The Dietary Phytochemical Chlorophyllin Alters E-Cadherin and β-Catenin Expression in Human Colon Cancer Cells

Chlorophyllin (CHL)5 is a water-soluble derivative of chlorophyll (Fig. 1) and has been used clinically in various capacities, including as an aid to wound healing (1). Previous work revealed that when administered during carcinogen exposure CHL is a potent antimutagen and anticarcinogen [see reference (1) for a review]. Mechanism studies showed that CHL acts as an effective blocking agent by virtue of its ability to form molecular complexes with planar aromatic compounds, thereby inhibiting their uptake and bioavailability from the gut (2–4). Because of this ability to serve as an interceptor molecule, CHL inhibited aflatoxin B1–induced liver tumorigenesis in rainbow trout (5) and attenuated significantly aflatoxin B1–DNA biomarkers in a human intervention trial (6). In studies with heterocyclic amine mutagens, such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, CHL also was an effective interceptor molecule and anticarcinogen in the rat (2,7,8). FIGURE 1 Comparison of the chemical structures of CHL and chlorophyll a. Interestingly, when given postinitiation, CHL promoted the formation of colon tumors or aberrant crypt foci in rats treated with the carcinogens IQ or 1,2-dimethylhydrazine (9,10). Subsequent studies revealed a concentration-dependent dysregulation of the homeostatic balance between cell birth and apoptosis in the colonic crypts (11). Specifically, the bromodeoxyuridine labeling index for the colonic crypts increased at all doses of CHL, whereas terminal deoxynucleotidyltransferase-mediated nick-end labeling was induced only at the higher concentrations of CHL. To investigate the mechanisms of apoptosis further, human colon cancer cells were treated with CHL (12). Cells underwent growth arrest and apoptosis after 24 h, with evidence for the formation of a sub-G1 peak in the attached cell population and nuclear condensation in the floating cell population. There was a concentration-dependent attenuation of mitochondrial membrane potential (Δψm) without the release of cytochrome c or activation of the caspase-9/caspase-3 pathway. However, apoptosis inducing factor (AIF) was released from mitochondria into the cytosol and nucleus, leading to cleavage of nuclear lamins. Upstream mediators of this apoptosis pathway were identified as caspase-8/caspase-6 and tBid acting in conjunction with other proapoptotic members of the Bcl-2 family, such as Bak. These findings suggested that CHL triggers apoptosis via interaction with so-called death receptors in the plasma membrane of cancer cells, leading to cleavage of procaspase-8, release of AIF from mitochondria, and activation of subsequent downstream events leading to the destruction of nuclear lamins. Importantly, E-cadherin and alkaline phosphatase, which are well-established indicators of cell differentiation, were strongly induced at all concentrations of CHL (12). The latter findings were based on immunoblot analyses of total cell lysates, and we sought to examine E-cadherin in more detail, because this protein is known to be induced by other cancer chemotherapeutic agents, such as sodium butyrate and vitamin D (13).