Application of asparaginase in preparation of medicine for treating pancreatic cancer

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of asparaginase in preparation of a medicine for treating pancreatic cancer. The asparaginase and the gemcitabine are combined for treating pancreatic cancer, and results show that the combination of the two medicines can promote apoptosis of pancreatic cancer cells, reduce proliferation and reduce activation of pancreatic stellate cells, so that an important scientific basis is provided for improving gemcitabine drug resistance, and meanwhile, a new application is provided for the asparaginase.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of asparaginase in preparing medicine for treating pancreatic cancer. Background Art

[0002] Pancreatic cancer is one of the malignant tumors with a high mortality rate, and most of them are already in the advanced stage when they are discovered. Gemcitabine (GEM) is one of the important drugs for the treatment of advanced pancreatic cancer, and its effect is restricted by the development of drug resistance. Compared with other malignant tumors, pancreatic cancer is a tumor with extensive and dense fibrous matrix, and the extracellular matrix limits the responsiveness of pancreatic cancer to chemotherapy. Single-drug or combined chemotherapy based on gemcitabine is an important treatment strategy for patients with advanced pancreatic cancer, but the gradually formed GEM resistance seriously restricts the effect of treatment. In addition to the cancer cells themselves, the rich stromal components in pancreatic cancer also promote the formation of drug resistance. Among them, cancer-associated fibroblasts and the extracellular matrix they deposit are important factors in the formation of extensive and dense fibrous matrix in pancreatic cancer.

[0003] In pancreatic cancer, most cancer-associated fibroblasts originate from the activation of pancreatic stellate cells (PSCs). Direct or indirect interactions between pancreatic cancer cells and PSCs may play an important role in GEM resistance. Therefore, exploring the communication and metabolic crosstalk between pancreatic cancer cells and PSCs can provide an important basis for targeting the pancreatic cancer stromal microenvironment and reversing GEM chemotherapy resistance. In view of this, it is urgent to develop new drugs to enhance the therapeutic effect of GEM on pancreatic cancer, improve chemotherapy sensitivity, and solve the problem of its easy development of chemotherapy resistance. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defect in the prior art that pancreatic cancer cells gradually develop gemcitabine resistance, which seriously restricts the effect of treatment, and to provide the use of asparaginase in the preparation of drugs for treating pancreatic cancer.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0006] The first aspect of the present invention provides the use of asparaginase in the preparation of a medicament for treating pancreatic cancer.

[0007] In some technical solutions of the present invention, the drug includes the asparaginase and a pancreatic cancer chemotherapy drug.

[0008] In some technical solutions of the present invention, the pancreatic cancer chemotherapy drug is gemcitabine.

[0009] In some technical schemes of the present invention, the asparaginase is used to reduce the drug resistance of pancreatic cancer cells to pancreatic cancer chemotherapy drugs.

[0010] In some technical solutions of the present invention, the drug is used to promote apoptosis of pancreatic cancer cells.

[0011] In some technical solutions of the present invention, the drug is used to reduce the proliferation of pancreatic cancer cells.

[0012] In some technical solutions of the present invention, the drug is used to reduce the activation of pancreatic stellate cells.

[0013] The second aspect of the present invention provides a drug for treating pancreatic cancer, comprising: the asparaginase and a pancreatic cancer chemotherapy drug.

[0014] In some technical solutions of the present invention, the pancreatic cancer chemotherapy drug is gemcitabine.

[0015] In some technical solutions of the present invention, the drug also includes pharmaceutically acceptable excipients.

[0016] In some technical solutions of the present invention, the drug is a liquid preparation.

[0017] In some technical solutions of the present invention, the concentration ratio of asparaginase to gemcitabine is 5-15 μg / mL:45-55 nmol / L.

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

[0019] In the present invention, asparaginase is used in combination with gemcitabine to treat pancreatic cancer. The results show that the combination of the two drugs can better promote the apoptosis of pancreatic cancer cells, reduce proliferation, and reduce the activation of PSCs, which provides an important scientific basis for improving GEM resistance and provides a new use for asparaginase.

