Method for determining paclitaxel concentration inside and outside non-small cell lung cancer cells

Abstract

The invention discloses a method for determining paclitaxel concentrations inside and outside non-small cell lung cancer cells, which comprises the following steps: preparing a paclitaxel series standard solution, a QC series standard solution and an internal standard solution by using a paclitaxel stock solution, a paclitaxel-d5 stock solution, methanol and deionized water; the method comprises the following steps: culturing and treating parent cells A459 and paclitaxel drug-resistant cells A459 / TAX of human non-small cell lung cancer to obtain a cell sample; extracting paclitaxel in the cell sample by adopting an LLE method to obtain a detection sample; setting chromatographic and mass spectrum conditions; sending a detection sample into a UPLC-MS / MS system, quantifying paclitaxel concentrations inside and outside A459 cells and A459 / TAX cells, and counting and analyzing quantitative results. According to the invention, the concentration of paclitaxel inside and outside cells is determined by innovatively adopting a UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) method, so that the important effect of ABCB1 mediated paclitaxel drug resistance in non-small cell lung cancer cells is clarified.

Description

Technical Field

[0001] This invention belongs to the field of tumor treatment, specifically relating to a method for determining the intracellular and extracellular paclitaxel concentration in non-small cell lung cancer. Background Technology

[0002] Lung cancer is one of the most common and deadliest malignant tumors worldwide. Non-small cell lung cancer (NSCLC) accounts for 80%–85% of all lung cancer cases and is characterized by high invasiveness, high recurrence rate, and poor treatment outcomes. Although treatment options for NSCLC include surgical intervention, radiotherapy, chemotherapy, molecular targeted therapy, and immunotherapy, chemotherapy remains the primary treatment method due to its broad-spectrum efficacy. Among chemotherapy drugs, paclitaxel (PTX) is widely considered a first-line drug for treating NSCLC. Notably, paclitaxel combined with immunotherapy shows significant efficacy in treating advanced lung cancer, particularly NSCLC lacking driver mutation genes (including lung adenocarcinoma and squamous cell carcinoma).

[0003] Paclitaxel was originally derived from the bark of the yew tree (Taxus brevifolia). Paclitaxel acts as a microtubule stabilizer by specifically binding to tubulin. This interaction stabilizes microtubules, thereby reducing their dynamic instability and inhibiting normal depolymerization and polymerization processes. Although paclitaxel is initially effective, long-term use often leads to the development of acquired resistance, ultimately reducing its therapeutic efficacy. Therefore, it is urgent to elucidate the molecular regulatory network of paclitaxel resistance development and evolution in order to identify novel targets for its reversal.

[0004] The following factors can all be attributed to the development of paclitaxel resistance: (1) Expression and activity of ATP-binding cassette (ABC) family members involved in multidrug efflux transporters; (2) Imbalance between apoptotic and anti-apoptotic proteins; (3) Cancer stem cell (CSC)-mediated maintenance of stemness; (4) Changes in microtubules and tubulin.

[0005] Multidrug resistance mediated by the p-glycoprotein encoded by ATP-binding cassette subfamily B member 1 (ABCB1) is the most significant obstacle to chemotherapy efficacy and has been reported in various types of cancer, including colorectal and breast cancer. Currently, several p-glycoprotein inhibitors have been developed to address chemoresistance induced by ABCB1 overexpression in cancer cells. P-glycoprotein inhibitors such as verapamil, tariquidar, and elacridar have been investigated as potential drugs to overcome multidrug resistance in cancer treatment by inhibiting ABCB1 transporter-mediated efflux of chemotherapeutic drugs. However, their clinical application is limited due to poor specificity, off-target effects, and significant toxicity. Currently, there is a lack of paclitaxel concentration data to confirm the role of ABCB1 in drug efflux. Furthermore, the efficacy of p-glycoprotein inhibitors in reversing chemoresistance remains uncertain because there is insufficient robust clinical evidence to support their ability to improve treatment outcomes. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a method for determining the intracellular and extracellular paclitaxel concentration in non-small cell lung cancer (NSCLC) cells based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). This method enables high-throughput and precise quantification of paclitaxel levels in NSCLC cells, elucidating the role of ABCB1-mediated paclitaxel resistance in NSCLC cells and contributing to the selection and application of p-glycoprotein inhibitors in overcoming paclitaxel resistance.

[0007] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution: A method for determining intracellular and extracellular paclitaxel concentrations in non-small cell lung cancer, based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), includes the following steps: Step 1) Using paclitaxel stock solution, paclitaxel-d5 stock solution, methanol and deionized water, prepare a series of paclitaxel standard solutions, a series of QC standard solutions and an internal standard solution (IS solution). Step 2) The parental cells (A459 cells) and paclitaxel-resistant cells (A459 / TAX cells) of human non-small cell lung cancer were cultured and treated to obtain the corresponding cell samples; Step 3) Use liquid-liquid extraction (LLE) to extract paclitaxel from the cell sample to obtain the test sample; Step 4) Set the chromatographic and mass spectrometric conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry system; Step 5) The test sample is sent into an ultra-high performance liquid chromatography-tandem mass spectrometry system to quantify the paclitaxel concentration inside and outside the parental cells (A459 cells) and paclitaxel-resistant cells (A459 / TAX cells); Step 6) Perform statistical analysis on the quantitative results.

