Boeravinone B a natural rotenoid exerts anticancer activity via inducing internalization and degradation of inactivated EGFR and ErbB2 in human colon cancer cells

Abstract

Background: Epidermal growth factor receptors (EGFR) are identified to be favorable targets for cancer treatment. In present work, we showed that Boeravinone B, a Rotenoid from natural origin has significant anticancer activity via internalization of ErbB2 and EGFR, and thereby resulting in destruction of the receptors.

Methods: For cell viability and apoptosis were done by MTT assay. Annexin V-FITC staining was done for determining the extent of apoptosis. Immunoblotting for expression of proteins in HT-29 cell lysates after exposing them to Boeravinone G. Immunofluorescence and Confocal microscopic analysis was done for HT-29 cells incubated with anti-EGFR or anti-ErbB2 antibodies. Surface biotinylation assay was done followed by western blot analysis for expression of proteins using antibodies against transferrin receptor, ErbB2 and EGFR.

Results: Exposure of HT-29 cells with Boeravinone B suppressed constitutive as well as ligand mediated phosphorylation of ErbB2, ErbB3 and EGFR. The treatment also inhibited the activation of mitogen-activated protein kinase (MAPK), Akt and Erk1/2 which are downstream signaling molecules. The treatment also bought about internalization of ErbB2 and EGFR causing destruction of receptors, Boeravinone B also caused apoptosis in HT-29 cells. Boeravinone B mediated degradation was halted by Chloroquine (lysosomal inhibitor). Boeravinone B caused nuclear translocation of apoptosis-inducing factor (AIF) and caused proteolytic processing of PARP along with caspase-3, confirming Boeravinone B may induce caspase-independent apoptosis in HT-29 cells.

Conclusion: The findings of present study provide first ever evidences for Boeravinone B suggesting anticancer activity via internalization and destruction of EGFR family receptors i.e. ErbB2 and EGFR in HT-29 cell lines.

Keywords: Boeravinone; EGFR; HT-29 cells; internalization.

Figure 1

Effect of Boeravinone B on human colon cancer cell viability. A. The human colon cancer cells were treated with Boeravinone B for 48 h followed by MTT assay for cell viability, results are percentage mean ± SD of the number cell of control (n = 2 experiments). B. Immunoblotting studies shows expression of ErbB3, ErbB3 and EGFR in selected three cell lines (SW-620, HCT-116 and HT-29), α-tubulin was used as loading control.

Figure 2

Effect of Boeravinone B on levels of p-ErbB2, p-ErbB3 and EGFR. A. The human colon cancer HT-29 cells were treated with 0, 1, 3 and 10 µM concentration of Boeravinone B for 24 h followed by western blot analysis for expression of ErbB2, ErbB3, EGFR and transferrin (TfR) B. The HT-29 cells were treated with Boeravinone B (10 µM) for 1, 3, 6, 12 and 24 h. The cell lysates were subjected to western blot analysis along with antibodies specific for ErbB2, p-ErbB2, ErbB3, p-ErbB3, EGFR, p-EGFR and TfR. C. The HT-29 cells were treated with predefined concentrations of Boeravinone B for 3 h. The cell lysates were subjected to western blot analysis using antibodies specific for AKT, pAKT, Erk1/2 and p-Erk1/2. In all the three experiments the blots were compared against loading control α-tubulin.

Figure 3

Effect of Boeravinone B on activation of ErbB2, ErbB3 and EGFR mediated by EGF and HRG. A. The HT-29 cells were pre-treated with Boeravinone B at defined concentrations for 3 h followed by treatment with EGF (50 ng/ml) for 15 min. The whole cell lysates were incubated with antibodies specific for ErbB2, and phospho-ErbB2 and EGFR, phospho-EGFR. B. The human colon cancer HT-29 cells were pre-exposed with 10 µM concentration of Boeravinone B for 3 h followed by treatment of 15 with EGF (50 ng/ml). The lysates were subjected to western blot analysis with antibodies specific for Erk1/2 pErk1/2, AKT and pAKT. C. The HT-29 cells after exposure to Boeravinone B for 3 h followed by treatment with HRG (50 ng/ml) for 15 min followed by stimulation with HRG (50 ng/ml). Western blot analysis was done for lysate along with antibodies specific for ErbB2, p-ErbB2, ErbB3 and p-ErbB3. D. The HT-29 cells were pre-exposed with 10 µM concentration of Boeravinone B for 3 h followed by treatment with HRG (50 ng/ml) for 15 min. The lysates were subjected to western blot analysis with antibodies specific for Erk1/2, pErk1/2, AKT and p-AKT.

Figure 4

Boeravinone B leads to internalization of ErbB2 and EGFR in human colon cancer HT-29 cells. A. The HT-29 cells were treated with 10 µM concentration of Boeravinone B for 24 h along with Lactacystin (5 µM) and Chloroquine (30 µM) followed by western blot analysis of lysates along with ErbB2 and EGFR antibodies. B. Western blot analysis for conforming internalization of ErbB2, EGFR and TfR by surface biotinylation assay in HT-29 cells treated with Boeravinone B, control and positive control (EGF treated for 0.5 h). C. The colon cancer HT-29 cells were treated with 10 µM of Boeravinone B for 0, 3 and 24 h, after fixation and incubating cells with anti-EGFR or anti-ErbB2 antibodies followed by subsequent exposure to Alexa 546-conjugated anti-rabbit secondary followed by microscopic analysis.

Figure 5

Effect of Boeravinone B on apoptosis. A. The HT-29 cells were treated with predefined concentrations of Boeravinone B for 48 h and stained with Annexin V-FITC and propidium iodide followed by flow cytometry analysis. B. The Annexin V positive cells shown as percentage of apoptotic cells, data is mean ± SD (n = 3). The *P < 0.05 and **P < 0.01 compared to control.

Figure 6

A. Boeravinone B inhibited proteolytic processing of PARP and caspase-3. The HT-29 cells were treated with Boeravinone B for 48 h and subjecting lysates for immunoblotting studies with anti-PARP and anti-caspase3. B. Boeravinone B caused significant rise in nuclear translocation of AIF. The HT-29 cells were treated with various concentrations of Boeravinone B for 48 h, the fraction Cytosol and nuclear were immunoblotted with AIF antibody. The Toposiomerase-I was employed as nuclear protein marker, whereas GAPDH as cytosol marker.