Behavior of Ultra Fine Particles in Electric Field.

The behavior of ultra fine particles in an electric field was measured in order to apply it to classification of fine particles. The sample particles used are spherical polystyrene particles with the average size of 0.03, 0.1, 1.0, 4.3 and 9.6 µm. The forces acting the particles in an electric field are considered to be electrical force, friction force and some other forces like the asymmetric effect and electrophoretic retardation effect which appear in ionic behavior. We found that the moving velocity of particles depends on the particle size. When the particle size is less than 1 µm, the velocity increases with increasing the particle size. On the other hand, the velocity deceases with an increase in the particle size, when the particle size is larger than 1 µm. We can apply the behavior to classification of fine particles. The phenomena could be explained by various forces acting the fine particles in the electric field.

Surgical stress response after colorectal resection: a comparison of robotic, laparoscopic, and open surgery.

BACKGROUND: The perioperative immune status of colorectal robotic surgery (RS), laparoscopic surgery (LS), and open surgery (OS) patients has not been compared. Our aim was to evaluate perioperative stress and immune response after RS, LS and OS. METHODS: This prospective study included 46 colorectal surgery patients from the Department of Surgical Oncology of the University of Tokyo Hospital. Peripheral venous blood samples were obtained preoperatively and on postoperative days 1, 3, and 6. We evaluated expression of HLA-DR (marker of immune competence), C-reactive protein (CRP) levels, and lymphocyte subset counts (natural killers, cytotoxic T cells and helper T cells). RESULTS: Fifteen, 23, and 8 patients underwent RS, LS and OS, respectively. HLA-DR expression was the lowest on day 1 and gradually increased on days 3 and 6 in all the groups. There was no significant difference in postoperative HLA-DR expression between the RS and LS group. However, on day 3, HLA-DR expression in the RS group was significantly higher than in the OS group (p = 0.04). On day 1, CRP levels in the LS group were significantly lower than in the RS group (p = 0.038). There were no significant perioperative changes in the lymphocyte subset cell count between the three groups. CONCLUSIONS: Perioperative surgical stress, as evaluated by immunological parameters, was comparable between robotic and laparoscopic surgery and higher with open surgery. Robotic surgery may be an alternative to laparoscopic surgery, as a minimally invasive surgery option for colorectal cancer.

Induction of interleukin-12 by Lactobacillus strains having a rigid cell wall resistant to intracellular digestion.

Some strains of lactobacilli can stimulate macrophages and dendritic cells to secrete IL-12, which plays a key role in activating innate immunity. We examined the IL-12-inducing ability of 47 Lactobacillus strains belonging to 10 species in mouse peritoneal macrophages, and characterized the properties important for the induction of IL-12. Although considerable differences in IL-12-inducing ability were observed among the strains tested, almost all strains belonging to the Lactobacillus casei group (L. casei, Lactobacillus rhamnosus, and Lactobacillus zeae) or to Lactobacillus fermentum induced high levels of IL-12. Phagocytosis of lactobacilli was necessary for IL-12 induction, and the strains with strong IL-12 induction were relatively resistant to lysis in the macrophages. The sensitivity of Lactobacillus strains to in vitro treatment with M-1 enzyme, a member of the N-acetylmuramidases, was negatively correlated with IL-12-inducing ability. Using a probiotic strain, L. casei strain Shirota (LcS), we showed that the cell wall of LcS could be digested by long-term treatment with a high dose of M-1 enzyme and that the IL-12-inducing ability was diminished according to the duration of the enzyme treatment. The soluble polysaccharide-peptidoglycan complex released from the cell wall of LcS did not induce IL-12, whereas the insoluble intact cell wall of LcS induced IL-12. These results suggest that the intact cell wall structure of lactobacilli is an important element in the ability to induce IL-12 and that Lactobacillus strains having a rigid cell wall resistant to intracellular digestion effectively stimulate macrophages to induce IL-12.

Phase I clinical study of a novel lipophilic platinum complex (SM-11355) in patients with hepatocellular carcinoma refractory to cisplatin/lipiodol.

