Department of Internal Medicine and Liver Research Institute, Seoul National University Hospital, College of Medicine, Seoul National University, Seoul 03080, Republic of Korea.
Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea.
Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, Republic of Korea.
Department of Biomedical Sciences, Korean Cell Line Bank, Laboratory of Cell Biology and Cancer Research Institute, College of Medicine, Seoul National University, Seoul 03080, Republic of Korea.
Dxome Co. Ltd., Seongnam 13558, Republic of Korea.
Abstract
Although gemcitabine-based regimens are widely used as an effective treatment for pancreatic cancer, acquired resistance to gemcitabine has become an increasingly common problem. Therefore, a novel therapeutic strategy to treat gemcitabine-resistant pancreatic cancer is urgently required. Piceamycin has been reported to exhibit antiproliferative activity against various cancer cells; however, its underlying molecular mechanism for anticancer activity in pancreatic cancer cells remains unexplored. Therefore, the present study evaluated the antiproliferation activity of piceamycin in a gemcitabine-resistant pancreatic cancer cell line and patient-derived pancreatic cancer organoids. Piceamycin effectively inhibited the proliferation and suppressed the expression of alpha-actinin-4, a gene that plays a pivotal role in tumorigenesis and metastasis of various cancers, in gemcitabine-resistant cells. Long-term exposure to piceamycin induced cell cycle arrest at the G0/G1 phase and caused apoptosis. Piceamycin also inhibited the invasion and migration of gemcitabine-resistant cells by modulating focal adhesion and epithelial-mesenchymal transition biomarkers. Moreover, the combination of piceamycin and gemcitabine exhibited a synergistic antiproliferative activity in gemcitabine-resistant cells. Piceamycin also effectively inhibited patient-derived pancreatic cancer organoid growth and induced apoptosis in the organoids. Taken together, these findings demonstrate that piceamycin may be an effective agent for overcoming gemcitabine resistance in pancreatic cancer.
Keywords
Alpha-actinin-4 (ACTN4)ApoptosisPancreatic cancerPatient-derived pancreatic cancer organoids (PDPCOs)Piceamycin
Figures
Fig. 1The chemical structure of piceamycin.Fig. 2(A, B) The Kaplan-Meier survival curve represents the overall survival (OS) (the patient’s number; low=43, high=134) and relapse-free survival (RFS) (the patient’s number; low=21, high=48) of patient with pancreatic cancer according to the ACTN4 expression level. (C) Effect of piceamycin on ACTN4 mRNA expression. RNA-seq assay was performed in gemcitabine-resistant AsPC-1 cells treated with piceamycin (1.25 μM) compared with control (PBS with DMSO-treated). Heatmap shows ACTN4 mRNA expression according to z-score. The color represents z-score (yellow; positive score; upregulation, blue; negative score; downregulation). / Normalized value of ACTN4 mRNA expression. Data are presented as mean ± SD (n=3). (D) Effect of piceamycin on ACTN4 protein expression in gemcitabine-resistant AsPC-1 cells. Gemcitabine-resistant AsPC-1 cells were treated with the indicated concentrations of piceamycin for 48 h, and the protein expression level of ACTN4 was determined by western blotting. α-Tubulin was used as an internal control. (E) Protein-Protein Interaction (PPI) for ACTN4 from the search tool (STRING) database. Interacting proteins for ACTNB (Actin beta) Gene: ACTB (Actin beta)–ACTG1 (Actin gamma1), Interacting proteins for ACTN4 (Actinin alpha4) Gene: ACTN4–VCN (Vinculin) (F) Effect of piceamycin on mRNA expression of actin polymerization related genes. The heatmap was visualized from the RNA-seq data (according to z-score of actin polymerization related genes) and displayed on the column (according to z-score; –2≤z≤2, AVE; average of z-score, STD; standard deviation of z-score, n=3). / Normalized value (mRNA level) of ACTB and ACTG1 from RNA-seq analysis treated with piceamycin (1.25 μM) versus control (PBS with DMSO-treated) in gemcitabine-resistant AsPC-1 cells. Data are presented as mean ± SD (n=3) (G) Modified KEGG pathway of the genes related to actin polymerization signaling pathway. Modified KEGG pathway from Kyoto Encyclopedia of Genes and Genomes (KEGG) treated with piceamycin (1.25 μM) for 48 h compared with control (PBS with DMSO-treated) in gemcitabine-resistant AsPC-1 cells. The box colors of modified KEGG pathway represent fold change. (H) Effect of piceamycin on the proteins related to actin polymerization (VCN and ACTB). The protein expression related to actin polymerization biomarkers were analyzed by western blotting. Piceamycin was treated on the gemcitabine-resistant AsPC-1 cells with indicated concentrations. α-Tubulin was used as an internal control.Fig. 3(A) Heatmap of mRNA expression related to the focal adhesion signaling pathway in gemcitabine-resistant AsPC-1 cells. Gemcitabine-resistant AsPC-1 cells were treated with piceamycin (PCM) (1.25 μM) versus control (PBS with DMSO-treated) for 48 h. The color represents mRNA level using z-score (yellow; positive score; upregulation, blue; negative score; downregulation) and displayed on the column (according to z-score; –2≤z≤2, AVE; average of z-score, STD; standard