Application of PDPN in the treatment of gemcitabine-resistant pancreatic cancer

Abstract

The application of PDPN in the treatment of gemcitabine-resistant pancreatic cancer belongs to the field of biological medicine, and the application of PDPN in the preparation of a reagent for detecting a gemcitabine-resistant pancreatic cancer product. The application of PDPN in the preparation of a drug for treating gemcitabine-resistant pancreatic cancer or increasing the sensitivity of pancreatic cancer to gemcitabine treatment. The application of PDPN in constructing a subcutaneous transplantation tumor cell model of gemcitabine-resistant pancreatic cancer and in constructing an orthotopic transplantation tumor model of gemcitabine-resistant pancreatic cancer. It is found through research that changing the expression level of PDPN can affect the sensitivity of gemcitabine-resistant pancreatic cancer cells to gemcitabine chemotherapy, which indicates that PDPN can be applied to the treatment and detection of gemcitabine-resistant pancreatic cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to the application of PDPN in the treatment of gemcitabine-resistant pancreatic cancer. Background Technology

[0002] Pancreatic cancer is a highly malignant and fatal digestive system tumor. It has an insidious onset, is asymptomatic in its early stages, and by the time symptoms appear, it is often already in an advanced stage and readily metastasizes to multiple organs, making surgical resection extremely difficult and resulting in a very high mortality rate. Even with recent advancements in medical technology, the 5-year survival rate for pancreatic cancer has only increased from 5% to 10%, and its treatment and prognosis remain unfavorable. Currently, chemotherapy is the primary treatment for patients with advanced pancreatic cancer, with gemcitabine (dFdC) monotherapy being the first-line treatment. However, most patients develop gemcitabine resistance, with an overall response rate of <20% for treating pancreatic cancer. Therefore, exploring the molecular mechanisms of gemcitabine resistance in pancreatic cancer, identifying therapeutic targets to reverse gemcitabine resistance, and developing new anti-tumor treatment strategies can help improve the treatment efficiency of pancreatic cancer, prolong patient survival, and improve patients' quality of life.

[0003] Podoplanin (PDPN) is a single-pass transmembrane receptor on the cell surface. The NCBI Gene ID for the human gene encoding this protein is 10630, and the NCBI Gene ID for the murine gene is 14726. Some studies have found that PDPN has potential application value in the diagnosis and treatment of lapatinib-resistant HER2-positive gastric cancer. However, current technologies do not utilize PDPN as a therapeutic target for gemcitabine-resistant pancreatic cancer.

[0004] CN115786515B, entitled "Application of PDPN in the Diagnosis and Treatment of Lapatinib-Resistant HER2-Positive Gastric Cancer," discloses the following: This invention discovered that the content of PDPN in the human gastric cancer lapatinib-resistant cell line HGC-27-LR is significantly higher than that in the human gastric cancer cell line HGC-27. Based on this, this invention provides the application of a reagent for detecting PDPN in the preparation of a product for diagnosing lapatinib-resistant HER2-positive gastric cancer, as well as a product for diagnosing lapatinib-resistant HER2-positive gastric cancer. This invention also found that reducing PDPN expression levels can inhibit the proliferation and invasion / migration ability of lapatinib-resistant HER2-positive gastric cancer cells and increase the drug sensitivity of lapatinib. Therefore, this invention provides the application of PDPN in the preparation of a pharmaceutical composition for treating lapatinib-resistant HER2-positive gastric cancer, as well as a pharmaceutical composition for treating lapatinib-resistant HER2-positive gastric cancer. This invention also provides a method for constructing a lapatinib-resistant gastric cancer model and the application of the gastric cancer model in screening drugs for treating lapatinib-resistant gastric cancer. The patent only identifies the effect of PDPN on lapatinib resistance. It does not cover gemcitabine.

[0005] As is well known, lapatinib has the following structural formula:

[0006]

[0007] Chemical name: N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[(2-methanesulfonylethylamino)methyl]-2-furanyl]quinazolin-4-amine, molecular formula: C 29 H 26 ClFN4O4S, lapatinib is an anti-tumor drug, mainly used in combination with capecitabine to treat ErbB-2 overexpression.

