Application of combination of cryptotanshinone and PARP inhibitor in preparation of medicine for treating tumors

Abstract

The invention provides an application of combination of cryptotanshinone and a PARP inhibitor in preparation of a medicine for treating tumors. The invention also provides a pharmaceutical composition for treating breast cancer, which contains cryptotanshinone and olaparib in a molar ratio of (4-32): (4-32) or (0.5-4): (2-16). The combination of the natural active product cryptotanshinone with slight toxic and side effects and olaparib is used for treating the triple-negative breast cancer, so that the DNA damage degree of tumor cells can be enhanced, and the apoptosis level of the tumor cells can be increased. Besides, cryptotanshinone can promote degradation of PARP inhibitor drug-resistant key protein RAD51, has a certain effect on inhibiting and reversing Olaparib drug resistance, and has a good application prospect in treatment of malignant triple negative breast cancer through synergistic interaction of Olaparib.

Description

Technical Field

[0001] The present invention relates to the use of cryptotanshinone in combination with a PARP inhibitor in preparing a drug for treating tumors, and belongs to the field of medicines. Background Art

[0002] Data from the World Health Organization's International Agency for Research on Cancer (IARC) indicates that breast cancer has become the most common malignant tumor worldwide. Triple-negative breast cancer (TNBC) is the most aggressive and lethal subtype of breast cancer, accounting for 10%-20% of breast cancer cases. It is defined as a deficiency of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER-2). Due to the lack of clear therapeutic targets, traditional tumor resection and chemotherapy often have limited efficacy.

[0003] Some TNBC patients have BRCA1 / 2 gene mutations, which lead to impaired homologous recombination repair in tumor cells. Poly (ADP-ribose) polymerase PARP is a key enzyme for repairing single-strand DNA damage. The use of PARP inhibitors for BRCA1 / BRCA2-deficient tumor cells can produce a synthetic lethal effect, leading to tumor cell death. Olaparib is the first PARP inhibitor to be marketed. The olympiAD clinical trial study showed that for patients with HER2-negative advanced breast cancer with BRCA1 / 2 mutations, olaparib monotherapy can significantly prolong patients' progression-free survival compared to standard chemotherapy (4.2 months vs. 7 months).

[0004] CN202111551555.1, Invention Name: Application of Olaparib in Inducing Nucleolar Stress, discloses that Olaparib triggers nucleolar stress by inhibiting the synthesis of ribosomal RNA (rRNA) precursors, thereby enhancing the interaction between ribosomal proteins (RP) RPL5 and RPL11 and MDM2, and knocking down RPL5 and RPL11 inhibits Olaparib-induced p53 activation. Olaparib can effectively inhibit the survival and proliferation of breast cancer and colorectal cancer cells by activating p53. CN202111675554., Invention Name: A method for combining anti-breast cancer tumor drugs, in vitro and in vivo verification methods, involving the application of Olaparib combined with CD47 neutralizing antibodies in anti-breast cancer tumors, mainly solving the technical problem that Olaparib is currently used less in combination with non-BRCA mutation breast cancer tumors. It has been officially approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with HER-2-negative advanced or metastatic breast cancer with BRCA mutations. Although single targeted drugs have made considerable progress, long-term use still results in a series of side effects such as nausea, vomiting, and fatigue, and a certain degree of drug resistance. Therefore, there is an urgent need to find drugs that can reduce the side effects of olaparib, enhance its efficacy, and reverse or delay the development of drug resistance in combination with it.

