Cucurbitacin B inhibits the stemness and metastatic abilities of NSCLC via downregulation of canonical Wnt/β-catenin signaling axis

Abstract

Lack of effective anti-metastatic drugs creates a major hurdle for metastatic lung cancer therapy. For successful lung cancer treatment, there is a strong need of newer therapeutics with metastasis-inhibitory potential. In the present study, we determined the anti-metastatic and anti-angiogenic potential of a natural plant triterpenoid, Cucurbitacin B (CuB) against non-small cell lung cancer (NSCLC) both in vitro and in vivo. CuB demonstrated a strong anti-migratory and anti-invasive ability against metastatic NSCLC at nanomolar concentrations. CuB also showed significant tumor angiogenesis-inhibitory effects as evidenced by the inhibition of migratory, invasive and tube-forming capacities of human umbilical vein endothelial cells. CuB-mediated inhibition of angiogenesis was validated by the inhibition of pre-existing vasculature in chick embryo chorio-allantoic membrane and matrigel plugs. Similarly, CuB inhibited the migratory behavior of TGF-β1-induced experimental EMT model. The CuB-mediated inhibition of metastasis and angiogenesis was attributable to the downregulation of Wnt/β-catenin signaling axis, validated by siRNA-knockdown of Wnt3 and Wnt3a. The CuB-mediated downregulation of Wnt/β-catenin signaling was also validated using 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis model in vivo. Collectively, our findings suggest that CuB inhibited the metastatic abilities of NSCLC through the inhibition of Wnt/β-catenin signaling axis.

Figure 1. CuB suppresses cellular migration, invasion and stemness in NSCLC cells at sub-IC₅₀ concentrations.

(A) Wounds were created in subconfluent A549 (Panel A), H1299 (Panel B) and H23 (Panel C) cells and cells were treated with indicated concentrations of CuB for 12, 24 and 48 h. Data were expressed as percent wound closure in treatment groups compared to control. (B) A549, H1299 and H23 cells were seeded in matrigel-coated chambers and treated with indicated concentrations of CuB for 48 h. Data were expressed as percent invasion in treatment groups compared to control (C) A549 and H1299 cells were seeded in ultra-low attachment 96-well plates in 10 replicates with variable concentrations of CuB. The numbers of tumorspheres were counted and represented as average number of tumorspheres per well. (D) The average number of CuB-treated A549 and H1299 colonies containing ≥50 cells were counted and represented as percent of control. Each treatment was repeated in triplicates. Results were obtained from three independent experiments, mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus control.

Figure 2. CuB inhibits angiogenesis in vitro and ex vivo.

(A) Wound healing assays were performed in the subconfluent HUVECs. Images of wound closure were captured at 0 h and 24 h after treatment. Data were expressed as percent wound closure compared to control. (B) HUVECs were seeded onto the upper surface of the matrigel-coated chambers with indicated concentrations of CuB for 24 h. Images of invaded cells were captured and total numbers of invaded cells were counted in 10 different microscopic fields. Each treatment was given in triplicates and data were plotted as percent invasion compared to control. (C) HUVECs were seeded in matrigel-precoated 96-well plates and treated with indicated concentrations of CuB for 6 h. Images of tube formation from each group was captured and total number of nodes and branches were calculated. Each treatment was given in triplicates and results were represented as average number of nodes and branches present in each treatment group. (D) The anti-angiogenic potential of CuB was determined by performing chorio-allantoic membrane (CAM) assay. Images x & y show the CAM membrane exposed after 8 days incubation of fertilized egg. Image z shows the effect of 25 nM CuB on the pre-existing vasculature after two days of exposure. The images are representative of three independent experiments. (E) The in vivo anti-angiogenic potential of CuB was determined by implanting matrigel plugs in the right flank of BALB/c mice, and then by treating the mice with 0.1 mg/kg and 0.2 mg/kg b.w. doses of CuB. Imatinib at 60 mg/kg b.w. dose was used as positive control. Matrigel plugs from different animal groups were excised and photographed. H& E staining was performed to visualize the vascular formation. The arrows in the histopathological sections indicate the presence of endothelial cells. The images are representative of each group. In each case, results were obtained from three independent experiments, mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus control.

Figure 3. CuB inhibits the expression and nuclear translocation of β-catenin in NSCLC cells.

A549 (Panel A) and H1299 (Panel B) cells were seeded on glass cover slips for 24 h and then treated with varying concentrations of CuB for 24 h. Endogenous cytoplasmic and nuclear β-catenin (FITC-green) localization was visualized by immunofluorescence followed by confocal imaging. DAPI was used as nuclear stain (blue). IgG control was used as the negative control. Images are representative of three-independent experiments. The graphs (Panels C,D) represent the raw integrated density as well as CTCF values in A549 and H1299 cells, respectively. The numbers in parenthesis are indicative of number of cells analyzed for each group.

