Novel method of measurement of in vitro drug uptake in OATP1B3 overexpressing cells in the presence of dextran.

BACKGROUND: In predictions about hepatic clearance (CLH), a number of studies explored the role of albumin and transporters in drug uptake by liver cells, challenging the traditional free-drug theory. It was proposed that liver uptake can occur for transporter substrate compounds not only from the drug's unbound form but also directly from the drug-albumin complex, a phenomenon known as uptake facilitated by albumin. In contrast to albumin, dextran does not exhibit binding properties for compounds. However, as a result of its inherent capacity for stabilization, it is widely used to mimic conditions within cells. METHODS: The uptake of eight known substrates of the organic anion-transporting polypeptide 1B3 (OATP1B3) was assessed using a human embryonic kidney cell line (HEK293), which stably overexpresses this transporter. An inert polymer, dextran, was used to simulate cellular conditions, and the results were compared with experiments involving human plasma and human serum albumin (HSA). RESULTS: This study is the first to demonstrate that dextran increases compound uptake in cells with overexpression of the OATP1B3 transporter. Contrary to the common theory that highly protein-bound ligands interact with hepatocytes to increase drug uptake, the results indicate that dextran's interaction with test compounds does not significantly increase concentrations near the cell membrane surface. CONCLUSIONS: We evaluated the effect of dextran on the uptake of known substrates using OATP1B3 overexpressed in the HEK293 cell line, and we suggest that its impact on drug concentrations in liver cells may differ from the traditional role of plasma proteins and albumin.

Microcystin-LR-induced epithelial-mesenchymal transition-like cells acquire resistance to multi-toxins.

The protein phosphatase inhibitor microcystin-LR (MC-LR), a hepatocyte-selective cyanotoxin, induces phenotypic changes in HEK293 OATP1B3-expressing (HEK293-OATP1B3) cells, which include cytoskeletal reorganization (HEK293-OATP1B3-AD) and anoikis resistance (HEK293-OATP1B3-FL) transformed cells, respectively. These cells acquire resistance to MC-LR and partial epithelial-mesenchymal transition (EMT) characteristics. In cancer cells, EMT is generally involved in multi-drug resistance. Here, we focused on the multi-drug resistance of HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. The MTT assay and immunoblotting were conducted to examine the responses of HEK293-OATP1B3, HEK293-OATP1B3-AD, and HEK293-OATP1B3-FL cells to multiple toxins and drugs that function as substrates for OATP1B3, including MC-LR, nodularin (Nod), okadaic acid (OA), and cisplatin (CDDP). HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells were more resistant to MC-LR, Nod, and OA than HEK293-OATP1B3 cells. Conversely, the three cell types were equivalently sensitive to CDDP. By using protein phosphatase assay, the reduction of the inhibitory effect of MC-LR and Nod on phosphatase activity might be one reason for the resistance to MC-LR and Nod in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Furthermore, the parental HEK293-OATP1B3 cells showed enhanced p53 phosphorylation and stabilization after MC-LR exposure, while p53 phosphorylation was attenuated in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Moreover, in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells, AKT phosphorylation was higher than that of the parental HEK293-OATP1B3 cell line. These results suggest that the multi-toxin resistance observed in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells is associated with AKT activation and p53 inactivation.

AMP-activated protein kinase re-sensitizes A549 to paclitaxel via up-regulating solute carrier organic anion transporter family member 1B3 expression.

Paclitaxel (PTX) is a common antineoplastic drug whose functionality is often restricted by drug resistance. Solute carrier organic anion transporter family member 1B3 (SLCO1B3) is a PTX influx transporter and its low expression has been proved to be relevant with PTX resistance. It has been widely reported that AMP-activated protein kinase (AMPK) could re-sensitize tumor cells to PTX. Our gene array result demonstrates AMPK up-regulated SLCO1B3. In this paper, we have tried to explain the relationships between PTX, SLCO1B3 and AMPK. First, we have verified the proliferative inhibition of PTX on A549 and found that PTX could inhibit A549 cells proliferation. Then, we have explored the relationship between SLCO1B3 and PTX: SLCO1B3 expression significantly decreased when A549 cells were treated with PTX or in A549 PTX resistant cells (A549-PTX) and the intracellular PTX concentration in A549-PTX was also lower. When treated with metformin/LKB1, both SLCO1B3 expression and intracellular PTX concentration have increased. Knockdown of AMPK has induced decreased SLCO1B3 expression. Moreover, in vitro and in vivo experiments have showed that metformin not only obviously inhibited A549-PTX tumor xenograft and A549-PTX proliferation alone, but also enhanced PTX efficacy to A549-PTX and this may be relevant to SLCO1B3. To verify it, we have treated A549 cells with AMPK both activators and an inhibitor, and then found that AMPK activators could weaken the PTX effect in inhibiting SLCO1B3 while its inhibitor has opposite effect. With knockdown of SLCO1B3, the effect of AMPK in re-sensitizing A549 to paclitaxel has decreased. To sum up, activation of AMPK can up-regulate SLCO1B3 expression, enhance the sensitivity of A549 cells to PTX, providing a new way to re-sensitize PTX resistance.

Transport of salvianolic acid B via the human organic anion transporter 1B1 in the liver.

Salvianolic acid B (SAB) has a high concentration in the liver, but the mechanism of its distribution in the liver is unclear. The aim of this study was to investigate the mechanisms of hepatic uptake of SAB. In this study, we first explored the uptake features of SAB in HepG2 cells and the effect of rifampicin on uptake. Then, we explored the effects of SAB on the uptake of pitavastatin in HepG2 cells. Finally, we established an HEK239T-OATP1B1 cell model to confirm whether OATP1B1 mediated the transport of SAB. Results showed that the uptake kinetic parameters Vmax and Km for SAB in HepG2 cells were 21.28 ± 2.06 pmol mg-1 per protein min-1 and 28.47 ± 7.36 µM, respectively. Rifampicin inhibited the uptake of SAB in HepG2 cells (IC50 was 5.85 ± 1.70 µM), and SAB affected the uptake of pitavastatin in HepG2 cells (IC50 was 27.67 ± 1.90 µM). The uptake kinetic parameters Vmax and Km for SAB in HEK239T-OATP1B1 were 60.03 ± 6.16 pmol mg-1 per protein min-1 and 87.24 ± 15.28 µM, respectively, whereas in EGFP-HEK293 cells were 14.04 ± 2.53 pmol mg-1 per protein min-1 and 56.53 ± 15.50 µM. The SAB had no effect of on the expression of OATP1B1 in HEK239T-OATP1B1 cells. In conclusion, this study demonstrated that OATP1B1 contributes to the transport and accumulation of SAB in the liver.