[0008] Furthermore, in step 1, the preparation method of the paclitaxel series standard solutions is as follows: First, the paclitaxel stock solution was dissolved in methanol to obtain a paclitaxel-methanol solution with a mass concentration of 1.0 mg / mL, which was then stored at -80℃. Next, it was quantitatively diluted with a 50:50 deionized water-methanol solution to prepare a series of paclitaxel standard solutions with mass concentrations of 10.0, 50.0, 100.0, 500.0, 1000.0, 5000.0, 10000.0, and 50000.0 ng / mL, which were then stored at -80℃ for later use.

[0009] Furthermore, in step 1, the preparation method of the QC series standard solutions is as follows: First, the paclitaxel stock solution was dissolved in methanol to obtain a paclitaxel-methanol solution with a mass concentration of 1.0 mg / mL, which was then stored at -80℃. Then, it was quantitatively diluted with a 50:50 deionized water-methanol solution to prepare QC series standard solutions with mass concentrations of 10.0, 1000.0, and 40000.0 ng / mL, which were then stored at -80℃ for later use.

[0010] Furthermore, in step 1, the method for preparing the internal standard solution (IS solution) is as follows: The paclitaxel-d5 stock solution was quantitatively diluted with a 50:50 deionized water-methanol solution to prepare an internal standard solution with a paclitaxel-d5 mass concentration of 1000.0 ng / mL, which was then stored at -80℃ for later use.

[0011] Furthermore, in step 2, the specific methods for culturing and treating the parental cells of non-small cell lung cancer and paclitaxel-resistant cells are as follows: Parental non-small cell lung cancer (NSCLC) cells and paclitaxel-resistant cells were seeded at equal densities in 6-well plates. After 24 hours, the parental NSCLC cells and paclitaxel-resistant cells were treated with a p-glycoprotein inhibitor in combination with different concentrations of paclitaxel, and then co-incubated for 24 hours. The culture medium was then collected and designated as the cell supernatant. After trypsinization, adherent cells were collected and resuspended in PBS to a volume equivalent to that of the cell supernatant. Finally, the cell suspension was rapidly frozen in liquid nitrogen, then thawed at room temperature for three cycles, subjected to sonication in an ice bath (10-second pulses, 5-second intervals), and stored at 4°C.

[0012] Furthermore, in step 3, the method for extracting paclitaxel from the sample is as follows: First, take 100 μL of cell sample, add 10 μL of internal standard solution and 1 mL of methyl tert-butyl ether, and then vortex the mixture for 5 minutes to homogenize. After centrifuging at 14000 rpm for 10 minutes, 900 μL of the upper organic layer was transferred into a tube and evaporated to dryness with nitrogen at 40 °C. Finally, the dried residue was reconstituted in 100 μL of mobile phase (water / methanol, 20 / 80, v / v or water / methanol / formic acid, 50 / 50 / 0.1, v / v / v) and centrifuged at 12000 rpm for 10 minutes. 5 μL of the supernatant was taken as the detection sample injected into the LC-MS / MS system.

[0013] Furthermore, in step 4, the chromatographic conditions are set as follows: The chromatographic column was a 2.1×50mm, 1.7μm ACQUITY UPLC™ BEH C18 column; The mobile phase is a solution of A and B prepared in a volume ratio (A: water with a formic acid mass fraction of 0.1%; B: methanol solution). Gradient elution was used: 0-1 minute, 10%→80%B; 1-2 minutes, 80%B; 2-3 minutes, 80%→10%B; The total processing time for each test sample is 3 minutes; The flow rate was 0.3 mL / min; The column temperature is 40℃; The injection volume was 5 μL.

[0014] Furthermore, in step 4, the mass spectrometry conditions are set as follows: The ion spray voltage is 5500V; The ion source temperature is 550℃; The declustering voltage (DP) is 60V; The input potential (EP) is 10V; The collision chamber exit voltage (EP) is 10V; Paclitaxel m / z 854.2→286.0; The paclitaxel declustering voltage (DP) is 30V; The collision energy (CE) of paclitaxel is 15 eV; Paclitaxel-d5 m / z 859.2→291.2; The declustering voltage (DP) of paclitaxel-d5 is 30V; The collision energy (CE) of paclitaxel-d5 is 20 eV.

[0015] Furthermore, in step 6, during statistical analysis, all numerical data are expressed as mean ± standard deviation (SD); t-tests, one-way ANOVA, or two-way ANOVA are used to assess statistical significance; significance levels are expressed as: *p < 0.05, **p < 0.01, ***p < 0.001.

[0016] The beneficial effects of this invention are as follows: This invention proposes a comprehensive pharmacological and analytical method to quantify paclitaxel treatment and overcome drug resistance in an A549 / TAX cell model. CCK-8 cell viability, colony formation, and apoptosis assays confirmed that A549 / TAX cells exhibited significant paclitaxel resistance relative to parental A549 cells. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) analysis showed that ABCB1 gene silencing or pharmacological inhibition by specific p-glycoprotein inhibitors Tariquidar and Elacridar restored intracellular paclitaxel levels and synergistically reduced cell viability as measured by CCK-8 assays.