[0020] The present invention discovered a new mechanism by which pancreatic cancer cell-derived EVs promote the activation of PSCs, and clarified the key role of pancreatic cancer cell-derived EVs in the metabolic crosstalk between pancreatic cancer and PSCs, that is, Asn promoted the activation of PSCs and the production of extracellular matrix proteins, which is beneficial to GEM resistance. Asn is a metabolic crosstalk substance between the two, and GEM combined with ASNase provides an important scientific basis for targeting tumor microenvironment metabolism and improving GEM resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effects of different concentrations of gemcitabine on human pancreatic cancer cell line MIAPaCa-2 and pancreatic cancer resistant cell line MIAPaCa-2 R IC50 curve of cytotoxicity experiment.

[0022] Figure 2 This is a diagram of the primary culture and identification of human pancreatic stellate cells; Figure 2 A in the figure indicates that the primary human pancreatic stellate cells isolated under ordinary light microscope show spindle-shaped or polygonal phenotype; Figure 2 B in the figure shows the result of oil red O staining showing the loss of lipid droplets in the cytoplasm of primary pancreatic stellate cells; Figure 2 C in the figure shows the result of cell immunofluorescence showing that primary pancreatic stellate cells express the specific marker α-SMA.

[0023] Figure 3 The expression of SV40LT and hTERT at the mRNA level after Primary hPSCs and imPSCs were transfected with immortalizing viruses. Figure 3 A in the figure represents the result of the relative expression level determination of SV40LT mRNA; Figure 3 B in the figure represents the result of the relative expression level determination of hTERT mRNA; Figure 3 C in the figure represents the result of the relative expression level determination of SV40LT mRNA; Figure 3 D in the figure shows the results of the relative expression level determination of hTERT mRNA.

[0024] Figure 4 Western blot of human pancreatic stellate cells transfected with immortalized virus.

[0025] Figure 5 This figure shows the expression of activation markers α-SMA, Fibronectin and Collagen-1 in primary and immortalized pancreatic stellate cells detected by qRT-PCR.

[0026] Figure 6 This figure shows the expression of activation markers α-SMA, Fibronectin and Collagen-1 in primary and immortalized pancreatic stellate cells detected by Western blot.

[0027] Figure 7 pancreatic cancer cell line MIAPaCa-2 and pancreatic cancer drug-resistant cell line MIAPaCa-2 R Transmission electron microscopy image of extracellular vesicles from cells; Figure 7 A represents the transmission electron microscopy image of MIAPaCa-2-derived extracellular vesicles; Figure 7 B stands for MIAPaCa-2 R Transmission electron micrograph of extracellular vesicles from the cells.

[0028] Figure 8This is a particle size detection diagram of extracellular vesicles derived from pancreatic cancer cell line MIAPaCa-2 and pancreatic cancer resistant cell line MIAPaCa-2R; Figure 8 A represents the particle size detection diagram of MIAPaCa-2-derived extracellular vesicles; Figure 8 B shows the particle size detection diagram of MIAPaCa-2R-derived extracellular vesicles.

[0029] Fig. 9 pancreatic cancer cell line MIAPaCa-2 and pancreatic cancer drug-resistant cell line MIAPaCa-2 R Protein marker detection diagram of extracellular vesicles.

[0030] Fig.10 This is a picture of PSCs cells taking up PKH26-labeled extracellular vesicles observed under a confocal microscope.

[0031] Fig.11 The qRT-PCR and Western blot images show that extracellular vesicles from pancreatic cancer cells and their drug-resistant cell lines activate PSCs and regulate the expression of extracellular matrix proteins. Fig.11 A represents the qRT-PCR graph of the activation of PSCs and regulation of the expression of extracellular matrix proteins by extracellular vesicles derived from pancreatic cancer cells and their drug-resistant cell lines; Fig.11 B represents a Western blot diagram showing the activation of PSCs and regulation of the expression of extracellular matrix proteins by extracellular vesicles derived from pancreatic cancer cells and their drug-resistant cell lines; Fig.11 C represents the grayscale analysis result of the Western blot image.

[0032] Fig.12 This is an immunofluorescence image of the activation of PSCs and regulation of extracellular matrix protein expression by extracellular vesicles from pancreatic cancer cells and their drug-resistant cell lines; Fig.12 A represents the immunofluorescence image of the effect of extracellular vesicles derived from pancreatic cancer cells and their drug-resistant cell lines on α-SMA, a marker of PSCs activation; Fig.12 B represents the immunofluorescence image of Fibronectin when extracellular vesicles derived from pancreatic cancer cells and their drug-resistant cell lines act on PSCs; Fig.12 C represents the immunofluorescence image of Collagen-1 when extracellular vesicles derived from pancreatic cancer cells and their drug-resistant cell lines act on PSCs.

