Use of PBI-TPE-11 in the preparation of a product for indicating tumor cell drug resistance

Abstract

The application belongs to the technical field of tumor treatment, and particularly relates to application of PBI-TPE-11 in preparation of a product for indicating drug resistance of tumor cells. The application aims to provide a new method for understanding drug resistance of tumor cells. The technical scheme of the application is that PBI-TPE-11 is applied in preparation of a product for indicating drug resistance of tumor cells. The application provides a substance PBI-TPE-11 which can be used for indicating drug resistance of tumor cells. The PBI-TPE-11 is an amphiphilic molecule which can self-assemble and aggregate in cells, and emit fluorescence under the action of excitation light; and can also be recognized by ABCG2 and transported to the outside of cells. Therefore, drug resistance of tumor cells can be indicated by observing and comparing signals and changes of cell fluorescence.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tumor treatment, and particularly relates to application of PBI-TPE-11 in preparation of a product for indicating drug resistance of tumor cells. BACKGROUND

[0002] Lung cancer is the highest incidence and mortality of cancer in China, and the prevalence is increasing year by year. Lung cancer is mainly non-small cell lung cancer (NSCLC), accounting for about 85% of all lung cancer cases. The heterogeneity of lung cancer cells, tumor stem cell characteristics (stemness) and multidrug resistance (MDR) are the main reasons for the difficulty of conventional chemotherapy and high recurrence of lung cancer. The efflux of chemotherapeutic drugs is one of the main mechanisms of tumor cell drug resistance, and the ATP-binding cassette (ABC) protein family is the main protein involved in the efflux of chemotherapeutic drugs. ABC family is a group of multiple transmembrane proteins that actively transport chemotherapeutic drugs out of cells. This protein family has multiple members, such as ABCA, ABCB, ABCC, ABCF, ABCG superfamily members. This family of proteins can transport various chemicals or small molecules from the cytoplasm to the outside of the cell by hydrolyzing ATP, especially ABC superfamily B member 1 (ABCB1) and ABC superfamily G member 2 (ABCG2), which can transport anticancer drugs (mitoxantrone, topotecan and methotrexate), antibiotics (cimetidine, prazosin and fluoroquinolones), porphyrin and tyrosine kinase inhibitors from the cytoplasm to the outside of the cell. ABCB1 is also known as P-glycoprotein (P-glycoprotein) or multidrug resistance protein 1 (MDR1), which is closely related to the drug resistance of various tumor cells and is also related to the excretion of steroid hormones and glucocorticoids. ABCC1 transporter is also known as multidrug resistance-associated protein 1 (MRP1), which can excrete a variety of chemotherapeutic drugs and excrete corticosterone, but not cortisol. ABCG2-positive lung cancer cells may be cancer stem cells (CSCs), which account for a small percentage of drug-resistant lung cancer cells and are also known as tumor initiating cells (TICs). These tumor cells may be in a dormant state at ordinary times, but under suitable conditions, these cells exhibit strong proliferation capacity, clonal formation, invasion ability, distant metastasis, chemotherapy resistance and multidrug resistance. In addition, cell membrane proteins CD133, CD44 and CD24, and cytoplasmic protein ALDHl are also markers of lung cancer stemness.Lung cancer cells exhibit high heterogeneity; not all cells in a tumor can proliferate indefinitely and differentiate into tumor tissue types. The development and progression of lung cancer are driven by a small number of cancer cells (CSCs) within the tumor, resulting in high chemotherapeutic insensitivity and resistance, which negatively impacts tumor development, progression, and recurrence. ABCB1 and ABCG2 are primarily located on the cell membrane. Specific inhibitors that block ABCB1 or ABCG2 proteins on the cell membrane can inhibit drug efflux, increase drug accumulation, and exert the effects of chemotherapeutic drugs, thus providing novel targets for antibody drugs. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide a new method for understanding the drug resistance of tumor cells.

[0004] The technical solution of this invention is the application of PBI-TPE-11 in the preparation of products indicating tumor cell drug resistance; the structure of PBI-TPE-11 is as follows: The arrow indicates the connection point of a saturated long carbon chain (undecaneous).

