Application of lumacapine in improvement of triple negative breast cancer taxol resistance

Abstract

The invention discloses an application of lumacatray in improvement of triple negative breast cancer paclitaxel resistance, and belongs to the technical field of biological medicines. The invention provides an application of a pharmaceutical composition in preparation of a drug for preventing and / or treating triple negative breast cancer with paclitaxel drug resistance. The active ingredients of the pharmaceutical composition are lumacapine and paclitaxel. According to the application, it is found for the first time that lumacatray can significantly enhance the killing effect of paclitaxel on TNBC drug-resistant cells, and the mechanism of lumacatray may be related to glycolysis pathway inhibition, cell cycle arrest recovery and apoptosis signal promotion. Experimental results prove that when the lumacatray is used as the GTM1 agonist, an AMPK-glycolysis pathway can be inhibited, and the generation of lactic acid is reduced; the compound is combined with paclitaxel in paclitaxel drug-resistant cells, so that the cell proliferation is obviously inhibited, and the influence in parent cells is not obvious. Therefore, the lumacatray can be used as a chemosensitizer for the paclitaxel-resistant triple-negative breast cancer, and has a clear mechanism and a good application prospect.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, in particular to application of lumaquatic acid in improving paclitaxel resistance of triple-negative breast cancer. BACKGROUND

[0002] Triple-negative breast cancer (TNBC) is a subtype of malignant breast cancer lacking estrogen receptor (ER), progesterone receptor (PR) and HER2 expression, accounting for about 15-20% of all breast cancers, and is characterized by strong invasiveness, rapid metastasis and poor prognosis. Due to the lack of a clear molecular target, TNBC mainly relies on cytotoxic drugs such as paclitaxel for chemotherapy. However, as the treatment time is prolonged, about 40-60% of TNBC patients will gradually develop paclitaxel resistance, showing decreased treatment effect, persistent tumor progression or recurrence, and seriously affecting the survival rate and quality of life of patients.

[0003] The main mechanism of paclitaxel is to stabilize microtubules, block mitosis and induce apoptosis. However, the emergence of drug resistance is often accompanied by the participation of multiple mechanisms, including: up-regulation of drug efflux proteins such as P-gp to accelerate drug clearance; abnormal expression of microtubule-associated proteins to reduce drug binding capacity; blocked apoptosis signaling pathway; and tumor metabolic reprogramming, especially enhanced glycolysis to provide energy support for drug-resistant cells and maintain their survival advantage. Notably, the AMPK signaling pathway, as a core regulator of cell metabolism, is abnormally activated in paclitaxel-resistant cells, and its downstream glycolytic enzyme ENO1 is also significantly up-regulated, forming an “AMPK-ENO1 axis” to further promote tumor glycolysis and enhance drug resistance.

[0004] In the prior art, various strategies have been used to reverse paclitaxel resistance, including the combined use of AMPK inhibitors, targeting the P-gp efflux pathway, or directly interfering with glycolytic enzymes. However, these methods have the following disadvantages: (1) the targets are mostly general metabolic pathways with poor selectivity and high toxicity; (2) there is a lack of stable and controllable small molecule agonists to regulate metabolic pathways from the upstream; (3) the AMPK signaling pathway exhibits dual regulation in different cancers, and direct inhibition of AMPK may cause systemic metabolic disorders. Therefore, it is urgent to find new, more specific and less toxic treatment entry points to break through the paclitaxel resistance of TNBC. SUMMARY

[0005] The application aims to provide the application of lumaquatic acid in improving paclitaxel resistance of triple-negative breast cancer, and overcomes paclitaxel resistance of breast cancer.

[0006] The application provides a use of a pharmaceutical composition in preparation of a drug for preventing and / or treating triple-negative breast cancer with paclitaxel resistance. The active ingredients of the pharmaceutical composition are lumarikat and paclitaxel.

