Anwulignan is a novel JAK1 inhibitor that suppresses non-small cell lung cancer growth

Abstract

Anwulignan is a monomer compound derived from Schisandra sphenanthera lignans. It has been reported to possess a spectrum of pharmacological activities, including anti-bacterial, anti-inflammatory, anticancer and hepatoprotective properties. However, its anticancer capacity and molecular mechanism(s) against non-small cell lung cancer (NSCLC) have not been fully elucidated. Anwulignan significantly inhibited cell growth and increased G1-phase cell cycle arrest in NSCLC cells. Anwulignan strongly attenuates the JAK1/STAT3 signalling pathway by directly targeting JAK1 protein kinase activity in vitro. The anticancer activity by Anwulignan is dependent upon the JAK1 protein expression. Remarkably, Anwulignan strongly inhibited tumour growth in vivo. In conclusion, Anwulignan is a novel JAK1 inhibitor that may have therapeutic implications for NSCLC management.

Keywords: Anwulignan; JAK1; NSCLC; STAT3; cell-derived xenograft.

FIGURE 1

Anwulignan inhibits NSCLC cell growth. A, Chemical structure of Anwulignan. B, Effect on the growth of NSCLC cells by Anwulignan treatment. Cell growth was analysed by the MTT assay. C, Effect on foci formation by Anwulignan treatment. Foci ability was determined by the foci formation assay. D, Effect on anchorage‐independent NSCLC cell growth by Anwulignan treatment. Colonies were imaged using a microscope and quantified with the Image‐Pro PLUS (v.6) computer software program. For B–D, data are shown as means ± SD of values from 3 independent experiments each with triplicate samples. One‐way ANOVA with Tukey's honestly significant difference (HSD) post hoc test was used to analyse the data. The asterisk (*) indicates a significant (P < 0.05) inhibitory effect of Anwulignan

FIGURE 2

Anwulignan induces G1‐phase cell cycle arrest. A, B, Effect of Anwulignan on cell cycle in A549 and H1299 NSCLC cells was examined by Fluorescence‐Activated Cell Sorting (FACS). Data are indicated as means ± SD of values from 3 independent experiments. One‐way ANOVA with Dunnett’s post hoc test was used to analyse the data. The asterisk (*) indicates a significant (P < 0.05) difference between Anwulignan‐treated cells and DMSO‐treated cells. C, Effect of Anwulignan on the expression of cell cycle marker proteins was examined by Western blotting. Band density was measured using the Image J (NIH) software program

FIGURE 3

Anwulignan strongly inhibits the JAK1/STAT3 signalling pathway. A, Effect of Anwulignan on various kinase signalling in NSCLC cells. Cells were treated with Anwulignan for 24 hours and then various signalling molecules were examined by Western blotting. Anwulignan directly binds to JAK1 in (B) an NSCLC cell lysate or (C) recombinant proteins. The cell lysate or recombinant protein was incubated with Anwulignan‐conjugated Sepharose 4B beads or with Sepharose 4B beads alone. Pulled down proteins were analysed by Western blotting. D, Effect of Anwulignan on JAK1 kinase activity was measured by an in vitro kinase assay. For all experiments, similar results were detected from three independent experiments. The asterisk (*) indicates a significant (P < 0.05) inhibitory effect of Anwulignan

FIGURE 4

JAK1 is a therapeutic target in NSCLC cells. A, Effect of JAK1 knockdown on NSCLC cell growth was determined by MTT assay. B, Effect of JAK1 knockdown on foci formation ability of NSCLC cells. Cells were incubated for 1 week and then foci number was counted. C, Effect on anchorage‐independent growth of NSCLC cells by JAK1 knockdown. After incubation for 2 weeks, colony number was counted. For all, data are shown as means ± SD of values from 3 independent experiments. One‐way ANOVA with Dunnett’s post hoc test was used to analyse the data. The asterisk (*) indicates a significant (P < 0.05) inhibitory effect of JAK1 knockdown

FIGURE 5

JAK1‐dependent anticancer activity of Anwulignan. A, B, JAK1‐dependent inhibitory effect of Anwulignan on anchorage‐dependent (A) and anchorage‐independent (B) NSCLC cell growth. shJAK1 or shControl cells were treated with or without with Anwulignan for 48 hours or 2 weeks, respectively. Cell growth was analysed by the MTT assay and soft agar assay. For all experiments, data are shown as means ± SD of values from 3 independent experiments. An un‐paired t test was used to analyse the data. The asterisk (*) indicates a significant effect (P < 0.05) of Anwulignan treatment between JAK1 knockdown cells and shControl cells

FIGURE 6

Anwulignan reduces H1975 cell‐derived xenograft tumour growth in vivo. The effect of Anwulignan on H1975 cell‐derived xenograft tumour growth was assessed. Tumour‐bearing mice (vehicle group or 40 mg/kg of Anwulignan group) were orally treated with Anwulignan or vehicle for 18 days. Effect of Anwulignan on (A) tumour volumes, (B) tumour weight and (C) mice body weight was measured on the indicated days. D, The effect of Anwulignan on the activity of AST or ALT was analysed. All data are shown as mean ± SE from each group. E, Anwulignan inhibits expression of Ki‐67 protein. F, Anwulignan reduces phosphorylated STAT3 expression. Non‐parametric test was used to analyse the data. The asterisk (*) indicates a significant (P < 0.05) inhibitory effect of Anwulignan treatment