[0033] Fig.13 This is a graph showing the asparagine content in pancreatic cancer cells and their drug-resistant cells; Fig.13 A represents the difference fold of asparagine content in extracellular vesicles from pancreatic cancer cells and drug-resistant cells; Fig.13B represents a bar graph of asparagine content in extracellular vesicles derived from pancreatic cancer cells and drug-resistant cells; Fig.13 C represents the content of asparagine in the supernatant of pancreatic cancer cells and gemcitabine-resistant cells; Fig.13 D represents the asparagine content in pancreatic cancer cells and gemcitabine-resistant cells.

[0034] Fig.14 The figure shows the effect of different concentrations of asparagine on the activity of PSCs cells.

[0035] Fig.15 Asparagine regulates the expression of PSCs activation markers α-SMA, Fibronectin, and Collagen-1; Fig.15 A represents the Western blot of activation markers α-SMA, Fibronectin, and Collagen-1 when PSCs were treated with asparagine or not with asparaginase; Fig.15 B represents the qRT-PCR graph of activation markers α-SMA, Fibronectin, and Collagen-1 when PSCs were treated with asparagine or not with asparaginase.

[0036] Fig.16 This is the immunofluorescence image of asparagine regulating PSCs activation markers α-SMA, Fibronectin, and Collagen-1; Fig.16 A represents the immunofluorescence image of α-SMA; Fig.16 B represents the immunofluorescence image of Fibronectin; Fig.16 C shows the immunofluorescence image of Collagen-1.

[0037] Fig.17 This is a diagram showing the effect of gemcitabine combined with asparaginase on apoptosis of pancreatic cancer cells; Fig.17 A represents the result of flow cytometry detection of the effect of gemcitabine and / or asparaginase on apoptosis of pancreatic cancer cells; Fig.17 B represents the results of Western blot detection of the effect of gemcitabine and / or asparaginase on the apoptosis of pancreatic cancer cells.

[0038] Fig.18 EdU graph showing the effect of gemcitabine and / or asparaginase on the proliferation of pancreatic cancer cells.

[0039] Fig.19 Figure 2 shows the effects of gemcitabine and / or asparaginase on the clonogenicity of pancreatic cancer cell proliferation.

[0040] Fig. 20This is an in vivo experiment of gemcitabine combined with asparaginase in the treatment of pancreatic cancer; Fig. 20 A represents the flow chart of in vivo experiments; Fig. 20 B shows the fluorescence images of tumor growth in mice treated with different drugs.

[0041] Fig.21 Immunohistochemistry of pancreatic cancer mice treated with gemcitabine and / or asparaginase.

[0042] Fig. 22 The figure shows the average radiation, tumor weight and survival time of pancreatic cancer mice treated with gemcitabine and / or asparaginase; Fig. 22 A represents the average radiation curve of the mouse bioluminescence image;

[0043] Fig. 22 B represents the tumor weight measurement graph; Fig. 22 C represents the survival analysis graph. DETAILED DESCRIPTION

[0044] The present invention is described in detail below in conjunction with specific examples, but it should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0045] Experimental Materials:

[0046] Asparagine was purchased from Sigma-Aldrich with the product number A4159.

[0047] Asparaginase was purchased from MCE with the catalog number HY-P1923.

[0048] The human pancreatic cancer cell lines MIAPaCa-2 and PANC-1 used in the present invention were purchased from the American Type Culture Collection (ATCC).

[0049] Embodiment 1:

[0050] In order to understand the effect of pancreatic cancer cell-derived EVs on PSCs in GEM resistance, the present invention first constructed pancreatic cancer GEM-resistant cells and immortalized human pancreatic stellate cells (such as Figure 1 First, by gradually increasing the concentration of GEM in the cell culture medium, a pancreatic cancer gemcitabine-resistant cell line was constructed. Then, immortalized human pancreatic stellate cells were constructed to prepare for subsequent experiments.

[0051] 1. The method for constructing pancreatic cancer GEM-resistant cells is as follows:

[0052] First, determine the IC50 value of gemcitabine (GEM) for parental cells MIAPaCa-2 and PANC-1. Use GEM (1 nM) less than IC50 as the initial drug concentration, and continue to subculture after the cells adapt to the drug concentration, gradually increasing the drug concentration to 500 nM. -1 μM, until the cells reached a state of resistance to gemcitabine, and the pancreatic cancer resistant cell line MIAPaCa-2 was successfully constructed R 、PANC-1 R .

