with 8 Gy irradiation, DSF exhibited a radiosensitizing effect that inhibited cell migration. Figure 7 Figure B presents the statistical analysis results of the relative reduction in scratch area. In both the HOS and K7M2 cell lines, the combined application of DSF and irradiation further inhibited cell migration ability compared to irradiation alone, and the results were statistically significant. ImageJ software was used to automatically calculate the scratch area. ns indicates no statistically significant difference between the two groups. *, **, and *** represent P < 0.05, 0.01, and 0.001, respectively, indicating a statistically significant difference between the groups. Numerical values ​​are expressed as mean ± standard deviation.

[0081] Apoptosis detection procedure: 1. HOS and K7M2 cells were evenly seeded into 12-well plates; 2. Two hours before the planned irradiation time, DMSO or DSF was added to each well, and irradiation was performed with 8 Gy of gamma rays; 3. Twenty-four hours after irradiation, the cells were digested and washed, and the proportion of apoptotic cells was detected using the Annexin-V FITC / PI apoptosis staining kit in conjunction with flow cytometry; 4. The additional increase in the proportion of apoptotic cells after combined treatment (%) = (mean percentage of apoptotic cells in the combined treatment group / mean percentage of apoptotic cells in the irradiation-only group) * 100% - 100%.

[0082] The results are as follows Figure 8 As shown, Figure 8This image shows the results of Annexin V / PI flow cytometry apoptosis detection in different osteosarcoma cell lines and their statistical representation. Based on... Figure 8 As shown in the flow cytometry apoptosis assay, in the absence of irradiation, DSF alone can increase the apoptosis levels of HOS and K7M2 cell lines to some extent, leading to an increase in the proportion of cells in the upper right and lower right quadrants. However, when combined with 8 Gy irradiation, DSF exhibits a radiosensitizing effect that significantly increases the apoptosis level, with a significant increase in the proportion of cells in the aforementioned quadrants. Figure 8 Figure B presents the statistical analysis results of the specific proportions of apoptotic cells in the two cell lines under different treatment factors. The combined treatment group significantly increased the proportion of apoptotic cells compared with the irradiation-only group, and the difference was statistically significant. Moreover, compared with irradiation alone, the combined application of DSF and irradiation can further increase the proportion of apoptotic cells by 72% (HOS cells) and 181.1% (K7M2 cells). *, **, and *** represent P < 0.05, 0.01, and 0.001, respectively, indicating statistical differences between groups. Values ​​are expressed as mean ± standard deviation.

[0083] Western blot detection steps: 1. Extract total protein from each group 24 hours after different treatments; 2. SDS-PAGE electrophoresis; 3. Transfer to membrane; 4. Antibody incubation; 5. Detect Western blot results using chemiluminescence; 6. Detect the gray values ​​of each target protein and actin internal reference protein bands using ImageJ software. The normalization ratio of each target protein = target protein gray value / corresponding actin internal reference gray value. The relative expression level of the target protein in each treatment group = normalization ratio of the target protein in the experimental group / normalization ratio of the target protein in the DMSO group.

[0084] The results are as follows Figure 9 As shown, Figure 9 This is a Western blot diagram showing the detection of apoptosis-related proteins BAX, BCL2, Caspase-3, and Cleaved Caspase-3, along with a statistical illustration of their relative protein expression levels. Based on... Figure 9 The Western blotting of apoptosis-related proteins and the bar chart showing the grayscale values ​​of each protein on the right indicate that, compared to the irradiation-only group, the combined application of DSF and IR did not affect the expression of unactivated Caspase-3 (in grayscale values), but significantly upregulated the expression of pro-apoptotic proteins BAX and cleaved Caspase-3 (in grayscale values) and downregulated the expression of anti-apoptotic protein BCL2 (in grayscale values). This suggests that activation of the apoptosis pathway is one of the key pathways through which DSF plays a role in enhancing the radiosensitization of osteosarcoma radiotherapy. ns indicates no statistically significant difference between the two groups. *, **, and *** represent P < 0.05, 0.01, and 0.001, respectively, indicating statistically significant differences between the groups. Values ​​are expressed as mean ± standard deviation.

[0085] Cell cycle detection steps: 1. Evenly seed HOS and K7M2 cells onto 12-well plates; 2. Add DMSO or DSF to each well 2 hours before the planned irradiation time and irradiate with 8 Gy of gamma rays; 3. Digest and wash cells at 0, 4, 8, 12, and 24 hours after irradiation, and then fix cells with 70% ethanol; 4. Prepare propidium iodide staining solution using a cell cycle detection kit and perform staining; 5. Detect cell cycle distribution using flow cytometry; 6. Calculate the proportion of cells in each cycle using CytExpert software and display it as a bar chart.

