Summary Neoplastic events are marked by uncontrolled cell proliferation. One major focus of cancer research has been to identify treatments that reduce or inhibit cell growth. Over the years, various compounds, both naturally occurring and chemically synthesized, have been used to inhibit neoplastic cell proliferation. Two such oncostatic agents, melatonin and retinoic acid, have been shown to suppress the growth of hormone-responsive breast cancer. Currently, separate clinical protocols exist for the administration of retinoids and melatonin as adjuvant therapies for cancer. Using the oestrogen receptor (ER)-positive MCF-7 human breast tumour cell line, our laboratory has studied the effects of a sequential treatment regimen of melatonin followed by all-trans retinoic acid (atRA) on breast tumour cell proliferation in vitro. Incubation of hormonally responsive MCF-7 and T47D cells with melatonin (10-9 M) followed 24 h later by atRA (10-9 M) resulted in the complete cessation of cell growth as well as a reduction in the number of cells to below the initial plating density. This cytocidal effect is in contrast to the growth-suppressive effects seen with either hormone alone. This regimen of melatonin followed by atRA induced cytocidal effects on MCF-7 cells by activating pathways leading to apoptosis (programmed cell death) as evidenced by decreased ER and Bcl-2 and increased Bax and transforming growth factor beta 1 (TGF-f1) expression. Apoptosis was reflected morphologically by an increase in the number of lysosomal bodies and perinuclear chromatin condensation, cytoplasmic blebbing and the presence of apoptotic bodies. The apoptotic effect of this sequential treatment with melatonin and atRA appears to be both cell and regimen specific as (a) ER-negative MDA-MB-231 and BT-20 breast tumour cells were unaffected, and (b) the simultaneous administration of melatonin and atRA was not associated with apoptosis in any of the breast cancer cell lines studied. Taken together, the results suggest that use of an appropriate regimen of melatonin and atRA should be considered for preclinical and clinical evaluation against ER-positive human breast cancer.Keywords: apoptosis; melatonin; retinoic acid; MCF-7; breast cancer Melatonin, the major hormonal product of the pineal gland, has repeatedly been shown to exert a negative growth-regulatory influence on the development and growth of hormone-responsive breast cancer (Blask et al, 1986(Blask et al, , 1991. In addition, our laboratory (Hill and Blask, 1988) as well as others (Cos and Sanchez-Barcel6, 1994) have shown that melatonin treatment can act directly on hormoneresponsive human breast cancer cells in vitro to suppress their proliferation. We have also recently reported that melatonin not only suppresses the expression of the oestrogen receptor (ER) gene (Molis et al, 1994), but also up-regulates steady-state mRNA levels of transforming growth factor beta (TGF-f) and the proto-oncogene, c-mync (Molis et al, 1995). Even though the effects of melatonin on various growth...

Normal young adult male rats were either force-fed or allowed to eat ad libitum a moderate carbohydrate diet for 3–4 weeks. The force-fed animals were given either the amount of diet consumed by the animals eating ad libitum (pair-fed) or 80% of this amount (underfed). After a 2-week period of observation, we found that the rats eating ad libitum gained 65 gm of body weight, the pair-fed, force-fed 62 gm and the underfed, force-fed 40 gm. On the basis of the water, fat and protein content of the skin, viscera and carcass of control animals killed at the beginning of the feeding regimen and of similar constituents of the experimental animals after 2 weeks of feeding, the composition of the newly formed tissues of the various groups of animals consisted of the following: a) the rat with free access to food—water = 67.8%, fat = 7.8% and protein = 22.4%; b) the pair-fed, force-fed animal—water = 55.5%, fat = 23.6% and protein = 17.7%; c) the underfed, force-fed animal—water = 64.4%, fat = 7.9% and protein = 20.0%. The ratio of calories retained in newly formed tissue to the calories ingested over the 2-week period was 11.9% for the animals eating ad libitum, 20.6% for the pair-fed, force-fed animals and 9.5% for the underfed, force-fed rats. Force feeding appears to change intermediary metabolic pathways in the direction of increased ‘efficiency’ with resultant greater fat deposition.

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Normal young adult male rats were allowed to eat a diet of a specified protein content ad libitum, or were pair-force-fed the same diet twice a day. At the end of 14 days of controlled feeding, the animals were killed and analyzed for total body lipids. These analyses showed that the fat content of the force-fed animals, relative to those that ate ad libitum, became progressively greater as the dietary protein was increased in quantity from 0 to 67% of the diet. Furthermore, it was observed that force-fed animals excreted about 37% more urea nitrogen over a 7-day period than pair-fed animals eating ad libitum. Abruptly reversing the feeding habits of the rats immediately reversed the relative amounts of urinary nitrogen they excreted. The results of both types of experiments suggest that the intermediary metabolism of protein is related to feeding frequency (or load of absorbed nutrients to be metabolized per unit time). With fewer feedings but with total 24-hr intake constant, less dietary protein appears to participate in protein anabolic reactions since larger amounts of nitrogen are lost in the urine.