[0053] like Figure 1 As shown in the cytotoxicity experiment IC50 curve, different concentration gradients of GEM treated pancreatic cancer parental cells MIAPaCa-2 and drug-resistant cells MIAPaCa-2 respectively. R After 72 hours, the cell viability curves of the two groups were well separated, with IC50 values ​​of 13.69nM and 628.0nM, respectively. Compared with the parental cells, the IC50 value of the resistant cell line was significantly higher. According to the formula RI = IC50 of resistant cell line / IC50 of parental cell line, the resistance coefficient of MIAPaCa-2R was 45.87, which was greater than 5, indicating that the pancreatic cancer resistant cell line MIAPaCa-2R was successfully constructed.

[0054] 2. Construction of immortalized human pancreatic stellate cells:

[0055] (1) Extracting and culturing primary human pancreatic stellate cells (hPSCs) using an in vitro growth method

[0056] Fresh pancreatic cancer tissue specimens were collected in the operating room. The pathological diagnosis was pancreatic ductal adenocarcinoma. After being brought back to the laboratory, the specimens were first rinsed several times with PBS containing penicillin / streptomycin double antibodies, and the tissues were cut into tissue blocks with a diameter of about 2-3 mm with sterile ophthalmic scissors. The blocks were inoculated in a six-well plate containing DMEM / F-12 (1:1) culture medium containing 10% FBS and 2 mmol / L glutamine. 3-5 tissues were added to each well and incubated at 37°C and 5% CO 2

[0057] Culture under the conditions of 1% PBS. Change the medium every 24 hours, change the plate every 48 hours, and change the medium every 2-3 days until the pancreatic stellate cells grow into tissue blocks and adhere to the well plate. When the cells reach about 80% to 90% confluence, digest them with 0.25% trypsin and continue to culture in T25 culture bottles.

[0058] (2) Immortalization of primary pancreatic stellate cells

[0059] ① Select the third generation hPSCs with good growth status and culture them at 5×10 4 / well inoculated in 6-well plates;

[0060] ②Using the 1 / 2 small volume infection method, add 1 mL of fresh culture medium to each well, add immortalized virus solution and pdybrene, and add another 1 mL of culture medium 4 hours after infection;

[0061] ③Observe the cell status 12 hours after transfection, replace the complete medium 24 hours later, and digest with trypsin after 72 hours before subculturing in T25 culture flasks;

[0062] ④ The cells were screened continuously for 14 days using a culture medium containing 2 μg / mL puromycin, and then maintained in culture using a complete culture medium containing 1 μg / mL puromycin. The cells were labeled as imPSCs.

[0063] (3) Identification of primary pancreatic stellate cells

[0064] ①Cell morphology: observe the morphology of primary cells under an ordinary optical microscope;

[0065] ② Oil Red O experiment: Detection of cytoplasmic lipid droplets. The cells were divided into two groups, PrimaryhPSC#1 and PrimaryhPSC#2. They were first fixed with 4% paraformaldehyde at room temperature for 15 minutes, rinsed with PBS, and then washed with 60% isopropanol three times. Oil Red O working solution (3 mL 0.5% dye solution plus 2 mL ddHO) was used. 2 O) staining for 15 minutes, rinsing with 60% isopropanol three times, then staining with hematoxylin for 1 minute, rinsing with water, drying, and observing and photographing under a microscope;

[0066] ③Cell immunofluorescence: detect the expression of primary pancreatic stellate cell marker α-SMA.

[0067] The results are as follows Figure 2 As shown in Figure A, after 4-10 days of human pancreatic cancer tissue blocks adhered to the wall, primary PSCs gradually crawled out from the edge of the tissue block and grew around the tissue block. The cells were large in size and were spindle-shaped or polygonal. Figure 2 As shown in Figure B, the Oil Red O experiment showed that there were no lipid droplets in the cytoplasm of primary stellate cells isolated from pancreatic cancer tissue, indicating that the stellate cells were activated. Figure 2 As shown in C, cell immunofluorescence staining showed that both primary cell lines expressed the stellate cell-specific marker α-SMA, proving that the cells isolated from pancreatic cancer tissues were PSCs.

