Phylogenetic relationships among basal Hexapods inferred from Bayesian analysis of protein-coding gene sequences.

<p>2 Crustacean species <i>Capitulum mitella</i> and <i>Pollicipes mitella</i> were used as the outgroup. Numbers denoted posterior probabilities of nodes.</p

Phylogenetic relationships among basal Hexapods inferred from Maximum parsimony of 13 protein-coding sequences.

<p>1 Protura species, 10 Collembola species and 3 Diplura species were used as the outgroup. Numbers denoted bootstrap values in percentages.</p

UDP-Glucuronosyltransferase 1A Determinates Intracellular Accumulation and Anti-Cancer Effect of β-Lapachone in Human Colon Cancer Cells

<div><p>β-lapachone (β-lap), an NAD(P)H:quinone oxidoreductase 1 (NQO1) targeting antitumor drug candidate in phase II clinical trials, is metabolically eliminated via NQO1 mediated quinone reduction and subsequent UDP-glucuronosyltransferases (UGTs) catalyzed glucuronidation. This study intends to explore the inner link between the cellular glucuronidation and pharmacokinetics of β-lap and its apoptotic effect in human colon cancer cells. HT29 cells S9 fractions exhibited high glucuronidation activity towards β-lap, which can be inhibited by UGT1A9 competitive inhibitor propofol. UGT1A siRNA treated HT29 cells S9 fractions displayed an apparent low glucuronidation activity. Intracellular accumulation of β-lap in HCT116 cells was much higher than that in HT29 cells, correlated with the absence of UGT1A in HCT116 cells. The cytotoxic and apoptotic effect of β-lap in HT29 cells were much lower than that in HCT116 cells; moreover, β-lap triggered activation of SIRT1-FOXO1 apoptotic pathway was observed in HCT116 cells but not in HT29 cells. Pretreatment of HT29 cells with UGT1A siRNA or propofol significantly decreased β-lap’s cytotoxic and apoptotic effects, due to the repression of glucuronidation and the resultant intracellular accumulation. In conclusion, UGT1A is an important determinant, via switching NQO1-triggered redox cycle to metabolic elimination, in the intracellular accumulation of β-lap and thereafter its cytotoxicity in human colon cancer cells. Together with our previous works, we propose that UGTs determined cellular pharmacokinetics is an important determinant in the apoptotic effects of NQO1 targeting substrates serving as chemotherapeutic drugs.</p></div

UGT1A activity affects β-lap -induced apoptosis to Colon Cancer Cells.

<p>Cells were pretreated with propofol (100 μM) for 1 h or UGT1A siRNA for 24 h. Then cells were exposed to β-lap (5 μM) for 24 h and collected. Cell apoptosis was assessed by TUNEL assay. (A) Cell apoptosis in HCT116 cells with propofol/NAC/catalase pretreatment; (B) Cell apoptosis in HT29 cells with propofol/NAC/catalase pretreatment; (C) Cell apoptosis in HT29 cells with siRNA/NAC/catalase pretreatment. Results are presented as mean ± 3 SEM of at least three independent experiments (*P<0.05, **P<0.01, β-lap treatment vs. control cells; #P<0.05, NAC pretreatment vs. corresponding β-lap only; $P<0.05, catalase pretreatment vs. corresponding β-lap only).</p

Phylogenetic relationships among basal Hexapods inferred from Bayesian analysis of 13 protein-coding sequences.

<p>1 Protura species, 10 Collembola species and 3 Diplura species were used as the outgroup. Numbers denoted posterior probabilities of nodes.</p

Intracellular accumulation and glucuronidation of β-lap in colon cancer cells.

<p>HT29 cells were pretreated with propofol (100 μM) for 1 h or UGT1A siRNA for 24 h. Cells were exposed to β-lap (5 μM) for indicated time. (A) Intracellular β-lap level in HCT116 is much higher than in HT29 cells; (B) Propofol or siRNA pretreatment lead to intracellular accumulation of β-lap in HT29 cells; (C) Propofol or siRNA pretreatment decreases intracellular M2 level in HT29 cells; (D) Propofol or siRNA pretreatment decreases M2 level in HT29 cell culture medium. Data are shown as mean 3 ± SEM of at least three independent experiments (*P<0.05, **P<0.01, ***P<0.001, propofol or UGT1A siRNA pretreatment vs. control cells).</p

AUC_{0–120 min} and C_max values of β-lap and its glucuronide (M2) with or without propofol pretreatment in HT29 and HCT116 cells and culture medium.

<p>Data are shown as mean ± SD of three independent experiments; **P<0.01, ***P<0.001, propofol pretreatment vs.β-lap only; ###P<0.001, HCT116 vs. HT29.</p><p>AUC_{0–120 min} and C_max values of β-lap and its glucuronide (M2) with or without propofol pretreatment in HT29 and HCT116 cells and culture medium.</p

UGT1A compromises β-lap-activated FOXO1 apoptosis pathway in colon cancer cells.

<p>Cells were pretreated with propofol (100 μM) for 1 h or UGT1A siRNA for 24 h. Then, cells were exposed to β-lap (5 μM) for 24 h and collected. Cell mRNA levels were assessed by PCR and protein levels by western blot assays. (A) Relative mRNA and protein levels in HCT116 cells with propofol/NAC/catalase pretreatment; (B) Relative mRNA and protein levels in HT29 cells with propofol/NAC/catalase pretreatment; (C) Relative mRNA and protein levels in HT29 cells with siRNA/NAC/catalase pretreatment. Results are presented as mean ± 3 SEM of at least three independent experiments (*P<0.05, **P<0.01, ***P<0.001, propofol pretreatment vs. control cells, or UGT1A siRNA pretreatment vs. scrambled siRNA pretreatment; #P<0.05, ###P<0.001, NAC pretreatment vs. corresponding β-lap only; $P<0.05,$ $P<0.01, catalase pretreatment vs. corresponding β-lap only).</p

UGT1A Diminishes β-lap -induced ROS Formation.

<p>Cells were pretreated with UGT1A siRNA or scrambled siRNA (negative control) for 24 h, or pretreated with propofol (100 μM)/ NAC (5 μM)/ catalase (4000 U) for 1 h. Then, cells were exposed to β-lap (5 μM) for 2 h and ROS assay or GSSG/GSH ratio assay was performed. (A) ROS formation and GSSG/GSH ratio in HCT116 cells pretreated with propofol/NAC/catalase; (B) ROS formation and GSSG/GSH ratio in HT29 cells pretreated with propofol/NAC/catalase; (C) ROS formation and GSSG/GSH ratio in HT29 cells pretreated with siRNA/NAC/catalase. Results are presented as mean ± 3 SEM of at least three independent experiments (*P<0.05, **P<0.01, ***P<0.001, β-lap treatment vs. control cells; #P<0.05, ##P<0.01, NAC pretreatment vs. corresponding β-lap only; $P<0.05,$$P<0.01,$ $$P<0.001, catalase pretreatment vs. corresponding β-lap only).</p

Glucuronidation of β-lap in HT29 cell S9 fractions.

<p>HT29 cells S9 were incubated with β-lap according to the details in methods. (A) A typical Michaelis-Menten kinetics of β-lap glucuronidation in HT29 cells S9 fractions; (B) UGT1A siRNA treated HT29 cells S9 fractions significantly decreases β-lap glucuronidation; (C) Inhibitory potency of propofol on β-lap glucuronidation in HT29 cell S9 fractions. Results are presented as mean ± 3 SEM of three independent experiments (***P<0.001, UGT1A siRNA treatment vs. negative control cells).</p