SM-11355 is a platinum complex developed to treat hepatocellular carcinoma (HCC). It is administered via the hepatic artery, using a carrier, lipiodol, that consists of ethyl esters of iodized poppy seed oil. We have performed a phase I clinical trial of an SM-11355-lipiodol formulation in 11 HCC patients, in order to investigate the maximum allowable dose and to maximize the efficacy and safety of the drug in the treatment of HCC. The SM-11355 arterial infusion suspension was administered at doses of 6, 12 and 20 mg ml(-1) in a maximum lipiodol volume of 6 ml. An antitumour efficacy rating of complete response was achieved for one patient and a partial response rating was achieved for a second patient, giving an overall response rate of 18.2%. Anorexia, nausea and vomiting, pyrexia, thrombocytopenia and increases in AST, ALT and total bilirubin were observed as adverse effects, but each was transient and each patient had recovered completely by 4 weeks after drug administration. Hence, we concluded that the maximum allowable dose was not reached in this study. Overall, our results suggest that SM-11355 is effective in treating HCC and we suggest that the dose for early phase II trials should be 20 mg ml(-1).

A potential use of a synthetic retinoid TAC-101 as an orally active agent that blocks angiogenesis in liver metastases of human stomach cancer cells.

TAC-101 (4-[3,5-bis(trimethylsilyl)benzamido]benzoic acid) is a novel, synthetic retinoid that is effective against liver metastases of human gastrointestinal cancer cells such as the human stomach carcinoma line AZ-521 in animal models, and is currently in use in phase I cancer trials. However, the mechanism of its antimetastatic action is still poorly understood. Tumor metastasis depends on angiogenesis, and various retinoids have been found to exhibit antiangiogenic activity. Based on these findings we here examined the antiangiogenic effects of TAC-101. Oral administration of TAC-101 (2-8 mg/kg/day) resulted in a drastic suppression of the AZ-521 cell-induced angiogenesis in a mouse dorsal air sac assay system, compared to the vehicle alone. Immunohistochemical analysis with antibody against the endothelial marker CD31 revealed a significant reduction in microvessel density in liver metastases from animals treated with TAC-101 (8 mg/kg p.o.), compared to liver metastases from the untreated control animals. The ability of TAC-101 (8 mg/kg p.o.) to prevent experimental liver metastasis of AZ-521 cells in athymic nude mice was comparable with that of the known angiogenesis inhibitor TNP-470 (30 mg/kg s.c.). TAC-101 also affected angiogenesis in chorioallantoic membranes and some functions of endothelial cells associated with angiogenesis, whereas the retinoid failed to suppress AZ-521 cell proliferation directly. These data suggest that the TAC-101 is an orally active antiangiogenic agent and that this antiangiogenic property may contribute to its efficacy against liver metastasis of human stomach cancer cells.

The induction of apoptosis and inhibition of AP-1 activity by TAC-101 (4-[3,5-bis(trimethylsilyl) benzamido] benzoic acid) may result in life prolonging effect in animals bearing metastasizing cancer.

BACKGROUND: The incidence of cancers of the digestive tract has been high among all of the cancers in Japan and the western hemisphere. The poor prognosis of patients, especially those with liver metastases, has become a great challenge for the development of a new drug to cope with this problem. MATERIALS AND METHODS: Mice implanted by intrasplenic injection of TMK-1, human gastric carcinoma cells, were used to examine the life-prolonging effect of TAC-101. To elucidate a mechanism of action of TAC-101, the drug-induced apoptosis was assessed by DNA ladder formation whilst the prevention of transcription factor AP-1 binding to its DNA recognition sequence was assessed by gel shift assay. RESULTS: TAC-101 showed the life prolonging effect in a model of experimental liver metastasis of TMK-1. The antitumor effect, expressed as T/C (%), was 201, 141 and 112%, for TAC-101 (2 mg/kg), ATRA (8 mg/kg) and 5-FU (19 mg/kg), respectively. The in vitro experiments revealed that the anticancer activity of TAC-101 is related to its ability to induce apoptosis within a short period of time in TMK-1 cells and human leukemic cells, HL-60. TAC-101-induced apoptosis was suppressed by the inhibitors of proteases, specifically by Z-Val-Ala-DL-Asp-fluoromethylketone, indicating the involvement of caspase activation. TAC-101 also inhibited, in a concentration-dependent manner, the binding of AP-1 to its DNA binding sites present in the promoter region of the genes involved in the control of cell migration, invasion, and angiogenesis. CONCLUSION: TAC-101 may suppress liver metastasis by the induction of an apoptotic mechanism(s) in cancer cells and possibly by controlling transcriptional activity of AP-1.