[0068] (4) Validation of immortalization of pancreatic stellate cells

[0069] qRT-PCR and Western blot were used to detect the expression of SV40LT, hTERT, α-SMA, Fibronectin and Collagen-1 at the mRNA and protein levels, respectively.

[0070] The third generation of primary human PSCs with good growth status were selected and transfected with immortalizing viruses SV40LT and hTERT at the same time. After continuous selection with puromycin for 14 days, the cells were stably subcultured. qRT-PCR and Western blot showed that compared with the primary cell PSCs in the control group, the immortalized PSCs (imPSCs) had a higher expression of mRNA ( Figure 3 AD in ) and proteins (as shown in Figure 4 SV40LT and hTERT were highly expressed in the mRNA (as shown in Figure 2), indicating that the immortalized viruses were transferred into primary pancreatic stellate cells and the immortalized pancreatic stellate cell line imPSCs was successfully constructed. Similarly, qRT-PCR and Western blot showed that in mRNA (as shown in Figure 2), SV40LT and hTERT were highly expressed in the mRNA (as shown in Figure 2), indicating that the immortalized viruses were transferred into primary pancreatic stellate cells and the immortalized pancreatic stellate cell line imPSCs was successfully constructed. Figure 5 A and B in Figure ) and protein (as Figure 6 At the same time, the pancreatic stellate cell activation markers α-SMA, Fibronectin and Collagen-1 were expressed in both primary hPSCs and immortalized cell lines imPSCs, indicating that the cell lines imPSCs stably transfected with immortalization viruses were in the same activated state as the primary cells. In summary, the immortalized pancreatic stellate cells imPSCs can be used for subsequent related research.

[0071] 3. Extraction of EVs from parental and GEM-resistant pancreatic cancer cells

[0072] like Figure 7 A and B in Figure 8 A and B in Fig. 9 As shown, cells were cultured in complete medium. When the cell confluence reached 80-90%, the complete medium was discarded, the cells were washed three times with PBS, and replaced with serum-free medium. The supernatant was collected after 48 hours of culture. The collected supernatant was extracted with differential ultracentrifugation, that is, after centrifugation at 500×g for 15 minutes, centrifugation at 10,000×g for 30 minutes, and then EVs were separated by ultracentrifugation at 110,000×g for 70 minutes, and washed in PBS under the same ultracentrifugation conditions. The extracted EVs (derived from MIAPaCa-2 / MIAPaCa-2 R and PANC-1 / PANC-1 R EVs, respectively, are called EV m / EV mR and EVP / EV PR) were dissolved in PBS and stored at -80°C. The concentration of EVs was determined by BCA method. Freshly extracted pancreatic cancer parental cells and GEM resistant cell EVs were appropriately diluted with PBS buffer and then identified by transmission electron microscopy and particle size distribution by Novo Bio. Western blot was used to detect EVs marker proteins CD63 and TSG101 and EVs negative protein calnexin.

[0073] 4. Effects of pancreatic cancer cell-derived EVs on PSCs

[0074] MIAPaCa-2 and MIAPaCa-2 R Derived EVs act on PSCs. First, MIAPaCa-2 and MIAPaCa-2 were separated by differential ultracentrifugation. R EVs were isolated from the supernatant and identified by electron microscopy and particle size detection (e.g. Figure 7 A and B in Figure 8 The extracellular vesicle protein markers CD63 and TSG101 were highly expressed in EVs, but very weak expression levels were detected in the corresponding cell lysates, while the negative protein marker Calnexin of EVs was the opposite (as shown in Figure 2A and B). Fig. 9 ), further indicating that EVs were successfully extracted.

[0075] Then, to confirm the uptake of EVs by PSCs, we added PKH26-labeled EVs to PSCs at 50 μg / mL, and the results showed that PSCs successfully took up MIAPaCa-2 / MIAPaCa-2 R Source of EVs( Fig.10 shown).

[0076] qRT-PCR and Western Blot results showed that pancreatic cancer cell-derived EVs could promote the upregulation of α-SMA in PSCs and the increase of extracellular matrix Collagen-1 and Fibronectin, among which the effect of GEM-resistant cell EVs was more significant. Immunofluorescence also showed the same effect (such as Fig.11 AC and Fig.12 ). Therefore, the applicant found that pancreatic cancer cell-derived EVs are closely related to PSCs activation.

