Akihiko Suto

Single‐cell Suspension Assay with an MTT End Point Is Useful for Evaluating the Optimal Adjuvant Chemotherapy for Advanced Gastric Cancer

One hundred and forty‐eight patients with gastric cancer admitted to Keio University Hospital between July 1988 and October 1992 underwent resection of the primary lesion, as well as single‐cell suspension assay of fresh surgical materials with 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT assay) for chemosensitivity evaluation. Fifty patients with histologically stage III or IV gastric cancer were enrolled in this study, among whom 10 received no chemotherapy after surgery while 40 received chemotherapy at equivalent dose levels after surgery. The patients given chemotherapy were divided into two groups consisting of an “Adapted” group treated with at least one agent identified as effective by the assay, and a “Non‐adapted” group treated with agents to which the cells were not sensitive in the assay, in order to identify the optimal cut‐off inhibition rate (IR) in MTT assay for evaluation of the appropriate adjuvant cancer chemotherapy after surgery. A cut‐off IR of 30% was optimal for differentiating the survival rates between the “Adapted” and “Non‐adapted” groups. Patients treated with drugs which showed more than 30% IR on their surgical specimens showed a better survival rate than patients treated with drugs which were ineffective in the assay.

Carcinosarcoma of the ampulla of Vater: a case report with immunohistochemical and ultrastructural studies.

Carcinosarcoma of the duodenum has not been reported previously, although this type of tumor has been detected in other organs. We present here a case of carcinosarcoma of the duodenum, including immunohistochemical and electron microscopical findings. An ulcerating tumor, located in the duodenal ampullary region, contained two divergent components: ordinary differentiated tubular adenocarcinoma, and sarcomatoid tissue composed of spindle tumor cells. Immunohistochemically, the adenocarcinoma cells were stained with antibodies against epithelial markers including keratin and CA19-9. In contrast, the sarcomatoid tissue was strongly positive for vimentin and was focally positive for myoglobin, keratin, and CA19-9. We speculate that the sarcomatoid element of the carcinosarcoma arose from part of the ordinary adenocarcinoma tissue.

Resistant mechanisms of anthracyclines — pirarubicin might partly break through the P-glycoprotein-mediated drug-resistance of human breast cancer tissues

Juliano and Ling initially reported the expression of a 170 kDa glycoprotein in the membrane of Chinese hamster ovarian cells in 1976, and named this glycoprotein P-glycoprotein (P-gp) based on its predicted role of causing “permeability” of the cell membrane. After much research on anthracycline-resistance, this P-gp was finally characterized as a multidrug-resistant protein coded by themdrl gene. Multidrug resistance associated protein (MRP) was initially cloned from H69AR, a human small cell-lung carcinoma cell line which is resistant to doxorubicin (DXR) but does not express P-gp. MRP also excretes substrates through the cell membrane using energy from ATP catabolism. The substrate of MRP is conjugated with glutathione before active efflux from cell membrane. Recently, membrane transporter proteins were re-categorized as members of “ATP-Binding Cassette transporter” (ABC-transporter) superfamily, as shown at http://www.med.rug.nl/mdl/humanabc. htm and http://www.gene.ucl.ac.uk/nomenclature/genefamily/abc.html. A total of ABC transporters have been defined, and MDR1 and multidrug resistance associated protein 1 (MRP1) were reclassified as ABCB1 and ABCC1, respectively. Their associated superfamilies include 11 and 13 other protein, in addition to ABCB and ABCC, respectively. Lung resistance-related protein (LRP) is not a member of the superfamily of ABC transporter proteins, because it shows nuclear membrane expression and transports substrate between nucleus and cytoplasm. LRP was initially cloned from a non-small cell lung carcinoma cell line, SW1573/2R120 which is resistant to DXR, vincristine, etoposide and gramicidin D and does not express Pgp. The mechanisms of resistance remains unclear, and why some resistant cell lines express P-gp and others express MRP and/or LRP is likewise unclear.