[0005] In recent years, natural products have attracted widespread attention due to their high efficiency and low toxicity, and are clinically used as auxiliary treatments for various diseases to achieve synergistic effects. Danshen, also known as red ginseng, is the dried root and rhizome of the Lamiaceae plant Danshen. It has the effects of promoting blood circulation and removing blood stasis, promoting menstruation and relieving pain, clearing the heart and eliminating restlessness, cooling blood and eliminating carbuncle. Cryptotanshinone is the main fat-soluble extract of Danshen, which has multiple pharmacological activities such as anti-fibrosis, anti-inflammatory, cardiovascular protection, and anti-tumor. Current studies have shown that cryptotanshinone has anti-cancer effects on various types of cancer such as breast cancer, lung cancer, and gastric cancer. Studies have also found that cryptotanshinone can synergistically exert anti-tumor effects when combined with traditional anticancer drugs. For example, cryptotanshinone can synergistically enhance the effect of paclitaxel in the treatment of tongue squamous cell carcinoma. CN202110838215.0, invention name: Cryptotanshinone derivatives, preparation methods and applications thereof, discloses cryptotanshinone derivatives, preparation methods and applications thereof. The present invention specifically discloses that cryptotanshinone derivatives have excellent inhibitory activity against triple-negative breast cancer cells. CN202210731322.8, invention name: Application of cryptotanshinone substances and their combined compositions, discloses the application of cryptotanshinone substances (such as cryptotanshinone) in the preparation of chemotherapy sensitization drugs for NQO1-overexpressing malignant tumors; and the application of cryptotanshinone substances (such as cryptotanshinone) and NQO1 activating drugs in combination to prepare therapeutic and / or preventive drugs, drug resistance reversal drugs and / or prognosis and recurrence prevention drugs for NQO1-overexpressing tumors.

[0006] So far, there are no reports on the combined use of cryptotanshinone and PARP inhibitors for the treatment of triple-negative breast cancer. Summary of the Invention

[0007] The technical solution of the present invention is to provide the use of cryptotanshinone in combination with a PARP inhibitor in the preparation of a drug for treating tumors. The present invention also provides a pharmaceutical composition for treating breast cancer.

[0008] The present invention provides the use of cryptotanshinone in combination with a PARP inhibitor in preparing a medicine for treating tumors.

[0009] Among them, the PARP inhibitors are Olaparib, Rucaparib, Niraparib, and Talazoparib.

[0010] Wherein, the medicine is a medicine for treating breast cancer.

[0011] Further preferably, the drug is a drug for treating triple-negative breast cancer; and the PARP inhibitor is olaparib.

[0012] Wherein, the molar ratio of cryptotanshinone to olaparib is: (4-32): (4-32) or (0.5-4): (2-16).

[0013] Preferably, the molar ratio of cryptotanshinone to olaparib is: (0.5-2): (2-16).

[0014] The present invention also provides a pharmaceutical composition for treating breast cancer, which contains cryptotanshinone and olaparib in a molar ratio of (4-32): (4-32) or (0.5-4): (2-16).

[0015] More preferably, it contains cryptotanshinone and olaparib in a molar ratio of 1:1 or 1:2.

[0016] The pharmaceutical composition of the present invention contains cryptotanshinone and olaparib as active ingredients, and is added with pharmaceutically acceptable excipients or auxiliary ingredients to prepare a commonly used pharmaceutical preparation.

[0017] Wherein, the preparation is an oral preparation or an injection preparation.

[0018] The present invention combines cryptotanshinone, a naturally active product with mild side effects, with olaparib to treat triple-negative breast cancer, enhancing the degree of DNA damage in tumor cells and increasing the level of apoptosis. Furthermore, cryptotanshinone can promote the degradation of RAD51, a key protein in PARP inhibitor resistance, and has a certain effect on inhibiting and reversing olaparib resistance. Cryptotanshinone has great application prospects in synergizing with olaparib in the treatment of malignant triple-negative breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 a is a line graph showing the killing effects of cryptotanshinone, olaparib, and cryptotanshinone + olaparib on MDA-MB-468 and SUMP149PT cells; b is a scatter plot of CI values ​​at different concentration combinations;

[0020] Figure 2 Figure 1 shows the results of cryptotanshinone synergistically inducing apoptosis in triple-negative breast cancer cells by olaparib, where a is a representative flow cytometry apoptosis image of MDA-MB-468 and SUMP149PT cells; b is the Western blot detection result of apoptosis proteins in MDA-MB-468 and SUMP149PT cells;

