Tohru Utsunomiya

Characterization of a side population of cancer cells from human gastrointestinal system.

A subset of stem cells, termed “side population” (SP) cells, has been identified and characterized in several mammalian tissues and cell lines. However, SP cells have never been identified or isolated from gastrointestinal cancers. We used flow cytometry and the DNA‐binding dye Hoechst 33342 to isolate SP cells from various human gastrointestinal system cancer cell lines. Fifteen of sixteen cancer cell lines from the gastrointestinal system contained 0.3%–2.2% SP cells. Next, we used an oligonucleotide microarray to analyze differentially expressed genes between SP and non‐SP cells of hepatoma HuH7. The expression of GATA6, which is associated with embryonic development and hepatocytic differentiation, was significantly upregulated in HuH7 SP cells. The expression of ABCG2, ABCB1, and CEACAM6, which are associated with chemoresistance, was also significantly increased in SP cells. In addition, some epithelial markers and mesenchymal markers were overexpressed in SP cells. Reverse transcription‐polymerase chain reaction and immunocytochemical staining validated these results and suggested a multilineage potential for HuH7 SP cells. In hepatoma HuH7 and colorectal SW480 cell lines, SP cells showed evidence for self‐renewal, generating both SP and non‐SP cells. Finally, chemoresistance to anticancer agents, including doxorubicin, 5‐fluorouracil, and gemcitabine, were compared between HuH7 SP and non‐SP cells using an ATP bioluminescence assay. The HuH7 SP cells expressed a higher resistance to doxorubicin, 5‐fluorouracil, and gemcitabine compared with non‐SP cells. These findings demonstrate that cancers of the gastrointestinal system do contain SP cells that show some characteristics of so‐called stem cells.

Postoperative liver failure after major hepatic resection for hepatocellular carcinoma in the modern era with special reference to remnant liver volume

BACKGROUND Postoperative liver failure is a life-threatening complication after hepatic resection. Because of recent advances in liver surgery technique and a more stringent patient selection, mortality after hepatic resection has steadily decreased, but its incidence still ranges from 10% to 20%. The factors linked to postoperative liver failure in major hepatic resection in the modern era should be reevaluated. STUDY DESIGN Of 80 patients with viral markers (hepatitis C viral antibody or hepatitis B surface antigen) who underwent major hepatic resections (no less than bisegmentectomies) for hepatocellular carcinoma between 1990 and 1996, 7 patients (8.8%) died of postoperative liver failure within 6 months after hepatectomy. The cause of liver failure was analyzed based on both the preoperative data and the intraoperative findings. In addition, since all the patients who died of liver failure underwent a right hepatic lobectomy, a further data analysis was also done in 47 patients who underwent a right lobectomy of the liver. A volumetric analysis by CT was then done to evaluate the remnant liver volume. RESULTS Between the patients with liver failure and those without liver failure who underwent a right lobectomy, there were no significant differences in preoperative data or intraoperative findings. Volumetric analysis revealed that the remnant liver volume of patients who died of liver failure was significantly smaller than that of patients who lived (p = 0.008). The incidence of liver failure in patients with a remnant liver volume of less than 250 mL/m2 was 7 of 20 (38%), while it was 0 of 27 in patients with a liver volume of no less than 250 mL/m2 (p = 0.0012). The only significant risk factor for liver failure in patients with a remnant liver volume of less than 250 mL/m2 was diabetes mellitus (p = 0.0072). CONCLUSIONS The expected remnant liver volume appears to be a good predictor for liver failure in patients who undergo a right lobectomy of the liver. In patients with diabetes mellitus and an expected remnant liver volume of less than 250 mL/m2, a major hepatectomy should be avoided. Careful patient selection based on volumetric analysis in major hepatectomy cases could help prevent the occurrence of postoperative liver failure.

Cancer stem cells in human gastrointestinal cancers.

The concept of cancer stem cell has developed in leukemia. Recently, it has expanded to include solid tumors such as brain or breast tumors. However, the descriptions are not recognized in human gastrointestinal cancers. We used flow cytometry and the DNA-binding dye (Hoechst 33342) to isolate side population (SP) cells from various human gastrointestinal system cancer cell lines. The SP cell fraction is considered to contain abundant stem cells. Fifteen of 16 cancer cell lines from the gastrointestinal system contained 0.3–2.2% SP cells. We studied the characteristics of the SP cells in hepatic or colon cancer cell lines. The results demonstrated that cancers of the gastrointestinal system do contain SP cells that show some characters of so-called stem cells. In this paper, we report our study results with a review of the literature.

Specific gene-expression profiles of noncancerous liver tissue predict the risk for multicentric occurrence of hepatocellular carcinoma in hepatitis C virus-positive patients.

