Application of combination of paclitaxel and cephalomannine in preparation of anti-breast cancer product

Abstract

The invention discloses an application of combination of paclitaxel and cephalomannine in preparation of an anti-breast cancer product, and particularly provides an application of combination of paclitaxel and cephalomannine in preparation of a medicine for treating and / or preventing breast cancer. The invention finds that the combination of paclitaxel and cephalomannine has the effect of enhancing the breast cancer treatment effect, not only can promote tumor cell death through a traditional apoptosis pathway, but also can enhance the anti-tumor effect by remarkably activating cell death pathways such as pyroptosis (NLRP3 / GSDMD) and necrotic apoptosis (RIPK1 / RIPK3 / MLKL). The treatment strategy of the synergistic effect of various cell death mechanisms can alleviate the reduction of the curative effect caused by the drug resistance of the traditional paclitaxel, overcomes the drug resistance problem in the traditional paclitaxel single-drug treatment, and has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly relates to the application of the combination of paclitaxel and cephalomannine in the preparation of anti-breast cancer products. Background Art

[0002] Breast cancer is the most common malignant tumor among women globally and is also one of the major diseases seriously threatening women's health. The heterogeneity of breast cancer leads to significant differences in its treatment and prognosis. Among them, triple-negative breast cancer (TNBC) is considered the most challenging subtype due to the lack of estrogen receptor (ER), progesterone receptor (PR), and HER2 expression, with high invasiveness and high recurrence rate. Since TNBC does not express the above receptors, traditional targeted therapy and endocrine therapy are ineffective for it, so chemotherapy has become the main treatment method. However, TNBC usually develops strong drug resistance to chemotherapy, especially in the recurrence and metastasis stages, and the efficacy of single chemotherapy is significantly reduced. There is an urgent need to find a combined treatment strategy with multiple mechanisms and multiple targets.

[0003] Paclitaxel is a taxane compound extracted from plants of the genus Taxus and is an important drug for the treatment of solid tumors. Paclitaxel can bind to tubulin and inhibit microtubule depolymerization, resulting in cell cycle arrest at the G2 / M phase, ultimately inhibiting the proliferation of tumor cells and inducing apoptosis. However, paclitaxel often faces problems of reduced efficacy and drug resistance in monotherapy, which limits its long-term application effect. Cephalomannine is another taxane compound derived from the branches and leaves of Taxus, with a relatively high content and good anti-tumor activity in Taxus. The anti-tumor mechanism of cephalomannine mainly includes inhibiting the expression of protein genes, inducing cell differentiation and apoptosis, etc., and shows great potential in inducing pyroptosis and necrosis. However, the effect of cephalomannine alone is weak, and it has not been developed into a related drug clinically.

[0004] Related studies have shown that there is extensive interaction and regulation among multiple programmed cell death pathways (such as apoptosis, pyroptosis, and necroptosis), forming a complex cell death network, which can provide a theoretical basis for multi-target intervention in anti-cancer treatment. Among them, apoptosis, pyroptosis, and necroptosis, as the most representative forms of programmed death, together constitute the panoptosis regulatory system. Apoptosis is the most classical form of programmed cell death, which regulates the permeability of the outer mitochondrial membrane through the Bax / Bcl-2 signaling pathway, induces the release of cytochrome C, and activates the caspase cascade reaction, ultimately leading to the degradation of the cytoskeleton and nuclear structure, thereby clearing damaged or abnormal cells. Pyroptosis is a form of programmed death dependent on caspase-1 / NLRP3, which causes the rupture of the cell membrane and the release of inflammatory factors such as IL-1β and IL-18, thereby further activating the immune response. Necroptosis depends on the RIPK1 / RIPK3 / MLKL signaling pathway and is regulated through the RIPK1 / RIPK3 / MLKL pathway, ultimately leading to the phosphorylation of MLKL, mediating cell membrane perforation and lysis. In this process, necroptosis can either be secondary to the failure of apoptosis or enhanced inflammatory stimulation, or can play a role in the immune response as a supplementary mechanism. Its unique activation of inflammatory signals further promotes the recognition and clearance of tumor cells by the immune system. These diverse cell death mechanisms provide multiple targets and treatment ideas for anti-cancer treatment. However, there are currently no drugs in clinical practice that specifically regulate panoptosis for breast cancer treatment.

[0005] Based on this, there is an urgent need to seek a drug that can inhibit the growth of breast cancer cells through the panoptosis regulatory system to improve the treatment effect of breast cancer. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention proposes the application of the combination of paclitaxel and cephalomannine in the preparation of anti-breast cancer products, which reveals a new use of the combination of paclitaxel and cephalomannine in the synergistic treatment of triple-negative breast cancer. The combination of the two can induce tumor cell death by regulating panoptosis. Specifically, the present invention finds that the combination of paclitaxel and cephalomannine has the effect of enhancing the treatment effect of breast cancer. It can not only promote tumor cell death through traditional apoptotic pathways (such as the Bcl-2 / Bax pathway, the JNK / p53 pathway), but also enhance the anti-tumor effect by significantly activating cell death pathways such as pyroptosis (NLRP3 / GSDMD) and necroptosis (RIPK1 / RIPK3 / MLKL). This treatment strategy that synergistically acts through multiple cell death mechanisms can not only effectively inhibit the proliferation of tumor cells, but also help to reduce the decline in treatment efficacy caused by drug resistance, and has broad application prospects.

[0007] In the first aspect of the present invention, there is provided the use of paclitaxel in combination with cephalomannine in the preparation of a medicament for treating and / or preventing breast cancer.

[0008] The use according to the embodiments of the present invention has at least the following beneficial effects:

[0009] Through experiments, the present invention found that, compared with the administration of paclitaxel or cephalomannine alone, the combination of paclitaxel and cephalomannine has a synergistic effect in anti-breast cancer, and can significantly improve the inhibitory effect on breast cancer cells. At the same time, the present invention found that the combined treatment of paclitaxel and cephalomannine can not only promote tumor cell death through traditional apoptotic pathways (such as the Bcl-2 / Bax pathway, the JNK / p53 pathway), but also enhance the anti-tumor effect by significantly activating cell death pathways such as pyroptosis (NLRP3 / GSDMD) and necroptosis (RIPK1 / RIPK3 / MLKL). This treatment strategy that synergistically acts through multiple cell death mechanisms can effectively inhibit the proliferation of tumor cells and alleviate the decline in therapeutic efficacy caused by drug resistance, overcoming the drug resistance problem in the previous single-agent treatment of paclitaxel, and is expected to be widely used in the treatment of tumors related to programmed death regulatory mechanisms.

