Application of allicin in preparation of antitumor drug sensitizer and tumor chemotherapy drug composition

Abstract

The invention belongs to the technical field of anti-tumor drugs, and particularly relates to application of allicin in preparation of an anti-tumor drug sensitizer and a tumor chemotherapy pharmaceutical composition, an A549 cell strain, a paclitaxel drug-resistant cell model A549 / Taxol and a tumor-bearing mouse model are established, and in-vivo and in-vitro pharmacodynamic research finds that the allicin can effectively inhibit cell proliferation and has a good anti-tumor effect. The apoptosis is induced, and the drug resistance of paclitaxel is reversed. The compound inhibits P-gp expression, reduces drug efflux and destroys cell autophagy. Particularly, allicin inhibits cathepsin B (CTSB), destroys lysosome functions, blocks autophagy flux, reduces mitochondrial membrane potential and enhances sensitivity to paclitaxel. The combination of allicin and paclitaxel can significantly inhibit the growth of tumor nodules and reduce the number of tumor nodules, shows the potential of reversing the drug resistance of paclitaxel in vivo, can significantly improve the chemotherapy effect of NSCLC, and has potential clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of tumor treatment, and in particular to the use of allicin in the preparation of anti-tumor drug sensitizers by regulating CTSB (cathepsin B) and a tumor chemotherapy drug composition. Technical Background

[0002] Lung cancer is the malignant tumor with the highest mortality rate, among which non-small cell lung cancer (NSCLC) is the most common pathological type of lung cancer, accounting for 80-85% of the incidence. In the 1960s, the chemotherapy drugs for lung cancer were mainly nitrogen mustard, cyclophosphamide, etc., but the efficacy was poor; after the 1990s, the third-generation chemotherapy drugs such as paclitaxel, docetaxel, vinorelbine, gemcitabine and other drugs have achieved significant clinical efficacy. At present, paclitaxel (Taxol) has become the first-line chemotherapy regimen for the treatment of advanced NSCLC. Although paclitaxel has a good therapeutic effect on NSCLC, the overall survival rate is still low, and its single-drug efficacy is only 50-70%. Nearly half of the patients are still insensitive to paclitaxel treatment. Tumor cells are primary or secondary resistant to paclitaxel, which greatly limits its use in the clinical treatment of NSCLC. Therefore, how to effectively improve the sensitivity of tumors to paclitaxel and reduce the occurrence of drug resistance is an urgent problem to be solved in the treatment of advanced NSCLC.

[0003] Studies have shown that the occurrence of paclitaxel resistance is related to changes in multiple molecular mechanisms in tumor cells. Among them, cathepsin B (CTSB) plays an important role in cancer cell invasion, migration, apoptosis inhibition and drug resistance. CTSB is a lysosomal protease that can affect the response of cancer cells to chemotherapeutic drugs by regulating multiple intracellular signaling pathways. Previous studies have shown that overexpression of CTSB is associated with drug resistance in a variety of cancers, including NSCLC. Therefore, inhibiting the expression or activity of CTSB may be an important strategy to reverse paclitaxel resistance.

[0004] Allicin, as a natural organosulfur compound, has multiple biological activities, including anti-cancer, antioxidant, and antibacterial effects. The anti-cancer mechanisms of allicin include inducing cancer cell apoptosis, inhibiting cell proliferation, and inhibiting metastasis.

[0005] In view of the multiple anticancer activities of allicin and its regulatory effects on tumor resistance mechanisms, it is of great significance to explore the potential of allicin in reversing paclitaxel resistance. Summary of the invention

[0006] Purpose of the invention: In view of the problems existing in the above-mentioned prior art, the present invention provides the use of allicin in the preparation of an anti-tumor drug sensitizer and a tumor chemotherapy drug composition, aiming to use allicin to inhibit the expression or activity of CTSB, especially to reverse the drug resistance of NSCLC to paclitaxel, to provide a new combined treatment strategy, and to improve the treatment effect of NSCLC patients.

[0007] Technical solution:

[0008] The first aspect of the present invention provides the use of allicin in the preparation of an anti-tumor drug sensitizer or reversal agent.

[0009] Furthermore, the allicin is a sensitizer or reversal agent of the anti-tumor drug paclitaxel.

[0010] Furthermore, the allicin reduces the drug resistance of tumor cells to paclitaxel by inhibiting the CTSB-related signaling pathways. The CTSB-related signaling pathways include lysosome-autophagy, mitochondrial function and other related pathways.

[0011] Furthermore, the tumors include non-small cell lung cancer, breast cancer, liver cancer, gastric cancer, malignant lymphoma, nasopharyngeal carcinoma, melanoma, esophageal cancer and intestinal cancer.

