Transport of the new chemotherapeutic agent beta-D-glucosylisophosphoramide mustard (D-19575) into tumor cells is mediated by the Na+-D-glucose cotransporter SAAT1

Abstract

For beta-D-glucosylisophosphoramide mustard (beta-D-Glc-IPM), a new alkylating drug in which isophosphoramide mustard is stabilized, a higher selectivity and lower myelotoxicity was observed than for the currently used cytostatic ifosfamide. Because beta-D-Glc-IPM is hydrophilic and does not diffuse passively through the lipid bilayer, we investigated whether a transporter may be involved in the cellular uptake. A variety of cloned Na+-sugar cotransporters were expressed in Xenopus oocytes, and uptake measurements were performed. By tracer uptake and electrical measurements it was found that beta-D-Glc-IPM was transported by the low-affinity Na+-D-glucose cotransporter SAAT1, which had been cloned from pig and is also expressed in humans. At membrane potentials between -50 and -150 mV, a 10-fold higher substrate affinity (Km approximately 0.25 mM) and a 10-fold lower Vmax value were estimated for beta-D-Glc-IPM transport than for the transport of D-glucose or methyl-alpha-D-glucopyranoside (AMG). Transport of beta-D-Glc-IPM and glucose by SAAT1 is apparently performed by the same mechanism because similar sodium dependence, dependence on membrane potential, electrogenicity, and phlorizin inhibition were determined for beta-D-Glc-IPM, D-glucose, and AMG. Transcription of human SAAT1 was demonstrated in various human carcinomas and tumor cell lines. In one of these, the human carcinoma cell line T84, phlorizin inhibitable uptake of beta-D-Glc-IPM was demonstrated with substrate saturation and an apparent Km of 0.4 mM. The data suggest that the Na+-D-glucose cotransporter SAAT1 transports beta-D-Glc-IPM into human tumor cells and may accumulate the drug in the cells. They provide an example for drug targeting by employing a plasma membrane transporter.

Figure 1

Chemical structure of [¹⁴C]β-d-Glc-IPM. The position of ¹⁴C label in the radioactive material is marked by asterisks.

Figure 2

Na⁺-sugar cotransporters homologous to SGLT1 were tested for their capability to transport [¹⁴C]β-d-Glc-IPM. Xenopus laevis oocytes were injected with 50 nl of water with or without 10 ng cRNA of SGLT1 (rabbit), SGLT1 (man), Hu14 (man), SMIT (dog), or SAAT1 (pig). After 3–6 days of incubation, the expression of the respective transporter was controlled by measuring the phlorizin-inhibitable uptake after 30 min of incubation with 50 μM [¹⁴C]AMG (SGLT1s, SAAT1), 1.25 mM [¹⁴C]AMG (Hu14), and 1 μM [³H]myo-inositol (SMIT). The employed phlorizin concentrations were 100 μM (SGLT1 from rabbit), 200 μM (SGLT1 from man, Hu14, SAAT1), or 500 μM (SMIT). The expressed phlorizin-inhibitable uptake rates of AMG and myo-inositol were 274 ± 22 (SGLT1, rabbit), 48 ± 5 (SGLT1, man), 25 ± 1 (Hu14), 6.5 ± 0.7 (SMIT), and 18 ± 2 (SAAT1) pmol × oocyte⁻¹ × h⁻¹, respectively. Under identical experimental conditions the same batches of injected oocytes were tested for phlorizin-inhibitable uptake of 100 μM [¹⁴C]β-d-Glc-IPM. Medians and SEM values from 8–10 parallel determinations without and with phlorizin are indicated.

Figure 3

Time course (a) and stereospecificity (b) of Glc-IPM uptake expressed by SAAT1. Xenopus oocytes were injected with 50 nl of water without (squares in a) or with 10 ng of SAAT1-cRNA (circles in a, and all experiments in b) and incubated for 6 days. Uptake of 200 μM [¹⁴C]β-d-Glc-IPM or [¹⁴C]β-l-Glc-IPM was measured after incubating the oocytes for different time periods (a) or for 30 min (b) either in Ori buffer or in Ori buffer containing 175 μM phlorizin. Uptake rates of [¹⁴C]β-d-Glc-IPM and [¹⁴C]β-l-Glc-IPM in SAAT1-cRNA-injected oocytes are shown in b. In oocytes injected with water identical uptake rates for [¹⁴C]β-d-Glc-IPM and [¹⁴C]β-l-Glc-IPM were measured. These rates were insensitive to phlorizin and not significantly different from those in oocytes injected with SAAT1-cRNA in the presence of phlorizin (data not shown). Medians from 8–10 oocytes and SEM are indicated. Typical experiments out of three are presented.

