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 Table of Contents  
Year : 2017  |  Volume : 8  |  Issue : 1  |  Page : 61-73

Caenorhabditis elegans organic cation transporter-2 is a novel drug uptake transporter that mediates induced mutagenesis by environmental genotoxic compounds

Department of Medicine, Maisonneuve-Rosemont Hospital Research Centre, Faculty of Medicine, University of Montreal, Quebec, Canada

Date of Web Publication1-Feb-2017

Correspondence Address:
Dindial Ramotar
Department of Medicine, Maisonneuve-Rosemont Hospital Research Centre, Faculty of Medicine, University of Montreal, Quebec
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jrcr.jrcr_6_17

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Uptake transporters are being studied for roles in the entry of therapeutic drugs into cells and thus can be exploited to improve the treatment of various diseases. The live whole model organism, Caenorhabditis elegans, offers an array of advantages to investigate the roles of these transporters. This organism possesses two organic cation transporters (OCTs), OCT1 and OCT2 that are involved in the uptake of clinically relevant genotoxic anticancer drugs such as doxorubicin and cisplatin into the animal. C. elegans lacking OCT1 displays a shortened lifespan, a decreased brood size, an increased susceptibility to oxidative stress, and certain DNA damaging agents. Remarkably, these phenotypes can be rescued by downregulating the OCT1 paralog, OCT2, leading to the suggestion that OCT1 exerts control on OCT2. Indeed, the loss of OCT1 led to the upregulation of OCT2. OCT2 is an uptake transporter involved in the influx of doxorubicin, as well as a number of other therapeutic agents and chemical compounds, some of which have been identified through ligand-protein docking analyses. The genotoxic compounds entering into C. elegans lead to DNA damage-induced apoptosis of germ cells, a process that can be attenuated by blocking OCT2 function. Thus, by combining the roles of the OCT1 and OCT2 transporters with defects in various DNA repair mechanisms, it is possible to engineer a set of supersensitive C. elegans strains that can serve as the most powerful living sensors to date. These tester C. elegans strains can be used to report on the cytotoxicities and genotoxicities of a battery of old and new drugs developed by pharmaceuticals, undocumented toxicants generated by various industries, and compounds that can cause cancers and are present in trace amounts in the environment around the world. These efforts are attainable as C. elegans can live in the soil and water, and a multitude of tools are available to monitor several readouts from the animals.

Keywords: Caenorhabditis elegans organic cation transporters, DNA damage and repair pathways, drug uptake and resistance, germ cells apoptosis

How to cite this article:
Ramotar D. Caenorhabditis elegans organic cation transporter-2 is a novel drug uptake transporter that mediates induced mutagenesis by environmental genotoxic compounds. J Radiat Cancer Res 2017;8:61-73

How to cite this URL:
Ramotar D. Caenorhabditis elegans organic cation transporter-2 is a novel drug uptake transporter that mediates induced mutagenesis by environmental genotoxic compounds. J Radiat Cancer Res [serial online] 2017 [cited 2020 Aug 7];8:61-73. Available from: http://www.journalrcr.org/text.asp?2017/8/1/61/199313

  Introduction Top

The treatment of cancers relies heavily on DNA damaging agents to eliminate cancer cells and decrease tumor burden.[1],[2],[3] These agents include ionizing radiation and the mainstay chemotherapeutics such as anthracyclines, cisplatin, and radiomimetic drugs. These therapies are often administered in combination leading to the activation of the DNA damage response pathway that detects and signals the presence of DNA damage to allow cell cycle arrest and DNA repair.[4],[5] Cancer cells are known to use compensatory DNA repair pathways to process the chemotherapy-induced DNA lesions to survive.[6],[7] Thus, to effectively combat cancer cells would require a multitude of additional approaches. One revolutionary approach involves the search for small molecule inhibitors that block the auxiliary DNA repair pathways with the aim of sensitizing cancer cells toward the chemotherapeutic regimens.[8] In circumstances involving combination therapy with chemotherapeutics and even with inhibitors of DNA repair pathways, there is a presumption that these molecules simply diffuse into cells. This possibility is highly unlikely for several reasons including (i) most chemotherapeutics are charged and structurally unrelated and cannot cross the plasma membrane barrier and (ii) a single chemotherapeutic agent does not show the same genotoxicity to different cell types. However, what seems clear is that when a patient is given an anticancer drug systemically, there are four pharmacokinetic factors, i.e., absorption, distribution, metabolism, and elimination that limit the amount of drug that reaches the tumor.[9] In the tumor, there are other parameters to be considered such as the drug activity on the cancer cell, which can be limited, for example, by poor drug influx or excessive efflux. The elevated level of plasma membrane ABC transporters is a well-established mechanism associated with excessive drug efflux leading to drug resistance.[10],[11],[12] These transporters include the multidrug-resistant efflux pump, MDR1/ABCB1, and the multidrug-resistant associated protein, MRP1, which are known to increase efflux of chemotherapeutic agents, thereby allowing tumor (and normal) cells to evade drug-induced cytotoxicity.[10],[11] In fact, drug efflux transporters are known to be upregulated, for example, in some acute myeloid leukemia patients and there is evidence suggesting that one of these efflux pumps, ABCB1 can expel daunorubicin, an anthracycline typically used for treating these cancer cells.[13] However, inhibition of ABCB1 with valspodar was rigorously tested and found to provide no improvement for the drug-resistant acute myeloid leukemia patients.[13] Thus, there must be other mechanisms that when defective would cause resistance to anthracyclines.