[0007] In the use, the molar ratio of the lumarikat and the paclitaxel is 1:0.1-1:100, preferably 1:0.5-1:5, and more preferably 1:1-1:2 or 1:1.5.

[0008] In the use, the lumarikat has the following structural formula: ; The paclitaxel has the following structural formula: .

[0009] The application also provides a use of a pharmaceutical composition in preparation of a product for inhibiting proliferation of paclitaxel-resistant cells. The active ingredients of the pharmaceutical composition are lumarikat and paclitaxel.

[0010] In the use, the molar ratio of the lumarikat and the paclitaxel is 1:0.1-1:100, preferably 1:0.5-1:5, and more preferably 1:1-1:2.

[0011] Thirdly, the application provides a paclitaxel treatment sensitizer for triple-negative breast cancer, and the active ingredient of the sensitizer is lumarikat.

[0012] Finally, the application provides a drug for preventing and / or treating triple-negative breast cancer with paclitaxel resistance, and the active ingredients of the drug are lumarikat and paclitaxel.

[0013] In the drug, the molar ratio of the lumarikat and the paclitaxel is 1:0.1-1:100, preferably 1:0.5-1:5, and more preferably 1:1-1:2.

[0014] In the application, the drug resistance is caused by an endocrine treatment drug paclitaxel.

[0015] The present application first identifies GTM1 (Glycolytic Transition Modulator 1) as a negative regulator in paclitaxel resistance, and the expression of the regulator can cause the activity of an AMPK-ENO1 axis to be enhanced and glycolytic metabolism to be increased, and the recovery of the expression of the regulator can inhibit the above metabolic processes and restore drug sensitivity. Therefore, the development of a small molecule drug capable of agonizing GTM1 becomes a new strategy for reversing paclitaxel resistance.

[0016] Lumacaftor is a small molecule drug approved by FDA for the treatment of cystic fibrosis, which has good oral bioavailability and safety. It is found for the first time that lumacaftor can significantly enhance the killing effect of paclitaxel on TNBC drug-resistant cells, and the mechanism may be related to the inhibition of glycolysis pathway, the recovery of cell cycle arrest and the promotion of apoptosis signal. As a low-toxicity, safe and orally available clinical drug, lumacaftor is expected to become an effective sensitizer in the treatment of paclitaxel-resistant TNBC, expand its clinical indications, and significantly improve the survival outcome of patients.

[0017] As a GTM1 agonist, lumacaftor can inhibit the AMPK-glycolysis pathway and reduce lactic acid production. In paclitaxel-resistant cells (MDA-MB-231PR, BT549PR), it can significantly inhibit cell proliferation (CCK-8, EdU) when combined with paclitaxel, but has no significant effect on parent cells. GTM1 knockdown can eliminate the above effects, and rescue can restore them, proving that the sensitization effect depends on GTM1. Therefore, lumacaftor can be used as a chemosensitizer for paclitaxel-resistant triple-negative breast cancer, with a clear mechanism and good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Molecular docking screening diagram of molecules as GTM1 agonists.

[0019] Figure 2 IC50 determination diagram of midostaurin, nilotinib, alectinib and lumacaftor on triple-negative breast cancer cells. 50

[0020] Figure 3 Measurement results of lactic acid production of cells treated with midostaurin, venetoclax, nilotinib, alectinib and lumacaftor.

[0021] Figure 4 Western blot (WB) detection results of the influence of midostaurin, nilotinib, alectinib and lumacaftor on GTM1 protein expression.

[0022] Figure 5 Western blot (WB) results of lumacaftor recovery experiment in shGTM1 background.

[0023] Figure 6 Influence of lumacaftor on parent cells MDA-MB-231 and BT549 and paclitaxel-resistant cells MDA-MB-231 PR and BT549 PR

[0024] Figure 7 ​​For the treatment of MDA-MB-231 cells and paclitaxel-resistant MDA-MB-231 cells by combining rumacato and paclitaxel. PR It has a proliferative inhibitory effect.

