Use of sildenafil in combination with tamoxifen for the preparation of an anti-breast cancer drug and composition

Abstract

The application discloses a use of sildenafil in preparation of an anti-breast cancer drug in combination with tamoxifen, and sildenafil can promote the entry of GAPDH into a nucleus and inhibit the proliferation of tamoxifen-resistant cells (MCF-7TR). The application also discloses an anti-breast cancer drug composition which can inhibit the proliferation of breast cancer tamoxifen-resistant cells (MCF-7TR) and thus reverse the sensitivity of the drug-resistant cells to tamoxifen, and the composition comprises sildenafil and tamoxifen. The application utilizes the targeted reversing effect of sildenafil on breast cancer cells resistant to tamoxifen, so that the breast cancer tamoxifen-resistant cells can be restored to be sensitive to tamoxifen, and a more economical, convenient and effective treatment option is brought to tumor patients.

Description

Technical Field

[0001] This invention relates to the use of sildenafil in the preparation of combination antitumor drugs, and more specifically, to the use of sildenafil in combination with tamoxifen in the preparation of anti-breast cancer drugs and compositions for use in combination anti-breast cancer treatment. Background Technology

[0002] According to the latest global cancer burden data released by the International Agency for Research on Cancer (IARC) of the World Health Organization in 2021, there were 2.26 million new cases of breast cancer worldwide, surpassing the 2.2 million cases of lung cancer, making breast cancer the leading cause of cancer death globally. Its mortality rate, however, is second only to lung cancer, making it the second leading cause of cancer death among women. Of the global new breast cancer cases, 420,000 were in China, accounting for 18.6% of the global total, making it the leading cause of cancer death among Chinese women.

[0003] Standardized treatments for breast cancer primarily include surgery, chemotherapy, radiotherapy, targeted therapy, and endocrine therapy. Endocrine therapy is mainly used for ER / PR positive patients, and approximately 60% of breast cancer patients require adjuvant endocrine therapy. Commonly used endocrine therapy drugs include estrogen receptor antagonists (tamoxifen, toremifene, fulvestrant) and aromatase inhibitors (AIs) (anastrozole, letrozole, exemestane, etc.). Aromatase inhibitors and fulvestrant are mainly used for postmenopausal patients and are expensive, with some drugs not covered by medical insurance. Tamoxifen treatment for 5-10 years is the standard endocrine therapy regimen recommended by major domestic and international guidelines for premenopausal hormone receptor-positive early breast cancer patients. Clinically, tamoxifen is currently the most widely used in major hospitals due to its affordability, high efficacy, and broad applicability (suitable for both premenopausal and postmenopausal patients).

[0004] Tamoxifen (Tam) is a synthetic nonsteroidal anti-estrogenic drug that inhibits estrogen-activated gene transcription and cell proliferation by competitively binding to estrogen receptors. It is suitable for patients with early and advanced ER+ breast cancer, especially premenopausal breast cancer. Tamoxifen can reduce mortality by 31% in ER+ premenopausal breast cancer patients. Although there are significant benefits to early treatment with tamoxifen, clinical application has shown that patients are prone to developing tamoxifen resistance, leading to tumor progression and metastasis. Reports indicate that the incidence of primary tamoxifen resistance in ER+ breast cancer patients is 30%, and the incidence of secondary tamoxifen resistance is 40%. Tamoxifen resistance (TR) can lead to breast cancer recurrence, metastasis, and even death. Studying the mechanism of tamoxifen resistance and finding ways to reverse it can not only guide the development of targeted drugs against tamoxifen resistance but also guide combination therapy regimens in the initial treatment selection, thereby avoiding primary resistance and treating secondary resistance. Current research suggests that tamoxifen resistance is associated with multiple factors, including: CYP2D6 gene polymorphism, androgen receptor expression, HER-2 expression, G protein-coupled estrogen receptors, miRNAs, lncRNAs, and various molecular pathways [ER signaling pathway, PI3K-AKT-mTOR signaling pathway, RTKS signaling pathway (HER2, EGFR, FGFR, and IGF1R), HH (Hedgehog) signaling pathway], etc. Recent studies have also suggested that metabolic reprogramming may be a significant cause of tamoxifen resistance. However, to date, there is still a lack of biomarkers that accurately indicate the efficacy of tamoxifen treatment and targets to overcome tamoxifen resistance.

[0005] Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) participates in glycolysis and is widely used as an internal control in protein, mRNA, and DNA studies as a product of housekeeping genes. The expression level of GAPDH in tumor tissues is not constant. Studies have shown that most tumor tissues highly express GAPDH, and the expression level varies among different tumor subtypes within the same tissue. The impact of GAPDH on tumor cell fate is of particular interest in tumor research; GAPDH in the nucleus and cytoplasm of tumor cells may have different functions. Many glycolysis-related enzymes, including GAPDH, are elevated in tumor tissues. GAPDH in the cytoplasm is considered to promote cell growth, proliferation, invasion, and drug resistance because it primarily promotes glycolysis.

