Application of phenethyl caffeate or pterostilbene in the preparation of adjuvant drugs for the treatment of drug-resistant tumors

Abstract

This invention discloses the application of phenethyl caffeate or pterostilbene in the preparation of adjuvant drugs for the treatment of drug-resistant tumors. The study found that phenethyl caffeate can significantly improve the efficacy of doxorubicin and docetaxel against drug-resistant breast cancer and non-small cell lung cancer cells, while pterostilbene can significantly improve the efficacy of doxorubicin against drug-resistant breast cancer. This invention provides a novel treatment method combining phenethyl caffeate or pterostilbene with clinical antitumor drugs, which can significantly improve the treatment effect against drug-resistant tumors. Because phenethyl caffeate and pterostilbene are abundant, safe, and low in cost, this combination is simple and convenient to administer, making it suitable for large-scale application.

Description

Technical Field

[0001] This invention relates to the application of phenethyl caffeate or pterostilbene in the preparation of adjuvant drugs for the treatment of drug-resistant tumors, specifically to the application of phenethyl caffeate or pterostilbene in the preparation of drugs for the treatment of drug-resistant breast cancer and drug-resistant non-small cell lung cancer, and belongs to the pharmaceutical field. Background Technology

[0002] Multidrug resistance (MDR) in tumors refers to the development of resistance in tumor cells to multiple other antitumor drugs, both structurally similar and different, with varying mechanisms of action, after resistance to one antitumor drug. This often occurs after prolonged and repeated exposure to antitumor drugs. The mechanisms of tumor resistance mainly include increased drug efflux, decreased drug uptake, target mutations, and phenotypic switching. Multidrug resistance is closely related to the ATP-binding cassette (ABC) transporter, which primarily includes multidrug resistance proteins such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance protein (MRP), or multidrug and toxic compound efflux transporter (MATE). Among these, P-glycoprotein (P-gp)-mediated multidrug resistance is a crucial mechanism. P-gp is a broad-spectrum transporter that mediates the efflux of multiple antitumor drugs. Overexpression of P-gp in tumor cells enhances the efflux of drugs from the cells, leading to drug resistance in tumor cells and is one of the important reasons for multidrug resistance in tumors.

[0003] Multidrug resistance has become a major cause of chemotherapy failure in malignant tumors. In today's society with a high incidence of cancer, multidrug resistance seriously affects the efficacy of anti-tumor drug treatment and is often a key factor in clinical anti-tumor failure. It is also a thorny problem that needs to be solved in clinical practice, and there is currently no targeted method.

[0004] Caffeic acid phenethyl ester (CAPE), CAS No.: 104594-70-9, molecular formula C17H16O4, molecular weight: 284.3, structural formula as follows:

[0005]

[0006] Caffeic acid phenethyl ester is a phenolic compound mainly extracted from propolis, and it has a variety of pharmacological activities such as antioxidant, anti-inflammatory, analgesic, immunomodulatory, antibacterial, and antiviral effects.

[0007] Pterostilbene (PTE), CAS No. 537-42-8, molecular formula C16H16O3, molecular weight 256.3, structural formula as follows:

[0008]

[0009] Pterocarpus santalinus is mainly derived from plants such as sandalwood, blueberry, grape, and rosewood, and has been reported to have anti-inflammatory, antioxidant, and analgesic effects. Both are polyphenolic acids with similar physicochemical and pharmacological properties; currently, there is no evidence that they can enhance the therapeutic effect of antitumor drugs against drug-resistant tumor cells.

[0010] There are currently no reports of either drug having an adjuvant effect on drug-resistant tumor cells. Summary of the Invention

[0011] Purpose of the Invention: Addressing the serious problems of drug resistance in chemotherapy and the poor efficacy of antitumor drugs in treating drug-resistant tumors, this invention aims to provide the application of caffeic acid phenethyl ester or pterostilbene in the preparation of adjuvant drugs for the treatment of drug-resistant tumors. It can significantly enhance the efficacy of docetaxel, doxorubicin, and other drugs against drug-resistant non-small cell lung cancer and breast cancer through drug combinations or compound preparations. Its advantages include that the adjuvant antitumor drugs are derived from natural health products or plants, vegetables, and fruits, resulting in significant adjuvant antitumor effects while being safe and economical.

