Evaluation of CYP2D6 Protein Expression and Activity in the Small Intestine to Determine Its Metabolic Capability in the Japanese Population.

CYP2D6 plays an important role in the metabolism of many drugs such as opioids and antidepressants. Polymorphisms of the CYP2D6 gene are widely observed in the Japanese population, and can affect the first-pass metabolism of orally administered drugs. Several CYP enzymes have been identified in the small intestine of Caucasians, but intestinal CYP enzymes have not been reported in the Japanese population, except for CYP3A4 and CYP2C19. In this study, we evaluated the CYP2D6 metabolic capacity by measurement of CYP2D6 mRNA and protein levels and activity in the small intestine of Japanese individuals. Normal jejunal tissues were obtained from 31 patients who had undergone pancreaticoduodenectomy, and the CYP2D6*10 variant was identified in these tissues. CYP2D6 mRNA and CYP2D6 protein levels were analyzed using real-time RT-PCR and Western blotting, respectively. Bufuralol 1'-hydroxylation, a marker of CYP2D6 activity, was analyzed using HPLC. Frequencies of the CYP2D6*1/*1, *1/*10, and *10/*10 genotypes in the jejunal tissue were 29.0% (n=9), 35.5% (n=11), and 35.5% (n=11), respectively. CYP2D6 protein and activity levels did not differ significantly between the genotypes. A positive correlation was found between CYP2D6 protein and activity levels. Furthermore, CYP2D6 protein levels and activity in the small intestine were significantly lower than those in the liver. These findings suggest that the metabolic capacity of CYP2D6 in the small intestine of the Japanese population has a relatively small effect on drug metabolism.

Comparative study of culture conditions for maintaining CYP3A4 and ATP-binding cassette transporters activity in primary cultured human hepatocytes.

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.

[A case of multiple liver metastases of sigmoid colon cancer responding well to UFT+Leucovorin therapy].

The subject was a 75-year-old female. For the treatment of multiple liver metastases from sigmoid colon cancer, the administration of UFT(300 mg/day)and oral Leucovorin(75 mg/day)was initiated after a resection of the sigmoid colon. A decrease in the liver metastases was observed in a CT scan 4 months after the start of this administration, and the disappearance of the metastatic focus in the liver was confirmed at 10 months thereafter. The administration of UFT and oral Leucovorin was continued for 2 months. Thereafter, at 12 months after the start of this administration, was continued with only the administration of UFT(300 mg/day)for 1 year. Now, 26 months have passed since the liver metastases disappeared and no recurrence has been found, even within the abdominal cavity. The condition of the patient has been excellent. The subject of this study was an elderly person, but an excellent QOL was secured without any adverse events, and the chemotherapy regimen could be maintained for 2 years. These findings suggest that UFT and oral Leucovorin can be expected to have an excellent therapeutic effect as an oral chemotherapeutic agent.

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.

Hepatopancreatoduodenectomy could be allowed for patients with advanced intrahepatic cholangiocarcinoma.

BACKGROUND/AIMS: Patients with advanced intrahepatic cholangiocarcinoma (ICC) have a poor outcome even if they undergo extended radical surgery. Hepatopancreatoduodenectomy (HPD; hepatectomy with pancreatoduodenectomy) for ICCs may be expected to provide a favorable outcome if curative resection is reasonable and patients can tolerate the radical major procedure. METHODOLOGY: Between January 1981 and March 2002, 152 hepatic resections were performed for ICC. Of these, 12 patients underwent HPD for ICC at the same institute of Gastroenterology, Tokyo Women's Medical University. HPD for ICC was indicated in patients who (1) require dissection of the peripancreatic lymph nodes, (2) exhibit direct invasion of intrapancreatic bile duct, (3) show signs of intrapancreatic bile ductal growth. RESULTS: Characteristics of the short-term survivors (died within 12 months), compared with long-term survivors (survived more than 12 months), indicated that they were more likely to be positive intrahepatic metastasis, to be positive lymph node metastasis, to be positive portal venous invasion, and margins of resected surface with residual tumor. The actuarial overall 1-, 3-, 5-, 10-year survival rates were 42%, 33%, 33%, and 23%, respectively. The 5-year survival rate in patients without lymph node metastasis was significantly better (p = 0.045) than that of patients with lymph node metastasis. The patients who underwent potentially curative resection had significantly better 5-year survival rates than those who underwent non-curative resection. Four patients survived for at least 5 years and two of these patients survived for more than 10 years. Nine patients developed recurrence after resection, and of these, 5 patients with recurrence died within 12 months after surgery. CONCLUSIONS: HPD is considered to be an efficacious procedure for advanced ICC and long-term survival may be possible in a selected group of patients.

Liver metastases with massive portal venous tumor thrombi from colorectal cancer: can be treated by surgical resection?

