Pengli Bu

Cytotoxicity assessment of lipid-based self-emulsifying drug delivery system with Caco-2 cell model: Cremophor EL as the surfactant

PURPOSE Caco-2 cells are used extensively for in vitro prediction of intestinal drug absorption. However, toxicity of excipients and formulations used can artificially increase drug permeation by damaging cell monolayers, thus providing misleading results. The present study aimed to investigate cytotoxicity of common lipid-based excipients and formulations on Caco-2 cells. METHODS Medium-chain monoglycerides alone or in mixture with the surfactant Cremophor EL, with and without a medium-chain triglyceride, were prepared and incubated with Caco-2 cells from a series of culture stages with varying maturity. Cell viability was evaluated and cell membrane integrity assessed. RESULTS Cytotoxicity of lipid-based formulations was influenced by the maturity of Caco-2 cells and formulation composition. One-day culture was most sensitive to lipids. When cultured for 5days, viability of Caco-2 cells was significantly improved. The 21-day Caco-2 monolayers maintained the highest survival rate. Microemulsion formulations exhibited significantly less cytotoxicity than neat lipids or surfactant at all stages of cell maturity, and microemulsions containing 1:1 mixtures of monoglyceride and triglyceride appeared to be best tolerated among all the formulations tested. Mechanistically, the observed cytotoxicity was partially due to lipid-induced rupture of cell membrane. CONCLUSIONS Microemulsions of lipid-surfactant mixtures have less cytotoxicity than lipid alone. Maturity of Caco-2 cells renders significant resistance to cytotoxicity, and monolayers with 21-day maturity are more relevant to in vivo conditions and appear to be a more accurate in vitro model for cytotoxicity assessment.

Histone Deacetylase (HDAC) Inhibition Induces IκB Kinase (IKK)-dependent Interleukin-8/CXCL8 Expression in Ovarian Cancer Cells

Overexpression of the pro-angiogenic chemokine IL-8 (CXCL8) is associated with a poor prognosis in several solid tumors, including epithelial ovarian cancer (EOC). Even though histone deacetylase (HDAC) inhibition has shown remarkable antitumor activity in hematological malignancies, it has been less effective in solid tumors, including EOC. Here we report results that may explain the decreased efficiency of HDAC inhibition in EOC, based on our data demonstrating that HDAC inhibition specifically induces expression of IL-8/CXCL8 in SKOV3, CAOV3, and OVCAR3 cells. Suppression or neutralization of vorinostat-induced IL-8/CXCL8 potentiates the vorinostat inhibitory effect on cell viability and proliferation. The IL-8/CXCL8 expression induced by vorinostat in EOC cells is dependent on IκB kinase (IKK) activity and associated with a gene-specific recruitment of IKKβ and IKK-dependent recruitment of p65 NFκB to the IL-8/CXCL8 promoter. In addition, HDAC inhibition induces acetylation of p65 and histone H3 and their IL-8/CXCL8 promoter occupancy. In vivo results demonstrate that combining vorinostat and the IKK inhibitor Bay 117085 significantly reduces tumor growth in nude mice compared with control untreated mice or either drug alone. Mice in the combination group had the lowest IL-8/CXCL8 tumor levels and the lowest tumor expression of the murine neutrophil [7/4] antigen, indicating reduced neutrophil infiltration. Together, our results demonstrate that HDAC inhibition specifically induces IL-8/CXCL8 expression in EOC cells and that the mechanism involves IKK, suggesting that using IKK inhibitors may increase the effectiveness of HDAC inhibitors when treating ovarian cancer and other solid tumors characterized by increased IL-8/CXCL8 expression.

Increased heme synthesis in yeast induces a metabolic switch from fermentation to respiration even under conditions of glucose repression