[0025] Figure 8 Rumacata combined with paclitaxel for the treatment of BT549 cells and paclitaxel-resistant BT549 cells PR It has a proliferative inhibitory effect. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0027] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0028] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] The biological materials, reagents, and kits used in the following examples are all commercially available, and the experimental techniques used are all standard procedures in the field or performed in accordance with the instructions of the corresponding products.

[0031] The English name of the paclitaxel used in the following examples is Paclitaxel, and its Chinese chemical name is diphenylacetic acid-3′-benzamide-4,10-diacetoxy-2α-benzoyloxy-5β,20-epoxy-1,2,4,7,13-pentahydroxytaxyl-11(15)-ene, with the molecular formula C 47 H 51 NO 14 The structural formula is as follows:

[0032] The rumacaftor used in the following examples is called 3-[6-[(1R)-1-(2,2-dimethylpropoxy)-2,2-dimethylethyl]pyridin-3-yl]benzoic acid, with CAS number 936727-05-8 and molecular formula C64. 24 H 25 F2NO5, structural formula as follows:

[0033] MDA-MB-231 and BT549 cell lines used in the following examples were from the Breast Cancer Diagnosis and Treatment Research Key Laboratory of the Affiliated Tumor Hospital of Shantou University Medical College. The construction method of paclitaxel-resistant MDA-MB-231 and BT549 cells is as follows: cultured in 1640 culture medium (Biological Industries) containing 10% (v / v) carbon adsorbed fetal bovine serum (Biological Industries) without phenol red / glutamine, first, logarithmically growing parent cells were inoculated in a 25T culture flask, 0.25 μM paclitaxel was added for 48-72 h, when the cells appeared 70%-80% rounding or the survival rate detected by trypan blue was ≤20%-30%, the drug solution was discarded and replaced with drug-free culture medium for continuous culture, when the cells recovered to 70%-80% confluence, the next round of induction was carried out; this was repeated for 3-6 rounds until the cells could be continuously passaged and stably grown at 0.25 μM. Subsequently, the drug concentration was gradually increased to 0.5, 1, 2 and 3 μM, respectively, and each concentration was screened according to the same "induction-recovery" cycle method until the cells could be stably passaged for ≥2 weeks at this concentration. Finally, when the cells could be continuously passaged for ≥8-12 weeks and stably grown in 3 μM paclitaxel culture medium, single clones were isolated by limiting dilution or cloning ring method, and clones with uniform morphology and stable proliferation were screened and expanded in 3 μM paclitaxel-containing culture medium to obtain paclitaxel-resistant cell lines. The whole induction-recovery-pressing-cloning process lasted about 12 months. To ensure the maintenance of drug resistance, 1 μM paclitaxel was used to maintain the pressure in daily culture, and the drug was stopped 7-14 days before the experiment. The above culture was carried out in a culture incubator at 37°C, 5.0% CO2. PR PR The construction method of paclitaxel-resistant MDA-MB-231 and BT549 cells is as follows: cultured in 1640 culture medium (Biological Industries) containing 10% (v / v) carbon adsorbed fetal bovine serum (Biological Industries) without phenol red / glutamine, first, logarithmically growing parent cells were inoculated in a 25T culture flask, 0.25 μM paclitaxel was added for 48-72 h, when the cells appeared 70%-80% rounding or the survival rate detected by trypan blue was ≤20%-30%, the drug solution was discarded and replaced with drug-free culture medium for continuous culture, when the cells recovered to 70%-80% confluence, the next round of induction was carried out; this was repeated for 3-6 rounds until the cells could be continuously passaged and stably grown at 0.25 μM. Subsequently, the drug concentration was gradually increased to 0.5, 1, 2 and 3 μM, respectively, and each concentration was screened according to the same "induction-recovery" cycle method until the cells could be stably passaged for ≥2 weeks at this concentration. Finally, when the cells could be continuously passaged for ≥8-12 weeks and stably grown in 3 μM paclitaxel culture medium, single clones were isolated by limiting dilution or cloning ring method, and clones with uniform morphology and stable proliferation were screened and expanded in 3 μM paclitaxel-containing culture medium to obtain paclitaxel-resistant cell lines. The whole induction-recovery-pressing-cloning process lasted about 12 months. To ensure the maintenance of drug resistance, 1 μM paclitaxel was used to maintain the pressure in daily culture, and the drug was stopped 7-14 days before the experiment. The above culture was carried out in a culture incubator at 37°C, 5.0% CO2.

