Gemcitabine-resistant Pancreatic Cancer Cell Line Sw1990/gz Enriches Pancreatic Cancer Stem Cells

Objective Establish a gemcitabine-resistant pancreatic cancer cell line SW1990/GZ, to explore the relationship between drug-resistant cell line SW1990/GZ and pancreatic cancer stem cell. Methods Gemcitabine-resistant pancreatic cancer cell line SW1990/GZ was obtained by treating parental cell line SW1990 in vitro with increasing dosage of gemcitabine in culture medium intermittently for 24 weeks. Stable cultures were obtained which IC50 was 232.2μM. Use an invert microscope to observe the cells'morphological changes. The cellular proliferation capacity of the two cell lines were detected by MTT assay and the resistant index was calculated. Doubling time was calculated and compared based on the growth curve of these two cell lines and cell cycles were assayed with flowcytometry method. Total RNA was isolated from cell lines SW1990 and SW1990/GZ separately. Real-time RT-PCR was done to detect the gene expression of human ABCB1/MDR1, ABCC1/MRP and ABCG2/BCRP,β-actin was used as a control. Western blot was performed to detect the expression of protein ABCB1, ABCC1 and ABCG2,β-actin was used as an internal control. Tumorigenic potential was performed by nude mice xenograft transplant experiments. Side population analysis and CD₂₄CD₄₄ positive cells explore were determined by flowcytometry to examine cancer stem cell proportion. Results After adding gemcitabine, the cells showed morphological changes, cells became larger, extended foot processes, similar to the nerve cell-like change. When the cells adapted the dose of gemcitabine, they turned shrinkage, and foot processes disappeared. The resistant index was 77.2. Compared with the parental cells, SW1990/GZ cells exhibited a smaller growth rate, delayed cell-doubling time. More cells were in G0/G1 cycle phase of SW1990/GZ than those of SW1990(P <0.01), and less cells in cycle phase S(P <0.01). SW1990/GZ had a high gene expression level of ABCB1/MDR1, ABCC1/MRP and ABCG2/BCRP (P <0.01) and expressed high level of protein ABCB1, ABCC1 and ABCG2 (P <0.01). Nude mice xenograft transplant experiments showed that only 1×10⁵ SW1990/GZ cells were sufficient for tumor formation, whereas an injection of 1×10⁵ SW1990 cells did not initiate tumors in 16 weeks. Flow cytometry analysis showed that SP proportion in SW1990/GZ was (11.03±0.98)% whereas in parental SW1990 it was (4.56±0.87)% , CD₄₄CD₂₄ positive cells was(8.73±0.81)% in SW1990/GZ whereas (1.13±0.35)% in SW1990. The CD₄₄⁺ subfraction was dramatically increased in SW1990/GZ cells compared with its parental cells (29.45±1.99% vs.93.16±2.46%). Conclusion Treating parental cell line SW1990 in vitro with increasing dosage of gemcitabine in culture medium intermittently could enhance acquired drug resistance of pancreatic cancer cell line SW1990. Gemcitabine-resistant cell line SW1990/GZ has a higher proportion of pancreatic cancer stem cells compared to its parental cell line SW1990. CD₄₄ is mainly responsible for acquired drug resistance, which can be a potential target to overcome acquired drug resistance in pancreatic cancer.