[0012] Preferably, the tumor is non-small cell lung cancer.

[0013] The second aspect of the present invention provides the use of allicin in the preparation of a drug for inhibiting cell protective autophagy.

[0014] Furthermore, the cell protective autophagy is formed through the lysosome-autophagy pathway mediated by CTSB.

[0015] The third aspect of the present invention provides a non-small cell lung cancer chemotherapy drug composition, comprising allicin and paclitaxel, and pharmaceutically acceptable excipients. The drug composition can be prepared into an oral preparation or an injection, but is not limited to the above dosage forms.

[0016] Preferably, the allicin includes its main body, and one or more of its pharmaceutically acceptable salts, stereoisomers and prodrug molecules, allicin derivatives and its pharmaceutically acceptable salts, stereoisomers and prodrug molecules.

[0017] Preferably, the effective concentration of allicin is 5-20 μM. The concentration of paclitaxel is not less than 5 nM, preferably 300 nM.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention proposes the use of allicin in the preparation of an anti-tumor drug sensitizer. By establishing an A549 cell line, a paclitaxel-resistant cell model A549 / Taxol and a tumor-bearing mouse model, in vitro and in vivo pharmacodynamic studies have shown that allicin can effectively inhibit cell proliferation, induce cell apoptosis and reverse paclitaxel resistance.

[0020] The experimental results showed that allicin reduced the expression of P-gp and its drug efflux function, enhanced the accumulation of drugs in cells, and further enhanced the cytotoxicity and apoptosis-inducing ability of Taxol. Allicin inhibited P-gp expression, reduced drug efflux, and disrupted cell autophagy. In particular, allicin inhibited cathepsin B (CTSB), disrupted lysosomal function, interfered with the formation of lysosome-autophagy and mitochondrial functional pathways, blocked autophagic flux, reduced mitochondrial membrane potential, and enhanced sensitivity to paclitaxel.

[0021] The present invention also provides a new pharmaceutical composition and a combined treatment scheme, wherein allicin and paclitaxel are combined to prepare a drug for treating non-small cell lung cancer, and the viability of A549 / Taxol resistant cells is significantly reduced, and the IC 50 Significantly reduce; allicin interferes with the degradation activity of lysosomes and the process of autophagy by inhibiting the expression and function of CTSB. The autophagy blockade in the reversal process is through the inhibition of CTSB formation, which can include direct inhibition of CTSB or indirect inhibition of CTSB pathway formation, significantly improving the inhibitory effect of Taxol on drug-resistant NSCLC cells. Enhance the sensitivity of tumor cells to paclitaxel, reduce the dosage of paclitaxel, and thus reduce the toxic side effects associated with chemotherapy. In addition, allicin combined with paclitaxel can significantly inhibit the growth of tumor nodules and reduce the number of tumor nodules, showing its potential to reverse paclitaxel resistance in vivo. It can significantly improve the chemotherapy effect of NSCLC.

[0022] In summary, allicin combined with Taxol can significantly inhibit the growth of paclitaxel-resistant tumors in mice. Therefore, allicin as an inhibitor of CTSB can effectively reverse the Taxol resistance of NSCLC and play a sensitizing role, which has potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figures are the correlation analysis of CTSB and paclitaxel resistance and survival of lung cancer patients; (a) is the analysis of CTSB protein level in paclitaxel-sensitive / resistant (A549, A549 / T) lung cancer cells, and the data are expressed as mean ± standard deviation (n = 3), ***p < 0.001 vs. A549 group; (b) is the Kaplan-Meier survival diagram based on the Cancer Genome Atlas (TCGA) database to analyze the correlation between patient survival rate and CTSB expression;

[0024] Figure 2 The expression analysis of CTSB protein (a) and mRNA (b) in A549 / Taxol cells by different shRNAs;

[0025] Figure 3 This is the result analysis diagram of the effect of different shRNAs on the survival rate of A549 / Taxol cells;

[0026] Figure 4 Figure 1 shows the results of the inhibition of cell proliferation and induction of apoptosis by paclitaxel when allicin and paclitaxel are used in combination; (A) shows the inhibitory effect of paclitaxel on A549 / Taxol cells enhanced by shCTSB-776 (a) and the IC of paclitaxel after shCTSB-776 treatment 50 Value (b); (B) is a fluorescence image of flow cytometry showing that CTSB inhibitor CA074 Me can promote apoptosis of A549 / Taxol cells through paclitaxel; (C) is a numerical analysis of the accumulation of Rho123 after shCTSB-776 interfered with A549 / Taxol cells;