[0008] Gemcitabine, structural formula:

[0009]

[0010] It is a novel cytosine nucleoside derivative with the chemical formula C9H. 11 F2N3O4. Like cytarabine, it is activated by deoxycytosine kinase after entering the human body and metabolized by cytidine deaminase. Gemcitabine is a pyrimidine antitumor drug with the same mechanism of action as cytarabine; its main metabolite is incorporated into DNA within cells and primarily acts on the G1 / S phase.

[0011] These are different drugs, and therefore they bind to proteins in different ways. Therefore, research is being conducted on proteins that are effective against gemcitabine resistance in order to provide drug formulations for treating gemcitabine-resistant cancers. Summary of the Invention

[0012] The purpose of this invention is to provide an application of PDPN in the treatment of gemcitabine-resistant pancreatic cancer.

[0013] The application of the PDPN of the present invention in the preparation of reagents for detecting gemcitabine-resistant pancreatic cancer.

[0014] Furthermore, the reagent is a primer that specifically amplifies the PDPN gene or a binder that specifically binds to the protein encoded by the PDPN gene.

[0015] Furthermore, the primers are:

[0016] PDPN-qPCR-F: 5'-AATGTCGGGAAGGTACTCGC-3';

[0017] PDPN-qPCR-R: 5'-AGGGCACAGAGTCAGAAACG-3'.

[0018] The application of the PDPN of the present invention in the preparation of medicaments for treating gemcitabine-resistant pancreatic cancer or increasing the sensitivity of gemcitabine treatment for pancreatic cancer.

[0019] Furthermore, the drug is an inhibitor of PDPN.

[0020] Furthermore, the inhibitors include agents that inhibit PDPN siRNA or locked nucleotide LNA-PDPN.

[0021] Furthermore, the inhibitor is used to inhibit the proliferation of gemcitabine-resistant pancreatic cancer cells or to increase gemcitabine sensitivity.

[0022] This invention includes reagents for detecting gemcitabine-resistant pancreatic cancer products, or kits for treating gemcitabine-resistant pancreatic cancer or increasing the sensitivity of gemcitabine to pancreatic cancer treatment.

[0023] The application of PDPN in constructing a sitabine-resistant subcutaneous xenograft model of pancreatic cancer, wherein the model construction method is as follows:

[0024] The cells were obtained by injecting gemcitabine-resistant pancreatic cancer cells subcutaneously into nude mice; the cells were BxPC-3-Gem cells obtained by long-term treatment of the pancreatic cancer cell line BxPC-3 with gemcitabine.

[0025] This invention relates to the application of PDPN in constructing gemcitabine-resistant pancreatic cancer orthotopic xenograft models, wherein the models include cell models and animal models; the cells in the cell models are gemcitabine-resistant pancreatic cancer cells BxPC-3-Gem-Luc labeled with fluorescein; and the animal models are obtained by injecting gemcitabine-resistant pancreatic cancer cells into the pancreas of nude mice.

[0026] The beneficial effects of this invention include:

[0027] This invention has discovered a molecular marker associated with the development and progression of gemcitabine-resistant pancreatic cancer—the PDPN gene. Research has shown that altering PDPN expression levels can affect the chemosensitivity of gemcitabine-resistant pancreatic cancer cells, suggesting that PDPN could be applied to the treatment and detection of gemcitabine-resistant pancreatic cancer. This invention also provides methods for constructing subcutaneous xenograft models of gemcitabine-resistant pancreatic cancer using PDPN, as well as for constructing orthotopic xenograft models of gemcitabine-resistant pancreatic cancer. Attached Figure Description

[0028] Figure 1 To detect the differential expression of PDPN in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells; 1A shows the MTT assay used to detect the sensitivity of pancreatic cancer cells to gemcitabine; Figure 1 B is a graph showing the expression of PDPN in pancreatic cancer cells as detected by qRT-PCR;

[0029] Figure 2 The silencing effect of siRNA on PDPN at different transfection times was detected by qRT-PCR.

[0030] Figure 3 A graph showing the sensitivity of pancreatic cancer cells to gemcitabine after silencing PDPN in the MTT assay;

[0031] Figure 4 The graph shows the effect of silencing PDPN on the clonogenic ability of gemcitabine-treated pancreatic cancer cells; 4A shows the effect of silencing PDPN on the proliferation ability of pancreatic cancer cells; 4B shows the statistical effect of silencing PDPN on the proliferation ability of pancreatic cancer cells.

