Junichi Koh

Human breast carcinoma (MCF-7) serially transplanted into nude mice

The tumor cells (0.5 ml, 1×107) of MCF-7 line were inoculated into the subcutaneous tissue or intraperitoneum of female BALB/c nude mice. Primarily tansplanted mice were treated with 17β-estradiol dipropionate (E2) in a dose of 5 mg/kg and 17α-hydroxy progesterone caproate (Pg) in a dose of 250 mg/kg once a week. After the transferable strain was established, tumors were transplanted into female and male mice treated with E2, Pg, and E2+Pg. The tumors treated with E2 or E2+Pg grew exponentially while tumors in the other group regressed. Pg was assumed to play some role in the growth of MCF-7, in the presence of estrogen. Although cytosol estrogen receptors (ERc), nuclear estrogen receptors (ERn), and progesterone receptors (PgR) were detected by dextran coatedcharcoal method and exchange assay in the growing tumors, ERn and PgR of regressing tumors was usually negative. This MCF-7 strain in nude mice may be a promising animal model for studying chemo-hormone therapy for human breast carcinomas.

Antitumor activity of paclitaxel against human breast carcinoma xenografts serially transplanted into nude mice

Paclitaxel (BMS‐181339: Taxol) is a promising agent against previously treated breast cancer. The antitumor activity of paclitaxel was evaluated using five human breast carcinoma xenografts in nude mice.

UCN-01 (7-hydroxystaurosporine) inhibits the growth of human breast cancer xenografts through disruption of signal transduction

Background7-Hydroxystaurosporine (UCN-01), originally isolated as a phospholipid-dependent protein kinase C inhibitor, has been shown to have antitumor activity against several human cancer cell lines. UCN-01 inhibits cell cycle progression from the G1 to S phase by inhibition of cyclin-dependent kinase (CDK) activity and induction of intrinsic CDK inhibitor protein, leading to dephosphorylation of retinoblastoma (Rb) protein.Materials and MethodsThe antitumor activity of UCN-01 has been investigated against three human breast carcinoma strains serially transplanted into nude mice, including estrogen-dependent MCF-7, Br-10, and estrogen-independent MX-1. When the inoculated tumors started growing exponentially, UCN-01 (7.5 mg/kg) was administered intraperitoneally on five consecutive days a week for 2 weeks. The antitumor effect was evaluated as the lowest T/C ratio (%) during the experiments, where T was the relative mean tumor weight of the treated group and C was that of the control group. At the end of UCN-01 administration expression of p21, a protein of the CDK inhibitor family, and phosphorylated and dephosphorylated Rb protein was detected by Western blotting using treated and control tumors.ResultsUCN-01 had activity against MCF-7 and Br-10, with the lowest T/C ratios of 25.0% and 27.0%, respectively, while MX-1 was resistant to UCN-01 with a T/C ratio of 65.9%. The antitumor spectrum of UCN-01 was different from that of other conventional agents such as doxorubicin and cyclophosphamide which were ineffective against Br-10 but were active against MX-1. Although p21 was induced in three tested strains by UCN-01, little dephosphorylated Rb protein was expressed in MX-1 compared with Br-10 and MCF-7 (in vitro).ConclusionUCN-01 appeared to be a promising agent for the treatment of breast cancer, with a different mode of action and antitumor spectrum from other currently available antitumor drugs.

Combined antitumor activity of 7-Hydroxystaurosporine (UCN-01) and Tamoxifen against human breast carcinomain Vitro andin Vivo

Background7-Hydroxystaurosporine (UCN-01) was originally isolated as a protein kinase C inhibitor and has shown antitumor activity against several human cancer cell lines. UCN-01 inhibits cell cycle progression from the G1to the S phase and is associated with inhibition of cyclin-dependent kinase (CDK) activity and induction of intrinsic CDK inhibitor p21, leading to dephosphorylation of retinoblastoma (Rb) protein. Tamoxifen (TAM) traps cancer cells in the Gl phase, suggesting that the mechanism of action of TAM is similar to that of UCN-01. The present study was conducted to assess the antitumor activity of UCN-01 combined with TAM against human breast carcinoma cellsin vitro andin vivo.Materials and MethodsMCF-7 cells were treated with UCN-01, TAM, or UCN-01 combined with TAM at various concentrationsin vitro. The antitumor effect was evaluated as the inhibition rate (I.R.%) by MTT assay. Two human breast carcinoma xenografts in nude mice, MCF-7 and Br-10, were treated with UCN-01, TAM or both agents together. The expression of p21 and the phosphorylation status of Rb protein in MCF-7 cells were detected by Western blotting.ResultsUCN-01 or TAM alone inhibited the proliferation of MCF-7 cells in a concentration-dependent manner. Combined treatment with UCN-01 followed by TAM inhibited the growth of MCF-7 cells synergistically and no significant differences in cytotoxicity were observed between the different sequences of UCN-01/TAM and TAM/UCN-01. Combination treatment with UCN-01 and TAM against MCF-7 and Br-10in vivo exhibited superior antitumor effects compared with either agent treatment alone. Although 0.1μg UCN-01 per ml (I.R.: 48.1%) or 2μM TAM (I.R.: 31%) induced p21 expression, phosphorylation of Rb protein was not inhibited. However, combination treatment with UCN-01 and TAM at the same concentrations resulted in an I.R. of 67% and dephosphorylation of Rb protein.ConclusionThe present study suggests that combining UCN-01 and TAM could result in augmented cytotoxicity because of their similar mechanism of action. This combination may have potential clinical applications for breast cancer treatment, by reducing the toxicity of UCN-01.