[0005] Specifically, the tumor cells are lung cancer cells.

[0006] The drug resistance is caused by the high expression of ABCG21 and / or ABCB in cells.

[0007] The present invention also provides a product for indicating tumor cell drug resistance, comprising PBI-TPE-11, the structure of which is as follows: The arrow indicates the connection point of a saturated long carbon chain (undecaneous).

[0008] Specifically, the tumor cells are lung cancer cells.

[0009] The drug resistance is caused by the high expression of ABCG2 and / or ABCB1 in cells.

[0010] Furthermore, the product also includes antibodies against ABCB1 and / or ABCG2.

[0011] This invention also provides a method for indicating drug resistance in tumor cells, comprising the following steps: treating tumor cells to be tested with PBI-TPE-11, performing flow cytometry analysis and immunofluorescence staining, and analyzing the positive rate and cell fluorescence intensity to determine whether the tumor cells to be tested have drug resistance; a positive rate of less than 50% can be judged as drug resistance caused by high expression of ABCG2 and / or ABCB1 in tumor cells.

[0012] Specifically, the processing time is 2 hours.

[0013] Furthermore, the flow cytometry analysis uses excitation light at a wavelength of 561-633 nm.

[0014] The beneficial effects of this invention: This invention provides a substance that can be used to indicate drug resistance in tumor cells, namely the ester molecule PBI-TPE-11. PBI-TPE-11 is an amphiphilic molecule that can aggregate and self-assemble in vitro, and can also enter cells through the cell membrane composed of a phospholipid bilayer, where it self-assembles and aggregates intracellularly. Due to its aggregation-enhancing effect, the aggregated ester molecules emit fluorescence under excitation light. Simultaneously, this amphiphilic molecule can also be recognized by ABCG2 and transported extracellularly. Therefore, by observing and comparing changes in cell positivity and fluorescence signals through flow cytometry and immunofluorescence staining, this provides a potentially effective tool for studying and analyzing drug resistance in tumor cells. Using the self-assembly and aggregation-enhancing PBI-11 ester molecule to treat tumor cells, combined with flow cytometry analysis of the percentage of PBI-11-positive cells, can differentiate between sensitive and drug-resistant tumor cell lines, simplifying the distinction between sensitive and drug-resistant lung cancer cell lines. In cases where drug resistance develops due to drug administration, PBI-TPE-11 can be used to process tumor cell samples for flow cytometry analysis and immunofluorescence staining. Positive rate and fluorescence intensity analysis can be performed. A positive rate below 50% indicates drug resistance in tumor cells due to high expression of ABCG2 and / or ABCB1. A targeted treatment regimen can then be developed based on this assessment. Compared to traditional IC50... 50 The detection method of this invention is simple to operate and saves time and effort. It reduces the time and numerous experimental operations required for conventional identification of drug-resistant cells and has good application value. Attached Figure Description

[0015] Figure 1 A correlation plot showing the relationship between A549 resistance and ABCG2 expression in lung cancer cells, specifically the IC50 values ​​of A549, A549-Res, and A549-OE-ABCG2 cell lines. 50 Tests. (a) (b) (c) are ICs for cisplatin. 50 Tests (d), (e), and (f) show the IC50 values ​​of etoposide. 50 test.

[0016] Figure 2 The graph shows the positive rate of ABCG2 expression in A549, A549-Res, and A549-OE-ABCG2 cell lines.

[0017] Figure 3 This is a structural diagram of the ester-like molecule PBI-TPE-11. The arrows indicate the connection points of the saturated long carbon chain (undecaneous carbon).

[0018] Figure 4 Immunofluorescence analysis of sensitive and drug-resistant A549 lung cancer cells. Comparison of fluorescence signals of ABCG2, ABCB1, and PBI-11 in sensitive A549 and drug-resistant A549-Res cells. Blue represents ABCG2, red represents PBI-11, green represents ABCB1, Merge represents a mixture of the three colors, and the scale bar is 15 micrometers.