This review will shed light on an emerging phenomenon whereby uptake transporters govern cellular responses to chemotherapeutic drugs and other compounds by regulating their entry into cells. The broader goal is to stimulate discussion on how these transporters can give rise to specific drug resistance. More importantly, while these transporters are being exploited for treating various illnesses and subjected to large-scale screening to hunt for new drugs as substrates, there is evidence emanating from my laboratory that activation of these transporters can have deleterious consequences on the cells. One such effect is the import of toxic compounds from the growth environment that can damage the DNA leading to genomic instability and hence carcinogenesis.

  Uptake Transporters Top

There are over 450 solute carriers or transporters in the human genome that are believed to facilitate the uptake of nutrients, ions, and drugs for treating various illnesses although the exact substrates for many of these transporters are unknown.[9] Among the 450 solute carriers, nearly 145 of these are mutated in human diseases. For example, SLC22A4 is mutated in Crohn's disease, a type of inflammatory bowel disease affecting the gastrointestinal tract, and mutations in SLC12A3 lead to Gitelman syndrome, defined by individuals suffering from low blood pressure.[14],[15] At least 12 of the transporters specifically transport drugs approved by the Food and Drug Administration (USA), for example, SLC6A3 transports methylphenidate used for treating psychiatric disorders.[9] In general, uptake transporters are now attracting additional attention because a few have been shown to mediate uptake of anticancer drugs. YM155, an anticancer drug in clinical evaluation, was found to be completely dependent on the SLC35F2 transporter for entry into human tumor cells.[16] Moreover, another recent study unequivocally established that epigenetic downregulation of the organic cation transporter (OCT) SLC22A2 (OCT2) contributes to oxaliplatin resistance in renal cell carcinoma.[17] The authors showed that DNA hypermethylation at the CpG island of the OCT2 promoter blocked the MYC factor from activating OCT2 by preventing the recruitment of MLL1, a methylase that catalyzes H3K4 trimethylation (H3K4 me3) to promote gene expression.[17] These authors were able to restore oxaliplatin sensitivity to the cells by preventing the hypermethylation on the OCT2 promoter by treating cells with decitabine that blocks DNA methyltransferases and rescues OCT2 expression.[17] A separate study documented that the activity level of the SLC22A1 transporter OCT1 in chronic phase chronic myeloid leukemia is a superior predictor for the responses of this disease toward the tyrosine kinase inhibitor imatinib.[18] These chronic myeloid leukemia patients with low activity levels of OCT1 have the poorest response to imatinib and correlated with lower overall survival as compared to patients with high levels of OCT1.[18] OCT1 has also been implicated in the transport of the diabetes drug metformin, thus underscoring the wide substrate recognition by some of the transporters.[19] On the basis of the foregoing evidence, it seems plausible that uptake transporters would play vital roles in the underlying mechanism causing resistance to anticancer drugs.

  Anthracyclines Are a Family of Anticancer Drugs Top

The anthracyclines consist of several members that include doxorubicin, daunorubicin, epirubicin, and idarubicin. Anthracyclines act by intercalating with the DNA and believed to mainly block the function of DNA topoisomerase leading to cell death.[20] These clinically important drugs have a high tissue penetration and retention in nucleated cells, and besides leukemia, they are used for treating other types of cancers including lymphomas, breast, lung, ovarian, and gastric and thyroid malignancies.[21] In the case of acute myeloid leukemia characterized by the rapid clonal expansion of immature blood cells and a major cause of mortality from hematological malignancies in adults,[22] the patients are given a standard induction chemotherapy consisting of continuous infusion of cytarabine, which acts by inhibiting DNA synthesis, for 7 consecutive days and with the anthracycline daunorubicin or idarubicin for 3 days.[22] Despite the induction therapy, a significant fraction (>50%) of older patients (>60 years) with acute myeloid leukemia do not achieve complete remission with anthracyclines due to drug resistance.[23],[24],[25] This poor outcome raises an important question as to how these patients become resistant to the chemotherapy. For years, it has been assumed that anthracyclines simply diffuse into cells to exert the genotoxic effects. If this was the case, then all patients would likely respond in a similar manner to anthracyclines. In fact, anthracyclines are hydrophilic in nature and must be actively transported across the plasma membrane into the cells. In an earlier report, only parsimonious evidence exists to support a transporter for anthracyclines.[26] However, with the advent of new tools, an uptake transporter, OCT1 (SLC22A1), was recently uncovered that allows entry of anthracyclines into cells.[27] This discovery, therefore, dismissed previous misconceptions that the drug action is mediated by passive diffusion. As such, it remains possible that a fraction of the acute myeloid leukemia patients that is resistant to anthracyclines might be due to a defect in OCT1 uptake function and causing the cells to evade the genotoxicity of the drugs.