[0012] Technical solution: The application of caffeic acid phenethyl ester or pterostilbene in the preparation of adjuvant therapy drugs for drug-resistant tumors.

[0013] Furthermore, the drug is a compound drug or a combination drug containing caffeic acid phenethyl ester or pterostilbene.

[0014] Furthermore, the dosage form of the drug is tablets, capsules, granules, injections, or oral liquid preparations.

[0015] Furthermore, the combined / adjuvant drugs of the drug include one or more of doxorubicin, docetaxel, tyrosine kinase inhibitors, antibiotics, nucleic acid drugs, or alkaloids.

[0016] Furthermore, the drug comprises phenethyl caffeate and doxorubicin, wherein the molar ratio of phenethyl caffeate to doxorubicin is 0.1–5:0.01–1.

[0017] Furthermore, the drug comprises phenethyl caffeate and docetaxel, wherein the molar ratio of phenethyl caffeate to docetaxel is 5-50:1-100.

[0018] Furthermore, the drug comprises pterostilbene and doxorubicin, wherein the molar ratio of pterostilbene to doxorubicin is 1–5:0.01–1.

[0019] Furthermore, the drug-resistant tumor is a drug-resistant tumor with high expression of multidrug resistance proteins, such as breast cancer and non-small cell lung cancer.

[0020] Furthermore, multidrug resistance proteins are P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance protein (MRP), or multidrug and toxic compound efflux transporter (MATE).

[0021] Furthermore, the caffeic acid phenethyl ester or pterostilbene is derived from propolis, vegetables or fruits, or synthesized using modern biotechnology and chemical methods.

[0022] Studies have found that phenethyl caffeate and pterostilbene alone have little antitumor effect on drug-resistant tumor cells. However, when used in combination with other antitumor drugs, they significantly enhance the activity of the combined antitumor drugs against drug-resistant tumor cells, thus exerting a significant antitumor effect on drug-resistant tumors. Phenethyl caffeate or pterostilbene can be used to prepare drugs for treating drug-resistant tumors or in combination with other antitumor drugs.

[0023] The study first revealed that, 24 hours after treatment with doxorubicin and caffeic acid phenethyl ester (monotherapy / combination therapy), cell viability was significantly lower in the doxorubicin combined with caffeic acid phenethyl ester group compared to other groups, as measured by the CCK-8 assay. Further analysis of intracellular ROS levels, 24 hours after treatment, showed that caffeic acid phenethyl ester significantly reduced the increase in ROS induced by chemotherapy drugs. Further evaluation of P-gp levels and function, using Western blotting and Rho123 efflux assays, showed a significant decrease in P-gp levels and function in MCF-7 / ADR cells, indicating increased entry of antitumor drugs into tumor cells. This suggests that caffeic acid phenethyl ester may regulate the high expression of P-gp after drug resistance by reducing intracellular ROS levels, thereby reducing the efflux of antitumor drugs from tumor cells and exerting a better antitumor effect. The combination of pterostilbene and doxorubicin also showed similar effects to the combination of doxorubicin and caffeic acid phenethyl ester, and its effect on inhibiting tumor cells was more obvious and more advantageous.

[0024] Furthermore, in lung adenocarcinoma A549 / DTX cells, the combined use of caffeic acid phenethyl ester and docetaxel showed a similar effect to that in breast cancer cells, namely, caffeic acid phenethyl ester significantly enhanced the antitumor effect of docetaxel on drug-resistant lung adenocarcinoma cells.