Regulation of mitochondrial biogenesis and respiration is a complex process that involves several signaling pathways and transcription factors as well as communication between the nuclear and mitochondrial genomes. Under aerobic conditions, the budding yeast Saccharomyces cerevisiae metabolizes glucose predominantly by glycolysis and fermentation. We have recently shown that altered chromatin structure in yeast induces respiration by a mechanism that requires transport and metabolism of pyruvate in mitochondria. However, how pyruvate controls the transcriptional responses underlying the metabolic switch from fermentation to respiration is unknown. Here, we report that this pyruvate effect involves heme. We found that heme induces transcription of HAP4, the transcriptional activation subunit of the Hap2/3/4/5p complex, required for growth on nonfermentable carbon sources, in a Hap1p- and Hap2/3/4/5p-dependent manner. Increasing cellular heme levels by inactivating ROX1, which encodes a repressor of many hypoxic genes, or by overexpressing HEM3 or HEM12 induced respiration and elevated ATP levels. Increased heme synthesis, even under conditions of glucose repression, activated Hap1p and the Hap2/3/4/5p complex and induced transcription of HAP4 and genes required for the tricarboxylic acid (TCA) cycle, electron transport chain, and oxidative phosphorylation, leading to a switch from fermentation to respiration. Conversely, inhibiting metabolic flux into the TCA cycle reduced cellular heme levels and HAP4 transcription. Together, our results indicate that the glucose-mediated repression of respiration in budding yeast is at least partly due to the low cellular heme level.

Assessment of cell viability and permeation enhancement in presence of lipid-based self-emulsifying drug delivery systems using Caco-2 cell model: Polysorbate 80 as the surfactant

Purpose Lipid‐based self‐emulsifying drug delivery systems (SEDDS) are commonly used for solubilizing and enhancing oral bioavailability of poorly water‐soluble drugs. However, their effects on viability of intestine epithelial cells and influence on membrane permeation are poorly understood. The present study was undertaken for safety assessment of lipid‐based formulations containing medium‐chain fatty acid esters as lipids and polysorbate 80 as the surfactant using the Caco‐2 in vitro model. Any possible paracellular permeation enhancement through Caco‐2 monolayers by the nontoxic formulations was also investigated. Methods Mixtures of monoglyceride (Capmul MCM EP or 708G) or propylene glycol monoester (Capmul PG‐8 NF) of medium chain fatty acids with polysorbate 80, with and without the incorporation of a medium‐chain triglyceride (Captex 355), were prepared. After suitable dilution with aqueous culture medium, the formulations were incubated with a series of Caco‐2 cultures of different maturity. Cell viability and membrane integrity were assessed. Any effects of nontoxic formulations on the transport of the fluorescent dye, Lucifer yellow, through Caco‐2 monolayers were also determined. Results Formulations containing 1:1 ratios of monoglyceride or propylene glycol monoester to triglyceride (30% polysorbate 80, 35% monoglyceride or monoester and 35% triglyceride) were best tolerated by Caco‐2 cells. Increased maturity obtained through longer culture durations rendered Caco‐2 cells greater tolerance towards lipid‐based formulations, and maximum tolerance to lipid‐based formulations was observed with Caco‐2 monolayers after being cultured for 21–23 days. Furthermore, extent of cell membrane rupture caused by lipid‐surfactant mixtures correlated positively with levels of cytotoxicity, suggesting a potential underlying mechanism. Permeation studies using Caco‐2 monolayer model revealed that certain formulations significantly enhanced paracellular transport activities. Conclusions Lipid‐based SEDDS containing mixtures of monoglyceride (or monoester) and triglyceride of medium chain fatty acids formed fine microemulsions and were significantly less toxic than other formulations. Fully differentiated Caco‐2 monolayer was more resistant to lipid‐surfactant mixtures than less mature cultures. Certain formulations were also capable of enhancing paracellular permeation.

Activation of GR but not PXR by dexamethasone attenuated acetaminophen hepatotoxicities via Fgf21 induction

Glucocorticoid receptor (GR) signaling is indispensable for cell growth and development, and plays important roles in drug metabolism. Fibroblast growth factor (Fgf) 21, an important regulator of glucose, lipid, and energy metabolism, plays a cytoprotective role by attenuating toxicities induced by chemicals such as dioxins, acetaminophen (APAP), and alcohols. The present study investigates the impact of dexamethasone (DEX)-activated GR on Fgf21 expression and how it affects the progression of APAP-induced hepatotoxicity. Our results showed that DEX dose/concentration- and time-dependently increased Fgf21 mRNA and protein expression in mouse liver as well as cultured mouse and human hepatoma cells. By using PXR-null mouse model, we demonstrated that DEX induced Fgf21 expression by a PXR-independent mechanism. In cultured mouse and human hepatoma cells, inhibition of GR signaling, by RU486 (Mifepristone) or GR silencing using GR-specific siRNA, attenuated DEX-induced Fgf21 expression. In addition, DEX increased luciferase reporter activity driven by the 3.0-kb mouse and human Fgf21/FGF21 gene promoter. Further, ChIP-qPCR assays demonstrated that DEX increased the binding of GR to the specific cis-regulatory elements located in the 3.0-kb mouse and human Fgf21/FGF21 gene promoter. Pretreatment of 2mg/kg DEX ameliorated APAP-induced liver injury in wild-type but not Fgf21-null mice. In conclusion, via GR activation, DEX induced Fgf21 expression in mouse liver and human hepatoma cells.