[0034] The culture, incubation or treatment described in the following examples were carried out in a culture incubator at 37°C, 5.0% CO2 unless otherwise specified.

[0035] GTM1 described in the following examples refers to human RSRP1 gene (Gene ID: 57035) retrieved from NCBI gene database.

[0036] Example 1 Molecular docking screening experiment of GTM1 agonist: first, download FDA small molecule drugs from ZINC database, optimize small molecule structure using openbable and UFF force field, prepare protein GTM1 and receptor using ADFRsuite1.0, and perform docking using vina1.2.5. Screen out the top five small molecules in docking binding energy, and the results are as follows: Figure 1 ​The drugs are midotutolin, nilotinib, venetum, alectinib, and rumacato. Among them, rumacato has better binding energy and conformational stability, suggesting its potential as a GTM1 agonist.

[0037] Example 2 CCK-8 method drug IC 50 Assay: To evaluate the inhibitory effect of different small molecule drugs on the proliferation of triple-negative breast cancer cells and to determine their IC50 values. 50 MDA-MB-231 and BT549 cells were seeded into 96-well plates, with 100 μL of cell suspension (1640 complete medium) added to each well, at a density of approximately 5000 cells / well. After cell adhesion, different concentrations of drugs were applied. Five drug groups were included: control (DMSO), midostaurin, nilotinib, alectinib, and lumacaftor. Five concentration gradients were set for each drug: 0 (DMSO control), 100 nM, 200 nM, 1 μM, and 2 μM, with six replicates per group. Drug treatment lasted for 0, 24, 48, and 72 hours. At each time point, the original culture medium was removed, and 100 μL of complete culture medium containing 10% CCK-8 reagent (CCK-8 and culture medium were prepared at a volume ratio of 1:9) was added to each well. After incubation at 37°C and 5% CO2 for 2 hours, the absorbance (OD) was measured at 450 nm using a microplate reader. 450 Cell viability curves were plotted based on OD values ​​at different concentrations and time points, and dose-response fitting was performed using 72-hour data to calculate the IC50 of each drug in MDA-MB-231 and BT549 cells. 50 The values ​​were used to assess its sensitivity and inhibitory effect on cell growth; the results are shown in [Figure Number]. Figure 2 Venetoclax was used at a concentration of 2 μM, according to literature references.

[0038] Depend on Figure 2 It was found that in MDA-MB-231 and BT549 cells, CCK-8 assay results showed that all five drugs could effectively inhibit cell proliferation. Among them, rumacata showed stable and significant inhibitory activity in both cell lines, with a half-maximal inhibitory concentration (IC50) of [value missing]. 50 The levels were at low micromolar levels. This result indicates that rumacato has a strong anti-proliferative effect on triple-negative breast cancer cells, providing experimental evidence for its potential as a candidate drug and for subsequent mechanism studies and combination therapy strategies.

