Application of 19-hydroxybufalin in preparation of medicine for treating prostatic cancer

Abstract

The invention discloses application of 19-hydroxybufalin in preparation of a medicine for treating prostatic cancer, and belongs to the technical field of pharmaceutical preparations. The 19-hydroxybufalin is found to be targeted in vivo and in vitro for the first time, and the growth of prostate tumors is remarkably inhibited, so that the survival basis of tumor cells is fundamentally weakened, programmed death of the tumor cells is triggered, and tumor regression is effectively induced; a brand-new treatment approach is provided, namely, a non-classical drug target, namely HELLS, is degraded in a targeted manner, so that a new treatment choice is expected to be provided for a patient lacking existing target mutation; the provided 19-hydroxybufalin can be used for treating advanced prostate cancer, especially castration-resistant prostate cancer, and a new candidate treatment drug with great potential and a brand new treatment strategy are provided for effectively solving the problem that chemotherapy drug resistance and targeted therapy selection are limited in the castration-resistant prostate cancer at present.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to the application of 19-hydroxybufalin in the preparation of a drug for treating prostate cancer. BACKGROUND

[0002] Prostate cancer is one of the most common malignant tumors in men worldwide. According to the data of the International Agency for Research on Cancer of the World Health Organization, its incidence ranks among the top three of all male malignant tumors in more than half of the countries. With the acceleration of population aging in China, the adjustment of dietary structure and the popularization of prostate-specific antigen screening, the incidence of prostate cancer in China is showing a rapid upward trend and has become the highest incidence of cancer in the male urinary system in China.

[0003] In recent years, although the diagnosis and treatment of prostate cancer has made continuous progress, for early-stage patients, radical surgery or radiotherapy can achieve good prognosis, but some patients have metastasis at the time of initial diagnosis. In addition, most of the advanced patients who receive initial endocrine therapy will progress to castration-resistant prostate cancer after 1-2 years, with a dramatic deterioration in prognosis and a significant decrease in 5-year survival rate.

[0004] With the development of molecular pathology, targeted therapy has become an important direction for the treatment of castration-resistant prostate cancer after endocrine therapy and chemotherapy. A large number of studies have confirmed that PARP inhibitors targeting specific gene mutations (such as HRR gene mutations of BRCA1 / 2) and new endocrine drugs with regulatory effects on tumor microenvironment exhibit significant efficacy for specific populations and are precise treatment strategies with clear clinical application prospects.

[0005] However, targeted therapy for prostate cancer still faces severe challenges. First, the currently effective targeted drugs are mainly limited to patients with specific gene mutations, and their applicability in the overall population is limited. Second, tumor heterogeneity and clonal evolution lead to secondary drug resistance in targeted therapy. SUMMARY

[0006] The purpose of the present application is to provide the application of 19-hydroxybufalin in the preparation of a drug for treating prostate cancer, which provides a new treatment drug and direction to solve the problems of chemotherapy resistance and limited selection of targeted therapy in the current traditional treatment methods for castration-resistant prostate cancer.

[0007] To achieve the above purpose, the present application provides the application of 19-hydroxybufalin in the preparation of a drug for treating prostate cancer, wherein the molecular formula of the 19-hydroxybufalin is C 24 H 34 O5; the drug for treating prostate cancer contains 19-hydroxybufalin as an effective ingredient.

[0008] Preferably, the drug for treating prostate cancer further comprises a surfactant, a flavoring agent, a solubilizer, an emulsifier and / or other pharmaceutically acceptable adjuvants.

[0009] Preferably, the emulsifier is dimethyl sulfoxide.

[0010] Preferably, the drug dosage form for treating prostate cancer is one of a capsule, a tablet, a granule, a powder, a dripping pill, a film and a coating.

[0011] Therefore, the application provides the use of 19-hydroxybufalin in the preparation of a drug for treating prostate cancer, and the specific technical effects are as follows: (1) The application first discovers that 19-hydroxybufalin can significantly inhibit the growth of prostate tumors and effectively induce tumor regression in vivo and in vitro; 19-hydroxybufalin targets the degradation of HELLS, a non-classical drug target, and opens up a new path for prostate cancer treatment that is different from existing endocrine therapy and PARP inhibitors, which is expected to provide new treatment options for patients lacking mutations in existing targets; (2) As a chromatin remodeling protein, HELLS protein plays a key role in maintaining genome stability and DNA damage repair; the application discovers that 19-hydroxybufalin can be used as a molecular glue or an inducer to directly target and promote the degradation of HELLS protein, destroy the integrity of chromatin, and exacerbate DNA damage and genomic instability, thereby fundamentally weakening the survival basis of tumor cells and triggering tumor cell programmed death; (3) The 19-hydroxybufalin provided by the application can be used for the treatment of advanced prostate cancer, especially castration-resistant prostate cancer, and provides a new extremely potential candidate therapeutic drug and a new treatment strategy for effectively solving the problems of limited chemotherapy resistance and limited targeted therapy options for castration-resistant prostate cancer.

