Fanfan Zhou

Molecular insights into the structure-function relationship of organic anion transporters OATs.

The organic anion transporter (OAT) family encoded by SLC22A mediates the absorption, distribution, and excretion of a diverse array of environmental toxins, and clinically important drugs, including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories, and therefore is critical for the survival of mammalian species. Several OATs have been identified: OAT1 (SLC22A6), OAT2 (SLC22A7), OAT3 (SLC22A8), OAT4 (SLC22A11), OAT5 (SLC22A19) OAT6 (SLC22A20) and URAT1 (SLC22A12). The expressions of these OATs have been detected in key organs such as kidney, liver, brain and placenta. OAT dysfunction in these organs may contribute to the renal, hepatic, neurological and fetal toxicity and diseases. In this review, we summarize, according to the work done by our laboratory as well as by others, the most updated molecular studies on these OAT members, especially on the aspect of their structure–function relationships. The functional roles of N-glycosylation, transmembrane domains and individual amino acids, cell surface assembly, as well as associating proteins will be discussed. In addition, we will show the recent analyses of coding region polymorphisms of OATs, which give us information on the genetic variants of OATs and their potential effects on OAT functions.

Protective Effect of Paeoniflorin on Aβ25–35-Induced SH-SY5Y Cell Injury by Preventing Mitochondrial Dysfunction

Alzheimer’s disease (AD) is a major neurodegenerative brain disorder affecting about 14 million people worldwide. Aβ-induced cell injury is a crucial cause of neuronal loss in AD, thus the suppression of which might be useful for the treatment of this disease. In this study, we aimed to evaluate the effect of paeoniflorin (PF), a monoterpene glycoside isolated from aqueous extract of Radix Paeoniae Alba, on Aβ25–35-induced cytotoxicity in SH-SY5Y cells. The results showed PF could attenuate or restore the viability loss, apoptotic increase, and ROS production induced by Aβ25–35 in SH-SY5Y cells. In addition, PF strikingly inhibited Aβ25–35-induced mitochondrial dysfunction, which includes decreased mitochondrial membrane potential, increased Bax/Bcl-2 ratio, cytochrome c release and activity of caspase-3 and caspase-9. Therefore, our study provided the first experimental evidence that PF could modulate ROS production and apoptotic mitochondrial pathway in model of neuron injury in vitro and which might provide new insights into its application toward Alzheimer’s disease therapy.

Protein kinase C regulates the internalization and function of the human organic anion transporting polypeptide 1A2

BACKGROUND AND PURPOSE The human organic anion transporting polypeptide 1A2 (OATP1A2) is expressed in cells from several regions of the human body, including the kidney, cholangiocytes and the blood‐brain barrier, and mediates the cellular flux of various anionic substances, including drugs in clinical use. Several related mammalian transporters have been shown to be subject to post‐translational regulation, including kinase‐induced internalization. In the present study the role of protein kinase C (PKC) in the regulation of OATP1A2 was investigated in an in vitro cell model.

Comparison of the Interaction of Human Organic Anion Transporter hOAT4 with PDZ Proteins between Kidney Cells and Placental Cells

PurposeTo compare the interaction of human organic anion transporter hOAT4 with PDZ proteins between kidney cells and placental cells.Materials and MethodsPDZ proteins PDZK1 and NHERF1 were transfected into kidney LLC-PK1 cells and placental BeWo cells expressing hOAT4 or hOAT4-Δ, which lacks the PDZ consensus binding site. The interaction of PDZK1 and NHERF1 with hOAT4 and hOAT4-Δ was investigated by measurement of [3H] estrone sulfate uptake, cell surface and total cell expression of hOAT4.ResultsPDZK1 and NHERF1 enhanced hOAT4 activity in LLC-PK1 cells by increasing the cell surface expression of the transporter. In contrasts, these two PDZ proteins had no effect on hOAT4 activity in BeWo cells.ConclusionThe interaction of PDZ proteins with hOAT4 may be cell-specific. In placenta, a different set of interacting proteins from PDZK1 and NHERF1 may be required to modulate hOAT4 activity.