[0025] The above research results clearly indicate that caffeic acid phenethyl ester can significantly enhance the efficacy of doxorubicin against drug-resistant breast cancer cells, significantly enhance the efficacy of docetaxel against docetaxel-resistant breast cancer cells, and styrax significantly enhances the efficacy of doxorubicin against drug-resistant breast cancer cells. Their mechanisms of action are all related to regulating cellular redox balance, downregulating drug resistance proteins, and increasing the entry of antitumor drugs into drug-resistant cells.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0027] (1) The present invention found that caffeic acid phenethyl ester can significantly improve the efficacy of doxorubicin and docetaxel against drug-resistant breast cancer and lung adenocarcinoma cells, and that styrax can significantly improve the efficacy of doxorubicin against drug-resistant breast cancer.

[0028] (2) This invention provides a novel treatment method combining caffeic acid phenethyl ester or pterostilbene with clinical antitumor drugs, which can significantly improve the treatment effect on drug-resistant tumors. Since caffeic acid phenethyl ester and pterostilbene are abundant, safe, and low in cost, the administration method of this combination is simple and convenient, and suitable for large-scale promotion and application. Attached Figure Description

[0029] Figure 1 The image shows the antitumor efficacy of caffeic acid phenethyl ester combined with doxorubicin against drug-resistant breast cancer cells in Example 1. Two-way ANOVA analysis was performed, and the CAPE+ADR group was compared with the ADR group. *p<0.05,***p<0.001.

[0030] Figure 2 The figure shows the effect of caffeic acid phenethyl ester on ROS, P-gp expression and function in drug-resistant breast cancer cells in Example 2; one-way ANOVA analysis was performed, and ***p<0.001.

[0031] Figure 3 The figure shows the effect of caffeic acid phenethyl ester on the intracellular doxorubicin concentration in drug-resistant breast cancer cells in Example 3; T-test analysis was used, and ***p<0.001.

[0032] Figure 4 The image shows the antitumor efficacy of caffeic acid phenethyl ester combined with docetaxel against drug-resistant non-small cell lung cancer in Example 4. Two-way ANOVA analysis was performed, and the CAPE+DTX group was compared with the DTX group. *p<0.05,***p<0.001.

[0033] Figure 5 The graph shows the expression and function of ROS and P-gp in drug-resistant non-small cell lung cancer by caffeic acid phenethyl ester in Example 5; one-way ANOVA analysis was performed, **p<0.01, ***p<0.001.

[0034] Figure 6 The figure shows the effect of caffeic acid phenethyl ester on the intracellular docetaxel concentration in drug-resistant non-small cell lung cancer cells in Example 6; T-test analysis was performed, *p<0.05.

[0035] Figure 7 The pharmacodynamics of Pterostilbene combined with doxorubicin against drug-resistant breast cancer in Example 7 is shown in the figure. Two-way ANOVA analysis was performed, and the PTE+ADR group was compared with the ADR group. ***p<0.001.

[0036] Figure 8 The figure shows the effect of Pterocarya stenoptera on the expression and function of ROS and P-gp in drug-resistant breast cancer cells in Example 8; one-way ANOVA analysis was performed, and ***p<0.001.

[0037] Figure 9 The figure shows the effect of pterostilbene on the intracellular doxorubicin concentration in drug-resistant breast cancer cells in Example 9; T-test analysis was used, and ***p<0.001. Detailed Implementation

[0038] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0039] Cell source and culture: Human breast cancer doxorubicin-resistant cell line MCF-7 / ADR (provided by Jiangsu Kaiji Biotechnology Co., Ltd.) was used. Cells were cultured at 37℃ in a 5% CO2 environment in RPMI 1640 medium containing 10% fetal bovine serum (FBS), streptomycin 100 mg / L, and penicillin 100 mg / L. Cells grew at 25 cm⁻¹. 2 In the culture dish, change the culture medium every 2-3 days, and passage the cells when they reach 90% confluence.

[0040] Cell source and culture: Following the method used for the doxorubicin-resistant human breast cancer cell line MCF-7 / ADR, the human non-small cell lung cancer cell line A549 was purchased from the Chinese Academy of Sciences Type Culture Collection. After laboratory induction to a docetaxel-resistant strain A549 / DTX, cells were cultured at 37°C in a 5% CO2 environment in RPMI 1640 medium containing 10% fetal bovine serum (FBS), streptomycin 100 mg / L, and penicillin 100 mg / L. Cells grew at 25 cm⁻¹. 2 In the culture dish, change the culture medium every 2-3 days, and passage the cells when they reach 90% confluence.