  Evidence That Organic Cation Transporter 1 Mediates the Uptake of Anthracyclines in Human Cells Top

Anthracyclines autofluorescence at 640 nm, which provides a notable physical property leading to the development of two in vitro assays to quantitatively monitor the uptake of daunorubicin into the cells over time using FACS analysis, as well as epifluorescent microscopy.[28],[29] Using these assays and a panel of cancer cells including the leukemia HL60 and the ovarian TOV222G2 cell lines, there was a rapid and a concentration dependent increased in the accumulation of daunorubicin (5 µM) into these cells reaching saturation by 60 min [Figure 1].[27] The rapid rate of daunorubicin uptake and the creation of a concentration gradient in these cancer cells are consistent with an active transport mechanism for the drug, again refuting previous assumption that anthracyclines can diffuse into cells.[27] A search was then initiated for natural substrates that would compete with the transporter for the uptake of daunorubicin. One clue came from a previous work claiming that the L-carnitine transporter SLC22A16 (hCT2 or OCT6) was involved in the uptake of doxorubicin.[26] hCT2 is a high-affinity transporter of L-carnitine, an amino acid that serves as a carrier for acetyl-CoA from the peroxisomes to the mitochondria.[30] Besides hCT2, there are other transporters of L-carnitine including OCTN1 and OCTN2.[30] Surprisingly, addition of L-carnitine in excess of 200-fold did not compete for the uptake of daunorubicin into the cells.[27] While the observation eliminates a role for hCT2 in the uptake of daunorubicin, it also excludes OCTN1 and OCTN2.[27] Attention was diverted to another family of OCT1, OCT2, and OCT3 that have been reported to transport choline but with different affinities.[27] Interestingly, when choline was added at equimolar (5 μM) amount as daunorubicin, it completely blocked the uptake of the drug into the cells [Figure 1], as well as protected the cells from the genotoxic effects of the drug [Figure 2]. Since there are several known choline transporters including OCT1, OCT2, and OCT3,[31] it was necessary to define whether one or more could be involved in anthracycline uptake. Among the OCT transporters, OCT1 has been implicated in the transport of the tyrosine kinase inhibitor imatinib for treating chronic myeloid leukemia.[18],[32] OCT1 is also involved in the transport of the diabetes drug metformin, thus underscoring the ability of OCT1 to recognize other substrates.[19] Using gene silencing approach, the knockdown of OCT1 expression by shRNA greatly diminished the level of the OCT1 protein in the ovarian cancer cells, as revealed by antibodies against OCT1.[27] These knockdown cells showed a dramatic reduction (~85%) in daunorubicin uptake, while the control shRNA had no effect [Figure 3].[27] The above findings were also observed with other cell types besides ovarian.[27] In addition, overproduction of OCT1 can stimulate daunorubicin uptake into the cancer cell lines [Figure 4],[27] suggesting that patients with a hyperactive OCT1 could be sensitized to daunorubicin. These in vitro cell line data provide the first compelling evidence, which strongly supports the notion that OCT1 functional level in patients might be a key determinant that will govern the effectiveness of anthracycline regimens. Patients who are refractory to anthracycline treatment may harbor a defect leading to a dysfunctional uptake transporter. It is noteworthy that efflux pumps may play little or no role in the resistant toward anthracyclines, as these drugs are tightly bound to the cellular DNA and may not exist in free forms to activate the efflux pumps.
Figure 1: Uptake of daunorubicin in HL60 is blocked by choline. Briefly, HL60 cells were incubated without (a) or with daunorubicin alone (b), or daunorubicin plus choline (added together) (c and d). After 1 h, samples were fixed and monitor for daunorubicin uptake using epifluorescence microscopy. Nuclear DNA stained with DAPI, Merge of DAPI, and daunorubicin

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Figure 2: Choline protects HL60 cells from daunorubicin cytotoxicity. Cells were treated with increasing daunorubicin alone for 16 h or together with choline (5 μM) and scored for viability by MTT assay using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide that forms a purple color reflecting viable cells. The data are averages of two experiments

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Figure 3: Organic cation transporter 1-shRNA blocked daunorubicin uptake into HL60 cells. HL60 cells were untransfected (b) or transfected with either control shRNA (c) or organic cation transporter 1-shRNA (d) for 72 h and cells processed for daunorubicin uptake (a-d) as in [Figure 1]

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Figure 4: Organic cation transporter 1 overproduction stimulates daunorubicin uptake in various cancer cell lines. Organic cation transporter 1 cDNA was cloned next to the cytomegalovirus promoter in the empty vector pEYFP, and time-dependent uptake of daunorubicin monitored by fluorescence-activated cell sorting with transiently transfected cells

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  Caenorhabditis Elegansas a Model to Facilitate Studies on Drug Uptake Transporters and the Association With Genomic Instability Top

C. elegans is an inexpensive model organism that offers a multitude of advantages over mammalian cells to rapidly study many biological processes that are highly conserved in nature.[33]C. elegans developed from eggs to the adult stage within 3 days spanning four larval stages. Genes can be readily downregulated in this organism by simply feeding the animals bacteria expressing double-stranded RNA from a segment of the coding region of the targeted gene. These animals are transparent and well suitable for microscopy to visualize germ and somatic cells. While C. elegans has been extensively used to understand DNA repair mechanisms and genetic stability, only limited studies have been performed with this organism to understand the functions of uptake transporters.[34] During the last decade, C. elegans has become instrumental in several drug discovery programs.[33],[35] However, in many high-throughput screens performed so far to identify novel small molecules, for example, those that act as antimicrobials, extend lifespan, inhibit oxidative stress, or prevent multidrug resistance; the yield of bioactive compounds is typically in the range of 0.03% to <1%.[33],[36] It is possible that the recovery rate could be higher if there is a greater influx of the molecules into the animal cells. High-throughput screens at higher initial concentrations of the small molecules may alleviate this issue but could be cost prohibitive. Thus, characterization of the function and substrate specificities of uptake transporters in C. elegans will be advantageous toward improving the strategies employed to identify novel bioactive molecules.

