Various Mechanisms Involve the Nuclear Factor (Erythroid-Derived 2)-Like (NRF2) to Achieve Cytoprotection in Long-Term Cisplatin-Treated Urothelial Carcinoma Cell Lines

Abstract

Therapeutic efficacy of cisplatin-based chemotherapy for advanced-stage urothelial carcinoma (UC) is limited by drug resistance. The nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway is a major regulator of cytoprotective responses. We investigated its involvement in cisplatin resistance in long-term cisplatin treated UC cell lines (LTTs). Expression of NRF2 pathway components and targets was evaluated by qRT-PCR and western blotting in LTT sublines from four different parental cells. NRF2 transcriptional activity was determined by reporter assays and total glutathione (GSH) was quantified enzymatically. Effects of siRNA-mediated NRF2 knockdown on chemosensitivity were analysed by viability assays, γH2AX immunofluorescence, and flow cytometry. Increased expression of NRF2, its positive regulator p62/SQSTM1, and elevated NRF2 activity was observed in 3/4 LTTs, which correlated with KEAP1 expression. Expression of cytoprotective enzymes and GSH concentration were upregulated in some LTTs. NRF2 knockdown resulted in downregulation of cytoprotective enzymes and resensitised 3/4 LTTs towards cisplatin as demonstrated by reduced IC50 values, increased γH2AX foci formation, and elevated number of apoptotic cells. In conclusion, while LTT lines displayed diversity in NRF2 activation, NRF2 signalling contributed to cisplatin resistance in LTT lines, albeit in diverse ways. Accordingly, inhibition of NRF2 can be used to resensitise UC cells to cisplatin, but responses in patients may likewise be variable.

Multifaceted Mechanisms of Cisplatin Resistance in Long-Term Treated Urothelial Carcinoma Cell Lines

“…Four cisplatin-resistant LTT sublines were generated from the UCCs RT-112, J82, 253J, and T-24 [ 16 , 33 ]. Resistance was quantified by determination of IC 50 values following 72 h cisplatin treatment, which were between 10-fold and 21-fold increased compared to their parental controls ( Figure 1 a).…”

“…Of note, we have previously reported that several other factors involved in cisplatin and glutathione metabolism, which are NRF2 targets, are also upregulated to different extents in the LTT lines, most prominently in RT-112-LTT and T24-LTT [ 16 ]. These data indicate that a number of different pre-target factors are implicated to various extents in cisplatin resistance in different sublines.…”

“…Overexpression of metallothioneins has been implicated in cisplatin resistance in several cancer types [ 12 , 13 ], including bladder cancer [ 14 , 15 ]. In addition, we observed increased GSH levels in most long-term cisplatin treated (LTTs) UC cell lines (UCCs) in our previous study [ 16 ]. Cisplatin–GSH conjugates are then exported by MRP2 (also known as cMOAT and ABCC2) [ 17 ].…”

“…Thus, our data support the idea that cisplatin resistance in UC rather originates from acquired resistance. We moreover identified NRF2 as one mediator of cisplatin resistance in UCCs, activated to different extents and by different mechanisms between the cell lines [ 16 ].…”

Stimulated upregulation of HO‑1 is associated with inadequate response of gastric and ovarian cancer cell lines to hyperthermia and cisplatin treatment

“…The response and resistance of cancer cells to chemotherapy and hyperthermia depend on the induction and expression of a number of cytoprotective proteins, including Hsp70 and Hsp27 (17)(18)(19). Therefore, the modulation of cytoprotective proteins may serve a crucial role in cancer treatment.…”