[0021] Figure 3 The immunofluorescence results and foci quantification images of triple-negative breast cancer cells after treatment with cryptotanshinone, olaparib, and cryptotanshinone + olaparib, where a is the immunofluorescence result image and b is the foci quantification image;

[0022] Figure 4 This is a Western blot result showing the expression of DNA damage repair proteins RAD51 and γH2AX in triple-negative breast cancer cells after treatment with cryptotanshinone, olaparib, and cryptotanshinone + olaparib;

[0023] Figure 5 The figure shows the qPCR results of RAD51 mRNA expression in triple-negative breast cancer cells after treatment with cryptotanshinone, olaparib, and cryptotanshinone + olaparib;

[0024] Figure 6Figure 1 shows Western blot results of RAD51 protein expression after treatment with CHX, CHX + cryptotanshinone, CHX + MG132, and CHX + MG132 + PA cryptotanshinone. (a) RAD51 protein levels in MDA-MB-468 and SUM149PT cells treated with CHX alone at different time points; (b) RAD51 protein levels in MDA-MB-468 and SUM149PT cells co-treated with CHX and cryptotanshinone at different time points; (c) RAD51 protein levels in MDA-MB-468 and SUM149PT cells co-treated with CHX and MG132 at different time points; and (d) RAD51 protein levels in MDA-MB-468 and SUM149PT cells co-treated with CHX, MG132, and cryptotanshinone at different time points. DETAILED DESCRIPTION

[0025] The structural formula of the compound cryptotanshinone used in the present invention is shown in formula (1). This compound has the characteristics of low cytotoxicity and high safety (Chinese patent document CN106699771A). This drug has broad development prospects in the field of downregulating the expression of DNA damage repair-related proteins and improving the efficacy of chemotherapy drugs in the treatment of triple-negative breast cancer. The compound represented by formula (1) is shown below:

[0026]

[0027] Explanation of terms:

[0028] RAD51: RAD51 is a core recombinase in the homologous recombination repair mechanism. When double-strand breaks occur, RAD51 wraps around single-stranded DNA to form nuclear filaments, searches for homologous templates, and initiates DNA strand invasion, thereby initiating DNA repair synthesis. Studies have found that high expression of RAD51 in BRCA-deficient cells is associated with resistance to PARP inhibitors.

[0029] γH2AX: γH2AX is a member of the chromosomal histone H2A family. In response to various physical and chemical stimuli, double-strand breaks (DSBs) occur in cellular DNA. Phosphatidylinositol 3-kinases, such as ATM and ATR, phosphorylate serine 139 on H2AX, forming phosphorylated H2AX, or γH2AX. γH2AX, produced by H2AX phosphorylation, serves as a biomarker that clearly reflects the extent of DNA damage and repair.

[0030] Cicloheximide (CHX): is an organic compound with the chemical formula C 15 H 23 NO4 can inhibit protein synthesis in eukaryotes.

[0031] MG132: Also known as benzyloxycarbonyl-leucine-leucine-leucine, it is a peptide aldehyde. It is a proteasome inhibitor that inhibits proteasome activity by reacting with the hydroxyl hemiacetal in the active site threonine residue of the 20S catalytic core of the proteasome.

[0032] Experimental Example 1 Effects of Cryptotanshinone and Olaparib on Survival Rate of Triple-Negative Breast Cancer and Their Synergistic Effects

[0033] 1. Materials

[0034] 1.1 Cell Source

[0035] Human breast cancer cells MDA-MB-468 and SUMP149PT were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0036] 1.2 Main reagents and their preparation methods

[0037] 1.2.1 Main reagents: complete culture medium, dimethyl sulfoxide (DMSO), PBS, trypsin, CCK8 test, cryptotanshinone, olaparib

[0038] 1.2.2 Preparation method

[0039] (1) Preparation of Cryptotanshinone and Olaparib Solution

[0040] Cryptotanshinone (S2286) was purchased from Selleck and dissolved in dimethyl sulfoxide (DMSO) to prepare a 30 mM stock solution. Olaparib (S1060) was purchased from Selleck and dissolved in DMSO to prepare a 200 mM stock solution.

