Jewell M. Graves

Evaluation of the mutagenic, cytotoxic, and antitumor potential of triptolide, a highly oxygenated diterpene isolated from Tripterygium wilfordii

Triptolide, a highly oxygenated diterpene isolated from Tripterygium wilfordii Hook f. (Celastraceae), has been shown to demonstrate potent antileukemic activity in rodent models at remarkably low treatment doses. A variety of other physiological responses are known to be mediated by this compound, including immunosuppressive and antifertility effects. We currently report that triptolide was not mutagenic toward Salmonella typhimurium strain TM677, either in the presence or absence of a metabolic activating system. Relatively potent but non-specific cytotoxicity was observed with a panel of cultured mammalian cell lines, and modest antitumor activity was observed when an i.p. dose of 25 microg was administered three times weekly to athymic mice carrying human breast tumors. Treatment regimens involving higher doses of triptolide (e.g. 50 microg/mouse three times weekly) were lethal.

Bacterial cupredoxin azurin as an inducer of apoptosis and regression in human breast cancer

Azurin, a copper-containing redox protein released by the pathogenic bacterium Pseudomonas aeruginosa, is highly cytotoxic to the human breast cancer cell line MCF-7, but is less cytotoxic toward p53-negative (MDA-MB-157) or nonfunctional p53 cell lines like MDD2 and MDA-MB-231. The purpose of this study was to investigate the underlying mechanism of the action of bacterial cupredoxin azurin in the regression of breast cancer and its potential chemotherapeutic efficacy. Azurin enters into the cytosol of MCF-7 cells and travels to the nucleus, enhancing the intracellular levels of p53 and Bax, thereby triggering the release of mitochondrial cytochrome c into the cytosol. This process activates the caspase cascade (including caspase-9 and caspase-7), thereby initiating the apoptotic process. Our results indicate that azurin-induced cell death stimuli are amplified in the presence of p53. In vivo injection of azurin in immunodeficient mice harboring xenografted human breast cancer cells in the mammary fat pad leads to statistically significant regression (85%, P=0.0179, Kruskal–Wallis Test) of the tumor. In conclusion, azurin blocks breast cancer cell proliferation and induces apoptosis through the mitochondrial pathway both in vitro and in vivo, thereby suggesting a potential chemotherapeutic application of this bacterial cupredoxin for the treatment of breast cancer.

Growth and metastasis of human breast carcinomas with Matrigel in athymic mice

SummaryImmunodeficient athymic mice with human tumor xenografts provide an importantin vivo experimental model for cancer research. However, only a limited number of tumor types grow in these animals. For human breast carcinomas, the incidence of tumor-take is 6–15%. Recently, increased incidence of xenograft development in mice has been reported for various human tumors when the tumors were coinjected with Matrigel. We studied the development of human breast carcinoma xenografts in athymic mice with and without coinjection of Matrigel. Tumors developed in only 7.3% of enzyme-dispersed tumors injected subcutaneously in saline solution alone. None of these tumors metastasized to distant sites. On the other hand, 50% of enzymedispersed tumors coinjected with Matrigel developed xenografts; four out of five of these tumors metastasized to distant sites. Our data from the recent study suggest that, in athymic mice, Matrigel not only enhanced breast tumor growth but also facilitated tumor metastasis.

Effect of vitamin D analog (1α hydroxy D5) immunoconjugated to Her‐2 antibody on breast cancer

We previously showed that a new vitamin D analog, 1α(OH)D5 (D5), induced differentiation and inhibited the growth of breast cancer cells. In this report, we examined whether D5 specifically delivered to breast cancer cells could have any therapeutic effect. D5 was linked to Her‐2 antibody using sulfosuccinimidyl 6‐4 azido nitrophenylamido hexanode (SANPAH) as a linker. The Her‐2 antibody selected in our study had no significant effect on the in vitro or in vivo growth of breast cancer cells; however, it had cell‐differentiating action. In vitro, D5‐Her‐2 antibody conjugate (IMC) showed the ability to specifically bind to Her‐2‐expressing cells, to compete with Her‐2 antibody for surface receptor and to cause internalization. IMC (equivalent to 5 μg Her‐2 antibody given intraperitoneally once weekly for 6 weeks) significantly inhibited the growth of BT‐474 cells transplanted into athymic mice. The in vivo growth‐inhibitory effect of IMC treatment was similar to that observed in animals receiving D5 continuously as a dietary supplement. These results show that the targeted delivery of D5 by immunoconjugation to cell surface receptor antibodies may be of potential therapeutic value for the treatment of Her‐2 positive breast cancer.

Development of a new metastatic human breast carcinoma xenograft line

Xenografts originated from human tumours offer the most appropriate research material for in vivo experimental research. However, primary human breast carcinomas are difficult to grow when transplanted in athymic mice: tumour take is less than 15%. Recently, we have achieved 60% tumour take by injecting tumour cell suspensions mixed with Matrigel. Human breast xenografts originated from primary breast carcinoma also frequently show the potential to metastasize spontaneously. In the present study, we generated a human breast carcinoma xenograft line (UISO-BCA-NMT-18) that shows 100% tumorigenicity and 80-100% lung metastasis when transplanted s.c. in athymic mice. We have studied in detail the characteristics of the xenograft and the patients tumour from which the xenograft line originated. Both the xenograft and the patients tumour showed intense staining for mutant p53 nuclear protein, and high expression of U-PA, PAI and u-PAR. In vivo growth of the xenograft is stimulated by exogenous supplementation of oestrogen. This xenograft is continuously growing in mice and has shown 80-100% metastasis for the last three successive in vivo passages. This well-characterized, oestrogen-responsive, metastatic breast carcinoma xenograft line will provide excellent research material for metastasis-related research.

Overexpression of mutant p53 and c-erbB-2 proteins and breast tumour take in mice

We established a panel of 17 xenografts from primary human breast carcinomas. We examined which characteristics of the original tumours and the xenografts facilitate growth in animals. Tumours expressing medium or strong immunoreactivity for p53 protein had significantly (P < 0.05) higher incidence (92%) of in vivo tumour take than those showing weak or negative immunoreactivity (9.1%). No such association was observed between either c-erbB-2 or epidermal growth factor receptor (EGFR) expression in the original tumours and their in vivo tumour take. Following subcutaneous (s.c.) transplantation of original breast tumours or established xenografts, 7/17 tumours showed metastatic disease spread to distant sites (mainly lungs). This study suggests that selective growth of highly aggressive tumours occurs during in vivo propagation of malignant tumours, and these tumours will be of particular interest in evaluating various chemotherapeutic agents for breast cancer management.