UFT and its metabolites inhibit cancer-induced angiogenesis. Via a VEGF-related pathway.

Treatment with UFT for spontaneous lung metastasis of murine renal carcinoma (RENCA) after resection of the primary tumor has resulted in significant prolongation of the life span of tumor-bearing animals. UFT inhibited the growth of metastatic nodules in the lung, apparently via decreased density of microvessels in the metastatic foci. Subsequent experiments used dorsal air sac assay to directly trace newly forming microvessels. UFT abrogated the process of angiogenesis, induced by the RENCA cells, in a dose-dependent manner. The inhibitory effect appeared to originate from tegafur, a component of UFT, and from its known metabolites: fluorouracil (5-FU), gamma-hydroxybutyric acid (GHB), and gamma-butyrolactone (GBL). The inhibition of angiogenesis by UFT appeared to be a common phenomenon, also observed in other human cancer cell lines characterized by an excessive production of vascular endothelial growth factor (VEGF)--such as gastric, lung, and colon cancers. In vitro analysis revealed that 5-FU and gamma-hydroxybutyric acid regulated VEGF-dependent responses of human umbilical vein endothelial cells. Dorsal air sac assay revealed that UFT, 5-FU, and gamma-hydroxybutyric acid strongly inhibited the angiogenesis induced by recombinant human VEGF. These data suggest that the antiangiogenic activity of UFT is at least partially associated with an ability of the metabolites of UFT to interfere with VEGF-dependent responses of vascular endothelial cells.

Anticancer effect of 4-[3,5-bis(trimethylsilyl)benzamido] benzoic acid (TAC-101) against A549 non-small cell lung cancer cell line is related to its anti-invasive activity.

We examined the effects of TAC-101 on the invasion and metastasis of human non-small cell lung cancer (NSCLC) cell lines. TAC-101 showed an ability to inhibit in vitro invasiveness of NSCLC at a non-cytotoxic concentration range of 3-10 microM; such concentration levels were easily achievable following oral administration of therapeutically effective doses. The inhibition of cell invasion at 10 microM of TAC-101 accounted for 58-69% when compared with control cells. Oral administration of TAC-101 (4 mg/kg/day) to mice bearing lung implanted A549 lung cancer resulted in significant life-prolonging effect (T/C: 143%). More pronounced life-prolonging effect was observed in the experimental liver metastasis model of A549, where T/C of 215% was observed following administration at 4 mg/kg/day of TAC-101. However, TAC-101 did not show the direct anti-tumor effect against the established A549 tumor xenografts after subcutaneous implantation. These findings suggest that TAC-101 interferes with cell-to-cell interaction processes leading, for instance, to the inhibition of the invasion of NSCLC cells. Taking into account the pharmacological properties of TAC-101, it is expected that TAC-101 may be a suitable candidate drug for the treatment of lung cancer patients, especially those with a predictable metastasizing potential.

Papillary serous carcinoma of the peritoneum: analysis of clonality of peritoneal tumors.

Papillary serous carcinoma of the peritoneum (PSCP) is a primary neoplasm of peritoneal origin, and is histologically difficult to differentiate from papillary serous carcinoma of the ovary (PSCO). PSCP is frequently accompanied by many peritoneal tumors, and has been managed as a disseminated disease. In previous reports, however, the clonality of the tumors has not been fully discussed. Recently, the significant roles of the p53 and BRCA1 genes in PSCP have been reported. In this study, we investigated immunohistochemical staining for p53 proteins, and investigated p53 gene mutations, using DNA sequencing analysis, to clarify the clonality of PSCP tumors. Immunohistochemically, all the tumor samples demonstrated nuclear overexpression of p53 proteins, and the DNA sequencing analysis of the p53 gene showed diverse point mutations at codons 167 and 192 in two of four anatomically different tumors. In conclusion, the possibility of polyclonality of PSCP tumors is suggested.