BackgroundHepatitis C virus (HCV) infection produces chronic hepatitis, cirrhosis, and, ultimately, hepatocellular carcinoma (HCC). A molecular analysis of the damaged liver tissues infected with HCV may identify specific gene-expression profiles associated with a risk for liver carcinogenesis.MethodsForty patients with HCV-positive HCC were classified into two groups: single nodular HCC group (n = 28) and multicentric HCC group (n = 12). Using a complementary DNA microarray, we compared the gene-expression patterns of the noncancerous liver tissue specimens between the two groups. We also identified the differentially expressed genes related to multicentric recurrence in the liver remnant. We then evaluated whether a specific gene-expression profile can accurately estimate the risk for multicentric hepatocarcinogenesis.ResultsWe selected the 230 differentially expressed genes in the multicentric HCC group. A hierarchical clustering analysis identified a cluster that might be closely associated with the multicentric occurrence of HCC. On the basis of the gene-expression profiling of the 36 genes commonly associated with both multicentric HCC and multicentric recurrence, we created a scoring system to estimate the risk for multicentric hepatocarcinogenesis. The prediction score of patients in the multicentric HCC group with multicentric recurrence (19.9 ± 9.2) was significantly higher (P < .05) than that in the single nodular HCC group without multicentric recurrence (−1.8 ± 12.7).ConclusionsSpecific gene-expression signatures in noncancerous liver tissue may help to accurately predict the risk for developing HCC.

MAL gene expression in esophageal cancer suppresses motility, invasion and tumorigenicity and enhances apoptosis through the Fas pathway

We isolated the MAL (T-lymphocyte maturation associated protein) gene from differentially expressed products of esophageal epithelium relative to esophageal carcinoma tissues. The Mal protein has been demonstrated as being a component of the protein machinery for apical transport in epithelial polarized cells. In this study, we describe the reduced expression of MAL in all 39 cases of esophageal carcinoma tested and 60 other human carcinomas. MAL gene transcription was induced in three out of 13 esophageal carcinoma cell lines by treatment with the demethylating agent 5-aza-2′-deoxycytidine (DAC), and in nine additional cell lines by simultaneous treatment with trichostatin A, an inhibitor of deacetylation, and DAC. We established a stable MAL gene transfectant whose expression was regulated by subcutaneous doxycycline injection in nude mice. Tumor growth was suppressed in cells expressing TE3-MAL compared with TE3 parent cells or cells not expressing TE3-MAL with doxycycline injection (20 μg/body) (P<0.01). Additionally, the TE3-MAL transfectant cells exhibited decreased cellular motility, a G1/S transition block and increased levels of apoptosis, concomitant with increased expression of Fas receptor in vitro. The apoptotic staining in MAL-expressing tumors was confirmed by TUNEL assay. Therefore, we conclude that expression of MAL was frequently decreased or diminished in gastrointestinal tract cancers, and that Mal expression confers reduced tumorigenicity in vivo to tumor TE3 cells through the induction of apoptosis via the Fas signaling pathway.

Increased survival after cecal ligation and puncture in mice consuming diets enriched with sesame seed oil.

ObjectivesLignans that present in the nonfat portion of sesame seed oil (SSO) can inhibit &Dgr;-5 desaturase activity, resulting in an increase in the accumulation of dihomo-&ggr;-linolenic acid and, subsequently, decrease the production of proinflammatory dienoic eicosanoids with a concomitant increase in the secretion of less inflammatory monoenoic eicosanoids. DesignFemale Balb/c mice were fed diets supplemented with 5wt% SSO or a physical mixture of oils (control) whose fatty acid composition resembled that of SSO for 3 wks. Measurements and Main Results During a 4-day observation period after cecal ligation and puncture, only 20% of the controls and as many as 65% in the SSO group survived. Furthermore, the levels of cytokines and dienoic eicosanoids produced in response to an intraperitoneal injection of a nonlethal dose (50 &mgr;g/mouse) of endotoxin were measured in both groups. The interleukin (IL)-10 levels were markedly higher in mice fed SSO diets compared with the controls. However, the plasma concentrations of prostaglandin E1 + 2, tumor necrosis factor-&agr;, IL-6, and IL-12 did not differ significantly between the two groups of mice. ConclusionsBecause the fatty acid composition is almost similar between the two diets, sesamin, sesamol and other lignans in SSO appear to be responsible for an increase in survival after cecal ligation and puncture and also for an increase in the IL-10 levels in response to a nonlethal dose of endotoxin in mice.

Characteristics of Multicentric Hepatocellular Carcinomas: Comparison with Intrahepatic Metastasis

Characteristics of multicentric hepatocellular carcinomas (HCCs) remain obscure. We therefore aimed to clarify them and compare them with HCC with intrahepatic metastases. A series of 118 patients who had definite hepatitis C viral status and multinodular HCC were divided into two groups: a multicentric occurrence (MO) group (n= 38), with multicentric HCCs; and an intrahepatic metastasis (IM) group (n= 80), with HCC having intrahepatic metastases. Clinicopathologic variables, including the patients survival and disease-free survival rates, were compared between the MO and IM groups. Univariate analysis revealed the presence of esophageal varices, the presence of hepatitis C virus infection, a platelet count of less than 10 × 104/μl, hepaplastin test, γ-globulin, the histologically active hepatitis, tumor size, des-γ-carboxy prothrombin > 0.1 AU/ml, positive portal vein invasion, and histologic grade as discriminating factors. The MO score to differentiate multicentric HCCs from intrahepatic metastatic HCCs was determined using the following four independent factors selected by a stepwise regression analysis: the presence of hepatitis C virus infection, a platelet count of less than 10 × 104/μl, tumor size, and histologic grade. The sensitivity and specificity of the MO scores using those factors were 84% and 70%, respectively, when the cutoff value was 0.4. The disease-free survival rate in the MO group was similar to that in the IM group, whereas the survival rate in the MO group was significantly better than that in the IM group. The multivariate analysis revealed the multicentric occurrence of HCC as one of the independent prognostic factors. Clinicopathologic factors differentiating multicentric HCCs from intrahepatic metastatic HCCs were the presence of hepatitis C virus infection, a platelet count of less than 10 × 104/μl, small tumor size, and low histologic grade.