[0010] In some embodiments of the present invention, the medicament for treating and / or preventing breast cancer has at least one of the uses (A1) to (A3):

[0011] (A1) Reducing the survival rate of breast cancer cells;

[0012] (A2) Inhibiting the proliferation of breast cancer cells;

[0013] (A3) Promoting the pan-apoptosis of breast cancer cells.

[0014] In some embodiments of the present invention, the inhibition of the proliferation of breast cancer cells includes promoting G2 / M phase arrest. The G2 / M phase is an important stage in the cell cycle, involving the preparation for cell division and the actual division process.

[0015] In some embodiments of the present invention, the pan-apoptosis includes pyroptosis, apoptosis and / or necroptosis.

[0016] In some embodiments of the present invention, the promotion of the pan-apoptosis of breast cancer cells includes at least one of (B1) to (B5):

[0017] (B1) Activating cell death signals by promoting DNA damage in breast cancer cells;

[0018] (B2) Promoting apoptosis of breast cancer cells through the JNK / p53 pathway;

[0019] (B3) Promoting apoptosis of breast cancer cells through the mitochondrial-dependent apoptotic pathway;

[0020] (B4), promoting necrosis of breast cancer cells through the RIPK1 / RIPK3 / MLKL pathway;

[0021] (B5), promoting the death of breast cancer cells through the Caspase-8 / NLRP3 / GSDMD pyroptosis signaling pathway.

[0022] In some embodiments of the present invention, the promotion of pan-apoptosis of breast cancer cells includes at least one of (C1) to (C5):

[0023] (C1), increasing the expression level of γ-H2AX;

[0024] (C2), increasing the expression levels of p-JNK / JNK, p53, p-DRP1, Cleaved Caspase7, Cleaved PARP, p21 and / or decreasing the relative expression level of Bcl-2 / Bax;

[0025] (C3), increasing the expression level of p-RIPK1, p-RIPK3, P-MLKL or HMGB1;

[0026] (C4), increasing the expression level of Caspase-8, NLRP3, GSDMD or IL-18;

[0027] (C5), increasing the extracellular secretion levels of IL-18, IL-1β or TNF-α.

[0028] In some embodiments of the present invention, the mass ratio of paclitaxel to cephalomannine is 1:0.1 to 10.

[0029] In some embodiments of the present invention, the mass ratio of paclitaxel to cephalomannine is 1:0.5 to 1.5.

[0030] In some preferred embodiments of the present invention, the mass ratio of paclitaxel to cephalomannine is 1:0.8 to 1.2. More preferably 1:1.

[0031] In some embodiments of the present invention, the breast cancer includes triple-negative breast cancer.

[0032] In a second aspect of the present invention, there is provided an anti-breast cancer pharmaceutical composition, the active substances of which comprise paclitaxel and cephalomannine.

[0033] The pharmaceutical composition according to the embodiments of the present invention has at least the following beneficial effects:

[0034] Through experiments, it is found in the present invention that the combined treatment of paclitaxel and cephalomannine has a synergistic effect in inhibiting the growth of triple-negative breast cancer cells and promoting cancer cell death, which is significantly better than single administration. Moreover, this pharmaceutical composition can regulate cell death through multiple pathways such as activating apoptosis, pyroptosis, and necroptosis, and can effectively enhance the anti-tumor efficacy through the regulation of pan-apoptosis, and overcome the shortcoming of weak efficacy of single-component treatment.

[0035] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0036] In some embodiments of the present invention, the pharmaceutically acceptable excipient is selected from at least one of disintegrants, diluents, lubricants, binders, wetting agents, flavoring agents, suspending agents, surfactants, and preservatives.

[0037] Preferably, the disintegrant is selected from at least one of corn starch, potato starch, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked carboxymethyl cellulose sodium, carboxymethyl cellulose, carboxymethyl cellulose calcium, and alginic acid;

[0038] Preferably, the diluent is selected from at least one of lactose, sucrose, mannitol, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose;

[0039] Preferably, the lubricant is selected from at least one of colloidal silicon dioxide, magnesium stearate, calcium stearate, stearic acid, talc powder, and anhydrous silica gel;

[0040] Preferably, the binder is selected from at least one of gum arabic, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose, and polyvinylpyrrolidone;

[0041] Preferably, the wetting agent is selected from sodium lauryl sulfate;

[0042] Preferably, the flavoring agent is selected from at least one of aspartame, stevioside, sucrose, maltitol, and citric acid;

[0043] Preferably, the suspending agent is selected from at least one of gum arabic, gelatin, methyl cellulose, sodium carboxymethyl cellulose, hydroxymethyl cellulose, and aluminum stearate gel;

[0044] Preferably, the surfactant is selected from at least one of lecithin, sorbitan monooleate, and glycerol monostearate;

[0045] Preferably, the preservative is selected from at least one of methyl p-hydroxybenzoate or propyl p-hydroxybenzoate.

[0046] In some embodiments of the present invention, the dosage form of the pharmaceutical composition is at least one of solid preparations, liquid preparations, and semi-solid preparations.

[0047] In some embodiments of the present invention, the solid preparations include tablets, granules, powders, and capsules;

[0048] In some embodiments of the present invention, the liquid preparations include injections;

[0049] Preferably, the semi-solid preparations include ointments and creams.

[0050] Other features and advantages of the present invention will be described in the subsequent specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The present invention will be further described below in conjunction with the drawings and examples, wherein:

[0052] Figure 1 are the chemical structures of paclitaxel (PubChem CID: 36314) and cephalomannine (PubChem CID: 6436208) of the present invention, where A is paclitaxel and B is cephalomannine.

[0053] Figure 2 are the experimental results of the effects of paclitaxel, cephalomannine and their combined treatment on the survival rate of MDA-MB-231 cells, where A-E correspond to the detection results under different concentration treatment conditions. For example, P0.5 represents the treatment concentration of paclitaxel is 0.5 ng / mL; C0.5 represents the treatment concentration of cephalomannine is 0.5 ng / mL, and so on.

[0054] Figure 3 are the experimental results of the effects of paclitaxel, cephalomannine and their combined treatment on the apoptosis of MDA-MB-231 cells. Among them, A is the flow cytometry analysis result of the control group, B is the flow cytometry analysis result of the P1 group, C is the flow cytometry analysis result of the C1 group, D is the flow cytometry analysis result of the P1C1 group (combined treatment group). P1 represents the treatment concentration of paclitaxel is 1 ng / mL; C1 represents the treatment concentration of cephalomannine is 1 ng / mL, and the data are expressed as mean ± standard deviation.