[0032] Figure 5 Figure 5A shows the effect of silencing PDPN on apoptosis of pancreatic cancer cells treated with gemcitabine, as observed under an optical microscope. Figure 5B shows the results of flow cytometry analysis of apoptosis in pancreatic cancer cells after silencing PDPN. Figure 5C is a statistical graph showing the results of apoptosis in pancreatic cancer cells after silencing PDPN.

[0033] Figure 6The figures show the expression of NF-κB in pancreatic cancer sensitive and drug-resistant cells after silencing PDPN; 6A shows the changes in NF-κB p65 subunit phosphorylation level in pancreatic cancer sensitive and drug-resistant cells detected by Western blot; 6B shows the effect of dual-luciferase reporter gene assay on NF-κB activity in pancreatic cancer sensitive and drug-resistant cells.

[0034] Figure 7 The figure shows the effect of targeted PDPN on gemcitabine chemotherapy resistance in pancreatic cancer in nude mice to verify subcutaneous tumor formation. Among them, 7A is a photograph of the tumor solid in nude mice treated with LNA-PDPN in combination with gemcitabine; 7B is a statistical graph of tumor volume in combination with LNA-PDPN and gemcitabine; and 7C is a statistical graph of tumor weight in combination with LNA-PDPN and gemcitabine.

[0035] Figure 8 Figure 8A shows the effect of targeted PDPN on gemcitabine resistance in nude mouse orthotopic pancreatic cancer xenografts. Figure 8B shows the difference between fluorescently labeled and unlabeled fluorescently labeled resistant pancreatic cancer cells. Figure 8C shows the fluorescence of the pancreas after abdominal dissection in nude mice, monitored by the IVIS small animal in vivo imaging system. Figure 8D shows the bioluminescence fluorescence intensity of nude mouse orthotopic pancreatic cancer xenografts treated with LNA-PDPN in combination with gemcitabine. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

[0037] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0038] Example 1: Expression level of PDPN in gemcitabine-resistant pancreatic cancer cells

[0039] 1. Detection of PDPN expression level in pancreatic cancer cell lines

[0040] Human pancreatic cancer gemcitabine-sensitive cell line BxPC-3 was cultured in 1640 medium containing 10% FBS and incubated at 37°C in a 5% CO2 incubator. By treating BxPC-3 cells with progressively increasing concentrations of gemcitabine, drug-resistant pancreatic cancer cell lines BxPC-3-Gem-1μM, BxPC-3-Gem-5μM, BxPC-3-Gem-10μM, and BxPC-3-Gem-20μM were obtained. These cells were seeded at 5000 cells per well in 96-well plates and treated with different doses of gemcitabine for 72 hours, followed by the addition of MTT (working concentration 0.5 mg / mL, Amresco). The resulting MTT product was dissolved in DMSO, and the optical density was measured at a wavelength of 490 nm using a microplate reader to calculate cell viability and plot survival curves. At the same time, total RNA was isolated from pancreatic cancer cells using Trizol (Invitrogen) and reverse transcribed into cDNA using ReverTra Ace (TOYOBO). qRT-PCR was performed using SYBR Premix ExTaq (TAKARA) and gene-specific primers. The reaction mixture consisted of 5 μL PCR enzyme Mix, 2 μL cDNA, 0.2 μL Primer F, 0.2 μL Primer R, and 2.6 μL ddH2O. The amplification program was as follows: first, pre-denaturation at 95℃ for 2 min, one cycle; then, the PCR reaction was performed at 95℃ for 5 s, followed by at 60℃ for 35 s, for a total of 45 cycles; next, the melting curve analysis was performed at 95℃ for 15 s, at 60℃ for 1 min, and at 95℃ for 1 s, one cycle; finally, the cooling phase was performed at 50℃ for 30 s, one cycle. Primer sequences are shown in Table 1. 2 -ΔΔCt The method calculates the relative expression level of the target gene.

