Nonclinical testing has served as a foundation for evaluating potential risks and effectiveness of investigational new drugs in humans. However, the current two‐dimensional (2D) in vitro cell culture systems cannot accurately depict and simulate the rich environment and complex processes observed in vivo, whereas animal studies present significant drawbacks with inherited species‐specific differences and low throughput for increased demands. To improve the nonclinical prediction of drug safety and efficacy, researchers continue to develop novel models to evaluate and promote the use of improved cell‐ and organ‐based assays for more accurate representation of human susceptibility to drug response. Among others, the three‐dimensional (3D) cell culture models present physiologically relevant cellular microenvironment and offer great promise for assessing drug disposition and pharmacokinetics (PKs) that influence drug safety and efficacy from an early stage of drug development. Currently, there are numerous different types of 3D culture systems, from simple spheroids to more complicated organoids and organs‐on‐chips, and from single‐cell type static 3D models to cell co‐culture 3D models equipped with microfluidic flow control as well as hybrid 3D systems that combine 2D culture with biomedical microelectromechanical systems. This article reviews the current application and challenges of 3D culture systems in drug PKs, safety, and efficacy assessment, and provides a focused discussion and regulatory perspectives on the liver‐, intestine‐, kidney‐, and neuron‐based 3D cellular models.

The solute carrier family 13 member 5 (SLC13A5), a sodium-coupled citrate transporter, plays a key role in importing citrate from the circulation into liver cells. Recent evidence has revealed that SLC13A5 deletion protects mice from high-fat diet–induced hepatic steatosis and that mutation of the SLC13A5 orthologues in Drosophila melanogaster and Caenorhabditis elegans promotes longevity. However, despite the emerging importance of SLC13A5 in energy homeostasis, whether perturbation of SLC13A5 affects the metabolism and malignancy of hepatocellular carcinoma is unknown. Here, we sought to determine whether SLC13A5 regulates hepatic energy homeostasis and proliferation of hepatoma cells. RNAi-mediated silencing of SLC13A5 expression in two human hepatoma cell lines, HepG2 and Huh7, profoundly suppressed cell proliferation and colony formation, and induced cell cycle arrest accompanied by increased expression of cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin B1. Furthermore, such suppressive effects were also observed on the growth of HepG2 cell-derived xenografts expressing SLC13A5-shRNA in nude mice. Metabolically, knockdown of SLC13A5 in HepG2 and Huh7 cells was associated with a decrease in intracellular levels of citrate, the ratio of ATP/ADP, phospholipid content, and ATP citrate lyase expression. Moreover, both in vitro and in vivo assays demonstrated that SLC13A5 depletion promotes activation of the AMP-activated protein kinase, which was accompanied by deactivation of oncogenic mechanistic target of rapamycin signaling. Together, our findings expand the role of SLC13A5 from facilitating hepatic energy homeostasis to influencing hepatoma cell proliferation and suggest a potential role of SLC13A5 in the progression of human hepatocellular carcinoma.

Constitutive androstane receptor (CAR) (NR1I3), a xenobiotic receptor, has long been considered a master mediator of drug disposition and detoxification. Accumulating evidence indicates that CAR also participates in various physiologic and pathophysiological pathways regulating the homeostasis of glucose, lipid, and bile acids, and contributing to cell proliferation, tissue regeneration and repair, as well as cancer development. The expression and activity of CAR can be regulated by various factors, including small molecular modulators, CAR interaction with other transcription factors, and naturally occurring genetic variants. Given that the influence of CAR has extended beyond the realm of drug metabolism and disposition and has expanded into a potential modulator of human diseases, growing efforts have centered on understanding its clinical relevance and impact on human pathophysiology. This review highlights the current information available regarding the contribution of CAR to various metabolic disorders and cancers and ponders the possible challenges that might arise from pursuing CAR as a potential therapeutic target for these diseases. SIGNIFICANCE STATEMENT The growing importance of the constitutive androstane receptor (CAR) in glucose and lipid metabolism as well as its potential implication in cell proliferation emphasizes a need to keenly understand the biological function and clinical impact of CAR. This minireview captures the clinical relevance of CAR by highlighting its role in metabolic disorders and cancer development.