Berberine-induced Inactivation of Signal Transducer and Activator of Transcription 5 Signaling Promotes Male-specific Expression of a Bile-acid Uptake Transporter

Sodium-taurocholate co-transporting polypeptide (Ntcp/NTCP) is the major uptake transporter of bile salts in mouse and human livers. In certain diseases, including endotoxemia, cholestasis, diabetes, and hepatocarcinoma, Ntcp/NTCP expression is markedly reduced, which interferes with enterohepatic circulation of bile salts, impairing the absorption of lipophilic compounds. Therefore, normal Ntcp/NTCP expression in the liver is physiologically important. Berberine is an herbal medicine used historically to improve liver function and has recently been shown to repress STAT signaling. However, berberine effects on Ntcp/NTCP expression are unknown, prompting use to investigate this possible connection. Our results showed that berberine dose-dependently increased Ntcp expression in male mouse liver and decreased taurocholic acid levels in serum but increased them in the liver. In mouse and human hepatoma cells, berberine induced Ntcp/NTCP mRNA and protein expression and increased cellular uptake of [3H] taurocholate. Mechanistically, berberine decreased nuclear protein levels of phospho-JAK2 and phospho-STAT5, thus disrupting the JAK2-STAT5 signaling. Moreover, berberine stimulated luciferase reporter expression from the mouse Ntcp promoter when one putative STAT5 response element (RE) (−1137 bp) was deleted and from the human NTCP promoter when three putative STAT5REs (−2898, −2164, and −691 bp) were deleted. Chromatin immunoprecipitation demonstrated that berberine decreased binding of phospho-STAT5 protein to the−2164 and −691 bp STAT5REs in the human NTCP promoter. In summary, berberine-disrupted STAT5 signaling promoted mouse and human Ntcp/NTCP expression, resulting in enhanced bile acid uptake. Therefore, berberine may be a therapeutic candidate compound for maintaining bile acid homeostasis.

Metformin as an Anticancer Agent

Metformin has been a frontline therapy for type 2 diabetes (T2D) for many years. Its effectiveness in T2D treatment is mostly attributed to its suppression of hepatic gluconeogenesis; however, the mechanistic aspects of metformin action remain elusive. In addition to its glucose-lowering effect, metformin possesses other pleiotropic health-promoting effects that include reduced cancer risk and tumorigenesis. Metformin inhibits the electron transport chain (ETC) and ATP synthesis; however, recent data reveal that metformin regulates AMP-activated protein kinase (AMPK) and the mechanistic target of rapamycin complex 1 (mTORC1) by multiple, mutually nonexclusive mechanisms that do not necessarily depend on the inhibition of ETC and the cellular ATP level. In this review, we discuss recent advances in elucidating the molecular mechanisms that are relevant for metformin use in cancer treatment.

Epalrestat Stimulated Oxidative Stress, Inflammation, and Fibrogenesis in Mouse Liver

