Improved safety of induced pluripotent stem cell-derived antigen-presenting cell-based cancer immunotherapy.

The tumorigenicity and toxicity of induced pluripotent stem cells (iPSCs) and their derivatives are major safety concerns in their clinical application. Recently, we developed granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing proliferating myeloid cells (GM-pMCs) from mouse iPSCs as a source of unlimited antigen-presenting cells for use in cancer immunotherapy. As GM-pMCs are generated by introducing c-Myc and Csf2 into iPSC-derived MCs and are dependent on self-produced GM-CSF for proliferation, methods to control their proliferation after administration should be introduced to improve safety. In this study, we compared the efficacy of two promising suicide gene systems, herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) and inducible caspase-9 (iCasp9)/AP1903, for safeguarding GM-pMCs in cancer immunotherapy. The expression of HSV-TK or iCasp9 did not impair the fundamental properties of GM-pMCs. Both of these suicide gene-expressing cells selectively underwent apoptosis after treatment with the corresponding apoptosis-inducing drug, and they were promptly eliminated in vivo. iCasp9/AP1903 induced apoptosis more efficiently than HSV-TK/GCV. Furthermore, high concentrations of GCV were toxic to cells not expressing HSV-TK, whereas AP1903 was bioinert. These results suggest that iCasp9/AP1903 is superior to HSV-TK/GCV in terms of both safety and efficacy when controlling the fate of GM-pMCs after priming antitumor immunity.

Generation of GM-CSF-producing antigen-presenting cells that induce a cytotoxic T cell-mediated antitumor response.

Immunotherapy using dendritic cells (DCs) is a promising treatment modality for cancer. However, the limited number of functional DCs from peripheral blood has been linked to the unsatisfactory clinical efficacies of current DC-based cancer immunotherapies. We previously generated proliferating antigen-presenting cells (APCs) by genetically engineering myeloid cells derived from induced pluripotent stem cells (iPSC-pMCs), which offer infinite functional APCs for broad applications in cancer therapy. Herein, we aimed to further enhance the antitumor effect of these cells by genetic modification. GM-CSF gene transfer did not affect the morphology, or surface phenotype of the original iPSC-pMCs, however, it did impart good viability to iPSC-pMCs. The resultant cells induced GM-CSF-dependent CD8+ T cell homeostatic proliferation, thereby enhancing antigen-specific T cell priming in vitro. Administration of the tumor antigen-loaded GM-CSF-producing iPSC-pMCs (GM-pMCs) efficiently stimulated antigen-specific T cells and promoted effector cell infiltration of the tumor tissues, leading to an augmented antitumor effect. To address the potential tumorigenicity of iPSC-derived products, irradiation was applied and found to restrict the proliferation of GM-pMCs, while retaining their T cell-stimulatory capacity. Furthermore, the irradiated cells exerted an antitumor effect equivalent to that of bone marrow-derived DCs obtained from immunocompetent mice. Additionally, combination with immune checkpoint inhibitors increased the infiltration of CD8+ or NK1.1+ effector cells and decreased CD11b+/Gr-1+ cells without causing adverse effects. Hence, although GM-pMCs have certain characteristics that differ from endogenous DCs, our findings suggest the applicability of these cells for broad clinical use and will provide an unlimited source of APCs with uniform quality.

5-Aminolevulinic acid regulates the immune response in LPS-stimulated RAW 264.7 macrophages.

BACKGROUND: Macrophages are crucial players in a variety of inflammatory responses to environmental cues. However, it has been widely reported that macrophages cause chronic inflammation and are involved in a variety of diseases, such as obesity, diabetes, metabolic syndrome, and cancer. In this study, we report the suppressive effect of 5-aminolevulinic acid (ALA), via the HO-1-related system, on the immune response of the LPS-stimulated mouse macrophage cell line RAW264.7. RESULTS: RAW264.7 cells were treated with LPS with or without ALA, and proinflammatory mediator expression levels and phagocytic ability were assessed. ALA treatment resulted in the attenuation of iNOS and NO expression and the downregulation of proinflammatory cytokines (TNF-α, cyclooxygenase2, IL-1ß, IL-6). In addition, ALA treatment did not affect the phagocytic ability of macrophages. To our knowledge, this study is the first to investigate the effect of ALA on macrophage function. Our findings suggest that ALA may have high potential as a novel anti-inflammatory agent. CONCLUSIONS: In the present study, we showed that exogenous addition of ALA induces HO-1 and leads to the downregulation of NO and some proinflammatory cytokines. These findings support ALA as a promising anti-inflammatory agent.