  Uptake Transporters in Caenorhabditis Elegans Top

There are far fewer OCTs in C. elegans following an analysis of the sequence information in the WormBase, and these include OCT1, OCT2, and PES-23. In addition, there are at least five other transmembrane transporters, i.e., OAT-1, SVOP-1, STR-176, HMT-1.1/1.2, and GEM-1. Among these transporters, C. elegans, OCT1, and OCT2 are counterparts of the human OCTs, and little is known about their roles and affinities toward distinct substrates. OCT1 was the first uptake transporter characterized from C. elegans and when expressed in mammalian cells was shown to mediate the transport of the organic cation tetraethylammonium, a prototypical substrate used for classifying OCTs.[37]C. elegans deleted for OCT1 exhibits a shortened lifespan and increased susceptibility to oxidative stress, which led to the proposition that OCT1 facilitates the import of antioxidants required to protect OCT1 mutant animals from oxidative stress.[34] However, uptake of ergothioneine, the purported antioxidant substrate of OCT1, was not reduced in OCT1 mutant animals as compared to the parent.[34] Therefore, it seems plausible that an alternative explanation could account for the OCT1 mutant animal phenotypes.

Expression of C. elegans OCT1 can restore uptake of the chemotherapeutic drug doxorubicin into Saccharomyces cerevisiae cells lacking the regulator Agp2, an amino acid transporter that when deleted blocked the expression of several target genes including the polyamine transporters Dur3 and Sam3.[29] No further studies were done to determine whether OCT1 substituted for the regulatory function of Agp2 or directly for the roles of Dur3 and Sam3, as both of these transporters also mediate the transport of doxorubicin.[29] At the time, no further studies were done to test directly or test whether OCT1 might mediate the transport of doxorubicin into C. elegans.

In addition to OCT1, C. elegans possesses another related member of the SLC22 OCT family, i.e., OCT2. OCT2 shares 22.56% identity with OCT1 but differs as it possesses an extended N-terminal of 172 amino-acid residues that is unrelated to OCT1. Previously, no function was assigned to this putative transporter OCT2 until very recently.[38] To date, there are no animals in the consortiums, USA or Japan, carrying either partial or complete deletion of the OCT2 gene. Thus, to determine the phenotypes caused by animals devoid of OCT2, RNA-interference (RNAi) feeding bacteria constructed to downregulate the OCT2 gene were used.[38] This approach revealed a number of novel findings regarding the OCT1 and OCT2 transporters of C. elegans. Unlike the downregulation of OCT1, depletion of OCT2 did not affect the lifespan of the animals. Remarkably, the downregulation of OCT2 rescued the shortened lifespan of animals already deleted for the OCT1 gene.[38] This observation was surprising and prompted the possibility that OCT1 might influence OCT2 function, whereby OCT1 downregulation leads to OCT2 upregulation, which in turn could be responsible for mediating uptake of toxic environmental compounds and account for the shortened lifespan of the OCT1 animals. This notion is based on the fact that OCT1 knockout mice manifest upregulation of two related transporter genes, OCT2 and OCT3.[39] Indeed, quantitative PCR analysis revealed that downregulation of OCT1 led to the upregulation of OCT2 expression, while several control genes were unaffected such as act-1.[38] It is unclear whether OCT1 downregulation influences the expression of the other transporters such as PES-23 although it would be an important mechanism to explore to understand the rationale for this regulation.

There is striking evidence that the upregulation of OCT2 can lead to the import of toxic compounds from the environment into the animals. This was demonstrated using a simple reporter animal that harbors within its genome the oxidative stress response gene GST-4 encoding glutathione S-transferase 4, where the promoter is fused to the green fluorescent protein (GFP) to create the fusion gene GST-4:GFP. GST-4 is a target for the conserved SKN-1/Nrf2 transcriptional activator that plays a role in the defense against oxidative stress.[40],[41] These animals showed a basal level of GST-4:GFP expression in the intestine. However, the level of the expression of the GST-4:GFP was greatly stimulated following RNAi downregulation of OCT1.[38] Since the GST-4:GFP reporter was not constructed in the OCT1 null animals, it was not possible to directly address whether the stimulation of the reporter could be the result of the OCT2 upregulation. In fact, downregulation of the OCT2 transporter gene by RNAi lowered the basal levels of GST-4:GFP expression in the reporter strain.[38] These findings are consistent with a model whereby OCT2 upregulation, through OCT1 depletion, allows entry of toxic compounds such as prooxidants into C. elegans, which in turn increase the oxidative stress of the animals and leading to a shortened lifespan.