[0027] Figure 5 The results of flow cytometry determination of intracellular Rho-123 uptake rate; (a) is a fluorescence image of flow cytometry showing that CTSB inhibitor CA074 Me can promote apoptosis of A549 / Taxol cells through paclitaxel; (b) is a numerical analysis of Rho123 accumulation after shCTSB-776 interfered with A549 / Taxol cells;

[0028] Figure 6 Abnormal fluorescence images of lysosomal degradation activity caused by CTSB knockdown detected by DQ-BSA assay, scale bar = 10 μm; and quantitative numerical analysis of the integrated optical density (IOD) of each cell;

[0029] Figure 7 Fluorescence images and numerical analysis graphs showing the effect of shCTSB-776 on autophagic vacuoles formation detected by GFP-LC3 assay;

[0030] Figure 8 Fluorescence images of A549 / Taxol cells infected with mRFP-GFP-LC3 lentivirus after interference with shCTSB-776, scale bar = 50 μm; and statistical analysis of the red dot level of each cell;

[0031] Fig. 9 To analyze the ultrastructural changes of cells by TEM, representative TEM images are shown; green indicates phagosomes; yellow indicates autophagosomes; red indicates autolysosomes, scale bar = 2 μM;

[0032] Fig.10 Western blot analysis of autophagy-related proteins in A549 / Taxol cells after CTSB knockout; data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 vs control group; #p < 0.05 vs paclitaxel; ns: not significant;

[0033] Fig.11 Typical morphological images of parental (a. A549), paclitaxel-resistant NSCLC (b. A549 / Taxol), and A549 / Taxol cells treated with allicin (10 μM) combined with paclitaxel (300 nM) (c), scale bar = 200 μm;

[0034] Fig.12 is the IC of paclitaxel against A549 (a) and A549 / Taxol (b) 50 Value, IC value of allicin on A549 / Taxol 50 Value (c), IC value of paclitaxel on A549 / Taxol after allicin (IC10) treatment 50 Value (d) Data analysis graph;

[0035] Fig.13 This is a graph and statistical analysis of the long-term inhibitory effect of allicin and paclitaxel on A549 / Taxol cells measured by clone formation assay;

[0036] Fig.14 Annexin-V-FITC / PI staining was performed for 48 hours to observe the apoptosis-inducing effects of paclitaxel, allicin, and allicin combined with paclitaxel on A549 / Taxol cells and statistical analysis;

[0037] Fig.15 The results of Hoechst 33342 staining of A549 / Taxol cells for 48 hours; strong blue fluorescence indicates apoptotic cells, scale bar = 100 μm; data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 vs control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs paclitaxel; ns: not significant;

[0038] Fig.16 Western blot analysis of allicin on P-gp expression and statistical analysis;

[0039] Fig.17 The graph and statistical analysis of the accumulation of Rho-123 in cells increased by allicin;

[0040] Fig.18The effect of CTSB inhibition on mitochondrial membrane potential was detected by JC-1 assay; data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs control group; #p < 0.05 vs paclitaxel; ns: not significant;

[0041] Fig.19 This is a diagram showing the inhibitory effect of allicin on CTSB enzyme activity;

[0042] Fig. 20 This is an analysis diagram of the inhibitory effect of different concentrations of allicin on CTSB detected by Elisa test;

[0043] Fig.21 Western blot and statistical analysis of CTSB protein in A549 / Taxol cells treated with different concentrations of allicin (a), and RT-qPCR analysis of CTSB mRNA expression in A549 / Taxol cells treated with allicin (b); Fig. 22 Western blot and statistical analysis of the effects of different drugs on CTSB in A549 / Taxol cells;

[0044] Fig.23 Figure 2 shows the detection of allicin-induced lysosomal degradation defect by DQ-BSA assay and statistical analysis, scale bar = 10 μm; the integrated optical density (IOD) of each cell was quantified, and the data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 vs control group; #p < 0.05 vs paclitaxel; ns: not significant;

[0045] Fig.24 Western blot and statistical analysis of autophagy-related proteins in A549 / Taxol cells;

[0046] Fig.25 To detect the fluorescence image of autophagic vacuoles formation inhibited by allicin through GFP-LC3 assay and to calculate and analyze the number of GFP-LC3 spots;

[0047] Fig.26 The ultrastructural changes of cells were analyzed by TEM; representative TEM images are shown; green represents phagocytic vacuoles; yellow represents autophagosomes; red represents autophagolysosomes, scale bar = 2 μM; data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 vs control group; ns: not significant;

[0048] Fig. 27 Schematic diagram of nude mice for tumor modeling and drug administration;