[0077] 5. Metabolomics sequencing of EVs in parental and GEM-resistant pancreatic cancer cells

[0078] To search for metabolic crosstalk between pancreatic cancer cells and PSCs, metabolomics sequencing was performed on EVs of parental and GEM-resistant pancreatic cancer cells. Fig.13As shown in A and B in Figure 1, the content of asparagine (Asn) in pancreatic cancer GEM-resistant cell EVs was significantly increased compared with that in parental cell EVs. Based on this, it is speculated that the supernatant of pancreatic cancer GEM-resistant cells also contains more Asn.

[0079] For further verification, the Asn concentrations in parental and GEM-resistant pancreatic cancer cells and their corresponding cell supernatants were measured, and the results showed that GEM-resistant cells and their conditioned medium contained more Asn (e.g. Fig.13 As shown in C and D). The control group was not added with Asn, and the experimental groups were different concentrations of Asn (250 μM, 500 μM, 750 μM) to determine the effects of different concentrations of Asn on the growth of PSCs. Fig.14 As shown, it was found that 250 μM Asn significantly promoted the proliferation of PSCs.

[0080] The conditioned medium of parental pancreatic cancer cells and GEM-resistant pancreatic cancer cells was collected as follows:

[0081] The parental pancreatic cancer cells with good growth status and GEM-resistant pancreatic cancer cells were selected and inoculated with 5×10 5 The cells were inoculated at a density of 1.54 × 10 / well in a 6-well plate. After the cells adhered to the wall, fresh complete medium was replaced. After 48 hours, the supernatant was collected and filtered through a 0.45 μm filter to obtain the conditioned medium.

[0082] 7. To evaluate whether Asn promotes the activation of PSCs, PBS was used to act on PSCs as the NC group, 250μM Asn was used to act on PSCs, and 250μM Asn plus asparaginase (Asparaginase, ASNase, 10μg / mL) was used to act on PSCs as the experimental groups, followed by qRT-PCR, Western Blot and immunofluorescence detection.

[0083] The results are as follows Fig.15 A and B in Fig.16 As shown, qRT-PCR, Western Blot, and immunofluorescence all demonstrated that Asn promoted the upregulation of α-SMA, Collagen-1, and Fibronectin, and this effect could be inhibited by the addition of ASNase.

[0084] In summary, it was found that Asn can promote the activation of PSCs and thus enhance the resistance of pancreatic cancer cells to gemcitabine, thus further proving the promoting role of ASNase and GEM in anti-pancreatic cancer cell growth and reversing GEM chemotherapy resistance. PBS was added to the cells as a control; 10 μg / mL ASNase was added to the cells as the ASNase group; 50nM GEM was added to the cells as the GEM group; 10 μg / mL ASNase and 50nmol / L GEM were added to the cells as the ASNase combined with GEM group.

[0085] like Fig.17 As shown in A and B, flow cytometry and Western blot detection of apoptosis-related proteins showed that compared with the control group, ASNase group and GEM group, ASNase combined with GEM promoted pancreatic cancer cell apoptosis more significantly.

[0086] Example 2: Effects of different drug treatments on the proliferation ability of pancreatic cancer cells

[0087] Experimental methods:

[0088] 1. Edu proliferation assay

[0089] Use UELandy according to the manufacturer's instructions. TM Edu staining was performed using the Edu cell proliferation kit. First, 10 μM Edu was added to the cells of the control group, ASNase group, GEM group, and ASNase combined with GEM group and incubated for 2 hours, followed by rinsing with PBS, fixation with 4% paraformaldehyde, permeabilization with 0.5% TritonX-100, and treatment of the cells with Click-iT working solution for 30 minutes. Finally, after incubation with DAPI solution, the cells were observed under a fluorescence microscope (Nikon, Japan).

[0090] 2. Clone formation experiment

[0091] Cells treated with different drugs were inoculated in 6-well plates at 500 cells per well, and the medium was changed every three days for 2 weeks. Then the medium was discarded, and the cells were fixed with 4% paraformaldehyde after rinsing with PBS. After discarding the fixative, the cells were washed once with PBS and then stained with 0.4% crystal violet. Finally, the colonies on the plate were photographed and counted.

[0092] Experimental results: Fig.18 and Fig.19 As shown, Edu proliferation assay and clone formation assay showed that ASNase combined with GEM had stronger proliferation inhibition ability than control or single drug use.

