N-Substituted Pyrido-1,4-Oxazin-3-Ones Induce Apoptosis of Hepatocellular Carcinoma Cells by Targeting NF-κB Signaling Pathway

Abstract

Hepatocellular carcinoma (HCC) is a fatal disease and ranked fifth in cancer related mortality. Persistent activation of NF-κB is responsible for the oncogenesis, metastasis, tumor evasion, anti-apoptosis, angiogenesis and proliferation in HCC. Therefore, designing of chemically novel, biologically potent small molecules that target NF-κB signaling cascade have gained prominent clinical interest. Herein we synthesized a novel class of 4-(substituted)-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one by reacting 2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one with various alkyl halides by using combustion derived bismuth oxide. We evaluated the antiproliferative efficacy of newly synthesized compounds against HCC cells and identified 4-(4-nitrobenzyl)-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one (NPO) as lead anticancer agent. In addition, we investigated the effect of NPO on the DNA binding ability of NF-κB and NF-κB regulated luciferase expression in HCC cells. The results demonstrated that NPO can induce significant growth inhibitory effects in HepG2, HCCLM3 and Huh-7 cells in dose and time-dependent manner. Interestingly, NPO induced significant downregulation in p65 DNA binding ability, p65 phosphorylation and subsequent expression of NF-κB dependent luciferase gene expression in diverse HCC cell lines. Further, in silico docking analysis suggested that NPO can show direct physical interaction with NF-κB. Finally, NPO was found to significantly abrogate tumor growth at a dose of 50 mg/kg in an orthotopic mouse model. Thus, we report the potential anticancer effects of NPO as a novel inhibitor of NF-κB signaling pathway in HCC.

Keywords: NF-κB; anticancer; apoptosis; hepatocellular carcinoma; oxazines.

FIGURE 1

Schematic representation for the synthesis of N-substituted pyrido1,4-oxazin-3-ones.

FIGURE 2

(A–C) NPO the lead compound significantly inhibits the viability of various HCC cells lines. NPO was identified as lead anticancer agent among the structural variants and NPO decreased the growth of HepG2, HCCLM3 and Huh-7 cells in dose- and time-dependent manner. (D) and (E), NPO can induce apoptosis in a dose and time-dependent manner in HCCLM3 cells. ^∗p < 0.05.

FIGURE 3

(A–C) NPO downregulates NF-κB DNA binding ability in HCC cells. HepG2, HCCLM3, and Huh-7 cells were treated with NPO for different time points, nuclear extracts were prepared, and 20 μg of nuclear extract protein was used for DNA binding assay. The NF-κB DNA binding was inhibited in time-dependent manner. (D) Effect of NPO on p65 phosphorylation in HCCLM3 cells. Nuclear extracts were prepared as described in Materials and Methods. HCCLM3 cells were treated with NPO at doses of 10, 25, and 50 μM for 24 h and expression of various proteins was analyzed by western blot analysis. ^∗p < 0.05.

FIGURE 4

NPO inhibits NF-κB dependent luciferase expression in HCC cells: with NF-κB responsive elements linked to a luciferase gene were transfected with wild-type or dominant-negative IκB and transfected (A) HepG2, (B) HCCLM3, and (C) Huh-7 cells were treated with NPO at indicated time points and reporter (luciferase) activity was measured. All luciferase experiments were done in triplicate and repeated twice (^∗p < 0.05).

FIGURE 5

Molecular interactions between NPO and the IκBα/NF-κB complex. (A) Molecular docking solution for NPO (in stick representation, and atomic coloring with orange) targeting IκBα/NF-κB complex. The p50 sub-unit is show in orange, p65 in cyan, and IκBα in green; (B) the interaction map of the complex formed between NPO and IκBα/NF-κB complex is shown.

FIGURE 6

Acute toxicity studies with NPO. (A) The effect of intraperitoneal administration of NPO on body weight change. The nude mice were treated with one single dose of NPO (5, 25, or 50 mg/kg). Error bars are means ± SD. Ordinary one-way ANOVA. (B,C) The effect of NPO on mice behavior study including feed consumption (B) and water intake (C). Error bars are means ± SD. Ordinary one-way ANOVA. (D–F) Effect of NPO on biochemical parameters such as ALT, AST, and BUN levels. Error bars are means ± SD. Ordinary one-way ANOVA.

FIGURE 7

NPO significantly abrogates tumor growth in an orthotopic mouse model. (A) Bioluminescence images of orthotopic tumor bearing mice. Ncr nude mice were orthotopically implanted with HCCLM3_Luc cells and then treated with 0.1% DMSO (n = 8) or 50 mg/kg NPO (n = 8) for 26 days. (B) The scatter plot represents the differences in tumor burden between control and NPO treated mice and is expressed as tumor burden relative to photon counts. The statistics was determined using unpaired t test with Welch’s correction. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.