[0049] Fig.28 This is a curve diagram of the changes in nude mouse body weight after drug administration;

[0050] Fig.29 Images of nude mouse xenograft A549 / Taxol lung samples based on Bouin's staining (a) (n=6), arrows and magnified parts indicate nodules in the lungs; and statistical analysis of the number of lung nodules in each group of mice (b) (n=6); statistical comparisons were performed using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test, and data are expressed as mean ± SD, ***p<0.001, ****p<0.0001vs control group, #p<0.01, ###p<0.0001vs paclitaxel;

[0051] Fig.30 Representative intravital imaging images of nude mouse xenografted A549 / Taxol lung samples;

[0052] Fig.31 H&E staining images of lung tissues in each group at different magnifications (400×), scale bar = 100 μm;

[0053] Fig.32 Expression images of P-gp, CTSB, p62 and LC3B in lung tissue images detected by immunohistochemistry (a) and statistical analysis of P-gp (b), CTSB (c), p62 (d) and LC3B (e); scale bar = 100 μm, data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs model; ns: not significant. DETAILED DESCRIPTION

[0054] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0055] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0056] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0057] Example 1 In vitro experiment

[0058] Part I Experimental Methods

[0059] 1.1 Cell culture and treatment

[0060] A549 cells, A549 / Taxol cells and A549 / Taxol-Luc cells were cultured in RPMI 1640 medium containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C and 5% CO2 in T25 culture flasks. A549 / Taxol cells and A549 / Taxol-Luc cells were maintained in medium containing 300 nM paclitaxel to maintain drug resistance and were switched to drug-free medium one week before the experiment.

[0061] 1.2 MTT assay for cell proliferation

[0062] 1.2.1 Drug resistance index (RI) and drug resistance reversal factor (RF)

[0063] A549 cells and A549 / Taxol cells were seeded into 96-well plates (8 × 103 cells / well) and treated with different concentrations of paclitaxel (1-1000 nM). IC was calculated using SPSS software. 50 The RI value was calculated as follows: RI = (IC of A549 / Taxol cells) 50 ) / (IC of A549 cells 50 ).

[0064] To determine the concentration that was not toxic to A549 / Taxol cells, the IC 10 The concentration of allicin and 300 nM paclitaxel were combined and applied to A549 / Taxol cells for 48 hours. Then the IC 50 The value change and reversal factor (RF) are as follows: RF = (IC 50 ) / (IC of allicin and paclitaxel combined 50 ).

[0065] 1.2.2 Effect of allicin on A549 / Taxol cell viability

[0066] A549 / Taxol cells were inoculated into 96-well plates (8×103 cells / well). When the cells reached 70-80% confluence, allicin was added in a concentration range of 1-1000 μM. After 48 hours of treatment, the absorbance was measured at 490 nm using a microplate reader. The inhibition rate (%) was calculated as follows: (1-OD value of the treatment group / OD value of the control group)×100%.

[0067] 1.3 Cell clone formation experiment

[0068] A549 / Taxol cells in the logarithmic growth phase were seeded into 6-well plates at a density of 1.0×103 cells per well. The cells were then treated with paclitaxel, allicin, and their combination for 24 hours. Subsequently, the cells were incubated in drug-free medium for 10 days, and the formed cell clones were fixed with methanol, stained with 0.1% (w / v) crystal violet, rinsed with PBS, air-dried, and imaged. Clone quantification was performed using ImageJ software.

[0069] 1.4 Apoptosis assay

[0070] 1.4.1 Flow cytometry

[0071] A549 / Taxol cells (2×105 cells / mL) were seeded into 6-well plates for 24 hours before staining. The cells were analyzed using a FACS Verse flow cytometer with a cell count of 10,000 and data were analyzed using FlowJO software.

[0072] 1.4.2 Hoechst 33342 staining

[0073] The cells were washed with pre-cooled PBS (2 times), stained with Hoechst 33342 for 15 minutes in the dark, and observed under a fluorescence microscope. Weak blue fluorescence indicates normal or necrotic cells, and strong blue fluorescence indicates apoptotic cells.

[0074] 1.5 Western blot analysis

[0075] The protein concentration was assessed using a BCA kit, and the proteins were separated by SDS-PAGE and transferred to a PVDF membrane, blocked with 5% skim milk powder, and incubated with the primary antibody at 4°C overnight, followed by incubation with the secondary antibody at room temperature for 1 hour. The membrane was rinsed three times with PBST buffer and developed with ECL luminescent solution.

