![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Linus Pauling Institute, Oregon State University, Corvallis, OR 97331
4To whom correspondence should be addressed. E-mail: Rod.Dashwood{at}oregonstate.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
|---|
KEY WORDS: • colon cancer • chemoprevention • adherins junctions
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).
|
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 (
m) 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).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
|---|
Human HCT116 colorectal cancer cells were obtained from the American Type Culture Collection. CHL was purchased from Sigma Chemical, and the concentrations reported here were normalized for total chlorin content. 4',6-diamidino-2-phenylindole (DAPI) and Alexa Fluor 488 were from Molecular Probes.
Cell culture
HCT116 cells were maintained in McCoys 5A medium with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich). Cells were placed in an incubator at 37°C under 5% CO2 conditions. Cells (1 x 106) were seeded in 60-mm plates and grown to 70% confluency, washed with PBS, and then treated with or without fresh solutions of CHL (in cell culture medium) under subdued lighting. Cells were harvested after 24 h, and cytoplasmic and nuclear fractions were isolated (see below).
Immunocytochemistry
Cells were washed in PBS and were immunostained for ß-catenin using rabbit polyclonal anti-ß-catenin (Abcam), followed by goat anti-rabbit IgG conjugated to Texas Red. E-cadherin was detected using mouse monoclonal antibody (HECD-1, Zeneca) followed by a goat antimouse IgG conjugated to Alexa Fluor 488. DAPI was used as a counterstain for nuclei. Cells were mounted and stored in the dark at –20°C until viewed with a Zeiss LSM 510 Meta confocal microscope.
Immunoblotting
Western analyses were performed essentially as reported (14,15), with the following modifications. Nuclear and cytoplasmic extracts were first obtained using NE-PER Extraction Reagents (Pierce Biotechnology). Proteins were separated on 4–12% bis-tris gels (Novex, Invitrogen), and they were transferred to nitrocellulose membranes (Invitrogen). Primary antibodies were mouse anti-ß-catenin monoclonal (C19220, BD Transduction), mouse monoclonal E-cadherin (HECD-1, Zeneca), or mouse monoclonal anti-histone H1 (SC8030, Santa Cruz), the latter serving as control for the clean separation of nuclear and cytoplasmic fractions. ß-Actin was used as the loading control (not shown). After incubation with primary antibody followed by secondary antibody conjugated to horseradish peroxidase, detection was by Western Lighting Chemiluminescence Reagents Plus (Perkin Elmer Life Science).
RT-PCR
Cells were seeded (1 x 106) and grown in 60-mm culture plates for 2 d. After reaching 70–80% confluency, cells were treated with various concentrations of CHL in fresh medium and then were incubated for another 24 h at 37°C. Harvest of cells was followed by 2 washes in PBS and centrifugation at 500 x g for 5 min. Isolation of mRNA, preparation of cDNA, and subsequent RT-PCR conditions were essentially as reported before, including the use of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping control (13,14).
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
|---|
Immunocytochemical localization of E-cadherin and ß-catenin in HCT116 cells
CHL induced several markers of terminal differentiation in HCT116 cells, including E-cadherin and alkaline phosphatase (12). In the case of E-cadherin, protein and RNA levels were increased 7- and 3-fold, respectively, 24 h after CHL dosing, whereas alkaline phosphatase mRNA expression was induced 6-fold. No changes were detected in ß-catenin, but total cell lysates were used in the latter studies, and we sought to clarify the distribution of E-cadherin and ß-catenin in different cellular compartments in response to CHL treatment. Using immunocytochemistry, we found that E-cadherin expression was immunolocalized at the cell periphery of control HCT116 cells (Fig. 2A), and this expression was markedly increased after treatment with CHL (Fig. 2B). As expected, we observed strong nuclear, cytoplasmic, and plasma membrane staining for ß-catenin, because HCT116 colon cancer cells contain a stabilized mutant form of ß-catenin (Fig. 2C). There was high expression of ß-catenin at the plasma membrane of cells treated with or without CHL (Fig. 2D).
|
Immunocytochemical studies showed that ß-catenin expression at the plasma membrane was high even after CHL treatment, and we next examined ß-catenin expression in other cellular compartments, namely cytoplasm and nucleus. No change in total cellular ß-catenin mRNA expression was detected across a broad range of CHL concentrations, with GAPDH used as the housekeeping control (Fig. 3A). Moreover, no change was detected in the cytosolic pool of ß-catenin protein, which was strongly expressed in all cells and at all concentrations of CHL (Fig. 3B). However, a concentration-dependent decrease in nuclear ß-catenin occurred with dose of CHL tested; expression of ß-catenin in the nucleus was attenuated >50% by CHL at 250 µmol/L relative to histone H1 (Fig. 3B).
|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 This work was supported in part by NIH grants CA65525, CA80176, and CA90890, and by Environmental Health Sciences Center grant P30 ES00210. Support for O.C. was provided, in part, by a postdoctoral fellowship, under grant number T32 ES07060 from the National Institute of Environmental Health Sciences
3 Supporting material is available from the posting of the article on www.nutrition.org.
5 Abbreviations used: AIF, apoptosis inducing factor; CHL, chlorophyllin; DAPI, 4',6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
|---|
1. Dashwood, R. (1997) Chlorophylls as anticarcinogens. Int. J. Oncol. 10:721-727.
2. Dashwood, R. H. (2002) Modulation of heterocyclic amine-induced mutagenicity and carcinogenicity: An ‘A-to-Z’ guide to chemopreventive agents, promoters, and transgenic models. Mutat. Res. 511:89-112.[Medline]
3. Dashwood, R. H., Yamane, S. & Larsen, R. (1996) A study of the forces stabilizing complexes between chlorophylls and heterocyclic amine mutagens. Environ. Mol. Mutagen. 27:211-218.[Medline]
4. Dashwood, R. H. & Guo, D. (1992) Inhibition of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-DNA binding by chlorophyllin: studies of enzyme inhibition and molecular complex formation. Carcinogenesis 13:1121-1126.
