The copper-chelating agent, trientine, suppresses tumor development and angiogenesis in the murine hepatocellular carcinoma cells.

Angiogenesis is now recognized as a crucial process in tumor development, including hepatocellular carcinoma (HCC). Since HCC is known as a hypervascular tumor, anti-angiogenesis is a promising approach to inhibit the HCC development. Trientine dihydrochloride (trientine) is used in clinical practice as an alternative copper (Cu)-chelating agent for patients with Wilson's disease of penicillamine intolerance. In our study, we examined the effect of Cu-chelating agents on tumor development and angiogenesis in the murine HCC xenograft model. Although both trientine and penicillamine in the drinking water suppressed the tumor development, trientine exerted a more potent inhibitory effect than penicillamine. In combination with a Cu-deficient diet, both trientine and penicillamine almost abolished the HCC development. Trientine treatment resulted in a marked suppression of neovascularization and increase of apoptosis in the tumor, whereas tumor cell proliferation itself was not altered. In vitro studies also exhibited that trientine is not cytotoxic for the tumor cells. On the other hand, it significantly suppressed the endothelial cell proliferation. These results suggested that Cu plays a pivotal role in tumor development and angiogenesis in the murine HCC cells, and Cu-chelators, especially trientine, could inhibit angiogenesis and enhance apoptosis in the tumor with consequent suppression of the tumor growth in vivo. Since trientine is already used in clinical practice without any serious side effects as compared to penicillamine, it may be an effective new strategy for future HCC therapy.

Chronic in vitro exposure to 3'-azido-2', 3'-dideoxythymidine induces senescence and apoptosis and reduces tumorigenicity of metastatic mouse mammary tumor cells.

Normal cells in culture divide a certain amount of times and undergo a process termed replicative senescence. Telomere loss is thought to control entry into senescence. Activation of telomerase in tumors bypasses cellular senescence and is thus a requirement for tumor progression. We reported previously the preferential incorporation of 3'-azido-2', 3'-dideoxythymidine (AZT) in telomeric sequences of immortalized cells in culture. In this work, we have investigated the effects of chronic in vitro AZT exposure on F3II mouse mammary carcinoma cells. We demonstrate, for the first time, that AZT-treated tumor cells have a reduced tumorigenicity in syngeneic BALB/c mice. Tumor incidence was reduced and survival was prolonged in animals inoculated with AZT-treated cells when comparing with control counterparts. The number and size of spontaneous metastases were also decreased in animals inoculated with AZT-treated cells. In addition, we present evidence of morphological and biochemical signs of senescence, as shown by the staining for senescence associated beta-galactosidase activity, and induction of programmed cell death, as demonstrated by an increase of caspase-3 activity, in tumor cells exposed to AZT. These data indicate that chronic exposure of mammary carcinoma cells to AZT may be sufficient to induce a senescent phenotype and to reduce tumorigenicity.

Irreversible telomere shortening by 3'-azido-2',3'-dideoxythymidine (AZT) treatment.

Telomeres shorten by 30 to 50 bp with each cell division. Germ line, tumor and stem cells overcome progressive shortening by elongating their telomeres with telomerase. Previously we demonstrated that 3'-azido-2',3'-dideoxythymidine (AZT), incorporates into telomeric DNA. To determine if telomeric AZT incorporation was a telomerase mediated phenomenon, we subjected tumor cells to long-term AZT exposure. Here we report the shortening of the telomeric sequences of HeLa cells cultured with 800 microM AZT for 15 passages. Southern blots of HeLa DNA cultured with AZT and digested with SAU 3AI, Alu I, and Rsa I revealed a progressive shortening of the telomeric repeats when probed with a human biotinylated telomeric probe. The shortened telomeric repeats did not elongate after culturing without AZT for an additional 25 passages. No evidence of senescence could be detected.

Fibrin formation induced by tumor procoagulants enhances urokinase activity produced by mammary carcinoma cells.

We have examined the relationship between the procoagulant activity of F3II mouse mammary carcinoma cells and the production of urokinase, a profibrinolytic serine protease involved in tumor invasion and hematogenous metastasis. F3II cells were capable of inducing the conversion of purified fibrinogen to fibrin in the presence of calcium and plasma traces. Immunocytochemical examination of semi-confluent monolayers demonstrated that F3II cells also synthesized high levels of urokinase. Although fibrinogen did not modify profibrinolytic activity produced by F3II monolayers, fibrin formation increased tumor-derived urokinase activity by two-fold. The present data provide new insights into the cooperative role of coagulation and fibrinolysis facilitating and perpetuating tumor invasion.

Inhibition of mammary tumor cell adhesion, migration, and invasion by the selective synthetic urokinase inhibitor B428.

We have analyzed the anti-invasive properties of the selective synthetic urokinase inhibitor 4-iodo benzo[b]thiopene-2-carboxamidine (B428) in the mouse mammary carcinoma model F3II. At non-cytotoxic concentrations (10-20 microM), B428 blocked secreted and cell-associated tumor-derived urokinase activity as well as whole cell plasminogen-dependent casein degradation. Pretreatment of F3II monolayers with B428 enhanced membrane bound uPA, suggesting that the compound may modify urokinase receptor mobilization and urokinase-dependent cell signaling. B428 exerted a dose-dependent inhibition of Matrigel invasion by F3II cells and also reduced tumor cell adhesion and migration using the same doses. Our data indicate that uPA and its cell surface receptor are involved in attachment, migration, and invasion of mammary tumor cells, and that the three processes can be blocked by a synthetic urokinase inhibitor.