[0041] (2) Preparation of complete culture medium: 89% DMEM high glucose medium + 1% penicillin-streptomycin double antibody solution + 10% fetal bovine serum.

[0042] 1.3 Cell culture conditions

[0043] The cell culture environment was: 37°C, 5% CO2 in a cell culture incubator.

[0044] 1.4 Main instruments and consumables

[0045] Clean bench, cell culture incubator, microplate reader, low-speed centrifuge, cell culture dish, 96-well cell culture plate, centrifuge tube, EP tube.

[0046] 2. Experimental Methods

[0047] 2.1 CCK8 assay for cell viability:

[0048] MDA-MB-468 and SUM149PT triple-negative breast cancer cells were seeded at 4,000 and 6,000 cells per well in 96-well plates. After cell attachment, the cells were treated with a control group, a cryptotanshinone-alone group, an olaparib-alone group, and a cryptotanshinone + olaparib combination group for 72 hours in a cell culture incubator. The culture medium was then discarded, and CCK8 reagent was used according to the manufacturer's instructions. After incubation at 37°C for 1 hour, the absorbance of each well was measured at 450 nm using a microplate reader. Each assay was repeated three times.

[0049] 2.2 Determination of synergistic effects:

[0050] The experimental method was the same as that for determining cell viability using CCK8. The absorbance values ​​were converted to calculate cell viability. Compusyn software was used to calculate the drug combination index (CI) for different drug concentration combinations. According to the software definition, a CI > 1 indicates an antagonistic effect between the two drugs; a CI = 1 indicates an additive effect between the two drugs; and a CI < 1 indicates a synergistic effect between the two drugs.

[0051] 3. Experimental results:

[0052] like Figure 1 As shown in a, MDA-MB-468 cells were treated with 4, 8, 16, and 32 μM olaparib and cryptotanshinone, either individually or in combination, for 72 hours. SUM149PT cells were treated with 2, 4, 8, and 16 μM olaparib and 0.5, 1, 2, and 4 μM cryptotanshinone, either individually or in combination, for 72 hours. Cell viability was measured using the CCK8 assay under different drug combinations. The results showed that the combination of olaparib and cryptotanshinone significantly inhibited cell proliferation. Figure 1 b. Compusyn software was used to calculate the drug combination index (CI) for each drug concentration combination. The results showed that the CI index for different concentrations of olaparib and cryptotanshinone in both cell lines was less than 1, indicating that olaparib and cryptotanshinone have a synergistic effect. As can be seen in Figure b, the synergistic effect weakened with increasing doses of cryptotanshinone. For MDA-MB-468 cells, the effect of cryptotanshinone was poor when the dose exceeded 32 μM, and the synergistic effect began to weaken when the dose exceeded 2 μM for SUM149PT cells.

[0053] Experimental Example 2: Cryptotanshinone and Olaparib induce apoptosis in triple-negative breast cancer cells

[0054] 1. Materials

[0055] 1.1 Cell samples

[0056] MDA-MB-468 triple-negative breast cancer cells were treated with cryptotanshinone 32 μM, olaparib 32 μM, or both for 72 hours to obtain cell samples.

[0057] SUM149PT triple-negative breast cancer cells were treated with 1 μM cryptotanshinone and 2 μM olaparib, respectively, or in combination for 72 hours to obtain cell samples.

[0058] 1.2 Main Reagents

[0059] 1.2.1 Reagents used in flow cytometry experiments: Annexin V-FITC / PI double staining apoptosis detection kit (AD10, Tongren), PBS, EDTA-free trypsin,

[0060] 1.2.2 Western blot reagents: protein lysis buffer, Caspase 3 antibody (9662S, CST), Cleaved Caspase 3 antibody (ab32042, Abcam), GAPDH (60004-1-Ig, Proteintech)

[0061] 1.2.3 Preparation of relevant solutions

[0062] Protein lysis buffer: Prepared using RIPA150: protease inhibitor (PI): sodium glycerophosphate: NaF in a ratio of 90:10:1:1

[0063] 10X electrophoresis buffer: Tris base 30 g, glycine 144 g, SDS 10 g, and dilute to 1 L with ddH2O.

