CN115137719A

CN115137719A - Application of small molecular compound in reversing drug resistance of colorectal cancer chemotherapy

Info

Publication number: CN115137719A
Application number: CN202210976725.9A
Authority: CN
Inventors: 陈巍; 李夏; 吴军舟; 倪茂巍; 戴五敏; 陈忠坚; 陈肖皖
Original assignee: Zhejiang Cancer Hospital
Current assignee: Zhejiang Cancer Hospital
Priority date: 2022-08-15
Filing date: 2022-08-15
Publication date: 2022-10-04

Abstract

The invention discloses an application of a small molecular compound in reversing drug resistance of colorectal cancer chemotherapy. 5-Fu drug-resistant CRC cells are constructed through drug concentration gradient culture, and the following results are found through MTT experiments and apoptosis experiments of in vitro cells: the 2-AAPA remarkably enhances the apoptosis of the drug-resistant CRC cells induced by the 5-Fu and remarkably increases the sensitivity of the drug-resistant CRC cells to the 5-Fu. Meanwhile, 2-AAPA can inhibit the growth of tumor in 5-Fu drug-resistant CRC cell xenograft tumor model by obviously enhancing 5-Fu induced CRC drug-resistant cell apoptosis in nude mice, and increase the sensitivity of 5-Fu drug-resistant CRC cells to 5-Fu in animal bodies. Therefore, the 2-AAPA can remarkably reverse 5-drug resistance of 5-Fu drug-resistant CRC cells in vitro and in vivo, and can be used for reversing the drug resistance of colorectal cancer to the 5-Fu.

Description

Application of small molecular compound in reversing drug resistance of colorectal cancer chemotherapy

Technical Field

The invention belongs to the fields of biomedicine and pharmacy, and particularly relates to application of a small molecular compound in reversing drug resistance of colorectal cancer chemotherapy.

Background

Colorectal cancer (CRC) is one of the most common cancers in the world. According to 2018 global cancer statistics, CRC ranks first in cancer-related deaths and third in morbidity.

5-fluorouracil (5-fluorouracil, 5-Fu) as a first-line drug for treating colorectal cancer is a basic drug in various new adjuvant chemotherapy, postoperative chemotherapy and palliative chemotherapy schemes, and can improve postoperative disease-free survival and overall survival of patients with stage III colorectal cancer. However, only 10-20% of patients with advanced colorectal cancer are sensitive to 5-Fu based chemotherapy regimens, and these patients who are initially sensitive to 5-Fu eventually develop resistance. Drug resistance to 5-Fu has been considered as one of the major obstacles to chemotherapy for advanced colorectal cancer, and there is an urgent need to increase the sensitivity of chemotherapy treatment for colorectal cancer 5-Fu and to reverse or prevent its chemotherapy resistance.

5-Fu is an analogue of uracil (the hydrogen atom at the C-5 position of uracil is replaced by a fluorine atom), and can be converted into various active metabolites in cells after entering into a human body, such as fluorouracil deoxynucleotide (FdUMP), fluorodeoxyuridine triphosphate (FdUTP), fluorouracil triphosphate (FUTP) and the like. FdUMP interferes with DNA synthesis by inhibiting Thymidylate Synthase (TS); FUTP is erroneously incorporated into RNA, affecting cellular metabolism in many ways, such as transcription, translation, and post-translational modification. 5-Fu influences nucleoside metabolism by inhibiting thymidylate synthase, interfering with DNA and RNA synthesis, and ultimately leading to cytotoxicity and apoptosis.

There are many factors reported by studies to cause 5-Fu resistance, including: tumors resist drug-induced apoptosis, and the like, against accelerated drug expulsion and decreased drug uptake, drug inactivation, drug target changes, increased cell proliferation, and the like. Therefore, 5-Fu drug resistance can occur in a plurality of biological processes, such as drug activation and inactivation, drug transport and excretion, drug action target point mutation, TS expression increase, deoxyuridine triphosphatase (DPD) activity increase, expression abnormality of related genes, posttranslational modification and the like.

In conclusion, 5-Fu exerts an anti-tumor effect mainly by inhibiting TS and influencing DNA and RNA synthesis, and the response rate of 5-Fu is low due to tumor cell drug resistance, but the molecular mechanism of 5-Fu drug resistance generation is very complex. Therefore, the drug resistance of 5-Fu is still a problem to be solved urgently in clinical application of fluorouracil drugs, and the finding of a compound with the effect of reversing the drug resistance of 5-Fu has a very important meaning.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of a small molecule compound in reversing drug resistance of colorectal cancer chemotherapy.