  Organic Cation Transporter-2 Mediates the Genotoxic Effects of the Anticancer Drug Doxorubicin Top

A series of experiments have been performed to gain insights into whether OCT1 regulation of OCT2 would be involved in the differential uptake of genotoxic compounds. One such experiment involves treating the worms with the chemotherapeutic drug doxorubicin and then monitor their survival by scoring the size of brood.[42] Doxorubicin has been chosen since its uptake depends on cationic transporters in S. cerevisiae[29] and mammalian cells,[27] as well as shown to trigger germ cell apoptosis in C. elegans.[42] The wild-type animals displayed ~55% decrease in brood size when treated with a fix concentration of doxorubicin (100 µM), while the OCT1 deletion mutants were more sensitive showing nearly 80% decrease in brood size and this was associated with a significant level of dead embryos. This observation was entirely unexpected since the loss of the uptake transporter should have caused resistance to the drug. However, based on the gene expression data, it seems that the enhanced doxorubicin sensitivity of the OCT1 deletion mutant animals can be explained by an increased uptake of the drug due to the upregulation of OCT2. This was validated when it was observed that RNAi-driven depletion of OCT2 in the OCT1 deletion mutant abolished the hypersensitivity of these animals toward doxorubicin, showing <20% reduction in brood size compared to 80% in the OCT1 null mutant fed control RNAi. Since these analyses were conducted with a fixed high concentration of doxorubicin, it is not clear whether OCT2 would function at lower concentrations of the drug. Therefore, no assignment could be made regarding the affinity of OCT2 toward the drug.

  Organic Cation Transporter-2 Allows the Accumulation of Doxorubicin in Caenorhabditis Elegans Tissues Top

An uptake transporter functions to concentrate its substrate against a gradient and as such when OCT2 is upregulated it is expected to accumulate doxorubicin in C. elegans tissues. In the design of the experiment, the physical property of doxorubicin, which emits fluorescence at wavelengths of λex 470− λem 585 nm, became a convenient mean to monitor its uptake through OCT1 and OCT2 in situ by imaging the pharynx. Uptake of the drug into the pharynx became the organ of choice as it is relatively large and allows food consumption [Figure 5]a. OCT2 is expressed at higher levels than OCT1 in nearly all tissues of the animal, and its expression can be stimulated nearly 2-fold in OCT1 null animals as compared to the wild type. Indeed, the OCT1 null mutant depicted a markedly stimulated fluorescence intensity of doxorubicin in the pharynx, as compared to the parent, and which was shown to be the result of the upregulated OCT2 [Figure 5]b.[38] The downregulation of OCT2 did not interfere with the uptake of fluorescein, which is anionic in nature as compared to the cationic drug doxorubicin.[38] It seems that OCT2 may recognize an overall positive charge, as well as aromatic groups, to display specificity in the uptake of substrates into C. elegans tissues.
Figure 5: Doxorubicin transport into the pharynx of Caenorhabditis elegans is stimulated by organic cation transporter 2 upregulation. (a) Model of organic cation transporter 2 localization at the terminal bulb of the pharynx and the fluorescence (red dots) where the drug is detected. (b) Images of the pharynx from untreated (control) and doxorubicin-treated animals. Differential interference contrast images are shown in the upper left corner of each panel. Images to the right of each pharynx depict a three-dimensional representation of the doxorubicin treatment signal intensity for the indicated genotypes. Fluorescence posterior to the pharynx is autofluorescence detected from the intestine. Reproduced with permission

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  Organic Cation Transporter-2-Mediated Uptake of Doxorubicin Is Blocked by Choline in the Pharynx Top

In human cells, OCT1 and OCT2 have been shown to transport other cationic molecules such as choline.[43] Test for substrates revealed that choline when mixed with equimolar amounts of doxorubicin, impeded uptake of the drug into the pharynx of the OCT1 null mutant, clearly indicating that OCT2 has the ability to recognize and compete for other cationic compounds.[38] It seems logical that the competition for doxorubicin uptake into the OCT1 null animals can be used as a screening tool to find compounds that are a putative substrate for OCT2. Some of these compounds could simply quench the fluorescence of doxorubicin leading to false positives as in the case of spermine that directly alters the fluorescent properties of doxorubicin. Likewise, stimulated- or inhibited-doxorubicin uptake into the OCT1 null animals can be used as a screening tool to find, respectively, activators or inhibitors of OCT2 uptake function.

  the Organic Cation Transporter-1 Mutant Animals Display Increased Spontaneous and Drug-Induced Germ Cell Death That Is Suppressed by Organic Cation Transporter-2 Downregulation Top

Analogous to many stem cell systems, C. elegans has a self-renewing germ cell population derived from a cellular niche located at the distal tip [Figure 5]a.[44] These germ cells progress through distinct stages of differentiation and must faithfully maintain the genome. They are very sensitive to genotoxic compounds and respond using conserved DNA repair mechanisms to maintain genomic stability.[45] Germ cells with excessive DNA damage undergo apoptosis and are unable to form viable embryos.[44],[46] Thus, germ cell apoptosis is an ideal in vivo experimental system to determine the extent of genotoxicity caused by natural compounds and those introduced to the growth environment of the animals. The use of germ cell apoptosis also has the advantage of allowing assessment of the uptake of genotoxic anticancer drugs that cannot be monitored directly such as those that do not emit fluorescence or unavailable in radioactively labeled form. Of importance is whether OCT2 level influences germ cell apoptosis?