[0064] 1X transfer buffer: glycine 28.8 g, Tris base 6 g, methanol 400 ml, and dilute to 2 L with ddH2O.

[0065] 10X TBS solution: 88 g sodium chloride, 24 g Tris base, 13 ml concentrated hydrochloric acid, and dilute to 1 L with ddH2O.

[0066] 1×TBST solution: Measure 100 ml of 10×TBS solution and mix with 1 ml of 20% Tween to make up to 1 L.

[0067] 1.3 Main instruments and consumables

[0068] 1.3.1 Flow cytometry related instrument consumables: flow cytometer, 6-well plate, centrifuge

[0069] 1.3.2 Western blot experiment related instruments and consumables: vertical electrophoresis tank, multifunctional imaging system, 24-well plate, bench scale, analytical balance, shaker, PVDF membrane, filter paper.

[0070] 2. Experimental methods:

[0071] 2.1 Flow cytometry detection of cell apoptosis

[0072] MDA-MB-468 and SUM149PT triple-negative breast cancer cells were seeded on 6-well plates. After the cells adhered, a control group, a cryptotanshinone-alone treatment group, an olaparib-alone treatment group, and a cryptotanshinone + olaparib combination treatment group were set up. The cells were treated in a cell culture incubator for 72 hours to digest the cells. After the treatment, the cells were centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, PBS was added to resuspend the cells, and the plates were washed and centrifuged at 1000 rpm for 3 minutes. The washing step was repeated once. Finally, the samples were collected and stained according to the supplier's reagent instructions, and the cell apoptosis was detected by flow cytometry.

[0073] 2.2 Western blot assay to detect protein levels

[0074] Drug-treated MDA-MB-468 and SUM149PT cells were lysed with protein lysis buffer, and 5X SDS loading buffer (1 / 4 the volume of the lysate) was added and mixed evenly. The protein samples were heated at 100°C for 8 minutes to completely denature them. The resulting proteins were then separated by SDS-PAGE polyacrylamide gel electrophoresis and transferred to a PVDF membrane (O162-0177, Bio-Rad). After incubation with the corresponding primary and secondary antibodies, protein bands were detected using a chemiluminescence kit (K22030, Abbkine).

[0075] 3. Experimental Results

[0076] like Figure 2 As shown in Figure a, triple-negative breast cancer cells were treated with different concentrations of cryptotanshinone and olaparib alone or in combination for 72 hours, and the cell apoptosis rate was measured by flow cytometry. The results showed that the cell apoptosis rate in the combination group was significantly increased compared with the single-drug group. Figure 2 As shown in Figure b, protein samples were collected after drug treatment, and changes in the expression levels of apoptosis-related proteins (caspase 3 and cleaved-caspase 3) were detected by Western blot. The results showed that compared with the monotherapy group, the expression level of caspase 3 protein did not change significantly in the combination group, while cleaved-caspase 3 was significantly upregulated. This suggests that cryptotanshinone can enhance olaparib-induced apoptosis in triple-negative breast cancer cells.

[0077] Experimental Example 3: Detection of DNA damage

[0078] 1. Materials

[0079] 1.1 Cell samples

[0080] MDA-MB-468 triple-negative breast cancer cells were treated with cryptotanshinone 32 μM, olaparib 32 μM, or both for 72 hours to obtain cell samples.

[0081] SUM149PT triple-negative breast cancer cells were treated with 1 μM cryptotanshinone and 2 μM olaparib, or treated in combination for 72 hours to obtain cell samples.

[0082] 1.2 Main Reagents

[0083] PBS, 4% paraformaldehyde, 0.1% Triton, 5% BSA, 1% BSA

[0084] The antibodies used for immunofluorescence analysis in the present invention are:

[0085]

[0086] 1.3 Main instruments and consumables

[0087] Ultraclean workbench, centrifuge, inverted fluorescence microscope.

