**3. Results**

#### *3.1. Effects of Ixazomib, Oprozomib, and Delanzomib on the Ubiquitination of OAT3*

Ixazomib, oprozomib, and delanzomib, as proteasome inhibitors, target the ubiquitinproteasome system for cancer therapy. First, we investigated their effects on the intracellular ubiquitination of OAT3 in OAT3-expressing COS-7 cells. OAT3-expressing cells were treated with ixazomib, oprozomib, or delanzomib for 6 h, then harvested and lysed. OAT3 was pulled down from cell lysate by anti-Myc antibody (Myc tag was fused onto OAT3, enabling immunodetection), followed by immunoblotting (IB) using anti-ubiquitin antibody to probe the ubiquitinated OAT3. The results (Figure 1) showed that incubating cells with ixazomib, oprozomib, or delanzomib resulted in a significant accumulation of ubiquitinated OAT3, which was not because of the difference in immunoprecipitated OAT3, since there were similar quantities of OAT3 pulled down from all samples. Further study showed that like lactacystin, a classical proteasome inhibitor, ixazomib, oprozomib, and delanzomib inhibited the 20S proteasome activity by 50% (95% confidence interval (CI): 46% to 54%), 87% (95% CI: 83% to 91%), and 61% (95% CI: 57% to 65%), respectively, after 6 h of treatment (Figure 2). Therefore, the accumulation of ubiquitinated OAT3 was attributed to the decreased proteasome activity, suggesting that ubiquitinated OAT3 can be modulated by interfering the ubiquitin–proteasome system in the cell model that we used.

**Figure 1.** Effect of proteasomal inhibitors ixazomib, oprozomib, or delanzomib on the accumulation of ubiquitinated OAT3. Top panel: OAT3-expressing COS-7 cells were treated with ixazomib (30 nM), oprozomib (200 nM), or delanzomib (30 nM) for 6 h. Treated cells were then lysed, and OAT3 was immunoprecipitated with anti-Myc antibody or mouse IgG (as negative control, lane 5), followed by IB with anti-Ub. Bottom panel: The same immunoblot from the top panel was reprobed with anti-Myc antibody to determine the amount of OAT3 immunoprecipitated.

**Figure 2.** Effect of ixazomib, oprozomib, or delanzomib on the 20S proteasome activity. OAT3- expressing COS-7 cells were treated with lactacystin (10 μM), a classical proteasome inhibitor as positive control, ixazomib (30 nM), oprozomib (200 nM), or delanzomib (30 nM) for 6 h. The 20S proteasome activity of cells was then performed. The 20S proteasome activity was expressed as the percentage of control cells from three independent experiments. Values are means ± S.D. (n = 3). \* *p* < 0.05.

#### *3.2. Cis-Effect of Ixazomib, Oprozomib, or Delanzomib on OAT3-Mediated Uptake of Estrone Sulfate*

As OAT3 has multispecificity toward multiple substrates, we investigated whether ixazomib, oprozomib, and delanzomib are inhibitors or inducers of OAT3 by performing a cis-inhibition assay. Estrone sulfate (ES) is a prototypical OAT3 substrate, and probenecid is a well-recognized competitive inhibitor of OAT3 [2,40]. We measured 3 min of uptake of [3H]ES (250 nM) into OAT3-expressing cells with or without probenecid, ixazomib, oprozomib, or delanzomib existing in the ES solution. The results (Figure 3) showed that probenecid inhibited OAT3-mediated transport of [3H]ES by 40% (95% CI: 34% to 46%), while ixazomib, oprozomib, and delanzomib did not have any effects, indicating that ixazomib, oprozomib, and delanzomib are not inhibitors or inducers of OAT3. Therefore, ixazomib, oprozomib, and delanzomib did not affect OAT3 function through direct interaction with the transporter.

**Figure 3.** Cis-effect of ixazomib, oprozomib, or delanzomib on OAT3-mediated uptake of [3H]ES. The uptake of [3H]ES (250 nM) in the presence of ixazomib (1 μM), oprozomib (1 μM), delanzomib (1 μM), or probenecid (5 μM) for 3 min was measured in OAT3-expressing COS-7 cells. Each data point represented only carrier-mediated transport after subtraction of values from parental cells. Uptake activity was expressed as the percentage of uptake measured in control cells from three independent experiments. Values are means ± S.D. (n = 3). \* *p* < 0.05; ns = not statistically significant.