[0041] Example 1: Study on the antitumor efficacy of caffeic acid phenethyl ester combined with doxorubicin against drug-resistant breast cancer cells.

[0042] Experimental method: MCF-7 / ADR cells in logarithmic growth phase were digested with 0.25% trypsin and seeded at 10,000 cells / well in 96-well culture plates. After the cells had basically adhered, the medium was replaced with drug-containing medium. The groups were as follows: control group (no drug administration, NC); caffeine phenethyl ester group (CAPE): only different concentrations of caffeine phenethyl ester solution (0, 0.1, 0.5, 1.0, 5.0, 10.0, 20.0 μmol / L / or μM) were administered; doxorubicin group (ADR): only different concentrations of doxorubicin solution (0, 0.01, 0.05, 0.1, 1.0, 5.0, 10.0 μmol / L) were administered; caffeine phenethyl ester + doxorubicin group (CAPE+ADR): different concentrations (0, 0.01, 0.05, 0.1, 1.0, 5.0, 10.0 μmol / L) of doxorubicin combined with a fixed concentration (10 μmol / L) of caffeine phenethyl ester. After incubation at 37℃ for 24 hours, 10 μL of CCK-8 solution was added, and the mixture was incubated at 37℃ for 30 min. The absorbance of each well was measured at 450 nm using a microplate reader and compared with the control group without the drug. The results are as follows: Figure 1 As shown.

[0043] Depend on Figure 1 The results showed that caffeic acid phenethyl ester alone had no significant inhibitory effect on drug-resistant breast cancer cells. Compared with the doxorubicin group, the combination of caffeic acid phenethyl ester and doxorubicin significantly enhanced the inhibitory effect of doxorubicin on MCF-7 / ADR-resistant cells at low concentrations (0.01-1.0 μmol / L). In other words, caffeic acid phenethyl ester can significantly enhance the antitumor efficacy of doxorubicin against drug-resistant tumor cells.

[0044] Example 2: Effects of caffeic acid phenethyl ester on ROS, P-gp expression and function in drug-resistant breast cancer cells

[0045] Experimental method: Following the method in Example 1, MCF-7 / ADR cells in the logarithmic growth phase were taken, digested with 0.25% trypsin, and then subjected to a reaction at a concentration of 3 × 10⁻⁶ cells / mL. 5 Cells were seeded per well in 12-well plates. Once cells had largely adhered, the medium was replaced with drug-containing medium. Groups were as follows: control group (no drug, NC); caffeic acid phenethyl ester group (CAPE, 10 μmol / L); doxorubicin group (ADR, 0.1 μmol / L); caffeic acid phenethyl ester (10 μmol / L) + doxorubicin (0.1 μmol / L) group (CAPE+ADR); cultured at 37°C for 24 hours.

[0046] A 10 μmol / L DCFH-DA solution was prepared using blank culture medium, and 1 mL / well was added to each well of the plate. Incubation was performed at 37°C for 60 minutes, with gentle shaking of the plate every 10 minutes to ensure probe contact. After incubation, cells were washed three times with PBS, lysed, and centrifuged at 6000 rpm for 5 minutes at 4°C. 200 μL of the supernatant was collected and read at 488-525 nm using a microplate reader to determine ROS levels. Additionally, cultured cells were collected, cell membrane proteins were extracted, and Western blotting experiments were performed, involving P-gp and Na+. + / K + After incubation with the ATPase-specific recognition antibody, the expression level of P-gp protein was detected by chemiluminescence immunoassay. Results are as follows: Figure 2 As shown.