[0055] Figure 4 are the experimental results of the effects of paclitaxel, cephalomannine and their combined treatment on the cell cycle distribution of MDA-MB-231 cells. A is the cell cycle distribution result of the control group, B is the cell cycle distribution result of the P1 group, C is the cell cycle distribution result of the C1 group, D is the cell cycle distribution result of the P1C1 group (combined treatment group), and E is the statistical result of the proportions of MDA-MB-231 cells in the G1, S and G2 / M phases in different treatment groups. The data are expressed as mean ± standard deviation.

[0056] Figure 5 Experimental results of the effects of paclitaxel, cephalomannine, and their combination treatment on the expression of DNA damage marker γ-H2AX in MDA-MB-231 cells. Among them, A is the Western blot detection results of γ-H2AX and H2AX, and B is the statistical analysis results of the relative expression levels of the ratio of γ-H2AX / H2AX. The data are expressed as mean ± standard deviation, **P<0.01 vs Con.

[0057] Figure 6 Experimental results of the effects of paclitaxel, cephalomannine, and their combination treatment on the expression of apoptosis-related proteins in MDA-MB-231 cells. Among them, A is the Western blot detection results of apoptosis-related proteins p-JNK / JNK, p53, Bcl-2, Bax, p-DRP1, DRP1, Cleaved Caspase7, Cleaved PARP, and p21. B is the statistical analysis results of the relative expression levels of p-JNK / JNK. C is the statistical analysis results of the relative expression levels of p53. D is the statistical analysis of the relative expression levels of the ratio of Bcl-2 / Bax. E is the statistical analysis results of the relative expression levels of the ratio of p-DRP1 / DRP1. F is the statistical analysis results of the relative expression levels of Cleaved Caspase7. G is the statistical analysis results of the relative expression levels of Cleaved PARP. H is the statistical analysis results of the relative expression levels of p21. The data are expressed as mean ± standard deviation, **P<0.01 vs Con, *P<0.05 vs Con, ##P<0.01 vs P1, #P<0.05 vs P1, &&P<0.01 vs C1, &P<0.05 vs C1.

[0058] Figure 7 Experimental results of the effects of paclitaxel, cephalomannine, and their combination treatment on the expression of necrosis-related proteins in MDA-MB-231 cells. Among them, A is the Western blot detection results of necrosis-related proteins p-RIPK1, p-RIPK3, P-MLKL, and HMGB1. B is the statistical analysis results of the relative expression levels of p-RIPK1. C is the statistical analysis results of the relative expression levels of the ratio of p-RIPK3 / RIPK3. D is the statistical analysis of the relative expression levels of the ratio of P-MLKL / MLKL. E is the statistical analysis results of the relative expression levels of HMGB1. The data are expressed as mean ± standard deviation, **P<0.01 vs Con, *P<0.05 vs Con, ##P<0.01 vs P1, #P<0.05 vs P1, &&P<0.01 vs C1, &P<0.05 vs C1.

[0059] Figure 8 Experimental results of the effects of paclitaxel, cephalomannine and their combined treatment on pyroptosis-related proteins and the secretion levels of extracellular IL-18, IL-1β and TNF-α in MDA-MB-231 cells. Among them, A shows the Western blot detection results of Caspase-8, pyroptosis-related proteins NLRP3, GSDMD and IL-18; B shows the statistical analysis of the relative expression level of Caspase-8; C shows the statistical analysis of the relative expression level of NLRP3; D shows the statistical analysis of the relative expression level of the ratio of n-GSDMD / GSDMD; E shows the statistical analysis of the relative expression level of HMGB1; F shows the ELISA detection results of extracellular IL-18; G shows the ELISA detection results of extracellular IL-1β; H shows the ELISA detection results of extracellular TNF-α. Data are expressed as mean ± standard deviation, **P<0.01 vs Con, *P<0.05 vs Con, ##P<0.01 vs P1, #P<0.05 vs P1, &&P<0.01 vs C1, &P<0.05 vs C1.

[0060] Figure 9 Experimental results of the effects of pyroptosis, apoptosis and necroptosis inhibitors on the survival rate of MDA-MB-231 cells after treatment with paclitaxel, cephalomannine and their combination. Among them, A shows the results of the effect of the pyroptosis inhibitor Disulfiram on the survival rate of MDA-MB-231 cells treated with paclitaxel, cephalomannine and their combination; B shows the results of the effect of the apoptosis inhibitor Z-VAD-FMK on the survival rate of MDA-MB-231 cells treated with paclitaxel, cephalomannine and their combination; C shows the results of the effect of the necroptosis inhibitor Necrostatin-1 on the survival rate of MDA-MB-231 cells treated with paclitaxel, cephalomannine and their combination. P represents paclitaxel, P1 represents a concentration of 1 ng / mL, C represents cephalomannine, and C1 represents a concentration of 1 ng / mL. Data are expressed as mean ± standard deviation, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, ns indicates no significant difference.

[0061] Figure 10 Experimental results of the effects of different treatment groups on tumors. Among them, A shows the statistical results of the food intake of different treatment groups; B shows the statistical results of the water intake of different treatment groups; C shows the statistical results of the kidney coefficients of different treatment groups; D shows the statistical results of the liver coefficients of different treatment groups; E shows the maximum diameter of the tumors in different treatment groups (mm 3) Distribution statistics results, F is the trend of tumor growth in different treatment groups, and G is the photo after tumor resection in different treatment groups. Data are expressed as mean ± standard deviation, **P<0.01 vs Con, *P<0.05 vs Con, ##P<0.01 vs P1, #P<0.05 vs P1, &&P<0.01 vs C1, &P<0.05 vs C1. Detailed implementation mode

[0062] The following will clearly and completely describe the concept and technical effects generated by the present invention in combination with the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0063] The terms "preferably", "more preferably", etc. in the present invention refer to the embodiments of the present invention that can provide certain beneficial effects in certain cases. However, in the same or other cases, other embodiments may also be preferred. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not available, nor is it intended to exclude other embodiments from the scope of the present invention.

[0064] When a numerical range is disclosed herein, the above range is considered continuous and includes the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to an integer, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0065] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the related listed items.

[0066] In the description of the present invention, the description of reference terms such as "one embodiment", "some embodiments", "schematic embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0067] In the embodiments of the present invention, the biochemical reagents used include MTT cell proliferation and cytotoxicity assay kits (Nanjing Jiancheng Bioengineering Institute, China). Mouse IL-1β ELISA Kit (1210122, Dayou, China). Mouse IL-18 ELISA Kit (MOES01225, Genie, USA). Mouse TNF-α Precoated ELISA Kit (1217202, Genie, USA). Cell Cycle and Apoptosis Analysis Kit (40301ES60, Yeasen, China). Annexin V-FITC / PI Apoptosis Detection Kit (40302ES60, Yeasen, China).