deviation of z-score, n=3). Normalized value (mRNA level) of Rac2, Rac3, and VAV3. (B) Modified KEGG pathway from Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of the genes related to focal adhesion signaling pathway from RNA-sequencing data. Gemcitabine-resistant AsPC-1 cells were treated with piceamycin (1.25 μM) for 48 h compared with control (PBS with DMSO-treated). The box colors of the Modified KEGG pathway indicate fold change of mRNA expression. (C) The protein expressions of focal adhesion-related biomarkers (FAK and Rac) were analyzed by western blotting in gemcitabine-resistant AsPC-1 cells. Piceamycin (PCM) was treated in the gemcitabine-resistant AsPC-1 cells with indicated concentrations. α-Tubulin was used as an internal control. (D) The protein expression levels of epithelial-mesenchymal transition (EMT)-related biomarkers (N-cadherin, Slug, and Snail) were analyzed by western blotting in gemcitabine-resistant AsPC-1 cells. Piceamycin was treated on the gemcitabine-resistant AsPC-1 cells with indicated concentrations. α-Tubulin was used as an internal control. (E) Effect of piceamycin on gemcitabine-resistant AsPC-1 cells migration. Monolayers of gemcitabine-resistant AsPC-1 cells were scratched mechanically and treated with piceamycin (indicated concentrations) for 48 h. Representative images of wound closure obtained under a light microscope. All data are presented as mean ± SD (n=3, *p<0.05, **p<0.01). (F) Effect of piceamycin on gemcitabine-resistant AsPC-1 cells invasion. The cells (treated with piceamycin) (at 0, 1, 2, and 5 μM concentration) that invaded to lower chambers were fixed, stained, imaged, and counted. All data are presented as mean ± SD (n=3, *p<0.05) (Scale bars, 50 μm).Fig. 4Piceamycin (PCM) induced G0/G1 cell cycle arrest in gemcitabine-resistant AsPC-1 cells. (A, B) Gemcitabine-resistant AsPC-1 cells were treated with piceamycin (at 0, 1, 2, and 5 μM concentration) for 24 h, stained with PI, and cell cycle arrest was detected by flow cytometry. All data are presented as mean ± SD (n=3, *p<0.05, **p<0.01, ***p<0.001). (C) Heatmap of the genes related to the cell cycle signaling pathway. Z-score comes from RNA-sequencing data treated with piceamycin (1.25 μM) for 48 h in gemcitabine-resistant AsPC-1 cells. The color represents z-score (yellow; positive score; upregulation, blue; negative score; downregulation). The column indicates z-score (–2≤z≤2) related with cell cycle signaling pathway. Normalized value (mRNA level) of CDK2 and Cyclin D1. All data are presented as mean ± SD (n=3). (D) The effect of piceamycin on the G0/G1 phase cell cycle arrest was confirmed by observing the cell cycle regulatory proteins by western blotting. Piceamycin was treated with indicated concentrations in the gemcitabine-resistant AsPC-1 cells. α-Tubulin was used as an internal control.Fig. 5Induction of apoptosis by piceamycin (PCM) in gemcitabine-resistant AsPC-1 cells. Gemcitabine-resistant AsPC-1 cells were treated with piceamycin at different concentrations (0, 0.1, 1, 2, 5, and 10 μM) for 48 h. (A) Cell apoptosis was measured by flow cytometry using Annexin V-FITC/PI staining. Representative images of Annexin V-FITC/PI staining. (B) Statistical analysis of the cell apoptosis rate at 48 h. All data are presented as mean ± SD (n=3, *p<0.05, **p<0.01). (C) Effect of piceamycin on caspase activity in gemcitabine-resistant AsPC-1 cells. Gemcitabine-resistant AsPC-1 cells (80% confluent) were treated with piceamycin (10 μM) for 48 h and treated with caspase 3/7 reagents. The enzymatic activity of piceamycin-treated AsPC-1 cells was evaluated. All data are presented as mean ± SD (n=3, **p<0.01). (D) The effect of piceamycin on the expression of apoptotic biomarkers in gemcitabine-resistant AsPC-1 cells. The cells were treated with piceamycin versus control (PBS with DMSO-treated) for 48 h and the expression levels of the mitochondrial apoptosis regulatory proteins were detected by western blotting. α-Tubulin was used as an internal control. (E) The effect of piceamycin on colony formation in gemcitabine-resistant AsPC-1 cells. Gemcitabine-resistant AsPC-1 cells were treated with various concentrations of piceamycin (0, 1, 5, and 10 μM) for 48 h. When colony formation was visible, the medium was removed, stained, and imaged. All data are presented as mean ± SD (n=3, *p<0.05) (Scale bars, 50 μm).Fig. 6Antiproliferative activity of piceamycin in PDPCOs.(A) Drug response (AUC) heatmap based on gemcitabine treatment. PDPCOs (SNU-4206, SNU-4305-TO, SNU-4425-TO, and SNU-4340-TO) were treated with gemcitabine (0.01 μM-100 μM) for 72 h and the drug sensitivity was measured using cell-titer glow, 3D reagent. Drug response heatmap (red; resistant, blue; sensitive). (B) PDPCOs (SNU-4206, SNU-4305-TO, SNU-4425-TO, and SNU-4340-TO) were treated with various concentrations of piceamycin (PCM) and detected by 3D cell titer-glow assay after 72 h. All data are presented as mean ± SD (n=3, *p<0.05, **p<0.01). (C) The level of cell apoptosis was determined by flow cytometry using Annexin V-FITC/ propidium iodide (PI) staining in PDPCO (SNU-4340-TO).
그림 출처: PubMed Central 공개 논문 (PMC10762279)
Conflict of interest statement
CONFLICT OF INTEREST . The authors declare there is no conflict of interest.