#### *3.3. Effects of Ixazomib, Oprozomib, or Delanzomib on OAT3-Mediated Uptake of Estrone Sulfate or P-Aminohippuric Acid*

Since ixazomib, oprozomib, and delanzomib can increase OAT3 ubiquitination, we further investigated their effect on the transport activity. OAT3-expressing cells were treated with ixazomib, oprozomib, or delanzomib for 6 h, then OAT3-mediated uptake of ES was measured. The results (Figure 4A–C) showed that ixazomib, oprozomib, and delanzomib all induced a dose-dependent stimulation of ES uptake in OAT3-expressing COS-7 cells. The OAT3 transport activity was stimulated by 72% (95% CI: 46% to 97%), 45% (95% CI: 33% to 56%), and 48% (95% CI: 31% to 64%) at 30 nM ixazomib, 200 nM oprozomib, and 30 nM delanzomib, respectively. Consistently, 6 h of treatment with classical proteasome inhibitors lactacystin or epoxomicin stimulated the uptake of ES (Figure 4D). Besides, paminohippuric acid (PAH) is another OAT3 substrate [41,42]. Like ES, our result (Figure 5) showed that all three proteasome inhibitor drugs also significantly stimulated PAH uptake in a substrate-independent manner. Similar stimulative effects also existed in OAT3- expressing HEK293 cells, excluding cell-specific effects of proteasome inhibitors (Figure 6). Further study showed that 10~40 nM ixazomib induced a dose-dependent inhibition of proteasome activity (Figure 7A), and there was a strongly association between transport activity and proteasomal activity (correlation coefficient was 0.98, Figure 7B). We selected the concentration of 30 nM ixazomib, 200 nM oprozomib, and 30 nM delanzomib for the following mechanisms study.

**Figure 4.** Effect of ixazomib, oprozomib, delanzomib, or classical proteasome inhibitors on OAT3 activity. OAT3-expressing COS-7 cells were treated with ixazomib ( **A**), oprozomib (**B**), delanzomib (**C**), or classical proteasome inhibitors lactacystin or epoxomicin ( **D**) at indicated concentrations for 6 h. The uptake of [3H]ES (250 nM) for 3 minu was then performed. Each data point represented only carrier-mediated transport after subtraction of values from parental cells. Uptake activity was expressed as the percentage of uptake measured in control cells from three independent experiments. Values are means ± S.D. (n = 3). \* *p* < 0.05; ns = not statistically significant.

**Figure 5.** Effect of ixazomib, oprozomib, or delanzomib on OAT3-mediated transport of p-aminohippuric acid. OAT3-expressing COS-7 cells were treated with ixazomib (30 nM), oprozomib (200 nM), or delanzomib (30 nM) for 6 h. The uptake of [3H]PAH (20 μM) for 3 min was then performed. Each data point represented only carrier-mediated transport after subtraction of values from parental cells. Uptake activity was expressed as the percentage of uptake measured in control cells from three independent experiments. Values are means ± S.D. (n = 3). \* *p* < 0.05.

**Figure 6.** Effect of ixazomib, oprozomib, or delanzomib on OAT3 activity in OAT3-expressing HEK293 cells. OAT3-expressing HEK293 cells were treated with ixazomib (30 nM), oprozomib (200 nM), or delanzomib (30 nM) for 6 h. The uptake of [3H]ES (250 nM) for 3 min was then performed. Each data point represented only carrier-mediated transport after subtraction of values from parental cells. Uptake activity was expressed as the percentage of uptake measured in control cells from three independent experiments. Values are means ± S.D. (n = 3). \* *p* < 0.05.

#### *3.4. Kinetic Analysis of the Effects of Ixazomib, Oprozomib, or Delanzomib on OAT3-Mediated Uptake of Estrone Sulfate*

To examine the mechanism of ixazomib-, oprozomib-, and delanzomib-induced stimulation of OAT3 activity, we determined [3H]ES uptake at a series of concentrations (0.3~10 μM). Eadie–Hofstee analyses of the derived data (Figure 8) showed that incubation of ixazomib (Figure 8A), oprozomib (Figure 8B), or delanzomib (Figure 8C) resulted in an increased maximum transport velocity Vmax (128 ± 3 pmol·mg<sup>−</sup>1·<sup>3</sup> min−<sup>1</sup> with untreated cells and 176 ± 7 pmol·mg<sup>−</sup>1·<sup>3</sup> min−<sup>1</sup> in the presence of ixazomib; 130 ± 2 pmol·mg<sup>−</sup>1·<sup>3</sup> min−<sup>1</sup> with untreated cells and 223 ± 4 pmol·mg<sup>−</sup>1·<sup>3</sup> min−<sup>1</sup> in the presence of oprozomib; 128 ± 11 pmol·mg<sup>−</sup>1·<sup>3</sup> min−<sup>1</sup> with untreated cells and 175 ± 11 pmol·mg<sup>−</sup>1·<sup>3</sup> min−<sup>1</sup> in the presence of delanzomib), with no significant change of substrate-binding-affinity Km for ES (4.2 ± 0.3 μM with untreated cells and 4.6 ± 0.4 μM in the presence of ixazomib; 4.6 ± 0.1 μM with untreated cells and 6.0 ± 0.2 μM in the presence of oprozomib; 4.3 ± 0.9 μM with untreated cells and 4.9 ± 0.7 μM in the presence of delanzomib). These results indicated that stimulated activity of ixazomib, oprozomib, and delanzomib resulted from an increase of the transport rate of OAT3, and not from an enhanced affinity at the substrate-binding site.