Enhancement of 5-aminolevulinic acid-based fluorescence detection of side population-defined glioma stem cells by iron chelation.

Cancer stem cells (CSCs) are dominantly responsible for tumor progression and chemo/radio-resistance, resulting in tumor recurrence. 5-aminolevulinic acid (ALA) is metabolized to fluorescent protoporphyrin IX (PpIX) specifically in tumor cells, and therefore clinically used as a reagent for photodynamic diagnosis (PDD) and therapy (PDT) of cancers including gliomas. However, it remains to be clarified whether this method could be effective for CSC detection. Here, using flow cytometry-based analysis, we show that side population (SP)-defined C6 glioma CSCs (GSCs) displayed much less 5-ALA-derived PpIX fluorescence than non-GSCs. Among the C6 GSCs, cells with ultralow PpIX fluorescence exhibited dramatically higher tumorigenicity when transplanted into the immune-deficient mouse brain. We further demonstrated that the low PpIX accumulation in the C6 GSCs was enhanced by deferoxamine (DFO)-mediated iron chelation, not by reserpine-mediated inhibition of PpIX-effluxing ABCG2. Finally, we found that the expression level of the gene for heme oxygenase-1 (HO-1), a heme degradation enzyme, was high in C6 GSCs, which was further up-regulated when treated with 5-ALA. Our results provide important new insights into 5-ALA-based PDD of gliomas, particularly photodetection of SP-defined GSCs by iron chelation based on their ALA-PpIX-Heme metabolism.

Dormant cancer cells accumulate high protoporphyrin IX levels and are sensitive to 5-aminolevulinic acid-based photodynamic therapy.

Photodynamic therapy (PDT) and diagnosis (PDD) using 5-aminolevulinic acid (ALA) to drive the production of an intracellular photosensitizer, protoporphyrin IX (PpIX), are in common clinical use. However, the tendency to accumulate PpIX is not well understood. Patients with cancer can develop recurrent metastatic disease with latency periods. This pause can be explained by cancer dormancy. Here we created uniformly sized PC-3 prostate cancer spheroids using a 3D culture plate (EZSPHERE). We demonstrated that cancer cells exhibited dormancy in a cell density-dependent manner not only in spheroids but also in 2D culture. Dormant cancer cells accumulated high PpIX levels and were sensitive to ALA-PDT. In dormant cancer cells, transporter expressions of PEPT1, ALA importer, and ABCB6, an intermediate porphyrin transporter, were upregulated and that of ABCG2, a PpIX exporter, was downregulated. PpIX accumulation and ALA-PDT cytotoxicity were enhanced by G0/G1-phase arrestors in non-dormant cancer cells. Our results demonstrate that ALA-PDT would be an effective approach for dormant cancer cells and can be enhanced by combining with a cell-growth inhibitor.

The effect of iron ion on the specificity of photodynamic therapy with 5-aminolevulinic acid.

Recently, photodynamic therapy using 5-aminolevulinic acid (ALA-PDT) has been widely used in cancer therapy. ALA administration results in tumor-selective accumulation of the photosensitizer protoporphyrin IX (PpIX) via the heme biosynthetic pathway. Although ALA-PDT has selectivity for tumor cells, PpIX is accumulated into cultured normal cells to a small extent, causing side effects. The mechanism of tumor-selective PpIX accumulation is not well understood. The purpose of the present study was to identify the mechanism of tumor-selective PpIX accumulation after ALA administration. We focused on mitochondrial labile iron ion, which is the substrate for metabolism of PpIX to heme. We investigated differences in iron metabolism between tumor cells and normal cells and found that the amount of mitochondrial labile iron ion in cancer was lower than that in normal cells. This finding could be because of the lower expression of mitoferrins, which are the mitochondrial iron transporters. Accordingly, we added sodium ferrous citrate (SFC) with ALA as a source of iron. As a result, we observed the accumulation of PpIX only in tumor cells, and only these cells showed sensitivity to ALA-PDT. Taken together, these results suggest that the uptake abilities of iron ion into mitochondria play a key role in tumor-selective PpIX accumulation. Using SFC as a source of iron might thus increase the specificity of ALA-PDT effects.

Effects of plasma membrane ABCB6 on 5-aminolevulinic acid (ALA)-induced porphyrin accumulation in vitro: tumor cell response to hypoxia.