[0088] 2. Experimental Methods

[0089] Immunofluorescence

[0090] MDA-MB-468 and SUM149PT cells were digested and centrifuged, and 1×10 5 Cells were seeded in confocal microplates and allowed to adhere. After 24 hours of drug intervention according to group, cells were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS, and permeabilized with 0.1% Triton on a shaker for 15 minutes, washed with PBS, and then blocked with 5% BSA for 1 hour before washing with PBS. The cells were then incubated with primary antibodies diluted in 1% BSA overnight at 4°C. The next day, fluorescently labeled secondary antibodies were added for 1 hour, and finally, DAPI was added for nucleus staining. Images were then collected under a laser confocal microscope.

[0091] 3. Experimental results:

[0092] Cryptotanshinone synergizes with olaparib to enhance DNA damage in triple-negative breast cancer cells. Figure 3As shown, MDA-MB-468 cells were treated with 32 μM olaparib and 32 μM cryptotanshinone, either alone or in combination, and SUM149PT cells were treated with 2 μM olaparib and 1 μM cryptotanshinone, either alone or in combination, for 72 hours. Immunofluorescence was used to measure the number of repair foci for the DNA double-strand damage marker γH2AX and the key HR repair protein RAD51. The results showed that compared with the single-drug groups, the number of γH2AX foci in the combination group was significantly increased, while the number of RAD51 foci did not increase significantly and even decreased. This suggests that the combination of cryptotanshinone and olaparib can increase DNA damage, which may be related to cryptotanshinone's inhibition of DNA damage repair.

[0093] Experimental Example 4: Expression of DNA damage repair-related gene proteins

[0094] 1. Materials and Methods

[0095] 1.1 Cell samples

[0096] MDA-MB-468 triple-negative breast cancer cells were treated with cryptotanshinone 32 μM, olaparib 32 μM, or both for 72 hours to obtain cell samples.

[0097] SUM149PT triple-negative breast cancer cells were treated with 1 μM cryptotanshinone and 2 μM olaparib, or treated in combination for 72 hours to obtain cell samples.

[0098] 1.2 Main reagents are the same as those in 1.2 of Test Example 2

[0099] 1.3 Main instruments and consumables are the same as those in 1.3 of Test Example 2

[0100] 2. Experimental Methods

[0101] Western blot analysis was the same as in Experiment 2.

[0102] The antibodies used for western blot analysis in this study are:

[0103]

[0104] 3. Experimental results:

[0105] like Figure 4As shown, protein samples were collected from triple-negative breast cancer cells treated with varying concentrations of olaparib and cryptotanshinone, either alone or in combination, for 72 hours. Western blot analysis was used to examine changes in the protein levels of related genes. The results showed that the combination of olaparib and cryptotanshinone significantly increased γH2AX protein levels compared to the monotherapy group, indicating that the combination of olaparib and cryptotanshinone can increase DNA double-strand damage. Simultaneously, it downregulated RAD51 expression, suggesting that cryptotanshinone's sensitization to olaparib may be related to its inhibition of DNA damage repair.

[0106] Experimental Example 5: mRNA expression of genes related to DNA damage and repair

[0107] 1. Materials

[0108] 1.1 Cell samples

[0109] MDA-MB-468 triple-negative breast cancer cells were treated with cryptotanshinone 32 μM, olaparib 32 μM, or both for 72 hours to obtain cell samples.

[0110] SUM149PT triple-negative breast cancer cells were treated with 1 μM cryptotanshinone and 2 μM olaparib, respectively, or in combination for 72 hours to obtain cell samples.

[0111] 1.2 Main Reagents

[0112] Trizol (15596026, Ambion), reverse transcription kit (R333-01, Vazyme).

[0113] 1.3 Main instruments and consumables

[0114] PCR instrument, fluorescence quantitative PCR instrument, clean bench, 24-well plate.