[0012] The technical solutions of the application will be further described in detail below with the aid of the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed to be used in the description of the embodiments of the application will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0014] Figure 1 The results of Western blot detection in Example 2 of the application; MG132 is a proteasome inhibitor, + indicates addition, and - indicates no addition; Figure 2 The test results of 19-hydroxybufalin in inducing apoptosis of prostate cancer PC3 cells in Example 2 of the present application; A is the flow cytometry detection result; B is the statistical chart of the apoptosis rate of PC3 cells; C is the result chart of the influence of 19-hydroxybufalin on the activity of key effector caspase-3 / 7 of cell apoptosis; D is the statistical chart of the percentage of PC3 cell death detected by trypan blue staining method; Figure 3 The test results of 19-hydroxybufalin in inducing DNA damage of prostate cancer PC3 cells in Example 2 of the present application; A is the result chart of the influence of 19-hydroxybufalin on the DNA damage marker γH2AX of PC3 cells detected by immunofluorescence staining; B is the statistical chart of the fluorescence intensity change of γH2AX; C is the detection result chart by single cell gel electrophoresis (comet assay); D is the statistical chart of the degree of DNA breakage (comet tail moment); Figure 4 The evaluation results of 19-hydroxybufalin on the apoptosis of prostate cancer LNCaP cells in Example 2 of the present application; A is the flow cytometry detection result chart; B is the statistical chart of the apoptosis rate of LNCaP cells; C is the result chart of the influence on the activity of LNCaP cell caspase-3 / 7; D is the statistical chart of the percentage of LNCaP cell death detected by trypan blue staining method; Figure 5 The test results of 19-hydroxybufalin on the DNA damage of prostate cancer LNCaP cells in Example 2 of the present application; A is the result chart of the influence of 19-hydroxybufalin on the DNA damage marker γH2AX of LNCaP cells detected by immunofluorescence staining; B is the statistical chart of the fluorescence intensity change of γH2AX; C is the evaluation result chart by single cell gel electrophoresis; D is the statistical chart of the degree of DNA breakage (comet tail moment); Figure 6 The test results of 19-hydroxybufalin on the growth of prostate cancer PC3 cell xenografts in vivo in Example 2 of the present application; A is the comparison chart of the mass of xenografts at the end of treatment; B is the growth curve of xenografts during treatment; C is the body weight change curve of mice during treatment; Figure 7 The test results of 19-hydroxybufalin in treating prostate cancer PC3 xenografts in multiple doses in Example 2 of the present application; A is a representative image of the appearance of subcutaneous xenografts of mice in each dose group at the end of treatment; B is the immunofluorescence result chart of tumor tissue sections after treatment with different doses; Figure 8 The test results of multiple immunofluorescence staining of prostate cancer PC3 xenografts in Example 2 of the present application; A is the immunofluorescence result of tumor tissue sections after treatment with multiple doses of 19-hydroxybufalin; B is the statistical analysis chart of fluorescence signal intensity. DETAILED DESCRIPTION

[0015] The technical solutions of the present application are further described below by means of the accompanying drawings and examples.

[0016] In order to make the purpose, technical solutions and advantages of the present application more clear, thorough and complete, the technical solutions of the present application are described clearly and completely below by means of the accompanying drawings and examples. The following detailed description is the description of examples, which aims to provide further detailed description of the present application. Unless otherwise specified, all technical terms used in the present application have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0017] The instrument equipment and reagent materials used in the examples are obtained through commercial channels. The 19-hydroxybufalin used in the examples is chromatographically pure 98.22%. The method steps not specifically described in the examples are conventional technical means in the art. The animal experiment scheme in the examples has been reviewed and approved by the Animal Ethics Committee of Huazhong Agricultural University. The protein HELLS is a lymph-specific helicase (chromatin remodeling protein). The protein γH2AX is a histone H2AX phosphorylated at serine 139. The protein Cleaved-capase-3 is activated caspase-3.