[0068] The antibodies used included β-actin (A1978, Sigma-Aldrich, USA), GAPDH (2118T, CST, USA), p21 Waf1 / Cip1 (E2R7A, CST, USA), NLRP3 (D4D8T, CST, USA), Gasdermin D (E9S1X, CST, USA), Cleaved Gasdermin D (Asp275) (E7H9G, CST, USA), MLKL (D2I6N, CST, USA), Phospho-MLKL (Ser358) (D6H3V, CST, USA), Caspase-1 (E2Z1C, CST, USA), Cleaved Caspase-1 (Asp297) (D57A2, CST, USA), SAPK / JNK (9252, CST, USA), Phospho-SAPK / JNK (Thr183 / Tyr185) (9251, CST, USA), RIP (D94C12) (3493T, CST, USA), Phospho-RIP (Ser166) (D1L3S, CST, USA), RIP3 (E1Z1D, CST, USA), Phospho-RIP3 (Ser227) (D6W2T, CST, USA), Histone H2A.X (D17A3) (7631T, CST, USA), Phospho-Histone H2A.X (Ser139) (20E3, CST, USA), DRP1 (Selleck, China), Phospho-DRP1 (Ser637) (Selleck, China), Caspase-8 (Selleck, China), PARP (Selleck, China), Cleaved PARP (Asp214) (Selleck, China), Caspase-3 (Selleck, China), Cleaved Caspase-9 (Asp315) (Selleck, China), Cleaved Caspase-7 (Asp198) (Selleck, China), Caspase-7 (Selleck, China), IL-18 (Selleck, China), NLRP3 (Selleck, China) and HMGB1 (Selleck, China).

[0069] The experimental consumables such as 10-cm cell culture plates, 6-well plates, 96-well plates, pipettes, and centrifuge tubes used were all purchased from Guangzhou Jet Bio-Filtration Co., Ltd. These experimental consumables play a fundamental role in cell culture and experiments.

[0070] The female nude mice (3 - 4 weeks old, weighing about 18 g) used were purchased from the Guangdong Provincial Center for Medical Laboratory Animals. Breeding conditions: Experimental Animal Center of Peking University Shenzhen Graduate School (SPF - level animal house certificate SYXK(Yue)2022 - 0172).

[0071] For those not specifying specific conditions in the examples, they were carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they were all conventional products that could be obtained through commercial purchase.

[0072] Example 1

[0073] This example explored the effects of the combination of paclitaxel and cephalomannine on the survival rate, apoptosis, and cell cycle of breast cancer cells. The specific experiments were as follows:

[0074] 1. Experimental materials:

[0075] (1) Compounds: Paclitaxel (PS0036 - 1000) and cephalomannine (PS2035 - 0025) used in the experiment were both purchased from Chengdu Purui Biotechnology (China), and their chemical structural formulas are as Figure 1 shown.

[0076] (2) Cell source: The breast cancer cell line MDA - MB - 231 was provided by the Cell Resource Center of the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences.

[0077] 2. Experimental methods:

[0078] (1) Cell culture and treatment: The MDA - MB - 231 cells used in the experiment were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody, and incubated in an incubator at 5% CO2 and 37 °C. When the cells reached 70% - 80% confluence, sub - culture was carried out to maintain the cells in the logarithmic growth phase.

[0079] (2) MTT assay: The MTT assay was used to evaluate the effects of different doses of paclitaxel and cephalotaxine and their combination on cell viability. The administration concentrations of paclitaxel were set at 0.091 ng / mL, 167 ng / mL, 0.33 ng / mL, 0.5 ng / mL, 0.66 ng / mL, 0.833 ng / mL, 0.909 ng / mL, 1 ng / mL, 5 ng / mL, or 10 ng / mL, and the administration concentrations of cephalotaxine were set at 0.091 ng / mL, 0.1 ng / mL, 167 ng / mL, 0.2 ng / mL, 0.33 ng / mL, 0.5 ng / mL, 0.66 ng / mL, 0.833 ng / mL, 0.909 ng / mL, 1 ng / mL, 5 ng / mL, or 10 ng / mL. The combination administration group was the combined administration of paclitaxel and cephalotaxine. MDA-MB-231 cells in the logarithmic growth phase were digested with 0.25% trypsin, centrifuged at 4°C and 800 r / min for 3 minutes to collect the cells, and resuspended with DMEM medium. The cells were adjusted to 5×10 3 / well. After 48 hours of drug treatment, MTT solution was added, and incubation was continued for 2 - 3 hours. The formazan crystals were dissolved with DMSO, and the absorbance value at 570 nm was measured.

[0080] (3) Determination of the combined medicinal index CI value: By calculating the IC 50 value of the single drug and entering it into the CompuSyn software, the CI value of the combination of paclitaxel and cephalotaxine was calculated to evaluate the synergistic effect of the drug combination. The dose - effect curve was plotted based on the MTT assay data, and the inhibitory effect of different drug combinations on cell proliferation was further analyzed, and the effect of their combined use was evaluated.

[0081] (4) Cell cycle and apoptosis assays: The administration concentrations of both paclitaxel and cephalotaxine were 1 ng / mL. The cell cycle was detected by PI staining, and the DNA content and sub - diploid peak were analyzed using a flow cytometer. Annexin V - FITC / PI staining was used to detect apoptosis.

[0082] The above experimental data were expressed as mean ± standard deviation (mean ± SD). The statistical significance of the data was tested by one - way analysis of variance (ANOVA). P < 0.05 was considered statistically significant. Otherwise, without special instructions, P represents paclitaxel, P1 represents the administration concentration of 1 ng / mL (and so on); C represents cephalotaxine, C1 represents the administration concentration of 1 ng / mL (and so on), P1 + C1 or P1C1 both represent combined administration, where the administration concentration of paclitaxel is 1 ng / mL and the administration concentration of cephalotaxine is 1 ng / mL (and so on).

[0083] 3. Experimental results:

[0084] Figure 2 The detection results of the viability of breast cancer cells are shown. It shows that the treatment with paclitaxel (P1) and cephalotaxine (C1) alone can both reduce the viability of MDA-MB-231 cells, while the combined treatment (such as P0.5C0.5, P1C1, etc.) further significantly reduces the cell survival rate, showing a significant inhibitory effect. And both the P0.909C0.091 combination administration group and the P0.091C0.909 combination administration group show a certain synergistic inhibitory effect.