Functional characterization of nonsynonymous single nucleotide polymorphisms in the human organic anion transporter 4 (hOAT4)

Background and purpose:  The human organic anion transporter (hOAT) family of transmembrane carrier proteins mediate the cellular flux of anionic substances, including certain hormones and anti‐cancer drugs. hOAT4 is highly expressed at the apical membrane of the renal tubular cell and facilitates drug re‐absorption in the kidney. In the present study, the impact of 10 nonsynonymous single nucleotide polymorphisms (SNPs) of hOAT4 on transport function in COS‐7 cells was characterized.

Cysteine residues in the organic anion transporter mOAT1

Mouse organic anion transporter 1 (mOAT1) belongs to a family of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, anti-tumour drugs, antibiotics, anti-hypertensives and anti-inflammatories. mOAT1-mediated transport of organic anion PAH ( p -aminohippurate) in HeLa cells was inhibited by the cysteine-modifying reagent PCMBS (p-chloromercuribenzenesulphonate). Therefore the role of cysteine residues in the function of mOAT1 was examined by site-directed mutagenesis. All 13 cysteine residues in mOAT1 were replaced by alanine, singly or in combination. Single replacement of these residues had no significant effect on mOAT1-mediated PAH transport, indicating that no individual cysteine residue is necessary for function. Multiple replacements at a C-terminal region (C335/379/427/434A; Cys(335/379/427/434)-->Ala) resulted in a substantial decrease in transport activity. A simultaneous replacement of all 13 cysteine residues (C-less) led to a complete loss of transport function. The decreased or lack of transport activity of the mutants C335/379/427/434A and C-less was due to the impaired trafficking of the mutant transporters to the cell surface. These results suggest that although cysteine residues are not required for function in mOAT1, their presence appears to be important for the targeting of the transporter to the plasma membrane. We also showed that, although all cysteine mutants of mOAT1 were sensitive to the inhibition by PCMBS, C49A was less sensitive than the wild-type mOAT1, suggesting that the modification of Cys49 may play a role in the inhibition of mOAT1 by PCMBS.

Ziyuglycoside II induces cell cycle arrest and apoptosis through activation of ROS/JNK pathway in human breast cancer cells.

Ziyuglycoside II, a triterpenoid saponin compound extracted from Sanguisorba officinalis L., has been reported to have a wide range of clinical applications including anti-cancer effect. In this study, the anti-proliferative effect of ziyuglycoside II in two classic human breast cancer cell lines, MCF-7 and MDA-MB-231, was extensively investigated. Our study indicated that ziyuglycoside II could effectively induce G2/M phase arrest and apoptosis in both cell lines. Cell cycle blocking was associated with the down-regulation of Cdc25C, Cdc2, cyclin A and cyclin B1 as well as the up-regulation of p21/WAF1, phospho-Cdc25C and phospho-Cdc2. Ziyuglycoside II treatment also induced reactive oxygen species (ROS) production and apoptosis by activating the extrinsic/Fas/FasL pathway as well as the intrinsic/mitochondrial pathway. More importantly, the c-Jun NH2-terminal kinase (JNK), a downstream target of ROS, was found to be a critical mediator of ziyuglycoside II-induced cell apoptosis. Further knockdown of JNK by siRNA could inhibit ziyuglycoside II-mediated apoptosis with attenuating the up-regulation of Bax and Fas/FasL as well as the down-regulation of Bcl-2. Taken together, the cell death of breast cancer cells in response to ziyuglycoside II was dependent upon cell cycle arrest and cell apoptosis via a ROS-dependent JNK activation pathway. Our findings may significantly contribute to the understanding of the anti-proliferative effect of ziyuglycoside II, in particular to breast carcinoma and provide novel insights into the potential application of such compound in breast cancer therapy.

