R. A. Hawkins

Apoptotic death of pancreatic cancer cells induced by polyunsaturated fatty acids varies with double bond number and involves an oxidative mechanism.

Polyunsaturated fatty acids (PUFAs), reported to be cytotoxic at micromolar concentrations for cancer cells in vitroand in vivo, are currently being tested in clinical trials as anti‐cancer agents. This study has shown that seven PUFAs all inhibited the growth in vitroof three pancreatic cancer cell lines and the HL‐60 leukaemic cell line. Five PUFAs induced cell death within 20–30 h, but two less potent PUFAs induced death between 50 and 75 h. Apoptosis was demonstrated to be the mode of cell death by light, UV fluorescence, and electron microscopy, together with studies of DNA fragmentation. In a time–course study of PUFA‐treated Mia‐Pa‐Ca‐2 cells, apoptosis accounted for an average of 80 per cent of the loss of viability, with ‘secondary necrosis’, a feature of late apoptosis, apparently accounting for the remainder. Correlations were found between the number of fatty acid double bonds and the proportion of cells undergoing apoptosis induced in both Mia‐Pa‐Ca‐2 cells (R=0·88, P=0·0001) and HL‐60 cells (R=0·85, P=0·0001) and inversely with the micromolar concentrations of PUFAs required for 50 per cent inhibition of growth (IC50) of Mia‐Pa‐Ca‐2 cells (R=−0·73, P=0·05). Cell death was preceded by progressively increasing lipid peroxidation. The extent of PUFA‐induced lipid peroxidation, measured as malondialdehyde (MDA), also correlated with the proportion of apoptosis induced in Mia‐Pa‐Ca‐2 cells (R=0·69, P=0·025) or HL‐60 cells (R=0·64, P=0·043), as well as with the number of fatty acid double bonds (R=0·82, P=0·0015). PUFA‐induced apoptosis was oxidative, being blocked by both vitamin E acetate and sodium selenite, the latter in a critically time‐dependent manner. The cytotoxic effects of exposure to a PUFA and to γ‐irradiation simultaneously with, or prior to, the addition of PUFA.

A HUMAN TUMOUR MODEL

An approach to the management of patients with large (greater than 4 cm) operable breast cancers is described. The conventional sequence of mastectomy followed by systemic therapy is reversed, allowing accurate measurements of response to individual forms of endocrine therapy or chemotherapy. Such a method not only permits individual selection of appropriate systemic therapy, but also allows clinical response to be related to histological and biochemical tumour parameters. A response was observed in eleven of twenty-three patients to endocrine treatment and in twelve of thirteen to combination chemotherapy. In five of the latter the response was histologically complete.

Relation between immunocytochemical estimation of oestrogen receptor in elderly patients with primary breast cancer and response to tamoxifen.

The relation between oestrogen receptor status of primary breast cancer as determined immuncytochemically on fine-needle aspirates and the response to tamoxifen therapy has been assessed in 52 patients. The proportion of cells staining correlated well with the likelihood of response, which emphasises the importance of tumour heterogeneity.

Oestrogen receptor concentration in primary breast cancer and axillary node metastases

SummaryThe primary tumour and 1–3 invaded, axillary nodes from each of 24 patients were examined both histologically for the proportion of the specimen constituted by malignant epithelial cells (‘cellularity index’) and biochemically for oestrogen receptor concentration. Both malignant epithelial cell content and oestrogen receptor concentration were significantly higher in the nodal metastases than in the primary tumours, malignant cells constituting approximately half of the former tissue and three quarters of the latter. On average, receptor concentrations were 1.6 × (protein basis) to 2.3 × (wet weight basis) higher in nodes than in the primary tumours, probably due at least in part to the difference in cellularity. When, to eliminate the effect of the latter, receptor concentration in each tumour deposit was ‘corrected’ using the appropriate ‘cellularity index’, the difference in receptor concentration between primary and node was significantly diminished, but not quite eliminated. In one patient, progestogen receptor concentrations were also studied and found to be higher in the nodes than in the primary tumour. If the actual quantity of receptor is to be used for predictive/prognostic purposes, then either a different ‘cut-off point’ should be used for invaded nodes from that used for assessment on the primary tumour, or receptor concentrations should be corrected for differences in cellularity.

Oestrogen receptor activity in intraduct and invasive breast carcinomas

SummaryBreast cancers analysed for oestrogen receptor activity over a ten-year period have been surveyed in order to select a group of intraduct carcinomas without invasion and a second, control group of invasive carcinomas without intraduct carcinoma. Examination of histological sections taken from the face of the tumour samples used for receptor analysis showed only 13 purely intraduct carcinomas without any invasion. Each of these was matched for age, menstrual status, hospital of origin, and approximate assay date with two purely invasive ductal carcinomas of no specialized type (26 invasive carcinomas in all).In invasive carcinomas, a significantly higher proportion of the specimen was occupied by malignant cells (mean 30%) than in the intraduct carcinomas (mean 15%), and receptors were detected more frequently (77% versus 46%) and at higher concentrations (mean 26 times on a wet weight basis, 19 times on a protein basis). When allowance was made for the difference in cellularity between the groups, the invasive carcinomas still contained significantly higher concentrations of receptor protein (mean = ten times more on a wet weight basis).These findings suggest that the expression of the gene encoding the receptor protein tends to be a property either maintained, or acquired upon progression to invasive disease. Further studies will be needed to determine whether or not the established prognostic and predictive values of receptor measurements apply to non-invasive disease, and to clarify the relationship between receptor expression in benign and malignant breast in relation to morphological changes.