In order to achieve the above objects, one aspect of the present invention provides a small molecule compound for use in preparing a medicament for improving sensitivity of 5-fluorouracil-resistant cells to 5-fluorouracil in colorectal cancer, wherein the small molecule compound is 2-AAPA, has CAS number 1133387-90-2, and has a structural formula shown in formula (I) below:

with respect to the above-mentioned use of the present invention, the 2-AAPA increases the sensitivity of colorectal cancer 5-fluorouracil-resistant cells to 5-fluorouracil by inducing apoptosis of colorectal cancer 5-fluorouracil-resistant cells.

For the above use of the present invention, the colorectal cancer 5-fluorouracil-resistant cell is a HCT8/5Fu or HCT15/5Fu cell.

The invention also provides application of the small molecule compound 2-AAPA shown in the formula (I) in preparing a medicine for reversing the drug resistance of colorectal cancer 5-fluorouracil drug-resistant cells to 5-fluorouracil.

For the above use of the present invention, the 2-AAPA reverses the resistance of colorectal cancer to 5-fluorouracil by inducing apoptosis of colorectal cancer 5-fluorouracil-resistant cells.

The invention also provides application of the small molecule compound 2-AAPA shown in the formula (I) in combination with 5-fluorouracil in preparing a pharmaceutical composition for treating 5-fluorouracil-resistant colorectal cancer.

For the above-mentioned use of the present invention, the 2-AAPA in combination with 5-fluorouracil induces apoptosis of colorectal cancer 5-fluorouracil-resistant cells.

For the above-mentioned use of the present invention, the induction of colorectal cancer 5-fluorouracil-resistant apoptosis is achieved by activating caspase 3/PARP-dependent apoptosis pathway.

In the invention, colorectal cancer cells HCT8 and HCT15 are used as parent cells, and corresponding drug-resistant HCT8/5Fu and HCT15/5Fu cells are obtained by culturing 5-Fu with a series of concentration gradients. MTT experimental results showed that the drug resistance indexes of HCT8/5Fu and HCT15/5Fu cells were 21.1 and 15.5, respectively, showing drug resistance to 5-Fu. HCT8/5Fu or HCT15/5Fu drug-resistant cells were treated with 5-Fu in the presence of 2-AAPA, and the IC of 5-Fu on the cells was found ₅₀ The values were significantly reduced, reversing fold 2.2 and 3.7 respectively, which means that 2-AAPA increased HCT8/5Fu or HCT15/5Fu drug-resistant cells 2-4 times sensitive to 5-Fu, and was able to reverse the resistance of drug-resistant cells to 5-Fu.

The apoptosis experiment shows that compared with a control group, a 2-AAPA single drug group or a 5-Fu single drug group, the apoptosis of HCT8/5Fu and HCT15/5Fu drug-resistant cells is obviously increased under the combined treatment of the 2-AAPA and the 5-Fu. Western blot analysis of the treated cells revealed that: combined treatment of 2-AAPA and 5-Fu induces cleavage of caspase3 and PARP in HCT8/5Fu and HCT15/5Fu cells. Further, experiments were designed in which HCT8/5Fu or HCT15/5Fu resistant cells were pretreated with Z-VAD-FMK for 1 hour prior to combined treatment with 2-AAPA and 5-Fu, and the results showed that: Z-VAD-FMK pretreatment effectively blocked apoptosis of HCT8/5Fu or HCT15/5Fu cells induced by combination therapy of 2-AAPA and 5-Fu. These demonstrate that combined treatment with 2-AAPA and 5-Fu can induce a significant increase in drug-resistant apoptosis by activating caspase 3/PARP-dependent apoptotic pathways, leading to increased sensitivity of drug-resistant cells to 5-Fu.

Further, an HCT15/5Fu cell xenograft tumor model was constructed on BALB/c nude mice, the therapeutic effect of 2-AAPA single drug, 5-Fu single drug and combination of 2-AAPA and 5-Fu on the HCT15/5Fu xenograft tumor model was evaluated, and proliferation indexes Ki67 and TUNEL positive tumor cells in tumor samples of tumor-bearing mice were examined. The following discovery: the tumor volume of the combination drug combination of 2-AAPA and 5-Fu was significantly reduced compared to the 5-Fu monotherapy group, which indicates that: the combination of 2-AAPA and 5-Fu can obviously increase the tumor inhibition rate. In the transplanted tumor tissue, compared with the control group, the 2-AAPA single drug group and the 5-Fu single drug group do not obviously inhibit the proliferation of tumor cells and do not obviously induce the apoptosis of the tumor cells, while the 2-AAPA and 5-Fu combined drug group obviously inhibits the proliferation of the tumor cells and obviously induces the apoptosis of the tumor cells. Description of the drawings: 2-AAPA can increase the sensitivity of the HCT15/5Fu cell xenograft tumor model to treatment with 5-Fu by increasing 5-Fu-induced apoptosis. 2-AAPA was shown to increase the sensitivity of 5-Fu drug resistant CRC cells to 5-Fu in animals.

