Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer

Abstract

We report herein the development, functional and molecular characterization of an isogenic, paired bladder cancer cell culture model system for studying platinum drug resistance. The 5637 human bladder cancer cell line was cultured over ten months with stepwise increases in oxaliplatin concentration to generate a drug resistant 5637R sub cell line. The MTT assay was used to measure the cytotoxicity of several bladder cancer drugs. Liquid scintillation counting allowed quantification of cellular drug uptake and efflux of radiolabeled oxaliplatin and carboplatin. The impact of intracellular drug inactivation was assessed by chemical modulation of glutathione levels. Oxaliplatin- and carboplatin-DNA adduct formation and repair was measured using accelerator mass spectrometry. Resistance factors including apoptosis, growth factor signaling and others were assessed with RNAseq of both cell lines and included confirmation of selected transcripts by RT-PCR. Oxaliplatin, carboplatin, cisplatin and gemcitabine were significantly less cytotoxic to 5637R cells compared to the 5637 cells. In contrast, doxorubicin, methotrexate and vinblastine had no cell line dependent difference in cytotoxicity. Upon exposure to therapeutically relevant doses of oxaliplatin, 5637R cells had lower drug-DNA adduct levels than 5637 cells. This difference was partially accounted for by pre-DNA damage mechanisms such as drug uptake and intracellular inactivation by glutathione, as well as faster oxaliplatin-DNA adduct repair. In contrast, both cell lines had no significant differences in carboplatin cell uptake, efflux and drug-DNA adduct formation and repair, suggesting distinct resistance mechanisms for these two closely related drugs. The functional studies were augmented by RNAseq analysis, which demonstrated a significant change in expression of 83 transcripts, including 50 known genes and 22 novel transcripts. Most of the transcripts were not previously associated with bladder cancer chemoresistance. This model system and the associated phenotypic and genotypic data has the potential to identify some novel details of resistance mechanisms of clinical importance to bladder cancer.

Fig 1. DNA damage as the critical step in Pt-induced cell death.

(A) The major pathways of platinum (Pt) drug-induced cell death. After administration, cellular uptake and efflux determines the intracellular accumulation of Pt agents, which can be inactivated by the intracellular thiol-containing molecules. Eventually, Pt agents induce DNA damage, including drug-DNA adducts, which triggers cell cycle arrest and DNA repair. DNA adduct formation and repair determines the fate of cells, although other factors also play important roles, such as pro- and anti-apoptotic proteins. (B) Diagram showing the formation of carboplatin- and oxaliplatin-DNA adducts and the positions of the radiocarbon labels on each drug used for this study in order to enable quantification of drug-DNA adduct formation and repair by accelerator mass spectrometry.

Fig 2. Drug uptake and efflux.

(A-B) Comparison of cell uptake and efflux. A. cell uptake of oxaliplatin. 5637R cells had decreased cell uptake. B: 5637 and 5637R had similar cell efflux rates. (C-D) Oxaliplatin and carboplatin cellular efflux differences between the two cell lines were not statistically significant.

Fig 3. Drug inactivation by cellular glutathione.

(A) Comparison of carboplatin-DNA adduct formation between 5637 and 5637R. (B) Comparison and correlation of IC₅₀ values with carboplatin-adduct AUC, adduct levels four hours after dosing and DNA repair. (C) Comparison of cell uptake and efflux of carboplatin between 5637 and 5637R cells.

Fig 4. Oxaliplatin- and carboplatin-DNA adduct formation and repair.

Comparison of oxaliplatin- and carboplatin-DNA adduct formation between 5637 and 5637R cells. The chemoresistant 5637R cells had higher oxaliplatin-DNA adduct levels at all time points compared to more treatment sensitive 5637 cells.

Fig 5. RNAseq and qRT-PCR show similar trends in gene expression levels for selected resistance-realated genes.

Four genes (TSPAN7, AKR1C2, AKR1C1, and CYR61) have increased levels in 5637R cells and two genes (HTRA1 and AQP3) have decreased levels in the resistant cells. (A) Fold changes in chemoresistance gene levels relative to the 5637 parental cells as determined by RNA-seq. (B) Fold change in chemoresistance gene transcript levels relative to the 5637 parental cells as determined by qRT-PCR.