Berberine Inhibits Fluphenazine-Induced Up-Regulation of <i>CDR1</i> in <i>Candida albicans</i>

Abstract

Over-expression of the Candida drug resistance gene CDR1 is a common mechanism generating azoleresistant Candida albicans in clinical isolates. CDR1 is transcriptionally activated through the binding of the transcription factor Tac1p to the cis-acting drug-responsive element (DRE) in its promoter. We previously demonstrated that the combination of fluconazole (FLC) and berberine (BBR) produced significant synergy when used against FLC-resistant C. albicans in vitro. In this study, we found that BBR inhibited both the up-regulation of CDR1 mRNA and the transport function of Cdr1p induced by fluphenazine (FNZ). Further, electrophoretic mobility shift assays suggested that the transcription activation complex of protein-DRE was disrupted by BBR, and electrospray ionization mass spectrometry analysis showed that BBR bound to the DRE of CDR1. Thus we propose that BBR inhibits the FNZ-induced transcriptional activation of CDR1 in C. albicans by blocking transcription factor binding to the DRE of CDR1. These results contribute to our understanding of the mechanism of synergistic effect of BBR and FLC. Key words Candida albicans; berberine; CDR1; drug-responsive element; fluphenazineCandida albicans is one of the major opportunistic fungal pathogens in humans, and causes candidiasis ranging from superficial mucosal infections to life-threatening systemic disorders.1-3) Triazole antifungal agents, such as fluconazole (FLC) and itraconazole, are considered to be the first-choice agents for treatment and prophylaxis because of their relatively low side effects and high effectiveness on mucosal infections. However, with prolonged exposure to azoles, drug resistance becomes a serious problem in clinical practice. 4,5) Several genes have been confirmed to contribute to the development of multidrug resistance including ERG11, CDR1, CDR2, MDR1, RTA2, Among them, upregulation of CDR1, a member of the ATP-binding cassette (ABC) transporter super-family in C. albicans, is a common occurrence in clinical C. albicans isolates resistant to antifungal azoles.13) Cdr1p pumps azoles out of C. albicans cells to reduce intracellular accumulation, which can be indirectly identified by the efflux of rhodamine 6G (R6G), which is a fluorescent substrate transported by Cdr1p. 14) In the promoter of CDR1, the basal expression element (BEE) (−960-−710) is responsible for basal transcription of CDR1, and the drugresponsive element (DRE) (−264-−244) is responsible for drug-induced transcription.15) The DRE (5′-CGG ATA TCG GAT ATT TTT TTT-3′) contains CGG triplets that are often recognized by Zinc finger proteins. 16,17) It has been demonstrated that the DRE of CDR1 can be recognized and bound by the transcription controller Tac1p that is characterized by a highly conserved Zn(II) 2 Cys 6 motif within the DNA binding domain (DBD). 15,16) Small molecules which can regulate the expression of CDR1 become a promising strategy to combat azole resistance in C. albicans. Berberine (BBR) is a plant-derived isoquinoline alkaloid from the roots and bark o...

Synergistic Effect of Berberine Hydrochloride and Fluconazole Against Candida albicans Resistant Isolates

“…Previous studies reported that Eucalyptal D (Xu et al, 2019) geraniol (Singh and Sharma, 2018) and magnolol (Sun L. M. et al, 2015) which are substrates for Cdr1p efflux pumps, exert synergistic effects by simultaneously upregulating CDR1 and CDR2 expression, while competitively inhibiting FLC efflux. Numerous studies suggest that synergy results from increased intracellular drug accumulation caused by downregulated efflux pump genes in FLC-resistant strains (Garcia-Gomes et al, 2012;Zhu et al, 2014;Shao et al, 2016). Although Rh6G efflux gradually increased in all groups, much lower Rh6G efflux and CDR1 expression were detected in the BBH + FLC group, confirming previous results.…”

“…Previous studies revealed that ergosterol synthesis inhibition and apoptosis induced by endogenous ROS augmentation contribute to the synergistic effect of berberine plus FLC against C. albicans ( Xu et al, 2009 ; Xu et al, 2017 ; Yang et al, 2018 ). Furthermore, this combination could also downregulate efflux pump genes CDR1 and CDR2 overexpression ( Zhu et al, 2014 ).…”

Antifungal Compounds againstCandidaInfections from Traditional Chinese Medicine

“…As an important constituent of many herbs of Berberidaceae family such as Berberis vulgaris , berberine exerts its antifungal action by induction of mitochondrial dysfunction and increased ROS generation, and its effects are in synergy with fluconazole, even in fluconazole-resistant clinical isolates [ 54 – 56 ]. Moreover, berberine treatment could also culminate in disruption of cell wall integrity in C. albicans [ 57 ] and inhibit the overexpression of drug resistance gene CDR1 induced by fluphenazine [ 58 ]. Although berberine could induce apoptosis in many human cells such as HL-60 leukemia cells and thyroid carcinoma cells [ 59 , 60 ], berberine could recover the mitochondrial function induced by high-fat feeding in a rat model and could decrease the triglyceride accumulation in the liver in mice [ 61 , 62 ].…”

Antioxidant Effect of Berberine and its Phenolic Derivatives Against Human Fibrosarcoma Cells

“…Berberine, an isoquinoline alkaloid, has been used for traditional medical systems in eastern countries. It possesses several biological activities including anti-inflammatory (Chu et al, 2014), anticancer (Yip and Ho, 2013;Cai et al, 2014), anti-diabetic (Rajendran et al, 2014), antibacterial (Wojtyczka et al, 2014), and antifungal functions (Zhu et al, 2014). The antioxidant capacity of berberine has demonstrated both direct (Jang et al, 2009;Bashir and Gilani, 2011) and indirect activities (Hwang et al, 2002;Tan et al, 2007).…”