[0039] Example 3 Lactate content detection experiment: MDA-MB-231 and BT549 cells were seeded into 6 cm dishes, with approximately 2 × 10⁶ cells per well. 5 Cells were collected and 2 mL of 1640 complete culture medium were added. After cell adhesion, candidate drugs Midostaurin (100 nM), Venetoclax (2 μM), Nilotinib (1 μM), Alectinib (100 nM), Lumacaftor (2 μM), and a DMSO control group were added, with 3 replicates for each group. After 72 hours of drug treatment, cells were digested with trypsin and collected into centrifuge tubes. Cells were washed with cold PBS, centrifuged, and the supernatant was discarded. Cells were then sonicated at a ratio of 1 mL / 5 million with Lactate Assay Buffer for 5 min on ice (200W, 3 s sonication, 7 s interval, repeated 30 times), followed by centrifugation at 12,000 g at 4°C for 5 min. The supernatant was collected and placed on ice for analysis. Lactate content was determined using CheKine (AbbKine Corporation). TM The lactate content assay kit (catalog number KTB1100) was used. The working solution (Lactate Assay Buffer, Cofactor, WST-8, Enhancer, and Lactate Dehydrogenase) was prepared according to the instructions. 50 μL of cell sample was added to each well and mixed with 50 μL of the working solution. After incubation at 37°C in the dark for 30 minutes, the absorbance was read at 450 nm using a microplate reader. A lactate standard curve (0-2 mM) was prepared before the experiment. The lactate production level in each group of samples was calculated based on the standard curve. The results are shown below. Figure 3 .

[0040] Depend on Figure 3 It can be seen that, compared with the DMSO control, only rumacato treatment significantly reduced cellular lactate production, suggesting that rumacato is more effective in inhibiting the glycolytic metabolic pathway; rumacato is the only candidate drug that can downregulate lactate, while the other drugs upregulate lactate.

[0041] Example 4 Western blot mechanism validation experiment: To further verify the regulatory effects of different small molecule drugs on GTM1 and its downstream signaling AMPK pathway, MDA-MB-231 cells were seeded in 6-well plates, with 1 × 10⁶ cells per well. 6Cells were cultured in 2 ml of 1640 complete medium. After cell attachment, cells were treated with DMSO (control group), midostaurin (100 nM), nilotinib (1 μM), alectinib (100 nM), and lumacaftor (2 μM) for 72 hours. Total protein was extracted using RIPA lysis buffer, quantified using the BCA method, and loaded onto an equal volume for SDS-PAGE (8% separating gel) electrophoresis and PVDF membrane transfer. Cells were incubated with antibodies against GTM1, p-AMPK (Thr172), total AMPK, ENO1, and β-Tubulin, and protein expression signals were recorded using chemiluminescence detection with a Bio-Rad imaging system. Results Figure 4 The results showed that in MDA-MB-231 cells, rumacaftor significantly upregulated GTM1 protein levels and inhibited p-AMPK (Thr172) and ENO1 expression; midostaurin, nilotinib, and alectinib did not show similar changes. Therefore, only the rumacaftor group significantly upregulated GTM1 expression and simultaneously inhibited p-AMPK and ENO1 expression, suggesting its unique mechanism of action: "activating GTM1, inhibiting AMPK, and downregulating glycolysis." No similar regulatory trends were observed in other drug groups.

[0042] To further verify the regulatory role of GTM1 on the AMPK signaling pathway under the action of rumacato, and whether its effect depends on the functional integrity of GTM1, five experimental groups were set up in MDA-MB-231 and BT549 triple-negative breast cancer cells: shScramble + DMSO group (negative control), shScramble + rumacato group (drug treatment control), shGTM1 + DMSO group (GTM1 knockdown), shGTM1 + rumacato group (drug treatment on knockdown background), and shGTM1 + rumacato + recovery group. All groups were treated with 2 μM rumacato for 72 hours. After cell lysis, proteins were extracted using RIPA lysis buffer, quantified using the BCA method, and loaded onto equal volumes. Separation was performed by 8% SDS-PAGE electrophoresis, and the proteins were transferred to PVDF membranes. Immunoblotting was performed using antibodies against GTM1, p-AMPK (Thr172), AMPK, ENO1, and β-Tubulin. Chemiluminescence detection was performed using the Bio-Rad imaging system. The results are as follows Figure 5As shown, in both MDA-MB-231 and BT549 cell lines, the shScramble + VX-809 group exhibited upregulation of GTM1 and downregulation of p-AMPK and ENO1, suggesting that GTM1 was activated by the drug and inhibited the AMPK-glycolysis axis; this trend disappeared in the shGTM1 + rumacato group; while in the Rescue group, after the GTM1 nonsense mutation was restored to expression, the effect of rumacato was effective again, further demonstrating that the regulation of the AMPK pathway and glycolysis by rumacato depends on the functional integrity of GTM1.

