Stephen G. Grant

Melatonin and breast cancer: cellular mechanisms, clinical studies and future perspectives

Recent studies have suggested that the pineal hormone melatonin may protect against breast cancer, and the mechanisms underlying its actions are becoming clearer. Melatonin works through receptors and distinct second messenger pathways to reduce cellular proliferation and to induce cellular differentiation. In addition, independently of receptors melatonin can modulate oestrogen-dependent pathways and reduce free-radical formation, thus preventing mutation and cellular toxicity. The fact that melatonin works through a myriad of signalling cascades that are protective to cells makes this hormone a good candidate for use in the clinic for the prevention and/or treatment of cancer. This review summarises cellular mechanisms governing the action of melatonin and then considers the potential use of melatonin in breast cancer prevention and treatment, with an emphasis on improving clinical outcomes.

Development, Characterization and Application of Predictive-toxicology Models

The adoption of SAR techniques for risk assessment purposes requires that the predictive performance of models be characterized and optimized. The development of such methods with respect to CASE/MULTICASE are described. Moreover, the effects of size, informational content, ratio of actives/inactives in the model on predictivity must be determined. Characterized models can provide mechanistic insights: nature of toxicophore, reactivity, receptor binding. Comparison of toxicophores among SAR models allows a determination of mechanistic overlaps (e.g., mutagenicity, toxicity, inhibition of gap junctional intercellular communication vs. carcinogenicity). Methods have been developed to combine SAR submodels and thereby improve predictive performance. Now that predictive toxicology methods are gaining acceptance, the development of Good Laboratory Practices is a further priority, as is the development of graduate programs in Computational Toxicology to adequately train the needed professional.

WNT10B/β-catenin signalling induces HMGA2 and proliferation in metastatic triple-negative breast cancer

Wnt/β‐catenin signalling has been suggested to be active in basal‐like breast cancer. However, in highly aggressive metastatic triple‐negative breast cancers (TNBC) the role of β‐catenin and the underlying mechanism(s) for the aggressiveness of TNBC remain unknown. We illustrate that WNT10B induces transcriptionally active β‐catenin in human TNBC and predicts survival‐outcome of patients with both TNBC and basal‐like tumours. We provide evidence that transgenic murine Wnt10b‐driven tumours are devoid of ERα, PR and HER2 expression and can model human TNBC. Importantly, HMGA2 is specifically expressed during early stages of embryonic mammogenesis and absent when WNT10B expression is lost, suggesting a developmentally conserved mode of action. Mechanistically, ChIP analysis uncovered that WNT10B activates canonical β‐catenin signalling leading to up‐regulation of HMGA2. Treatment of mouse and human triple‐negative tumour cells with two Wnt/β‐catenin pathway modulators or siRNA to HMGA2 decreases HMGA2 levels and proliferation. We demonstrate that WNT10B has epistatic activity on HMGA2, which is necessary and sufficient for proliferation of TNBC cells. Furthermore, HMGA2 expression predicts relapse‐free‐survival and metastasis in TNBC patients.

The potent microtubule-stabilizing agent (+)-discodermolide induces apoptosis in human breast carcinoma cells--preliminary comparisons to paclitaxel.

(+)–Discodermolide, a sponge–derived natural product, stabilizes microtubules more potently than paclitaxel despite the lack of any obvious structural similarities between the drugs. It competitively inhibits the binding of paclitaxel to tubulin polymers, hypernucleates microtubule assembly more potently than paclitaxel, and inhibits the growth of paclitaxel–reslstant ovarian and colon carcinoma cells. Because paclitaxel shows clinical promise for breast cancer treatment, its effects in a series of human breast cancer cells were compared to those of (+)–discodermolide. Growth inhibition, cell and nuclear morphological, and electrophoretic and flow cytometric analyses were performed on (+)–discodermolide–treated MCF–7 and MDAMB231 cells. (–f)–Discodermolide potently inhibited the growth of both cell types (IC50<2.5 nM) at concentrations similar to those observed with paclitaxel. Complete inhibition of growth occurred with 10 nM or greater of each drug and was not reversed by removal. (+)–Discodermolide–treated cells exhibited condensed and highly fragmented nuclei. Flow cytometric comparison of cells treated with either drug at 10 nM, a concentration well below that achieved clinically with paclitaxel, showed both caused cell cycle perturbation and induction of a hypodiploid cell population. (+)–Discodermolide caused these effects more extensively and at earlier time points. The timing and type of high molecular weight DNA fragmentation induced by the two agents was consistent with induction of apoptosis. The results suggest that (+)–discodermolide has promise as a new chemotherapeutic agent against breast and other cancers.