[0017] The experimental results of this invention show that ATP-binding box subfamily B1 (ABCB1) is highly expressed in paclitaxel-resistant A549 cells. Silencing ABCB1 enhances sensitivity to paclitaxel. Furthermore, this invention successfully developed and established an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) method, innovatively applied to detect intracellular and extracellular paclitaxel concentrations. By directly measuring paclitaxel concentrations, this invention verified the effect of ABCB1 expression on the paclitaxel sensitivity of NSCLC cells, elucidating the important role of ABCB1-mediated paclitaxel resistance in non-small cell lung cancer cells. The effects of the p-glycoprotein-specific inhibitors Tariquidar and Elacridar on resistant cells were further evaluated. Using the UPLC-MS / MS method, this invention observed a significant increase in intracellular paclitaxel concentration after the application of these inhibitors.

[0018] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0019] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings: Figure 1 This is a schematic diagram showing the intracellular and extracellular paclitaxel drug concentrations in paclitaxel-resistant non-small cell lung cancer cells determined using UPLC-MS / MS in an embodiment of the present invention.

[0020] Figure 2 This invention provides evidence that A549 / TAX cells are paclitaxel-resistant cells in non-small cell lung cancer; wherein... Figure 2 In the figure, A represents the analysis diagram of paclitaxel sensitivity of A549 cells and A549 / TAX cells using the CCK-8 activity assay. Figure 2 In the figure, B represents the effect of paclitaxel on the proliferation level of A549 cells and A549 / TAX cells measured by colony formation assay, and the statistical analysis graph. Figure 2 The "C" in the figure represents the apoptosis analysis diagram used to detect the effect of paclitaxel on the apoptosis levels of A549 cells and A549 / TAX cells through apoptosis assays.

[0021] Figure 3 This invention demonstrates that ABCB1 is considered a key factor promoting paclitaxel resistance in NSCLC cells; wherein... Figure 3 In the diagram, A represents the differential gene volcano plot of A549 and A549 / TAX obtained through RNA sequencing and analysis; Figure 3 In the figure, B represents a heatmap of protein expression analysis of ABCB1 and its family members in A549 cells and A549 / TAX cells obtained through differential protein expression analysis. Figure 3 In the figure, C represents the result of Western blot analysis of ABCB1 expression between A549 cells and A549 / TAX cells.

[0022] Figure 4 The chromatograms and calibration curves of paclitaxel and paclitaxel-d5 are shown in the embodiments of the present invention; wherein Figure 4 In the figure, A represents the detection curve of the retention time of paclitaxel and paclitaxel-d5; Figure 4 In the figure, B represents the typical regression equation curve for paclitaxel concentration determination by UPLC-MS / MS. Figure 4 In the graph, C represents the detection limit of quantitation for paclitaxel and paclitaxel-d5.

[0023] Figure 5 This figure shows the results of quantifying intracellular and extracellular paclitaxel concentrations in A549 and A549 / TAX cells using the UPLC-MS / MS method in an embodiment of the present invention.

[0024] Figure 6 In this embodiment of the invention, knocking down ABCB1 in A549 / TAX cells can improve paclitaxel drug sensitivity; wherein Figure 6 In the diagram, A represents the analysis of p-glycoprotein expression in A549 / TAX cells by different siRNAs; Figure 6In the diagram, B represents the analysis of ABCB1 mRNA expression in A549 / TAX cells by different siRNAs; Figure 6 The "C" in the diagram represents the analysis of paclitaxel resistance sensitivity after transfection and knockdown of ABCB1 in A549 / TAX cells.

[0025] Figure 7 In the embodiments of this invention, the p-glycoprotein inhibitors Tariquidar or Elacridar can improve the sensitivity of A549 / TAX cells to paclitaxel; wherein Figure 7 The figure shows the analysis of Tariquidar and Elacridar in the evaluation of toxicity, and the selection of non-toxic concentrations of Tariquidar and Elacridar for subsequent combined administration experiments with paclitaxel. Figure 7 In the figure, B represents the analysis diagram of the effect of two p-glycoprotein inhibitors combined with paclitaxel on the IC50 of A549 / TAX cells.

[0026] Figure 8 In this embodiment of the invention, the p-glycoprotein inhibitor Tariquidar or Elacridar, combined with paclitaxel, inhibits A549 / TAX cell proliferation and promotes A549 / TAX cell apoptosis; wherein... Figure 8 In the figure, A represents the colony formation results of the effect of administration of p-glycoprotein inhibitor alone or in combination with paclitaxel on the proliferation capacity of A549 / TAX cells. Figure 8 The B in the figure represents the apoptosis analysis diagram of the p-glycoprotein inhibitor's effect on paclitaxel-induced A549 / TAX cell toxicity.

[0027] Figure 9 This is a graph showing the results of quantifying intracellular and extracellular paclitaxel concentrations in A549 / TAX cells after treatment with p-glycoprotein inhibitors and paclitaxel using UPLC-MS / MS in an embodiment of the present invention; wherein... Figure 9 In the figure, A represents the results of quantifying the intracellular paclitaxel concentration in A549 / TAX cells treated with different doses of paclitaxel using UPLC-MS / MS. Figure 9 In the figure, B represents the results of quantifying the extracellular paclitaxel concentration in A549 / TAX cells treated with different doses of paclitaxel using UPLC-MS / MS. Detailed Implementation

[0028] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings to provide a clearer understanding of the invention's purpose, features, and advantages. It should be understood that the embodiments shown in the drawings are not intended to limit the scope of the invention, but are merely illustrative of the essential spirit of the invention's technical solutions. Furthermore, the technical features involved in the different embodiments of the invention described below can be combined with each other as long as they do not conflict with each other.

