Angiotensin II mediates cell hypertrophy in vascular smooth muscle cultures from hypertensive Ren-2 transgenic rats by an amiloride- and furosemide-sensitive mechanism

Matrix metalloproteinases (MMP) are enzymes responsible for extracellular matrix degradation, a critical component influencing the growth and metastatic potential of cancer. The purpose of this study was to determine the in vitro effects of MMP inhibition on human pancreatic cancer cells and to document its effect on cancer growth in vivo. The effect of MMP inhibition was determined using the MMP inhibitor BB-94 and a moderately differentiated pancreatic cancer cell line (HPAC). In vitro, a dose response curve was generated over 5 days utilizing the MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. In vivo, using an established orthotopic model for pancreatic cancer (LD100 = 80 days), 22 nude mice with orthotopic tumors (30 were implanted) received either BB-94 or vehicle beginning 4 days prior to implantation and continuing to death or sacrifice on Day 70. Mice were weighted weekly. At death/sacrifice, tumors were weighted, volume determined, and metastases/ distant spread documented. In vitro, BB-94 had little effect on HPAC proliferation at 40 ng/ml but achieved progressively greater to near complete inhibition at doses up to 4000 ng/ml while maintaining cell viability. In vivo, BB-94 significantly increased length of survival (69 +/- 0.1 days vs. 56 +/- 3.1 days) and necropsy weight (25.7 +/- 1.67 g vs. 19.8 +/- 1.14 g) while decreasing metastatic rate (1 vs. 20) and tumor size (0.14 +/- 0.02 g vs. 0.65 +/- 0.1 g). MMP inhibition limits HPAC proliferation in a dose-dependent fashion without direct cytotoxic effects in vitro. Mice harboring orthotopic tumors treated with BB-94 demonstrated significant reductions in tumor weight, volume, and metastases which corresponded to increased animal weight and prolonged survival.     

Modulation of tumor cell response to chemotherapy by the organ environment

The outcome of cancer metastasis depends on the interaction of metastatic cells with various host factors. The implantation of human cancer cells into anatomically correct (orthotopic) sites in nude mice can be used to ascertain their metastatic potential. While it is clear that vascularity and local immunity can retard or facilitate tumor growth, we have found that the organ environment also influences tumor cell functions such as production of degradative enzymes. The organ microenvironment can also influence the response of metastases to chemotherapy. It is not uncommon to observe the regression of cancer metastases in one organ and their continued growth in other sites after systemic chemotherapy. We demonstrated this effect in a series of experiments using a murine fibrosarcoma, a murine colon carcinoma, and a human colon carcinoma. The tumor cells were implanted subcutaneously or into different visceral organs. Subcutaneous tumors were sensitive to doxorubicin (DXR), whereas lung or liver metastases were not. In contrast, sensitivity to 5-FU did not differ between these sites of growth. The differences in response to DXR between s.c. tumors (sensitive) and lung or liver tumors (resistant) were not due to variations in DXR potency or DXR distribution. The expression of the multidrug resistance-associated P-glycoprotein as determined by flow cytometric analysis of tumor cells harvested from lesions in different organs correlated inversely with their sensitivity to DXR: increased P-glycoprotein was associated with overexpression of mdr1 mRNA. However, the organ-specific mechanism for upregulating mdr1 and P-glycoprotein has yet to be elucidated.     

Comparison between intraperitoneal and intravenous 5-fluorouracil administration using pancreatic cancer model of nude mouse

Carcinoma of the pancreas is a virulent malignancy. The purpose of this study was to evaluate the efficiency of 5-FU intraperitoneal administration for this malignancy. We developed a pancreatic cancer model whereby a human pancreatic cell line, MIA PaCa-2, was orthotopically transplanted to the pancreas of nude mice as cell suspension (1 x 10(6) cells). IP group (n = 6) received 5 times IP administration (4, 7, 12, 16, 20 days after implantation) of 5-FU 50 mg/kg, 1.5 ml. IV group (n = 6) received 5 times IV therapy (the same dates as IP group) of 5-FU 50 mg/kg, 0.2 ml. Control group (n = 6) received no treatment. The mice were sacrificed 42 days after implantation. The weight of the tumors of IP, IV and Control group was 0.332 +/- 0.143 g, 0.138 +/- 0.047 g and 0.329 +/- 0.085 g. Significant differences were found between IP and IV, and control. There was no difference between IP and control. This experiment demonstrated that 5 FUIP therapy for primary pancreatic cancer showed no effect and 5 FUIV therapy was much more effective.     