[0006] GAPDH is not only present in the cytoplasm but can also translocate into the nucleus to exert its function. In tumor research, GAPDH has been observed to localize to the nucleus. Cytoplasmic GAPDH can bind to SIAH1 (Seven in absentia homolog 1) and enter the nucleus. SIAH1 is an E3 ligase with a nuclear ligase sequence; after entering the nucleus, it degrades nuclear proteins, inducing apoptosis. Cytoplasmic GAPDH can not only form a binding complex with SIAH1 and translocate into the nucleus but also promote the degradation of nuclear proteins by SIAH1, thus exerting its pro-apoptotic effect. Therefore, nuclear localization of GAPDH is an important pathway for inducing apoptosis. Some post-translational modifications can increase the binding capacity of cytoplasmic GAPDH to SIAH1, promoting its translocation into the nucleus and inducing apoptosis, such as nitric oxide (NO)-dependent nitrosylation. Other modifications can prevent GAPDH from entering the nucleus, thereby inhibiting apoptosis; AKT phosphorylation of GAPDH belongs to this category.

[0007] NO, as a messenger molecule, plays important functions in the body and cells, including energy metabolism and cell death. NO synthase (NOS) in cells can synthesize NO from L-arginine (L-Arg). Endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) have been reported to be expressed in breast tumor cells. In the Tamm-resistant ZR-75-1 (ZR-75-9a1) breast cancer cell line, the expression of both NOS was significantly reduced, suggesting that low concentrations of NO may be associated with Tamm resistance. The role of low concentrations of NO in promoting breast cancer cell growth may be related to its activation of AKT and / or ERKMAPK. It has also been reported that high concentrations of NO can inhibit the transcriptional activity of ERα through nitrosylation, but whether this affects Tamm sensitivity has not been investigated. To date, studies on the subcellular localization of GAPDH in breast tumors have only found that the loss of nuclear localization of GAPDH in triple-negative breast cancer cell lines can lead to resistance to chemotherapeutic drugs. In addition, GAPDH in the nuclei of rat mammalian tissue cells gradually increases during pregnancy. During this process, nuclear GAPDH, as a target gene-specific ERα coactivator, participates in the transcription of the estrogen-induced neutral amino acid transporter SNAT2 (Sodium-coupled neutral amiNOacid transporter 2) gene.

[0008] On the other hand, despite improvements in our understanding of the prevention, diagnosis, treatment, and prognosis of human diseases, translating this knowledge into new drugs is happening much slower than anticipated. New drug development projects are typically driven primarily by unmet clinical needs. Initial efforts often involve generating data in academia to support a hypothesis that could lead to the identification of new targets or treatments for a specific disease. However, drug discovery and development is resource- and time-intensive and highly multifaceted; recent estimates suggest it takes over 10 years and approximately $2 billion to bring a new drug to market. There is increasing pressure to develop cheaper and more efficient methods to bring safe and effective drugs to market. Within this framework, the drug discovery process is constantly evolving and adapting to improve efficiency, productivity, and profitability. Against this backdrop, the so-called "drug repurposing" is gaining increasing attention. This strategy involves identifying new therapeutic applications that differ from the original regulatory indications of already approved or investigational drugs. The benefits of this strategy include significant time and money savings, as the risk of failure is low since most preclinical and clinical trials, safety assessments, and drug formulations are already completed. Finally, and equally importantly, repurposing drugs can highlight new targets and pathways for further investigation. A prominent example is zidovudine, initially developed as an anticancer drug, but later identified in an in vitro screening library as the first FDA-approved treatment for HIV. Other notable examples include thalidomide, initially developed to treat morning sickness, which was later approved for the treatment of erythema nodosum leprosy and multiple myeloma based on pharmacological analysis.

[0009] Sildenafil is a selective inhibitor of phosphodiesterase type 5 (PDE5). Developed by Pfizer Inc., it was launched in June 1998 under the brand name Viagra. It is the main active ingredient in Viagra, and its chemical name is 1-[4-ethoxy-3-[5-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3d]pyrimidine)]benzenesulfonyl]-4-methylpiperazine citrate. Its molecular formula is C1. 22 H 30 N6O4S, molecular weight 474.57600. Sildenafil is an FDA-approved clinical drug with high safety, good prospects for development and utilization, few toxic side effects, and low price.

[0010] Sildenafil is a NO donor drug, currently mainly used clinically to treat erectile dysfunction in men. It promotes the release of nitric oxide (NO) by inhibiting phosphodiesterase type 5 (PDE5), which breaks down cGMP in the corpus cavernosum. NO activates guanylate cyclase, leading to increased levels of cyclic guanosine monophosphate (cGMP), causing relaxation of the smooth muscle in the corpus cavernosum and allowing blood flow. The role of sildenafil in breast cancer treatment is increasingly attracting the attention of researchers. Baravalle et al., through molecular and cellular studies, found that sildenafil can bind to human aromatase and inhibit its activity, potentially serving as a new target for breast cancer treatment. Ji Heng et al. found that certain concentrations of sildenafil homologues can inhibit the efflux function of P-gp protein in breast cancer MCF-7 / ADR cells and significantly reverse their resistance to doxorubicin. Fariba Hassanvand et al. found that sildenafil combined with cisplatin can enhance the cytotoxicity and anticancer effects of cisplatin. Sildenafil, as a PDE-5 inhibitor, can also be combined with cisplatin as an adjuvant therapy to reduce the dosage and side effects of cisplatin. Khaled Greish et al. found that when sildenafil was added to doxorubicin, it showed synergistic anticancer activity against 4T1 breast cancer cells in vitro. Adding 1, 30, and 100 μM Viagra to 1 μM doxorubicin increased the cytotoxic effect by 1.8-fold, 6.2-fold, and 21-fold, respectively, which was statistically significant. The concentrations of the fluorescent dye DiI and doxorubicin and its nano-formulations in the tumor tissue of 4T1 tumor-bearing mice increased by 2.7-fold. Compared with doxorubicin alone, animals treated with a combination of sildenafil citrate and doxorubicin showed a statistically significant 4.7-fold reduction in tumor size. These research results are all at the cellular or animal level and have not yet entered clinical application. Researchers in China have also begun to develop the use of sildenafil in the treatment of malignant tumors. For example:

[0011] Chinese invention application 202110255303.8 discloses the use of sildenafil as an antitumor drug sensitizer in the preparation of tumor chemotherapy drugs. It discovers that by using sildenafil as a sensitizer, and combining sildenafil with antitumor drugs such as oxaliplatin, irinotecan, or capecitabine through a carrier, an antitumor composition can be prepared, which can be used to effectively kill tumor cells such as colorectal cancer or drug-resistant tumor cells. It confirms that sildenafil as a sensitizer has a synergistic effect on the treatment of cancers such as colorectal cancer, gastric cancer, liver cancer, breast cancer, and prostate cancer.

[0012] Chinese invention application 201610884154.0 also discloses the application of low-dose sildenafil as an anti-tumor drug, with a daily oral dose of 2-25 mg, or an injection dose of 2-10 mg, or a topical dose containing less than 1% by weight of sildenafil citrate, applied to the treatment of malignant tumors, with an efficiency of more than 50% in inhibiting tumor growth, while without producing significant toxic side effects.

[0013] The above indicates that sildenafil does have a certain effect in anti-tumor activity. However, sildenafil is not a panacea or applicable to all types of cancer. For example, the role of sildenafil in tamoxifen-resistant breast cancer has not yet been reported.

[0014] Therefore, it is necessary to determine the specific indications for sildenafil in anti-tumor treatment. Summary of the Invention

[0015] One of the objectives of this invention is to provide a method that can overcome or reduce tamoxifen resistance in the treatment of breast cancer.

[0016] Another objective of this invention is to provide an anti-breast cancer drug composition capable of inhibiting the proliferation of tamoxifen-resistant breast cancer cells (MCF-7TR) and thereby reversing the sensitivity of the resistant cells to tamoxifen.

[0017] On the one hand, in order to achieve the above-mentioned objectives, the present invention provides the use of sildenafil in combination with tamoxifen in the preparation of an anti-breast cancer drug.

[0018] The above-mentioned technical solution of the present invention is based on the following mechanism of action:

[0019] The inventors discovered that in monolayers of MCF-7 cells obtained from Mammosphere, the level of GAPDH protein increased more than threefold compared to the original MCF-7 cells, suggesting that GAPDH expression may be related to the development and progression of breast cancer. The inventors further found that GAPDH nuclear localization not only independently indicates a better prognosis in ERα-positive patients but also significantly correlates with PR expression, which is a clinically used biomarker to indicate tam (Tam) sensitivity in breast cancer patients. These experimental results suggest that the absence of GAPDH nuclear localization may be used to indicate tam resistance.

[0020] Next, the inventors compared the subcellular localization of GAPDH in MCF-7 and MCF-7 TR cells using immunofluorescence technology. The results showed that compared with MCF-7 cells, the nuclear localization of GAPDH in MCF-7 TR cells was significantly reduced (or absent). These experimental results suggest that the loss of nuclear localization of GAPDH in breast cancer cell lines is also related to Tam resistance.

[0021] GAPDH first requires NO-dependent nitrosylation before it can bind to SIAH1 and translocate to the nucleus. The inventors examined the amount of NO and the expression level of SIAH1 in MCF-7 and MCF-7 TR cells. The results showed that compared to MCF-7 cells, the expression level of SIAH1 mRNA was increased in MCF-7 TR cells, while the amount of NO was significantly reduced. These experimental results suggest that the decrease in NO in cells may be the reason for the loss of GAPDH nuclear localization in MCF-7 TR cells.

[0022] To further verify that reduced NO levels may lead to the loss of GAPDH nuclear localization in the MCF-7 TR cell line, and that this loss may lead to Tam resistance, the inventors treated MCF-7 and MCF-7 TR cells with NO carriers, sildenafil, and / or Tam. Immunofluorescence staining results showed that sildenafil could drive GAPDH translocation into the nucleus in MCF-7 TR cells, and that MCF-7 TR cells were more sensitive to sildenafil than MCF-7 cells. This experimental result also suggests that strategies using NO donors or targeting GAPDH nuclear translocation could be used to intervene in Tam resistance.

[0023] Therefore, the absence of nuclear GAPDH can serve as a biomarker for tamoxifen resistance, while sildenafil, as a NO donor drug, can serve as a targeted drug to reverse tamoxifen resistance in breast cancer cells, thereby restoring their sensitivity to tamoxifen.