It can be seen that 2-AAPA can significantly reverse 5-Fu drug resistance in 5-Fu drug resistant CRC cells in vitro and in vivo.

Compared with the prior art, the invention has the following beneficial technical effects:

the invention discovers that: 2-AAPA can enhance 5-Fu induced apoptosis by activating caspase3/PARP dependent apoptosis pathway, and increase sensitivity of 5-Fu drug resistant CRC cells to 5-Fu, and drug resistance of colorectal cancer to 5-Fu can be reversibly transferred by combining 2-AAPA and 5-Fu in vitro or in vivo.

Drawings

FIG. 1A shows the control of cytotoxicity of 5-Fu treatment at different concentrations on parental HCT8 and 5-Fu drug-resistant HCT8/5Fu in vitro.

FIG. 1B shows the control of cytotoxicity of 5-Fu in vitro against parent HCT15 and 5-Fu drug-resistant HCT15/5Fu at different concentrations.

FIG. 2A shows the results of flow cytometry to detect apoptosis of HCT8/5Fu cells treated in groups according to the method of 2.1.

Fig. 2B is a histogram quantifying the apoptotic results of fig. 2A, wherein denotes P <0.001.

FIG. 2C shows the results of flow cytometry to detect apoptosis of HCT15/5Fu cells treated in groups according to the method of 2.2.

Fig. 2D is a histogram quantifying the apoptotic results of fig. 2C, wherein x represents P <0.01.

FIG. 2E shows the results of flow cytometry to detect apoptosis of HCT8/5Fu cells treated in groups according to the method of 2.3.

Fig. 2F is a histogram quantifying the apoptotic results of fig. 2E, where denotes P <0.05.

FIG. 2G shows the results of flow cytometry to detect apoptosis of HCT15/5Fu cells treated in groups according to the method of 2.4.

Fig. 2H is a histogram quantifying the apoptotic results of fig. 2G, where x represents P <0.01.

FIG. 2I shows the results of Western blot detection of apoptosis-related proteins in the grouped-treated cells for HCT8/5Fu cells.

FIG. 2J shows the results of Western blot detection of apoptosis-related proteins in the grouped-treated cells for HCT15/5Fu cells.

Figure 3A is a picture of tumors in HCT15/5Fu cell nude mouse xenograft tumor model 28 days after treatment via different cohorts.

Fig. 3B is data of changes in tumor volume measured during tumor loading of nude mice treated via different groups, wherein: p <0.05 compared to control; and, # denotes: p <0.05 compared to the 5-Fu group.

Figure 3C is a graph comparing body weights of tumor-bearing mice treated by different groups.

Figure 3D shows representative IHC-Ki67 images of four tumor samples grouped together.

Fig. 3E is the result of quantitative processing of the staining in fig. 3D with ImageJ software, wherein: p <0.05 compared to control; # denotes: p <0.05 compared to the 5-Fu group.

Fig. 3F shows representative IHC-TUNEL images of four tumor samples grouped.

Fig. 3G is the result of quantitative processing of the fluorescent staining in fig. 3F using ImageJ software, wherein: p <0.01 compared to control; and # # denotes: p <0.001 compared to the 5-Fu group.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and specific examples. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

The following examples do not specify particular techniques or conditions, according to the techniques or conditions described in the literature in the field or according to the product description. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.

In the following examples, the data below are all the mean ± sd of three independent experiments.

In the examples, the concentration unit is written by the conventional writing mode in the field: m, mM or. Mu.M, representing mol/L, mmol/L or. Mu. Mol/L, respectively.

Description of materials:

2-AAPA is commercially available from Sigma or can be purified from the reaction of terephthalocyanuric acid and N-acetyl-L-cysteine, and is specifically prepared in the following references: seefeldt, Y.ZHao, W.Chen, A.S.Raza, L.Carlson, J.Herman, A.Stoebner, S.Hanson, R.Foll, X.Guan, characterisation of a novel dithiocarbamate synthase inhibitor and its use as a tool to a modified intracellular synthase, the Journal of biological chemistry 284,2729-2737 (2009).