Alteratioon in proliferative and endocrine responsiveness of human mammary carcinoma cells by prototypic tumor-suppressing agents

The experiments performed in this study were designed to establish that (1) acquisition of anchorage-independent growth, a biological characteristic of tumorigenically transformed phenotype, can be modulated by prototypic tumor-suppressing agents, and (2) modulation of growth is influenced by the metabolic competence of the cells to biotransform estradiol, MCF-7 human breast carcinoma cells exhibited linear cell proliferative kinetics with a 41-hour population doubling time, and a 15% colony-forming efficiency in 0.33% agar. Indole-3-carbinol (13C), a naturally occurring tumor-suppressive agent; tamoxifen (TAM), an antiestrogenic agent; and 4-hydroxytamoxifen (4-OHTAM), a metabolite of TAM, demonstrated 73.7%, 72.5%, and 89.9% suppression in anchorage-independent growth of MCF-7 cells, respectively. At the metabolic level, 13C and 4-OHTAM induced 2.3-fold (P < 0.0001) and 1.3-fold increase (P = 0.001) relative to their own controls in the extent of 2-hydroxylation of estradiol. The results indicate that growth inhibition by 13C, TAM, and 4-OHTAM may in part be due to altered estradiol metabolism in MCF-7 cells. Thus, anchorage-independent growth and altered biotransformation of estradiol may constitute useful cellular and endocrine markers to evaluate the biological response of chemosuppressive agents.

Genotoxic Damage and Aberrant Proliferation in Mouse Mammary Epithelial Cells

Publisher Summary This chapter discusses genotoxic damage and aberrant proliferation in the mouse mammary epithelial cells. Conversion to the fully transformed tumorigenic phenotype is the end result of a series of molecular, metabolic, and cellular events leading to the neoplastic state. This series of events has been reported in vivo as well as in vitro during mammary carcinogenesis. In addition to intermediate biomarkers, such as deregulated expression of oncogenes, mutagenic changes, and altered proliferation, changes in estrogen metabolism may be a biomarker for target tissue susceptibility to tumorigenic changes. Increases in DNA repair have been extensively validated as a marker for genotoxic DNA damage. Using hydroxyurea to suppress replicative DNA synthesis, one can readily measure DNA repair activity. 16α-OHE1 can directly interact with cellular DNA and thereby function as an initiator. The hyperproliferation of the epithelial component has been reported to occur in tissues at increased risk for tumors or following exposure to carcinogens and is implicated as an intermediate biomarker preceding transformation. The ability to acquire anchorage-independent growth has been correlated with treatment with various initiators of tumorigenic transformation and may be a cellular marker for tumor genesis. Preliminary studies have shown that inhibiting the formation of 16α-OHE1 by treatment with indole-3-carbinol decreases the incidence of tumors in susceptible mice.

The Japanese Guidelines for Breast Cancer Screening

OBJECTIVE The incidence of breast cancer has progressively increased, making it the leading cause of cancer deaths in Japan. Breast cancer accounts for 20.4% of all new cancers with a reported age-standardized rate of 63.6 per 100 000 women. METHODS The Japanese guidelines for breast cancer screening were developed based on a previously established method. The efficacies of mammography with and without clinical breast examination, clinical breast examination and ultrasonography with and without mammography were evaluated. Based on the balance of the benefits and harms, recommendations for population-based and opportunistic screenings were formulated. RESULTS Five randomized controlled trials of mammographic screening without clinical breast examination were identified for mortality reduction from breast cancer. The overall relative risk for women aged 40-74 years was 0.75 (95% CI: 0.67-0.83). Three randomized controlled trials of mammographic screening with clinical breast examination served as eligible evidence for mortality reduction from breast cancer. The overall relative risk for women aged 40-64 years was 0.87 (95% confidence interval: 0.77-0.98). The major harms of mammographic screening were radiation exposure, false-positive cases and overdiagnosis. Although two case-control studies evaluating mortality reduction from breast cancer were found for clinical breast examination, there was no study assessing the effectiveness of ultrasonography for breast cancer screening. CONCLUSIONS Mammographic screening without clinical breast examination for women aged 40-74 years and with clinical breast examination for women aged 40-64 years is recommended for population-based and opportunistic screenings. Clinical breast examination and ultrasonography are not recommended for population-based screening because of insufficient evidence regarding their effectiveness.

In Vitro and In Vivo Modulation of Growth Regulation in the Human Breast Cancer Cell Line MCF-7 by Estradiol Metabolites.

