Tetsuya Kusumoto

Cytokeratin-positive cells in bone marrow for identifying distant micrometastasis of gastric cancer.

Direct evidence of tumour seeding in distant organs at the time of surgery for gastric cancer is not available. An immunocytochemical assay for epithelial cytokeratin protein may fill this gap since it is a feature of epithelial cells that would not normally be present in bone marrow. The bone marrow of 46 patients with primary gastric cancer was examined for tumour cells, using immunocytochemical techniques and antibody reacting with cytokeratin, a component of the intracytoplasmic network of intermediate filaments. The monoclonal antibody CK2 recognises a single cytokeratin polypeptide (human cytokeratin no. 18) commonly present in epithelial cells. The expression of tumour-suppressor genes p53 and RB for the primary lesion was also determined using the monoclonal antibodies PAb 1801 and 3H9 respectively, and the proliferating activity was determined by the Ki-67 antigen labelling index for MIB-1 antibody staining. Of these 46 patients, 15 (32.6%) presented with cytokeratin-positive cells at the time of primary surgery. The positive findings were related to the undifferentiated tissue type and to the prominent depth of invasion, but not to other clinicopathological factors. In 2 of 15 (13.3%) patients, the depth of invasion was limited to the mucosa. The metastatic potential to bone marrow did not relate to expressions of p53 and RB genes, or to the proliferating activity of MIB-1 staining for the primary lesion of gastric cancer. As tumour cells in bone marrow are indicative of the general disseminative capability of an individual tumour, this technique may be useful for identifying patients at high risk of metastasis from a gastric tumour.

Expression of multidrug‐resistance‐associated protein (MRP) and chemosensitivity in human gastric cancer

Evidence has accumulated that, in addition to the MDRI gene‐coded P‐glycoprotein (Pgp), multidrug resistance‐associated protein (MRP) also mediates the multidrug resistance (MDR) of various human tumors. In the case of gastric cancer, there is little or no involvement of P‐glycoprotein, and the mechanisms of MDR remain to be understood. To search for a possible relationship between expression of MRP and sensitivity to anti‐cancer agents in gastric cancer, 4 gastric cancer cell lines, 43 human gastric carcinomas and 17 adjacent normal gastric tissue samples were analyzed. Expression of MRP mRNA was evaluated using reverse transcription PCR (RT‐PCR) and Southern hybridization. Sensitivity of the test samples to the anti‐cancer drugs cisplatin (CDDP), doxorubicin (DXR) and etoposide (VP‐16) was examined using the MTT{3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl [2H]‐tetrazolium bromide} assay. Immunohistochemical staining with the use of the MRP antibody (MRPrI) was done to confirm the findings regarding the expression of mRNA levels. The MRP expression evaluated with RT‐PCR and Southern hybridization as well as with immunohistochemical staining revealed that 23 of 43 gastric‐cancer tissues (53.5%), 15 of 17 normal gastric tissues (88%) and 3 of 4 gastric‐cancer cell lines (75%) were positive. The MTT assay showed that DXR was significantly more sensitive (p < 0.01) in gastric carcinoma tissues lacking MRP expression than in those with positive expression. The same tendency was seen with the other agents used. Of the cell lines, one which showed no MRP expression also had a higher sensitivity to CDDP, DXR and VP‐16 than the other positive cases. These results show that MRP expression is involved in MDR of human gastric cancer and is inversely related to the chemosensitivity of tumor cells against some anticancer drugs.

Multidrug resistance-associated protein expression in clinical gastric carcinoma

We examined the relationship between the expression of a multidrug resistance‐associated protein (MRP) and the biologic factors regarding invasion and metastasis of human gastric cancer.

Correlation between sialyl Tn antigen and lymphatic metastasis in patients with Borrmann type IV gastric carcinoma.