[0018] Example 1 An animal tumor model was constructed as follows: (1) Tumor cell culture.

[0019] Logarithmic growth phase (90% confluence) human prostate cancer PC3 cell line was taken and placed in a 15 cm culture dish, about 2×10 7 cells per dish, 3 mL of 0.25% Trypsin-EDTA solution was added, and after gentle mixing, it was digested at 5% CO2, 37°C for 2 minutes.

[0020] Then 3 mL of RPMI 1640 complete culture medium was added to terminate the digestion reaction, and then 1 volume of phosphate buffered saline was added, centrifuged at 1000 rpm for 5 minutes, and the phosphate buffered saline centrifugal washing was repeated twice, then the cells were resuspended with ice-bath pre-cooled, phenol red-free RPMI 1640 medium, and the cell concentration was adjusted to 5×10 7 cells / mL.

[0021] (2) PC3 cells were inoculated into NSG mice.

[0022] Male NSG mice (severe immunodeficient mice, 5 weeks old, body weight 20±2 g) were adaptively fed under the condition of SPF animal experiment facility for 7 days, then the resuspended cells obtained in step (1) were mixed with an equal volume of Matrigel™ matrix gel pre-cooled in ice bath, and then subcutaneously injected into the groin area of the right hind limb of the mice, 0.1 mL (2.5×10 6 PC3 cells) per mouse, and then normally raised, free to drink and drink water. When the tumor could be touched (volume about 50-100 mm 3 ), it was considered that the modeling was successful.

[0023] Example 2 The therapeutic effect of 19-hydroxyl bufalin was evaluated as follows: (1) Preparation of 19-hydroxyl bufalin preparation.

[0024] 25 mg of 19-hydroxyl bufalin was dissolved in 500 μL of dimethyl sulfoxide to prepare a storage mother liquor with a concentration of 50 mg / mL. According to the required injection concentration of the experiment, the mother liquor was diluted with dimethyl sulfoxide. Then 45% PEG300 was added and mixed uniformly until clear, 5% Tween-80 was added and mixed uniformly, and finally 44% normal saline was added to obtain a stable preparation that can be used for intraperitoneal injection.

[0025] (2) Evaluation using human prostate cancer PC3 cell line.

[0026] PC3 cells were cultured in RPMI 1640 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. When the cell confluence reached 90%, 19-hydroxyl bufalin at a final concentration of 10 μM or an equal volume of dimethyl sulfoxide (solvent control) was used for treatment, and the cells were cultured for another 48 hours. Subsequently, the effects of 19-hydroxyl bufalin on PC3 cell apoptosis and DNA damage were evaluated by Annexin V / PI double staining flow cytometry, trypan blue staining, immunofluorescence, single cell gel electrophoresis, and Western blotting, respectively.

[0027] 1) 19-hydroxyl bufalin promotes HELLS protein degradation through the ubiquitin-proteasome pathway, as follows: PC3 cells were treated with 10 μM 19-hydroxyl bufalin alone or in combination with 10 μM proteasome inhibitor MG132 for 48 hours. Cell lysates were collected to detect the expression level of HELLS protein by Western blotting.

[0028] The results are as follows: Figure 1As shown, 19-hydroxybufalin can significantly reduce the expression of HELLS protein, while the combined treatment of MG132 can reverse this degradation effect and restore the level of HELLS protein. The results show that 19-hydroxybufalin promotes the degradation of HELLS protein through the ubiquitin-proteasome pathway.

[0029] 2) 19-hydroxybufalin effectively induces apoptosis of prostate cancer PC3 cells, as follows: Flow cytometry detection: After 19-hydroxybufalin treatment for 48 hours, the cells were collected and washed with pre-cooled PBS. After trypsin digestion without EDTA, centrifugation, resuspension with phosphate buffer solution and counting (density about 5×10 6 After that, 195 μL of 1×binding buffer (included in the Annexin V-FITC / PI Apoptosis Detection Kit) was resuspended, 5 μL of Annexin V-FITC and 10 μL of PI dye were added, and the mixture was gently mixed and incubated at room temperature for 20 minutes. Subsequently, flow cytometry was used to detect and analyze apoptosis under the FITC and PI channels.

[0030] Caspase-3 / 7 activity detection: PC3 cells were seeded in a 96-well plate at a density of 3×10 4 After 19-hydroxybufalin treatment for 48 hours, the caspase-3 / 7 detection working solution was prepared, 100 μL was added to each well, and incubated at room temperature for 1 hour. The fluorescence intensity was detected using a microplate reader at an excitation wavelength of 490 nm and an emission wavelength of 525 nm.