Figure 4

Substrate dependence (a), phlorizin inhibition (b), and Na⁺ dependence (c) of the expressed [¹⁴C]β-d-Glc-IPM uptake by SAAT1. Xenopus oocytes were injected with 50 nl of water without or with 10 ng of SAAT1-cRNA and incubated for 5 days. In both types of oocytes initial uptake rates of [¹⁴C]β-d-Glc-IPM were measured after 30 min of incubation, and the expressed uptake rates were calculated. (a) The expressed [¹⁴C]β-d-Glc-IPM uptake measured in Ori buffer containing different concentrations of [¹⁴C]β-d-Glc-IPM. (b) The expressed uptake of 0.8 mM [¹⁴C]β-d-Glc-IPM is shown, which was measured in Ori buffer containing different concentrations of phlorizin. (c) The expressed uptake rate of 0.8 mM [¹⁴C]β-d-Glc-IPM measured in the presence of different sodium concentrations. Here sodium in the Ori buffer was replaced by tetramethylammonium.

Figure 5

Potential dependence of SAAT1-mediated currents induced by different concentrations of β-d-Glc-IPM in the presence of Na⁺ (a and c) or by different Na⁺ concentrations in the presence of β-d-Glc-IPM (b and d). Ten nanograms of SAAT1-cRNA was injected into Xenopus oocytes, and the oocytes were incubated for 5 days. (a) The currents are shown that were measured when oocytes clamped at different membrane potentials were superfused with different concentrations of β-d-Glc-IPM in the presence of 100 mM Na⁺. The measurements from three oocytes were normalized to their maximal currents at −150 mV. The Michaelis–Menten equation was fitted to the data obtained at the respective membrane potential, and the K_m values were plotted against the different membrane potentials (c). The currents measured after superfusion of the oocytes with 1 mM β-d-Glc-IPM in the presence of different concentrations of Na⁺ are shown in b. Here measurements from three oocytes were normalized to their current in the presence of 100 mM Na⁺ at −100 mV. Means and SEM values are presented, and the Michaelis–Menten equation was fitted to the currents obtained at each membrane potential. The K_0.5 values for Na⁺ activation at different membrane potentials are presented in d.

Figure 6

Transcription of SAAT1 in human tumor cells. Total RNA was isolated from differentially graded human renal carcinomas (lane c, nuclear grade-1 tumor; lane d, nuclear grade-2 tumor; lane e, nuclear grade-3 tumor), from mouse tumor xenografts (lane f, human colon carcinoma; lane g, human ovary carcinoma), and from the human carcinoma cell lines KTCTL30 (lane h), KTCTL104 (lane i), or T84 (lane k). The RNAs and a control without RNA (lane b) were reverse transcribed, and PCRs were performed with primers that were specific for the cloned fragment of the human SAAT1. The amplification products, size markers, and the control were separated on an agarose gel and stained with ethidium bromide (Upper) or hybridized with an internal primer specific for the human SAAT1 (Lower).

Figure 7

[¹⁴C]β-d-Glc-IPM uptake into suspended human carcinoma T84 cells, which is mediated by a phlorizin-sensitive transporter. (a) The time course of the phlorizin-inhibitable uptake of 0.2 mM [¹⁴C]β-d-Glc-IPM measured in the presence of sodium. (b) The substrate dependence of the initial phlorizin-inhibitable uptake rate of [¹⁴C]β-d-Glc-IPM, which was measured in the presence of Na⁺. Mean values ± SEM from four separate determinations are shown. In the presence of 0.5 mM phlorizin, nonspecific uptake of [¹⁴C]β-d-Glc-IPM was measured, which increased linearly with time and substrate concentration. The nonspecific uptake rate of [¹⁴C]β-d-Glc-IPM was 73 ± 2 pmol × mg ⁻¹ mM⁻¹ min⁻¹.