BACKGROUND/AIMS: The surgical treatments for liver metastases from colorectal cancer with massive portal venous tumor thrombi were evaluated. METHODOLOGY: Five patients, among the 142 patients who underwent hepatic resection for liver metastases from colorectal cancer from 1989 to 1998, were included in this study. The tumor thrombi in the main portal vein were removed by the following procedures; (1) the circumferential incision of the first branch of the portal vein and removal of the exposed tumor thrombi with ring forceps and suction, (2) temporary clamping of the distal end, (3) dilatation of the round ligament and the venous cannula was inserted into the umbilical portion, (4) washing out of the residual tumor thrombi, (5) declamping of the distal end and closing suture of the cut end of the portal branch. RESULTS: All patients had metachronous metastases and underwent resections of the primary tumor within 2 years. The surgical procedures performed were as follows: two cases that underwent right hepatectomies with portal venous tumor thrombectomies, one right trisectionectomy with portal venous tumor thrombectomy, one right hepatectomy plus limited resection of the contralateral lobe, and one left lateral sectionectomy with limited resection of the right lobe. All patients had no major postoperative complications and returned to their social lives within 1 month after operation. The intra-arterial catheter devices were implanted in four patients in order to receive adjuvant chemotherapy. One patient survived the 36-month period after liver resection, although 4 patients died of liver recurrence within 12 months. The mean survival time was 14.4 months and the overall 1-year survival rate was 20.0 percent. CONCLUSIONS: Surgical resection for this disease may bring longer survival rates for some patients, but not be an effective therapeutic option in our series. We should create other adjuvant therapies to improve these survival rates.

Step classification is useful for the determination of indications for systematized hepatectomy in hepatocellular carcinoma.

BACKGROUND/PURPOSE: The effectiveness of systematized hepatectomy with transection of Glisson's pedicle at the hepatic hilus has not been clarified in detail in relation to previous staging systems. Outcomes after systematized hepatectomy in patients with hepatocellular carcinoma (HCC) were examined in relation to our new staging system. METHODS: We retrospectively studied 955 patients with HCC who underwent hepatectomy from 1989 through 2002. We classified patients with HCC into four groups according to the pathological findings (pathological step [p-step]): p-step 1, HCC with absence of vascular invasion and absence of intrahepatic metastasis; p-step 2, HCC with vascular invasion and/or intrahepatic metastasis; p-step 3, HCC with major vascular invasion and/or intrahepatic metastasis to both lobes of the liver; and p-step 4, HCC with distant metastasis, including lymph node metastasis or ruptured HCC). We separated the liver into three segments (Takasaki's liver segments). Systematized hepatectomy was classified as systematized segmentectomy or larger resection, and partial segmentectomy. Segmentectomy refers to resection of one of Takasaki's segments. RESULTS: Systematized segmentectomy did not affect recurrence-free survival, by univariate analysis, in patients with p-step 1, p-step 3, or p-step 4. However, systematized segmentectomy or larger resection was significantly associated with patient recurrence-free survival, by univariate analysis, in patients with p-step 2. Multivariate analysis also showed systematized segmentectomy or larger resection as a significant independent prognostic factor in patients with p-step 2. CONCLUSIONS: Systematized segmentectomy is suitable for patients with p-step 2 HCC according to this step classification.

Histopathologic differentiation of the main nodule determines outcome after hepatic resection for synchronous multicentric hepatocellular carcinomas.

BACKGROUND/AIMS: Clinicopathological features and outcome after surgery in patients with synchronous multicentric hepatocellular carcinoma were examined in relation to the histopathological grade of differentiation of the main nodule. METHODOLOGY: Two hundred and sixty-five patients with synchronous multicentric hepatocellular carcinoma (total, 683 nodules) who had undergone curative hepatectomy from 1988 through 1999 were studied retrospectively. In multicentric occurrences of hepatocellular carcinoma, the tumor with the largest dimension was defined as the main nodule, and the others as accessory nodules. RESULTS: The histopathological grade of differentiation of the main nodule was assessed to be well differentiated in 72 patients (27.2%), moderately differentiated in 160 patients (60.4%), and poorly differentiated in 33 patients (12.4%). Tumor size of the main nodule was significantly smaller in patients with well differentiated hepatocellular carcinoma than in patients with moderately or poorly differentiated hepatocellular carcinoma. Alpha-fetoprotein levels were significantly lower in cases in which the main nodule was diagnosed to be well differentiated hepatocellular carcinoma than in other cases. The 5-year survival rate and recurrence-free survival rate were significantly greater in cases in which the main nodule showed well differentiated hepatocellular carcinoma (78.1% and 33.8%, respectively) than in other cases [moderately differentiated 49.0% (p<0.0001), 11.6% (P=0.0002); poorly differentiated 37.4% (p<0.0001), 8.3% (P=0.0002), respectively]. Multivariate analysis identified the histopathological grade of the main nodule as significant independent prognostic factors. CONCLUSIONS: There were differences in surgical outcome in relation to the histopathological grade of differentiation of the main nodule in patients with synchronous multicentric hepatocellular carcinoma.