Estrogen agonistic/antagonistic effects of miproxifene phosphate (TAT-59)

PURPOSE: We evaluated miproxifene phosphate (TAT-59) to elucidate its efficacy in antiestrogen therapy for breast cancer patients and to assess its tissue-selective estrogenic/antiestrogenic activity. METHODS: Using DP-TAT-59, a major and active metabolite of TAT-59, an in vitro cell growth inhibition test was performed. Antitumor activity was determined using TAT-59 against human tumor xenografts of the MCF-7 and the Br-10 cell lines and MCF-7-derived tamoxifen-resistant cell lines, R-27 and FST-1. The antitumor activity of DP-TAT-59 and DM-DP-TAT-59, major metabolites of TAT-59 found in human blood following a TAT-59 dose, was also examined after intravenous administration to experimental animals. The residual estrogenic activity of TAT-59, evaluated in terms of bone and lipid metabolism in ovariectomized rats, was then compared with that of tamoxifen. RESULTS: DP-TAT-59 significantly inhibited the proliferation of estrogen receptor-positive MCF-7 and T-47D tumor cells in the presence of 1 nM estradiol. TAT-59, given to mice bearing MCF-7 or Br-10 xenografts, at the dose level of 5 mg/kg, exerted a significant growth inhibitory effect that was stronger than that of tamoxifen. Moreover, R-27 and FST-1 tumors, which show a resistance to tamoxifen, responded strongly to TAT-59, suggesting that TAT-59 might be effective against tumors resistant to tamoxifen. The metabolites of TAT-59, DP-TAT-59 and DM-DP-TAT-59, showed similar antitumor activity. Both TAT-59 and tamoxifen suppressed the decrease in bone density and reduced the blood cholesterol levels in ovariectomized rats, suggesting that the estrogenic activity of TAT-59 is comparable to that of tamoxifen. CONCLUSIONS: On the basis of the above results, one may expect TAT-59 to become an effective drug in patients with tumors less sensitive to tamoxifen, while its estrogenic activity as determined by bone and lipid metabolism is similar to that of tamoxifen.

[Anti-metastatic and anti-angiogenic activity of UFT in a lung spontaneous metastasis model in mice].

We investigated the effect of UFT on a pulmonary metastasis after excision of the primary lesion, which was induced by implantation of murine renal carcinoma cells, RENCA. The cells were implanted into the left kidney of Balb/cA mice, and nephrectomy of the left kidney with a primary tumor was performed on day 10 after implantation. Administration of antitumor drugs, was started on day 13 [UFT (20 mg/kg, p.o., 5'-DFUR (24.6 mg/kg, p.o.), 5-FU (19 mg/kg, i.v.), TNP-470 (30 mg/kg, s.c.)]. In this metastasis model, the estimated mean survival time of the control group was 41.3 +/- 2.9 days. A significant life-prolonging effect was observed for UFT and 5-FU (T/C: 160.8%, 125.7%, respectively). An inhibitory effect on the growth of metastatic tumors in the lung was detected for both UFT and TNP-470 (TWI: 55.5%, 48.7%, respectively), but not 5'-DFUR. In a dorsal air sac (DAS) model, UFT abrogated angiogenesis induced by RENCA in a dose-dependent manner. These data suggest that the life-prolonging effect of UFT results from the continuous exposure of a tumor to its cytotoxic effects and anti-angiogenic activity.

Inhibition of angiogenesis and intrahepatic growth of colon cancer by TAC-101.