[0047] In addition, 10,000 cells / well were seeded into 96-well culture plates. After treatment, the cells were washed twice with D-Hanks. 100 μL of 1 μmol / L Rho123 was added to each well, and the plates were incubated for 40 min. The cells were then washed three times with D-Hanks, and 100 μL of D-Hanks was added. Live-cell imaging was performed to acquire images. After adjusting the brightness and contrast to the same values, the images were exported. P-gp function was detected using the Rho123 method. The results are as follows: Figure 2 As shown.

[0048] Figure 2 The diagram shows the effects of caffeic acid phenethyl ester on ROS, P-gp expression and function in drug-resistant breast cancer cells in Example 2. In the diagram, A shows the effect of caffeic acid phenethyl ester combined with doxorubicin on intracellular ROS expression, B shows the effect of caffeic acid phenethyl ester combined with doxorubicin on intracellular P-gp protein expression, and C shows the effect of caffeic acid phenethyl ester combined with doxorubicin on the function of drug-resistant breast cancer cells.

[0049] Depend on Figure 2 It can be seen that: the combination of caffeic acid phenethyl ester and doxorubicin significantly reduced intracellular ROS levels ( Figure 2 A) P-gp protein expression level ( Figure 2 B), and at the same time, the intracellular Rho123 fluorescence intensity increased ( Figure 2 C) indicates that P-gp function is suppressed.

[0050] Example 3: Effect of caffeic acid phenethyl ester on intracellular doxorubicin concentration in drug-resistant breast cancer cells

[0051] Experimental Methods: Following the method in Example 2, MCF-7 / ADR cells in the logarithmic growth phase were seeded into plates. After reaching 80-90% confluence, cells were administered doxorubicin (ADR, 0.1 μmol / L) alone or in combination with caffeine phenethyl ester (CAPE+ADR, 10 μmol / L). Cells were collected 24 h after administration, washed three times with pre-cooled PBS, and 500 μL of ultrapure water was added. Cells were scraped off and sonicated. 100 μL of the supernatant was added to 300 μL of acetonitrile containing the internal standard to precipitate proteins. The mixture was shaken for 10 min, centrifuged at 18000 rpm and 4℃ for 10 min, and 200 μL of the supernatant was collected. The supernatant was centrifuged at 18000 rpm and 4℃ for 5 min, and 80 μL of the supernatant was transferred to an intracellular catheter. 10 μL of the supernatant was injected for analysis to determine the intracellular doxorubicin concentration in MCF-7 / ADR cells, investigating the effect of combined administration of caffeine phenethyl ester. Results are as follows: Figure 3 As shown.

[0052] Depend on Figure 3 The results showed that the intracellular concentration of doxorubicin significantly increased after administration of caffeic acid phenethyl ester in combination with doxorubicin. This suggests that the synergistic effect of caffeic acid phenethyl ester on doxorubicin is due to the increased concentration of the latter in resistant cells.

[0053] Example 4: Study on the antitumor efficacy of caffeic acid phenethyl ester combined with docetaxel against drug-resistant non-small cell lung cancer.

[0054] Experimental Methods: A549 / DTX cells in logarithmic growth phase were digested with 0.25% trypsin and seeded at 6000 cells / well in 96-well culture plates. After basic cell attachment, the medium was replaced with drug-containing medium. Groups were as follows: Control group (NC, no drug administered); Caffeine phenethyl ester group (CAPE): administered only with different concentrations of caffeine phenethyl ester solution (0, 5, 10, 20, 30, 40, 50 μmol / L); Docetaxel group (DTX): administered only with different concentrations of docetaxel solution (0, 0.1, 0.5, 2, 10, 50, 100 μmol / L); Caffeine phenethyl ester + Docetaxel group (CAPE+DTX): different concentrations (0, 0.1, 0.5, 2, 10, 50, 100 μmol / L) of docetaxel combined with a fixed concentration (40 μmol / L) of caffeine phenethyl ester. After incubation at 37℃ for 24 hours, 10 μL of LCK-8 solution was added, and the mixture was incubated at 37℃ for 30 minutes. The absorbance of each well was measured using a microplate reader at a wavelength of 450 nm. The results are as follows: Figure 4 As shown.