Hepatic arterial chemotherapy for liver cancer over a period of 8 years

Hepatic arterial chemotherapy was performed for 27 patients with primary (3), metastatic liver cancer (21), and 3 other cases, over a period of 8 years. Chemotherapy was performed by intermittent hepatic arterial infusion of 5-FU or FAM (in case of metastatic tumor from colorectal cancer), FAM (from gastric cancer), and CDDP or Farmorubicin (HCC). Hepatic resection was performed in 10 cases of metastatic tumor from colorectal cancer, and 8 cases of 10 were curative operation. The 5-year survival rates of curative liver resection group, and non-curative liver resection or non-resection group were 57.1% and 12.5%, respectively. As is the case with metastatic cancer from gastric cancer, pancreatic cancer, and hepatocellular carcinoma (HCC), the prognosis was poor except for one CR case of HCC. We concluded that hepatic arterial chemotherapy may be recommended for a curative resected case of liver metastasis from colorectal cancer.     

Reference ranges for haematology parameters in pregnancy derived from patient populations

Using immunodeficient mice human tumor cell lines, which metastasize to specific organs from orthotopic implantation sites but not from ectopic sites, have been established. We have been studying mechanisms of the regulation of tumor metastasis by organ specific microenvironments, and have recently found significant differences in interactions between metastatic tumor cells and fibroblasts from different tissues. Fibroblasts isolated from orthotopic organ tissues of nude mice enhanced invasion of metastatic human colon carcinoma cells. In contrast, fibroblasts derived from a subcutaneous tissue suppressed carcinoma cell invasion. These divergent effects were found in part due to differential expression of cytokines and growth factors by fibroblasts from different tissues.     

Gene therapy for primary and metastatic pancreatic cancer with intraperitoneal retroviral vector bearing the wild-type p53 gene

BACKGROUND: Metastatic pancreatic cancer is uniformly fatal because no effective chemotherapy is available. Mutations in the p53 tumor suppressor gene are found in up to 70% of pancreatic adenocarcinomas. We examined the efficacy of a retroviral vector containing the wild-type p53 gene on metastatic pancreatic cancer in a nude mouse model. METHODS: Bxpc3 human pancreatic cancer cells were transduced with either a retroviral p53 vector or an LXSN empty vector. Cells were examined for incorporation of tritiated thymidine to determine the effect of p53 retroviral transduction on DNA synthesis, and a TACS2 assay for apoptosis was performed. The functional activity of p53 in transduced cells was assessed by Western blot analysis with an antibody to WAF1/p21. In vivo effects of intraperitoneal injections of the p53 vector were examined in a nude mouse model of peritoneal carcinomatosis. RESULTS: Cells treated with the p53 vector exhibited a 59% to 85.5% reduction in cell number compared with the control cells (P < .05). p53-treated cells demonstrated decreased incorporation of tritiated thymidine (12.7% +/- 0.7% vs 17.5% +/- 1.4%; P = .002), increased staining for apoptosis, and increased expression of the WAF1/p21 protein. Treatment of nude mice with the retroviral p53 vector resulted in a significant inhibition of growth of the primary pancreatic tumor, as well as the peritoneal tumor deposits, compared with the LXSN control vector. CONCLUSIONS: Intraperitoneal delivery of a retroviral p53 vector may provide a novel treatment approach for peritoneal carcinomatosis from pancreatic cancer.     