[0041] Table 1 Primer sequences

[0042] PDPN-qPCR-F 5'-AATGTCGGGAAGGTACTCGC-3' PDPN-qPCR-R 5'-AGGGCACAGAGTCAGAAACG-3'

[0043] 2. Experimental Results

[0044] Compared to pancreatic cancer-sensitive cells BxPC-3, BxPC-3-Gem-1μM, BxPC-3-Gem-5μM, BxPC-3-Gem-10μM, and BxPC-3-Gem-20μM showed significantly enhanced resistance to gemcitabine with progressively increasing concentrations (e.g., ...). Figure 1 A) PDPN is highly expressed in gemcitabine-resistant pancreatic cancer cells, especially in the most resistant BxPC-3-Gem-20μM cells (e.g., ...). Figure 1B), which shows that PDPN can be used as a molecular marker for detecting gemcitabine resistance in pancreatic cancer.

[0045] Example 2: Effect of PDPN on gemcitabine-resistant pancreatic cancer cells

[0046] 1. To investigate the effect of PDPN on the proliferation ability of pancreatic cancer cells and their sensitivity to gemcitabine.

[0047] Gemcitabine-resistant pancreatic cancer cells (BxPC-3-Gem) were transfected with siRNA targeting PDPN. The siRNA sequence is shown in Table 2 and was synthesized by Ribobio Technology Co., Ltd. RNA was collected at 24h, 48h, and 72h, and the corresponding cDNA was obtained by reverse transcription. PDPN expression was detected by qRT-PCR. Simultaneously, si-PDPN was transiently transfected into BxPC-3-Gem pancreatic cancer cells to silence PDPN expression. Forty-eight hours after transfection, cells were seeded into 96-well plates at a density of 5000 cells per well. After overnight cell adhesion, cells were treated with different concentrations of gemcitabine for 72 hours. The absorbance of the cells was measured using a microplate reader at 490 nm, and cell viability was calculated. Simultaneously, si-PDPN was transiently transfected into BxPC-3-Gem pancreatic cancer cells. Cells were seeded at 500 cells / well in 6-well plates and pretreated with a low concentration of gemcitabine for 24 hours, then cultured in normal medium for approximately 14 days. After fixation and staining, the number of cell colonies was counted. Simultaneously, si-PDPN was transiently transfected into BxPC-3-Gem pancreatic cancer cells, and after gemcitabine treatment for 72 hours, cells were collected. Apoptosis of pancreatic cancer cells was detected by flow cytometry using Annexin V-PI double staining.

[0048] Table 2 siRNA sequences

[0049] si-PDPN 5'-UGUACACAUUCUGGUCUAGdTdT-3'(sense) si-NC 5'-UGGUUUACAUGUCGACUAAdTdT-3'(sense)

[0050] 2. Detection of the effect of PDPN on NF-κB expression in pancreatic cancer cells

[0051] Following protein mass spectrometry analysis of gemcitabine-resistant pancreatic cancer cells BxPC-3-Gem and their parental gemcitabine-sensitive pancreatic cancer cells BxPC-3, KEGG pathway analysis revealed enrichment of the NF-κB signaling pathway. Therefore, the effects of PDPN on the phosphorylation level of the NF-κB p65 subunit and its influence on NF-κB activity in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells were investigated.

[0052] Transiently transfecting drug-resistant pancreatic cancer cells BxPC-3-Gem with si-PDPN, and extracting proteins using cell lysis buffer. Western blot analysis was performed to detect the expression of PDPN, phos-p65 (Ser536), p65, and β-actin. Simultaneously, si-PDPN, NF-κB-Luc reporter plasmid, and Renilla luciferase reporter plasmid were co-transfected into drug-resistant pancreatic cancer cells BxPC-3-Gem. Fluorescence was detected using a dual-luciferase reporter gene assay kit, and the fluorescence values ​​of firefly luciferase and Renilla luciferase were recorded; the ratio was considered the relative luciferase fluorescence level.

[0053] 3. Experimental Results

[0054] In gemcitabine-resistant pancreatic cancer cells, si-PDPN effectively silenced PDPN expression at 24h, 48h, and 72h after transfection (e.g., Figure 2 Therefore, si-PDPN was used in subsequent experiments; the results showed that silencing PDPN expression enhanced the sensitivity of pancreatic cancer cells to gemcitabine chemotherapy, manifested as decreased cell viability (e.g., Figure 3 ), reduced clone-forming ability (e.g. Figure 4 Increased number of apoptotic cells (e.g.) Figure 5 Simultaneously, silencing PDPN leads to downregulation of p65 expression and decreased phosphorylation of the p65 Ser536 subunit, an indicator of NF-κB activation, in gemcitabine-resistant pancreatic cancer cells (e.g., ...). Figure 6 A) Decreased NF-κB activity (e.g.) Figure 6 B).

