Involvement of the Pleiotropic Drug Resistance Response, Protein Kinase C Signaling, and Altered Zinc Homeostasis in Resistance of Saccharomyces cerevisiae to Diclofenac

Abstract

Diclofenac is a widely used analgesic drug that can cause serious adverse drug reactions. We used Saccharomyces cerevisiae as a model eukaryote with which to elucidate the molecular mechanisms of diclofenac toxicity and resistance. Although most yeast cells died during the initial diclofenac treatment, some survived and started growing again. Microarray analysis of the adapted cells identified three major processes involved in diclofenac detoxification and tolerance. In particular, pleiotropic drug resistance (PDR) genes and genes under the control of Rlm1p, a transcription factor in the protein kinase C (PKC) pathway, were upregulated in diclofenac-adapted cells. We tested if these processes or pathways were directly involved in diclofenac toxicity or resistance. Of the pleiotropic drug resistance gene products, the multidrug transporter Pdr5p was crucially important for diclofenac tolerance. Furthermore, deletion of components of the cell wall stress-responsive PKC pathway increased diclofenac toxicity, whereas incubation of cells with the cell wall stressor calcofluor white before the addition of diclofenac decreased its toxicity. Also, diclofenac induced flocculation, which might trigger the cell wall alterations. Genes involved in ribosome biogenesis and rRNA processing were downregulated, as were zinc-responsive genes. Paradoxically, deletion of the zinc-responsive transcription factor Zap1p or addition of the zinc chelator 1,10-phenanthroline significantly increased diclofenac toxicity, establishing a regulatory role for zinc in diclofenac resistance. In conclusion, we have identified three new pathways involved in diclofenac tolerance in yeast, namely, Pdr5p as the main contributor to the PDR response, cell wall signaling via the PKC pathway, and zinc homeostasis, regulated by Zap1p.

N-acetylcysteine potentiates diclofenac toxicity inSaccharomyces cerevisiae: stronger potentiation in ABC transporter mutant strains

“…This latter dose caused 22.6% and 55.8% inhibition at 3h and 6h treatment, respectively ( Figure 1a). The inhibitory effect of DCF was partially abolished after 24 hourtreatment reflecting a previously described process of adaption of yeast cells to this drug (figure 1b (van Leeuwen et al, 2011b).…”

“…Moreover, increased DCF toxicity occurs after deletion of genes of components of the cell wall stress-responsive PKC pathway or the Zinc (Zn)responsive transcription factor Zap1p (Zn-responsive activator protein). In addition, Zn-chelator increases DCF toxicity (van Leeuwen et al, 2011b).…”

Optical and impedimetric study of genetically modified cells for diclofenac sensing

“…However, in the whole-cell sensor device, S. cerevisiae BY4741 p426PDR5-tGFP cells were used because of the faster folding of tGFP compared to eGFP (Evdokimov et al, 2006). Cells transformed with the multicopy plasmid p426PDR5-tGFP, applying the 5' untranslated region (UTR) of the PDR5 gene, show a diclofenac-dependent expression of the turbo green fluorescent protein (tGFP) (Schuller et al, 2017;Van Leeuwen et al, 2011b).…”

“…In yeast, diclofenac targets are the mitochondrial respiratory chain subunits Rip1p of complex III and Cox9p of complex IV (Van Leeuwen et al, 2011a). Involved in diclofenac resistance in S. cerevisiae are cell wall signaling via the protein kinase C (PKC) pathway, altered zinc homeostasis, and the induction of PDR (Van Leeuwen et al, 2011b). Diclofenac induces transcription of the PDR5 gene encoding a plasma membrane ATP-binding cassette (ABC) transporter that acts as the main contributor in this PDR response (Van Leeuwen et al, 2011b).…”

“…Involved in diclofenac resistance in S. cerevisiae are cell wall signaling via the protein kinase C (PKC) pathway, altered zinc homeostasis, and the induction of PDR (Van Leeuwen et al, 2011b). Diclofenac induces transcription of the PDR5 gene encoding a plasma membrane ATP-binding cassette (ABC) transporter that acts as the main contributor in this PDR response (Van Leeuwen et al, 2011b). Hence, the diclofenac-responsive PDR5 promoter is an appropriate candidate for a promoter engineering approach (Schuller et al, 2017).…”