5-Fu was purchased from Sigma. The pan-cysteine aspartic protease inhibitor (Z-VAD-FMK) was purchased from Sigma. FBS, DMEM culture and pancreatin were purchased from Gibco, and antibodies (primary antibody and secondary antibody) were purchased from Proteinteck. DMEM complete medium is DMEM medium containing 10% FBS, 100U/mL penicillin and 100g/mL streptomycin.

Human colorectal cancer cell lines HCT8 and HCT15 were purchased from ATCC cell banks.

BALB/c nude mice, 6 weeks old, female, purchased from shanghai slyke laboratory animals ltd, laboratory animal license number: SCXK (Shanghai) 2017-0005, and all experimental animals are placed in SPF animal houses for feeding.

1. Construction of drug-resistant cell lines HCT8/5Fu and HCT15/5Fu

The HCT8 cells were passaged into 24-well plates, about 20000 cells were seeded per well, and the cells were allowed to stand at 5% CO ₂ An incubator, culturing for 24 hours in DMEM complete culture solution at 37 ℃; adding 20 mu M of 5-Fu into DMEM complete culture solution, and continuing to culture for 72 hours; then, changing fresh DMEM complete culture solution to continue culturing for 24 hours, adding 30 mu M of 5-Fu into the DMEM complete culture solution, and continuing culturing for 72 hours; the culture process was repeated by adjusting the concentration of 5-Fu in the DMEM complete medium (20. Mu.M → 30. Mu.M → 40. Mu.M → 50. Mu.M → 60. Mu.M → 70. Mu.M → 80. Mu.M → 90. Mu.M → 100. Mu.M in the following order) to obtain a 5-Fu-resistant HCT8/5Fu cell line.

The HCT15 cells were passaged into 24-well plates, about 20000 cells were seeded per well, and the cells were allowed to stand at 5% CO ₂ An incubator, culturing for 24 hours in DMEM complete culture solution at 37 ℃; adding 2 mu M of 5-Fu into DMEM complete culture solution, and continuing to culture for 72 hours; then, changing fresh DMEM complete culture solution to continue culturing for 24 hours, adding 3 mu M of 5-Fu into the DMEM complete culture solution, and continuing culturing for 72 hours; the above culture process was repeated by adjusting the concentration of 5-Fu in the DMEM complete medium (in this order, 2. Mu.M → 3. Mu.M → 4. Mu.M → 5. Mu.M → 6. Mu.M → 7. Mu.M → 8. Mu.M → 9. Mu.M → 10. Mu.M as the final concentration), to obtain a 5-Fu-resistant HCT15/5Fu cell strain.

The HCT8/5Fu and HCT15/5Fu drug-resistant cell strains are cultured in DMEM complete culture solution and maintained by 5-Fu at 100 mu M and 10 mu M respectively to ensure the stability of drug resistance.

2. MTT assay for in vitro cytotoxicity

Cells in the logarithmic growth phase were taken, digested with 0.25% trypsin and blown up as a cell suspension, and the cells were seeded in a 96-well cell culture plate with a final volume of 200. Mu.L per well (cell number 5000/well). Transferring 96 well plates to 37 ℃ C. 5%O ₂ After culturing in a saturated humidity incubator for 24h to adhere to the wall, discarding the culture solution, and replacing with DMEM complete culture solution containing the specified medicine with gradient concentration for culturing. 4 replicate wells per concentration, at 37 deg.C, 5% ₂ The incubation was continued for 48h in a saturated humidity incubator. MTT solution was added to each well and incubated at 37 ℃ for 4 hours. And detecting the respective absorbances, calculating the activity of each group of cells, and drawing a curve. IC calculation Using GraphPad Prism software ₅₀ The value is obtained. The Resistance Index (RI) is calculated according to the following formula: IC of RI =5-Fu drug-resistant CRC cells ₅₀ IC of non-drug resistant CRC parent cell ₅₀ 。

1.1 determination of 5-Fu cytotoxicity on respective cell lines in vitro by MTT method

The indicated drugs containing the gradient concentrations were the following concentration gradients of 5-Fu drugs: 0, 7.8. Mu.M, 15.6. Mu.M, 31.3. Mu.M, 62.5. Mu.M, 125. Mu.M, 250. Mu.M, 500. Mu.M, 1000. Mu.M.

The cells were: parental cells HCT8, HCT15,5-Fu drug-resistant cells HCT8/5Fu and HCT15/5Fu.

