Application of strepsterol in preparation of liver cancer radiotherapy sensitizing drug

Abstract

The invention provides application of strepsterol in preparation of a liver cancer radiotherapy sensitizing drug, and belongs to the technical field of biological medicine. The invention discloses a technical scheme for reducing the generation of macrophage chain sterol in radiation-resistant liver cancer tissues and promoting the sensitization of liver cancer radiotherapy by using chain sterol treatment for the first time. Wherein the CD8 < + > T cells serve as anti-tumor immune core effector cells, the killing function of the CD8 < + > T cells directly affects the curative effect, and the strepsterol can promote infiltration of the cells into tumor tissues and improve secretion of killing molecules such as perforin and granzyme, so that radiation-resistant hepatoma carcinoma cells are effectively eliminated. Streptosterol treatment not only reduces radiotherapy resistance factor expression, but also amplifies the radiotherapy killing effect, finally realizes efficient radiotherapy sensitization and significantly improves the tumor recession rate, provides a brand new efficient scheme for radiation-resistant liver cancer treatment, and has great clinical and social values.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a sterol in the preparation of a radiosensitizing drug for liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC), a highly prevalent and deadly malignant tumor, has long been among the leading causes of disease burden globally. In the multidisciplinary comprehensive treatment system for HCC, radiation therapy (RT), with its precise targeting and strong local control capabilities, has become an irreplaceable and crucial treatment method. Especially for patients with inoperable, postoperatively recurrent or metastatic, or advanced-stage HCC with vascular invasion, radiotherapy is a key treatment option for prolonging survival and improving quality of life. In recent years, with the rapid development of medical imaging, computer technology and radiation physics, advanced radiotherapy technologies, represented by image-guided radiation therapy (IGRT), intensity-modulated radiation therapy (IMRT), stereotactic body radiation therapy (SBRT) and proton and heavy ion therapy, have been widely used in the clinical treatment of liver cancer and have achieved significant breakthroughs in efficacy. Among them, IGRT can achieve dynamic correction of the radiotherapy target area by acquiring real-time imaging information of the tumor and surrounding normal tissues, effectively reducing the impact of liver motion on radiotherapy accuracy; IMRT can precisely adjust the radiation intensity according to the three-dimensional morphology of the tumor, ensuring that the tumor receives sufficient irradiation while minimizing the radiation dose to surrounding normal liver tissue, gastrointestinal tract, kidneys and other sensitive organs; SBRT has the core advantages of "high precision, high dose and fewer fractions", and its local control rate can reach 80%-90% for small hepatocellular carcinomas or oligometastases with a diameter ≤5cm, with a short treatment cycle and good patient tolerance; proton and heavy ion therapy, as one of the most advanced radiotherapy technologies, utilizes the Bragg peak effect of charged particles to ensure that the radiation energy is precisely released when it reaches the tumor target area, with almost no damage to the normal tissue in front of the target area, and the tissue behind the target area is completely unaffected by radiation, providing a safer treatment option for hepatocellular carcinoma patients with cirrhosis and poor liver function reserve. However, the existence of radioresistance remains a core challenge hindering further improvements in the efficacy of radiotherapy for liver cancer patients. Clinical studies show that approximately 30%-50% of liver cancer patients experience local recurrence or distant metastasis after receiving standard radiotherapy due to decreased tumor cell sensitivity to radiation. The median survival of these patients is typically less