We demonstrated in this study that inhibition of intra-hepatic growth of colon cancer by TAC-101 is mediated by inhibition of angiogenesis. In vitro experiments showed that TAC-101 inhibited the proliferation of murine hepatic sinusoidal endothelial (HSE) cells induced by coculture with murine colon 26-L5 (L5) cells. HSE cell proliferation was also enhanced by conditioned medium of L5 cells (CM-L5), and this enhancement of proliferation was abrogated by anti-vascular endothelial growth factor antibody. CM-L5 also induced in vitro tube formation of HSE cells on Matri-gel, and this activity of CM-L5 was abrogated by TAC-101 in a concentration-dependent manner. On the other hand, p.o. administration of TAC-101 inhibited tumor-induced angiogenesis in vivo and decreased the weights of L5 tumors in the mouse liver. Reverse transcriptase-PCR analysis using in vivo tumor tissue suggested that repression of vascular endothelial growth factor expression by TAC-101 was associated with the antiangiogenic activity. TAC-101 alone and 5-fluorouracil (5-FU)/D,L-leucovorin (LV) significantly inhibited the intrahepatic growth of L5 tumors (P = 0.002 and 0.001, respectively), whereas 5-FU alone did not (P = 0.088). When TAC-101 was administered with 5-FU/LV, marked enhancement of antitumor activity was observed (95% inhibition; P<0.001). This enhanced antitumor effect was also observed in experiments using Co-3 human colon adenocarcinoma. Concurrent treatment with TAC-101 and 5-FU/LV and sequential treatment with 5-FU/LV followed by TAC-101 resulted in significant augmentation of antitumor activity against Co-3 (overall P = 0.007 and 0.015, respectively). These findings indicate that TAC-101 inhibits tumor angiogenesis and suggest that it may be effective against hepatic metastasis of colon cancer.

Regulation of tumour necrosis factor (TNF) induced apoptosis by soluble TNF receptors in Helicobacter pylori infection.

BACKGROUND: Tumour necrosis factor (TNF) is a predominant cytokine produced in the gastric mucosa of patients with Helicobacter pylori infection. TNF induces apoptosis in a variety of cells. The soluble TNF receptors (sTNF-Rs) can be divided into sTNF-RI and sTNF-RII, both of which inhibit TNF activity. However, their precise mechanisms remain unclear. AIM: To investigate the role of sTNF-Rs in H pylori infection. METHODS: In 40 patients, production of TNF and sTNF-Rs in gastric mucosa was measured using biopsy specimens. In addition, in gastric epithelial cells, sTNF-R release in response to TNF and the protective effect of sTNF-Rs against the cytotoxic and apoptotic activities of TNF were examined. RESULTS: TNF and sTNF-R expression was significantly higher in H pylori positive than H pylori negative patients. TNF dose-dependently induced sTNF-RI release from gastric epithelial cells. sTNF-RII was also released from the cells. TNF decreased cell viability, but the effect was very small. A combination of anti-sTNF-RI and anti-sTNF-RII monoclonal antibodies significantly increased TNF induced cytotoxicity and apoptosis of gastric epithelial cells. CONCLUSIONS: These results show that sTNF-Rs are actively produced in H pylori infected gastric mucosa. sTNF-Rs appear to protect gastric epithelial cells from TNF induced apoptosis in H pylori infection.

Hepatic arterial infusion chemotherapy with continuous low dose administration of cisplatin and 5-fluorouracil for multiple recurrence of hepatocellular carcinoma after surgical treatment.

To date, conventional treatments for multiple intrahepatic recurrence of hepatocellular carcinoma (HCC) after surgery are unsuccessful. The aim of this retrospective study was to evaluate the prognostic effectiveness of a new infusion chemotherapy of cisplatin (CDDP) and 5-fluorouracil (5-FU) via hepatic artery for HCC with multiple intrahepatic recurrence. Fifty-two patients, who had postoperative multiple recurrence of HCC (more than 3 tumors), were enrolled in this study. Thirty-one patients were treated by hepatic arterial infusion chemotherapy via a subcutaneously implanted injection port. A one-week course of this treatment consisted of daily administration of cisplatin (10 mg for 1 h on days 1-5) and subsequent daily administration of 5-fluorouracil (250 mg for 5 h on days 1-5). Three to six sequential one-week courses were performed (the CDDP,5-FU group). Twenty-one patients underwent conventional interventional therapies including transcatheter arterial chemoembolization, lipiodolization (the conventional group). The complete response rate and the effective response rate in the CDDP,5-FU group were 29.0% and 71.0%, respectively. The 5-year survival rate in this group was 45.7%, which was significantly better than that in the conventional group. Based on multivariate analysis, CDDP,5-FU hepatic arterial infusion chemotherapy was found to be significant in prolonging survival, and this treatment achieved favorable therapeutic results for multiple recurrence of HCC. As part of a multidisciplinary approach this treatment is expected to improve the prognosis of patients with advanced HCC.

