Establishment and characterization of the gemcitabine-resistant human gallbladder cancer cell line NOZ GemR

Abstract

Background: Patients with gallbladder cancer (GBC) generally receive gemcitabine as the standard treatment; however, its efficacy is often limited owing to the development of resistance.

Methods: To identify the mechanisms underlying gemcitabine resistance in GBC, a gemcitabine-resistant GBC cell line (NOZ GemR) was established by exposing the parental NOZ cell line to increasing concentrations of gemcitabine. Morphological changes, growth rates, and migratory and invasive capabilities were evaluated. Protein expression was detected using western blotting.

Results: The results demonstrated that the IC₅₀ of NOZ and NOZ GemR was 0.011 and 4.464 μM, respectively, and that the resistance index ratio was 405.8. In comparison, NOZ GemR cells grew slower and had significantly lower migration and invasion abilities than NOZ cells. There were altered levels of epithelial-mesenchymal transformation markers in NOZ GemR cells, as well as increased levels of the Akt/mTOR pathway protein.

Conclusion: The NOZ GemR cell line could be used as an effective in vitro model to improve our understanding of gemcitabine resistance in GBC.

Keywords: chemotherapy; drug resistance; gallbladder cancer; gemcitabine.

Figure 1

Characteristics of gemcitabine-resistant gallbladder cancer cells. A. Dose-response curves for NOZ and NOZ GemR cells following 72 h of treatment with the indicated doses of gemcitabine (***P<0.001). B. Bright-field images showing the morphological characteristics of NOZ and NOZ GemR cells (100× magnification left and 400× magnification right), and fusiform-shaped cells are indicated by black arrowheads.

Figure 2

Inhibition of proliferation, colony formation of NOZ GemR cells. A. CCK-8 assays were used to detect cell growth upon NOZ and NOZ GemR cells (**P<0.01). B. Colony formation assay was performed to examine the colony formation ability of gemcitabine-resistant gallbladder cancer cells (**P<0.01).

Figure 3

NOZ GemR cells exhibit changes in EMT marker expression and the Akt/mTOR signaling pathway. A. Transwell assay was performed to detect the migration and invasion of gemcitabine-resistant GBC cells (100× magnification) (**P<0.01). B. The expression of β-Actin, Akt, p-Akt (Ser473), p-p70 S6K (Thr389), p-mTOR (Ser2448), vimentin, Slug and Snail in NOZ and NOZ GemR cells were examined by western blotting, each group was replicated three times (*P<0.05, **P<0.01).