than 12 months, far lower than the more than 30 months of radiosensitive patients. More seriously, research on the mechanisms of radioresistance in liver cancer is still in the exploratory stage, and a clear molecular regulatory network has not yet been established. Furthermore, there is a significant gap in effective intervention strategies for reversing radioresistance and improving radiosensitivity in clinical practice. Existing research largely focuses on intrinsic factors within tumor cells themselves, such as enhanced DNA damage repair capacity, abnormal cell cycle regulation, and altered oxidative stress levels, but insufficient attention has been paid to the regulatory role of the tumor microenvironment in radioresistance. Therefore, systematically revealing the potential regulatory factors of radioresistance in liver cancer, especially the interaction mechanism between the tumor microenvironment and tumor cells, identifying key radiosensitization targets with clinical translational value, and developing safe and effective combination therapies have become major scientific problems urgently needing to be solved in the field of liver cancer radiotherapy. This has significant clinical and social value for further improving the efficacy of radiotherapy and the overall prognosis of liver cancer patients. The tumor microenvironment, as the "soil" for tumor cell survival, is complex and dynamically changing. The functional state of immune cells plays a crucial regulatory role in tumor development, treatment response, and prognosis. Previous studies have shown that macrophages (Macs) are the most numerous immune cells in the liver's immune microenvironment, accounting for 20%-25% of total liver immune cells, and are also the main source of tumor-associated macrophages (TAMs). In the liver cancer microenvironment, macrophages can participate in regulating biological processes such as tumor angiogenesis, cell proliferation, invasion and metastasis, and immunosuppression by secreting various cytokines (such as IL-6, TNF-α, and TGF-β), chemokines (such as CCL2 and CXCL8), and growth factors (such as VEGF and EGF). In recent years, with the cross-integration of metabolomics and tumor immunology, studies have found that macrophages undergo significant metabolic reprogramming under the stimulation of the tumor microenvironment. This reprogramming involves altering their own glucose, lipid, amino acid, and mitochondrial metabolism patterns to achieve a functional state transition, thereby regulating the malignant progression and treatment resistance of tumors.For example, in solid tumors such as colorectal cancer and breast cancer, macrophages can produce large amounts of lactic acid by enhancing glycolysis, providing an energy source for tumor cells and inhibiting the anti-tumor activity of T cells, thereby reducing the sensitivity of tumors to radiotherapy. In pancreatic cancer, enhanced fatty acid oxidation metabolism of macrophages can promote their polarization towards the M2 anti-inflammatory phenotype, further exacerbating the immunosuppressive state of the tumor microenvironment and leading to the formation of radioresistance. However, the role and mechanism of macrophage metabolic reprogramming in radioresistance in hepatocellular carcinoma (HCC) remain unclear compared to other solid tumors. Currently, only a few studies suggest that macrophages in the HCC microenvironment may enhance the DNA damage repair capacity of tumor cells by upregulating the expression of key glycolytic enzymes (such as hexokinase 2 and lactate dehydrogenase A). However, related research still lacks systematicity and depth; the specific metabolic regulatory pathways, key effector molecules, and interaction mechanisms with tumor cells remain undefined. Therefore, in-depth exploration of the regulatory role of macrophage metabolic reprogramming in HCC radioresistance can not only provide a new research perspective for revealing the molecular mechanisms of HCC radioresistance but also potentially provide important theoretical basis and potential targets for developing novel radiosensitization strategies for HCC. Summary of the Invention