[0031] Trypan blue detection: After 19-hydroxybufalin treatment for 48 hours, the cell suspension was mixed with 0.4% trypan blue solution at a ratio of 1:1. After standing at room temperature for 3 minutes, the cell counting plate was counted under a light microscope. The total cell count and non-viable (blue) cell count were counted respectively, and the cell death rate was calculated (at least three independent fields were counted and averaged).

[0032] The results are shown in Figure 2 Compared with the dimethyl sulfoxide control group, 19-hydroxybufalin treatment can significantly promote apoptosis of PC3 cells: the early apoptosis rate increased from 1.55% to 3.15%, and the late apoptosis rate increased from 1.81% to 15.31%. At the same time, the fluorescence signal of caspase-3 / 7 was significantly enhanced, and the cell death rate shown by trypan blue staining was also increased by about 8 times. The above results consistently show that 19-hydroxybufalin can effectively induce apoptosis of prostate cancer PC3 cells.

[0033] 3) 19-hydroxybufalin induces DNA damage in prostate cancer PC3 cells, as follows: Immunofluorescence assay: PC3 cells grown on cell slides were treated with 19-hydroxypodophyllotoxin, fixed with 4% paraformaldehyde, and permeabilized with 0.3% triton X-100. Then, the cells were blocked with blocking buffer (containing 0.5% bovine serum albumin and 0.3% triton X-100) for 1 hour at room temperature. The primary antibody against γH2AX was added and incubated overnight at 4°C. The next day, the cells were washed with phosphate-buffered saline and incubated with the corresponding secondary antibody (Alexa Fluor 488-labeled rabbit anti or Alexa Fluor 594-labeled mouse anti) for 1 hour at room temperature in the dark. After washing, the nuclei were counterstained with DAPI. The slides were observed under a confocal microscope and images were captured.

[0034] Single cell gel electrophoresis (comet assay): The cells to be tested, which were treated with 19-hydroxypodophyllotoxin for 48 hours, were collected at a concentration of about 1 x 10 5 The cells were mixed with 0.7% low-melting-point agarose and placed on ice. Clean glass slides were coated with preheated 1% normal-melting-point agarose, and then the second layer of 0.7% low-melting-point agarose containing the cells was coated. After solidification, the third layer of 0.7% low-melting-point agarose was coated, and the gel was soaked in lysis buffer overnight. Then, electrophoresis was performed under alkaline conditions at 20 V and 200 mA for 20 minutes, followed by three washes in phosphate-buffered saline. Finally, the nuclei were stained with 50 μg / mL propidium iodide, and comet images were captured under a fluorescence microscope.

[0035] The results are shown in Figure 3 Compared with the control group, the fluorescence signal of the DNA damage marker γ-H2AX in the PC3 cells treated with 19-hydroxypodophyllotoxin was significantly enhanced, and the comet assay showed that the DNA breakage was significantly increased. The above results indicate that 19-hydroxypodophyllotoxin can effectively induce DNA damage in prostate cancer PC3 cells.

[0036] (3) Prostate tumor cells LNCaP were used for evaluation.

[0037] LNCaP cells were cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. When the cell confluence reached about 90%, the cells were treated with 19-hydroxypodophyllotoxin at a final concentration of 10 μM or an equal volume of dimethyl sulfoxide (solvent control), and cultured for another 48 hours. Then, the effects of 19-hydroxypodophyllotoxin on LNCaP cell apoptosis and DNA damage were evaluated by Annexin V / PI double staining flow cytometry, trypan blue staining, immunofluorescence, and single cell gel electrophoresis.

[0038] The results are shown in Figures 4-5As shown, compared with dimethyl sulfoxide control group, 19-hydroxybufalin treatment can significantly increase the apoptosis rate of LNCaP cells, enhance caspase-3 / 7 activity, and increase the number of trypan blue positive cells. This treatment also significantly up-regulates the expression of DNA damage marker γH2AX, and causes more significant DNA breakage. The above results show that 19-hydroxybufalin can effectively induce apoptosis and DNA damage in prostate cancer LNCaP cells.

[0039] (4) Evaluation using the mouse model constructed in Example 1.

[0040] 1) In vivo tumor inhibition experiment.