Regional delivery of an adenovirus vector containing the Escherichia coli cytosine deaminase gene to provide local activation of 5-fluorocytosine to suppress the growth of colon carcinoma metastatic to liver

To evaluate the hypothesis that regional delivery of an adenovirus vector containing the Escherichia coli cytosine deaminase gene (AdCMV.CD) together with systemic 5-FC could suppress the growth of metastatic colon cancer in the liver, the AdCMV.CD vector was injected 0.8-1 cm from the site of a human colon cancer tumor in the livers of nude mice. The growth of the human colon cancer cells was quantified by dot blot analysis of genomic DNA extracted from tumor-bearing liver, hybridized with a human-specific Alu probe. The combination of regional AdCMV.CD plus systemic 5-fluorocytosine (5-FC) suppressed the growth of the metastatic tumors over the 21 days of evaluation following vector administration. Histologic evaluation showed necrosis at the site of the tumor in the livers of mice treated with AdCMV.CD/5-FC, but not in control groups. Evaluation of the potential toxicity of AdCMV.CD plus 5-FC on the normal liver showed only mild, self-limited dose-related inflammation, with no deaths. These data suggest that the regional administration of AdCMV.CD together with systemic 5-FC may be a safe and effective strategy to suppress the growth of metastases of colorectal carcinoma in the liver.     

Combination hepatic intra-arterial 5-fluorouracil and CDDP administration with oral regimen in patients with colorectal cancer metastasis to the liver

We investigated therapeutic effectiveness and side effects of a combination weekly high-dose 5-FU plus one shot CDDP HAI (WHF + CDDP method) with oral regimen in patients with colorectal cancer metastasis to the liver. All 24 patients enrolled in this study showed 54% efficacy whereas patients combined HAI with oral regimen over one week obtained 83% efficacy for multiple liver metastasis. They showed good quality of the life during combination chemotherapy without any symptoms of metastatic lesions. The WHF + CDDP method combined with oral regimen is a promising treatment for colorectal cancer metastasis to the liver as well as extrahepatic distant organs, and this protocol may be satisfactorily accepted by most colorectal cancer laden patients because of negligible side effects.     

Biliary lipid composition in idiopathic bile acid malabsorption

Human colon cancer frequently develops liver metastasis. Matrilysin (MMP-7), the smallest member of the matrix metalloproteinase (MMP) family, is commonly produced by human colon carcinoma cells and has been suggested to be involved in the progression and metastasis of this type of cancer. In the present study, we tested the effect of a matrilysin-specific antisense phosphorothioate oligonucleotide on liver metastasis of the human colon carcinoma cell line WiDr in nude mice. In culture, the antisense oligonucleotide moderately inhibited the secretion of matrilysin by WiDr cells. Injection of WiDr cells into the spleen of nude mice produced many metastatic tumor nodules in the liver. When the antisense oligonucleotide was injected daily into the mice for 11 days, the formation of the metastatic tumor nodules was strongly inhibited in a dose-dependent manner. An inhibition of liver metastasis of over 70% was obtained at a dose of 120 micrograms of the oligonucleotide per mouse. The antisense oligonucleotide did not inhibit tumor growth in spleen and in liver. A scrambled control oligonucleotide had no effect on liver metastasis of WiDr cells. Our results demonstrate an important role of matrilysin in liver metastasis of human colon cancer and the therapeutic potential of matrilysin antisense oligonucleotides for the prevention of metastasis.     

Metal exposure studies: role of toxicology and epidemiology in public health policy

Cell motility is an important factor in the process of invasion and metastasis of tumor. In this study, the relationship between cell motility and experimental metastatic potential was examined using two human pancreatic cancer cell lines, SW1990 and PANC-1. Serum-free conditioned medium from the highly metastatic cell line SW1990 was found to contain a factor that stimulated the migration of and induced a fibroblast-like morphological change in the weakly metastatic cell line PANC-1. Preincubation of PANC-1 cells with SW1990 conditioned medium (SW-C.M.) induced liver metastasis following splenic injection of PANC-1 cells in nude mice, although no liver metastasis was observed without pretreatment of SW-C.M. This factor, temporarily termed PDMF (pancreatic cancer-derived motility factor) is a heparin non-binding protein having a molecular weight of 40 kDa calculated by gel-filtration HPLC which acts not only chemotactically but also chemokinetically, and also acts mainly in a paracrine fashion. However, this factor had no effect on the proliferation of PANC-1 cells; it therefore appears to be a so-called motility factor. Only TGF-beta1 and IL-6 were recognized in the SW-C.M. among cytokines thought to stimulate cell motility. These cytokines stimulated the motility of PANC-1 cells, but differed from PDMF in the neutralizing test with antibody against these cytokines. Results of characterization and preliminary purification suggest that this factor may be a novel motility factor. The above findings suggest that this motility factor may play an important role in the invasion and metastasis of pancreatic cancer, and complete purification of it will be useful in elucidating the mechanism of progression of cancer and designing a strategy for inhibition of invasion and metastasis of pancreatic cancer.     

