STX64 (Irosustat, 667COUMATE) was kindly supplied by Prof. Barry Potter (University of Oxford, UK). G1 and G15 were purchased from Torcis Bioscience (Abingdon, UK). E₂, tamoxifen, fulvestrant, and BSP were purchased from Sigma-Aldrich (Dorset, UK).

The CRC cell lines Colo205, HCT116, and HT29 were purchased from the American Type Culture Collection, USA; Caco2 and JEG3 cells were purchased from The European Collection of Cell Cultures (ECACC). Prior to experiments, all cell lines were authenticated by short tandem repeat profiling and were used between passages 10 and 35. Furthermore, all experiments were performed during the exponential growth phase of the cell line. HCT116 and HT29 were routinely cultured in McCoy’s 5a modified medium (Gibco, Life Technologies, USA) with 10% v/v heat inactivated FBS (Sigma-Aldrich, UK). Colo205 cells were culture in RPMI with 10% FBS; Caco2 cells were cultured in MEM with 10% FBS. JEG3 cells were cultured in DM-F12 (Gibco, USA) with 10% FBS. All culture mediums were supplemented with 2 mM L-glutamine (Sigma-Aldrich, UK) and 1% PenStrep (Gibco, USA). JEG3 cells were used as control as they exhibit high STS activity.

For experimental conditions, cells were initially starved of estrogens for 72 h by placing them in their appropriate phenol-red free medium plus 10% charcoal stripped FBS (sFBS) (Sigma-Aldrich, UK). After starvation, HCT116 and HT29 cells were treated with E₂ (100 nM), G1 (100 nM), G15 (1 μM), Tamoxifen (10 nM) or fulvestrant (1 μM) in stripped medium for 24 h prior to measuring STS activity.

In vitro STS activities of cell lines were measured as previously described (Purohit et al., 1997). Briefly, in intact cell assays, cells were incubated with appropriate medium containing [6,7-³H] E₁S (4 × 10⁵ dpm, Perkin-ElmerLS, Boston, MA, USA) adjusted to a final concentration of 20 μM with unlabeled E₁S (Sigma-Aldrich, UK). [4-¹⁴C] E₁ (1 × 10⁴ dpm, Perkin-Elmer) was also included in the reaction mixture to monitor procedural losses. Samples were incubated for 18 h at 37°C after which the product, E₁, was separated from E₁S by partition with toluene. A toluene aliquot was removed and ³H and ¹⁴C radioactivity measured by liquid scintillation spectrometry. The mass of E₁S hydrolyzed was calculated from the ³H counts detected corrected for procedural losses. A protein measurement was obtained for the cells by lysing the cells with RIPA buffer (Sigma-Aldrich, UK) followed by a BCA assay (Thermo Fisher Scientific, UK).

To determine STS activity in cell lysates, cells were first lysed with RIPA buffer and protein content determined using a BCA assay. Subsequently, 100 μg of cell protein was incubated for 4 h with PBS containing [6,7-³H] E₁S (4 × 10⁵ dpm) adjusted to a final concentration of 20 μM with unlabeled E₁S. [4-¹⁴C] E₁ (1 × 10⁴ dpm) was again used to monitor procedural losses. E₁ was separated from E₁S by partition with toluene and ³H and ¹⁴C radioactivity measured by liquid scintillation spectrometry. Results for both intact and cell lysates were determined as pmol E₁ formed/h/mg protein.

From cells mRNA was purified from T75 flasks or six well plates at approximately 80% confluency using RNeasy kits (QIAGEN, Crawley, UK) and stored at -80°C. Aliquots containing 5 μg of mRNA were reverse transcribed in a final volume of 20 μl to form cDNA using Tetro cDNA Synthesis Kit (Bioline Reagents, Ltd, UK). RT-PCR reactions were performed in a ‘Rotor Gene 2000 Real-Time Cycler’ (Corbett Life Science, Cambridge, UK) with 1 μl cDNA in a final volume of 12 μl, using Taqman universal PCR master mix and Taqman expression assays containing primers and probes for OATP1A2, OATP1B1, OATP1B3, OATP2B1, OATP3A1, OATP4A1, and for the endogenous control gene, RPLP0 (Applied Biosystems, UK). In CRC, RPLPO is considered the most reliable single standard gene to examine (Sørby et al., 2010). The conditions were as follows: 95°C for 10 min; followed by 40 cycles of 95°C for 15 s, and 60°C for 60 s. Relative mRNA expression was calculated using the comparative quantisation algorithm in the Rotor Gene 6 software (Corbett Life Science).