[0076] 1.6 Intracellular Rho-123 uptake

[0077] The effect of CTSB inhibition on P-gp function was evaluated by intracellular Rho-123 uptake assay. 5 / well were inoculated in a six-well plate and incubated overnight. After 48h of inhibition of CTSB (CTSB inhibitor CA074 Me group, CTSB knockout group and allicin group) or verapamil treatment, Rho-123 (15μM) was added to the culture medium and co-treated in the dark for 3h. The cells were washed twice with pre-cooled PBS, fixed with 4% paraformaldehyde for 30min, permeabilized with 0.1% TritonX-100, and incubated with DAPI for 15 minutes. Images were acquired using a confocal microscope. The cells were then collected, washed twice with pre-cooled PBS, and resuspended using a flow cytometer to detect the accumulation of Rho-123.

[0078] 1.7 DQ-BSA test

[0079] The function of lysosomes in degrading cellular components was assessed using the DQ-BSA assay, and samples were stained with DAPI to visualize the nuclei and analyzed using a laser confocal microscope and ImageJ software.

[0080] 1.8 GFP-LC3 assay

[0081] GFP-LC3-transfected A549 / Taxol cells were treated with paclitaxel, allicin, CQ and their combination, and images were taken with a fluorescence microscope after fixation, and the number of GFP-LC3 spots was counted using ImageJ software.

[0082] 1.9 mRFP-GFP-LC3 assay

[0083] A549 / Taxol cells were transfected with mRFP-GFP-LC3 lentivirus and then treated with paclitaxel, allicin, CQ and their combination, and fluorescent LC3 spots were observed using confocal microscopy 48 hours later.

[0084] 1.10 JC-1 test

[0085] The samples were treated with JC-1 and incubated for 15 min at 37° C. After washing with PBS, the fluorescence signals of JC-1 monomers and polymers were measured by flow cytometry using FL-1 and FL-2 detectors.

[0086] 1.11 Transmission electron microscopy (TEM) observation of ultrastructure

[0087] Growing A549 / Taxol cells were treated with paclitaxel, allicin, CA-074Me and their different combinations for 48 hours. After centrifugation and treatment steps, the samples were stained with lead acetate and uranyl acetate for TEM observation.

[0088] 1.12 Real-time quantitative PCR (RT-qPCR)

[0089] Total RNA was extracted from A549 / Taxol cells using Trizol and converted into cDNA using the 1st-Strand cDNA Synthesis Kit as described. Green master mix was used for quantitative real-time PCR under the following conditions: denaturation step, 95°C for 5 minutes, 45 amplification cycles, 95°C for 5 seconds; 60°C for 30 seconds; 72°C for 30 seconds. β-actin was used as an internal reference. 2-ΔΔ The relative expression level was detected by Ct method.

[0090] 1.13 RNA transfection

[0091] A549 / Taxol cells were seeded into 6-well plates at a density of 5×105 cells / well overnight. When the cells grew to 70%, shCTSB-199, shCTSB-776 and shCTSB-913 were transfected into the cells for 48 hours, and RT-qPCR was used to verify the knockdown efficiency of shRNA in the A549 / Taxol cell line (Table 1). All shRNAs were synthesized on the easy to lab platform, and Western blot was used to detect the expression level of CTSB protein.

[0092] Table 1 Primer design

[0093]

[0094]

[0095] 1.14 Statistical analysis

[0096] Data are presented as mean ± standard deviation (SD). Statistical analysis One-way analysis of variance (ANOVA) was performed using GraphPad Prism software, followed by Tukey and Dunnett tests. Independent sample t-tests were used to compare differences between experimental groups. The significance level was set at p < 0.05.

[0097] Part II In vitro experimental results

[0098] 2.1 Role of CTSB in paclitaxel resistance in NSCLC

[0099] Western blot analysis showed that CTSB was upregulated in drug-resistant A549 / Taxol cells, suggesting that it plays an important role in paclitaxel resistance ( Figure 1 a). Kaplan-Meier survival analysis based on The Cancer Genome Atlas (TCGA) database showed that CTSB expression was negatively correlated with the survival of lung cancer patients ( Figure 1b). This indicates that inhibiting the expression or activity of CTSB can reverse paclitaxel resistance.