Frequent development of murine T-cell lymphomas with TcR alpha/beta+, CD4-/8- phenotype after implantation of human inflammatory breast cancer cells in BALB/c nude mice

Tumors developed quite frequently in some of the visceral organs, including spleen and liver, in BALB/c nude mice upon subcutaneously xenografting surgical specimens from five different inflammatory breast cancer patients. All of these tumors developed within two and a half months to one year after the subcutaneous inoculation of surgical specimens. From these tumors, five independent transplantable tumors, including tMK-2, tHK-1, tYK-1, tYK-2 and tTY-1 have been established. Chromosome analysis, morphologic studies by light and electron microscopy and phenotype analysis indicated that these tumors are of mouse origin. The tMK-2 tumor was highly metastatic to the spleen and liver when it was subcutaneously transplanted into the right scapular region. In addition, the region where the tMK-2 tumor cells were subcutaneously inoculated showed an apparently inflammatory process represented by erythema. After subcutaneous inoculation into the right scapular region, tHK-1, tYK-1, 2, and tTY-1 tumors also metastasized to some of the visceral organs, including spleen and liver. From these tumors, in vitro cell lines were established. The cells grew in a stromal-cell dependent manner under in vitro culture conditions. The cells were again tumorigenic at the inoculated region and metastasized to various organs, including liver and spleen, of BABL/c nude mice. Histological examination revealed that the tumors showed features of malignant lymphoma. Phenotypically, these five tumors expressed early T lymphocyte markers as revealed by anti-mouse anti-TcR alpha/beta, anti-CD3, CD4 and CD8 monoclonal antibodies. To our knowledge, these cell lines are the first T-cell lines showing the phenotype of extrathymically differentiated T-cells in the liver.     

Effect of 5-fluorouracil and UFT on experimental liver metastasis model of colorectal cancer using mouse colon 26 cells

Effects of 5-fluorouracil (5-FU) and UFT on an experimental liver metastasis model were compared at equi-effective dosage levels against subcutaneous tumor of mouse colon 26. 5-FU at the dosage level of 40 mg/kg suppressed the subcutaneous tumor growth by 70.0% and 45.0% on day 13 and day 18, respectively, and UFT at 20 mg/kg provided almost equal suppression (63.0% and 48.0%). In the liver metastasis model, 5-FU at 40 mg/kg showed more potent prevention of the formation of metastatic foci (94.9%) than did UFT (60.4%) at 20 mg/kg. 5-FU at 40 mg/kg produced a much higher peak serum level of 5-FU than did UFT at 20 mg/kg and also showed a much higher AUC (area under the curve) level in the portal blood. These results suggest that oral administration of 5-FU might be useful in prevention of liver metastasis of colorectal cancer.     