Changes in the hormone receptors of human breast carcinoma xenografts in nude mice by treatment with cytotoxic agents

We examined the effect of chemotherapeutic agents on the estrogen receptors (ER) of breast carcinomasin vivo using human breast carcinoma strains (Br-10, T-61) serially transplanted into nude mice. When the tumor size reached approximately 1×1×1 cm, mitomycin C (MMC) at doses of 1, 2 and 4.5 mg/kg and cyclophosphamide (CPA) at a dose of 120 mg/kg, were administered once intraperitoneally, and the ERs of the tumors were measured sequentially by the dextran-coated charcoal method. Four days after the MMC administration at above doses, the binding sites of ER in Br-10 were not reduced and binding affinity was not affected. When the changes in ER content with time after the treatment with 4.5 mg/kg MMC and 120 mg/kg CPA were investigated, the ER content was found to be stable until 4 days after the treatment with both drugs, although the growth of T-61 had been significantly inhibited by the drugs. From these findings, it seems reasonable to initiate chemotherapy before endocrine therapy, since the chemotherapeutic agents did not reduce the ER content of the breast cancer strains.

The use of an ileostomy connector to diminish the frequency of defecation prior to ileostomy closure in patients with a pelvic pouch

A new method for allowing stool passage into the pelvic pouch before ileostomy closure to verify the defecation state and diminish stool frequency is reported herein. This was accomplished by fitting an ileostomy connector connecting the proximal and distal openings of the diverting loop stoma. The ileostomy connector was initially in place for 6 h a day, the length of time being gradually increased until it was able to be left in for 24 h a day over a 3-month period. The calculated daily frequency of stools decreased from 24 to 6 or 7 times, and the mean daily frequency immediately after ileostomy closure was 6.5 times. Physiological study also showed an improvement, with squeeze pressure increasing from 35 cmH2O to 116 cmH2O and the maximum tolerated volume increasing from 35 ml before, to 90 ml 3 months following the use of an ileostomy connector. Thus, we conclude that an ileostomy connector may be useful to predict postoperative functional outcome and its complications, and to diminish the frequency of defecation before ileostomy closure in patients with a covering loop stoma.

Mode of Action of Estra-1,3,5(10)-triene-3,17β-diol 3-Benzoate 17-((4-(4-Bis(2-chloroethyl)amino)phenyl)-1-oxobutoxy)acetate) on Human Breast Carcinoma Xenografts in Nude Mice

To elucidate the mode of action of busramustine (KM2210), 17β‐ and α‐busramustine, estradiol and chlorambucil were used for experimental chemo‐ and endocrino‐therapy against hormone‐dependent (T‐61) and independent (MX‐1) human breast carcinomas serially transplanted into BALB/cA female nude mice. Busramustine was administered po daily for 3 weeks at doses of 12.5–300 mg/kg for the β‐isomer and 25–300 mg/kg for the α‐isomer. Five to 50 mg of estradiol per kg was administered im once, and 3 to 6 mg of chlorambucil per kg was administered po daily for 3 weeks. All of the compounds were effective against estrogen receptor‐positive T‐61 with a clear dose‐response relationship, while estrogen receptor‐negative MX‐1 was sensitive to all of the agents except estradiol. Since the α‐isomer of busramustine was effective against both tumor lines, the mode of action of 17β‐busramustine may not be related to estrogenic action by estradiol released from the maternal compound. However, 17bT‐busramustine generated the estrogen receptor system of T‐61 tumor and resulted in the endometrial hyperplasia of tumor‐bearing nude mice, suggesting that this compound also has estrogenic action on transplanted human breast carcinoma and tumor‐bearing host mice, besides non‐estrogenic antitumor activity on human breast carcinoma xenografts.

Combined cytotoxic and endocrine therapy for human breast carcinoma (Br-10) serially transplanted into nude mice

Cytotoxic and endocrine therapy on a human breast carcinoma (Br-10) serially transplanted into nude mice was given with reference to the sequence of drug administration. Mitomycin C (MMC) was combined with 2.5 mg/kg of tamoxifen (TAM). MMC was dissolved in 0.2 ml of physiological saline and administered intraperitoneally once weekly. TAM was dissolved in 0.1 ml of sesame oil and administered intramuscularly twice weekly. Both drugs were administered in the reverse sequence for 2 or 3 weeks. Cytosol estrogen receptor (ERc), nuclear estrogen receptor (ERn) and progesterone receptor (PgR), and3H-thymidine uptake labeling index (L.I.) were assayed after the treatment. When 1.5 mg/kg of MMC was combined with TAM, statistically significant differences were nil between the different sequential administrations. When the MMC administration was reduced to 0.75 mg/kg and 2 weeks, respectively, the MMC→TAM sequence was more effective than the reversed sequential administration. MMC preserved ERc and depressed L.I. to almost half of that of the control tumor. TAM generated the ER systems and slightly depressed L.I. These different modes of action between MMC and TAM on ER systems and L.I. may explain the antitumor effects of different sequential administrations.