[0029] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0030] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0031] 1. Cell culture: Parental cells of human non-small cell lung cancer (NSCLC) (hereinafter referred to as A549 cells) were incubated in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco). Paclitaxel-resistant cells of NSCLC purchased from Fuheng Biotechnology (hereinafter referred to as A549 / TAX cells) were incubated in Ham's F-12K medium supplemented with 10% FBS and 200 ng / mL paclitaxel. Both A549 cells and A549 / TAX cells were incubated at 37°C and 5% CO2.

[0032] 2. CCK-8 assay: Cultured A549 cells and A549 / TAX cells (2×10⁶) 3 The paclitaxel ( / well) was seeded into the corresponding 96-well plates. After 24 hours, continuous concentrations of paclitaxel were added to each well and incubated for 48 hours. Then, 10% CCK-8 reagent (DOJINDO) was added to each well and incubated in the dark at 37°C for 2 hours. Next, the absorbance was measured at 450 nm using a microplate reader (Bio Tek). The dose-response curve was fitted using a GraphPad Prism 10 to determine the half-maximal inhibitory concentration (IC50).

[0033] Paclitaxel sensitivity in two cell lines, A549 and A549 / TAX, was assessed using CCK-8 viability assay. See also... Figure 2As shown in Figure A, the results indicate that the IC50 of A549 cells was 72.42 ± 22.83 ng / mL, while the IC50 of A549 / TAX cells was 6170.00 ± 2528.19 ng / mL.

[0034] 3. Colony formation experiment: A549 cells and A549 / TAX cells (8×10) were used. 2 Cells were seeded into 6-well plates (each well) and cultured in complete medium, which was changed every 3 days. Paclitaxel was added on day 3 of culture. After 14 days, colonies were fixed with 4% paraformaldehyde (Servicebio) for 40 minutes, stained with crystal violet solution (Biosharp) for 40 minutes, and washed with water. Colony numbers were quantified using ImageJ.

[0035] The resistance of A549 / TAX cells to paclitaxel was measured using a colony formation assay. See also... Figure 2 As shown in Figure B, the results indicate that when the paclitaxel concentration exceeds 1.6 μg / mL, the proliferation of A549 / TAX cells is significantly higher than that of A549 cells.

[0036] 4. Apoptosis assay: The cultured A549 cells and A549 / TAX cells (5×10⁶) were then cultured. 5 (Each well) was seeded into a fully cultured 6-well plate. After 24 hours, paclitaxel was added and incubated for 48 hours. Floating and adherent cells were collected, stained with Annexin V-FITC and propidium iodide (Elabscience), and the apoptosis level was detected by flow cytometry. The apoptosis rate was calculated using flow JO.

[0037] The effect of paclitaxel on the apoptosis levels of A549 cells and A549 / TAX cells was investigated using apoptosis assays. (See also...) Figure 2 As shown in Figure C, the results indicate that, in the absence of paclitaxel treatment, there was no significant difference in apoptosis between the parental A549 cell line and the paclitaxel-resistant A549 / TAX cell line. However, with increasing paclitaxel concentration, the proportion of apoptotic cells in the parental A549 cells was higher than that in the paclitaxel-resistant A549 / TAX cells.

[0038] The CCK-8 assay, colony formation assay, and apoptosis assay confirmed that paclitaxel-resistant A549 / TAX cells showed significantly higher resistance to paclitaxel than their parental A549 cells.

[0039] 5. RNA sequencing: Cultured A549 cells and A549 / TAX cells (7 × 10⁶ each) 6The samples were harvested in three batches. Total RNA was extracted using TRIzol reagent (Invitrogen) and sent to Azenta for further sequencing and analysis.

[0040] The molecular mechanisms of paclitaxel resistance phenotypes were investigated through RNA sequencing and analysis, and genes associated with paclitaxel resistance were identified. See also... Figure 3 As shown in Figure A, RNA sequencing results indicated that, with the application of conditional screening cutoff (|Log2FC|> 1, p < 0.05), 1268 differentially expressed genes (DEGs) positively correlated with paclitaxel resistance were identified. Among these genes, ABCB1 ranked first and had the highest Log2FC value.

[0041] See Figure 3 As shown in Figure B, the results of differential protein expression analysis indicate that, in addition to ABCB1, ABCG2, ABCA4, and ABCD1 are also involved in this process.

[0042] 6. Western Blot Validation: Cultured A549 and A549 / TAX cells were lysed using RIPA buffer (Beyotime) containing the protease inhibitor Roche. Protein concentration was determined using the biquinolinic acid (BCA) method. Total protein was separated by SDS-PAGE and transferred to methanol-activated polyvinylidene fluoride (PVDF) (Millipore) membranes. The membranes were blocked with 5% bovine serum albumin (BSA) at room temperature and incubated overnight at 4°C with primary antibody (ABCB1, Abcam, ab170904; GAPDH, Proteintech, 60004-1-1). On the second day, the membranes were washed with Tween-20 buffered saline (TBST) and incubated with secondary antibody according to the manufacturer's instructions. Protein bands were observed using enhanced chemiluminescence immunoassay and quantified using ImageJ.

[0043] See Figure 3 As shown in Figure C, Western blot analysis revealed that ABCB1 was upregulated in drug-resistant A549 / TAX cells, suggesting that it plays an important role in paclitaxel resistance.

[0044] Through RNA sequencing transcriptome analysis, followed by Western blot validation, we found that the expression of ATP-binding cassette B subfamily member 1 (ABCB1) (encoding a protein called p-glycoprotein) was significantly upregulated in drug-resistant cells.