Indirect antiproliferative effect of the somatostatin analog octreotide on MIA PaCa-2 human pancreatic carcinoma in nude mice

Analogs of somatostatin (SRIF) such as octreotide exert antiproliferative effects that are mediated directly by tumoral SRIF receptors or indirectly by down-modulation of factors that stimulate tumor growth. Direct and indirect antiproliferative effects have been demonstrated in certain SRIF receptor-positive and -negative human breast cancer models in nude mice, respectively. These antiproliferative mechanisms are also being explored in other cancer types including pancreatic cancer. While clinical pilot studies have indicated that a fraction of pancreatic adenocarcinomas respond to high-dose octreotide treatment, it is known from receptor autoradiographic and scintigraphic studies that human pancreatic carcinomas fail to express SRIF receptors, in contrast to rat pancreatic carcinomas or human endocrine pancreatic cancer. Studies on the potential anticancer effect of octreotide on the growth of experimental human pancreatic cancer and its SRIF receptor status have been controversial. Therefore, we investigated in vivo the effects of octreotide on the growth of MIA PaCa-2 human pancreatic carcinomas raised from cultured cells with a low passage number after receipt from the American Type Culture Collection. Nude mice bearing MIA PaCa-2 tumors were treated with a single injection of the recently developed octreotide long-acting release formulation, "SMS pa LAR." This treatment was well tolerated and resulted in a highly significant inhibition of tumor growth during weeks three and eight after administration. MIA PaCa-2 tumors were removed after eight weeks and processed for RT-PCR analysis using probes specific for each of the five somatostatin receptor subtypes sst1-sst5. This analysis revealed that MIA PaCa-2 tumors, like human pancreatic adenocarcinomas, do not express any of the five SRIF receptor subtypes, suggesting an indirect mode of tumor growth inhibition. In summary, the depot formulation SMS pa LAR exerted long-lasting antiproliferative effects in SRIF receptor-negative human pancreatic carcinomas in nude mice.     

Antitumor effect of a neutralizing antibody to vascular endothelial growth factor on liver metastasis of endocrine neoplasm

Distant metastasis of gastrointestinal endocrine neoplasm is resistant to currently available treatments. Because hematogenic metastasis is dominant, anti-angiogenic drugs are expected to be a novel therapy for this neoplasm. In the present study, the therapeutic effect of vascular endothelial growth factor neutralizing antibody (VEGFAb) on liver metastasis of an endocrine neoplasm was investigated experimentally. Cecal transplantation into nude mice of small pieces of EN-1, a xenotransplanted human intestinal endocrine neoplasm, resulted in liver metastasis. A treated group (n = 19) received 100 micrograms/mouse of VEGFAb intraperitoneally on alternate days from day 10 after tumor transplantation, and the control group (n = 19) received saline. Five of the 19 control mice died of tumor progression, of which 2 could not be evaluated. The cecal tumor weighed 6316 +/- 2333 mg (n = 17) in the control group and 1209 +/- 837 mg (n = 19) in the treated group (P < 0.01) 6 weeks after transplantation. Liver metastasis developed in 16 of 17 control mice and in 2 of 19 treated mice (P < 0.01). The VEGF level of the whole cecal tumor in the control group was significantly higher than that in the treated group (305.1 +/- 174.1 vs. 54.7 +/- 41.2 mg; P < 0.001). VEGFAb did not cause any body weight loss (28.52 +/- 1.63 in the control vs. 28.44 +/- 1.71 g in the treated group). These results indicate that VEGFAb may be a novel therapeutic agent for endocrine neoplasm with distant metastasis.     

Antitumor activity of 1 M tegafur-0.4 M 5-chloro-2,4-dihydroxypyridine-1 M potassium oxonate (S-1) against human colon carcinoma orthotopically implanted into nude rats