Elevated Frequency of Glycophorin A Mutations in Erythrocytes from Chernobyl Accident Victims

In 1986, when an explosion accident occurred at the Chernobyl, Ukraine nuclear power plant, a large number of people were exposed to significant amounts of ionizing radiation. During the time between 1986 and 1992, peripheral blood samples were obtained from 102 people who either were on site during the emergency or were brought to Chernobyl shortly thereafter to assist in the cleanup of radioactive contaminants and isolate the damaged reactor from the environment. These blood samples plus samples from 13 unexposed Soviet individuals were analyzed by flow cytometry using the allele-loss somatic mutation assay for glycophorin A. Results of these assays show that the frequency of N/O variant red cells increased in proportion to the estimated radiation exposure of each individual. The radiation dose-response function derived from this population closely resembles that determined previously for atomic bomb survivors whose blood samples were obtained and analyzed 40 years after their exposure. This suggests comparable mutation induction per unit dose for these two populations and long-term persistence of the mutational damage. In addition, measurements on multiple blood samples from each of 10 donors taken over a 7-year period showed no significant changes in N/O variant cell frequencies, confirming the persistence of radiation-induced somatic mutations in long-lived bone marrow stem cells.

Nucleotide excision repair deficiency is intrinsic in sporadic stage I breast cancer

The molecular etiology of breast cancer has proven to be remarkably complex. Most individual oncogenes are disregulated in only approximately 30% of breast tumors, indicating that either very few molecular alterations are common to the majority of breast cancers, or that they have not yet been identified. In striking contrast, we now show that 19 of 19 stage I breast tumors tested with the functional unscheduled DNA synthesis assay exhibited a significant deficiency of DNA nucleotide excision repair (NER) capacity relative to normal epithelial tissue from disease-free controls (n = 23). Loss of DNA repair capacity, including the complex, damage-comprehensive NER pathway, results in genomic instability, a hallmark of carcinogenesis. By microarray analysis, mRNA expression levels for 20 canonical NER genes were reduced in representative tumor samples versus normal. Significant reductions were observed in 19 of these genes analyzed by the more sensitive method of RNase protection. These results were confirmed at the protein level for five NER gene products. Taken together, these data suggest that NER deficiency may play an important role in the etiology of sporadic breast cancer, and that early-stage breast cancer may be intrinsically susceptible to genotoxic chemotherapeutic agents, such as cis-platinum, whose damage is remediated by NER. In addition, reduced NER capacity, or reduced expression of NER genes, could provide a basis for the development of biomarkers for the identification of tumorigenic breast epithelium.

Permanently Blocked Stem Cells Derived From Breast Cancer Cell Lines

Cancer stem cells (CSCs) are thought to be resistant to standard chemotherapeutic drugs and the inimical conditions of the tumor microenvironment. Obtaining CSCs in sufficient quantities and maintaining their undifferentiated state have been major hurdles to their further characterization and to the identification of new pharmaceuticals that preferentially target these cells. We describe here the tagging of CSC‐like populations from four human breast cancer cell lines with green fluorescent protein (GFP) under the control of the Oct3/4 stem cell‐specific promoter. As expected, GFP was expressed by the CSC‐enriched populations. However, an unanticipated result was that these cells remained blocked in a CSC‐like state and tended to be resistant to chemotherapeutic drugs as well as acidotic and hypoxic conditions. These CSC‐like cells possessed several other in vitro attributes of CSCs and were able to reproducibly generate tumors in immunocompromised mice from as few as 100 cells. Moreover, the tumors derived from these cells were comprised almost exclusively of pure CSCs. The ability of the Oct3/4 promoter to block CSC differentiation underscores its potential general utility for obtaining highly purified CSC populations, although the mechanism by which it does so remains undefined and subject to further study. Nonetheless, such stable cell lines should be extremely valuable tools for studying basic questions pertaining to CSC biology and for the initial identification of novel CSC‐specific chemotherapeutic agents, which can then be verified in primary CSCs. STEM Cells 2010;28:1008–1018