FIG. 1A shows the in vitro cytotoxicity control of the parent cell HCT8 and the drug-resistant cell HCT8/5Fu due to different concentrations of 5-Fu treatment, and FIG. 1B shows the in vitro cytotoxicity control of the parent cell HCT15 and the drug-resistant cell HCT15/5Fu due to different concentrations of 5-Fu treatment. In FIG. 1A, the cell viability of the drug-resistant cell HCT8/5Fu was higher compared to the parental cell HCT8 under different concentration treatments, indicating a significant decrease in the sensitivity to 5-Fu; in FIG. 1B, the cell viability of the drug-resistant HCT15/5Fu cell was higher compared to the parent HCT15 cell at different concentrations of treatment, indicating that its sensitivity to 5-Fu was significantly reduced.

At the same time, the IC of 5-Fu on HCT8, HCT15 cells, HCT8/5Fu, HCT15/5Fu cells was calculated ₅₀ The values are shown in tables 1 and 2 below. It can be seen that: IC of 5-Fu on HCT8/5Fu and HCT15/5Fu cells compared to HCT8 and HCT15 cells ₅₀ The values were significantly increased respectively. RI values for HCT8/5Fu and HCT15/5Fu cells were 21.1 and 15.5, respectively, indicating that HCT8/5Fu and HCT15/5Fu cells are resistant to 5-Fu.

1.2 detection of 2-AAPA cytotoxicity to drug-resistant cell line in vitro by MTT method

In the MTT test (see 1.1) of the HCT8/5Fu drug-resistant cell line, in the DMEM complete culture medium containing the drug (5-Fu) of the given concentration in a gradient manner, 40. Mu.M 2-AAPA was added to perform combined treatment for 48 hours, and in vitro cytotoxicity was detected.

In the MTT test (see 1.1) of the HCT15/5Fu drug-resistant cell line, in DMEM complete culture medium containing the drug (5-Fu) at a gradient concentration, 45. Mu.M 2-AAPA was added to the medium and combined for 48 hours to detect in vitro cytotoxicity.

IC of 5-Fu on HCT8/5Fu and HCT15/5Fu cells at the indicated 2-AAPA dose was calculated from MTT assay results ₅₀ The value is obtained. At the same time, IC of 5-Fu on HCT8/5Fu and HCT15/5Fu cells without addition of 2-AAPA ₅₀ Values were compared and fold reversal was calculated. Fold Reversal (RF) = (IC of 5-Fu on cells without 2-AAPA) ₅₀ ) /(IC of 5-Fu against cells with 2-AAPA) ₅₀ ). Specifically, as shown in the following tables 1 and 2, wherein RI is a drug resistance index; RF is the inverse multiple.

TABLE 1 determination of IC of HCT8/5Fu cells versus 5-Fu ₅₀ Value of

TABLE 2 determination of IC of HCT15/5Fu cells versus 5-Fu ₅₀ Value of

As can be seen from Table 1, the sensitivity of HCT8/5Fu cells to 5-Fu was increased by a factor of 2.2 in the presence of 40. Mu.M of 2-AAPA. That is, 2-AAPA can reverse the resistance of HCT8/5Fu cells to 5-Fu.

As can be seen from Table 2, the sensitivity of HCT15/5Fu cells to 5-Fu was increased by a factor of 3.7 in response to 45. Mu.M of 2-AAPA. That is, 2-AAPA can reverse the resistance of HCT15/5Fu cells to 5-Fu.

2. Apoptosis assay

Taking the drug-resistant cells in logarithmic growth phase, digesting into single cell suspension according to the ratio of 2 multiplied by 10 ⁵ Cells/well seeded in 6-well cell culture plates, at 37 ℃ and 5% CO ₂ Culturing in incubator with saturated humidity for 24 hr, grouping, adding DMEM complete culture solution containing different grouping drugs, and adding CO at 37 deg.C and 5% ₂ And continuously culturing for 48h in an incubator with saturated humidity, and collecting cells of different groups. Apoptosis was detected in different groups using Annexin V-FITC/PI kit from BD company by flow cytometry.

2.1 apoptosis assay of drug-resistant cells HCT8/5Fu

The drug-resistant cells were HCT8/5Fu cells, and the grouping was as follows: (1) DMEM complete culture solution without drugs is used as a blank Control group (marked as Control in the figure); (2) 2-AAPA single drug group (marked as 2-AAPA in the figure): adding 2-AAPA with the concentration of 40 mu M into DMEM complete culture solution; (3) 5-Fu single drug group (marked as 5-Fu in the figure): adding 5-Fu with the concentration of 400 mu M into DMEM complete culture solution; (4) the Combination (Combination in the figure) was prepared by adding 2-AAPA at a concentration of 40. Mu.M and 5-Fu at a concentration of 400. Mu.M to DMEM complete medium.