Cells were seeded at 200,000 cells per well in 6-well plates and allowed to acclimatize for 24 h. Appropriate phenol red free medium containing 10% sFBS plus [6,7-³H] E₁S (4 × 10⁵ dpm) was placed on the cells and was subsequently removed after 2, 5, 10, 15, 20, 30 min. For OATP inhibition studies, the competitive OATP inhibitor BSP (at 1 μM) was added to the medium and therefore was present throughout the 30 min uptake time. The cells were then washed twice in PBS, lysed using RIPA buffer, and the intracellular ³H radioactivity measured by liquid scintillation spectrometry. Cell protein content was also determined with a BCA assay. Results are expressed as E₁S uptake pmol/mg protein.

Protein concentration was determined from cell lysates using the BCA assay, and 15 μg samples were separated by electrophoresis under reducing conditions on 4–12% Bis-Tris 10% SDS-PAGE gels (Invitrogen, Paisley, UK) before being transferred to PVDF membranes. Membranes were immunoblotted with either ERα (1:1000), ERβ (1:1000), GPER (1:800) (all from Santa Cruz, UK), or β-actin (1:50,000) (Sigma-Aldrich, UK) monoclonal antibodies in incubation buffer containing 0.1% milk (Marvel; Premier Brands UK Ltd, Lincolnshire, UK) in TBST. For full details of antibodies and conditions used see Table 1. Bound antibody was detected with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies and chemiluminescence detection.

Twenty four hours after seeding HCT116 cultures were transfected with OATP-specific and control siRNA (Thermo Fisher Scientific, UK) by lipofectamine-2000 (Invitrogen) using standard protocols. 72 h post-transfection, E₁S uptake studies were performed over 30 min and total E₁S uptake calculated.

The panel of four CRC cell lines, plus the positive control JEG3, were investigated and demonstrate a wide range of STS activity (Figures 2A,B). In intact (not lysed) cells, Caco2 exhibited the highest STS activity (65.47 ± 28.51 pmol/mg/h), with HCT116 cells having the lowest activity (1.65 ± 0.14 pmol/mg/h). STS activity could be almost completely inhibited in intact cells using 1 μM of the specific STS inhibitor STX64 (Figure 2A).

When lysed, the intracellular STS activity was much greater in all cell lines examined compared to their intact state (Figure 2B). STS activity was again greatest in the Caco2 cell lines (452.90 ± 56.34 pmol/mg/h) with HCT116 cell showing the lowest STS activity (11.87 ± 6.41 pmol/mg/h). These results demonstrate that E₁S uptake kinetics most likely dictate the ability of CRC to hydrolyze E₁. Therefore, as E₁S is transported through the cell membrane by OATPs the expression of these transporters was next determined.

The mRNA expression of OATPs known to transport E₁S across the plasma membrane was determined in the panel of CRC cell lines (Figure 3). Of the six OATPs examined only OATP4A1 was present in all four cell lines. OATP2B1 expression was also notably high in Caco2 cells. The HT29 cells expressed five of the six OATPs determined (OATP1B3 was not present in HT29 cells), with the Caco2 cells expressing four (OATP1A1, OATP1B3, OATP2B1, OATP4A1) out of six. Our results are roughly consistent with the expression patterns obtained from the CellMiner database¹, as shown by average transcript log² intensities (see Table 2).

When the ability of CRC cells to transport E₁S into the cells was determined, Colo205 demonstrated the quickest uptake rate, with HCT116 cells having the slowest uptake (Figure 4A). Inhibition of OATP transport with the non-specific OATP inhibitor BSP significantly reduced E₁S uptake in all four cell lines (Figures 4B–E). Caco2 cells had the most rapid with HCT116 cells exhibited the slowest E₁S transport. E₁S uptake after 30 min demonstrated that Colo205 (89.41 ± 16.80 pmol/mg) and Caco2 (61.78 ± 10.80 pmol/mg) cells exhibited the most intracellular E₁S transport, with HCT116 (16.73 ± 6.80 pmol/mg) and HT29 (34.59 ± 5.63 pmol/mg) cells showing lower E₁S transport (Figure 4F). BSP significantly (p < 0.001) inhibited E₁S transport in all four cell lines.