[0055] Depend on Figure 4The results showed that within the experimental concentration range, caffeic acid phenethyl ester itself had no significant inhibitory effect on drug-resistant non-small cell lung cancer cells. Compared with the docetaxel group, caffeic acid phenethyl ester significantly enhanced the inhibitory effect of docetaxel on A549 / DTX cells at the experimental concentration. That is, caffeic acid phenethyl ester can significantly enhance the antitumor efficacy of docetaxel against drug-resistant non-small cell lung cancer cells.

[0056] Example 5: Effects of caffeic acid phenethyl ester on ROS and P-gp expression and function in drug-resistant non-small cell lung cancer

[0057] Experimental method: Referring to the method in Example 2, A549 / DTX-resistant non-small cell lung cancer cells in the logarithmic growth phase were taken, digested with 0.25% trypsin, and then subjected to a 1×10⁻⁶ solution. 5 Cells were seeded in 12-well plates. After basic cell attachment, the medium was replaced with drug-containing medium. Groups were as follows: control group (no drug, NC); caffeic acid phenethyl ester group (CAPE, 40 μmol / L); docetaxel group (DTX, 2.0 μmol / L); caffeic acid phenethyl ester (40 μmol / L) + docetaxel group (2.0 μmol / L) (CAPE+DTX). Cells were cultured at 37°C for 24 hours. Cell samples were collected, ROS concentration was measured, mRNA was extracted and reverse transcribed, and P-gp expression levels were measured by Q-RT-PCR. Results are as follows. Figure 5 As shown.

[0058] Further analysis of P-gp function was performed using the Rho123 method: 7000 cells / well were seeded into 96-well plates. After treatment, cells were washed twice with D-Hanks. 100 μL of 1 μmol / L Rho123 was added to each well, and the plates were incubated for 40 min. Cells were then washed three times with D-Hanks, and 100 μL of D-Hanks was added. Live-cell imaging was performed, and the brightness and contrast were adjusted to the same values ​​before exporting the results. Figure 5 As shown.

[0059] Figure 5 This is a graph illustrating the effects of caffeic acid phenethyl ester on ROS, P-gp expression, and function in drug-resistant non-small cell lung cancer (NSCLC) in Example 5. In this graph, A shows the effect of caffeic acid phenethyl ester combined with docetaxel on intracellular ROS levels; B shows the effect of caffeic acid phenethyl ester combined with docetaxel on intracellular P-gp gene expression levels; and C shows the effect of caffeic acid phenethyl ester on the function of drug-resistant NSCLC cells. Figure 5 The results showed that the combination of caffeic acid phenethyl ester and docetaxel significantly reduced intracellular ROS levels. Figure 5 A) Downregulates P-gp gene expression levels ( Figure 5 B); and the fluorescence intensity of Rho123 increased after the two drugs were used in combination (B); Figure 5C) indicates that the P-gp efflux function is inhibited.

[0060] Example 6: Effect of caffeic acid phenethyl ester on intracellular docetaxel concentration in drug-resistant lung adenocarcinoma cells

[0061] Experimental Methods: Following the method in Example 5, A549 / DTX-resistant non-small cell lung cancer cells in the logarithmic growth phase were seeded into 6-well culture plates. After reaching 80-90% confluence, docetaxel (DTX, 2.0 μmol / L) was administered alone or in combination with caffeoyl phenethyl ester (CAPE+DTX, 40 μmol / L). Cells were collected after 24 hours, washed three times with pre-cooled PBS, and 400 μL of ultrapure water was added. Cells were scraped off and sonicated. 100 μL of the supernatant was added to 300 μL of methanol containing the internal standard to precipitate proteins. The mixture was shaken for 10 min, centrifuged at 18000 rpm and 4°C for 10 min, and 200 μL of the supernatant was collected. The supernatant was centrifuged at 18000 rpm and 4°C for 5 min, and 80 μL of the supernatant was transferred to an inner tube. 20 μL of the supernatant was injected to determine the intracellular docetaxel concentration. Results are as follows: Figure 6 As shown.