[0115] 2. Experimental Methods

[0116] Real-time fluorescence quantitative PCR

[0117] The cells were lysed using Trizol reagent and total RNA was extracted. 300 ng of RNA was then converted to cDNA using a reverse transcription kit. The mRNA expression levels of the relevant genes were then detected using a fluorescent quantitative PCR instrument. The primer sequences used in this invention are as follows:

[0118] RAD51 forward primer: 5'-CGAGCGTTCAACACAGACCA-3'

[0119] RAD51 reverse primer: 5'-TGACGGAAGGGCACCACCAG-3'

[0120] 18S forward primer: 5'-TGACGGAAGGGCACCACCAG-3',

[0121] 18S reverse primer: 5′-GCACCACCACCCACGGAATC-3′.

[0122] 3. Experimental Results

[0123] like Figure 5 As shown in the study, MDA-MB-468 and SUM149PT cells were treated with different concentrations of olaparib and cryptotanshinone, either alone or in combination, for 72 hours. qPCR was then used to detect RAD51 mRNA expression levels in the cells. The results showed no significant changes in RAD51 mRNA expression in the combination group compared to the cryptotanshinone monotherapy group. This suggests that the effect of cryptotanshinone on RAD51 protein expression is unrelated to changes in gene expression.

[0124] Experimental Example 6 Effect of Cryptotanshinone on RAD51 Protein Levels under the Action of CHX and MG132

[0125] 1. Materials

[0126] 1.1 Cell samples

[0127] MDA-MB-468 and SUM149PT triple-negative breast cancer cells were treated with CHX (40 μg / mL) for 0-8 h to obtain cell samples;

[0128] MDA-MB-468 triple-negative breast cancer cells were treated with CHX (40ug / mL) and cryptotanshinone 32μM for 0-8h to obtain cell samples.

[0129] Cell samples were obtained by treating SUM149PT triple-negative breast cancer cells with CHX (40ug / mL) and cryptotanshinone 1μM for 0-8h.

[0130] MDA-MB-468 and SUM149PT cells were treated with CHX (40 ug / mL) and MG1322 (20 μM) for 0-8 h to obtain cell samples;

[0131] MDA-MB-468 triple-negative breast cancer cells were treated with CHX (40 ug / mL), MG1322 (20 μM), and cryptotanshinone 32 μM for 0-8 h to obtain cell samples;

[0132] SUM149PT triple-negative breast cancer cells were treated with CHX (40ug / mL), MG1322 (20μM) and cryptotanshinone 1μM for 0-8h to obtain cell samples.

[0133] 1.2 Main reagents and preparation methods

[0134] The CHX (66-81-9) and MG13 (S2619) used in the experiment were purchased from Selleck. The drug powder was centrifuged and dissolved in PBS to prepare a stock solution with a final concentration of 15 mg / mL and 200 mM. The stock solution was then aliquoted and frozen at -80°C for storage. The rest of the steps were the same as those in 1.2.2 of Experimental Example 2.

[0135] 1.3 Main instruments and consumables

[0136] Same as 1.3 in Test Example 2

[0137] 3. Experimental Results

[0138] like Figure 6 As shown in a, MDA-MB-468 cells and SUM149PT cells were treated with protein synthesis inhibitor CHX for 2, 4, 6, and 8 hours, and protein samples were collected. The changes in RAD51 protein expression levels were detected by WB. The results showed that the expression of RAD51 protein decreased with time, indicating that CHX played a role in protein degradation. On this basis, cryptotanshinone at a certain drug concentration was added, and the expression of RAD51 protein was detected after the same treatment time. Figure 6 As shown in Figure b, after adding cryptotanshinone, the expression level of RAD51 protein was lower at the same time point, and the protein degradation rate was faster, indicating that cryptotanshinone can promote the degradation of RAD51 protein.

[0139] At the same time, cells were treated with protein synthesis inhibitor CHX and proteasome inhibitor MG132, and protein samples were collected at the same time points to detect changes in RAD51 protein levels. Figure 6 As shown in Figure c, after the addition of MG132, protein expression levels no longer changed over time, suggesting that MG132 can inhibit protein degradation. Furthermore, the same concentration of cryptotanshinone was added to the cells for the same treatment time, and protein expression was measured. Figure 6 d As can be seen, in the presence of MG132, the addition of cryptotanshinone has no effect on protein expression, indicating that cryptotanshinone degrades RAD51 protein through the ubiquitin-protease pathway.