Characterization of the YbBa2Cu3O7-y and YBa2Cu3O7-y thin superconducting films prepared by chemical solution deposition on MgO(001) substrate.

Thin superconducting films of the YbBa2Cu3O7-y (Yb123) and YBa2Cu3O7-y (Y123), prepared by post-deposition annealing of the metal naphthenates gels spin-coated on MgO(001) substrate, have been characterized by cross-sectional high-resolution electron microscopy and X-ray diffraction. It was found that the c-axis Yb123 films were epitaxially grown on the MgO(001) substrate at the temperature range from 700 degrees C to 775 degrees C in a gas mixture containing Ar and O2 with the oxygen partial pressure of p(O2) = 10(-4) atm. In contrast to the Yb123 films, it was found that Y123 films could be derived at a wider temperature range from 750 degrees C to 950 degrees C. Randomly oriented Y123 films were also grown on the MgO(001) substrate besides the majority of in-plane c-axis oriented growth.

TAC-101 (4-[3,5-bis(trimethylsilyl)benzamido]benzoic acid) inhibits spontaneous mediastinal lymph node metastasis produced by orthotopic implantation of Lewis lung carcinoma.

The anti-tumor and anti-metastatic effects of 4-[3,5-bis(trimethylsilyl)benzamido]benzoic acid (TAC-101) were investigated using our established lung cancer model. Orthotopic implantation of Lewis lung carcinoma (LLC) cells into the lung parenchyma produced a solitary tumor nodule in the lung followed by mediastinal lymph node metastasis. Daily oral administration of TAC-101 at doses ranging from 4 to 16 mg/kg resulted in a significant inhibition of lymphatic metastasis (inhibition rate=57 to 76%), while only the dose of 16 mg/kg significantly inhibited tumor growth at the implanted sites (inhibition rate=46%). Combined treatment with cis-diamminedichloroplatinum (CDDP) and TAC-101 (8 mg/kg, p.o., daily) enhanced the anti-tumor effect of CDDP (7 mg/kg, i.v., bolus) against both the growth of implanted tumor and lymphatic metastasis. In addition, this combined treatment significantly prolonged the survival time of LLC tumor-bearing mice as compared to treatment with each agent alone. The anti-activating protein-1 (AP-1) activity of TAC-101 caused inhibition of LLC cell invasion through the repression of expression of urokinase-type plasminogen activator and its receptor. The anti-invasive activity of TAC-101 may be involved in its in vivo anti-metastatic activity. These findings suggest that TAC-101 is a novel anti-cancer agent that may improve the therapeutic modalities for lung cancer patients with metastatic disease.

A case of intraabdominal bleeding following pancreatoduodenectomy successfully treated by transcatheter arterial embolization.

A 58-year-old male underwent pancreatoduodenectomy based on a diagnosis of middle bile duct cancer. Because abdominal drainage revealed bile leakage 5 days postoperatively, leakage at the site of cholangiojejunostomy was diagnosed, and continuous aspiration was performed. Seventeen days postoperatively, pus was discharged through the abdominal drain. Because bleeding was detected by abdominal drainage, and shock ensued 20 days postoperatively, emergency abdominal angiography was carried out to identify the bleeding site. A false aneurysm in the proper hepatic artery and extravasation from the gastroduodenal artery stump were recognized, and therefore, the proper hepatic artery and common hepatic artery were embolized at a site distal to the false aneurysm using microcoils. Celiac arteriography after transcatheter arterial embolization (TAE) did not show extravasation, and revealed blood flow from the right inferior phrenic artery to the liver. Liver function was normal after TAE, and the patient recovered and was discharged from the hospital 54 days postoperatively. This paper presents a patient in whom intraabdominal bleeding due to leakage at the site of cholangiojejunostomy complicated by infection was successfully treated by hemostasis with TAE.