[0003] The purpose of this invention is to provide an application of sterols in the preparation of radiosensitizing drugs for liver cancer, which can enhance CD8 expression through sterols. + T-cell killing function, inhibiting radioresistance in liver cancer, and promoting radiosensitization in liver cancer.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of sterols in the preparation of radiosensitizing drugs for liver cancer.

[0005] Furthermore, the sterol can enhance CD8. + The killing function of T cells enhances the sensitization of liver cancer to radiotherapy.

[0006] Furthermore, the radiosensitizing drug for liver cancer can be added with a pharmaceutically acceptable carrier.

[0007] Furthermore, the dosage form of the radiosensitizing drug for treating liver cancer is one or more of the following: powder, injection, capsule, granule, tablet, emulsion, suspension, spray, powder, liposome, oral liquid, and drop pill.

[0008] Furthermore, the administration method of the radiosensitizing drug for liver cancer is one or more of the following: intramuscular injection, subcutaneous injection, intravenous injection, oral administration, sublingual administration, delivery to the lesion or brain or implantation, and spray administration.

[0009] The beneficial effects of this invention compared to the prior art are as follows: This invention discloses for the first time a technical solution that reduces the production of streptosterols in macrophages of radioresistant liver cancer tissue, and that treatment with streptosterols can promote radiosensitization in liver cancer. Among these solutions, CD8... + As core effector cells in anti-tumor immunity, T cells' cytotoxic function directly impacts treatment efficacy. Styrosterols can promote T cell infiltration into tumor tissue and increase the secretion of cytotoxic molecules such as perforin and granzymes, effectively eliminating radioresistant liver cancer cells. Styrosterol treatment both reduces the expression of radioresistance factors and amplifies the cytotoxic effect of radiotherapy, ultimately achieving highly efficient radiosensitization and significantly improving tumor regression rates. This provides a novel and highly effective treatment option for radioresistant liver cancer, possessing significant clinical and social value. Attached Figure Description

[0010] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1 This diagram illustrates the construction of a radioresistant hepatic orthotopic tumor in Example 1 of this invention; where A is a schematic diagram of the construction of a radioresistant hepatocellular carcinoma Hep53.4-R; B is a schematic diagram of the construction of a radioresistant orthotopic tumor model of hepatocellular carcinoma; and C is a gross image of a mouse hepatic orthotopic tumor and a bar chart of liver weight and liver weight / body weight, scale bar: 1 cm (n=6). *** P <0.001, **** P <0.0001, ns:nosignificance; Figure 2 This is a graph showing the chain sterol content in macrophages of radiation-resistant hepatic orthotopic tumors in Example 1 of the present invention; where A is a volcano diagram of differential metabolites in macrophages from hepatic orthotopic tumors sorted by magnetic beads (n=5); B is a graph showing the chain sterol content in macrophages (n=6); *** P <0.001; Figure 3 This is a graph showing the content of chain sterols in the tumor and serum of liver cancer patients in Example 1 of the present invention, where A represents the content of chain sterols in the tumor stroma fluid of mice (n=6); B represents the content of chain sterols in the serum of mice (n=6); **** P <0.0001; Figure 4This is a graph showing the levels of chain sterols in the tumor and serum of mice after radiotherapy for liver cancer in Example 2 of this invention, indicating disease progression (PD) and partial remission (PR). A is a waterfall plot evaluating the efficacy of radiotherapy for liver cancer in mice (n=30); B is the level of chain sterols in the tumor interstitial fluid of mice (n=6); C is the level of chain sterols in the serum of mice (n=6). The graphs represent: Complete remission (CR), Partial remission (PR), Stable disease (SD), Progressive disease (PD), **** P <0.0001; Figure 5 The chain sterols in Example 3 of this invention are for CD8 + The graph shows the effects of T cell killing function, where A represents IFN-γ expression levels, B represents GZMB expression levels, and C represents PD-1 expression levels. P <0.05,** P <0.01, **** P <0.0001, ns: no significance; Figure 6 This is a diagram illustrating the effect of sterols on radiosensitizing liver cancer in Example 4 of the present invention. A is a schematic diagram of model construction; B is a gross image of liver tumors in situ in four groups of mice; C is a bar chart of mouse liver weight and liver weight / body weight; D is a flow cytometry analysis of CD8 infiltrating liver tumors. + T cell proportion and IFN-γ + PD-1 + CD8 + T cell percentage chart (n=6);* P <0.05,** P <0.01, *** P <0.001, **** P <0.0001, ns: no significance. Detailed Implementation

[0012] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0013] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0014] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0015] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0016] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0017] Example 1

