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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.

Abstract. The aim of this study was to determine suitable culture conditions for maintaining the activity of cytochrome p450 (CYP) 3A4 and drug transporters in primary cultured human hepatocytes. Human hepatocytes were isolated using the two-step collagenase perfusion technique and were cultured with four different media, serum-free William's E medium (serum-free WEM), WEM containing fetal calf serum (FCS-WEM), WEM with human serum (HS-WEM), and Lanford's medium. The albumin levels were maintained for 7 days in hepatocytes. Although CYP3A4 mRNA levels gradually decreased from 3 days, CYP3A4 and hepatocyte nuclear factor-4α alpha protein levels and activities were maintained for 7 days in hepatocytes cultured with serum-free WEM and Lanford's but not in those with FCS-WEM and HS-WEM. Furthermore, CYP3A4 protein levels were significantly increased by the addition of rifampicin and dexamethasone to the culture media, indicating that the induction potential was maintained. The protein levels of P-glycoprotein, multi-drug-resistance-2, and breast cancer-resistance protein were maintained for 7 days in all media. Serum-free WEM and Lanford's also maintained protein levels of CYP2C19, CYP2D6, and organic anion transporter polypeptide in the hepatocytes. Serum-free WEM and Lanford's may be appropriate culture media for maintaining CYP3A4 and drug transporter protein levels in primary cultured hepatocytes.

In 115 patients (68 with liver cirrhosis and 47 without) who underwent curative resection of hepatocellular carcinoma (HCC) caused by hepatitis C virus (HCV)-related chronic liver diseases, we separated the liver into three segments (right, middle, and left) according to the three secondary branches of the Glissonean pedicle. We examined the weight of each resected segment. We also examined the histological findings of the segments in the same liver in 24 other patients with HCV-related chronic liver diseases. The average weight of the segments did not vary significantly in patients without liver cirrhosis. However, the average weight of the segments was significantly different in patients with liver cirrhosis (P = 0.0414) and the weight of the middle segment was lower than that of the other segments. In another group, of 246 patients with curative resection of HCC, of the 90 patients with single nodular HCCs, 45 nodules (50%) were located in the middle segment (P = 0.0004); in the 156 patients with synchronous multicentric HCCs (total, 401 nodules), 220 nodules (54.9%) were located in the middle segment. In 74 of the 156 patients with synchronous multicentric HCCs (47.4%), the HCCs were located in the same segment. The grade, stage of hepatitis, and number of sites of irregular regeneration were significantly different in each segment (P < 0.05), and the middle segment had more advanced hepatitis than the other segments. The rate of occurrence of HCC in the middle segment was higher than that in the other segments. The difference among the segments of the liver in regard to the degree of damage done by hepatitis may be related to the differences in HCC occurrence among the liver segments.

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