[0085] In addition, it is worth noting that compared with the low-dose combination group (such as P1C1), the higher-dose combinations (such as P5C5 and P10C10) did not show a significant difference in cell survival rate, indicating that the lower dose has reached the saturation state of the inhibitory effect. To balance the experimental effect and drug toxicity control, P1C1 was selected as the optimal dose for further analysis and verification in the subsequent experiments.

[0086] Furthermore, the combined medicinal index CI was used to evaluate whether there is a synergistic inhibitory effect between the two. The detection results are shown in Table 1.

[0087] Table 1: The combined medicinal index of the PC combination

[0088]

[0089]

[0090] Note: The dose ratio of P and C in the table is 1:1.

[0091] The results show that when the total dose of paclitaxel and cephalotaxine is 2 - 20 ng / mL, the combined use of paclitaxel and cephalotaxine can produce a synergistic effect, that is, the inhibitory effect of the combined use of the two drugs is better than that of the single use.

[0092] Figure 3 The detection results of the effect of the combined drug use on the apoptosis of breast cancer cells are shown. The flow cytometry analysis shows that compared with the control group, paclitaxel (P1) and cephalotaxine (C1) significantly induce cell apoptosis. Among them, the early and late apoptosis rates of the P1 group are 15.4% and 26.3% respectively, and those of the C1 group are 17.5% and 28.8% respectively. The early and late apoptosis rates of the combined treatment group (P1C1) reach 23.3% and 42.6% respectively, which are significantly higher than those of the single drug treatment group. Paclitaxel and cephalotaxine may act on the apoptosis signaling pathway through different targets and synergistically enhance the apoptosis effect under the condition of combined treatment.

[0093] Figure 4The flow cytometry analysis results are shown. The results show that treatment with paclitaxel (P1) mainly leads to cell arrest in the G2 / M phase, while treatment with cephalotaxine (C1) induces S-phase arrest. The combined treatment group (P1C1) further aggravates the G2 / M phase arrest and significantly reduces the proportion of cells in the S phase (A-D in Figure 4 ). Further statistical results show that compared with the control group, the proportion of cells in the G2 / M phase in the combined treatment group increases to 72.5%, while the proportion of cells in the S phase decreases to 10.8%, indicating that the two drugs interfere with DNA replication and mitosis processes through different mechanisms, thereby inhibiting cell cycle progression (E in Figure 4 ).

[0094] The above results show that the combination of paclitaxel and cephalotaxine can synergistically reduce the survival rate of breast cancer cells, can significantly induce apoptosis of breast cancer cells, and can interfere with DNA replication and mitosis processes, thereby inhibiting cell cycle progression.

[0095] Example 2

[0096] DNA damage is a key upstream signal for tumor cell death induced by the combined treatment of paclitaxel and cephalotaxine. γ-H2AX is the H2AX protein phosphorylated after DNA double-strand breakage and is an important marker of DNA damage. In this example, the effects of paclitaxel and cephalotaxine on MDA-MB-231 cells were further explored by detecting the expression of the DNA damage marker γ-H2AX. The specific experimental methods are as follows:

[0097] The culture and drug treatment of MDA-MB-231 cells were carried out according to the method of Example 1. During the Western blot experiment, the drug treatment concentrations of paclitaxel and cephalotaxine were both 1 ng / mL. Cells were lysed with cell lysis buffer, proteins were collected by centrifugation, electrophoresed using SDS-PAGE, and detected with the target antibody after transfer membrane, specifically referring to the well-known methods in the art.

[0098] The effects of paclitaxel, cephalotaxine and their combined treatment on the expression of the DNA damage marker γ-H2AX in MDA-MB-231 cells are as shown in Figure 5 . Among them, A is the Western blot detection result of the expression of γ-H2AX and H2AX, and B is the statistical analysis result of the relative expression level of the ratio of γ-H2AX / H2AX. The data are expressed as mean ± standard deviation, **P<0.01 vs Con, *P<0.05 vs Con, ##P<0.01 vs P1, #P<0.05 vs P1, &&P<0.01 vs C1, &P<0.05 vs C1. The detection results show that after treatment with paclitaxel and cephalotaxine alone, the expression level of γ-H2AX increased, while the combined treatment group showed a more significant enhancement effect.

[0099] The above results show that paclitaxel and cephalomannine can activate cell death signals through DNA damage.

[0100] Example 3

[0101] In this example, the pathways of the combination of paclitaxel and cephalomannine in inhibiting the proliferation and promoting apoptosis of MDA-MB-231 cells were studied. The specific experimental methods are as follows:

[0102] The culture and drug administration of MDA-MB-231 cells were carried out according to the method of Example 1. During the Western blot experiment, the drug administration concentrations of paclitaxel and cephalomannine were both 1 ng / mL. Cells were lysed with cell lysate, proteins were collected by centrifugation, electrophoresed using SDS-PAGE, and detected with the target antibody after membrane transfer. Specifically, it was carried out with reference to the methods well known in the art.

[0103] The results of the effects of paclitaxel, cephalomannine and their combined treatment on the expression of apoptosis-related proteins in MDA-MB-231 cells are as Figure 6 shown, where Figure 6 results A and B show that paclitaxel and cephalomannine significantly increased the expression of p-JNK respectively, and the expression of p-JNK in the combination treatment group was more significant. It is known in the art that JNK (c-Jun N-terminal kinase) is an important component of the MAPK signaling pathway, participating in cell stress response and apoptosis regulation. Its phosphorylated form (p-JNK) can promote programmed cell death by activating transcription factors and pro-apoptotic proteins. In this experiment, the more significant expression of p-JNK in the combination treatment group indicates that the combination of paclitaxel and cephalomannine helps to better promote programmed cell death.

[0104] Figure 6 Result C in

[0105] Figure 6The D result in [study] showed that when paclitaxel and cephalomannine were used to treat MDA-MB-231 cells alone, the Bcl-2 / Bax ratio decreased significantly, and the Bcl-2 / Bax ratio in the combination treatment group decreased further. It is known in the art that during the regulation of apoptosis, Bcl-2 family proteins play a key role in regulating the permeability of the mitochondrial membrane and the transmission of apoptotic signals. The expression ratio of Bcl-2 to Bax directly affects the permeability of the outer mitochondrial membrane and the release of cytochrome C, thereby activating the downstream apoptotic cascade reaction. Therefore, the two drugs can enhance the permeability of the mitochondrial membrane through synergistic effects, promote the release of cytochrome C, and thus initiate the downstream apoptotic cascade reaction.