Steroid metabolism and oestrogen receptors in human breast carcinomas

Metabolism of7α[3H] testosterone and oestrogen receptor activity have been measured in54 human breast cancers. All tumors converted testosterone to Δ4 androstenedione,5α dihydrotestosterone and5α androstanediol, but unequivocal evidence for production of oestradiol was obtained only29 of the tumours. Thirty-seven tumours were classified as oestrogen receptor ‘positive’ containing levels in excess of5 fmol/mg cytosol protein. Although mean conversions to Δ4 androstenedione,5α dihydrotestosterone and5α androstanediol were all higher in oestrogen receptor negative tumours as compared with receptor positive group, the differences did not reach statistical significance. There was, however, a significant trend for oestradiol synthesis to be associated with oestrogen receptor positive tumours (P < 0.025). All tumours with very high level of receptors converted testosterone to oestradiol.

The apoptosis-inducing effects of polyunsaturated fatty acids (PUFAs) on benign and malignant breast cells in vitro

Three breast cell lines, the malignant MCF-7 and MDA-MB-231 and the immortalized but non-malignant HBL-100 lines, were growth-inhibited in vitro by the polyunsaturated fatty acid, eicosapentaenoic acid at concentrations of 5–20 μM, inhibition being mild for the two malignant lines but strong for the immortalized HBL-100 line. Eicosapentaenoic acid (50 μM) induced moderate lipid peroxidation and extensive loss of viability in the HBL-100 cells, high levels of lipid peroxidation with slight loss of viability in MDA-MB-231 cells and neither peroxidation nor loss of viability in MCF-7 cells. In HBL-100 cells, all 7 polyunsaturated fatty acids tested inhibited growth: cis-parinaric, arachidonic, γ-linolenic and docosahexacnoic acids were the most potent, being effective (IC50) at <1 μM, whereas the mono-unsaturated fatty acids, erucic acid and oleic acid either had no effect, or stimulated growth. Growth-inhibition by polyunsaturated fatty acids was due to induction of cell death with characteristic morphological features of apoptosis. These findings suggest another possible reason why polyunsaturated fatty acids such as a γ-linolenic acid are effective in benign disease including mastalgia; they may inhibit the growth of some proliferating epithelial cells via the induction of apoptosis and this raises the prospect that other polyunsaturated fatty acids may be even more effective.

Oestrogen receptor activity in breast cancer detected at a prevalence screening examination

SummaryIn view of the possible introduction of screening programmes, this study compares oestrogen receptor (ER) levels in a series of women whose primary tumour was detected by screening and an age-matched consecutive series of women whose tumours were diagnosed after symptomatic presentation. Because of missing data and other statistical considerations, the comparison was made using T1 and T2 categories of tumour only. Some differences were found: the distribution of ER levels was significantly different in the two groups, with more extreme values in the symptomatic series; the screening series, however, had more moderate/rich ER levels than the symptomatic group. Tumours of special pathological type (for example, tubular, cribriform, lobular, medullary, and mucoid) were more likely to be ER-moderate or -rich, and there were more of these tumours in the screening series. The relationship of these findings to tumour growth rate is discussed. The study highlights the difficulty of obtaining sufficient tissue for conventional DCC biochemical assays from the small non-invasive tumours found by screening, and suggests that newer alternative methods employing monoclonal antibodies may be required for such types of tumour.

Cellularity and the quantitation of estrogen receptors

Fifty estrogen receptor (ER)-positive breast cancers have been studied to determine the best way of correcting for differences in cellularity when expressing ER concentration. ER concentration expressed on wet weight and tumour cytosol protein bases showed a positive correlation with tumour cellularity. In contrast, ER concentrations expressed on a DNA basis were not significantly related to cellularity. Although such a mode of correcting for differences in cellularity was imperfect, it did yield a receptor concentration which was less dependent upon tissue cellularity and which may reflect more accurately the inherent receptor status of the tumour cells.Fifty estrogen receptor (ER)-positive breast cancers have been studied to determine the best way of correcting for differences in cellularity when expressing ER concentration. ER concentration expressed on wet weight and tumour cytosol protein bases showed a positive correlation with tumour cellularity. In contrast, ER concentrations expressed on a DNA basis were not significantly related to cellularity. Although such a mode of correcting for differences in cellularity was imperfect, it did yield a receptor concentration which was less dependent upon tissue cellularity and which may reflect more accurately the inherent receptor status of the tumour cells.

Effects of drugs associated with hyperprolactinemia on plasma steroids and on steroid receptors and metabolism in human breast cancer

SummaryCertain commonly taken pharmaceutical preparations induce increased levels of plasma prolactin. The effects of these drugs on (a) tumor steroid receptors and metabolism, and (b) plasma hormones and hormone binding proteins have been studied in postmenopausal women with breast cancer. Two groups have been compared, 18 patients on drug treatment for at least 2 months and 15 subjects with no history of drug ingestion. Patients taking medication had significantly higher levels of plasma prolactin compared with control women. No significant difference was observed between the groups with regard to the plasma concentrations of dehydroepiandrosterone (DHA) and its sulphate (DHS), testosterone, estrone, estradiol-17β, sex hormone binding globulin (SHBG), and albumin. Similarly, no difference was observed between these two groups with regard to estrogen receptor (ER), progestogen receptor (PR), or androgen receptor (AR) levels in the tumors nor their ability to metabolize (7−3H) testosterone. It is considered that the ingestion of these drugs does not affect tumor mechanisms involving steroids.