[0045] 7. Transient knockout test: The cultured A549 / TAX cells were seeded into 6-well plates. When the confluence reached 60%–80%, each well was filled with a siRNA transfection mixture containing 300 μL of Opti-MEM (Gibco), 9 μL of Lipofectamine RNAiMAX (Thermo), and 30 pmol.

[0046] The siRNA sequence of ABCB1 is as follows: siABCB1 negative control: 5'-UUCUCCGAACGUGUCACGU-3'; siABCB1-1:5''-CGACAGAAUAGUAACUUGUU-3'; siABCB1-2:5''-GCAGCAAUUAGAACUGUGAUU-3'; siABCB1-3:5''-CCGAACACAUUGGAAGGAAAU-3'.

[0047] One to two days later, cells were collected to prepare for further functional experiments (including but not limited to LC-MS / MS detection, as well as Western Blot, qPCR, and CCK-8 assays).

[0048] 8. Real-time quantitative PCR (qRT-PCR): Total RNA was extracted using the RNA-quick purification kit. The reverse transcription PCR reaction system consisted of 4 μL 5×PrimeScript Buffer, 1 μL PrimeScript RT Enzyme Mix, 1 μL Oligo dT Primer, 1 μL Random 6 mers, and 1 μg RNA, with a final volume of 20 μL. Real-time PCR detection was performed using the PrimeScript RT Reagent Kit (Takara).

[0049] The PCR primers for ABCB1 and GAPDH are as follows: ABCB1 positive: 5'-GGGAGCTTAACACCCGACTTA-3'; ABCB1 reverse: 5'-GCCAAAATCACAAGGGTTAGCTT-3'; GAPDH forward: 5'-GGAGCGAGATCCCTCCAAAAT-3'; GAPDH reverse: 5'-GGCTGTTGTCATACTTTCTCATGG-3'.

[0050] The relative gene expression levels of A549 / TAX cells after ABCB1 knockout were calculated using the cycle threshold (CT) value.

[0051] In summary, the above data indicate that ABCB1 is a key factor in paclitaxel resistance in NSCLC cells and a key molecule involved in paclitaxel resistance in non-small cell lung cancer, providing potential for targeting this molecule to overcome paclitaxel resistance.

[0052] 9. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS): 9.1 Reagents and Chemicals: Paclitaxel and Paclitaxel-d5 were purchased from Alta Scientific Co., Ltd. First Standard.

[0053] HPLC-grade methanol and acetonitrile were purchased from Sigma-Aldrich.

[0054] Deionized water is obtained by filtration through a Millipore Milliq system.

[0055] 9.2 Preparation of paclitaxel series standard solutions, QC series standard solutions, and internal standard solutions: First, the paclitaxel stock solution was dissolved in methanol to obtain a paclitaxel-methanol solution with a mass concentration of 1.0 mg / mL, which was stored at -80℃. Then, it was quantitatively diluted with a 50:50 deionized water-methanol solution to prepare a series of paclitaxel standard solutions with mass concentrations of 10.0, 50.0, 100.0, 500.0, 1000.0, 5000.0, 10000.0, and 50000.0 ng / mL, and a series of QC standard solutions with mass concentrations of 10.0, 1000.0, and 40000.0 ng / mL, all of which were stored at -80℃ for later use. The paclitaxel-d5 stock solution was quantitatively diluted with a 50:50 deionized water-methanol solution to prepare an internal standard solution (hereinafter referred to as IS solution) with a mass concentration of 1000.0 ng / mL, which was stored at -80℃ for later use.

[0056] 9.3 Preparation and processing of cell samples: A549 cells and A549 / TAX cells were seeded at equal densities in 6-well plates. After 24 hours, parental non-small cell lung cancer cells and paclitaxel-resistant cells were treated with a p-glycoprotein inhibitor in combination with different concentrations of paclitaxel, respectively, followed by co-incubation for 24 hours. The culture medium was then collected and designated as cell supernatant; after trypsinization, adherent cells were collected and resuspended in PBS to a volume equivalent to that of the cell supernatant. Finally, the cell suspension was rapidly frozen in liquid nitrogen, then thawed at room temperature for three cycles, subjected to sonication in an ice bath (10-second pulses, 5-second intervals), and stored at 4°C to prepare cell samples.

[0057] 9.4 Extraction of paclitaxel: First, take 100 μL of cell sample, add 10 μL of IS solution and 1 mL of methyl tert-butyl ether, and vortex the mixture for 5 minutes. Then, centrifuge at 14000 rpm for 10 minutes, transfer 900 μL of the upper organic layer into a tube, and evaporate to dryness with nitrogen at 40 °C. Finally, reconstitute the dried residue in 100 μL of mobile phase (water / methanol, 20 / 80, v / v or water / methanol / formic acid, 50 / 50 / 0.1, v / v / v), centrifuge at 12000 rpm for 10 minutes, and take 5 μL of the supernatant as the detection sample for injection into the UPLC-MS / MS system.

[0058] 9.5. Instruments and UPLC-MS / MS conditions: The UPLC-MS / MS analysis of this invention uses the Waters Xevo TQD system (Waters, Milford, MA, USA), which consists of an ACQUITY UPLC Class I system.