Clinicopathologic features and postoperative prognosis of multicentric small hepatocellular carcinoma.

BACKGROUND Assessment of clinicopathologic characteristics and postoperative prognoses for patients with multicentric hepatocellular carcinoma (HCC) is important to determine not only a need to operate, but also an appropriate treatment after hepatic resection. STUDY DESIGN Between May 1990 and April 1998, among 116 patients with an initial hepatectomy for HCC measuring 3 cm or less in maximum diameter, 34 patients had multicentric HCC (MC group), and 82 patients had single nodular HCC (SN group). To clarify the clinicopathologic features of patients in the MC group versus the SN group, we compared both the clinicopathologic parameters and the postoperative prognosis after curative hepatectomy between the two groups. RESULTS The percentages of patients positive for hepatitis B surface antigen and hepatitis C virus antibody were not significantly different between the two groups. No differences were noted in pathologic characteristics of the main tumor or tumor markers. On the other hand, in the MC group, the percentage of patients evaluated in a Childs classification as either B or C was significantly higher (p < 0.05) than that of patients in the SN group, indicating that patients with multicentric HCC have a poor hepatic functional reserve. Both survival and disease-free survival of patients in the MC group who underwent a curative hepatectomy did not differ statistically from those in the SN group. CONCLUSIONS Our results indicate that hepatic resection is useful, even for patients with multicentric HCC, if a curative hepatectomy can be performed and liver function can be saved, despite their poor hepatic functional reserve.

Dietary fish oil and cytokine and eicosanoid production during human immunodeficiency virus infection

BACKGROUND Dietary fish oil (FO) has been shown to modulate the immune system. The purpose of this study was to explore the effects of FO supplementation on the production of dienoic eicosanoids and cytokines in patients with human immunodeficiency virus (HIV) infection. METHODS This was a randomized, prospective, double-blind study that included homosexual males with HIV infection. Patients were asked to consume voluntarily five food bars daily containing FO (n = 10) or safflower oil (SO) (n = 9) for 6 weeks. At baseline and week 6, plasma was obtained to measure incorporation of omega-3 fatty acids. At baseline, week 3, and week 6, measurements were made of changes in dienoic eicosanoids and cytokines from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMC) or spontaneously releasing cells. RESULTS In the FO group but not the SO group, there was increased incorporation of the omega-3 fatty acid docosahexaenoic acid (DHA) into the phospholipids of the fatty acids of the plasma. In the FO group, there was a significant decrease (p = .01) in 6-keto prostaglandin (PG) F1 alpha released from PBMC. There was a significant increase (p = .01) in interleukin (IL)-6 released from the PBMC in the FO group between baseline and week 3 and between week 3 and week 6. At week 6, there was significantly more IL-6 (p = .01) released from the PBMC in the FO group compared with the SO group. There was no change in CD4 cell counts by analysis of variance. CONCLUSIONS The FO-containing food bars were well tolerated and allowed incorporation of omega-3 fatty acids to occur. Despite evidence of significant metabolic effects on eicosanoid and cytokine production, widespread clinical use of FO for HIV-infected patients is not warranted without further study, particularly given the trend toward a decline in CD4 cell numbers at this dose and with this type of fish oil.

Clinicopathologic features of hepatocellular carcinoma in young patients

To investigate clinicopathologic characteristics of hepatocellular carcinoma (HCC) in young adults, excised tumors from 21 patients younger than 45 years (young group) were compared with findings in tumors from 204 patients older than 45 (old group). in the young group HCC showed (1) a high incidence of positive hepatitis B virus surface antigen (HBsAg) (young 71.4% versus old 20.1%); (2) relatively wellpreserved hepatocellular function (indocyanine green test; young 10.7 ± 8.8% versus old 20.6 ± 10.8%); (3) low incidence of histologically verified concomitant cirrhosis (young 52.4% versus old 78.4%); and (4) a more advanced stage of the disease in TNM classification (Stage III; young 52.4% versus old 18.1%). With respect to survival rates achieved by surgery, there was no statistically significant difference between the two groups. Thus, hepatitis B virus may relate to the occurrence of HCC in the young patients. Despite the advanced stage in the young group, survival rate after surgery was comparable with that achieved in the old group. These observations mean that a close periodic surveillance of young adults with a positive HBsAg is required to detect HCC at an early stage. Treatment of patients with HBsAg using interferon or vidarabine and hepatitis B vaccine should be made to convert HBsAg to negative in these individuals.