5. Dashwood, R. H., Negishi, T., Hayatsu, H., Breinholt, V., Hendricks, J. D. & Bailey, G. S. (1998) Chemopreventive properties of chlorophylls towards aflatoxin B1: a review of the antimutagenicity and anticarcinogenicity data in rainbow trout. Mutat. Res. 399:245-253.[Medline]
6. Egner, P. A., Wang, J. B., Zhu, Y. R., Zhang, B. C., Wu, Y., Zhang, Q. N., Quian, G. S., Kuang, S. Y. & Gange, S. J., et al (2001) Chlorophyllin intervention reduces aflatoxin-DNA adducts in individuals at high risk for liver cancer. Proc. Natl. Acad. Sci. U.S.A. 98:14601-14606.
7. Guo, D., Horio, D., Grove, J. & Dashwood, R. H. (1995) Inhibition by chlorophyllin of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced tumorigenesis in the male F344 rat. Cancer Lett. 95:161-165.[Medline]
8. Hasegawa, R., Hirose, M., Kato, T., Hagiwara, A., Boonyaphiphat, P., Nagao, M., Ito, N. & Shirai, T. (1995) Inhibitory effect of chlorophyllin on PhIP-induced mammary carcinogenesis in female F344 rats. Carcinogenesis 16:2243-2246.
9. Xu, M., Orner, G. A., Bailey, G. S., Stoner, G. D., Horio, H. T. & Dashwood, R. H. (2001) Post-initiation effects of chlorophyllin and indole-3-carbinol in rats given 1,2-dimethylhydrazine or 2-amino-3-methylimidazo[4,5-f]quinoline. Carcinogenesis 22:309-314.
10. Nelson, R. L. (1992) Chlorophyllin, an antimutagen, acts as a tumor promoter in the rat-dimethylhydrazine colon carcinogenesis model. Anticancer Res. 12:737-740.[Medline]
11. Dashwood, R. H., Xu, M., Orner, G. A. & Horio, D. T. (2001) Colonic cell proliferation, apoptosis, and aberrant crypt development in rats given 2-amino-3-methylimidazo[4,5-f]quinoline and treated post-initiation with chlorophyllin. Eur. J. Cancer Prev. 10:139-145.[Medline]
12. Díaz, G. D., Li, Q. & Dashwood, R. H. (2003) Caspase-8 and AIF mediate a cytochrome c-independent pathway of apoptosis in human colon cancer cells induced by the dietary phytochemical chlorophyllin. Cancer Res. 63:1254-1261.
13. Díaz, G. D., Paraskeva, C., Thomas, M. G., Binderup, L. & Hague, A. (2000) Apoptosis is induced by the active metabolite of vitamin D3 and its analogue EB1089 in colorectal adenoma and carcinoma cells: Possible implications for prevention and therapy. Cancer Res. 60:2304-2312.
14. Blum, C. A., Xu, M., Orner, G. A., Fong, A. T., Bailey, G. S., Stoner, G. D., Horio, D. T. & Dashwood, R. H. (2001) ß-catenin mutation in rat colon tumors initiated with 1,2-dimethylhydrazine or 2-amino-3-methylimidazo[4,5-f]quinoline, and the effect of post-initiation treatment with chlorophyllin and indole-3-carbinol. Carcinogenesis 22:315-320.
15. Dashwood, W.-M., Orner, G. A. & Dashwood, R. H. (2002) Inhibition of ß-catenin/Tcf activity by white tea, green tea, and epigallocatechin-3-gallate (EGCG): minor contribution of H2O2 at physiologically relevant EGCG concentrations. Biochem. Biophys. Res. Commun. 296:584-588.[Medline]
16. Dashwood, R. H. (2004) Adherins junction remodeling—a novel target for cancer chemoprevention and chemotherapy?. Int. J. Cancer Prev. 1:15-18.
17. Pece, S. & Gutkind, J. S. (2002) E-cadherin and Hakai: signalling, remodeling or destruction?. Nature Cell Biol. 4:E72-E74.[Medline]
This article has been cited by other articles:
|
|
 |
|
  G. Calviello, F. Resci, S. Serini, E. Piccioni, A. Toesca, A. Boninsegna, G. Monego, F. O. Ranelletti, and P. Palozza Docosahexaenoic acid induces proteasome-dependent degradation of {beta}-catenin, down-regulation of survivin and apoptosis in human colorectal cancer cells not expressing COX-2 Carcinogenesis, June 1, 2007; 28(6): 1202 - 1209. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
