(T1530-05-25) Chloroquine, Hydroxychloroquine Modulates Protein Expression, Transport Function and Ubiquitination of Organic Anion Transporter 3

Purpose: Chloroquine (CQ) and Hydroxychloroquine (HCQ), well-known as anti-malarial drugs, have been grabbing public attention through their values. In past clinical practices, CQ and HCQ have led to a considerable reduction in death caused by malaria, while currently, many preclinical studies have reported their positive therapeutic effect against cancer. Since 2000, they have been widely used in rheumatic disorders treatment. In the scientific research, both two drugs are recognized and well-established as lysosome or autophagy inhibitors. Both CQ and HCQ are predominantly excreted through kidney. Organic anion transporter 3 (OAT3) is expressed in the kidney proximal tubule cells and plays a critical role in drug elimination. The purpose of this study is to explore the regulatory effects of CQ/HCQ on OAT3 and the mechanism behind it.

Methods: OAT3-expressed COS-7 cells were treated with CQ/HCQ at the physiological related concentrations. Treated cells were examined for OAT3 transport function by measuring the uptake of [³H] estrone sulfate (ES), protein expression was assessed by Biotinylation approach in combination with immunoblotting and Ubiquitination of OAT3 was measured by immunoprecipitation (IP).

Results: CQ/HCQ treatment could enhance OAT3 uptake activity in both dose-dependent manner and time-dependent manner. For the dose-dependent course, with 2µM to 10 µM treatment, the uptake value showed 25% to 50% improvement. Under treatment time of 2 hours and 4 hours, an increase of ~30% and ~50%, respectively, was observed. The improved OAT3 transport function was accompanied by increases of OAT3 expression in both cell surface (CQ: ~30% and HCQ: ~35%) and total cell lysate. For the Ubiquitination study, an accumulation of ubiquitinated-OAT3 was observed after the CQ/HCQ treatment.

Conclusion: The results demonstrated that CQ/HCQ upregulate the protein expression and transport function of OAT3. The previous studies from our lab exhibited that the Ubiquitin-Proteasome Pathway (UPP) plays a vital role in the degradation of the ubiquitinated OAT3. The accumulation of ubiquitinated-OAT3 caused by CQ and HCQ in the current study indicates a decreased proteasome activity, suggesting that CQ and HCQ, although classically used as lysosome inhibitors, may surprisingly have proteasome inhibition effect. Furthermore, the upregulation of the OAT3 activity could be associated with the blocked degradation of the transporter.

OAT3 uptake activity in dose-dependent manner



OAT3 expression with 10uM 4hrs treatment(upper: cell surface expression, lower: total cell expression)



Ubiquitination of OAT3