[0062] Depend on Figure 6 The results showed that, compared with docetaxel alone, the combination of caffeic acid phenethyl ester and docetaxel significantly increased the intracellular concentration of docetaxel, suggesting that the reason why caffeic acid phenethyl ester enhances the effect of docetaxel is that it increases the concentration of the latter in drug-resistant cells.

[0063] Example 7: Study on the antitumor efficacy of pterostilbene combined with doxorubicin against drug-resistant breast cancer cells.

[0064] Experimental method: Following the method in Example 1, drug-resistant breast cancer cells MCF-7 / ADR in the logarithmic growth phase were taken, digested with 0.25% trypsin, and seeded at 10,000 cells / well in 96-well culture plates. After the cells had basically adhered, the medium was replaced with drug-containing medium. The groups were as follows: control group (NC, no drug administered); Pterocarya oleifera group (PTE): administered only with different concentrations of Pterocarya oleifera solution (0, 1, 2, 5, 10, 20, 50 μmol / L); Doxorubicin group (ADR): administered only with different concentrations of doxorubicin solution (0, 0.01, 0.05, 0.1, 1.0, 5.0, 10.0 μmol / L); Pterocarya oleifera + Doxorubicin group (PTE+ADR): administered with different concentrations (0, 0.01, 0.05, 0.1, 1.0, 5.0, 10.0 μmol / L) of doxorubicin combined with a fixed concentration (5 μmol / L) of Pterocarya oleifera. After incubation at 37℃ for 24 hours, wash twice with 200 μL PBS, discard the PBS, add 10 μL CCK-8 solution, and incubate at 37℃ for 30 min. Measure the absorbance of each well at 450 nm using a microplate reader. Results are as follows: Figure 7 As shown.

[0065] Depend on Figure 7 The results showed that at low concentrations (<5 μmol / L), stigmataecarpa itself had no significant inhibitory effect on drug-resistant breast cancer cells. Compared with the doxorubicin group, stigmataecarpa significantly enhanced the inhibitory effect of low concentrations (0.01–0.1 μmol / L) of doxorubicin on MCF-7 / ADR-resistant cells. In other words, stigmataecarpa can significantly enhance the antitumor efficacy of doxorubicin against drug-resistant tumor cells.

[0066] Example 8: Effects of Pterocarya stenoptera on ROS, Pgp (P-gp) expression and function in drug-resistant breast cancer cells

[0067] Experimental method: Following the method in Example 2, drug-resistant breast cancer cells MCF-7 / ADR in the logarithmic growth phase were taken, digested with 0.25% trypsin, and then subjected to 3×10⁻⁶ mol / L hydrochloride solution. 5 Cells were seeded per well in 12-well plates. After the cells had largely adhered, the medium was replaced with drug-containing medium. The groups were as follows: control group (no drug administration, NC); Pterostilbene group (PTE, 5 μmol / L); Doxorubicin group (ADR, 0.1 μmol / L); Pterostilbene (5 μmol / L) + Doxorubicin (0.1 μmol / L) group (PTE + ADR); cultured at 37°C for 24 hours.

[0068] ROS assay: Prepare a 10 μmol / L DCFH-DA solution using blank culture medium, add 1 mL / well to the plate, and incubate at 37°C for 60 minutes. Gently shake the culture plate every 10 minutes to ensure the probe is fully in contact with the cells. After incubation, wash the cells three times with PBS, lyse the cells, centrifuge at 4°C and 6000 rpm for 5 minutes, and take 200 μL of the supernatant for reading at 488-525 nm using a microplate reader.

[0069] Western blotting: Cells were collected, total cellular protein was extracted, and Western blotting was performed. After incubation with P-gp and GAPDH specific recognition antibodies, the antibody signals were detected by chemiluminescence.

[0070] Rho123 assay for P-gp function: 10,000 cells / well were seeded into 96-well plates. After treatment, cells were washed twice with D-Hanks. 100 μL of 1 μmol / L Rho123 was added to each well, and the plates were incubated for 60 min. Cells were washed three times with D-Hanks, and 100 μL of D-Hanks was added. Live-cell imaging was performed to acquire images, and the brightness and contrast were adjusted to the same values ​​before exporting. Results are as follows: Figure 8 As shown.