[0086] The results are as follows Figure 10 As shown, Figure 10 A bar chart showing the cell cycle detection results for different osteosarcoma cell lines. Figure 10 This study demonstrates cell cycle changes in HOS and K7M2 cell lines within 24 hours after irradiation. In the absence of irradiation, DSF alone did not significantly affect the cell cycle. However, after applying 8 Gy of gamma ray irradiation, both HOS and K7M2 cell lines showed a decreasing trend in the G1 phase. Interestingly, the combined application of DSF and IR significantly increased the proportion of cells in the G1 phase, suggesting G1 phase arrest.

[0087] The results are as follows Figure 11 As shown, Figure 11 Western blotting plot showing the changing trends of cycle-related proteins P53, P21, CDKN2C, CDK4, CDK6, and Cyclin D1 within 24 hours after IR. Figure 11 This study presents the changes in cell cycle-related protein expression trends in HOS cells from two groups (DMSO+IR and DSF+IR) after 8 Gy irradiation over 4 hours. As shown in the figure, CDK6 and Cyclin D1 expression were upregulated in the DMSO+IR group 6-12 hours after IR, while CDK4 showed significant upregulation 8-12 hours after IR. All these protein expressions declined after 24 hours. The Western blot results show the protein expression trends in the DMSO+IR group and... Figure 10The observed cycle change trends correspond to these findings. In contrast, the DSF group did not show significant upregulation of CDK4 / 6 and Cyclin D1 expression 6-12 hours after IR. In the DSF group, the cycle repressor molecule P53 began to be upregulated 4 hours after IR, peaking at 8 hours. Its downstream molecule P21 was also upregulated 6-12 hours after IR. In addition to the P53 / P21 / CDK repressor pathway, CDKN2C from the INK4 family was also found to be involved in the regulation of CDK4 / 6, showing significant upregulation 8-12 hours after IR. However, the aforementioned cycle repressor molecules did not show an upregulation trend after IR in the DMSO+IR group.

[0088] Overall, DSF induces post-irradiation cell cycle arrest by upregulating the expression of the P53 / P21 / CDK inhibitory pathway and the cell cycle inhibitory molecule CDKN2C. At the same time, DSF also upregulates the activation of the post-irradiation apoptosis pathway. It is this series of mechanisms that enable DSF to exert its radiosensitizing effect on osteosarcoma radiotherapy in vivo and in vitro.

[0089] This invention is the first to discover and demonstrate the novel use of disulfiram in enhancing the radiosensitivity of malignant tumors, significantly inhibiting the proliferation of osteosarcoma cells after radiotherapy and promoting cell death in vitro. It also shows outstanding efficacy in inhibiting the growth of subcutaneously implanted osteosarcoma in mice after radiotherapy.

[0090] In this invention, DSF's different effects in regulating the radiosensitivity of benign and malignant tissues allow it to minimize radiation damage to adjacent benign tissues while increasing tumor killing, making it particularly suitable as a radiosensitizing drug for malignant tumor radiotherapy.

[0091] This invention demonstrates the in vitro and in vivo radiosensitization effect of DSF on osteosarcoma through in vitro and in vivo experiments, and further confirms that this effect is based on DSF enhancing the activation of apoptosis pathways after radiotherapy and inhibiting cycle progression after radiotherapy.

[0092] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The use of a disulfiram or its pharmaceutical salt in the preparation of a radiosensitizer, characterized in that, The structure of the disulfiram is shown below: ； The radiosensitizer mentioned refers to a radiosensitizer for malignant tumors; The malignant tumor referred to is HOS osteosarcoma cells; The radiation involved in the radiosensitizer is gamma rays.

2. The application of disulfiram or its pharmaceutical salt according to claim 1 in the preparation of radiosensitizers, characterized in that, The pharmaceutical salt is an acid addition salt formed by disulfiram with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, or mandelic acid.

3. The application of disulfiram or its pharmaceutical salt according to claim 1 in the preparation of radiosensitizers, characterized in that, A pharmaceutical preparation made from the aforementioned disulfiram or its pharmaceutical salt and medically acceptable excipients.

4. The application of disulfiram or its pharmaceutical salt according to claim 3 in the preparation of radiosensitizers, characterized in that, The dosage forms of the pharmaceutical preparations include injections, capsules, tablets, granules, pills, microspheres, and nanoparticles.

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