[0100] RT-qPCR and Western blot results showed that silencing of the CTSB gene (shCTSB-776) significantly reduced the protein (a) and mRNA (b) expression levels of CTSB ( Figure 2 ), the CTSB protein expression of the shCTSB-776 group decreased by about 80%, and the mRNA expression decreased by about 75% (p<0.05). MTT experiments showed that silencing CTSB alone had little effect on the survival rate of A549 / Taxol cells ( Figure 3 , cell survival rate>95%). MTT and flow cytometry assays showed that allicin combined with 300nM paclitaxel and shCTSB-776 significantly enhanced the inhibitory effect of paclitaxel on cell proliferation ( Figure 4 Aa, cell survival rate decreased to about 40%) and induced apoptosis ( Figure 4 Ba, the apoptosis rate increased to about 40%), and the IC 50 The value decreased to 83.56nM ( Figure 4 Ab); CTSB inhibitor CA-074Me further verified the role of CTSB in enhancing the apoptosis-promoting effect of paclitaxel ( Figure 4 Bb). In addition, flow cytometry was used to measure the intracellular uptake rate of Rho-123, and it was found that inhibition of CTSB increased the accumulation of Rho-123 in A549 / Taxol cells, indicating that CTSB inhibition can improve drug retention ( Figure 5 , Rho-123 accumulation increased by about 2-fold).

[0101] 2.2 Effects of CTSB knockout on lysosomal function and autophagy in A549 / Taxol cells

[0102] The inhibition of CTSB can lead to the impairment of lysosomal degradation function in A549 / Taxol cells as determined by the DQ-BSA method, with a reduction of about 50% in intracellular red fluorescence ( Figure 6 ). The effect of inhibiting CTSB on cell autophagy was evaluated by GFP-LC3 and mRFP-GFP-LC3 experiments. The results showed that after CTSB silencing, LC3II accumulated and the green fluorescence dots increased by about 3 times ( Figure 7 ), autophagosome-lysosome fusion was blocked, and the red and yellow fluorescence decreased by about 70% ( Figure 8 ). Using TEM to observe the generation and quantity changes of autophagosomes, autolysosomes and autophagosomes in cells, it was found that inhibiting CTSB can significantly reduce the generation of autophagosomes ( Fig. 9). Western blot analysis showed that the expression of p62 and LC3 II / I increased by about 2 times, indicating that the autophagic flux decreased ( Fig.10 ).

[0103] 2.3 Role of allicin in overcoming paclitaxel resistance in A549 / Taxol cells

[0104] 2.3.1 Allicin enhances the inhibitory effect of paclitaxel on A549 / Taxol cells

[0105] By microscopic observation, compared with A549 cells ( Fig.11 -a) compared with A549 / Taxol cells ( Fig.11 -b) presents an elongated spindle-shaped appearance and can be passaged in a medium containing paclitaxel (300nM). After treatment with allicin (10μM) and paclitaxel (300nM), A549 / Taxol cells undergo significant deformation and present cell shrinkage and other morphologies ( Fig.11 -c). MTT assay, IC of A549 and A549 / Taxol 50 The values ​​were 2.303nM( Fig.12 -a) and 971.80nM( Fig.12 -b), and the drug resistance index was 4121.97. MTT assay was used to determine that allicin had a concentration-dependent inhibitory effect on A549 / Taxol cells (IC 50 =51.67μM, IC 10 =10.22 μM)( Fig.12 -c); Allicin treatment reduced the IC of paclitaxel in A549 / Taxol cells 50 Reduced to 98.54nM, the reversal factor was 9.86 ( Fig.12 -d). The long-term cytotoxic effect of allicin on A549 / Taxol cells was evaluated by cell clone formation test. The results showed that 115 colonies were formed in the control group, 108 colonies were formed in the paclitaxel group, 99 colonies were formed in the allicin group, and only 27 colonies were formed in the allicin combined with paclitaxel group, indicating that allicin can significantly enhance the inhibitory effect of paclitaxel on A549 / Taxol cells in the long term ( Fig.13 ).

[0106] 2.3.2 Effect of allicin combined with paclitaxel on apoptosis of A549 / Taxol cells

[0107] Annexin V-FITC / PI staining was detected by flow cytometry, and it was found that after treating A549 / Taxol cells with allicin (10 μM) and paclitaxel (300 nM) for 48 h, the apoptosis rate of A549 / Taxol cells increased significantly to 41.18% ( Fig.14 ), compared with 7.616% and 9.198% apoptosis rates in the paclitaxel and allicin groups treated alone, respectively. Hoechst 33342 staining was used to detect the characteristics of cell apoptosis, and it was found that allicin combined with paclitaxel could cause a decrease in cell number, blurred morphology, chromatin condensation, and nuclear fragmentation ( Fig.15 ).