[0019] Figure 5 PBI-11 signaling patterns in flow cytometry analysis of drug-resistant and sensitive A549 and H460 lung cancer cell lines. Lung cancer cells without PBI-11 treatment served as controls. The blue and red peaks represent the percentages of PBI-11-negative and positive cells, respectively.

[0020] Figure 6 This image shows the PBI-11 signal in A549 lung cancer cells treated with ABCG2 antibody, analyzed by flow cytometry. The image shows the signal analysis of PBI-11 positive cells after treatment with ABCG2 antibody in cisplatin- or etoposide-resistant lung cancer cells. The upper part of the image shows cisplatin-resistant cell lines, and the lower part shows etoposide-resistant cell lines.

[0021] Figure 7 Flow cytometry analysis of TPE-11-treated lung cancer cell lines sensitive and resistant to the drug: A549 and H460 cisplatin-sensitive and drug-resistant cell lines were treated with the ester-like molecule TPE-11 for 2 hours.

[0022] Figure 8 Flow cytometry analysis of lung cancer cell lines sensitive and resistant to graphene quantum dots (GQDs). Detailed Implementation

[0023] The applicant discovered through preliminary experiments that tumor cells overexpressing ABCG2 exhibit significant drug resistance. Drug resistance in tumor cells is a root cause of poor chemotherapy efficacy in cancer patients. Common chemotherapy drugs such as cisplatin or etoposide are small inorganic or organic molecules; however, these molecules lack the property of self-assembly and aggregation within cells, and therefore cannot aggregate to emit fluorescence, hindering observation and analysis at the cellular level. Therefore, the applicant sought to screen for a substance that can be recognized by ABCG2 and emit fluorescence within cells, which could be used to detect whether tumor cells overexpress ABCG2, thereby indicating the drug resistance of tumor cells.

[0024] In cell labeling experiments, this application screened an ester molecule, PBI-TPE-11, containing self-assembling groups perylene bisimides (PBI) and tetraphenylethene (TPE), as well as two saturated long carbon chains (undecyl groups). This ester molecule contains saturated long carbon chains and both lipophilic and hydrophilic groups, hence it is called an amphiphilic group. The tetraphenylethene and perylene bisimides groups in this ester molecule can not only self-assemble but also exhibit aggregation-induced emission (AIE). Cell experiments showed that PBI-TPE-11 (hereinafter referred to as PBI-11) can enter various cells and emit fluorescence.

[0025] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0026] Example 1: Correlation analysis between drug resistance in different cell lines and ABCG2 expression

[0027] The CCK8 assay for cell viability / proliferation was used to detect A549-sensitive lung cancer cells, multidrug-resistant cells (A549-Res) resistant to cisplatin or etoposide, and A549 cells overexpressing ABCG2 (A549-OE-ABCG2). A549-sensitive and drug-resistant cells (A549-Res) were purchased from the cell bank of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The A549 strain overexpressing ABCG2 (A549-OE-ABCG2) was obtained by infecting A549-sensitive cell lines with lentivirus. Specifically, to obtain an A549 cell line overexpressing ABCG2, the full-length ABCG2 cDNA plasmid BCRP / ABCG2cDNA ORF Clone, Human, untagged (HG17375-UT) was purchased from Sinocare. After amplifying the full-length ABCG2 gene by PCR, the product was double-digested with restriction endonucleases NheI and BamHI, and then inserted into the plasmid vector pHS-B-0089. This constructed a lentiviral vector using the EF-1A promoter, overexpressing (OE) ABCG2, and selected with puromycin. The A549 cell line was infected with the prepared lentivirus and screened by puromycin to obtain an A549 cell line overexpressing ABCG2 (A549-OE-ABCG2).

[0028] The drug inhibition assay (CCK8 assay) is performed as follows:

[0029] 1) Seed the cells to be tested into 96-well plates (the wells around the edge are not used for experiments; add PBS to reduce evaporation).

[0030] 2) After culturing for 12 hours, add the drug according to the set concentration gradient (generally at least 8 consecutive equal gradients), and make at least 3 replicates for each concentration;

[0031] 3) After the specified culture time, replace 100 μL of culture medium in each well, add 10 μL of CCK8 reagent (Beyotime Biotechnology Co., Ltd.), mix thoroughly, and then incubate at 37°C in the dark for 2 hours.