[0059] A. Chromatographic conditions: Chromatographic column: ACQUITY UPLC™ BEH C18 column (2.1×50mm, 1.7μm); mobile phase: a solution of A and B prepared in a volume ratio (A: water with 0.1% formic acid; B: methanol solution); gradient elution: 0-1 min, 10%→80%B; 1-2 min, 80%B; 2-3 min, 80%→10%B; total run time for each sample: 3 min; flow rate: 0.3 mL / min; column temperature: 40℃; injection volume: 5 μL.

[0060] B. Mass spectrometry conditions: Mass spectrometry detection was performed using an ESI electrospray ionization source in positive ion multiple reaction monitoring (MRM) mode. Specific mass spectrometry parameters were as follows: ion spray voltage 5500 V; ion source temperature 550 °C; declustering voltage (DP) 60 V; inlet potential (EP) 10 V; collision chamber outlet voltage (EP) 10 V; paclitaxel m / z 854.2→286.0; paclitaxel declustering voltage (DP) 30 V; paclitaxel collision energy (CE) 15 eV; internal isotopic standard (IS) paclitaxel-d5 m / z 859.2→291.2; paclitaxel-d5 declustering voltage (DP) 30 V; paclitaxel-d5 collision energy (CE) 20 eV.

[0061] 10. Statistical Analysis: All numerical data are expressed as mean ± standard deviation (SD). Statistical significance was assessed using t-tests, one-way ANOVA, or two-way ANOVA. All statistical analyses were performed using GraphPad Prism 10. A p-value < 0.05 is considered statistically significant. Significance levels are expressed as: * p < 0.05, ** p < 0.01, *** p < 0.001.

[0062] This invention further validates the developed method for determining intracellular and extracellular paclitaxel concentrations in non-small cell lung cancer based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS): 1. Specificity: The retention times of paclitaxel and paclitaxel-d5 were 1.82 min and 1.83 min, respectively. Interfering components in the blank sample had no significant effect on the analyte reaction, which meets the requirements. See [link to relevant documentation]. Figure 4 As shown in Figure A, the peaks of paclitaxel and paclitaxel-d5 are well-defined and completely separated.

[0063] 2. Quantitative linearity and lower limit: Calibration curves were plotted using the ratio of paclitaxel and paclitaxel-d5 concentrations to nominal concentrations. The regression equations were fitted using a weighted least squares model (weight factor 1 / ×2) to obtain the best linearity and least squares residuals. See [link to documentation]. Figure 4 As shown in B, the typical regression equation for paclitaxel concentration is: y = 0.0183448×x - 0.0143468. The linear range of the standard curve for paclitaxel concentration is 1~5000 ng / mL, and R0 is... 2 A correlation coefficient greater than 0.999 demonstrates that the method exhibits good linearity. See also... Figure 4 As shown in C, the lower limit of quantification for paclitaxel is 1 ng / mL.

[0064] 3. Precision and accuracy: As per regulations, three batches of spiked samples (five replicates at low, medium, and high QC) were selected for testing to determine the accuracy and precision of the method of this invention. As shown in Table 1, the intraday and extraday precision of the quantitation limit were 7.40% and 6.60%, respectively, indicating that the quantitation limit for paclitaxel in cells can reach 1 ng / mL. The results show that the accuracy and precision of the method of this invention meet the requirements for biological sample analysis.

[0065] Table 1

[0066] 4. Residual effects: To assess the residual effect of the method of this invention, three double-blank samples were injected directly after the ULOQ sample. The first double-blank reaction showed that there was no peak in the plasma paclitaxel retention time (2.95% of LLOQ).

[0067] 5. Stability: The analyte exhibited good stability under both low and high dose quality control conditions: 4 hours at room temperature, 3 freeze-thaw cycles, and storage at −80℃ for 30 days. As shown in Table 2, no significant deviation was found between the measured paclitaxel concentration and the nominal concentration; both met the requirements.

[0068] Table 2

[0069] This invention innovatively establishes an LC-MS / MS method for the precise quantification of paclitaxel concentration. This LC-MS / MS method measures intracellular and extracellular paclitaxel levels and mechanistically confirms the role of ABCB1-driven paclitaxel efflux in NSCLC.

[0070] This invention measures the uptake and excretion of paclitaxel in A549 and A549 / TAX cells at different concentrations: The activity of paclitaxel was indirectly assessed by quantifying its intracellular and extracellular concentrations. To further elucidate the role of ABCB1 in mediating paclitaxel resistance, this invention established a UPLC-MS / MS method to quantify paclitaxel concentration. After treating A549 and A549 / TAX cells with different concentrations of paclitaxel for 24 hours, the PTX concentration in A549 and A549 / TAX cells was detected by UPLC-MS / MS. See also Figure 5 Tables 3 and 4 show that the p-glycoprotein in A549 / TAX cells maintains its transport function across a paclitaxel concentration range (from 80 ng to 8000 ng), indicating that this transporter can expel at least 8000 ng of paclitaxel. This suggests that high expression of the p-glycoprotein in A549 / TAX cells may promote the efflux of paclitaxel from the intracellular to the extracellular compartment.

[0071] Table 3

[0072] Table 4

[0073] This invention also verified that knocking down ABCB1 in A549 / TAX cells can improve paclitaxel sensitivity: See Figure 6 As shown in A and 6B, the knockout efficiency of the three independent siRNAs targeting ABCB1 was verified by Western blot and qRT-PCR. See also Figure 6 As shown in C, the CCK-8 assay showed that transfection with siABCB1-1 reduced the IC50 of A549 / TAX cells from 6437.00±2263.97 ng / mL to 4722.00±1883.11 ng / mL, indicating that ABCB1 significantly promoted paclitaxel resistance.