The expression of sialyl Tn (STn) antigen in 180 patients with Borrmann type IV gastric carcinomas was examined immunohistochemically. The rate of positive STn staining was 32% (57/180) for the primary tumours, and this positive staining correlated well with tumour extension, lymph node metastasis (P < 0.05) and peritoneal dissemination (P < 0.01). One-third (5/15) of patients with positive STn-staining cancer cells had a high level of serum STn. Lesions with positive STn staining were related to a lower survival rate for the patients (P < 0.05). Proliferative activity of the tumour, as measured by proliferating nuclear antigen (PCNA) labelling percentage and argyrophilic nucleolar organiser region (AgNOR) count, was significantly higher (41.5 +/- 13.0%, 3.78 +/- 0.98) in the STn-positive group than in the STn-negative group (34.2 +/- 13.2%, 3.48 +/- 0.85) (P < 0.01, P < 0.05 respectively). Estimating STn antigen may be useful for predicting the likelihood of lymph node metastasis or peritoneal dissemination and the clinical prognosis for patients with Borrmann type IV gastric carcinoma.

Flavone Acetic Acid Increases the Antitumor Effect of Hyperthermia in Mice

The combined effects of flavone acetic acid (FAA), a synthetic flavonoid, and hyperthermia on B16 melanoma cells were investigated. In vitro, FAA alone at concentrations below 100 micrograms/ml was not cytotoxic with a 60-min exposure at 37 degrees C. Hyperthermia at 43 degrees C for 60 min enhanced the cytotoxicity of FAA only at concentrations over 100 micrograms/ml. Inhibition of the growth of B16 melanoma solid tumor by FAA and/or hyperthermia was examined in vivo. FAA (100-200 mg/kg) inhibited tumor growth in a dose-dependent manner. The combined treatment of FAA (200 mg/kg) and hyperthermia (43 degrees C, 15 min) significantly inhibited tumor growth compared to a treatment of FAA or hyperthermia alone. The maximum antitumor effect of FAA combined with hperthermia was obtained when FAA was administered 2 or 4 h before heat. The significantly increased cytotoxicity of FAA combined with hyperthermia seems to relate to specific decreases in tumor blood flow, a reduction in tumor pH, and an increased tumor temperature, without altering pH in the normal tissues. This combined treatment of FAA and hyperthermia warrants further study for treating subjects with solid tumors.

Estrogen-receptor-negative breast cancer tissue is chemosensitive in vitro compared with estrogen-receptor-positive tissue.

The chemosensitivities of 18 estrogen-receptor-positive (ER+) tissues were compared with that of 38 estrogen-receptor-negative (ER-) tissues, using the in vitro succinate dehydrogenase inhibition test. These human breast tissue were exposed to six antitumor drugs: carboquone, adriamycin, mitomycin C, aclacinomycin A, cisplatin and 5-fluorouracil. Decrease in succinate dehydrogenase activity was noted in ER- compared to ER+ tissues, exposed to six antitumor drugs, in particular to adriamycin (p less than 0.001) and aclacinomycin A (p less than 0.05). The sensitive rates were higher in ER- than in ER+ tissues, against all six antitumor drugs. The resistance rates to all drugs tested were 25% in ER- and 45% in ER+ tissues. A higher chemosensitivity is associated with the absence of ER. It appeared that the ER status in case of breast cancer is an important predictor of the response to chemotherapy.

Predictive value of preoperative carcinoembryonic antigen levels for the prognosis of patients with well-differentiated gastric cancer. A multivariate analysis

Serum carcinoembryonic antigen (CEA) levels were determined preoperatively in 221 patients with well-differentiated gastric cancer. The mean preoperative serum CEA level was 15.9 +/- 88.5 ng/ml (1.0-1,133.0 ng/ml) for all patients, and the incidence of an elevated CEA (> 5 ng/ml) was 11.8% (26/221). The CEA-positive patients had larger tumors, a more prominent serosal invasion, more frequent lymphatic and vascular involvement, less expansive tumor growth and higher rates of lymph node and hepatic metastases than did the CEA-negative patients. Thus, the CEA-positive patients had a more advanced stage of disease, and 61.5% underwent noncurative resection (vs. 11.3% in CEA-negative patients). The survival rate of the CEA-positive patients was lower than that of the CEA-negative ones (p < 0.01). As the multivariate analysis revealed the preoperative CEA level to be an independent prognostic factor for survival, an assay for this antigen prior to surgery is to be recommended.