BACKGROUND: Currently, 5-aminolevulinic acid-based photodynamic diagnosis (ALA-PDD) is used to detect tumors during surgery and exploit tumor-specific accumulation of protoporphyrin IX (PpIX) after administration of ALA. In a recent study, we showed that the human ATP-binding cassette transporter ABCG2 plays a key role in the regulation of PpIX as a specific exporter. However, coproporphyrin III (CPIII) was also detected in urine after ALA administration in patients with tumor, indicating the presence of a CPIII transporter. METHODS: We used two lines of human gastric cancer cells to measure the ALA-induced porphyrin metabolism. Intracellular and extracellular porphyrin levels and expressions of transporter were determined. RESULTS: In the present study, we showed that although ABCG2 did not transport CPIII, plasma membrane ABCB6 did. Moreover, under conditions of hypoxia, the expression of ABCB6 in plasma membrane was upregulated, resulting in increased extracellular CPIII concentrations. CONCLUSION: These data indicate that the expression of ABCB6 in plasma membrane is important for porphyrin accumulation after ALA administration, including hypoxic conditions.

The effects of the heme precursor 5-aminolevulinic acid (ALA) on REV-ERBα activation.

The nuclear receptor, REV-ERBα, has a key role in circadian rhythms and requires heme as its ligand. The present study determined whether the heme precursor, 5-aminolevulinic acid (ALA), affects REV-ERBα and its target genes. When exposed to ALA, the human lung diploid cell line, WI-38, exhibited activation of REV-ERBα and repression of the transcription of REV-ERBα target genes, including BMAL1, an essential component of the circadian oscillator. Moreover, co-incubation of sodium ferrous citrate (SFC) and ALA also activated REV-ERBα and repressed the transcription of REV-ERBα target genes. These results indicate that ALA regulates human circadian rhythms via REV-ERBα.

The heme precursor 5-aminolevulinic acid disrupts the Warburg effect in tumor cells and induces caspase-dependent apoptosis.

Our previous study demonstrated that 5-aminolevulinic acid (ALA) administered to mice stimulates oxidative phosphorylation by upregulation of the mitochondrial respiratory chain complex IV enzyme cytochrome c oxidase (COX). The present study investigated whether ALA disrupts the Warburg effect, which represents a shift in ATP generation from oxidative phosphorylation to glycolysis, protecting tumor cells against oxidative stress-mediated apoptosis. The human lung carcinoma cell line A549 exposed to ALA exhibited enhanced oxidative phosphorylation, which was indicated by an increase in COX protein expression and oxygen consumption. Furthermore, ALA suppressed glycolysis-mediated acidosis. This normalization of the ATP metabolic pathways significantly increased the generation of superoxide anion radical (O2•-) and the functional expression of active caspase-3, leading to caspase-dependent apoptosis. These data demonstrate that ALA inhibits the Warburg effect and induces cancer cell death. Use of this endogenous compound might constitute a novel approach to cancer therapy.

Access to a novel near-infrared photodynamic therapy through the combined use of 5-aminolevulinic acid and lanthanide nanoparticles.

BACKGROUND: There have been considerable efforts to develop photodynamic therapy (PDT) for cancer, in which photoirradiation of a sensitizer delivered near cancer cells results in the conversion of oxygen into active species, causing cell destruction. Aiming at the best cancer selectivity, one PDT method employed protoporphyrin IX (PPIX), which selectively accumulated in cancer cells after oral administration of 5-aminolevulinic acid (ALA). The drawback, however, is that blue incident lights are required to excite PPIX, resulting in low tissue penetrability, and therefore limiting its application to surface cancers. METHODS: To overcome the low penetrability of the incident light, we employed a light energy upconverter, lanthanide nanoparticle (LNP), which, upon irradiation with highly penetrative near-infrared (NIR) radiation, emits visible light within the Q-band region of PPIX absorbance allowing its sensitization. To discover the optimum conditions for the LNP-assisted PDT, the cytotoxicity and PPIX-sensitizability of LNPs were first studied. Then, the LNP-assisted PDT was validated using the MKN45 cell line: cells were pretreated with ALA and LNP, irradiated with a 975-nm diode laser, and subjected to MTT assay to measure cell viability. RESULTS: The singlet oxygen generation on NIR-irradiation of the PPIX-LNP mixture was proved, indicating that the emission from LNP could excite the PPIX sensitizer. An intermittent NIR-irradiation for 32 min of MKN45, pretreated with LNP (1mg/mL) and ALA (2mM), caused 87% cell destruction. CONCLUSIONS: The potential applicability of the NIR-irradiation PDT with ALA- and LNP-pretreated cancer cells was demonstrated.

