Irinotecan-induced apoptosis is inhibited by increased P-glycoprotein expression and decreased p53 in human hepatocellular carcinoma cells.
Irinotecan, a DNA topoisomerase I inhibitor, is widely used in cancer chemotherapy. However, little is known of the mechanisms of its antitumor effects and the development of drug resistance in human hepatocellular carcinoma (HCC). In this study, we investigated the effects of short-term culture with SN-38, the active metabolite of irinotecan, on apoptosis in Huh7 cells. The cells were cultured with SN-38 for 24, 72, and 120 h, and apoptosis was determined using the terminal dUTP nick-end labeling (TUNEL) assay. The expressions of p53, apoptosis-related proteins, and P-glycoprotein (P-gp), a protein conferring the multidrug-resistant phenotype, were analyzed using Western blotting. Induced expression of P-gp was detected using fluorescence microscopy. SN-38 significantly induced apoptosis in Huh7 cells at 24 h. SN-38 also increased the expression of p53, Bax, and caspase-9 and decreased Bcl-xL expression in Huh7 cells. SN-38 decreased p53 expression and increased P-gp expression after 120 h, resulting in inhibition of apoptosis. This inhibition was reversed by the addition of verapamil to the culture medium during 120 h incubation. SN-38-induced P-gp expression was additionally enhanced by p53 decoy oligodeoxynucleotide. The changes in P-gp expression were directly moderated by p53 gene downregulation, suggesting that it plays a role in the mechanism of drug resistance. These results suggest that the accumulation of irinotecan in HCC leads to the development of drug resistance.
Takeba
Yuko
Y
Department of Pharmacology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan. yakuri@marianna-u.ac.jp
Sekine
Susumu
S
Kumai
Toshio
T
Matsumoto
Naoki
N
Nakaya
Sachiko
S
Tsuzuki
Yashimitsu
Y
Yanagida
Yohei
Y
Nakano
Hiroshi
H
Asakura
Takeshi
T
Ohtsubo
Takehito
T
Kobayashi
Shinichi
S
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000502:physiology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D006801:Humans; D020287:In Situ Nick-End Labeling; D000077146:Irinotecan; D008113:Liver Neoplasms / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009841:Oligonucleotides / Q000494:pharmacology; D012334:RNA, Neoplasm / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis* / Q000502:physiology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000972:Antineoplastic Agents, Phytogenic; D009841:Oligonucleotides; D012334:RNA, Neoplasm; D016159:Tumor Suppressor Protein p53; D000077146:Irinotecan; D002166:Camptothecin