[0032] 4) Remove the 96-well plate, gently shake to mix, and then use a microplate reader to measure the absorbance at 450 nm. The value is directly proportional to the number of surviving cells. By comparing with the control group, the cell viability at different concentrations can be calculated. The IC50 value can be calculated and plotted using the research software Graphpad.

[0033] Inhibition assays of cisplatin showed that the A549-sensitive strain, A549-Res, and A549-OE-ABCG2 had the lowest half-maximal inhibitory concentration (IC50) for cisplatin. 50 The values ​​were 6.08 μmol / L, 20.65 μmol / L, and 41.90 μmol / L, respectively; the IC50 values ​​of these three tumor cell types for etoposide were... 50 The values ​​were 1.36 μmol / L, 3.80 μmol / L, and 7.05 μmol / L, respectively. Figure 1 a~f).

[0034] like Figure 2 As shown, overexpression of the ABC family protein ABCG2 in the A549 sensitive cell line can improve the IC50 of the therapeutic drugs cisplatin and etoposide. 50 The ABCG2 positivity rates in lung cancer cells A549, multidrug-resistant cell lines A549-Res (cisplatin or etoposide), and A549 cells overexpressing ABCG2 (A549-OE-ABCG2) were 29.39%, 96.52%, and 98.97%, respectively, with a significantly higher positivity rate in the multidrug-resistant cell line ABCG2-Res. In other words, drug resistance to cisplatin or etoposide multidrug-resistant cells (A549-Res) and significantly increased ABCG2 expression were observed in lung cancer cells A549. A549-OE-ABCG2 cells exhibited multidrug resistance to cisplatin and etoposide, demonstrating a correlation between high ABCG2 expression and chemotherapy resistance.

[0035] Example 2 Preliminary Screening Experiment

[0036] In this application, four compounds were initially screened during cell labeling experiments, and an ester molecule, PBI-TPE-11 (whose structure is shown below), was incidentally discovered. Figure 3 As shown, this ester molecule contains self-assembling groups perylene bisimides (PBI) and tetraphenylethene (TPE), as well as two saturated long carbon chains (undecyl groups). This ester molecule contains saturated long carbon chains and both lipophilic and hydrophilic groups, hence the term amphiphilic. The tetraphenylethene and perylene bisimides groups in this ester molecule can not only self-assemble but also exhibit aggregation-induced emission (AIE). Cellular experiments have shown that PBI-TPE-11 (abbreviated as PBI-11) can enter various cell types and emit fluorescence.

[0037] Cellular Experiment Procedure: Cells (A549, A549-Res, and A549-OE-ABCG2 cell lines) were cultured in DMEM containing 10% fetal bovine serum (Hyclone). Cells were seeded in glass-bottom confocal dishes (In VitroScientific, USA) or 6-well plates. After cell attachment, the cells were incubated overnight. PBI-11 was added to the cell culture medium, and the cells were cultured at 37°C for 2 hours. The cells were then carefully washed three times with phosphate-buffered saline (PBS), and the original medium was replaced with fresh medium without PBI-11. The cells were then cultured for another 3 hours. Immunofluorescence or flow cytometry analysis was performed according to the experimental design.

[0038] The flow cytometry analysis was performed using Gallios (Beckman Coulter, USA) and BD CaliburFacs. Prior to measurement, PBI-11-treated cells were isolated from culture dishes using trypsin, collected in tubes, and washed twice with PBS containing 0.5% FBS. A control group consisting of untreated cells was included. Approximately 10,000 cells were analyzed on the flow cytometer, fluorescence signals were acquired and counted, and analysis and plotting were performed using Flowjo 7.6. Results are as follows: Figure 2 . Figure 2 The study demonstrated a positive correlation between high expression of ABCG2 and drug-resistant cells A549, indicating that PBI-TPE-11 (hereinafter referred to as PBI-11) can enter various cells and emit fluorescence, and can be detected by flow cytometry.