[0041] When the tumor volume of tumor-bearing mice grows to about 50-100 mm 3 , they are randomly divided into two groups, the test group and the control group, with 6 mice in each group. The mice in the test group are injected intraperitoneally with 0.1 mL of the 19-hydroxybufalin stable preparation prepared in step (1) every 48 hours. The mice in the control group are injected intraperitoneally with the same amount of dimethyl sulfoxide. During the experiment, the body weight and tumor growth of the mice are continuously measured for 14 days. After the experiment, the mice are euthanized and the subcutaneous tumors are isolated and weighed.

[0042] Hematoxylin-eosin staining: The formalin-fixed mouse tumor tissue is paraffin-embedded and sectioned. After dewaxing with xylene and water gradient hydration, the sections are subjected to hematoxylin staining, hydrochloric acid ethanol differentiation, ammonia water blue recovery, and eosin re-staining, followed by gradient ethanol dehydration, xylene transparency, and finally mounting with neutral resin. The sections are observed under an optical microscope and images are collected.

[0043] The results, as shown in Figures 6-7 , show that 19-hydroxybufalin can significantly inhibit the mass and volume of subcutaneously transplanted tumors in mice in a concentration-dependent manner, and has no significant effect on the body weight of mice in each group. Hematoxylin-eosin staining of tumor tissue shows that as the concentration of the drug increases, the tumor cells arrange in disorder and the tissue structure tends to be loose, indicating that 19-hydroxybufalin can effectively inhibit the growth of prostate cancer.

[0044] 2) Multiple immunofluorescence analysis of tumor tissue.

[0045] The paraffin-embedded tumor tissue sections are dewaxed, hydrated, and heat-mediated antigen-repaired, and then blocked with 1% bovine serum albumin at room temperature for 1 hour. Then, mixed primary antibodies (HELLS, γH2AX, cleaved-caspase-3) are added and incubated at 4°C overnight. The next day, the sections are washed and incubated with corresponding fluorescent secondary antibodies (Alexa Fluor 488, Cy3, Cy5 labeled) at room temperature for 1-2 hours in the dark. After washing again, the nuclei are stained with DAPI, and finally mounted with an anti-fluorescence quenching mounting agent, and observed and imaged under a fluorescence microscope or a confocal microscope.

[0046] Results as shown in Figure 8 Fig. 6, with the increase of the concentration of 19-hydroxybufalin, the expression of HELLS protein in tumor tissues was gradually down-regulated, while the expression of cleaved-caspase-3 (apoptosis marker) and γH2AX (DNA damage marker) were significantly up-regulated. The above results collectively indicated that 19-hydroxybufalin could promote the degradation of HELLS protein in vivo, and then induce DNA damage and cell apoptosis, thereby exerting the anti-prostate cancer effect.

[0047] Therefore, the present application first discovered that 19-hydroxybufalin could significantly inhibit the growth of prostate tumor in vivo and in vitro, thereby fundamentally weakening the survival basis of tumor cells, triggering the programmed death of tumor cells, and then effectively inducing tumor regression; and provided a brand new treatment approach - by targeting the degradation of HELLS, a non-classical drug target, which is expected to provide new treatment options for patients lacking mutations in existing targets; the provided 19-hydroxybufalin can be used for the treatment of advanced prostate cancer, especially castration-resistant prostate cancer, and provides a new highly potential candidate therapeutic drug and brand new treatment strategy for effectively solving the current problems of chemotherapy resistance and limited options of targeted therapy for castration-resistant prostate cancer.

[0048] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application rather than limit it, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: it can still modify or equivalently replace the technical solutions of the present application, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. Use of 19-hydroxybufalin for the preparation of a medicament for the treatment of prostate cancer, characterized in that: The 19-hydroxycinobufagin has a molecular formula of C 24 H 34 O5; the drug for treating prostate cancer has 19-hydroxycinobufagin as an effective component.

2. Use of 19-hydroxyl bufalin for the preparation of a medicament for the treatment of prostate cancer according to claim 1, characterized in that: The drug for treating prostate cancer also includes a surfactant, a flavoring agent, a solubilizer, an emulsifier and / or other pharmaceutically acceptable adjuvants.

3. Use of 19-hydroxyl bufalin for the preparation of a medicament for the treatment of prostate cancer according to claim 1, characterized in that: The emulsifier is dimethyl sulfoxide.

4. The use of 19-hydroxyl bufalin according to claim 1 for the preparation of a medicament for the treatment of prostate cancer, characterized in that: The dosage form of the drug for treating prostate cancer is one of a capsule, a tablet, a granule, a powder, a dripping pill, a film and a coating.

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