Epalrestat (EPS), an aldose reductase inhibitor, is widely prescribed to manage diabetic neuropathy. It is generally believed that EPS is beneficial to diabetic patients because it can protect endothelial cells, Schwann cells, or other neural cells from oxidative stress. However, several clinical studies revealed that EPS therapy led to liver dysfunction, which limited its clinical applications. Currently, the underlying mechanism by which EPS causes liver dysfunction is unknown. This study aimed to investigate the mechanism responsible for EPS-induced liver injury. In mouse liver, EPS 1) increased oxidative stress, indicated by increased expression of manganese superoxide dismutase, Ho-1, and Nqo1, 2) induced inflammation, indicated by infiltration of inflammatory cells, and induced expression of tumor necrosis factor-alpha, CD11b, and CD11c, as well as 3) predisposed to induce fibrosis, evidenced by increased mRNA and protein expression of early profibrotic biomarker genes procollagen I and alpha-smooth muscle actin, and by increased collagen deposition. In cultured mouse and human hepatoma cells, EPS treatment induced oxidative stress, decreased cell viability, and triggered apoptosis evidenced by increased Caspase-3 cleavage/activation. In addition, EPS increased mRNA and protein expression of cytoglobin in mouse liver, indicating that EPS activated hepatic stellate cells (HSCs). Furthermore, EPS treatment in cultured human HSCs increased cell viability. In summary, EPS administration induced oxidative stress and inflammation in mouse liver, and stimulated liver fibrogenesis. Therefore, cautions should be exercised during EPS therapy.

Reciprocal Regulation of AMPK/SNF1 and Protein Acetylation

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) serves as an energy sensor and master regulator of metabolism. In general, AMPK inhibits anabolism to minimize energy consumption and activates catabolism to increase ATP production. One of the mechanisms employed by AMPK to regulate metabolism is protein acetylation. AMPK regulates protein acetylation by at least five distinct mechanisms. First, AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC) and thus regulates acetyl-CoA homeostasis. Since acetyl-CoA is a substrate for all lysine acetyltransferases (KATs), AMPK affects the activity of KATs by regulating the cellular level of acetyl-CoA. Second, AMPK activates histone deacetylases (HDACs) sirtuins by increasing the cellular concentration of NAD+, a cofactor of sirtuins. Third, AMPK inhibits class I and II HDACs by upregulating hepatic synthesis of α-hydroxybutyrate, a natural inhibitor of HDACs. Fourth, AMPK induces translocation of HDACs 4 and 5 from the nucleus to the cytoplasm and thus increases histone acetylation in the nucleus. Fifth, AMPK directly phosphorylates and downregulates p300 KAT. On the other hand, protein acetylation regulates AMPK activity. Sirtuin SIRT1-mediated deacetylation of liver kinase B1 (LKB1), an upstream kinase of AMPK, activates LKB1 and AMPK. AMPK phosphorylates and inactivates ACC, thus increasing acetyl-CoA level and promoting LKB1 acetylation and inhibition. In yeast cells, acetylation of Sip2p, one of the regulatory β-subunits of the SNF1 complex, results in inhibition of SNF1. This results in activation of ACC and reduced cellular level of acetyl-CoA, which promotes deacetylation of Sip2p and activation of SNF1. Thus, in both yeast and mammalian cells, AMPK/SNF1 regulate protein acetylation and are themselves regulated by protein acetylation.

Hormonal and Chemical Regulation of the Glut9 Transporter in Mice

Glucose transporter (Glut) 9 plays an important role in maintaining the homeostasis of uric acid in the body. Although the physiologic functions of Glut9 have been well established, the regulation of Glut9 expression is less well understood. In this study, we showed that the mRNA and protein expression of Glut9 in mouse liver and kidney are female predominant. Ontogeny studies further revealed that the female-predominant Glut9 expression in mouse liver only occurs in adult mice, which is primarily attributable to the fact that Glut9 expression sustains in females but gradually decreases in males after it reaches the peak level at day 22. Hormone replacement studies in gonadectomized mice, lit/lit mice, and hypophysectomized mice demonstrated that female-predominant Glut9 expression in mouse liver and kidney are primarily due to the inhibitory effects of male-pattern growth hormone secretion, but not sex hormones. In silico analysis of DNA sequences revealed that conserved response elements of signal transducer and activator of transcription 5b, which is an established relay molecule in the growth hormone signaling pathway, are present in mouse and human Glut9/GLUT9 gene promoters, suggesting that Glut9/GLUT9 is a potential target gene of growth hormone. Analysis of mice treated with a panel of chemicals revealed that agonists of the aryl hydrocarbon receptor and the peroxisome proliferator–activated receptor α induced Glut9 mRNA expression in the liver, which is further supported by the presence of conserved xenobiotic response elements and direct repeat 1 DNA motifs in the mouse Glut9 gene promoter. In summary, Glut9 expression is downregulated by male-pattern growth hormone secretion but is upregulated by activation of aryl hydrocarbon receptor and peroxisome proliferator–activated receptor α signaling in mice.