A Mathematical Model of in Vitro Cancer Cell Growth and Treatment with the Antimitotic Agent Curacin A

A mathematical model of cancer cell growth and response to treatment with the experimental antimitotic agent curacin A is presented. Rate parameters for the untreated growth of MCF-7/LY2 breast cancer and A2780 ovarian cell lines are determined from in vitro growth studies. Subsequent growth studies following treatments with 2.5, 25 and 50 nanomolar (nM), concentrations of curacin A are used to determine effects on the cell cycle and cell viability. The models system of ordinary differential equations yields an approximate analytical solution which predicts the minimum concentration necessary to prevent growth. The model shows that cell growth is arrested when the apoptotic rate is greater than the mitotic rate and that the S-phase transition rate acts to amplify this effect. Analysis of the data suggests that curacin A is rapidly absorbed into both cell lines causing an increase in the S-phase transition and a decrease in the M-phase transition. The model also indicates that the rate of apoptosis remains virtually constant for MCF-7/LY2 while that of A2780 increases 38% at 2.5 nM and 59% at 50 nM as compared to the untreated apoptotic rate.

Analysis of somatic cell mutations at the glycophorin A locus in atomic bomb survivors: a comparative study of assay methods.

The glycophorin A (GPA) assay for in vivo somatic cell mutations was performed on blood samples from 39 survivors of the atomic bomb at Hiroshima. Parallel analyses were performed at two laboratories using three different GPA assay methods to enumerate cells lacking expression of either the M- or N-allele of GPA. All assay methods yielded significant dose-dependent increases in hemizygous GPA variant cell frequencies (VFs) and smaller increases in homozygous VFs. The slopes of the fitted linear dose-response functions did not differ significantly among assay methods used in the present study, or from slopes obtained in a study reported previously. The version of the assay described most recently (BR6) appears best suited for future studies because the assay has a higher precision than earlier methods. Variant frequencies from different assay methods measuring the same variant cell type agreed with each other better than with the estimated dose, suggesting that the imprecision in the assay is not primarily responsible for VFs that differ from the fitted dose response. Consistent deviations from the dose response were seen for some individuals, suggesting either errors in dose estimates for these individuals or interindividual differences in susceptibility or other exposures. For the study population as a whole, however, discrepancies between assays for M-loss and N-loss variants suggest stochastic factors may have an important effect on individual VFs for A-bomb survivors.

Radiobiological evaluation of immigrants from the vicinity of Chernobyl.

Eighty individuals (55 adults and 25 children) who were residents of four cities (Kiev, Mozyr, Gomel and Bobrujsk) located 100-200 km from Chernobyl at the time of the accident in 1986 were tested after immigrating to the US from 1989-1991. A whole-body counter was employed to quantitate radiocesium content. In addition, two biological measures of radiation effects, namely, chromosomal integrity using the micronucleus assay and somatic mutation analysis of erythrocytes at the glycophorin A (GPA) locus, were applied to this group. Radiocesium activity in the body ranged from 0 to 56.8 Bq/kg with a mean and standard deviation of 5.0 +/- 8.2 and a median value of 2.0 Bq/kg. Mean radiocesium content by groups was highest in adult males (9.0 +/- 11.7; range 0.21-56.8 Bq/kg) followed by adult females (3.3 +/- 4.5; range 0-21.3 Bq/kg), male children (3.0 +/- 5.7; range 0-20.2 Bq/kg) and lowest in female children (1.6 +/- 3.5; range 0-12.7 Bq/kg). Individuals with the highest radiocesium content in each group belonged to one family that lived in Mozyr (100 km from Chernobyl) until emigrating in 1989. The frequency of lymphocyte micronuclei and erythrocyte GPA allele-loss (O/N) somatic mutations were both significantly correlated with radiocesium content (r=0.57, p=0.002; r=0.75, p=0.002, respectively). The micronucleus frequency also correlated with the estimated internal absorbed dose from radiocesium in a subset of 20 immigrants for whom this calculation was possible (r=0.71, p=0.0005). Altogether, the biomonitoring data indicate that some subjects had radiation doses sufficient to produce gene and chromosomal mutations in blood cells, although these effects cannot be attributed solely to radiocesium exposure.