To examine if OATP inhibition by BSP reduces E₁S intracellular substrate availability to STS, we examined the effect of BSP on STS activity in intact cells and cell lysates. We selected Caco2 and HCT116 cells as representative of CRC cells with high and low STS activity, respectively. In intact cells, treatment with BPS (1 μM) non-significantly reduced STS activity in Caco2 cells but had no effect on HCT116 STS activity (Figure 5A). When BPS (1 μM) was tested in cell lysates it had no effect on STS activity in both Caco2 and HCT116 cells, suggesting it does not directly inhibit the STS enzyme (Figure 5B). These results imply that OATP transport into cells plays a rate-limiting step on E₁S STS hydrolysis. The discrepancy between Caco2 and HCT116 cells in response to BSP most likely represents the difference between the STS activity in the cell lines. When STS activity is high (Caco2), limiting E₁S OATP transport limits intracellular E₁S availability thus reducing E₁ hydrolysis. When STS activity is low (HCT116), limiting E₁S OATP transport does not directly translate to decreased hydrolysis as there is less STS activity, and thus limiting substrate availability via OATP inhibition and where enzyme activity is already low does not translate to reduced E₁S hydrolysis.

As OATP2B1 and OATP4A1 were expressed at the highest concentrations compared to the other OATPs, we next performed siRNA knockdown of these two transporters in HCT116 cells to determine their importance on E₁S uptake. We used HCT116 cells as they had high OATP2B1 and OATP4A1 expression without high expression of any other OATPs. Furthermore, HCT116 cells are readily transfected by lipofectamine for siRNA delivery. For both OATPs siRNA gave < 95% knockdown as measured by qRT-PCR (Figure 6A). OATP2B1 knockdown did not significantly affect E₁S uptake (Figure 6B), however OATP4A1 knockdown significantly reduced E₁S uptake from 21.02 ± 3.12 to 8.93 ± 2.07 pmol/mg/h (p < 0.01), suggesting this transporter may play a key role in E₁S transport in CRC.

As steroid metabolism and estrogen action in the colon is poorly defined we next speculated whether estrogens may influence steroid sulfatase activity, as it has been reported to do in other malignancies (Zaichuk et al., 2007). Thus we next examined how E₂ starvation and subsequent E₂ supplementation effects STS activity. HT29 cell treated with sFBS medium (i.e., estrogen starvation) demonstrated a trend toward reduction in STS activity, with this effect reversed when supplemented with E₂ (Figure 7A). In HCT116 cells, 24 h of E₂ (100 nM) treatment significantly (p < 0.01) induced STS activity compared to sFBS controls (Figure 7B).

We next attempted to inhibit this E₂-induced increase in STS activity by treating HCT116 with Tamoxifen or fulvestrant co-administered with E₂. Surprisingly, both Tamoxifen and fulvestrant significantly increased STS activity in HCT116 cells (Figure 7C) and neither compound had any effect on E₂-induced STS activity. Tamoxifen (at 10 nM) increased STS activity to 6.43 ± 0.95 pmol/mg/h and fulvestrant (1 μM) increase activity to 7.84 ± 1.36 pmol/mg/h compared to 2.43 ± 0.31 pmol/mg/h.

Examination of the CRC cell lines ER status demonstrated that ERα protein expression was present in Caco2 cells, and lowly expressed in Colo205 cells. HCT116 and HT29 cells did not express ERα (Figure 7D). None of the cell lines expressed ERβ (Figure 7D). We also assessed the GPER status in our CRC cell lines. HCT116 and HT29 cells expressed GPER (Figure 7E), as did Caco2 and Colo205 (data not shown). Unedited immunoblots are shown in Supplementary Figure 1. As both Tamoxifen and fulvastrant have been shown to be GPER agonist (Prossnitz and Barton, 2014) we next examined whether the increased STS activity induced by these compounds could be inhibited by G15, a specific GPER antagonist (Dennis et al., 2009). In HCT116 cells, G15 (1 μM) significantly inhibited the increase of STS activity induced by 24 h treatment with E₂ (100 nM), the GPER agonist G1 (100 nM), Tamoxifen (10 nM), and fulvestrant (1 μM) (Figure 8). This suggests a potential novel positive estrogen metabolism feedback loop through GPER stimulation is present in CRC.