[0018] Example 1 of this invention detected the content of chain sterols in macrophages, tumor interstitial fluid, and serum of mice with radiation-resistant liver cancer. The specific steps are as follows: (1) Construction of a radioresistant in situ tumor model of liver cancer 1) According to Figure 1 The procedures shown in A and B are used to construct radioresistant liver tumors in situ: As shown in Figure A, a subcutaneous hepatocellular carcinoma model was constructed by injecting Hep53.4 liver cancer cells subcutaneously into C57BL / 6 mice. The size of the subcutaneous tumor was measured every 3 days, and the tumor was allowed to grow to 200-300 mm. 3 The mice were subjected to 8 Gy (8 Gy x 2 F) irradiation for two consecutive days. After the tumors recurred, the tumors were removed and implanted subcutaneously into new C57BL / 6 mice. The mice were then subjected to 8 Gy irradiation twice more. After the tumors recurred, the tumors were removed and implanted subcutaneously into new mice. The mice were then subjected to 8 Gy irradiation twice more. This process was repeated three times. The mice were then sacrificed, and the tumor tissue was isolated to obtain radioresistant subcutaneous tumor tissue Hep53.4-R.

[0019] 2) C57BL / 6 mice were randomly divided into two groups: the Hep53.4 group and the Hep53.4-R group. According to... Figure 1 As shown in Figure B, subcutaneous tumor tissue from the control group Hep53.4 (Hep53.4 group) and subcutaneous tumor tissue from Hep53.4-R (Hep53.4-R group) were implanted into the liver of C57BL / 6 mice to obtain the Hep53.4 group liver orthotopic tumor model and the Hep53.4-R group liver orthotopic tumor model, which was recorded as day 0.

[0020] (2) Half of the mice in the Hep53.4 group and the Hep53.4-R group were irradiated with 8 Gy on days 14 and 15, while the other half were not irradiated. The mice were sacrificed 4 weeks later, and their body weight and liver weight were recorded and the liver weight / body weight ratio was calculated. The results are as follows: Figure 1 As shown in C.

[0021] Figure 1 The results showed that the hepatocellular carcinoma in situ constructed using Hep53.4-R was radioresistant.

[0022] (3) Hepatic orthotopic tumors from the Hep53.4 group and the Hep53.4-R group after irradiation were dispersed into single-cell suspensions. Macrophages in the orthotopic tumors were sorted using magnetic beads and subjected to non-target metabolomics sequencing to obtain differential metabolite volcano maps. The content of chain sterols in macrophages was detected using an ELISA kit. The results are as follows: Figure 2 As shown.

[0023] Figure 2 The study showed that macrophages produced less streptosterol in radiation-resistant liver cancer tissue.

[0024] (4) The levels of streptosterols in mouse tumor stroma fluid and serum were detected using an ELISA kit. The results are as follows: Figure 3 As shown.

[0025] Figure 3 The results showed that radiation-resistant liver cancer tumors had reduced levels of chain sterols in the interstitial fluid and serum.

[0026] Example 2

[0027] Example 2 of this invention detected the levels of chain sterols in tumors and serum of mice with disease progression (PD) and partial remission (PR) after radiotherapy for liver cancer. The specific steps are as follows: Hep53.4 mouse hepatocellular carcinoma cells were injected orally into the livers of C57BL / 6 mice. Fourteen days after injection, tumor size was measured and recorded using MRI. The tumors were then irradiated with a dose of 8 Gy for three consecutive days, once daily. Four weeks after irradiation, tumor size was measured again using MRI, and the efficacy of radiotherapy was assessed using the mRESIST 1.1 standard, plotted as a waterfall graph (n=30). The levels of streptosterol in tumors and serum of mice with disease progression (PD) and partial remission (PR) after radiotherapy were detected using an ELISA kit. Results are shown below. Figure 4 As shown.

[0028] Figure 4 The results showed that, compared with mice in partial remission (PR), mice with disease progression (PD) after radiotherapy for liver cancer had reduced levels of chain sterols in the tumor and serum.