The results of flow cytometry to detect HCT8/5Fu apoptosis are shown in FIG. 2A. The corresponding histogram of apoptosis quantification is shown in fig. 2B. As can be seen from fig. 2A and 2B: compared with the control group, the 2-AAPA single drug group and the 5-Fu single drug group, the Apoptosis (Apoptosis) of the combined drug group is obviously increased, and the difference is statistically different (P < 0.001). Description of the drawings: the combination of 2-AAPA and 5-Fu significantly enhanced the induction of apoptosis in HCT8/5Fu cells.

2.2 apoptosis assay of drug-resistant cells HCT15/5Fu

The drug-resistant cells were HCT15/5Fu cells, and the grouping was as follows: (1) DMEM complete culture solution without drugs is used as a blank Control group (marked as Control in the figure); (2) 2-AAPA single drug group (marked as 2-AAPA in the figure): adding 2-AAPA with the concentration of 45 mu M into DMEM complete culture solution; (3) 5-Fu single drug group (marked as 5-Fu in the figure): adding 5-Fu with the concentration of 100 mu M into DMEM complete culture solution; (4) the Combination (Combination in the figure) was prepared by adding a Combination of 2-AAPA at a concentration of 45. Mu.M and 5-Fu at a concentration of 100. Mu.M to DMEM complete medium.

The results of flow cytometry to detect apoptosis in HCT8/5Fu cells are shown in fig. 2C, and the corresponding histogram of apoptosis quantification is shown in fig. 2D. As can be seen from fig. 2C and 2D: the Apoptosis (Apoptosis) was significantly increased in the combination group compared to the control group, 2-AAPA group and 5-Fu group, and the difference was statistically different (P < 0.01). Description of the drawings: the combination of 2-AAPA and 5-Fu significantly enhanced the induction of apoptosis in HCT15/5Fu cells.

2.3 apoptosis assay of drug-resistant cells HCT8/5Fu pretreated with Z-VAD-FMK

The drug-resistant cells were HCT8/5Fu cells, and the grouping was as follows: (1) DMEM complete culture solution without drug is used as a blank Control group (marked as Control in the figure); (2) Z-VAD-FMK single drug group (denoted as Z-VAD in the figure): treating HCT8/5Fu cells with DMEM complete medium containing 10 μ M Z-VAD-FMK for 1h; (3) a first combination group (2-AAPA + 5-Fu) prepared by adding 2-AAPA at a concentration of 40 μ M and 5-Fu at a concentration of 400 μ M to DMEM complete medium (treatment for 48 hours); (4) the second Combination (Combination) was performed by pretreating HCT8/5Fu cells with DMEM complete medium containing 10. Mu.M Z-VAD-FMK for 1h, and then with DMEM complete medium containing 2-AAPA at a concentration of 40. Mu.M in Combination with 5-Fu at a concentration of 400. Mu.M (48 h).

The results of flow cytometry to detect apoptosis in HCT8/5Fu cells are shown in fig. 2E, and the corresponding histogram of apoptosis quantification is shown in fig. 2F. As can be seen from fig. 2E and 2F: the HCT8/5Fu cells apoptosis was significantly reduced in the second combination compared to the first combination with a statistical difference (P < 0.05). Description of the invention: the apoptosis inhibitor Z-VAD-FMK blocks the HCT8/5Fu cell apoptosis induced by the combination therapy of 2-AAPA and 5-Fu.

Apoptosis assay of drug-resistant cells HCT15/5Fu pretreated with Z-VAD-FMK

The drug-resistant cells were HCT15/5Fu cells, and the grouping was as follows: taking (1) DMEM complete culture solution without drugs as a blank Control group (marked as Control in the figure); (2) Z-VAD-FMK single drug group (denoted as Z-VAD in the figure): treating HCT15/5Fu cells with DMEM complete medium containing 10. Mu.M Z-VAD-FMK for 1h; (3) a first combination group (2-AAPA + 5-Fu) prepared by adding 2-AAPA at a concentration of 45 μ M and 5-Fu at a concentration of 100 μ M to DMEM complete medium (treatment for 48 hours); (4) the second Combination (Combination) was performed by pretreating HCT15/5Fu cells with DMEM complete medium containing Z-VAD-FMK at a concentration of 10. Mu.M for 1h, and then with DMEM complete medium containing 2-AAPA at a concentration of 45. Mu.M in Combination with 5-Fu at a concentration of 100. Mu.M (48 h).