[0108] 2.4 Effects of allicin on inhibition of P-gp expression and reduction of mitochondrial membrane potential

[0109] Western blot and RT-qPCR were used to detect the effects of allicin on P-gp protein expression and gene expression. The results showed that the expression of P-gp in A549 / Taxol cells was significantly reduced after allicin treatment ( Fig.16 ), the P-gp expression in the control group and the paclitaxel group was significantly higher than that in the other groups, the P-gp expression in the allicin treatment group decreased by about 50%, and the P-gp expression in the VRP and allicin combined with paclitaxel group decreased by about 70%. At the same time, flow cytometry was used to detect the effect of allicin on Rho-123 uptake in A549 / Taxol cells, and the results showed that allicin treatment significantly increased the accumulation of Rho-123 ( Fig.17 ), VRP and allicin combined with paclitaxel group Rho-123 accumulation increased by about 2 times. JC-1 test showed that allicin and paclitaxel combined treatment significantly reduced mitochondrial membrane potential by about 60% ( Fig.18 ).

[0110] 2.5 Role of allicin in inhibiting CTSB and inducing lysosomal dysfunction

[0111] Elisa, Western blot, and RT-qPCR were used to determine the effect of allicin on CTSB. The results showed that the half inhibitory concentration of allicin on CTSB was 9.83 μM ( Fig.19 ), concentration-dependently reduced the expression of CTSB in A549 / Taxol cells; at 5μM and 10μM concentrations, allicin could inhibit the expression of CTSB, and the expression of CTSB decreased by about 14.37ng / mL and 29.58ng / mL, respectively; at 20μM concentration of allicin, the expression of CTSB decreased by about 37.63ng / mL ( Fig. 20 ).

[0112] RT-qPCR and Western blotting results showed that the relative protein and mRNA expression levels of CTSB decreased significantly after allicin treatment, decreasing by about 60% and 70%, respectively ( Fig.21 ). 10At 10 μM concentration, allicin and CA-074Me significantly reduced CTSB expression by about 50% ( Fig. 22 ). DQ-BSA test showed that allicin treatment significantly reduced lysosomal protein degradation activity, and the red fluorescence decreased by about 70% ( Fig.23 ).

[0113] In summary, the effective concentration of allicin is preferably 5-20 μM.

[0114] 2.6 Effect of allicin on inhibiting autophagy in A549 / Taxol cells

[0115] Western blot analysis showed that allicin treatment reduced the LC3Ⅱ / LC3Ⅰ ratio by about 50% and increased p62 expression by about 2-fold ( Fig.24 ). GFP-LC3 assay showed that the green fluorescence spots in cells treated with allicin increased by about 3 times ( Fig.25 TEM analysis showed that allicin combined with paclitaxel treatment significantly reduced autophagy-related structures, the number of autophagosomes decreased by about 60%, and mitochondrial swelling and rupture of the inner cristae were observed ( Fig.26 ). These results indicate that allicin significantly enhanced the inhibitory effect of paclitaxel on A549 / Taxol cells by inhibiting CTSB and lysosomal function, reversed drug resistance, and further increased the sensitivity of cells to paclitaxel by inhibiting the autophagy process.

[0116] Example 2 In vivo experiment

[0117] Part I Experimental Methods

[0118] Establishment of a paclitaxel-resistant human non-small cell lung cancer orthotopic tumor model in nude mice: An orthotopic tumor model was established in BALB / c nude mice (4-6 weeks old, weighing 18-22 g) using bronchial instillation. Nude mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (Beijing, China) and maintained in a specific pathogen-free (SPF) environment. A549 / Taxol cells were adjusted to 1.0×10 7 cells / mL concentration, and each mouse was injected with 50μL of cell suspension. The mice were divided into five groups: blank group, saline group, paclitaxel group, allicin group, and allicin + paclitaxel group, with 6 mice in each group. All drugs were diluted with saline, filtered through a 0.22μm filter membrane, and administered by intraperitoneal injection every two days. Body weight was measured daily, and mice were killed after 21 days. Lung tissue was fixed with Bouin solution for further analysis. In addition, A549 / Taxol-luc cells were collected, and 200mg / kg D-luciferin was injected intraperitoneally into each mouse. The same method was used to monitor tumor development and perform bioluminescence imaging.