The purpose of this study was to establish a nude rat orthotopic (organ-specific) human colorectal cancer model as an in vivo secondary screen for general evaluation of new anticancer agents against colorectal cancer and to evaluate practically the antitumor activity of 1 M tegafur-0.4 M 5-chloro-2,4-dihydroxypyridine-1 M potassium oxonate (S-1), a new p.o. fluoropyrimidine, in comparison to 1 M tegafur-4 M uracil [(UFT) effective on colorectal tumor in clinical]. After implantation of KM12C, a human colorectal cancer cell line, into the subserosal layer of the colon as a single-cell suspension, extensive local tumor growth and invasion to both the mucosal and the serosal sides were observed in all rats. Metastatic foci were also formed in both lymph nodes and lungs following local tumor growth in all of them. Using this method, an equitoxic dose of S-1 (15 mg/kg/day) and UFT (30 mg/kg/day) was administered p.o. for 14 consecutive days from 7 days after tumor cell implantation. S-1 showed a higher tumor growth inhibition than UFT did [S-1, 57% (significantly different from the tumor weight of the untreated group at P < 0.05) and UFT, 18% (P > 0.05)]. When both drugs were administered to nude rats bearing KM12C injected into the cecal wall for 28 consecutive days at equitoxic doses, the mean survival in the S-1 group was 16 days longer than that in the untreated group (P < 0.01) but that in the UFT group was only 8 days longer (P > 0.05). After the administration of an equitoxic dose of both drugs, S-1 gave the higher levels than UFT in various pharmacokinetic parameters as follows: area under the curve 0-24 h of 5-fluorouracil in plasma (3.5-fold), area under the curve 0-24 h of 5-fluorouracil incorporated into RNA in the tumor (1.3-fold), and thymidylate synthase inhibition rate (percentage) in the tumor (about 20%). Collectively, these findings suggested that this orthotopic human colorectal tumor model in nude rats is useful to evaluate the clinical therapeutic efficacy of drugs or therapies for colorectal cancer, and that S-1 had a higher therapeutic effect on human colorectal tumor than UFT did.     

Animal experiment on gene therapy of ovarian cancer by adenovirus-mediated thymidine kinase gene transduction and ganciclovir administration in vivo

OBJECTIVE: The efficacy and toxicity of adenovirus-mediated transduction of herpes simplex virus thymidine kinase gene started by Rous sarcoma virus (ADV/RSV-tk) followed by administration of ganciclovir (GCV) were studied in vivo. METHODS: An animal model of human epithelial ovarian cancer was established in nude mice using the serous ovarian adenocarcinoma cell lines Ov-ca-2774, then mice were treated by ADV/RSV-Tk and GCV, or GCV and HSV-tk respectively. The average survival time of mice and toxicity were assessed. RESULTS: The mice treated with GCV or HSV tk alone died from 14.4 +/- 1.7 to 19.3 +/- 3.5 days after treatment. The survival time had no difference with control group. The mice treated with ADV/RSV-tk followed by GCV lived at least two times longer than controls and the difference in both groups was significant. The earlier the treatment began, the longer the average survival time was. Treatment efficacy was dependent on dose of ADV/RSV-tk and tumor burden of mice. CONCLUSION: ADV/RSV-tk gene therapy is a safe and efficient approach to ovarian cancer treatment in the experiment.     

Hepatocellular carcinoma in an orthotopic mouse model metastasizes intrahepatically in cirrhotic but not in normal liver

Prognosis of hepatocellular carcinoma (HCC) still remains poor mainly because of intrahepatic metastasis. In the majority of cases, HCC is found in conjunction with liver cirrhosis. It is, therefore, of great importance to investigate the invasive and metastatic behavior of HCC in cirrhotic liver. To examine this, a liver cirrhosis model was produced by injecting thioacetamide i.p. into mice. Murine HCC cells were labeled with the fluorescent carbocyanine dye, DiI, and implanted directly under the capsule of cirrhotic and normal livers of syngeneic mice. DiI-labeled HCC cells in the liver were observed under fluorescent and confocal microscopy. Histological analysis of cirrhotic and normal livers revealed that implanted HCC cells migrated to and invaded the adjacent periportal regions, but not the adjacent centrolobular areas. This characteristic behavior of HCC was more evident in cirrhotic liver than in normal liver. Furthermore, intrahepatic metastasis to unimplanted hepatic lobes was observed in cirrhotic liver as early as 7 days after implantation, while it was not detected in normal liver even 4 weeks later. Thus, an orthotopic animal model for HCC with cirrhosis described here may be suitable for investigating the invasive and metastatic behavior of HCC. Importantly, labeling tumor cells with a fluorescent dye before orthotopic implantation may be a convenient and useful method to investigate the invasive and metastatic behavior of various types of cancer.     