[0024] In this invention, the English name of the tamoxifen used is Tamoxifen, and its Chinese chemical name is (Z)-2-[4-(1,2-diphenyl-1-butene)phenoxy]-N,N-dimethylethylamine, with the molecular formula C. 26 H 29 NO, its structural formula is:

[0025]

[0026] In this invention, the English name of the sildenafil used is Sildenafil, and its Chinese chemical name is 1-[4-ethoxy-3-[5-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3d]pyrimidine)]benzenesulfonyl]-4-methylpiperazine citrate, with the molecular formula C. 22 H 30 N6O4S has the following structural formula:

[0027]

[0028] In the use of sildenafil in combination with tamoxifen to prepare an anti-breast cancer drug, sildenafil can promote GAPDH nucleus entry.

[0029] In the use of sildenafil in combination with tamoxifen to prepare an anti-breast cancer drug, sildenafil is able to inhibit the proliferation of tamoxifen-resistant cells (MCF-7 TR).

[0030] The inventors of this application discovered that sildenafil can inhibit the proliferation of tamoxifen-resistant breast cancer cells (MCF-7 TR) at a concentration of 2-20 μM, and reverse the sensitivity of the resistant cells to tamoxifen.

[0031] On the other hand, in order to achieve the above-mentioned objective of the present invention, the present invention also provides an anti-breast cancer drug composition capable of inhibiting the proliferation of tamoxifen-resistant breast cancer cells (MCF-7 TR) and thereby reversing the sensitivity of the resistant cells to tamoxifen, the composition comprising sildenafil and tamoxifen.

[0032] The compositions of the present invention may further include at least one pharmaceutically acceptable excipient.

[0033] In the compositions of the present invention, the composition may be in the form of an oral preparation, an injectable preparation, or a topical preparation. Preferably, the compositions of the present invention are in the form of an oral preparation, such as various oral tablets, capsules, and injections.

[0034] Preferably, the oral dose of sildenafil is 50 mg.

[0035] Preferably, the injection dose of sildenafil is 10 mg.

[0036] Through extensive experiments, the inventors of this invention discovered that sildenafil has a targeted reversal effect on tamoxifen-resistant breast cancer cells, and can restore the sensitivity of tamoxifen-resistant breast cancer cells to tamoxifen, providing cancer patients with a more economical, convenient and effective treatment option.

[0037] Compared with the prior art, in this invention, sildenafil at a concentration of 2-20 μM can inhibit the proliferation of tamoxifen-resistant breast cancer cells (MCF-7 TR) by promoting GAPDH nuclear translocation, thereby achieving the effect of reversing tamoxifen resistance and effectively controlling tumor growth.

[0038] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. However, these specific embodiments are merely descriptions of certain specific implementations of the present invention and are not intended to limit the present invention. Attached Figure Description

[0039] Figure 1 The expression and localization of GAPDH in breast tumor tissues are shown, including:

[0040] (a) No GAPDH expression; (b) GAPDH expression only in the cytoplasm; (c) GAPDH expression only in the nucleus; (d) GAPDH expression in both the cytoplasm and nucleus; (e) No expression in adjacent normal tissues;

[0041] Figure 2 The study showed the overall survival (OS) of GAPDH and ERα-positive breast cancer, where: (a) the difference in expression in the cytoplasm had no effect on patient survival; and (b) patients expressing GAPDH in the cell nucleus had better survival.

[0042] Figure 3 The immunofluorescence assay shows the localization of GAPDH in MCF-7 and MCF-7 TR cells;

[0043] Figure 4 Showing the mRNA expression level of SIAH1 in MCF-7 and MCF-7 TR;

[0044] Figure 5 The flow cytometry analysis showed the NO content in MCF-7 and MCF-7 TR.

[0045] Figure 6 The results showed that MCF-7 TR cells were abnormally sensitive to sildenafil, which can drive GAPDH translocation into the nucleus, including: (A) immunofluorescence detection of GAPDH expression and localization in MCF-7 TR cells after treatment with 10 μM sildenafil for 48 hours; (B) CCK-8 assay of cell proliferation capacity of MCF-7 and MCF-7 TR cells after treatment with 100 nM 4-OH TAM and / or 10 μM sildenafil for 48 hours.

[0046] Figure 7 The results show the inhibitory effect of different concentrations of sildenafil on MCF-7 TR cells;

[0047] Figure 8 The figure shows the results of sildenafil significantly enhancing the killing effect of tamoxifen on human breast cancer drug-resistant cells (MCF-7TR) in vitro;

[0048] Figure 9 The results show the effect of sildenafil significantly enhancing the killing effect of tamoxifen on human breast cancer resistant cells (MCF-7TR) in vivo, including: (a) growth of organoid culture cells from human breast cancer tamoxifen resistant specimens; (b) quantitative graph of organoid cell proliferation capacity. Detailed Implementation Plan

[0049] 1. The materials, equipment, and reagents used in this invention are as follows:

[0050] 1.1 Breast cancer cells

[0051] The tamoxifen-resistant breast cancer cells (MCF-7 TR) and MCF-7 cells used in this embodiment were obtained from the Central Laboratory of the Affiliated Cancer Hospital of Shantou University Medical College.