Apoptotic germ cells can be quantified in vivo by examining for corpses in the proximal zone of the gonad arm [Figure 5]a by utilizing differential interference contrast (DIC) microscopy and staining with the DNA dye acridine orange.[47] In wild-type animals, typically there is one to four germ cell corpses [Figure 6]a.[48] In contrast, when OCT1 is downregulated the animals depicted an average of five to eight apoptotic cells [Figure 6]a. It is believed that this 2-fold increase of apoptotic cells in the gonads of the OCT1 downregulated animals may result from increase import of prooxidants by the elevated levels of the OCT2 transporter and thus cause damage to the genome of the germ cells leading to dead embryos.[38] Interestingly, RNAi-driven depletion of OCT2 in the OCT1 null mutant sharply reduced germ cell death to the point where it is even undetectable in some animals [Figure 6]a.
Figure 6: Organic cation transporter 2-mediated transport of genotoxic compounds induces germ cell death in Caenorhabditis elegans. (a) Representative images of acridine orange-stained and differential interference contrast (lower left) of control and doxorubicin-induced apoptotic cell corpses of the indicated genotypes. Apoptotic cell corpses were identified as bright spots correlating with raised-bottom-like refractive corpses shown on differential interference contrast images. Posterior is right and dorsal is top. Scale bar = 15 μm. (b) Representative images of control and doxorubicin-induced bcls39 (CED-1: green fluorescent protein) clusters around apoptotic cell corpses indicated by white arrows. Reproduced with permission

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Besides staining with acridine orange, there are other methods to evaluate if germ cells are undergoing apoptosis. One such method involves following a downstream step of the apoptotic pathway, i.e., the engulfment of apoptotic cells by the CED-1 protein to signal phagocytic degradation.[49],[50] In this approach, the animals must carry CED-1 tagged with GFP to serve as a reporter of engulfed apoptotic cells.[51] As in the case of acridine orange staining, the CED-1-GFP showed engulfment of 1-3 physiological apoptotic cells [Figure 6]b, whereas downregulation of OCT1, and not OCT2, engendered increased engulfment recapitulating the enhanced germ cell apoptosis observed in the OCT1 null mutant [Figure 6]. Taken together, it seems that OCT2 possesses the ability to transport toxic compounds such as prooxidants that cause germ cell death. These prooxidants are likely to cause damages to various tissues and may, therefore, account for the reduced lifespan observed in the OCT1 null animals.[38] In fact, these OCT1 deficient animals produce an elevated level of dead embryos under normal growth conditions, which are believed to be the result of the accumulation of genotoxic DNA lesions. As expected, studies are currently underway to address whether these OCT1 deficient animals gather mutations over several generations.

Treatment of wild-type worms with doxorubicin elevated the levels of apoptotic cell corpses as visualized by both acridine orange staining [Figure 6]a and CED-1-GFP engulfment of the cells [Figure 6]b. As predicted, the OCT1 deletion mutants treated with doxorubicin showed substantially higher levels of acridine orange-stained apoptotic cells, and which paralleled the increase in CED-1-GFP engulfment of the cells. The increased engulfment of apoptotic cells by CED-1-GFP did not occur in doxorubicin-exposed animals downregulated for OCT2. These observations led to several conclusions: (i) OCT2 has a predominant role over OCT1 in the uptake of doxorubicin, (ii) doxorubicin uptake induces germ cell death that correlates with decrease survival, and (iii) both the drug uptake and the induced germ cell death are OCT2 dependent.

  Organic Cation Transporter-2 Function Mediates Cisplatin-Induced Germ Cell Death Top

The germ cell death assays can be used to monitor the uptake of genotoxic drugs that are not readily available as either fluorescently or radioactively labeled form. Since the human OCT1 has been shown to transport members of the platinum family of anticancer drugs that act by creating intra- and inter-strand DNA cross-links,[52] it seems reasonable that the C. elegans OCT1 and OCT2 might be endowed with similar transport ability. As cisplatin is not available in labeled forms, the germ cell apoptotic assay provides a remarkable reporter for the uptake of the drug. It was shown that cisplatin induced an increase level of germ cell death in the wild type animal, and which was further stimulated in the OCT1 null mutants.[38] Downregulation of OCT2 in the OCT1 null mutants prevented cisplatin-induced germ cell death indicating that OCT2 plays a central role in mediating the genotoxic effects of cisplatin.[38] It is noteworthy that γ-rays that create multiple DNA lesions also induced germ cell death, but independently of OCT1 and OCT2 transporter functions.[38] Thus, the transporter function of OCT2 is not directly involved in the mechanism leading to germ cell death triggered by DNA damaging agents.

  Organic Cation Transporter-2-Dependent Transport of Doxorubicin or Cisplatin Stimulates Germ Cell Death in Caenorhabditis elegansmutants Defective in Dna Repair Top