[0106] Figure 6 The E result in [study] showed that both paclitaxel and cephalomannine significantly upregulated the expression of p-DRP1, and the p-DRP1 level in the combination treatment group was higher. It is known in the art that mitochondrial fission also plays an important role in the process of apoptosis. p-DRP1 is a key regulator of mitochondrial fission, and its activation is usually accompanied by changes in mitochondrial morphology and the occurrence of apoptosis. In this experiment, the higher p-DRP1 level in the combination treatment group indicates that the combination of the two drugs can better promote mitochondrial fission by activating p-DRP1, enhance the sensitivity of cells to apoptotic signals, and thus further promote cell death.

[0107] Figure 6 The F and G results in [study] showed that whether it was paclitaxel or cephalomannine, single or combined treatment significantly increased the expression of Cleaved Caspase7 and Cleaved PARP. It is known in the art that the execution of the apoptosis process is achieved through the activation of Cleaved Caspase7 and Cleaved PARP. The activation of Caspase7 is one of the hallmarks of the apoptosis process, and the production of the cleavage product Cleaved PARP of PARP, as a DNA repair enzyme, further indicates that the cell has entered an irreversible apoptosis stage. In this experiment, the significant increase in the expression of Cleaved Caspase7 and Cleaved PARP in single or combined treatment indicates that both can promote apoptosis.

[0108] Figure 6The H results in [study object] showed that paclitaxel and cephalomannine significantly upregulated the expression of p21, and the p21 level in the combination treatment group was significantly higher than that in the single-drug group. During the processes of tumor cell proliferation and apoptosis, the regulation of the cell cycle is also crucial. As a cyclin-dependent kinase inhibitor, the expression level of p21 is regulated by p53. p21 inhibits the cyclin / CDK complex, blocks the cell cycle process, and provides a time window for cells to repair DNA or initiate an apoptotic response. In this experiment, the significantly higher p21 level in the combination treatment group than in the single-drug group indicates its synergistic effect in blocking the cell cycle process.

[0109] The above results indicate that the combination of paclitaxel and cephalomannine can synergistically inhibit the proliferation and promote the apoptosis of MDA-MB-231 cells through the JNK / p53 pathway and the mitochondrial-dependent apoptosis pathway.

[0110] Example 4

[0111] As a form of programmed cell death, cell necrosis is characterized by the rupture of the cell membrane and the leakage of cell contents, usually accompanied by an inflammatory response. The RIPK1 / RIPK3 / MLKL signaling pathway is a key signaling pathway for cell necrosis. In this example, the effect of the combination of paclitaxel and cephalomannine on promoting the necrosis of MDA-MB-231 cells was explored. The specific method is as follows:

[0112] The culture and drug administration of MDA-MB-231 cells were carried out according to the method of Example 1. During the Western blot experiment, the drug administration concentrations of paclitaxel and cephalomannine were both 1 ng / mL. Cells were lysed with cell lysate, proteins were collected by centrifugation, electrophoresed using SDS-PAGE, and detected with the target antibody after membrane transfer, specifically referring to the well-known methods in the art.

[0113] The effects of paclitaxel, cephalomannine, and their combination treatment on the expression of necrosis-related proteins (p-RIPK1, p-RIPK3, P-MLKL, and HMGB1) in MDA-MB-231 cells are as Figure 7 shown, where the data are expressed as mean ± standard deviation, **P<0.01 vs Con, *P<0.05 vs Con, ##P<0.01 vs P1, #P<0.05 vs P1, &&P<0.01 vs C1, &P<0.05 vs C1. The results show that the combined use of paclitaxel and cephalomannine can synergistically promote the necrosis of MDA-MB-231 cells by activating the RIPK1 / RIPK3 / MLKL signaling pathway.

[0114] Among them, Figure 7A in the figure is the Western blot detection result of necrosis-related proteins, and B is the statistical analysis of the relative expression of p-RIPK1. The results show that when paclitaxel or cephalomannine is used alone, the phosphorylation levels of p-RIPK1 and p-RIPK3 are low, while the phosphorylation levels in the combined treatment group are significantly increased. RIPK1 and RIPK3 are core molecules of the RIPK1 / RIPK3 / MLKL pathway, and their interaction and phosphorylation are crucial initial steps in the necrosis process. In this study, Western blot analysis showed that paclitaxel and cephalomannine can effectively activate the RIPK1 / RIPK3 signaling pathway and promote cells to enter the necrosis program. RIPK1 and RIPK3 interact to form necrosomes and provide the necessary conditions for the activation of MLKL. Studies have shown that RIPK3 is a key upstream molecule of cell necrosis, and it activates the downstream effector molecule MLKL through phosphorylation after binding to RIPK1. Therefore, the combined treatment of paclitaxel and cephalomannine can activate the RIPK1 / RIPK3 signaling pathway, providing an initial signal for the advancement of the cell necrosis process.

[0115] Figure 7 C in the figure is the statistical analysis result of the relative expression of the ratio of p-RIPK3 / RIPK3, and D is the statistical analysis result of the relative expression of the ratio of P-MLKL / MLKL, which shows that the phosphorylation level of MLKL in the combined treatment group increased significantly. This result indicates that paclitaxel and cephalomannine promote the translocation of MLKL on the cell membrane by enhancing the phosphorylation of MLKL. The role of MLKL in the RIPK1 / RIPK3 signaling pathway is crucial. As a downstream effector molecule, the phosphorylation of MLKL promotes its transfer from the cytoplasm to the cell membrane and forms pores on the membrane, leading to cell membrane rupture and cell death. The activation of MLKL is not only a key event in the necrosis process, but also a hallmark step in cell death. The activation of MLKL directly leads to the collapse of the cell membrane, and this process plays a decisive role in the occurrence of necrosis.

[0116] Figure 7 E in the figure is the statistical analysis result of the relative expression of HMGB1, which shows that the combined treatment of paclitaxel and cephalomannine significantly upregulated the release of extracellular HMGB1 (a classic necrosis marker molecule). This finding indicates that paclitaxel and cephalomannine further promote the occurrence of cell necrosis by activating the RIPK1 / RIPK3 / MLKL signaling pathway, and may induce immune response in the tumor microenvironment by releasing HMGB1, thereby enhancing the therapeutic effect. The leakage of HMGB1 is not only a sign of cell necrosis, but also one of its potential mechanisms of action in tumor treatment.

[0117] The above results indicate that the combination of paclitaxel and cephalomannine can synergistically promote the necrosis of MDA-MB-231 cells through the RIPK1 / RIPK3 / MLKL pathway.