[0052] 1.2 Instruments

[0053] SWCJCO Clean Bench: Suzhou Purification Equipment Co., Ltd.

[0054] BB16 CO2 Incubator: Heraeus Corporation, Japan

[0055] TD5A-WS Large Capacity Centrifuge: Hunan Saite Xiangyi Co., Ltd.

[0056] GS-15R High-Speed ​​Tabletop Refrigerated Centrifuge: Eppendorf, Germany

[0057] DNP-9052BS Electric Heating Biochemical Incubator: Shanghai Xinmiao Medical Instrument Manufacturing Co., Ltd.

[0058] Thermostatic Magnetic Stirrer: Shanghai Sile Instrument Factory

[0059] LEICADMI 3000 B Inverted Microscope: Olympus Corporation, Japan

[0060] Cell culture flasks / plates: Corning Incorporated, USA

[0061] 96 / 12 / 6-well plate: Corning Corporation, USA

[0062] 15 / 50ml centrifuge tubes: Corning Corporation, USA

[0063] Pipettes (5ml): Corining Corporation, USA

[0064] RNase-free pipette tips: Axygen, USA

[0065] LEICA fluorescence microscope: Olympus Corporation, Japan

[0066] BDAccuri TM C6 analytical (four-color) flow cytometer: BD Biosciences, USA

[0067] Leica DM2000 All-Purpose Microscope: Leica GmbH, Germany

[0068] Olympus CX21 Microscope: Olympus Corporation, Japan

[0069] ABI 9700 PCR instrument: Applied Biosystems, USA

[0070] ABI 7300 Quantitative PCR Instrument: Applied Biosystems, USA

[0071] ELx800™ Fully Automated Microplate Reader: BioTek Instruments, USA

[0072] 1.3 Reagents

[0073] DMEM culture medium (Gibco)

[0074] Fetal bovine serum (FBS) Gibco

[0075] Penicillin-Streptomycin Mixture (100X) Beijing Solarbio Science & Technology Co., Ltd.

[0076] Thermo Fisher Scientific

[0077] NewSaiMei Biotechnology Co., Ltd.

[0078] Medical alcohol (75%, 95%) - Jiangxi Huikang Industrial Co., Ltd.

[0079] Anhydrous ethanol Tianjin Guangcheng Chemical Reagent Co., Ltd.

[0080] CCK8 Reagent Kit Beyotime Biotechnology Co., Ltd.

[0081] Immunofluorescence secondary antibody Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0082] DAPI dyes, Beyotime Biotechnology Co., Ltd.

[0083] GAPDH Antibody, ABCAM Antibody Company (UK)

[0084] Triton X-100 Thermo Fisher Scientific

[0085] Ready-to-use normal goat serum Wuhan Boster Biological Engineering Co., Ltd.

[0086] Immunohistochemical secondary antibody Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0087] Zhongshan Jinqiao Biotechnology Co., Ltd.

[0088] DAB chromogenic reagent kit, Zhongshan Jinqiao Biotechnology Co., Ltd.

[0089] DAF-FM DA (NO fluorescent probe) Beyotime Biotechnology Co., Ltd.

[0090] PrimeScript TMRT reagent kit with gDNAERaser (TAKARA)

[0091] Sildenafil Citrate (UK-92480 citrate) MCE Biotechnology, USA

[0092] 4-Hydroxytamoxifen, MCE Biotechnology, USA

[0093] Collagenase Type II, Thermo Fisher Scientific, USA

[0094] Cultrex RGF BME,Type 2 R&D Systems

[0095] Advanced DMEM / F12, Thermo Fisher Scientific, USA

[0096] DMEM GlutaMAX, Thermo Fisher Scientific, USA

[0097] Penicillin-streptomycin, Thermo Fisher Scientific, USA

[0098] GlutaMAX, Thermo Fisher Scientific, USA

[0099] BSA, fatty acid free, Sigma-Aldrich (USA)

[0100] DPBS, Thermo Fisher Scientific, USA

[0101] HEPES 1M Thermo Fisher Scientific, USA

[0102] Y-27632dihydrochloride AbMole Bioscience

[0103] Recovery cell culture freezing medium Thermo Fisher Scientific

[0104] Fetalbovine serum(FBS),heatinactivated Thermo Fisher Scientific

[0105] Red blood cell lysis buffer (Sigma-Aldrich, USA)

[0106] Dimethyl sulfoxide, Sigma-Aldrich (USA)

[0107] Breast cancer organoid culture medium from Kepuruisheng Biopharmaceutical Technology Co., Ltd.