Expression levels of PEPT1 and ABCG2 play key roles in 5-aminolevulinic acid (ALA)-induced tumor-specific protoporphyrin IX (PpIX) accumulation in bladder cancer.

BACKGROUND: A detection method widely used of late in cancer surgery is 5-aminolevulinic acid-based photodynamic diagnosis (ALA-PDD), which relies on the tumor-specific accumulation of photosensitizing protoporphyrin IX (PpIX) after the administration of ALA. In this regard, we recently reported that peptide transporter PEPT1 and human ATP-binding cassette transporter ABCG2 are key players in regulating intracellular PpIX levels. In the present study, we re-evaluated in vivo the expression of genes involved in the porphyrin biosynthesis pathway. METHODS: Using quantitative real-time (qRT)-PCR, we measured the mRNA levels in a clinical specimen of bladder cancer from a patient who had been subjected to ALA-PDD. RESULTS: We confirmed that PEPT1 and ABCG2 are major contributors to the regulation of tumor-specific PpIX accumulation. qRT-PCR analysis revealed a predominantly high level of PEPT1 mRNA and a very low level of ABCG2 mRNA in the bladder cancer, corresponding to the roles of these genes in vitro. These findings were further confirmed by immunohistochemical studies with PEPT1- and ABCG2-specific antibodies. CONCLUSION: The induction of PEPT1 gene and the suppression of ABCG2 gene expression are among the key molecular mechanisms underlying tumor-specific PpIX accumulation after the administration of ALA in bladder cancer.

Improvement of aminolevulinic acid (ALA)-mediated photodynamic diagnosis using n-propyl gallate.

BACKGROUND: Photodynamic diagnosis (PDD) using aminolevulinic acid (ALA) is widely used in clinical fields. In PDD, protoporphyrin IX (PpIX) is generated from ALA in tumors, allowing the detection of the tumors by PpIX fluorescence. However, it is well known that PpIX is bleached by light irradiation (photobleaching) resulting in reduced PDD efficiency. In this study, n-propyl gallate (NPG) was investigated as an enhancer of PDD efficiency. METHODS: Tumor cells were incubated with NPG after treatment with ALA, and reactive oxygen species and PpIX fluorescence were measured. RESULTS: The antioxidant NPG suppressed the production of reactive oxygen species from light-irradiated porphyrins and ameliorated photobleaching of PpIX generated from ALA in vitro and in vivo. CONCLUSION: Incubation with NPG decreased the production of reactive oxygen species from PpIX and suppressed PpIX photobleaching. These results indicate that the antioxidant NPG may significantly improve PDD efficiency.

Pivotal roles of peptide transporter PEPT1 and ATP-binding cassette (ABC) transporter ABCG2 in 5-aminolevulinic acid (ALA)-based photocytotoxicity of gastric cancer cells in vitro.

BACKGROUND: Recently, 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is being widely used in cancer therapy owing to the tumor-specific accumulation of photosensitizing protoporphyrin IX (PpIX) after the administration of ALA. In the present study, by focusing on genes involved in the porphyrin biosynthesis pathway, we aimed to explore biomarkers that are predictive for the efficacy of ALA-PDT. METHODS: We used five lines of human gastric cancer cells to measure the ALA-based photocytotoxicity. ALA-induced production of PpIX in cancer cells was quantified by fluorescence spectrophotometry. To examine the potential involvement of PEPT1 and ABCG2 in the ALA-PDT sensitivity, stable cell lines overexpressing PEPT1 were established and ABCG2-specific siRNA used. RESULTS: We observed that three cell lines were photosensitive, whereas the other two cell lines were resistant to ALA-based photocytotoxicity. The ALA-based photocytotoxicity was found to be well correlated with intracellular PpIX levels, which suggests that certain enzymes and/or transporters involved in ALA-induced PpIX production are critical determinants. We found that high expression of the peptide transporter PEPT1 (ALA influx transporter) and low expression of the ATP-binding cassette transporter ABCG2 (porphyrin efflux transporter) determined ALA-induced PpIX production and cellular photosensitivity in vitro. CONCLUSION: PEPT1 and ABCG2 are key players in regulating intracellular PpIX levels and determining the efficacy of ALA-based photocytotoxicity against gastric cancer cells in vitro. Evaluation of the expression levels of PEPT1 and ABCG2 genes could be useful to predict the efficacy of ALA-PDT. Primers specific to those target genes are practical and useful biomarkers for predicting the photo-sensitivity to ALA-PDT.