Investigation of Gallic Acid Induced Anticancer Effect in Human Breast Carcinoma MCF‐7 Cells

Gallic acid (GA), a polyhydroxylphenolic compound abundantly distributed in plants, fruits, and foods, has been reported to have various biological activities including an anticancer effect. In this study, we extensively investigated the anticancer effect of GA in human breast carcinoma MCF‐7 cells. Our study indicated that treatment with GA resulted in inhibition of proliferation and induction of apoptosis in MCF‐7 cells. Then, the molecular mechanism of GAs apoptotic action in MCF‐7 cells was further investigated. The results revealed that GA induced apoptosis by triggering the extrinsic or Fas/FasL pathway as well as the intrinsic or mitochondrial pathway. Furthermore, the apoptotic signaling induced by GA was amplified by cross‐link between the two pathways. Taken together, our findings may be useful for understanding the mechanism of action of GA on breast cancer cells and provide new insights into the possible application of such compound and its derivatives in breast cancer therapy.

Functional analysis of novel polymorphisms in the human SLCO1A2 gene that encodes the transporter OATP1A2.

The solute carrier organic anion transporting polypeptide 1A2 (OATP1A2, SLCO1A2) is implicated in the cellular influx of a number of drugs. We identified five novel single nucleotide polymorphisms (SNPs) in coding exons of the SLCO1A2 gene in a cohort of subjects: G550A, G553A, G673A, A775C, and G862A, that encoded the OATP1A2 variants E184K, D185N, V255I, T259P, and D288N, respectively. The function and expression of these variant transporters were assessed in HEK-293 cells. We found that the novel variants, E184K, D185N, T259P, and D288N, were associated with impaired estrone-3-sulfate, imatinib, and methotrexate transport (∼20–50% of wild-type control); function was retained by OATP1A2-V255I. From biotinylation assays, the decreased function of these variants was due, at least in part, to impaired plasma membrane expression. The four loss-of-function variants were studied further using mutagenesis to produce variants that encode residues with different charges or steric properties. From immunoblotting, the replacement of negatively charged residues at amino acid positions 184 and 185 impaired membrane expression, while either a positive or negative charge at residue 288 supported the correct membrane targeting of OATP1A2. Replacement of T259 with bulky residues disrupted transporter stability. From molecular models, E184, D185, and D288 were located near several charged residues such that intramolecular ionic interactions may stabilize the transporter structure. Individuals who carry these novel SNPs in the SLCO1A2 gene may be at risk from impaired efficacy or enhanced toxicity during treatment with drugs that are substrates for OATP1A2.

The inhibitory effects of the bioactive components isolated from Scutellaria baicalensis on the cellular uptake mediated by the essential solute carrier transporters.

Solute carrier transporters (SLCs), in particular the organic anion transporters (OATs), OAT polypeptides (OATPs), and organic cation transporters (OCTs/OCTNs), are the important membrane proteins responsible for the cellular influx of various drugs. Baicalein (BA), baicalin (BG), and wogonin (WG) are the three major bioactive components of Scutellaria baicalensis. In this study, we evaluated the inhibitory effects of BA, BG, and WG on the cellular uptake of specific substrates mediated by the essential SLCs in human embryonic kidney-293 cells. Our data demonstrated that BA and WG significantly inhibit the OAT1-, OAT3-, and OATP1B3-mediated uptake; BG effectively reduces the influx of substrates of OAT3, OAT4, OATP1B3, and OATP2B1; WG is a potent inhibitor of OCT3. Our further kinetic analysis derived the IC50 values of these compounds with pronounced inhibitory effects on SLCs, particularly the inhibitions of WG on OAT1 and OCT3 and that of BA and WG on OAT3. Our study comprehensively evaluated the inhibitory effects of three bioactive components of Scutellaria baicalensis on the uptake of specific substrates mediated by the essential SLC transporters, which suggested that precautions will be needed when coadministrating drugs with Scutellaria baicalensis so as to prevent the unfavorable drug-drug/herb interactions in human.