[0074] This invention also verified the synergistic effect of p-glycoprotein inhibitors Tariquidar and Elacridar with paclitaxel: See Figure 7 As shown in Figure A, the toxicity of Tariquidar and Elacridar was evaluated using the CCK-8 assay, and non-toxic concentrations of Tariquidar (25 ng / mL) and Elacridar (1.5 μg / mL) were selected for subsequent experiments. CCK-8 activity assays showed that Elacridar reduced the IC50 of A549 / TAX cells from 6319.00 ± 2947.05 ng / mL to 1698.00 ± 1052.72 ng / mL, a 3-fold reduction, while Tariquidar similarly reduced the IC50 to 287.80 ± 158.80 ng / mL, approximately a 21-fold reduction. See also... Figure 7 As shown in Figure B, both of these p-glycoprotein-specific inhibitors can reduce the IC50 value of A549 / TAX cells. See also... Figure 8 As shown in Figure A, colony formation assays revealed that increasing the paclitaxel concentration from 50 ng / mL to 150 ng / mL did not affect the proliferation of resistant cells, while the addition of Tariquidar (25 ng / mL) and Elacridar (1.5 μg / mL) significantly reduced cell proliferation. See also... Figure 8 As shown in B, the apoptosis assay results indicate that at a paclitaxel concentration of 1 μg / mL, the p-glycoprotein inhibitors Tariquidar and Elacridar enhanced the paclitaxel-induced cytotoxicity of A549 / TAX cells.

[0075] In summary, these results demonstrate that Tariquidar and Elacridar significantly reversed paclitaxel chemoresistance in A549 / TAX cells and sensitized NSCLC cells by inhibiting p-glycoprotein function, thus identifying ABCB1 as a viable therapeutic target for overcoming paclitaxel resistance.

[0076] This invention also verified the effect of the p-glycoprotein inhibitors Tariquidar and Elacridar in combination with paclitaxel on the intracellular and extracellular concentrations of A549 / TAX cells: The effects of the p-glycoprotein inhibitors Tariquidar and Elacridar in combination with paclitaxel on intracellular and extracellular paclitaxel concentrations in A549 / TAX cells were quantified by UPLC-MS / MS. See also Figure 9 As shown in Tables 5 and 6, after 24 hours of interaction between the inhibitor group and paclitaxel, we found that the addition of Tariquidar and Elacridar reduced the concentration of paclitaxel in the culture supernatant and increased the concentration of paclitaxel in the cell suspension. The effect of 1.5 μg / mL Elacridar on increasing the intracellular concentration was twice that of 25 ng / mL Tariquidar.

[0077] Table 5

[0078] Table 6

[0079] In summary, both Tariquidar and Elacridar can reduce paclitaxel efflux and increase paclitaxel accumulation in cells; secondly, Elacridar at 1.5 μg / mL is significantly more effective than Tariquidar at 25 ng / mL in inhibiting p-glycoprotein.

[0080] Intracellular and extracellular paclitaxel concentrations can serve as indirect indicators of paclitaxel resistance. To achieve precise quantification of paclitaxel levels in NSCLC cells, this invention innovatively employs ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). By directly measuring intracellular and extracellular paclitaxel concentrations, this invention demonstrates that ABCB1 overexpression promotes the efflux of intracellular paclitaxel, thereby reducing its cytotoxic accumulation, verifying the effect of ABCB1 expression on the paclitaxel sensitivity of NSCLC cells, and elucidating the role of ABCB1-mediated paclitaxel resistance in NSCLC cells. This invention, using UPLC-MS / MS, determined that pharmacological inhibition by gene silencing ABCB1 or the p-glycoprotein-specific inhibitors Tariquidar and Elacridar can restore intracellular paclitaxel levels and synergistically reduce NSCLC cell viability. Furthermore, this invention accurately determined the extracellular and intracellular paclitaxel concentrations in NSCLC cells treated with paclitaxel in combination with the p-glycoprotein-specific inhibitors Tariquidar and Elacridar. The efficacy of these p-glycoprotein inhibitors in reversing paclitaxel resistance and their optimal dosage were rigorously evaluated.

[0081] These findings elucidate a clear pharmacological mechanism: ABCB1-mediated drug efflux is a crucial mechanism for paclitaxel resistance in NSCLC, and UPLC-MS / MS is a sensitive analytical method for detecting paclitaxel concentrations. Inhibiting ABCB1 is a promising therapeutic strategy that could resensitize paclitaxel-resistant tumors to chemotherapy. These findings provide strong theoretical data to support the selection and application of p-glycoprotein inhibitors in overcoming paclitaxel resistance.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for determining the intracellular and extracellular paclitaxel concentration in non-small cell lung cancer, characterized in that, Detection based on ultra-high performance liquid chromatography-tandem mass spectrometry includes the following steps: Step 1) Using paclitaxel stock solution, paclitaxel-d5 stock solution, methanol and deionized water, prepare a series of paclitaxel standard solutions, a series of QC standard solutions and an internal standard solution; Step 2) The parental cells and paclitaxel-resistant cells of human non-small cell lung cancer are cultured and treated to obtain the corresponding cell samples; Step 3) Use liquid-liquid extraction to extract paclitaxel from the cell sample to obtain the test sample; Step 4) Set the chromatographic and mass spectrometric conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry system; Step 5) The test sample is sent into an ultra-high performance liquid chromatography-tandem mass spectrometry system to quantify the concentration of paclitaxel inside and outside the parental cells and paclitaxel-resistant cells; Step 6) Perform statistical analysis on the quantitative results.