5‐fluorouracil and uft‐sensitive gastric carcinoma has a high level of thymidylate synthase

The authors examined the relationship between the level of thymidylate synthase (TS) and the sensitivity to 5‐fluorouracil (5‐FU) and UFT, a combined oral preparation of 1‐(2‐tetrahydrofuryl)‐5‐fluorouracil (tegafur) and uracil in a molar ratio of 1:4. For the studies we used the subrenal capsule (SRC) assay and 15 human gastric cancer tissues. The TS levels were assayed by the ligand‐binding technique, using [6–3H]FdUMP. The relative variation of tumor size (ΔTuS/TuS0) was calculated to be as follows: ΔTuS/TuS0 = [(TuS6 – TuS0/TuS0] X 100 (%), where TuS6 was the tumor size on day 6 and TuS0 on day 0. The chemosensitivity was considered to be positive when ΔTuS/TuS0 in the treated group decreased to below –10%. Decrease in tumor size was marked in case of exposure to UFT (‐19.8 ± 13.0%) (mean ± standard deviation), compared with that to 5‐FU (‐9.0 ± 7.2%), with a statistically significant difference (P < 0.001). The TS level varied from 1.7 to 30.8 pmol/g gastric cancer tissue and the mean was 7.1 ± 7.2 pmol/g tissue. A correlation was noted between the TS level and decrease in size of the tumor exposed to 5‐FU (r = −0.671) or UFT (r = −0.758): gastric cancer tissue with higher level of TS is more sensitive to 5‐FU and UFT than is that with a lower TS level. These findings show that the sensitivity to 5‐FU and UFT of gastric cancer tissue is related to the TS level and that UFT shows promise for the treatment of patients with gastric cancer.

The p53 tumor suppressor gene in anticancer agent-induced apoptosis and chemosensitivity of human gastrointestinal cancer cell lines

Purpose: While the target of many anticancer agents has been identified, the processes leading to killing of the cancer cells and the molecular basis of resistance to the drugs are not well understood. We used human gastrointestinal cancer cell lines and examined how anticancer agents induced cell killing and how the chemosensitivity of these lines was determined. Methods: Twelve gastrointestinal cancer cell lines were examined for the presence of either a wild-type or mutant p53 gene by direct sequencing. We also determined whether or not cell killing would occur when the cell lines were exposed to anticancer drugs. The sensitivity to the anticancer agents was determined based on colony formation. Results: All 12 gastrointestinal cancer cell lines carried either a wild-type or mutant p53 gene. Three lines, MKN45, MKN74 and COLO320, carried the wild-type p53 gene, and nine carried the mutant p53 gene. When three lines were exposed to the anticancer agents etoposide, doxorubicin (DXR) or 5-fluorouracil (5-FU), cell death ensued. In these cells, the population of cells in G1 phase increased after exposure to high-dose anticancer agents, but cells in G2 phase increased when exposed to low-dose anticancer agents. Our observations support the concept that cells carrying the wild-type p53 gene tend to be sensitive to etoposide and DXR and, in particular, deletion of the p53 function results in a greater resistance to anticancer agents. Conclusion: Based on our findings, human gastrointestinal cancer-related cell death apparently occurs via a p53-dependent pathway. A relationship was observed between the induction of cell death and chemosensitivity.

Sodium Succinate Enhances the Colorimetric Reaction of the in vitro Chemosensitivity Test: MTT Assay

We compared the colorimetric reactions between the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl 2H-tetrazolium bromide (MTT) assay and the succinate dehydrogenase inhibition (SDI) test, in order to evaluate the usefulness of the SDI test for in vitro chemosensitivity testing. The addition of sodium succinate enhanced the colorimetric absorbance at 565 nm in the MTT assay in a dose- and a time-dependent manner, in mouse sarcoma-180 (S-180) cells. At 10 microM of sodium succinate, a dose used in the SDI test, the absorbance of the MTT assay increased by about 2.5-fold in the S-180 cells and in 10 human tumor tissues. The absorbance in the SDI test correlated well with the viable cell number of S-180 cells (r = 0.9993). These results show that the SDI test, using MTT as a tetrazolium salt, has a higher sensitivity for predicting cell viability, compared to the MTT assay.