[0039] Example 3: Cell Immunofluorescence Assay

[0040] After A549 and A549-Res cells adhered overnight, PBI-11 was added to the cell culture medium (the final concentration of PBI-11 after mixing with cells was 25 µg / mL). Cells were cultured at 37°C for 2 hours and then washed three times with phosphate-buffered saline (PBS). Immunofluorescence staining was performed using specific antibodies against ABCG2 and ABCB1 (mouse anti-human ABCG2 or ABCB1 monoclonal antibodies purchased from Abcam), and the cell fluorescence intensity was analyzed.

[0041] Immunofluorescence assay: Immunofluorescence was performed using 2% paraformaldehyde at room temperature for 30 min, followed by blocking with 2% horse serum at room temperature for 1 hour. Mouse anti-human ABCG2 or ABCB1 monoclonal antibodies (Abcam) were incubated overnight at 4°C at a dilution of 1:200. Secondary antibodies were goat anti-rabbit Alexa 488 or goat anti-mouse Alexa 405 (Beyotime) incubated at room temperature for 60 min. After washing three times with PBS, two-dimensional (2D) images were observed using a laser confocal microscope (TCS-SP8, Leica, Germany). Analysis was performed using "Leica Application Suite X" software to observe and analyze the fluorescence intensity and cellular localization of PBI-11 with these molecules.

[0042] Compared to the drug-resistant cell line A549-Res, the sensitive cell line A549 showed lower expression of ABCG2 and ABCB1, but stronger fluorescence signal of PBI-11; conversely, the drug-resistant cell line showed higher expression of ABCG2 and ABCB1, but weaker fluorescence signal of PBI-11. Figure 4 The results indicate that PBI-11 ester molecules accumulate in large quantities and exhibit strong luminescence in drug-sensitive A549 tumor cell lines, but accumulate in small quantities and exhibit weak luminescence in drug-resistant A549-Res cell lines.

[0043] Example 4: Flow Cytometry Analysis Experiment

[0044] Previously, the resistance of lung cancer cells A549 to cisplatin and etoposide was tested using the traditional cell proliferation CCK8 assay. It was found that the expression of ABCG2 or ABCB1 was higher in the resistant cell lines, and the fluorescence signal of PBI-11 was weaker.

[0045] Based on this, sensitive and resistant cell lines (A549 and H460) were treated with PBI-11 for 2 hours, washed three times with phosphate-buffered saline, and analyzed using a CytoFLEX flow cytometer (Beckman Coulter). The sensitive and resistant H460 cell lines were purchased from the cell bank of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. As a control, the function of the ABCG2 protein on the cell membrane of the A549 resistant cell line was blocked using ABCG2 mouse monoclonal antibody (mAb). Sensitive and resistant cell lines (A549) were treated with PBI-11 for 2 hours, washed three times with phosphate-buffered saline, and analyzed using a CytoFLEX flow cytometer (Beckman Coulter).

[0046] The results are as follows Figure 5 As shown, the percentages of PBI-11 positive cells in cisplatin-sensitive and cisplatin-resistant A549 cell lines were 87.20% and 26.57%, respectively; the percentages in etoposide-sensitive and etoposide-resistant A549 cell lines were 77.22% and 29.86%, respectively. The percentages of PBI-11 positive cells in etoposide-sensitive and etoposide-resistant H460 cell lines were 61.38% and 24.23%, respectively. Figure 5 If 50% is used as the cutoff, a positivity rate of 50% or higher can be considered a sensitive strain, while a rate below 50% can be considered a resistant strain. PBI-11 positive cell percentage: Using a negative control (without PBI-11 added), the percentage of PBI-11 fluorescently positive cells excited at 561-633 nm by flow cytometry is the PBI-11 positive cell percentage. The 50% positivity rate is derived from the results of several flow cytometry analyses.