Adenovirus-mediated transduction of Escherichia coli uracil phosphoribosyltransferase gene sensitizes cancer cells to low concentrations of 5-fluorouracil

5-fluorouracil (5-FU), although a widely used chemotherapeutic agent, has a limited effect in the treatment of human solid tumors due to their resistance to the cytotoxic effects of 5-FU. Escherichia coli uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage enzyme that catalyzes the synthesis of UMP from uracil and 5-phosphoribosyl-alpha-1-diphosphate. The present study demonstrates that adenovirus-mediated transduction of E. coli UPRT gene results in marked sensitization of colon, gastric, liver, and pancreas cancer cell lines to low concentration of 5-FU in vitro. The in vitro bystander effect was observed when only 10% of the hepatoma Hep3B cells were infected with UPRT-expressing adenovirus. In addition, 5-FU treatment of human hepatoma or gastric cancer xenografts in nude mice transduced with UPRT was demonstrated to result in significant in vivo antitumor effects. The adenovirus vector transduction of the UPRT gene followed by 5-FU administration is representative of a new chemosensitization strategy for cancer gene therapy.     

Networking rural health resources. The woman who cried wolf

OBJECTIVE: To study the genetic stability and to establish a method for detection of micrometastasis using microsatellite DNA in human breast cancer xenograft in nude mice. METHODS: Fresh tissue of human breast cancer was xenotransplanted in nude mice or thotopically. Genomic DNA extracted from tissues of human breast cancer, xenotransplanted tumors and metastatic foci in nude mice were PCR amplified at three microsatellite loci (D14S68, D18S69, D20S199) and were analysed by electrophoresis and silver stain on PAGE. RESULTS Microsatellite DNA in genome of the xenotransplanted tumors and metastatic foci in nude mice were identical with that of the human breast cancer. CONCLUSION: This study has demonstrated in nude mice the xenotransplanted tumors and metastatic foci that originated from human breast cancer. The genetic stability in human breast cancer is evident in the processes of xenotransplantation, serial passages in nude mice, metastasis and in vitro culture. This method is sensitive and specific for the discrimination of metastatic tumor cells.     

Experimental study on intraperitoneal sequential MTX/5-FU therapy for peritoneal seeding in comparison with intravenous administration

Sequential MTX/5-FU therapy (intravenous route) is powerful chemotherapy especially for poorly differentiated adenocarcinoma of the stomach and its peritoneal metastases. The authors had proposed the idea of intraperitoneal sequential MTX/5-FU chemotherapy for potential peritoneal metastases and micrometastases from advanced gastric carcinoma. This experimental study was planned to confirm this experimentally. Peritoneal seeding model of nude mice was made by the intraperitoneal inoculation of human gastric cancer cell line MKN-45. Control group (n = 5) had no treatment. The intraperitoneal (i.p.) group and intravenous (i.v.) group underwent the treatments on the 7th, 14th, and 21st day after cell implantation. Experimental chemotherapies consisted of intraperitoneal injection of MTX (15 mg/kg, 1.5 ml saline) and 5-FU (50 mg/kg, 1.0 ml saline) for i.p. group and intravenous injection of MTX (15 mg/kg, 0.2 ml saline) and 5-FU (50 mg/kg, 0.2 ml saline) for i.v. group. Interval time between MTX and 5-FU administration was 2 hours. On the 35th day after the cell implantation necropsies were performed. Counting of peritoneal metastatic nodules revealed the number of nodules of control group. (14.2 +/- 6.7) > i.v. group (5.3 +/- 4.1) > i.p. group (0.41 +/- 0.7) (p < 0.05). Weight of omental tumors showed Control group (0.246 +/- 0.136 g) > i.v. group (0.140 +/- 0.068 g) > i.p. group (0.051 +/- 0.017 g) (i.v.-i.p., p < 0.01). The mouse body weight decrease less in the i.p. group than in the i.v. group (p < 0.05) throughout this experiment. The results of this experiment demonstrated intraperitoneal sequential MTX/5-FU therapy was more effective than intravenous sequential MTX/5-FU therapy for potential peritoneal seeding and peritoneal micrometastases from the gastric cancer. Moreover, the side effect of intraperitoneal administration was milder than by the intravenous route.