Further investigation of the modifying effect of various chemopreventive agents on apoptosis and cell proliferation in human colon cancer cells.
OBJECTIVE
Recent preclinical assays using animal models have shown that naturally-occurring and synthetic chemicals such as auraptene (AUR), nobiletin (NOB), hesperidin (HE), diosmin (DIO), indole-3-carbinol (I3C), 1'-acetoxychavicol acetate (ACA), 2,5-di-O-acetyl-D-1,4-glucaro-6,3-dilactone (ACE), D-glucuronic acid gamma-lactone (GL), chlorogenic acid (CGA), protocatechuic acid (PA), and sinigrin (SIN) are possible preventive agents against the development of cancer. However, the mode of action of such preventive agents remains to be elucidated. The current study, therefore, was conducted to analyze whether these agents induce apoptosis and/or inhibit DNA synthesis in human colorectal cancer cell lines.


METHODS
We performed an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay to evaluate the modifying effects of the chemicals on cell viability as the first screening. Then, induction of apoptosis was detected by means of a DNA fragmentation assay, a quantitative enzyme immunoassay, and morphological analysis using 4-diamidino-2-phenylindole staining. In addition, the modulating effects of the compounds on DNA synthesis of the cells with fixed doses of the compounds were analyzed by scoring the 5-bromo-2'-deoxyuridine labeling index.


RESULTS
AUR, NOB, I3C, ACA, and ACE had apoptosis-inducing effects in a concentration- and time-dependent manner, some of which were followed by a reduction in replicating DNA synthesis. CGA, PA, SIN, GL, DIO, and HE had little modulating effect on cell viability, apoptosis, and DNA synthesis in this cell system.


CONCLUSIONS
Our results suggest that AUR, I3C, ACA, NOB, and ACE might exert tumor-preventive action through apoptosis- and/or cell proliferation-dependent mechanisms and, on the other hand, CGA, PA, SIN, HE, DIO, and GL might be apoptosis- and cell proliferation-independent. These assays provided an initial tool for further mechanical studies of tumor-preventive agents and future applications to mechanism-based chemopreventive studies.
Zheng
Qiao
Q
First Department of Pathology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan. e2101002@guedu.cc.gifu-u.ac.jp
Hirose
Yoshinobu
Y
Yoshimi
Naoki
N
Murakami
Akira
A
Koshimizu
Koichi
K
Ohigashi
Hajime
H
Sakata
Keiko
K
Matsumoto
Yuji
Y
Sayama
Yoshikatsu
Y
Mori
Hideki
H
D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001973:Bromodeoxyuridine; D002455:Cell Division / Q000187:drug effects*; D018890:Chemoprevention; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000473:pathology* / Q000517:prevention & control; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D014407:Tumor Cells, Cultured / Q000187:drug effects*
D016588:Anticarcinogenic Agents; D004247:DNA; D001973:Bromodeoxyuridine