[0055] Example 3: In vivo experimental detection of the effect of PDPN on gemcitabine chemotherapy resistance in pancreatic cancer.

[0056] 1. Nude mouse subcutaneous xenograft tumor model

[0057] 1×10 5 One BxPC-3-Gem cell was subcutaneously injected into the right hind limb of 4-5 week old nude mice. The tumor volume was increased to 50 mm². 3 After calculating the volume using the formula: major axis × minor axis × minor axis / 2, mice were randomly divided into 4 groups of 3 mice each: (a) LNA-NC + saline, (b) LNA-PDPN + saline, (c) LNA-NC + gemcitabine, and (d) LNA-PDPN + gemcitabine. Gemcitabine (100 mg / kg body weight) was administered intraperitoneally once a week, and LNA (200 mg / kg) was administered via tail vein once a week. Four weeks after tumor implantation, mice were sacrificed, and the tumors were collected and photographed. LNA is a locked nucleotide with the same sequence as the siRNA sequence in Table 2, synthesized by Ribobio Technology Co., Ltd.

[0058] 2. Nude mouse orthotopic tumor model

[0059] Nude mice aged 4-5 weeks were anesthetized with isoflurane via inhalation, and a small incision (1.5 cm) was made in the left abdominal wall. The spleen was then removed along with the underlying pancreas. Approximately 5 × 10⁶ cells were harvested. 5 BxPC3-Gem-Luc cells stably expressing firefly luciferase were suspended in a PBS:Matrigel (2:3) mixture and slowly injected into the pancreatic tail. The wound was then sutured with nylon sutures. Fluorescence signals in mice were monitored using a small animal in vivo imaging system after intraperitoneal injection of the luciferase substrate D-Lucifer. Fourteen days after tumor cell injection, mice were randomly divided into four groups of three mice each: (a) LNA-NC + saline, (b) LNA-PDPN + saline, (c) LNA-NC + gemcitabine, and (d) LNA-PDPN + gemcitabine. Gemcitabine (100 mg / kg) was administered intraperitoneally once a week, and LNA (200 mg / kg) was administered via tail vein once a week. On day 56 after tumor cell injection, mice were sacrificed, and pancreatic tumors were harvested and photographed.

[0060] 3. Experimental Results

[0061] In a nude mouse subcutaneous xenograft model: there was no statistically significant difference in tumor volume and weight between the LNA-PDPN group and the LNA-NC group. Gemcitabine itself has a certain degree of anti-tumor effect, and its anti-tumor effect is more pronounced after being combined with LNA-PDPN. Results regarding the volume, size, and weight of solid tumors are as follows: Figure 7 As shown.

[0062] In a nude mouse orthotopic tumor model: fluorescently labeled gemcitabine-resistant pancreatic cancer cells BxPC3-Gem-Luc were injected into the pancreas of nude mice. Figure 8 As shown in Figure A, the fluorescence images of nude mice in different body positions and the disappearance of bioluminescence after pancreas removal indicate that the model was successfully established (e.g., Figure 8 B, C). Experimental results show that gemcitabine combined with LNA-PDPN can significantly reduce the fluorescence intensity of tumors and the size of solid tumors (e.g., Figure 8 D).

[0063] The specific embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific embodiments described above. For those skilled in the art, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. The application of a PDPN inhibitor in combination with gemcitabine in the preparation of a drug for treating gemcitabine-resistant pancreatic cancer, or the application of a PDPN inhibitor in the preparation of a drug that increases the sensitivity of pancreatic cancer to gemcitabine treatment; wherein the PDPN inhibitor is an siRNA that inhibits PDPN, and the siRNA's positive strand sequence is 5'-UGUACACAUUCUGGUCUAGdTdT-3'.

2. A kit for treating gemcitabine-resistant pancreatic cancer or increasing the sensitivity of pancreatic cancer to gemcitabine treatment, said kit comprising an siRNA inhibitor that inhibits PDPN, said siRNA having a positive strand sequence of 5'-UGUACACAUUCUGGUCUAGdTdT-3'.

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