[0029] Example 3

[0030] Example 3 of this invention detected the effect of sterols on CD8. + The effects of T cell killing ability are explained in the following steps: Solution solutions of sterols with concentrations of 0, 0.5, 1.0, 1.5, and 2.0 μg / mL were prepared, and primary CD8+ cells from the spleens of C57BL / 6 mice were separated using a magnetic bead sorting kit. + T cells were cultured with a gradient concentration of steroidal solutions for 48 h, and then the effects of the gradient concentration of steroidal solutions on CD8 were detected by flow cytometry. + The effects of T cell activation markers IFN-γ, GZMB, and exhaustion marker PD-1 on the results. Figure 5 As shown.

[0031] Figure 5 The results showed that sterol treatment could increase CD8 + The expression levels of T cell activation markers IFN-γ and GZMB were reduced, while the expression level of exhaustion marker PD-1 was decreased. This indicates that steroids can enhance CD8 expression. + The killing function of T cells can thus inhibit radioresistance in liver cancer.

[0032] Example 4

[0033] Example 4 of this invention tested the effect of sterols on radiosensitizing liver cancer in mice. The specific steps are as follows: (1) Experimental grouping Mice were divided into four groups of six each: a control group, a control group plus radiation treatment group (Control+IR), a desmosterol treatment group, and a desmosterol plus radiation treatment group (Desmosterol+IR). (2) According to Figure 6 Process modeling shown in Figure A The radioresistant tumor tissue Hep53.4-R prepared in Example 1 was implanted into the liver of C57BL / 6 mice in situ. The desmosterol+IR group was irradiated with 8 Gy once daily on days 14, 15, and 16, and was also administered desmosterol solution by gavage at 5 mg / kg on days 7, 10, 13, 16, 19, and 22. The desmosterol group was not irradiated, and was administered desmosterol solution by gavage at 5 mg / kg on days 7, 10, 13, 16, 19, and 22.

[0034] The control group (with radiation treatment) received 8 Gy irradiation once daily on days 14, 15, and 16, and was given an equal volume of physiological saline by gavage on days 7, 10, 13, 16, 19, and 22. The control group (without irradiation treatment) was given an equal volume of physiological saline by gavage on days 7, 10, 13, 16, 19, and 22.

[0035] Four groups of mice were fed until day 47, at which point they were sacrificed. Body weight and liver weight were recorded, and the liver weight / body weight ratio was calculated. Flow cytometry was used to detect CD8 infiltrating hepatic orthotopic tumors. + T cell proportion and IFN-γ + PD-1 + CD8 + T cell proportion, results as follows Figure 6 As shown.

[0036] Figure 6 The results showed that sterol treatment could enhance CD8 + The killing function of T cells enhances the sensitization of liver cancer to radiotherapy.

[0037] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a sterol in the preparation of a radiosensitizing drug for liver cancer.

2. The application of the sterol according to claim 1 in the preparation of radiosensitizing drugs for liver cancer, characterized in that, The sterol can enhance CD8 + The killing function of T cells enhances the sensitization of liver cancer to radiotherapy.

3. The application of the sterol according to claim 2 in the preparation of radiosensitizing drugs for liver cancer, characterized in that, The radiosensitizing drugs for liver cancer can be added with pharmaceutically acceptable carriers.

4. The application of the sterol according to claim 3 in the preparation of radiosensitizing drugs for liver cancer, characterized in that, The dosage form of the radiosensitizing drug for treating liver cancer is one or more of the following: powder, injection, capsule, granule, tablet, emulsion, suspension, spray, powder, liposome, oral liquid, and drop pill.

5. The application of the sterol according to claim 3 in the preparation of radiosensitizing drugs for liver cancer, characterized in that, The administration methods of the radiosensitizing drugs for liver cancer radiotherapy are one or more of the following: intramuscular injection, subcutaneous injection, intravenous injection, oral administration, sublingual administration, delivery to the lesion or brain or implantation, and spray administration.

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