[0118] Example 5

[0119] Caspase-8 is a key initiating enzyme in the pyroptosis pathway, and its activation is an initial marker of pyroptosis. In this example, it was explored whether the combination of paclitaxel and cephalomannine could regulate the death of MDA-MB-231 cells through the Caspase-8 / NLRP3 / GSDMD pyroptosis signaling pathway. The specific method is as follows:

[0120] The culture and drug administration of MDA-MB-231 cells were carried out according to the method of Example 1. During the Western blot experiment, the drug administration concentrations of paclitaxel and cephalomannine were both 1 ng / mL. The cells were lysed with cell lysate, the proteins were collected by centrifugation, electrophoresed using SDS-PAGE, and detected with the target antibody after membrane transfer, specifically referring to the well-known methods in the art.

[0121] The effects of paclitaxel, cephalomannine and their combination treatment on the pyroptosis-related proteins (Caspase-8, NLRP3, GSDMD and IL-18) in MDA-MB-231 cells and the secretion levels of extracellular IL-18, IL-1β and Caspase-1 are as Figure 8 shown, indicating that the combination treatment of paclitaxel and cephalomannine can significantly regulate the expression of pyroptosis-related proteins in MDA-MB-231 cells by activating the pyroptosis signaling pathway, and further deepen the pyroptosis effect during the cell death process.

[0122] Among them, Figure 8 A is the Western blot detection results of Caspase-8, NLRP3, GSDMD and IL-18, and B is the statistical analysis result of the relative expression level of Caspase-8. It shows that in the combination treatment group, the relative expression level of Caspase-8 is significantly higher than that in the single treatment group. This result suggests that paclitaxel and cephalomannine can promote the conduction of pyroptosis signals and cell death by synergistically activating Caspase-8. The initial role of Caspase-8 in pyroptosis is consistent with its importance in the inflammatory response.

[0123] Figure 8In which, C is the statistical analysis result of the relative expression level of NLRP3, showing that the combined treatment of paclitaxel and cephalotaxine significantly promoted the upregulation of NLRP3. NLRP3 is the core molecule for the formation of pyroptotic bodies. The activation of NLRP3 promotes its binding to ASC protein to form pyroptotic bodies, which further activates Caspase-1. The activation of Caspase-1 further cleaves Gasdermin D (GSDMD) to produce cleavage products, which insert into the cell membrane to form pores, leading to the rupture of the cell membrane and the leakage of cell contents, thus triggering cell pyroptosis. Therefore, the expression level of NLRP3 in the combined treatment group was significantly higher than that in the single treatment group, suggesting that the combined treatment is more conducive to enhancing the activation of NLRP3 and promoting the formation of pyroptotic bodies and the initiation of downstream signals.

[0124] Figure 8 In which, D is the statistical analysis result of the relative expression level of the ratio of n-GSDMD / GSDMD, showing that the combined treatment of paclitaxel and cephalotaxine significantly upregulated the relative expression level of the cleavage product of GSDMD, indicating that the combined treatment promoted the pyroptosis process by enhancing the cleavage of GSDMD. As an essential execution protein in the pyroptosis process, the cleavage product of GSDMD can form cell membrane pores, leading to the rupture of the cell membrane and triggering cell death. Therefore, the expression level of GSDMD in the combined treatment group was significantly higher than that in the single treatment group, suggesting that the combined treatment is more conducive to promoting cell pyroptosis.

[0125] Figure 8 In which, E is the statistical analysis of the relative expression level of IL-18, and F is the ELISA detection result of extracellular IL-18, showing that the combined treatment of paclitaxel and cephalotaxine significantly increased the relative expression level and secretion level of IL-18 (*P<0.01 vs Con, ##P<0.01 vs P1). IL-18 is an important inflammatory factor released during pyroptosis, and it plays a key regulatory role in cell death and tumor immune responses.

[0126] Figure 8 In which, G is the ELISA detection result of extracellular IL-1β, and H is the ELISA detection result of extracellular TNF-α, showing that the combined treatment of paclitaxel and cephalotaxine significantly increased the secretion of extracellular IL-1β and TNF-α. As key pro-inflammatory cytokines, IL-1β and TNF-α play important roles in pyroptosis and immune responses. The secretion levels of these two factors in the combined treatment group were significantly higher than those in the single treatment group (*P<0.01 vs Con, ##P<0.01 vs P1, &&P<0.01 vs C1), indicating that the combined treatment not only promoted cell death through the activation of the pyroptosis pathway but also may enhance the anti-tumor immune effect by activating the immune response.

[0127] The above results indicate that the combination of paclitaxel and cephalomannine regulates MDA-MB-231 cell death through the Caspase-8 / NLRP3 / GSDMD pyroptosis signaling pathway.

[0128] Example 6

[0129] To further verify the effect of the combined treatment of paclitaxel and cephalomannine in inducing tumor cell death through multiple pathways such as apoptosis, pyroptosis, and necrosis, in this example, the apoptosis inhibitor Disulfiram, the pyroptosis inhibitor Z-VAD-FMK, and the necrosis inhibitor Necrostatin-1 were used to analyze the effects of different pathway inhibitors on cell viability. The specific method is as follows:

[0130] The experimental method of the inhibitor refers to the MTT experimental method.

[0131] The effects of pyroptosis, apoptosis, and necroptosis inhibitors on the viability of MDA-MB-231 cells after treatment with paclitaxel, cephalomannine, and their combination are shown as Figure 9 follows. Among them, A shows the effect of the pyroptosis inhibitor Disulfiram on the viability of MDA-MB-231 cells treated with paclitaxel, cephalomannine, and their combination; B shows the effect of the apoptosis inhibitor Z-VAD-FMK on the viability of MDA-MB-231 cells treated with paclitaxel, cephalomannine, and their combination; C shows the effect of the necroptosis inhibitor Necrostatin-1 on the viability of MDA-MB-231 cells treated with paclitaxel, cephalomannine, and their combination. P represents paclitaxel, P1 represents a concentration of 1 ng / mL, C represents cephalomannine, and C1 represents a concentration of 1 ng / mL. The data are expressed as mean ± standard deviation, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns indicates no significant difference. The detection results show that in all treatments with pathway inhibitors, the combined treatment of paclitaxel and cephalomannine significantly reduced the viability of MDA-MB-231 cells, and the combined treatment showed a stronger cell death effect under the action of each pathway inhibitor. This indicates that paclitaxel and cephalomannine act on multiple cell death pathways through synergy to further enhance their anti-tumor effect.

[0132] The above results indicate that the combined treatment of paclitaxel and cephalomannine can regulate tumor cell death through cell death pathway inhibitors.