Figure 4. CuB inhibits the Wnt/β-catenin signaling in the NSCLC cells.

A549 (Panel A) and H1299 cells (Panel B) were treated with 0–100 nM of CuB for 24 h and whole cell lysates were prepared. Western blot analysis was performed to analyze the expression of Wnt/β-catenin pathway proteins. Blots are representative of three independent experiments. Graphs in (Panel C,D) represent relative band intensities of proteins in A549 and H1299 cells normalized to β-actin. The results were obtained from three independent experiments, mean ± SEM. *p < 0.05 versus Control.

Figure 5. CuB treatment abrogates TGβ-1-induced EMT in A549 cells.

(A) A549 cells were treated with TGF-β1 (5 ng/mL) for 24 h. TGF-β1 treated and untreated A549 cells were treated with 25 and 50 nM concentrations of CuB for 24 h. Morphological changes are shown in light microscopic images (100× magnification). (B,C) Wounds were created in the sub-confluent cultures of A549 cells and then cells were treated with TGF-β1 (5 ng/mL) and 25 and 50 nM CuB. Photomicrographs were captured at 0, 12 and 24 h after treatment and wound area was measured. Data were expressed as percent wound closure in the treatment groups compared to control. The results were obtained from three independent experiments, mean ± SEM. **p < 0.01 and ***p < 0.001 compared to untreated control, ^##p < 0.01 and ^###p < 0.001 compared to respective TGF-β1-treated control group. (D) A549 cells were treated as in (Panel A), and whole cell lysates were prepared. Western blot analysis was performed to analyze the expression of EMT markers. Blots are representative of three independent experiments.

Figure 6. Wnt/β-catenin signaling is involved in the inhibition of metastatic progression of NSCLC A549 cells.

A549 (Panels A–D) were subjected to treatment with 40 nM of Wnt3 or Wnt3a siRNA, respectively. Control- and Wnt3/3a-siRNA transfected cells were treated with the indicated concentrations of CuB for 24 h. Cell lysates were prepared and protein expressions were analyzed by western blot. Blots are representative of three independent experiments. Graphs represent the relative band intensities normalized with β-actin. A549 (Panels E,F) cells transfected with Wnt3/3a-siRNA were treated with indicated concentrations of CuB for 24 h. Cellular migration was determined and plotted against percent wound closure in control group. Results were obtained from three independent experiments, mean ± SEM, Statistical significance, *p < 0.05, **p < 0.01 and ***p < 0.001 against control siRNA group.

Figure 7. Wnt/β-catenin signaling is involved in the inhibition of metastatic progression of NSCLC H1299 cells.

H1299 cells (Panels A–D) were subjected to treatment with 40 nM of Wnt3 or Wnt3a siRNA, respectively. Control- and Wnt3/3a-siRNA transfected cells were treated with the indicated concentrations of CuB for 24 h. Cell lysates were prepared and protein expressions were analyzed by western blot. Blots are representative of three independent experiments. Graphs represent the relative band intensities normalized with β-actin. H1299 (Panels E,F) cells transfected with Wnt3/3a-siRNA were treated with indicated concentrations of CuB for 24 h. Cellular migration was determined and plotted against percent wound closure in control group. Results were obtained from three independent experiments, mean ± SEM, Statistical significance, *p < 0.05, **p < 0.01 and ***p < 0.001 against control siRNA group.

Figure 8. CuB inhibits NNK-induced lung tumorigenesis and Wnt/β-catenin/MMP-2 signaling.

(A) Representative images of vehicle-treated, NNK-induced CuB-untreated as well as 0.1 mg/Kg and 0.2 mg/Kg b.w. CuB-treated mice lungs. (B) Effects of CuB on lung tumor incidence, lung tumor frequency and relative lung wet weights in NNK-treated mice. mean ± SEM. ^###p < 0.001 compared to vehicle-treated lungs, **p < 0.01 and ***p < 0.001 compared to NNK-induced lungs. (C) Tissue lysates from each animal group were used to analyze the expression of different Wnt/β-catenin signaling markers. β-actin was used as equal loading control. Blots are representative of three independent experiments.

Figure 9. Schematic representation of the mechanism of action of CuB against the metastatic abilities of NSCLC.

CuB inhibits the expression of canonical Wnt ligands, which leads to the degradation or inactivation of β-catenin. This results in the reduction of nuclear translocation of β-catenin leading to inhibition of metastasis and angiogenesis.