Improvement of tumor localization of photosensitizers for photodynamic therapy and its application for tumor diagnosis.

Photodynamic therapy (PDT) and photodynamic diagnosis of cancer are widely used in clinical fields. These are performed using photosensitizers. Many metalloporphyrin-related compounds have been developed as photosensitizers for use in PDT, and these tumor localization ability have been improved in recent research. Moreover, the precursor of porphyrin 5-aminolevulinic acid is used in fluorescence diagnosis using its tumor localization ability. In this review, these applications of photosensitizers in cancer therapy and diagnosis are summarized.

Porphyrins in urine after administration of 5-aminolevulinic acid as a potential tumor marker.

BACKGROUND: Tumor markers are commonly used for cancer screening and as indicators of therapeutic effects. Certain types of tumor have been known to produce a variety of porphyrins after 5-aminolevulinic acid (ALA) administration. In this study, porphyrins in tumor-bearing mouse urine were analyzed after oral administration of ALA in order to identify new tumor markers excreted in the urine. METHODS: Porphyrin concentrations in the urine of tumor-bearing mice were measured after administration of 1.0mg of ALA (approximately 50mgkg(-1)). RESULTS: Porphyrin concentrations in the urine of tumor-bearing mice increased after administration of ALA. HPLC analysis of the urine revealed the existence of uroporphyrin (UP) and coproporphyrin (CP) in the urine of ALA-treated tumor-bearing mice. Furthermore, at 3h after ALA administration, UP concentrations in the urine of tumor-bearing mice significantly increased compared to those in the urine of normal mice. CONCLUSION: These results suggest that UP as a precursor of heme detected in the urine of tumor-bearing mice after ALA administration is a potential marker of tumor development.

Enhanced expression of coproporphyrinogen oxidase in malignant brain tumors: CPOX expression and 5-ALA-induced fluorescence.

In photodynamic diagnosis, 5-aminolevulinic acid (5-ALA) is widely used for the fluorescence-guided resection of malignant brain tumors, where 5-ALA is converted to protoporphyrin IX, which exhibits strong fluorescence. Little is known, however, about the detailed molecular mechanisms underlying 5-ALA-induced fluorescence. To resolve this issue, we analyzed transcriptome profiles for the genes encoding enzymes, transporters, and a transcription factor involved in the porphyrin-biosynthesis pathway. By quantitative real-time (qRT)-PCR, we measured the mRNA levels of those genes in a total of 20 tumor samples that had been surgically resected from brain tumor patients at the Department of Neurosurgery of Osaka Medical College from 2008 to 2009. We selected 10 tumor samples with no 5-ALA-induced fluorescence, among which 2 were glioblastomas and 8 were metastatic brain tumors. Another 10 tumor samples were selected with strong fluorescence, among which 7 were glioblastomas and 3 were metastatic brain tumors. The qRT-PCR analysis study of these latter 10 samples revealed predominantly high levels of the mRNA of the coproporphyrinogen oxidase (CPOX) gene. The high mRNA level of CPOX expression was significantly well correlated with the phenotype of strong 5-ALA-induced fluorescence (P = .0003). These findings were further confirmed by immunohistochemical studies with a CPOX-specific antibody. It is concluded that induction of CPOX gene expression is one of the key molecular mechanisms underlying the 5-ALA-induced fluorescence of malignant brain tumors. The induction mechanism for the CPOX gene in brain tumors remains to be elucidated.

The effect of 5-aminolevulinic acid on cytochrome c oxidase activity in mouse liver.

BACKGROUND: 5-Aminolevulinic acid (ALA) is a precursor of heme that is fundamentally important in aerobic energy metabolism. Among the enzymes involved in aerobic energy metabolism, cytochrome c oxidase (COX) is crucial. In this study, the effect of ALA on cytochrome c oxidase activity was measured. FINDINGS: c57BL/6N species of mice were administered ALA orally for 15 weeks. After ALA administration, mice were sacrificed and livers were obtained. COX activity in mitochondria from ALA-administered mouse livers was 1.5-fold higher than that in mitochondria from PBS-administered mouse livers (P < 0.05). Furthermore, ATP levels in ALA-administered mouse livers were much higher than those in PBS-administered mouse livers. These data suggest that oral administration of ALA promotes aerobic energy metabolism, especially COX activity. CONCLUSIONS: This is the first report of a drug that functions in aerobic energy metabolism directly. Since COX activity is decreased in various diseases and aging, the pharmacological effects of ALA will be expanding.