[0108] Organoid detection kit CellTiter-Glo 3D CellViabilityAssay Promega

[0109] 2. The experimental methods used in this invention are as follows:

[0110] 2.1 Immunohistochemistry and patient survival analysis:

[0111] (1) Dewaxing: Xylene A, B, and C for 10 minutes each;

[0112] (2). Hydration: Hydrate with high-concentration to low-concentration alcohol for 5 minutes each, then rinse with double-distilled water for 5 minutes each time, x3 times;

[0113] (3). Microwave repair for 15 minutes (pH 6.0 citric acid solution), cool to room temperature, and rinse with PBS for 5 minutes x 3 times;

[0114] (4) Block endogenous peroxidase: 3% H2O2 for 30 minutes, followed by PBS washing for 5 minutes x 3 times;

[0115] (5) Block with 10% goat serum for 1 hour;

[0116] (6) Wipe dry, add primary antibody (GAPDH antibody, 1:200), incubate overnight at 4°C;

[0117] (7) Remove on the second day, allow to warm to room temperature for 1 hour, and rinse with PBS for 5 minutes x 3 times;

[0118] (8) Dry the container, add secondary antibody for 1 hour, and rinse with PBS for 5 minutes x 3 times.

[0119] (9). DAB staining (DAB preparation method: double distilled water: solution A: solution B = 20:1:1), observe the staining degree under the microscope until satisfactory, and then stop with tap water;

[0120] (10) Stain with hematoxylin for 3 minutes, wash with water, differentiate with 1% hydrochloric acid alcohol for 2 seconds, wash with water for 10 minutes, dehydrate with alcohol from low concentration to high concentration for 3 minutes each, clear with xylene A and B for 5 minutes each, and mount with neutral resin. Read the slide under a microscope, score and photograph for storage.

[0121] Both the cytoplasm and nucleus of GAPDH cells were stained. Results were semi-quantitatively analyzed based on staining intensity and the percentage of positive cells. Staining intensity was graded into four levels: negative, weak, moderate, and strong, scored as 0, 1, 2, and 3 points respectively. The percentage of positive cells was also graded into four levels: <5% (0 points), 5–25% (1 point), 26–50% (2 points), 51–75% (3 points), and >75% (4 points). The total score was calculated by multiplying the two scores, ranging from 0 to 12 points. In this study, ≤6 points were defined as low GAPDH expression, while ≥7 points were defined as high GAPDH expression. Outpatient follow-up and telephone follow-up were conducted. Kaplan-Meier survival analysis was used for disease-free survival and overall survival analysis. Since the relationship between GAPDH protein expression and prognosis in human breast cancer specimens has not been reported, FAMR collected tumor specimens from 338 breast cancer patients treated and followed up in detail at our facility. We then investigated the expression and subcellular localization of GAPDH using immunohistochemistry (IHC) (results are shown in...). Figure 1 The study found that GAPDH cell nuclear localization could independently indicate a better prognosis in ERα-positive patients (HR: 0.465, 95% CI: 0.253-0.855, P = 0.014). (See attached results). Figure 2 The expression of PR was significantly correlated with PR expression (χ2=5.705, P=0.017), which is a clinically used biomarker to indicate the sensitivity of breast cancer patients to Tam. Figure 1 and Figure 2 It can be seen that patients with GAPDH nuclear deletion have shorter overall survival, and the difference is statistically significant compared with those with nuclear expression. In this invention, overall survival is defined as the time from initial diagnosis to last follow-up or tumor-related death. P ≤ 0.05 is defined as statistically significant.

[0122] 2.2 Immunofluorescence assay

[0123] (1) Prepare the experimental materials and reagents. After drawing circles on the anti-detachment slides with an oil-based pen, soak them in alcohol for 30 minutes, then place them in a clean bench in a humidified, light-protected environment and irradiate with UV light for 30 minutes. Around 6 PM, drop the cells into the circles and spread them evenly. (Mix 5ml DMEM + 500ul trypsin, then add 500ul to 2ml DMEM, 300ul / slide)

[0124] (2) At 8 a.m. the next day, the slide was placed in PBS for 3 min x 3 times, and then fixed with 4% paraformaldehyde for 15 minutes.

[0125] (3) After 3 min x 3 times with PBS, 0.5% (100 ml PBS + 0.5 ml Triton X-100) Triton X-100 was used for 20 min perforation.

[0126] (4) After PBS for 3 min x 3 times, absorb the PBS with absorbent paper and block with goat serum for 30 min (60ul / tablet).

[0127] (5) After blotting the blocking solution with absorbent paper, do not wash. Add primary antibody (100ul) or PBS as a control directly and humidify overnight at 4°C.

[0128] (6) On the second day, PBST was applied 3 times for 3 minutes. After blotting with absorbent paper, the secondary antibody (1:500) was added and then incubated at room temperature in the dark for 1 hour.

[0129] (7) PBST 3min x 3 times, DAPI 5ul staining nucleus for 5min, long coverslip soaked in 95% alcohol dried and then sealed, gently pressed with tweezers.

[0130] (8) Use absorbent paper to dry the water on both sides of the slide, store it in the dark at 4 degrees Celsius or observe and photograph it immediately with a fluorescence microscope.

[0131] The inventors compared the results of MCF-7 and MCF-7 TR using immunofluorescence technology (see results). Figure 3 The subcellular localization of GAPDH in cells was investigated, and the results showed that compared with MCF-7 cells, the nuclear localization of GAPDH in MCF-7 TR cells was significantly reduced (or absent).