One prediction from the above findings is that the elevated uptake of genotoxic agents into animals defective in DNA repair should result in lethal phenotypes. There are several mutants in the C. elegans consortium carrying defects in the major DNA repair pathways and suitable to test whether OCT2 upregulation can trigger sensitization of the animals to DNA damage-induced germ cell death. For example, the RAD-51 deletion mutant lacking the RAD-51 protein needed for DNA strand invasion during homologous recombination-dependent double strand break repair, exhibited higher endogenous levels of apoptotic cell death due to spontaneous unrepaired meiotic breaks as compared to the wild type [Figure 7].[53] Treatment of the RAD-51 mutant with doxorubicin was shown to greatly stimulate apoptosis upon downregulation of OCT1 [Figure 7]c. In contrast, depletion of OCT2 by RNAi in the RAD-51 mutant animals suppressed the high level of apoptotic cells observed in this mutant upon exposure to doxorubicin [Figure 7]c. These data indicate that upregulation of OCT2 burdens the RAD-51 mutant animals with doxorubicin-induced DNA lesions leading to the enhancement of germ cell death. In a similar manner, the APN-1 mutant animals lacking the key enzyme APN-1, required for removing a variety of DNA lesions including oxidized bases through the base excision DNA repair pathway, showed enhance germ cell death by doxorubicin when OCT2 expression was stimulated [Figure 7]e.[54] This induced apoptosis was strongly attenuated the following depletion of OCT2 by RNAi [Figure 7]e. While the base-excision DNA repair pathway in C. elegans is also involved in processing doxorubicin-induced oxidative DNA lesions, it is clear that OCT2 controls the toxicity of the drug in the APN-1 mutant animals.[55] It is interesting that the APN-1; OCT1 (RNAi) genotype showed a significant increase in spontaneous germ cell death [Figure 7]d, raising the possibility that OCT2 might mediate uptake of prooxidants leading to oxidative DNA lesions that must be repaired by the base excision DNA repair pathway.[54] Collectively, the published work suggests that by combining defects in DNA repair pathways with functional OCTs such as OCT2, it is possible to determine whether an unknown compound has genotoxic effects and the type of lesions it may create.
Figure 7: Downregulation of organic cation transporter-1 sensitizes Caenorhabditis elegans DNA repair deficient mutants to drug-induced apoptotic cell death. (a) Wild type. (b and c) The homologous recombination mutant RAD-51 downregulated for organic cation transporter 1 shows stimulated doxorubicin-induced apoptotic cell death. D and E, The base excision repair mutant APN-1 downregulated for organic cation transporter 1 displays enhanced spontaneous, as well as doxorubicin-induced apoptotic cell death. Apoptotic cell corpses were analyzed in the young adults, and untreated animals are depicted as white boxes. Reproduced with permission

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  Predicted Ligands of Organic Cation Transporter-1 and Organic Cation Transporter-2 and Experimental Validation Top

So far, the roles of transporters in the uptake of a vast majority of genotoxic cationic drugs have not been tested and C. elegans offers an advantage to assess this rapidly. A number of known drugs were selected as potential substrates for uptake by OCT1 and/or OCT2 based on two criteria: (i) Mechanism of action and (ii) biological response. The docking ability of each compound onto OCT1 and OCT2 was assessed sequentially by first making predictions of the structure of the transporters by inputting the respective primary protein sequences into the I-TASSER protein structure prediction server for in silico analyses.[56] The I-TASSER server employed known Protein Data Bank (PDB) structures as threading templates to predict the OCT1 and OCT2 structures. Both OCT1 and OCT2 were modeled based on the X-ray diffraction structure of the glucose transporter GLUT3/SLC2A3 from Homo sapiens (PDB ID: 5c65) and validated with the glucose transporters GLUT1-4 structures (PDB ID: 4 gc0).[57] Overall, the analysis predicted three-dimensional (3D) structures for both OCT1 and OCT2, featuring the entire 12 transmembrane domain helices and belonging to solute carrier transporter family.[38] These predicted 3D structures of OCT1 and OCT2 were then employed to look for drug interactions using the BSP-SLIM and COACH algorithms to approximate the amino-acid residues of the transporters constituting the ligand-protein docking sites.[58],[59] Such in-depth analysis provides a computed docking score. From 19 tested ligands, four resulted with a docking score of zero, whereas the remaining 15 revealed a docking score >3.5 favoring robust binding with OCT2, as opposed to OCT1 [Supplemental Table S1]. Among the15 ligands, some possess distinct biological effects such as creating different types of DNA lesions and capable of triggering high levels of apoptotic cell death when OCT2 was upregulated [Supplemental Table S1]. There are ligands that can damage the DNA and induce apoptotic corpses such as melphalan and methoxyamine, but these do not dock onto OCT2 and are believed to use alternative transporters to enter the animal [Supplemental Table S1]. Thus, it is possible to perform protein-ligand modeling studies to predict and uncover novel substrates for these uptake transporters.

  Organic Cation Transporter-2 Transports the Rad51 Inhibitor B02 Top

The chemical compound 3-benzyl-2-([E]-2-pyridin-3-ylethenyl) quinazolin-4-one (B02, CID: 5738263) was shown to interfere with the DNA strand exchange and nuclear focus formation catalyzed by the human DNA repair protein RAD51 in response to DNA damage.[60],[61],[62] However, the pharmacological effect of B02 has not been previously tested in C. elegans. Protein-ligand analysis revealed that B02 has the ability to interact OCT2, but not with OCT1, and yielding a docking score of 5.4 [Supplemental Table S1 and [Figure 8]a, [Figure 8]b. Interestingly, in the OCT1 null mutant, the B02 inhibitor caused sterility [Figure 8]c by decreasing the number of viable animals in a manner analogous to the RAD-51 homozygotes [Figure 8]e.[63] As predicted from the model, downregulation of OCT2 in the OCT1 null mutant prevented the B02 ligand from causing sterility [Figure 8]d and thus restoring the number of broods to nearly untreated levels [Figure 8]e. These compelling data revealed for the first time that B02 can inhibit RAD51 function in C. elegans through uptake by the OCT2 transporter.[38] Thus, the docking scores of ligands and proper readouts from the targeted pathway provide valuable tools to monitor transporter-mediated drug uptake into C. elegans. This approach is particularly suitable for newly developed drugs that cannot be readily labeled or lack fluorescent properties for uptake studies.
Figure 8: The RAD-15 inhibitor B02 induces sterility in Caenorhabditis elegans when organic cation transporter 2 is upregulated. (a and b) Ligand-protein docking of organic cation transporter-1 and organic cation transporter-2 modeled with B02 and binding of the drug to organic cation transporter 2. (c) B02 causes embryonic lethality (diminishing number of germ cells) in the organic cation transporter 1 null mutant. (d) Organic cation transporter 2 downregulation in the organic cation transporter 1 null mutant animals restores fertility. (e) B02 diminishes the number of viable animals when organic cation transporter 2 is expressed. Brood size analysis of the indicated genotypes without and 5 μM B02. (f) The RAD-51 inhibitor, B02, mode of action in Caenorhabditis elegans germline. Reproduced with permission

