Novel Indole-fused benzo-oxazepines (IFBOs) inhibit invasion of hepatocellular carcinoma by targeting IL-6 mediated JAK2/STAT3 oncogenic signals

Abstract

Inspired by the well-documented tumor protecting ability of paullones, recently, we synthesized novel paullone-like scaffolds, indole-fused benzo-oxazepines (IFBOs), and screened them against hepatocellular carcinoma (HCC) specific Hep-G2 cells. Three of the synthesized compounds significantly attenuated the progression of HCC in vitro. By computational studies, we further discovered that IFBOs exhibited a stable binding complex with the IL-6 receptor. In this context, we investigated in vivo study using the nitrosodiethyl amine (NDEA)-induced HCC model, which strengthened our previous findings by showing the blockade of the IL-6 mediated JAK2/STAT3 oncogenic signaling pathway. Treatment with IFBOs showed remarkable attenuation of cellular proliferation, as evidenced through a decrease in the number of nodules, restoration of body weight, oxidative stress parameters, liver marker enzymes and histological architecture. Interestingly, using a metabolomic approach we further discovered that IFBOs can restore the perturbed metabolic profile associated with the HCC condition to normalcy. Particularly, the efficacy of compound 6a for an anti-HCC response was significantly better than the marketed chemotherapeutic drug, 5-fluorouracil. Altogether, these remarkable findings open up possibilities of developing IFBOs as novel future candidate molecules for plausible alternatives for HCC treatment.

Figure 1

Structure of indole-fused benzo-oxazepines (IFBOs): 6a (12-(4-Fluorophenyl)-12,12a-dihydro-5H-benzo[2,3][1,4]oxazepino[5,6-b]indole), 10a (5-(12,12a-Dihydro-5H-benzo[2,3][1,4]oxazepino[5,6-b]indol-12-yl)-2-methoxyphenol) and 15a (12-(2-Bromophenyl)-12,12a-dihydro-5H-benzo[2,3][1,4]oxazepino[5,6-b]indole).

Figure 2

Effects of IFBOs after oral administration of 10 mg/kg for 15 days in NDEA-exposed rats (A) Body weight, (B) Enzyme levels of AST, ALT, ALP and LDH in serum, (C) Catabolic by-product (bilirubin and biliverdin), and (D) Anti-proliferative biomarkers IL-2, IL-6, IL-10 and IL-1β in liver carcinogenic tissue. Normal control (NC), Carcinogen control (CC: NDEA), Positive control (PC: NDEA + 5-FU), Treatment T1 (NDEA + 6a), Treatment T2 (NDEA + 10a), Treatment T3 (NDEA + 15a). Results are expressed as mean ± SD (n = 8). Statistically significant differences were observed between carcinogen control (CC) group and test groups (PC, T1, T2 and T3). *p < 0.001, **p < 0.01 and ***p < 0.05, when compared to the carcinogen control (CC) group [one way-ANOVA followed by Bonferroni multiple comparison test].

Figure 3

The histopathological changes (40 × , Scale bar 50 µm) and scanning electron microscopy (X2000) of the liver tissue samples in NDEA-induced HCC rats (A) Normal control (NC), (B) Carcinogen control (CC: NDEA), (C) Positive control (PC: NDEA + 5-FU), (D) T1 (NDEA + 6a), (E) T2 (NDEA + 10a) and (F) T3 (NDEA + 15a). Normal nucleus (N), degenerated nucleus (dN), ruptured hepatic cells (RC), Tumor anaplastic cells (TA), Kupffer cells (K). Induction of carcinogenesis by NDEA is clearly visualized in the diseased group (B). Treated groups (C–F) images show the restoration of liver cell architecture by IFBOs.

Figure 4

(A) mRNA expression levels of IL-6. qRT-PCR analysis confirms IFBOs potential to regulate the expression of IL-6. Results are expressed as mean ± SD (n = 8). Statistically significant differences were observed between carcinogen control (CC) group and test groups (PC, T1, T2 and T3). *p < 0.001, when compared to the carcinogen control (CC) group [Paired T-test] (B) Protein expression levels of IL-6, JAK2, p-JAK2, STAT3 and p-STAT3. Immunoblot analysis confirms IFBOs potential to regulate the expression of IL-6/JAK2/STAT3 signaling. (C) The plausible antitumor activity of IFBOs by the inhibition of IL-6-mediated JAK2/STAT3 activation: Firstly, IL-6 is released strongly in response to various inflammatory stimuli. IL-6 serves as potent JAK2/STAT3 activator by inducing the tyrosine phosphorylation (P) of JAK2 and STAT3. The activated p-STAT3 forms the homo- or heterodimers, which translocate to nucleus, bind to DNA and transcript several oncogenes to accelerate the tumor progression. IFBOs can inhibit the over-expression of IL-6 and IL-6 mediated JAK2 and STAT3.

Figure 5

Stack plot of representative 1D ¹H CPMG NMR spectra of rat serum obtained from different groups. Normal control (NC), Carcinogen control (CC: NDEA), Positive control (PC: NDEA + 5-FU), T1 (NDEA + 6a), T2 (NDEA + 10a), T3 (NDEA + 15a). The abbreviations used are: LDL/VLDL: Low/very-low density lipoproteins; N-acetylglycoprotein: NAG, O-acetyl glycoprotein: OAG; Unsat. Lipids: Unsaturated lipids.

Figure 6

Combined and pair-wise 2D OPLS-DA analysis of 1D ¹H CPMG NMR spectra score plot (A) for the groups: NC (Normal control), CC (Carcinogen control: NDEA), PC (Positive control: NDEA + 5-FU), and (B) for the groups: NC (Normal control), T1 (NDEA + 6a), T2 (NDEA + 10a), T3 (NDEA + 15a), CC (Carcinogen control: NDEA). The potential discriminatory metabolites identified from VIP scores are derived from PLS-DA modeling of complete data matrix, and resulted VIP scores for top 20 metabolites are shown in increasing order of VIP score values to highlight their discriminatory potential.

Figure 7

Metabolic effects of 6a, 10a and 15a treatments: The box-cum-whisker plots are showing relative variations in quantitative profiles of serum metabolites relevant in the context of the pathophysiology of liver cancer. In the box plots, the boxes denote interquartile ranges, horizontal line inside the box denote the median, and bottom and top boundaries of boxes are 25th and 75th percentiles, respectively. Lower and upper whiskers are 5th and 95th percentiles, respectively. NC (Normal control), CC (Carcinogen control: NDEA), T1 (NDEA + 6a), T2 (NDEA + 10a) and T3 (NDEA + 15a).