2. The method for determining the intracellular and extracellular paclitaxel concentration in non-small cell lung cancer according to claim 1, characterized in that, In step 1, the preparation method of the paclitaxel series standard solutions is as follows: First, the paclitaxel stock solution was dissolved in methanol to obtain a paclitaxel-methanol solution with a mass concentration of 1.0 mg / mL, which was then stored at -80℃. Next, it was quantitatively diluted with a 50:50 deionized water-methanol solution to prepare a series of paclitaxel standard solutions with mass concentrations of 10.0, 50.0, 100.0, 500.0, 1000.0, 5000.0, 10000.0, and 50000.0 ng / mL, which were then stored at -80℃ for later use.

3. The method for determining the intracellular and extracellular paclitaxel concentration in non-small cell lung cancer according to claim 1, characterized in that, In step 1, the preparation method of the QC series standard solutions is as follows: First, the paclitaxel stock solution was dissolved in methanol to obtain a paclitaxel-methanol solution with a mass concentration of 1.0 mg / mL, which was then stored at -80℃. Then, it was quantitatively diluted with a 50:50 deionized water-methanol solution to prepare QC series standard solutions with mass concentrations of 10.0, 1000.0, and 40000.0 ng / mL, which were then stored at -80℃ for later use.

4. The method for determining the intracellular and extracellular paclitaxel concentration in non-small cell lung cancer according to claim 1, characterized in that, In step 1, the method for preparing the internal standard solution is as follows: The paclitaxel-d5 stock solution was quantitatively diluted with a 50:50 deionized water-methanol solution to prepare an internal standard solution with a paclitaxel-d5 mass concentration of 1000.0 ng / mL, which was then stored at -80℃ for later use.

5. The method for determining the intracellular and extracellular paclitaxel concentration in non-small cell lung cancer according to claim 1, characterized in that, In step 2, the specific methods for culturing and treating parental cells and paclitaxel-resistant cells of non-small cell lung cancer are as follows: Parental non-small cell lung cancer cells and paclitaxel-resistant cells were seeded at equal densities in 6-well plates. After 24 hours, the parental non-small cell lung cancer cells and paclitaxel-resistant cells were treated with a p-glycoprotein inhibitor in combination with different concentrations of paclitaxel, and then co-incubated for 24 hours. The culture medium was then collected and designated as cell supernatant. After trypsinization, adherent cells were collected and resuspended in PBS to a volume equivalent to that of the cell supernatant. Finally, the cell suspension was rapidly frozen in liquid nitrogen, then thawed at room temperature for three cycles, sonicated in an ice bath, and stored at 4°C.

6. The method for determining the intracellular and extracellular paclitaxel concentration in non-small cell lung cancer according to claim 1, characterized in that, In step 3, the method for extracting paclitaxel from the sample is as follows: First, take 100 μL of cell sample, add 10 μL of internal standard solution and 1 mL of methyl tert-butyl ether, and then vortex the mixture for 5 minutes to homogenize. After centrifuging at 14000 rpm for 10 minutes, 900 μL of the upper organic layer was transferred into a tube and evaporated to dryness with nitrogen at 40 °C. Finally, the dried residue was reconstituted in 100 μL of mobile phase and centrifuged at 12,000 rpm for 10 minutes. 5 μL of the supernatant was taken as the detection sample injected into the LC-MS / MS system.

7. The method for determining the intracellular and extracellular paclitaxel concentration in non-small cell lung cancer according to claim 1, characterized in that, In step 4, the chromatographic conditions are set as follows: The chromatographic column was a 2.1×50mm, 1.7μm ACQUITY UPLC™ BEH C18 column; The mobile phase is a solution of A and B prepared in a volume ratio, wherein A is water with a formic acid mass fraction of 0.1% and B is a methanol solution; Gradient elution was used: 0-1 minute, 10%→80%B; 1-2 minutes, 80%B; 2-3 minutes, 80%→10%B; The total processing time for each test sample is 3 minutes; The flow rate was 0.3 mL / min; The column temperature is 40℃; The injection volume was 5 μL.

8. The method for determining the intracellular and extracellular paclitaxel concentration in non-small cell lung cancer according to claim 1, characterized in that, In step 4, the mass spectrometry conditions are set as follows: The ion spray voltage is 5500V; The ion source temperature is 550℃; The declustering voltage is 60V; The input potential is 10V; The voltage at the collision chamber outlet is 10V; Paclitaxel m / z 854.2→286.0; The paclitaxel decomposition voltage is 30V; The collision energy of paclitaxel is 15 eV; Paclitaxel-d5 m / z 859.2→291.2; The declustering voltage of paclitaxel-d5 is 30V; The collision energy of paclitaxel-d5 is 20 eV.

9. The method for determining the intracellular and extracellular paclitaxel concentration in non-small cell lung cancer according to claim 1, characterized in that, In step 6, during statistical analysis, all numerical data are expressed as mean ± standard deviation; t-test, one-way ANOVA, or two-way ANOVA are used to assess statistical significance; significance levels are expressed as: *p < 0.05, **p < 0.01, ***p < 0.001.

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