BackgroundThe natural estrogen 17β-estradiol (E2) functions as a potent tumor promoter during tumorigenic transformation of the mammary gland. From amongst the various pathways of E2 metabolism upregulation of C16α-hydroxylation of E2 has been associated with carcinogenesis. In the present studyin vitro andin vivo experiments were performed on estrogen receptor positive human breast cancer MCF-7 cells to examine whether the natural estrogen E2 and its metabolites 16α-hydroxyestrone (16α-OHEi) and 2-hydroxyestrone (2-OHE1) function as modulators of tumor cell growth.MethodsAn anchorage-independent growth assay was used forin vitro study by counting the number of tri-dimensional colonies formed by MCF-7 cells suspended in 0.33% agar.In vivo experiments examined the effect of implanting metabolite material pellets into female nude mice.ResultsIn the anchorage-independent growth assay (AIG), continuous 14-day exposure to E2 and to 16α-OHE1 at 200 ng/ml induced a 59.4% and a 105.9% increase (P=0.001) respectively in the number of colonies of MCF-7 cells. Identical treatment with 2-OHE1, however, failed to increase AIG relative to that seen in the solvent treated control cultures. In thein vivo tumorigenicity assay, treatment of nude mice with 1.5 mg E2 or 16α-OHE1 resulted in a 335.4% and a 384.1% increase (P<0.0002) in tumor growth, while identical treatment with 2-OHE1 failed to exhibit any increase relative to the control group.ConclusionsThese results suggest that the 16α- and 2-hydroxylated metabolites of E2 may directly affectin vitro growth of MCF-7 cells via an autocrine mechanism andin vivo growth via paracrine mechanisms. Thus, E2-mediated growth regulation in MCF-7 cells may in part be due to distinct effects of specific E2 metabolites on the breast cancer cells.

Nude mouse resists hepatic metastasis of the allogeneic tumor, colon-26

The ability of the host-immune defense mechanism of nude mice and their immunocompetent littermates to prevent liver metastases from the murine colon carcinoma, colon-26, was assessed. Give thousand tumor cells suspended in 0.05 ml of Hanks balanced salt solution were inoculated into the spleens of BALB/c nu/+and BALB/c nu/nu mice. On the 21st day after inoculation, all the mice were sacrificed, and the liver metastases counted and the livers weighed. All the BALB/c nu/+mice were found to have developed hepatic metastases with a mean of 10 nodules, whereas no hepatic metastases were observed in any of the 10 BALB/c nude mice. On the other hand, 4 of 6 nude mice developed hepatic metastases after treatment with anti-asialo GM1 antibody. These results indicate that the BALB/c nude mouse has an excellent host-immune defense mechanism for preventing liver metastasis, with NK cells in the liver and/or blood circulation perhaps playing an important role.

Human breast carcinoma (ZR-75-1) serially transplanted into nude mice--with reference to estradiol dependency and sensitivity to tamoxifen.

Tumor cells of the ZR-75-1 line (1×107 in 0.5 ml) were inoculated into the right thigh muscles of BALB/c female nude mice which were, at the same time, given 1.0 mg of estradiol subcutaneously. After the transferable strain had been established, the tumors were then transplanted into female and male nude mice either with or without estradiol treatment. Although no exponential tumor growth was observed in the untreated male and female nude mice, no complete rejection was found during the experiments. The estrogen receptor of this strain was positive and the growth of ZR-75-1 was dependent on exogenous estradiol. ZR-75-1 in the nude mouse was insensitive to tamoxifen treatment, given as 5 mg/kg intramuscularly twice a week, suggesting that dependency on estradiol is not necessarily correlated with endocrine sensitivity to tamoxifen.

The influence of stromal cells on the MTT assay (I)—In vitro chemosensitivity of the tumor and stromal cells to mitomycin C—

To clarify the influence of stromal cells on the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assay (MTT assay), a gastric carcinoma cell line (KATO-III) and a human fibroblast cell line (IMR-90) were subjected to a colorimetric assay, in which the chemosensitivity to mitomycin C was assessed in different mixtures of the cell lines. KATO-III was found to be highly sensitive to mitomycin C at 10 μg/ml, whereas IMR-90 was insensitive to mitomycin C at the same concentration. When the mixtures of these two cell lines were tested by the assay, a mixture of more than 25 per cent stromal cells reduced the sensitivity of KATO-III to mitomycin C. This suggested that the stromal cells in fresh surgical specimens might reduce the apparent sensitivity of the tumor cells.