A novel approach using C. elegans DNA damage-induced apoptosis to characterize the dynamics of uptake transporters for therapeutic drug discoveries

Abstract

Organic cation transporter (OCT) function is critical for cellular homeostasis. C. elegans lacking OCT-1 displays a shortened lifespan and increased susceptibility to oxidative stress. We show that these phenotypes can be rescued by downregulating the OCT-1 paralogue, OCT-2. Herein, we delineate a biochemical pathway in C. elegans where uptake of genotoxic chemotherapeutics such as doxorubicin and cisplatin, and subsequent DNA damage-induced apoptosis of germ cells, are dependent exclusively upon OCT-2. We characterized OCT-2 as the main uptake transporter for doxorubicin, as well as a number of other therapeutic agents and chemical compounds, some identified through ligand-protein docking analyses. We provide insights into the conserved features of the structure and function and gene regulation of oct-1 and oct-2 in distinct tissues of C. elegans. Importantly, our innovative approach of exploiting C. elegans uptake transporters in combination with defective DNA repair pathways will have broad applications in medicinal chemistry.

The tales of two organic cation transporters, OCT-1 and OCT-2, in C. elegans

“…We used the gst::gfp reporter animals to monitor oxidative stress caused by toxic compounds and showed that these animals expressed a basal level of GST-4::GFP in the intestine. However, the expression level of the GST-4::GFP was greatly stimulated following RNAi downregulation of oct-1 that led to the upregulation of oct-2 [33]. Consistent with this observation, downregulation of the oct-2 transporter gene by RNAi lowered the basal levels of GST-4::GFP expression in the reporter strain [33].…”

“…First, unlike the oct-1 null animals, downregulation of oct-2 did not affect the lifespan of the animals. Second, perhaps most interesting, the depletion of OCT-2 rescued the shortened lifespan of animals already deleted for the oct-1 gene [33]. This striking observation prompted the possibility that OCT-1 might influence OCT-2 function, whereby oct-1 downregulation leads to oct-2 upregulation.…”

“…This notion is based on the fact that OCT1 knockout mice manifest upregulation of two related transporter genes, OCT2 and OCT3 [34]. And third, quantitative PCR analysis indeed revealed that downregulation of oct-1 led to the upregulation of oct-2 expression in several tissues of the animals, while several control genes, such as act-1 encoding actin, were unaffected [33]. As such, we proposed that OCT-2 upregulation could be responsible for triggering uptake of toxic environmental compounds into the animals and thus account for the shortened lifespan displayed by the oct-1 animals.…”

“…However, the existence of another related member of the organic cation transporter family, namely OCT-2, in C. elegans, suggests that there needs to be a strategy to test the contribution of each transporter towards doxorubicin uptake. Prior to our work, there was no function assigned to this putative transporter OCT-2 [33]. Moreover, there are no animals in the consortiums, USA or Japan, carrying either partial or complete deletion of the oct-2 gene, as in the case of oct-1.…”

“…Moreover, there are no animals in the consortiums, USA or Japan, carrying either partial or complete deletion of the oct-2 gene, as in the case of oct-1. Thus, to examine whether OCT-1 or OCT-2 or both have roles in the uptake of doxorubicin in C. elegans, we used RNA-interference (RNAi) feeding bacteria to generate animals with different genotypes, in particular, either downregulated for one or both transporters [33].…”