Transporter-Mediated Drug Interaction Strategy for 5-Aminolevulinic Acid (ALA)-Based Photodynamic Diagnosis of Malignant Brain Tumor: Molecular Design of ABCG2 Inhibitors.

Photodynamic diagnosis (PDD) is a practical tool currently used in surgical operation of aggressive brain tumors, such as glioblastoma. PDD is achieved by a photon-induced physicochemical reaction which is induced by excitation of protoporphyrin IX (PpIX) exposed to light. Fluorescence-guided gross-total resection has recently been developed in PDD, where 5-aminolevulinic acid (ALA) or its ester is administered as the precursor of PpIX. ALA induces the accumulation of PpIX, a natural photo-sensitizer, in cancer cells. Recent studies provide evidence that adenosine triphosphate (ATP)-binding cassette (ABC) transporter ABCG2 plays a pivotal role in regulating the cellular accumulation of porphyrins in cancer cells and thereby affects the efficacy of PDD. Protein kinase inhibitors are suggested to potentially enhance the PDD efficacy by blocking ABCG2-mediated porphyrin efflux from cancer cells. It is of great interest to develop potent ABCG2-inhibitors that can be applied to PDD for brain tumor therapy. This review article addresses a pivotal role of human ABC transporter ABCG2 in PDD as well as a new approach of quantitative structure-activity relationship (QSAR) analysis to design potent ABCG2-inhibitors.

Key Role of Human ABC Transporter ABCG2 in Photodynamic Therapy and Photodynamic Diagnosis.

Accumulating evidence indicates that ATP-binding cassette (ABC) transporter ABCG2 plays a key role in regulating the cellular accumulation of porphyrin derivatives in cancer cells and thereby affects the efficacy of photodynamic therapy and photodynamic diagnosis. The activity of porphyrin efflux can be affected by genetic polymorphisms in the ABCG2 gene. On the other hand, Nrf2, an NF-E2-related transcription factor, has been shown to be involved in oxidative stress-mediated induction of the ABCG2 gene. Since patients have demonstrated individual differences in their response to photodynamic therapy, transcriptional activation and/or genetic polymorphisms of the ABCG2 gene in cancer cells may affect patients' responses to photodynamic therapy. Protein kinase inhibitors, including imatinib mesylate and gefitinib, are suggested to potentially enhance the efficacy of photodynamic therapy by blocking ABCG2-mediated porphyrin efflux from cancer cells. This review article provides an overview on the role of human ABC transporter ABCG2 in photodynamic therapy and photodynamic diagnosis.

Transport-metabolism interplay: LXRalpha-mediated induction of human ABC transporter ABCC2 (cMOAT/MRP2) in HepG2 cells.

Human ATP-binding cassette (ABC) transporter ABCC2 (cMOAT/MRP2) plays a crucial role in the hepatobiliary transport of sulfate-, glucuronide-, and glutathione-conjugated metabolites as well as a variety of amphiphilic organic anions derived from hepatic metabolism. Molecular mechanisms underlying the induction of this hepatic ABC transporter are of great interest to understand the transport-metabolism interplay in vivo. In the present study, to gain insight into the mechanism of ABCC2 induction, we tested a total of 46 structurally diverse compounds, including nuclear receptor ligands, antibiotics, bile salts, phytochemicals, and anticancer drugs. Among them, we found that LXRalpha ligands, i.e., T0901317, paxilline, and 22(R)-hydroxycholesterol, acted potently to induce the expression of ABCC2 at both mRNA and protein levels in human hepatocellular carcinoma HepG2 cells. The ABCC2 induction by T0901317 was dose- and time-dependent, where the induction pattern of ABCC2 was very similar to that of ABCG1, one of the target genes of LXRalpha. The ABCC2 induction by T0901317 was more strongly elicited when the LXRalpha gene was transiently transfected into HepG2 cells. In contrast, ABCC2 induction by T0901317 was attenuated by transient transfection of a dominant negative LXRalpha variant, suggesting that LXRalpha is involved in ABCC2 induction. Interestingly, RXR, a heterodimer partner of LXRalpha, affected the mRNA levels of ABCC2 and ABCG1 differently. ABCC2 induction by T0901317 was enhanced by RXR siRNA treatment, whereas ABCG1 induction was suppressed by the same treatment. This is the first report demonstrating that LXRalpha is potentially involved in ABCC2 induction.