A case of atypical aortic coarctation with refractory congestive heart failure--treatment with axillo-iliac artery bypass.

Coarctation of the aorta is rare. We present a case of atypical coarctation due to aortitis syndrome with congestive heart failure refractory to conventional medical treatment. Although indications for surgical treatment are not well established and, in general, treatment of choice is an aorto-aortic bypass, we selected axillo-iliac bypass with subcutaneous tunnel because of severe heart failure. Postoperatively, the pressure gradient disappeared and heart failure was improved.

Detection and quantitation of thymidylate synthase mRNA in human colon adenocarcinoma cell line resistant to 5-fluorouracil by competitive PCR.

BACKGROUND: Thymldylate synthase (TS) is an important target of cancer chemotherapeutic agents, such as 5-fluorouracil (FU). To investigate mechanisms of resistance to FU, we tried to detect TS mRNA in the human colon adenocarcinoma cell lines. MATERIALS AND METHODS: SNU-C1 (C1) and its FU-resistant cell line, SNU-C1/FU (C1/FU) were used for this study. Total RNA was isolated by the AGPC method, then competitive PCR and northern blot were done to detect TS mRNA. RESULTS: Using sets of primers covering the 3'-untranslated region of TS mRNA, PCR products were amplified from cDNA prepared from both C1 and C1/FU in their logarithmic growth phases. However, only cDNA prepared from C1/FU was amplified in the stationary phase. The amount of mRNA was quantified by competitive PCR technique in both cell lines, using another set of primer to amplify the product in the stationary phase. The amount of TS mRNA in C1/FU was found to be four times more than that found in C1. In addition, TS catalytic activity of C1/FU was approximately 2-times higher than that of C1. Southern blot analysis revealed that no TS gene amplification or rearrangement in genomic DNA was detected in these cell lines. CONCLUSIONS: This PCR technique is applicable for detecting TS mRNA, and the TS mRNA level was found to be increased 1.5-fold (as detected by northern blot analysis) and 4-fold (measured by competitive PCR), leading to enhanced TS catalytic activity in C1/FU in contrast to its parent cell line, C1; thus accounting for one possible resistant mechanism to FU.

[Antitumor activity of miproxifene phosphate (TAT-59) against human mammary carcinoma].

DP-TAT-59, an active metabolite of miproxifene phosphate (TAT-59), showed a strong anti-proliferating activity against ER-positive human mammary carcinoma cell lines, MCF-7 and T-47D, in the presence of 1 nM of estradiol. The ED50 value of DP-TAT-59 for each cell line was 30-fold lower than that of tamoxifen. TAT-59 suppressed the growth of mammary carcinoma, MCF-7 and Br-10, xenografted into nude mouse at a dose of 5 mg/kg/day, which is equivalent to 20 mg/body of daily dose to the patients. TAT-59 inhibited the growth of tamoxifen-resistant breast cancer cell lines, R-27 and FST-1, but not tamoxifen, suggesting the possible efficacy of TAT-59 for tamoxifen-refractory patients. DP-TAT-59 and DM-DP-TAT-59, major metabolites of TAT-59 detected in blood after oral administration in the patients, exhibited equal growth-inhibitory activity against human mammary tumor xenograft, meaning the antitumor activity of TAT-59 may equally depend on these two metabolites. In uterotrophic testing using both immature mice and ovariectomized rats, while the effective dose of TAT-59 was lower than that of tamoxifen, TAT-59 showed dose-dependent estrogenic activity against their uteri, similar to tamoxifen. These results suggested that TAT-59 had a stronger antagonistic activity against estrogen-dependent mammary tumor than tamoxifen. We expect that TAT-59 will become an effective therapeutic agent for patients with high estrogen levels in their blood, such as premenopausal women, and the patients with whom the tamoxifen modality failed.