[0093] Example 3: Animal experiment to evaluate the therapeutic effect of ASNase combined with GEM on pancreatic cancer

[0094] 1. Experimental animals:

[0095] BALB / c nude mice, 4-6 weeks old female mice, weighing 15-20 g, were divided into 5 groups, 8 mice in each group. BALB / c nude mice were purchased from the East Campus of Sun Yat-sen University.

[0096] 2. Experimental methods:

[0097] The experimental process is as follows Fig. 20 As shown in A, 2×10 6 MIA PaCa-2 cells transfected with luciferase reporter gene were injected into the pancreas of 6-week-old female BALB / c nude mice. Fluorescent images of tumor-bearing mice were captured using a small animal in vivo fluorescence imaging system (Maestro 3.0.0, USA) before treatment. When in vivo fluorescence imaging showed that the tumor was visible (about 3 weeks after cell implantation), the mice were randomly assigned to one of the following groups: PBS group, ASNase group, GEM group, and ASNase+GEM group. Mice were intraperitoneally injected with ASNase (0.2 mg / mouse), GEM (100 mg / kg), ASNase (0.2 mg / mouse) + GEM (100 mg / kg), and PBS (control) twice a week, and small animal in vivo fluorescence imaging was performed weekly to observe tumor progression. Tumors were obtained after reaching the experimental endpoint for immunohistochemistry and other tests.

[0098] 3. Experimental results:

[0099] An orthotopic transplant tumor mouse model was used to evaluate the therapeutic effect of ASNase combined with GEM on pancreatic cancer compared with the control group or single drug group.

[0100] Small animal live imaging was performed at the indicated time after orthotopic injection of pancreatic cancer cells MIAPaCa-2 into the pancreas of mice. Fig. 20 As shown in B, it was observed that the combination of ASNase (0.2 mg / mouse) and GEM (100 mg / kg) significantly slowed down tumor growth compared with the other groups.

[0101] Immunohistochemical staining of mouse tumors Fig.21 As shown in Figure 2, the combination of the two drugs can promote apoptosis of pancreatic cancer cells, reduce proliferation, and reduce the activation of PSCs, which is consistent with the average radiation of small animal in vivo imaging and mouse tumor weight (such as Fig. 22 A and B in FIG.

[0102] In order to better evaluate the effect of GEM combined with or without ASNase on the survival of pancreatic cancer mice, the mice were observed until week 10. Kaplan-Meier analysis showed that the ASNase combined with GEM group had a longer overall survival (OS) compared with the control group, the ASNase group alone, and the GEM group alone (e.g. Fig. 22 (as shown in C in the figure).

[0103] Therefore, these results illustrate the key role of pancreatic cancer cell-derived EVs in the metabolic crosstalk between pancreatic cancer and PSCs, namely, promoting the activation of PSCs and the production of extracellular matrix proteins through Asn, which is beneficial to GEM resistance. The present invention discovered a new mechanism by which pancreatic cancer cell-derived EVs promote the activation of PSCs. Asn is a metabolic crosstalk substance between the two. GEM combined with ASNase provides an important scientific basis for targeting tumor microenvironment metabolism and improving GEM resistance.

[0104] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0105] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of asparaginase in the preparation of drugs for treating pancreatic cancer.

2. The use according to claim 1, characterized in that The drug includes the asparaginase and a pancreatic cancer chemotherapy drug.

3. The use according to claim 2, characterized in that The pancreatic cancer chemotherapy drug is gemcitabine.

4. The use according to claim 2, characterized in that The asparaginase is used to reduce the drug resistance of pancreatic cancer cells to pancreatic cancer chemotherapy drugs.

5. The use according to claim 1, characterized in that The drug is used for promoting apoptosis of pancreatic cancer cells.

6. The use according to claim 1, characterized in that The medicament is used to reduce the proliferation of pancreatic cancer cells.

7. The use according to claim 1, characterized in that The medicament is used to reduce the activation of pancreatic stellate cells.

8. A drug for treating pancreatic cancer, characterized in that: include: Asparaginase and pancreatic cancer chemotherapy drugs as claimed in claim 1.

9. The drug according to claim 8, characterized in that The pancreatic cancer chemotherapy drug is gemcitabine.

10. The drug according to claim 8, characterized in that The drug also includes pharmaceutically acceptable excipients.

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