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  Perspectives Top

So far, OCT1 appears to have no direct role in the transport of the chemotherapeutic drugs such as anthracyclines and cisplatin; however, it exerts control on OCT2 expression, and it is OCT2 that is primarily involved in the uptake of these agents. Since no additional drug resistance was observed in the OCT1 null, OCT2 (RNAi) double mutant animals as compared to ones depleted for OCT2 alone, a role for OCT1 in the uptake of these chemotherapeutic drugs can be excluded. Nonetheless, it has not been excluded yet whether OCT1 could act as a transporter for selective ligands. The observations to date underscore the importance of uptake transporters in regulating the entry of chemotherapeutic drugs into cells and raise the possibility that the drug-resistance and drug-sensitive responses exhibited by cancer patients could be governed at the level of drug uptake.

The downregulation of OCT1 leading to the upregulation of OCT2 was an unexpected finding and provides a compelling argument that the animal has evolved tight regulation of OCT2. So how might OCT1 depletion lead to the activation of OCT2? One possibility is that OCT1 might belong to the recent characterized class of surface sensors that act as nontransporting transceptors by sensing the availability of nutrients and signal the regulation of downstream plasma membrane transporters.[64],[65] In this model, when nutrients become scarce, OCT1 might serve as a sensor to promote the upregulation of OCT2 to scavenge limiting resources. Conversely, when nutrients are plentiful OCT1 might function to sustain the basal expression of OCT2. Precedence for this mode of regulation exists in S. cerevisiae, Drosophila melanogaster, and H. sapiens.[66] For instance, in S. cerevisiae, the Ssy1 sensor, a plasma membrane protein belonging to the amino acid permease family, is endowed with limited or no transport function.[67] Ssy1 senses amino acid availability by direct interaction with extracellular amino acids and transmitting the signal to zinc-finger transcription factors that trigger the expression of several downstream target genes encoding amino acid permeases.[67],[68],[69] Similar sensors exist in mammalian cells, for example, the SGLT3 glucose sensor that binds but does not transport sugar molecules.[70] Thus, in view of the increasing number of sensors that are currently being identified, it is plausible that OCT1 may indeed act either as a nontransporting or transporting sensor leading to regulation of OCT2 expression. The exact role by which OCT1 exerts control on OCT2 will need further investigation, but it is noteworthy that a similar regulation appears to occur in mice where deletion of OCT1 causes a significant upregulation of mRNA transcripts of its homologs OCT2 and OCT3.[39]

The observation that OCT2 downregulation sharply decreased the spontaneous apoptosis seen in the OCT1 deletion mutant can be explained if OCT2 indiscriminately transports toxic compounds such as prooxidants. Although the source of these compounds is unknown, i.e. whether they originate from the feeding bacteria or re-adsorption of metabolites secreted by C. elegans, they are capable of inducing DNA lesions that must be removed by DNA repair mechanisms. In fact, the base excision repair defective APN-1 deletion mutant exhibited higher levels of spontaneous apoptosis when OCT2 expression is upregulated.[38] These findings raise a very important concern regarding genetic variations leading to hyperactivation of uptake transporters as previously reported.[71] Based on the studies in C. elegans, one would expect that hyperactive uptake transporters are likely to cause accumulation of abnormally high concentrations of genotoxic compounds and metabolites into cells. Consequently, such toxic agents could induce substantial levels of DNA damage over the lifetime of an individual causing genomic instability and eventually cancer.

The evidence so far suggests that exploiting OCT2 in C. elegans could have far-reaching applications and supersede other whole model systems in drug discovery programs with respect to cost and time. Thus, maintaining the OCT2 transporter at optimal levels by deleting OCT1 should represent a useful step for incorporation into any high-throughput screens to improve the efficiency of identifying cationic bioactive molecules from chemical libraries. A key aspect of this strategy is that the overexpressed OCT2 is expected to operate at significantly lower concentrations of the chemicals. Thus, the previous barriers posed by C. elegans to find bioactive molecules could be explained by the lack of an activated mechanism to efficiently take up the compounds at lower concentrations. In short, C. elegans offers a comprehensive readout of the OCT2 functional selectivity toward cationic molecules that have deleterious effects and therefore provide a foundation to understand the regulatory control of drug uptake to circumvent genotoxicities.

Financial support and sponsorship

This work was funded by the research grant (RGPIN/202432–2012) to D.R. from the Natural Science and Engineering Research Council of Canada.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]


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