[0043] The shScramble + DMSO group (negative control) consisted of cells infected with shRNA lentivirus carrying irrelevant sequences, and stable shScramble transfected cells obtained by puromycin selection; an equal volume of DMSO (final concentration ≤0.1%) was added under normal culture conditions, without the addition of any drugs, and this served as a baseline reference. The shScramble + rumacato group (drug treatment control) was formed by adding 2 μM rumacato to the logarithmic growth phase of a stable shScramble mutant to evaluate the effect of rumacato under normal GTM1 expression conditions. The shGTM1 + DMSO group (GTM1 knockdown) was prepared by infecting cells with lentiviruses carrying shRNA targeting GTM1, and selecting stable shGTM1 transfectants with puromycin; an equal volume of DMSO was added without any drugs to evaluate the effect of continuous GTM1 knockdown on related proteins / pathways. The shGTM1 + rumacato group (drug treatment under knockdown background) was formed by adding 2 μM rumacato to the shGTM1 stable transgenic lineage to detect changes in the role of rumacato under GTM1 deletion / low expression conditions. The shGTM1 + rumacato + recovery (GTM1-rescue) group consisted of a stable shGTM1 transgenic strain with 2 μM rumacato added, followed by exogenous recovery of GTM1. GTM1 cDNA tolerant to shRNA was used, i.e., a synonymous mutation was introduced at the shRNA target site to avoid re-silencing. The expression was detected after recovery was stable (e.g., 48–72 h). This was used to verify whether the restoration of GTM1 reversed the rumacato effect caused by knockdown, thus demonstrating the necessity of GTM1 in the drug mechanism of action.

[0044] Example 5 CCK-8 drug susceptibility assay: As previously described, MDA-MB-231 and BT549, and their corresponding paclitaxel-resistant cells, were seeded in 96-well plates, approximately 3000 cells per well, with about 100 μL of suspension (1640 complete medium). Each cell type was divided into four groups: a vector control group, a paclitaxel group, a rumacato group, and a combination therapy (paclitaxel + rumacato) group. Each group was prepared in 6-8 replicates. After cell attachment, the corresponding drug solvent was added to the vector control group, 3 μM paclitaxel to the paclitaxel group, 2 μM rumacato to the rumacato group, and 3 μM paclitaxel and 2 μM rumacato to the combination therapy group, for 0, 24, 48, and 72 hours, respectively. Remove the existing culture medium from the 96-well plate. Add 100 μL of culture medium containing 10% CCK-8 working solution (Beyotime) to each well (prepared in advance according to the required total volume; the volume ratio of CCK-8 to culture medium is 1:9). Incubate for 2 hours. Measure the absorbance at 0, 24, 48, and 72 hours. Set the microplate reader to 450 nm and measure the absorbance. Obtain the average value of the readings from replicates. Plot a growth curve with time (h) on the x-axis and cell viability (absorbance at 450 nm) on the y-axis. Results are shown below. Figure 6 As shown, rumacato had no significant effect on the paclitaxel sensitivity of parental cells (MDA-MB-231 and BT549), while rumacato significantly enhanced the paclitaxel sensitivity of paclitaxel-resistant cells (MDA-MB-231). PR and BT549 PR ) paclitaxel sensitivity.