[0119] Part II In vivo experimental results

[0120] The effect of allicin in reversing drug resistance was verified by using a paclitaxel-resistant NSCLC nude mouse orthotopic tumor model. Fig. 27 During the treatment, the tumor-bearing nude mice showed normal activities and mental states, without discomfort or abnormal behavior. Daily monitoring of body weight showed that the body weight of the blank control group, allicin group, and allicin combined with paclitaxel group increased steadily, while the body weight of the model group and paclitaxel group decreased after the 10th day ( Fig.28 ). The lung tissue of normal mice was smooth and without pathological changes ( Fig.29 ). In contrast, the lung tumors in the saline group, paclitaxel group, and allicin group were significantly enlarged and protruding, with uneven surfaces and many tumor nodules of varying sizes. Allicin combined with paclitaxel significantly inhibited tumor growth, making the lung tissue surface relatively smooth and tumor nodules significantly reduced, with better results than using paclitaxel and allicin alone, indicating that allicin enhanced the in vivo therapeutic effect of paclitaxel. In vivo imaging showed that the fluorescence signal at the tumor site in the model group and paclitaxel group gradually increased, indicating that the tumor continued to grow ( Fig.30 ). However, the increase rate of fluorescence signal in the allicin group and the allicin combined with paclitaxel group was significantly slowed down, indicating that allicin treatment had a positive effect on inhibiting tumor growth.

[0121] Hematoxylin-eosin (HE) staining was used to evaluate the pathological changes in the lung tissues of mice. Fig.31 The results showed that the tumor cells in the allicin and paclitaxel groups were densely arranged, with few intercellular spaces, scattered fascicles, and large and darkly stained nuclei. In contrast, the tumor cells in the allicin combined with paclitaxel group were fewer and more dispersed, while the cells in the blank control group were sparsely arranged.

[0122] The expression of CTSB, P-gp, p62 and LC3B in orthotopic transplanted tumors was detected by immunohistochemistry. Fig.32 ). The expression of CTSB and P-gp increased in the model group and the paclitaxel group, while the expression of p62 and LC3B decreased. However, the allicin combined with paclitaxel group reduced the expression of CTSB and P-gp, while increasing the expression of p62 and LC3B, indicating that allicin reversed paclitaxel resistance by inhibiting autophagy, reducing the dosage of paclitaxel, and thus reducing chemotherapy-related toxic side effects. These results strongly support the therapeutic potential of allicin.

[0123] In summary, in vitro and in vivo efficacy studies have shown that the allicin proposed in the present invention can be used to prepare a sensitizer or reversal agent for the anti-tumor drug paclitaxel. The combined use of allicin and paclitaxel can effectively inhibit cell proliferation, induce cell apoptosis, enhance sensitivity to paclitaxel, reverse paclitaxel resistance, reduce the dosage of paclitaxel, significantly improve the chemotherapy effect of NSCLC, and reduce chemotherapy-related toxic side effects.

[0124] The allicin described in the present invention is not limited to allicin itself, but also includes one or more of its pharmaceutically acceptable salts, stereoisomers and prodrug molecules, allicin derivatives and pharmaceutically acceptable salts, stereoisomers and prodrug molecules. The combined treatment of allicin and paclitaxel is suitable for in vitro cell experiments, in vivo animal models or clinical applications, and is used to treat non-small cell lung cancer resistant to paclitaxel.

[0125] The allicin described in the present invention is not limited to the use as a sensitizer or reversal agent in the preparation of drugs for reversing NSCLC paclitaxel resistance, but is applicable to tumors involving inhibition of CTSB-related signaling pathways, and may also include reversal of drug resistance in breast cancer, liver cancer, gastric cancer, malignant lymphoma, nasopharyngeal carcinoma, melanoma, esophageal cancer, intestinal cancer, etc.

[0126] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Application of allicin in the preparation of anti-tumor drug sensitizers or reversal agents.

2. The use according to claim 1, characterized in that: The allicin is a sensitizer or reversal agent of the anti-tumor drug paclitaxel.

3. The use according to claim 2, characterized in that: The allicin reduces the drug resistance of tumor cells to paclitaxel by inhibiting the CTSB-related signaling pathway.

4. The use according to claim 1 or 2, characterized in that: The tumors include non-small cell lung cancer, breast cancer, liver cancer, gastric cancer, malignant lymphoma, nasopharyngeal carcinoma, melanoma, esophageal cancer and intestinal cancer.

5. The use according to claim 1 or 2, characterized in that: The tumor is non-small cell lung cancer.

6. Application of allicin in the preparation of drugs for inhibiting cell protective autophagy.

7. The use according to claim 6, characterized in that: The cell-protective autophagy is formed through the lysosomal-autophagic pathway mediated by CTSB.

8. A non-small cell lung cancer chemotherapy drug composition, characterized in that: The invention comprises allicin, paclitaxel and pharmaceutically acceptable auxiliary materials.

9. The composition according to claim 8, characterized in that The allicin includes its main body, and one or more of its pharmaceutically acceptable salts, stereoisomers and prodrug molecules, allicin derivatives and its pharmaceutically acceptable salts, stereoisomers and prodrug molecules.

10. The composition according to claim 8, characterized in that The effective concentration of the allicin is 5-20 μM.

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