Molecular Characterization and Enhancement of Anticancer Activity of Caffeic Acid Phenethyl Ester by γ Cyclodextrin

Abstract

Caffeic Acid Phenethyl Ester (CAPE) is a key component in New Zealand propolis, known for a variety of health promoting and therapeutic potentials. We investigated the molecular mechanism of anticancer and anti-metastasis activities of CAPE. cDNA array performed on the control and CAPE-treated breast cancer cells revealed activation of DNA damage signaling involving upregulation of GADD45α and p53 tumor suppressor proteins. Molecular docking analysis revealed that CAPE is capable of disrupting mortalin-p53 complexes. We provide experimental evidence and demonstrate that CAPE induced disruption of mortalin-p53 complexes led to nuclear translocation and activation of p53 resulting in growth arrest in cancer cells. Furthermore, CAPE-treated cells exhibited downregulation of mortalin and several other key regulators of cell migration accountable for its anti-metastasis activity. Of note, we found that whereas CAPE was unstable in the culture medium (as it gets degraded into caffeic acid by secreted esterases), its complex with gamma cyclodextrin (γCD) showed high efficacy in anti-tumor and anti-metastasis assays in vitro and in vivo (when administered through either intraperitoneal or oral route). The data proposes that CAPE-γCD complex is a potent anti-cancer and anti-metastasis reagent.

Keywords: CAPE; Propolis; anti-metastasis.; anticancer; complex; p53; upregulation; γCD.

Figure 1

Cytotoxicity of CAPE to human cancer cells. Dose dependent loss in cell viability in cells treated with CAPE is shown. IC50 for A549, HT1080, G361 and U20S were ~100, 5, 20, 60 μM respectively (A). Effect of CAPE on cell viability (B) and colony forming efficiency (C) of MCF-7, MDA-MB-231 cells and their mortalin-overexpressing metastatic deriatives showing that CAPE inhibited cell proliferation in both short (A) and long term (B) viability assays. (D) Cell cycle analysis of control and CAPE-treated cells showing G1 arrest both in MCF-7 and MDA-MB-231 treated with low dose (2 μM and 10 μM CAPE, respectively) of CAPE. (E) High dose (10 μM and 40 μM CAPE, respectively) caused apoptosis.

Figure 2

Instability of CAPE in cell culture medium and its complex with γCD. (A) Phase contrast images of MCF-7 cells treated with CAPE either one time or regularly (every 24 h) for total of 72 h. Whereas healthy dividing cells appeared in culures treated for only once, the regularly treated cultures showed growth arrest/apoptosis. (B) Reactive sites of CAPE, such as ester bond, α, β-unsaturated carbonyl and catechol group, that accounts for its unstability are shown. (C) The binding constant (K) for the complexation of CAPE with βCD, as by a UV/Vis-spectroscopic-titration method is shown. The analysis of the titration curve revealed 1:1 complexation provided a K value of (2.52 ± 0.04) x 10³ M^-1 (inset). (D) Binding constant (K) values of complex of CAPE with αCD or γCD.

Figure 3

Characterization of CAPE-γCD complex and its stability to various adverse conditions. (A) Thermostability analyis at 140^oC at 24 h showing color change and melting of CAPE, but not CAPE-γCD. The enhanced thermostability was also observed from the analysis of contents of CAPE in heat-treated samples by HPLC. (B) Stability of CAPE and CAPE-γCD against hydrolysis by α-Chymotrypsin (ChT) for 3 h at pH 7.4 and 25^oC. Decrease in CAPE and increase in caffeic acid were concurrently observed (a). CAPE-γCD indicated much higher stability than CAPE. Stabilization of CAPE against Michael 1,4-addition reaction with cysteine by γCD (is shown (b). (C) Stability of CAPE and CAPE-γCD against oxidation as monitored by addition of potassium iodate and UV/Vis-spectroscopy. Large proportion of CAPE (80%) reacted with potassium iodate in incubation. In case of CAPE-γCD, 40% CAPE reacted and 60% was protected in the same condition.

Figure 4

Anticancer potential of CAPE and CAPE-γCD complex. (A) Effect of CAPE, γCD and CAPE-γCD on viability, migration and invasion of MCF7 and MDA-MB-231 cells. Phase contrast images (A), cell viability (B) migration in wound scratch (C&D) and cell invasion assay (E&F) are shown.

Figure 5

Characterization of CAPE cytotoxicity by cDNA array analysis. (A) Pathways derived from cDNA array analysis; genes upregulated (Red) and downregulated (green) in CAPE treated as compared to the control cells. (B) Genes upreguleated in CAPE treated cells as determined by cDNA array analysis. (C) Immunostaining of GADD45α in control and CAPE treated cells showing increase in the latter. Increase in GADD45α in CAPE and CAPE-γCD treated cells detected by Western blotting (D) and RT-PCR. (E) 53BP1 immunostaining showing increase in CAPE-γCD tretaed cells.

Figure 6

Activation of p53-p21 pathway by CAPE. (A) Immunostaining of p53 and p21 in control and CAPE treated cells showing their increase. (B) Increase in p53 and p21 as determined by Western blotting with specific antibodies. (C) p53-dependent reporter assay showing increase in transcriptional activation function of p53 in CAPE-treated than in control cells.

Figure 7

Molecular docking analysis of CAPE and mortalin. (A) Amino acid residues of mortalin participating in hydrogen bond (pink), hydrophobic and van der Waal interactions (orange) wih CAPE after molecular docking. (B) Graph showing deviation of the docked complex from its initial conformation during the complete simulation run. (C) Molecular interaction pattern between mortalin and CAPE after simulation. Residues were mainly involved in hydrophobic and van der Waal interactions (shown in orange). Change in position of CAPE can be observed. The ligand showed variations in the orientation and hence an average representative structure from the stable time frame was used for interaction analysis. (D) p53 immunostaining of MCF7 control and CAPE treated cells showing intense nuclear staining in the later. (E) Relative amount of p53 in mortalin-p53 complex in control and CAPE-treated cells showing decrease in mortalin-p53 complex in the latter.

Figure 8

Downregulation of mortalin and other metastasis-regulatory proteins by CAPE. Effect of CAPE on mortalin and cell migration regulatory proteins. Decrease in mortalin, vimentin, MMP-2 was obserevd by Western blotting (A), immunostaining (B) and RT-PCR (C). Decrease in mortalin, vimentin, β-catenin, TGF-β and Wnt-3α as determined by qPCR is shown (D).

Figure 9

In vivo anti-tumor and anti-metastasis activties of CAPE and CAPE-γCD complex. Effect of CAPE and CAPE-γCD on in vivo progression of tumors generating either by subcutaneous xenografts or tail vein injections of HT1080 in nude mice. Tumor volumes showed decrease in mice treated with CAPE and CAPE-γCD complex (either peritoneal injections or gavage). CAPE-γCD treated mice showed higher degree of tumor suppression in both models. No effect on the body weight was observed.