[0047] The expression rate and intensity of ABCG2 in the drug-resistant cell line A549-Res were significantly higher than those in the sensitive cell line A549. Figure 2 To analyze the possible mechanisms of this phenomenon, A549 lung cancer cells were treated with a mouse monoclonal antibody (mAb) of ABCG2. Following the same procedure, the lipid molecule PBI-11 was added, and the positive rate of PBI-11 signal in the cells was analyzed by flow cytometry. The results showed that after the function of cisplatin- or etoposide-resistant A549 cell lines was inhibited by ABCG2 monoclonal antibody, the positive rate of PBI-11 signal in cisplatin-resistant lung cancer cells increased from 26.57% to 70.02%, and the positive rate of PBI-11 in etoposide-resistant lung cancer cells increased from 29.86% to 56.93%. Figure 6 The results suggest that inhibiting or blocking the efflux function of ABCG2 reduces the efflux of intracellular PBI-11, which can significantly increase intracellular PBI-11 signaling.

[0048] Comparative Example 1

[0049] A similar experiment was conducted using TPE-11, an ester molecule containing tetraphenylethylene. The results showed that both sensitive and drug-resistant strains of tumor cells A549 and H460 exhibited strong TPE-11 fluorescence with no significant difference in intensity. No other molecules have been found to have this function. The sensitive and drug-resistant strains of H460 were purchased from the cell bank of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0050] Using cells without TPE-11 treatment as a control, cells treated with the lipid molecule TPE-11 for 2 hours were washed three times with PBS. Flow cytometry was then used to analyze the positivity rate of cell fluorescence signals. The results showed that the TPE-11 fluorescence positivity rate was 81.0% for both the A549 sensitive and resistant strains; the TPE-11 fluorescence positivity rates for the H460 sensitive and resistant strains were 82.7% and 81.4%, respectively. There was no significant difference in fluorescence signals between the sensitive and resistant strains. Figure 7 ).

[0051] Comparative Example 2

[0052] Two types of graphene quantum dots (GQDs), GQD-FA and CSCNP-R-CQDs, were used to treat A549 cells, including sensitive and resistant strains A549-Res, for 2 hours. After washing three times with PBS, the positivity rate of cell fluorescence signals was analyzed by flow cytometry. The results showed that the positivity rates of GQD-FA fluorescence in the sensitive and resistant A549 cells were 99.8% and 99.4%, respectively; the positivity rates of CSCNP-R-CQDs fluorescence in both the sensitive and resistant H460 cells were 100%, with no significant difference in fluorescence signals between the two strains. Figure 8 The cells were treated with the ester molecule TPE-11 for 2 hours, then washed three times with PBS. Flow cytometry was then used to analyze the positive rate of TPE-11 fluorescence signals. The results showed that the TPE-11 fluorescence positivity rate was 81.0% for both the A549 sensitive and resistant strains; and 82.7% and 81.4% for the H460 sensitive and resistant strains, respectively. There was no significant difference in fluorescence signals between the sensitive and resistant strains. Figure 8For GQD-FA, please see Shuhua Li, Shixin Zhou, Yunchao Li, et al. ACS Applied Materials & Interfaces. 2017; 9 (27), 22332-22341. For CSCNP-R-CQDs, please see Wen Su, Ruihua Guo, Fanglong Yuan, et al. The Journal of Physical Chemistry Letters, 2020, 11 (4): 1357-1363.

[0053] The above experiments demonstrate that ABCG2 has the function of effluxing small molecule drugs or compounds. Due to the high expression of ABCG2 in the drug-resistant cell line A549, the intracellular PBI-11 level was significantly elevated through monoclonal antibody inhibition assays, suggesting that the drug-resistant cell line can efflux the lipid molecule PBI-11. Since cellular fluorescence signals can be directly observed and analyzed, using the correlation between cellular fluorescence signal and PBI-11 intensity to indicate sensitive and drug-resistant tumor cell lines is simpler and more time-saving than the traditional IC50 detection method. Determining the drug resistance of tumor cells by observing and comparing cellular PBI-11 fluorescence signals provides a potentially practical tool for studying and analyzing tumor cell drug resistance, and has potential application value.

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of PBI-TPE-11 in the preparation of products indicating tumor cell drug resistance, characterized in that: The structure of PBI-TPE-11 is as follows: The arrow indicates the connection point of the saturated long carbon chain with an eleventh carbon atom; the tumor cells are lung cancer cells; the drug resistance is caused by the high expression of ABCG2 in the cells.

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