The results of detecting HCT15/5Fu apoptosis by flow cytometry are shown in FIG. 2G, and the corresponding histogram of apoptosis quantification is shown in FIG. 2H. As can be seen from fig. 2G and 2H: the second combination group had a significant decrease in HCT15/5Fu apoptosis compared to the first combination group, with a statistical difference (P < 0.01). Description of the drawings: Z-VAD-FMK blocks HCT15/5Fu apoptosis induced by the combination of 2-AAPA and 5-Fu therapy.

2.5 Western blot analysis of apoptosis-related proteins

For each of the grouped cells processed in the above methods 2.1 and 2.2, cell lysate was added and lysed in ice bath for 30 minutes, 16000 Xg, centrifuged at 4 ℃ for 30min to collect supernatant, and after determining protein content, an equal amount of sample was separated from protein by SDS-PAGE. Transferring the protein onto PVDF membrane after electrophoresis, sealing with 5% skimmed milk powder, sealing with primary antibody (apoptosis-related protein caspase-3, shear type caspase-3, PARP and shear type PARP) overnight, sealing with horseradish peroxidase-labeled secondary antibody sealing solution for 2h, developing with Diaminobenzidine (DAB) solution, and scanning for analysis.

FIG. 2I shows WB results for grouped-treated cells of HCT8/5Fu cells, and FIG. 2J shows WB results for grouped-treated cells of HCT15/5Fu cells. A similar situation appears in fig. 2I and 2J: compared with a control group, a 2-AAPA single-drug group and a 5-Fu single-drug group, the combined drug group has the advantages that caspase3 and PARP are obviously reduced, and the cut caspase3 and PARP are obviously increased. It is demonstrated that the combined treatment of 2-AAPA and 5-Fu induces the cleavage of the apoptosis-related proteins capase3 and PARP in HCT8/5Fu cells and HCT15/5Fu cells.

In summary, it is shown that: the combination of 2-AAPA and 5-Fu significantly enhances the induction of apoptosis in HCT8/5Fu and HCT15/5Fu cells by activating caspase3/PARP dependent apoptotic pathways, thereby significantly enhancing the sensitivity of HCT8/5Fu and HCT15/5Fu cells to 5-Fu.

3. In vivo sensitization experiment of xenograft tumor model of HCT15/5Fu cell

A HCT15/5Fu xenograft tumor model is constructed on BALB/c nude mice, and the treatment effect of 2-AAPA single drug, 5-Fu single drug and the combination of 2-AAPA and 5-Fu on HCT15/5Fu xenograft tumor is evaluated.

The specific steps for constructing the HCT15/5Fu xenograft tumor model are as follows: HCT15/5Fu cells were injected subcutaneously (5X 10) into nude mice ⁶ Cell/cell) until the tumor grows to 100mm ³ Tumor-bearing mice were randomized into groups and given daily according to the following groups: (1) control group (20% PEG200 physiological saline solution), (2) 2-AAPA single drug group (35 mg/kg body weight), (3) 5-Fu single drug group (25 mg/kg body weight), (4) 2-AAPA (35 mg/kg body weight) +5-Fu (25 mg/kg body weight) combined drug group. Tumor volumes were measured 2-3 times per week, mice weighed, and data recorded.

Tumor volume (V) was calculated as: v =1/2 × a × b ² Wherein a and b represent the length and width of the tumor mass, respectively.

FIG. 3A is a picture of tumors in tumor-bearing mice grown for 28 days. Fig. 3B is data of changes in tumor volume measured during tumor loading in nude mice. Fig. 3A and 3B show: after 28 days of drug action, the combined drug group of 2-AAPA and 5-Fu has much smaller tumor volume than the 5-Fu single drug group. This indicates that: the combination of 2-AAPA and 5-Fu can obviously increase the tumor inhibition rate.

Figure 3C shows a comparison of body weights of the four groups of mice, showing: there was no significant difference in body weight of each group of nude mice. The results show that the tumor-bearing mice can well tolerate the medicaments and have no symptoms such as weight loss and the like.

In addition, proliferation index Ki67 and TUNEL positive tumor cells were also examined in tumor samples from different groups of tumor-bearing mice grown for 28 days as described above.

Figure 3D shows representative IHC-Ki67 images of tumor samples from tumor-bearing mice in four cohorts with drug exposure for 28 days, and figure 3E is the result of quantitative processing of the staining in figure 3D using ImageJ software. As can be seen from fig. 3D and 3E, in the transplanted tumor tissue, the 2-AAPA single drug group and the 5-Fu single drug group did not significantly inhibit the proliferation of tumor cells, while the 2-AAPA and 5-Fu combination drug group significantly inhibited the proliferation of tumor cells, as compared to the control group.