[0140] In summary, the present invention provides a use of a natural product, cryptotanshinone, to synergistically enhance the anti-tumor effect of olaparib. A series of drug concentration gradients of cryptotanshinone (0.5-32 μM) and olaparib (2-32 μM) are set, and triple-negative breast cancer cells are treated with the drugs alone or in combination for 72 hours. Afterwards, the cell state is observed under a microscope and the cell viability is detected using the cell viability assay reagent CCK8 to calculate the cell survival rate. It was found that compared with the single treatment, the combined treatment significantly enhanced the killing power of tumor cells. Subsequently, the synergistic score of the two drugs was calculated using synergistic scoring software to determine the presence of a synergistic effect between the two drugs. According to the synergistic score calculation results, the concentration combination with the best synergistic effect was selected to treat triple-negative breast cancer cells. After 72 hours, samples were collected and flow cytometry experiments showed that the cell apoptosis rate increased after the combination of cryptotanshinone and olaparib. In addition, immunofluorescence experiments found that the combination of cryptotanshinone and olaparib can increase DNA damage, and cryptotanshinone can inhibit RAD51-mediated DNA damage repair. Subsequent Western blot experiments showed that the combination of cryptotanshinone and olaparib can increase the protein level of the DNA damage marker γH2AX, while downregulating the protein expression of RAD51, which is consistent with the immunofluorescence results, indicating that cryptotanshinone can enhance the DNA damage of tumor cells caused by olaparib by downregulating the protein expression of RAD51, thereby promoting tumor cell apoptosis.

[0141] Triple-negative breast cancer cells were treated with the protein synthesis inhibitor CHX alone or in combination with CHX and cryptotanshinone to observe RAD51 degradation. Cryptotanshinone significantly promoted RAD51 degradation. Furthermore, the proteasome inhibitor MG132 was combined with CHX or CHX and cryptotanshinone to observe RAD51 degradation for 0-8 hours. Cryptotanshinone significantly slowed or halted RAD51 degradation, suggesting that cryptotanshinone promotes RAD51 protein degradation in triple-negative breast cancer cells via the ubiquitin-proteasome pathway.

Claims

1. Use of cryptotanshinone in combination with PARP inhibitors in the preparation of drugs for treating tumors.

2. The use according to claim 1, characterized in that: The PARP inhibitors are Olaparib, Rucaparib, Niraparib, and Talazoparib.

3. The use according to claim 1 or 2, characterized in that: The medicine is a medicine for treating breast cancer.

4. The use according to claim 3, characterized in that: The drug is a drug for treating triple-negative breast cancer; the PARP inhibitor is olaparib.

5. The use according to claim 4, characterized in that: The molar ratio of cryptotanshinone to olaparib is: (4-32): (4-32) or (0.5-4): (2-16); preferably, the molar ratio of cryptotanshinone to olaparib is: (0.5-2): (2-16).

6. The use according to claim 5, characterized in that: The molar ratio of cryptotanshinone to olaparib is 32:32 or 2:

1.

7. A pharmaceutical composition for treating breast cancer, characterized in that: It contains cryptotanshinone and olaparib, and the molar ratio thereof is: (4-32): (4-32) or (0.5-4): (2-16); preferably, the molar ratio of the cryptotanshinone to olaparib is: (0.5-2): (2-16).

8. The pharmaceutical composition according to claim 7, characterized in that: The invention contains cryptotanshinone and olaparib in a molar ratio of 32:32 or 2:

1.

9. The pharmaceutical composition according to claim 7 or 8, characterized in that: The drug contains cryptotanshinone and olaparib as active ingredients, and is added with pharmaceutically acceptable excipients or auxiliary ingredients to prepare a commonly used pharmaceutical preparation.

10. The pharmaceutical composition according to claim 9, characterized in that: The preparation is an oral preparation or an injection preparation.

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