[0045] Example 6 EdU-based cell proliferation assay: As described above, MDA-MB-231 and BT549 cells, along with their corresponding paclitaxel-resistant cells, were seeded into 24-well plates. After reaching approximately 70% confluence, each cell type was divided into four groups: a vector control group, a paclitaxel group, a rumacato group, and a combination therapy (paclitaxel + rumacato) group. The corresponding drug solvent was added to the vector control group, 3 μM paclitaxel to the paclitaxel group, 2 μM rumacato to the rumacato group, and 3 μM paclitaxel and 2 μM rumacato to the combination therapy group. Treatment time was 0 or 72 hours. 2× EdU working solution (Beyotime) was prepared and preheated to 37°C. For a 24-well plate, the culture medium volume per well was 500 μL, and the final EdU concentration was 10 μM (1×). After adding the preheated working solution, the cells were incubated for another 2 hours. Remove the culture medium and add 500 μL of fixative (Beyotime) to each well, incubate at room temperature for 15 minutes. Remove the fixative and wash each well three times with 500 μL of washing buffer (Beyotime), 3-5 minutes each time. Remove the washing buffer and add 500 μL of permeabilization buffer (Beyotime) to each well, incubate at room temperature for 10-15 minutes. Remove the permeabilization buffer and wash each well twice with 500 μL of washing buffer, 3-5 minutes each time. Prepare the reaction solution according to the manufacturer's instructions. For a 24-well plate, the reaction solution volume is 100 μL, and the reaction solution should be used within 15 minutes of preparation. After removing the washing buffer, add 100 μL of the prepared reaction solution to each well, gently shake the plate to ensure the reaction mixture evenly covers the sample, and incubate at room temperature in the dark for 30 minutes. Remove the reaction solution and wash three times with washing buffer, 3-5 minutes each time. Cell nuclei were stained using Hoechst 33342 (Beyotime). Hoechst 33342 was diluted 1000× with PBS at a volume ratio of 1:1000. After removing the washing buffer, 500 μL of 1× Hoechst 33342 was added to each well and incubated at room temperature in the dark for 10 minutes. The staining solution was then removed, and the cells were washed three times with washing buffer for 3-5 minutes each time, followed by fluorescence detection. Hoechst 33342 showed blue fluorescence with a maximum excitation wavelength of 346 nm and a maximum emission wavelength of 460 nm. EdU, labeled with Alexa Fluor 488, showed green fluorescence with a maximum excitation wavelength of 495 nm and a maximum emission wavelength of 519 nm. Results were analyzed using ImageJ software. Figure 7 and Figure 8 As shown, in parental cells (MDA-MB-231 and BT549), rumacato combined with paclitaxel showed no significant difference in inhibitory effect on breast cancer cell proliferation compared to paclitaxel alone; however, in paclitaxel-resistant cells (MDA-MB-231...), the effect was not significantly different. PR and BT549 PR Rumacato combined with paclitaxel has a significant inhibitory effect on the proliferation of breast cancer cells compared with paclitaxel alone.

Claims

1. The use of a pharmaceutical composition in the preparation of a drug for the prevention and / or treatment of paclitaxel-resistant triple-negative breast cancer; The active ingredients of the pharmaceutical composition are rumacato and paclitaxel.

2. The application according to claim 1, characterized in that: The molar ratio of rumacato to paclitaxel is 1:0.1 to 1:

100.

3. The application according to claim 1 or 2, characterized in that: The structural formula of the Rumacato is as follows: ； The structural formula of paclitaxel is as follows: 。 4. The use of a pharmaceutical composition in the preparation of a product that inhibits the proliferation of paclitaxel-resistant cells; The active ingredients of the pharmaceutical composition are rumacato and paclitaxel.

5. The application according to claim 4, characterized in that: The molar ratio of rumacato to paclitaxel is 1:0.1 to 1:

100.

6. The application according to claim 5, characterized in that: The molar ratio of rumacato to paclitaxel is 1:0.5 to 1:

5.

7. The application according to claim 6, characterized in that: The molar ratio of rumacato to paclitaxel is 1:1 to 1:

2.

8. Paclitaxel sensitizer for triple-negative breast cancer, the active ingredient of which is rumacato.

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