[0132] 2.3 Quantitative Real-Time PCR (RT-qPCR):

[0133] Cell RNA extraction

[0134] (1) Pour out the culture medium and wash once with 1×PBS. Add 1 ml of RNAiso Plus to every 10 cm2 of cultured DISH cells, and gently shake to ensure that the lysis buffer is evenly distributed on the cell surface;

[0135] (2) Transfer the lysate containing cells to a centrifuge tube and repeatedly pipette until there is no obvious precipitate in the lysate;

[0136] (3) Let stand at room temperature for 5 minutes, then isolate RNA from nucleoprotein;

[0137] (4) Add 200 μL of chloroform, tighten the cap, and shake for 15 seconds.

[0138] (5) Let stand at room temperature for 5 minutes;

[0139] (6). Centrifuge at 4℃, 12000 rpm for 15 minutes;

[0140] (7) Aspirate the supernatant into another EP tube, add an equal volume of isopropanol to the supernatant, and let stand at room temperature for 10 minutes;

[0141] (8). Centrifuge at 4℃, 12000 rpm for 10 minutes;

[0142] (9) Add 1 ml of 75% ethanol, centrifuge at 4°C and 12000 rpm for 5 minutes;

[0143] (10) Aspirate the supernatant, dry in a clean bench for 5 minutes after opening the lid, add 50ul DEPC water to each tube, measure the concentration, and store in a -80 degree refrigerator.

[0144] Reverse transcription to synthesize single-stranded cDNA

[0145] (1). Prepare mix in a 0.2 mL RNase-free microcentrifuge tube:

[0146]

[0147] (2) Add 0.8 μg of RNA to each of the above microcentrifuge tubes;

[0148] (3) Make up the remaining volume to 10 μL with RNase-free double-distilled water;

[0149] (4). After brief centrifugation, the product is placed on the machine at 37°C for 15 minutes, then at 85°C for 5 seconds, and at 4°C for 20 minutes.

[0150] (5) After reverse transcription is completed, add 50 μL of double-distilled water and store at -20°C.

[0151] Real-Time PCR

[0152] (1) Protect from light, when preparing the mix:

[0153]

[0154] (7) After a short centrifugation, dispense 9 μL / well into 0.2 μL EP tubes, add 1 μL of cDNA sample to each well, and then a short centrifuge to mix.

[0155] (8) The PCR reaction conditions were: 95℃ for 30 seconds, 95℃ for 5 seconds, 60℃ for 30 seconds, 95℃ for 15 seconds, 60℃ for 30 seconds, 95℃ for 15 seconds, for a total of 40 cycles.

[0156] (9) Specificity detection of PCR products was performed using 1.5% agarose gel electrophoresis. Amplified products were loaded at 6 μL per well, and 0.5×TBE electrophoresis buffer was added. Agarose gel electrophoresis was performed at a constant voltage of 5 V / cm for 45 min. Amplified bands were observed using a gel imaging system, and images were saved. The CT values ​​of each sample were exported using the software included with the ABI 7300 system, and then analyzed using 2... -△△ctThe method was used to analyze the differential expression of the SIAH1 gene.

[0157] The inventors examined the levels of NO and SIAH1 expression in MCF-7 and MCF-7 TR cells. Real-time quantitative PCR (RT-PCR) results showed that SIAH1 mRNA expression was increased in MCF-7 TR cells compared to MCF-7 cells (see results below). Figure 4 The amount of NO decreased significantly (see results). Figure 5 These results suggest that the decrease in NO in cells may be the cause of the loss of GAPDH nuclear localization in MCF-7 TR cells.

[0158] The inventors treated MCF-7 and MCF-7 TR cells with sildenafil and / or Tam, using NO carriers in cells. Immunofluorescence staining results showed that sildenafil could drive GAPDH translocation into the nucleus in MCF-7 TR cells; moreover, MCF-7 TR cells were more sensitive to sildenafil than MCF-7 cells (see results). Figure 6 This result also suggests that strategies involving NO donors or targeting GAPDH into the nucleus may be used to intervene in Tam resistance.

[0159] 2.4 Flow cytometry:

[0160] (1) Digestion with 0.25% trypsin to break down the cells into individual units;

[0161] (2). Centrifuge to collect cells, discard the supernatant, and wash the cells twice with pre-cooled PBS;

[0162] (3) Add 3 ml of pre-cooled 70% ethanol to the cell pellet and fix overnight at 4°C;

[0163] (4) Centrifuge to collect cells, wash cells twice with 3 mL PBS, and add 500 μL PBS containing DAF-FM DA (concentration of 5 μmol / L).

[0164] (5) Invert and mix every 3-5 minutes to ensure the probe and cells are in full contact. Incubate at 37°C in the dark for 20 minutes.

[0165] (6) Perform flow cytometry and analyze the results.

[0166] 2.5 Cytotoxicity assay

[0167] CCK8 Principle: WST-8 is the main chemical in the CCK8 kit. In living cells, it is oxidized and reduced by NAD+ to form a water-soluble yellow formazan product. The more living cells there are, the more formazan is produced, and the darker the color will be. Therefore, the darker the stain on the cells, the more living cells there are. It is often used to detect drug toxicity to cells and to plot cell proliferation-time curves.