**Figure 7.** Dose-effect of ixazomib on the 20S proteasome activity. ( **A**) OAT3-expressing COS-7 cells were treated with ixazomib at indicated concentrations for 6 h. The 20S proteasome activity of cells was then performed. The 20S proteasome activity was expressed as the percentage of control cells from three independent experiments. Values are means ± S.D. (n=3). \* *p* < 0.05. (**B**) Correlation analysis was performed between transport activity from Figure 4A and proteasomal activity from Figure 7A after ixazomib treatment.

**Figure 8.** Effect of ixazomib, oprozomib, or delanzomib on the kinetics of hOAT3-mediated estrone sulfate transport. OAT3-expressing COS-7 cells were treated with 30 nM ixazomib ( **A**), 200 nM oprozomib (**B**), or 30 nM delanzomib ( **C**) for 6 h, and initial uptake (3 min) of [3H]ES was measured at the concentration of 0.3~10 μM. The data represent uptake into hOAT3-expressing cells minus uptake into mock cells (parental COS-7 cells). Values are means ± S.D. (n = 3). V = velocity; S = substrate concentration.

#### *3.5. Effect of Ixazomib, Oprozomib, or Delanzomib on OAT3 Expression*

As ixazomib, oprozomib, and delanzomib did not alter the binding affinity of OAT3, the increase of transport activity may mainly result from the increased expression on the plasma membrane. OAT3-expressing cells were treated with ixazomib, oprozomib, or delanzomib for 6 h, and OAT3 expression on the plasma membrane and in the whole cell lysates were investigated. The result showed that treatment with ixazomib, oprozomib, or delanzomib all caused an increase of OAT3 expression on the cell membrane (Figure 9A,B) and in the whole cell lysate (Figure 9C,D), which was not because of the overall interference in cellular proteins, as there were similar quantities of membrane fraction marker E-Cadherin (Figure 9A) and whole cellular fraction marker β-actin (Figure 9C) in all samples.

**Figure 9.** Effect of ixazomib, oprozomib, or delanzomib on OAT3 expression. (**A**) Top panel: OAT3- expressing COS-7 cells were treated with ixazomib (30 nM), oprozomib (200 nM), or delanzomib (30 nM) for 6 h. Cell-surface biotinylation was performed. Biotinylated (cell surface) proteins were separated with using streptavidin agarose resin and analyzed by IB with an anti-Myc antibody. Bottom panel: The same blot from the top panel was reprobed with an anti-E-Cadherin antibody. E-Cadherin is an integral membrane protein marker. (**B**) Densitometry plot of results from (**A**), top panel, as well as from other experiments. Values are means ± S.D. (n = 3). \* *p* < 0.05. (**C**) Top panel: OAT3-expressing COS-7 cells were treated with ixazomib (30 nM), oprozomib (200 nM), or delanzomib (30 nM) for 6 h. Cells were then lysed, followed by IB with anti-Myc antibody. Bottom panel: The same blot from the top panel was reprobed with an anti-β-actin antibody. β-actin is a cellular protein marker. (**D**) Densitometry plot of results from (**C**), top panel, as well as from other experiments. Values are means ± S.D. (n = 3). \* *p* < 0.05.

#### *3.6. Effect of Ixazomib, Oprozomib, and Delanzomib on OAT3 Degradation*

The ubiquitin–proteasome pathway ultimately affects the degradation of targeted proteins, therefore the degradation of cell-membrane OAT3 was investigated by biotinylation and isolation of cell-surface proteins. OAT3-expressing cells were first labeled with sulfo-NHS-SS-biotin on all membrane proteins at 4 ◦C, then biotinylated cells were incubated with ixazomib, oprozomib, or delanzomib for 3 and 6 h at 37 ◦C. At the time points, those cells were harvested and lysed, and cell-membrane proteins were enriched in streptavidin agarose beads, followed by immunoblotting detection of OAT3 using anti-Myc antibody.

The results (Figure 10) revealed that compared to control, the degradation of cell membrane OAT3 was reduced markedly after 6 h incubation of the three drugs, and without effect at 3 h, indicating that ixazomib, oprozomib, and delanzomib chronically enhanced the stability of membrane OAT3.

**Figure 10.** Effect of ixazomib, oprozomib, or delanzomib on OAT3 stability. (**A**) OAT3-expressing COS-7 cells were biotinylated with membrane-impermeable biotinylation reagen<sup>t</sup> sulfo-NHS-SSbiotin. Labeled cells were then treated with ixazomib (30 nM), oprozomib (200 nM), or delanzomib (30 nM) at 37 ◦C for 3 and 6 h, respectively. Treated cells were lysed, and cell-surface proteins were isolated using streptavidin-agarose resin, followed by IB with anti-Myc antibody. (**B**) Densitometry plot of results from (**A**), as well as from other experiments. The amount of undegraded cell-surface hOAT3 was expressed as % of total initial cell-surface hOAT3 pool. Values are means ± S.D. (n=3). \* *p* < 0.05; ns = not statistically significant. Two-way ANOVA Tukey's test was applied for statistical analysis.