Phenobarbital (PB), a broadly used antiseizure drug, was the first to be characterized as an inducer of cytochrome P450 by activation of the constitutive androstane receptor (CAR). Although PB is recognized as a conserved CAR activator among species via a well-documented indirect activation mechanism, conflicting results have been reported regarding PB regulation of the pregnane X receptor (PXR), a sister receptor of CAR, and the underlying mechanisms remain elusive. Here, we show that in a human CAR (hCAR)-knockout (KO) HepaRG cell line, PB significantly induces the expression of CYP2B6 and CYP3A4, two shared target genes of hCAR and human PXR (hPXR). In human primary hepatocytes and hCAR-KO HepaRG cells, PB-induced expression of CYP3A4 was markedly repressed by genetic knockdown or pharmacological inhibition of hPXR. Mechanistically, PB concentration dependently activates hPXR but not its mouse counterpart in cell-based luciferase assays. Mammalian two-hybrid assays demonstrated that PB selectively increases the functional interaction between the steroid receptor coactivator-1 and hPXR but not mouse PXR. Moreover, surface plasmon resonance binding affinity assay showed that PB directly binds to the ligand binding domain of hPXR (K_D = 1.42 × 10⁻⁰⁵). Structure-activity analysis further revealed that the amino acid tryptophan-299 within the ligand binding pocket of hPXR plays a key role in the agonistic binding of PB and mutation of tryptophan-299 disrupts PB activation of hPXR. Collectively, these data reveal that PB, a selective mouse CAR activator, activates both hCAR and hPXR, and provide novel mechanistic insights for PB-mediated activation of hPXR.

The constitutive androstane receptor (CAR) is a nuclear receptor that plays a crucial role in regulating xenobiotic metabolism and detoxification, energy homeostasis, and cell proliferation by modulating the transcription of numerous target genes. CAR activation has been established as the mode of action by which phenobarbital-like nongenotoxic carcinogens promote liver tumor formation in rodents. This paradigm, however, appears to be unrelated to the function of human CAR (hCAR) in hepatocellular carcinoma (HCC), which remains poorly understood. Here, we show that hCAR expression is significantly lower in HCC than that in adjacent nontumor tissues and, importantly, reduced hCAR expression is associated with a worse HCC prognosis. We also show overexpression of hCAR in human hepatoma cells (HepG2 and Hep3B) profoundly suppressed cell proliferation, cell cycle progression, soft-agar colony formation, and the growth of xenografts in nude mice. RNA-Seq analysis revealed that the expression of erythropoietin (EPO), a pleiotropic growth factor, was markedly repressed by hCAR in hepatoma cells. Addition of recombinant EPO in HepG2 cells partially rescued hCAR-suppressed cell viability. Mechanistically, we showed that overexpressing hCAR repressed mitogenic EPO–EPO receptor signaling through dephosphorylation of signal transducer and activator of transcription 3, AKT, and extracellular signal–regulated kinase 1/2. Furthermore, we found that hCAR downregulates EPO expression by repressing the expression and activity of hepatocyte nuclear factor 4 alpha, a key transcription factor regulating EPO expression. Collectively, our results suggest that hCAR plays a tumor suppressive role in HCC development, which differs from that of rodent CAR and offers insight into the hCAR–hepatocyte nuclear factor 4 alpha–EPO axis in human liver tumorigenesis.

A prodrug is a molecule that lacks pharmacological activity, but upon enzymatic bioactivation, it can generate a therapeutically active molecule. The primary reason behind the design of a prodrug is to help circumvent challenges associated with the physicochemical properties of a drug molecule, such as solubility, absorption, distribution, and instability. Chemotherapy has been at the forefront of cancer treatment for over 70 years due to its ability to target rapidly proliferating tumor cells. However, a major concern with conventional chemotherapy is the lack of selectivity and its associated side toxicity, which can severely impact patients’ quality of life. In oncology, prodrugs have been explored to enhance the bioavailability, improve efficacy, and minimize systemic toxicity of chemotherapeutic agents. Prodrugs activated by enzymes unique to a tumor microenvironment can significantly increase targeted delivery of chemotherapeutic drugs. This review aims to highlight commonly used chemotherapeutic prodrugs, including both alkylating and non-alkylating agents, and discuss their clinical relevance, mechanisms of bioactivation, and toxicity concerns.