Figure 3F shows representative TUNEL images of tumor samples from tumor-bearing mice in four groups with drug exposure for 28 days, and figure 3G is the result of quantitative processing of the fluorescent staining in figure 3F using ImageJ software. As can be seen from fig. 3F and 3G, in the transplanted tumor tissue, compared with the control group, neither the 2-AAPA single drug group nor the 5-Fu single drug group significantly induced apoptosis of tumor cells, while the number of TUNEL-positive tumor cells in the tumor sample in the 2-AAPA and 5-Fu combined drug group was significantly increased, confirming that 2-AAPA significantly enhanced apoptosis induced by 5-Fu.

The results indicate that 2-AAPA can increase the sensitivity of the HCT15/5Fu cell xenograft tumor model to treatment with 5-Fu by increasing 5-Fu induced apoptosis. 2-AAPA was shown to increase the sensitivity of 5-Fu drug resistant CRC cells to 5-Fu in animals.

As can be seen, our studies indicate that 2-AAPA can significantly reverse 5-Fu drug resistance in 5-Fu drug-resistant CRC cells in vitro and in vivo.

Therefore, in the present invention, 5-Fu drug-resistant CRC cells were constructed by concentration gradient, and MTT assay and apoptosis assay were performed. The experimental results show that: under the action of the specified non-cytotoxic dose of 2-AAPA, the sensitivity of drug-resistant CRC cells to 5-Fu is increased by about 2 to 4 times; under the action of the combination of the 2-AAPA and the 5-Fu, the apoptosis induction of the drug-resistant CRC cell is obviously enhanced, so that the 2-AAPA can increase the apoptosis of the drug-resistant CRC cell induced by the 5-Fu, and the combined application of the 2-AAPA and the 5-Fu has obvious in-vitro reversal effect on the drug resistance of the 5-Fu. In addition, in vivo studies found: the 2-AAPA can obviously enhance 5-Fu induced apoptosis, inhibit the growth of tumors in a 5-Fu drug-resistant CRC cell nude mouse transplanted tumor model, and increase the treatment sensitivity of the nude mouse transplanted tumor model to 5-Fu. 2-AAPA was shown to increase the sensitivity of 5-Fu drug resistant CRC cells to 5-Fu in animals. It can be seen that 2-AAPA can significantly reverse 5-Fu drug resistance in 5-Fu drug resistant CRC cells in vitro and in vivo. Thus, 2-AAPA can be used for reversal of 5-Fu resistance in colorectal cancer.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that the invention is described with reference to exemplary embodiments, but rather the words used therein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.

Claims

1. Application of 2-AAPA with structural formula shown as formula (I) in preparation of medicine for improving sensitivity of colorectal cancer 5-fluorouracil drug-resistant cells to 5-fluorouracil

2. The use according to claim 1, wherein the 2-AAPA increases the sensitivity of colorectal cancer 5-fluorouracil-resistant cells to 5-fluorouracil by inducing apoptosis of colorectal cancer 5-fluorouracil-resistant cells.

3. The use of claim 2, wherein said colorectal cancer 5-fluorouracil-resistant cells are HCT8/5Fu or HCT15/5Fu cells.

4. Application of 2-AAPA with structural formula shown as formula (I) in preparation of drug for reversing drug resistance of colorectal cancer 5-fluorouracil drug-resistant cells to 5-fluorouracil

5. The use according to claim 4, wherein the 2-AAPA reverses 5-fluorouracil resistance in colorectal cancer by inducing apoptosis of 5-fluorouracil-resistant cells in the colorectal cancer.

6. The use of claim 5, wherein the colorectal cancer 5-fluorouracil-resistant cells are HCT8/5Fu or HCT15/5Fu cells.

7. Application of 2-AAPA combined with 5-fluorouracil with structural formula shown in formula (I) in preparation of pharmaceutical composition for treating 5-fluorouracil-resistant colorectal cancer

8. The use of claim 7, wherein the 2-AAPA in combination with 5-fluorouracil induces apoptosis in colorectal cancer 5-fluorouracil-resistant cells.

9. The use of claim 8, wherein said induction of colorectal cancer 5-fluorouracil-resistant apoptosis is achieved by activating caspase3/PARP dependent apoptosis pathway.

10. The use of claim 8 or 9, wherein the colorectal cancer 5-fluorouracil-resistant cells are HCT8/5Fu or HCT15/5Fu cells.