Mutation and aberrant expression of apoptotic proteins are hallmarks of cancer. These changes prevent proapoptotic signals from being transmitted to executioner caspases, thereby averting apoptotic death and allowing cellular proliferation. Caspase-3 is the key executioner caspase, and it exists as an inactive zymogen that is activated by upstream signals. Notably, concentrations of procaspase-3 in certain cancerous cells are significantly higher than those in noncancerous controls. Here we report the identification of a small molecule (PAC-1) that directly activates procaspase-3 to caspase-3 in vitro and induces apoptosis in cancerous cells isolated from primary colon tumors in a manner directly proportional to the concentration of procaspase-3 inside these cells. We found that PAC-1 retarded the growth of tumors in three different mouse models of cancer, including two models in which PAC-1 was administered orally. PAC-1 is the first small molecule known to directly activate procaspase-3 to caspase-3, a transformation that allows induction of apoptosis even in cells that have defective apoptotic machinery. The direct activation of executioner caspases is an anticancer strategy that may prove beneficial in treating the many cancers in which procaspase-3 concentrations are elevated.

Genistein is an isoflavone with oestrogenic activity that is present in a variety of soy products as a constituent of complex mixtures of bioactive compounds, whose matrix profiles play an important role in determining the overall oestrogenic bioactivity of genistein. We review data on how the profile of soy bioactive compounds can modulate genistein-stimulated oestrogen-dependent tumour growth. Our research has focused on the effects of dietary genistein on the growth of oestrogen (E)-dependent mammary tumours both in vitro and in vivo. Genistein enhances the proliferation of E-dependent human breast cancer tumour growth. In a similar manner, dietary genistein stimulates tumour growth in the chemically-induced (NMU) mammary cancer rodent model. Genistin, the glycoside of genistein, simulates growth similar to that of genistein and withdrawal of either genistein or genistin results in tumour regression. The extent of soy processing modulates the effects of dietary genistein in vivo as soy protein isolate, a highly purified and widely used source of protein that is processed to contain low, medium, and high amounts of isoflavones, stimulate the growth of the E-dependent mammary tumours in a dose dependent manner. In contrast to the more purified diets, studies with soy flour of equivalent genistein levels did not stimulate the growth of E-dependent breast cancer tumours in vivo. However, the size of these tumours also did not regress as is observed in control groups in which oestrogen and genistein have been withdrawn. The expression of the oestrogen-target genes of pS2, progesterone receptor, and cyclin D1 correlates with the growth of E-dependent tumours and has been consistently observed to be induced in response to treatment with dietary genistein. To evaluate whether dietary genistein interacts with current anti-oestrogen breast cancer therapies such as tamoxifen (TAM), we implanted E-dependent tumours into ovariectomized athymic mice and administered oestradiol, oestradiol plus TAM, or oestradiol, TAM, and dietary genistein. In these studies dietary genistein was able to negate the inhibitory effect of TAM on E-stimulated tumour growth. In summary, genistein can act as an oestrogen agonist resulting in proliferation of E-dependent human breast cancer tumours in vivo and its activity can be modulated by the presence of other bioactive components in complex soy foods. Additionally, dietary genistein can negate the inhibitory effects of TAM on E-stimulated growth of MCF-7 cell tumours implanted into ovariectomized athymic mice.

In situ synthesis of estrogens by breast cancer tissue provides a potential explanation for the high concentrations of estradiol in mammary neoplasms in postmenopausal women. A major metabolic pathway for estrogen biosynthesis is the conversion of androstenedione to estrone via the enzyme aromatase. Biochemical studies have demonstrated aromatase in tumor tissue, but at relatively low and not clearly biologically significant levels. The present study tested the hypothesis that tumor levels of aromatase, albeit low, could be biologically important if present in high concentrations in focal clusters of specific cell types. A pilot study used an immunohistochemical method in frozen sections of fresh breast tumors as an optimal means to detect aromatase. Twelve of 18 tumors contained aromatase-positive cells, some with highly intense staining. A follow-up study then attempted to precisely define the types of cells containing aromatase and correlate the immunohistochemical findings with biochemical aromatase activity. A modified H-score (histological scoring system) was used to semiquantitate the amount of aromatase staining in tumor epithelial, stromal spindle, stromal inflammatory, and normal breast epithelial cells. We found that immunohistochemical staining for aromatase predominated in stromal spindle cells with a median H-score of 13, whereas tumor epithelial, stromal inflammatory, and normal breast elements contained lesser amounts (median H-scores of 4.8, 0.03, and 0.5, respectively). The H-score for stromal spindle cells, but not those for other cell types, correlated highly with the biochemical aromatase assay (P < 0.01). Using a cut-off parameter estimated by a sensitivity/specificity (receiver operating curve) analysis, 62% of tumors were classified as aromatase positive based on stromal spindle cell staining. A similar number were also positive by biochemical assay, with concordance between the two methods of 77%. These observations provide substantial evidence for the presence of aromatase in human breast tumors, particularly in stromal spindle cells, and support the biological importance of aromatase for in situ production of estradiol.

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that is best known for its role in mediating the toxicity of many environmental contaminants such as 2,3,7,8 tetrachlorodibenzo-p-dioxin. However, the endogenous role of AHR, especially with respect to the apoptotic process, is largely unknown and contradictory. In this report, we have used a mouse hepatoma cell line (Hepa1c1c7) and its AHR-deficient derivative (LA1) to examine the effect of differing AHR levels on apoptosis susceptibility, in particular, apoptosis regulated by the intrinsic pathway. Toward this end, the cells were subjected to UV irradiation, hydrogen peroxide, and serum starvation. Analyses of a number of different endpoints of apoptosis revealed that the LA1 cells were more sensitive to these stresses than the wild-type cells, indicating that the AHR plays a cytoprotective role in the face of stimuli that initiate the intrinsic apoptotic pathway. A direct role of the AHR in mediating this effect was confirmed using both pharmacological and molecular approaches. Further analyses imply that lack of the AHR leads to an impaired survival response mediated by phosphatidylinositol 3Ј-OH kinase-Akt/protein kinase B and, to a lesser degree, epidermal growth factor receptor activation. These findings indicate that exploring the use of the AHR antagonist as agents that enhance the proapoptotic actions of cancer therapies may be a valid approach.