[0133] Example 7

[0134] To evaluate the effect of the combined treatment of paclitaxel and cephalotaxine on tumor growth in vivo, this example conducted a detailed analysis of different treatment groups (G1 control group, G2 paclitaxel group, G3 cephalotaxine group, G4 combined PC group) in terms of tumor growth. The specific method is as follows:

[0135] Twenty-eight female Balb / c nude mice aged 3-4 weeks and weighing about 18 g were purchased and randomly divided into 4 groups (G1 control group, G2 paclitaxel group, G3 cephalotaxine group, G4 combined PC group), with 6 mice in each group. Intragastric drug treatment was carried out using paclitaxel and cephalotaxine, where:

[0136] (1) G1 control group: 25 mL of soybean oil.

[0137] (2) G2 paclitaxel group: 7.25 mg of paclitaxel was dissolved in soybean oil, made up to 25 mL, and ground in a mortar.

[0138] (3) G3 cephalotaxine group: 7.25 mg of cephalotaxine was dissolved in soybean oil, made up to 25 mL, and ground in a mortar.

[0139] (4) G4 combined PC group: 7.25 mg of paclitaxel + 7.25 mg of cephalotaxine was dissolved in soybean oil, made up to 25 mL, and ground in a mortar.

[0140] A nude mouse xenograft model of MDA-MB-231 breast cancer cells was established and treated by intragastric administration to evaluate the anti-tumor effect of the combined drug treatment.

[0141] Figure 10 The comparison results of different treatment groups (G1 control group, G2 paclitaxel group, G3 cephalotaxine group, G4 combined PC group) in terms of tumor growth and tumor size are shown. Among them, the food and water intake of the mice and the weight coefficients of tumor-related organs (kidney, liver) showed that there were no significant differences in these indicators among all treatment groups (such as Figure 10 A-D in), suggesting that the combined treatment of paclitaxel and cephalotaxine did not cause obvious toxicity or damage to the whole body organs of the experimental animals. The measurement results of tumor size showed that the combined treatment group (G4) had a significant inhibitory effect on tumor growth. In the tumor size( Figure 10 E in) and the change trend of daily tumor volume( Figure 10 F in), the combined application of paclitaxel and cephalotaxine significantly slowed down the growth of tumors. Compared with the control group, the tumor volume of the G4 group was significantly reduced. Especially on the 10th day after treatment, the tumor volume growth of the G4 group was significantly lower than that of the other single-drug groups (G2 and G3 groups). Photos after tumor resection( Figure 10G) in it further verified the effect of the combination therapy. The tumors in Group G4 were significantly smaller and fewer in number, showing a significant advantage of the combination therapy in inhibiting tumor growth. In contrast, the therapeutic effects of using paclitaxel alone (Group G2) and cephalotaxine (Group G3) were somewhat inhibitory, but still appeared to be relatively limited.

[0142] The above results indicate that, compared with single use, the combination therapy of paclitaxel and cephalotaxine can significantly inhibit the growth of transplanted tumors.

[0143] In summary, the present invention provides an application of the combination of paclitaxel and cephalotaxine in the preparation of anti-breast cancer products. Through experiments, the present invention found that, compared with the single administration methods of paclitaxel or cephalotaxine (mainly involving inhibiting cell proliferation and promoting cell apoptosis), the combined use of paclitaxel and cephalotaxine has a synergistic effect and can significantly enhance the therapeutic effect. In addition, the present invention found that the combination therapy of paclitaxel and cephalotaxine can not only promote tumor cell death through traditional apoptotic pathways (such as the Bcl-2 / Bax pathway, the JNK / p53 pathway), but also enhance the anti-tumor effect by significantly activating cell death pathways such as pyroptosis (NLRP3 / GSDMD) and necroptosis (RIPK1 / RIPK3 / MLKL). This treatment strategy that synergistically acts through multiple cell death mechanisms can effectively inhibit the proliferation of tumor cells and alleviate the decline in therapeutic effect caused by drug resistance, overcoming the drug resistance problem in the previous single-agent paclitaxel treatment, and is expected to be widely applied to the tumor treatment related to the pan-apoptosis regulation mechanism.

[0144] The above has made a detailed description of the embodiments of the present invention. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the gist of the present invention within the knowledge scope of those of ordinary skill in the art. In addition, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

Claims

1. Use of paclitaxel in combination with cephalomannine in the preparation of a drug for treating and / or preventing breast cancer.

2. The use according to claim 1, characterized in that: The drug for treating and / or preventing breast cancer has at least one of the uses (A1) to (A3): (A1), reduce the survival rate of breast cancer cells; (A2), inhibiting breast cancer cell proliferation; (A3) Promotes pan-apoptosis of breast cancer cells.

3. The use according to claim 1, characterized in that: The method for promoting pan-apoptosis of breast cancer cells includes at least one of (B1) to (B5): (B1), activating cell death signals by promoting DNA damage in breast cancer cells; (B2), promoting apoptosis of breast cancer cells via the JNK / p53 pathway; (B3) Promote apoptosis of breast cancer cells through the mitochondria-dependent apoptosis pathway; (B4), promoting breast cancer cell necrosis via the RIPK1 / RIPK3 / MLKL pathway; (B5) Promote breast cancer cell death through the Caspase-8 / NLRP3 / GSDMD pyroptosis signaling pathway.

4. The use according to claim 1, characterized in that: The method for promoting pan-apoptosis of breast cancer cells includes at least one of (C1) to (C5): (C1), increasing the expression level of γ-H2AX; (C2), increase the expression levels of p-JNK / JNK, p53, p-DRP1, Cleaved Caspase7, Cleaved PARP, and p21, and / or decrease the relative expression levels of Bcl-2 / Bax; (C3), increasing the expression levels of p-RIPK1, p-RIPK3, P-MLKL or HMGB1; (C4), increasing the expression levels of Caspase-8, NLRP3, GSDMD or IL-18; (C5) Increase the secretion level of extracellular IL-18, IL-1β or TNF-α.

5. The use according to any one of claims 1 to 4, characterized in that: The mass ratio of paclitaxel to cephalomannine is 1:0.1-10.

6. The use according to claim 5, characterized in that: The breast cancer includes triple-negative breast cancer.

7. An anti-breast cancer pharmaceutical composition, characterized in that: The active ingredients include paclitaxel and cephalomannine.

8. The anti-breast cancer pharmaceutical composition according to claim 7, characterized in that: The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

9. The anti-breast cancer pharmaceutical composition according to claim 8, characterized in that: The pharmaceutically acceptable excipient is selected from at least one of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a suspending agent, a surfactant, and a preservative.

10. The anti-breast cancer pharmaceutical composition according to claim 9, characterized in that: The dosage form of the pharmaceutical composition is at least one of a solid preparation, a liquid preparation, or a semisolid preparation.

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