[0168] (1) Plating: Take MCF-7 TR cells in the logarithmic growth phase, digest and resuspend them with trypsin, count the cells under an inverted microscope, and add them into two different 96-well plates, so that the number of cells in each well is about 4000, and put them in a cell culture incubator overnight.

[0169] (2) Preparation of culture medium: Add sildenafil to serum-containing culture medium to make a final concentration of 1 μM. Then take 9 centrifuge tubes and prepare 6 different concentration gradients of culture medium with 10 μM CPT medium: 1, 10, 100, 1000, 2000, 4000 nM.

[0170] (3) On the second day, take out the 96-well plate, aspirate the old culture medium and wash it twice with sterile physiological saline, discard the waste liquid, add 200 μl of prepared drug-containing culture medium to each well, and leave 6 blank wells without any cells or drugs as blank controls. Put the 96-well plate back into the incubator to continue culturing.

[0171] (4) After 48 hours, remove the 96-well plate and prepare a medium containing 10% CCK8 in advance. Aspirate the old medium and add 100 μl of the solution (including the original 6 blank wells) to each well using a pipette. Place the plate in an incubator and continue culturing. After two and a half hours, remove the plate and measure the absorbance.

[0172] (5) Calculation formula: Inhibition rate of the drug on cells = (OD value of experimental group - OD value of blank control group) / (OD value of control group - OD value of blank control group) × 100%, and the result is as follows: Figure 6 , 7 Figures 8 and 9 both indicate that sildenafil has an inhibitory effect on MCF-7 TR. Figure 7 The results show the inhibitory effect of different concentrations of sildenafil on MCF-7 TR cells. Figure 8 The figure shows the results of sildenafil significantly enhancing the killing effect of tamoxifen on human breast cancer drug-resistant cells (MCF-7 TR) in vitro.

[0173] 2.6 Organoid Experiments

[0174] Breast cancer tissue was removed intraoperatively. Each tissue sample (<3-4 cm3) was transferred to a 15 ml centrifuge tube containing D-BSA culture medium and stored at 4°C for 24 hours for tissue separation. The tissue was placed in a sterile culture dish, and necrotic tissue, fat, and blood vessels were removed. The tissue was divided into individual small pieces (0.5-1 mm3) using two scalpels. The tissue fragments and D-BSA culture medium were aspirated into a 15 ml centrifuge tube and repeatedly blown up and down. The tube was allowed to stand at room temperature for 3 minutes. Approximately 5 ml of culture medium remained, and the supernatant was discarded. The volume was then increased to 10 ml, and the tissue was repeatedly washed by blowing and aspirating. This process was repeated 3 times. After standing at room temperature for 3 minutes, aspirate the supernatant and add approximately 5 ml of breast cancer organoid extension culture medium, 250 μl of collagenase II (20 mg / ml stock solution, final concentration 1 mg / ml), and 5 μl of ROCK inhibitor (10 mM stock solution, final concentration 10 μM). Tilt the centrifuge tube 15° and place it on a track shaker at 37°C (140 rpm) for 30-40 minutes. Add 1 ml of fetal bovine serum to terminate the digestion reaction. Mechanically separate the tissue using a pre-washed 5 ml pipette by pipetting up and down at least 20 times. Filter the tissue suspension through a 100 μm filter. For any unfiltered tissue, aspirate and add D-BSA, repeating the pipetting and aspiration process to mechanically separate the tissue. Then, aspirate the tissue suspension again and filter, repeating this process 3-4 times to minimize tissue waste. Centrifuge the filtrate at 500g for 10 minutes at 4°C, and aspirate as much supernatant as possible. Add 60-80 μL of melted BME, gently mix to avoid air bubbles, and repeat. Transfer 15 μL of the mixture to each well of a 24-well plate (2 drops per well). Immediately invert the plate and incubate at room temperature for 5 minutes to prevent organoid clumps from settling. Incubate at 37°C for 30 minutes to solidify the BME. After incubating the BME upright, add 500 μL of breast cancer organoid extension medium to each well and incubate at 37°C with 5% CO2. Change the extension medium every 2-4 days. Passage the organoids into 96-well plates. After 24 hours of culture, treat each well with tamoxifen. Perform tamoxifen treatment at 24, 72, 120, and 168 hours post-treatment, with three replicates for each treatment. Follow the manufacturer's instructions and add 100 μL of tamoxifen to each well. Mix 3DReagent vigorously up and down for 5 minutes to lyse cells, incubate at room temperature for 25 minutes, and record the fluorescence intensity of each well using a fluorescence spectrophotometer. Please refer to the experimental results. Figure 9 As can be seen, with the administration of the drug, the growth of tumors in tamoxifen-resistant patients was significantly inhibited.

Claims

1. The use of sildenafil in combination with tamoxifen in the preparation of anti-breast cancer drugs, among which, The sildenafil can inhibit the proliferation of tamoxifen-resistant breast cancer cells (MCF-7 TR), and the concentration of sildenafil is 2-20 μM.

2. An anti-breast cancer pharmaceutical composition capable of inhibiting the proliferation of tamoxifen-resistant breast cancer cells (MCF-7 TR) and thereby reversing the sensitivity of the resistant cells to tamoxifen, the composition comprising sildenafil, tamoxifen and at least one pharmaceutically acceptable excipient.

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