[0071] Figure 8This is a graph illustrating the effects of *Pterocarya stenoptera* on ROS, P-gp expression, and function in drug-resistant breast cancer cells, as shown in Example 8. In Figure A, the effect of *Pterocarya stenoptera* combined with doxorubicin on intracellular ROS levels; in Figure B, the effect of *Pterocarya stenoptera* combined with doxorubicin on intracellular P-gp protein expression levels; and in Figure C, the effect of *Pterocarya stenoptera* on the function of drug-resistant non-small cell lung cancer cells. Figure 8 The results show that the combination of pterostilbene and doxorubicin significantly reduced intracellular ROS levels. Figure 8 A) P-gp protein expression level ( Figure 8 B), and at the same time, the intracellular Rho123 fluorescence intensity increased ( Figure 8 C) indicates that P-gp function is suppressed.

[0072] Example 9: Effect of Pterostilbene on Intracellular Doxorubicin Concentration in Drug-Resistant Breast Cancer Cells

[0073] Experimental Methods: Following the method in Example 3, MCF-7 / ADR resistant breast cancer cells in the logarithmic growth phase were seeded into plates. The experimental method was the same as in Example 8. After reaching 80-90% confluence, cells were treated with doxorubicin (ADR, 0.1 μmol / L) alone or in combination with pterostilbene (PTE+ADR, 5 μmol / L). After 24 h of treatment, cells were collected, washed three times with pre-cooled PBS, and 400 μL of ultrapure water was added. Cells were scraped off and sonicated. 100 μL of the supernatant was added to 300 μL of acetonitrile containing the internal standard to precipitate proteins. The mixture was shaken for 10 min, centrifuged at 18000 rpm and 4℃ for 10 min, and 200 μL of the supernatant was collected. The cells were centrifuged at 18000 rpm and 4℃ for 5 min, and 80 μL of the supernatant was transferred to an intracellular catheter. 10 μL of the supernatant was injected for analysis. The effect of combined administration of pterostilbene and doxorubicin was investigated by measuring the intracellular doxorubicin concentration in MCF-7 / ADR cells. Results are as follows: Figure 9 As shown.

[0074] Depend on Figure 9 The results showed that the intracellular concentration of doxorubicin significantly increased after administration of pterostilbene in combination with doxorubicin. This suggests that the synergistic effect of pterostilbene on doxorubicin is due to the increased concentration of the latter in resistant cells.

[0075] In summary, this invention found that phenethyl caffeate can significantly enhance the efficacy of doxorubicin against MCF-7 / ADR-resistant tumor cells and significantly enhance the efficacy of docetaxel against A549 / DTX-resistant tumor cells; stigmata can also significantly enhance the efficacy of doxorubicin against MCF-7 / ADR-resistant tumor cells. The mechanisms of these enhanced effects on drug-resistant tumor cells are all related to regulating cellular redox balance, downregulating drug resistance proteins, and increasing the entry of antitumor drugs into drug-resistant cells. This suggests that phenethyl caffeate and stigmata can assist clinical antitumor drugs in significantly enhancing their efficacy against drug-resistant tumor cells.

Claims

1. The use of caffeic acid phenethyl ester and docetaxel in the preparation of antitumor drugs for drug-resistant non-small cell lung cancer, wherein, The molar ratio of caffeic acid phenethyl ester and docetaxel is 5-50:1-100.

2. Use according to claim 1, characterized in that, The medicine is a compound medicine or a combined medicine containing caffeic acid phenethyl ester and docetaxel.

3. Use according to claim 2, characterized in that, The dosage form of the medicine is a tablet, a capsule, a granule, an injection or an oral liquid preparation.

4. Use according to any one of claims 1 to 3, characterized in that, The source of the caffeic acid phenethyl ester is extracted from propolis, a vegetable or a fruit, or synthesized by modern biotechnology or a chemical method.

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