Effects ofMLT and RA on NMUinduced Rat Mammary Tumors

Both MLT and RA have been reported to individually repress the development and growth of carcinogen-induced rat mammary tumors (11,62). To determine if the antiproliferative and apoptotic effects of the sequential regimen of MLT and RA which we observed in vitro could be translated to in vivo conditions, we conducted a series of pre-clinical trials using the NMU-induced rat mammary tumor model to examine

Melatonin Receptor

Figure 4. The temporal effects of the sequential regimen of MLT and atRA on Bcl-2 and Bak protein expression in MCF-7 cells. MCF-7 cells were incubated with diluent (control) or a regimen of MLT followed 24 h later by atRA (M + atRA) for 1, 2, 3, 4, or 5 days. For each time point, 25 ^g of total cellular protein per lane was fractionated on 12.5% polyacrylamide gels and transferred to nitrocellulose membranes. Western blots were probed with polyclonal antibodies specific for Bcl-2 and Bak, and proteins were visualized after incubation with a horseradish peroxidase-conjugated secondary antibody and chemiluminescent substrate. Actin protein levels were used to monitor protein loading. Fluorographs from Western blot analyses of the time-course of Bak and Bcl-2 proteins in response to the sequential treatment of melatonin and atRA were quantified by scanning densitometry and normalized to actin protein levels. Results are presented graphically as percent of control (n = 3 independent experiments). * p < 0.05 vs controls.

Figure 4. The temporal effects of the sequential regimen of MLT and atRA on Bcl-2 and Bak protein expression in MCF-7 cells. MCF-7 cells were incubated with diluent (control) or a regimen of MLT followed 24 h later by atRA (M + atRA) for 1, 2, 3, 4, or 5 days. For each time point, 25 ^g of total cellular protein per lane was fractionated on 12.5% polyacrylamide gels and transferred to nitrocellulose membranes. Western blots were probed with polyclonal antibodies specific for Bcl-2 and Bak, and proteins were visualized after incubation with a horseradish peroxidase-conjugated secondary antibody and chemiluminescent substrate. Actin protein levels were used to monitor protein loading. Fluorographs from Western blot analyses of the time-course of Bak and Bcl-2 proteins in response to the sequential treatment of melatonin and atRA were quantified by scanning densitometry and normalized to actin protein levels. Results are presented graphically as percent of control (n = 3 independent experiments). * p < 0.05 vs controls.

the effects of MLT and RA on the development and growth of mammary tumors (Figure 6). Five treatment groups (n = 20 rats per group) were studied: control, MLT (500 ^g/day s.c.), 9cRA (30 mg/kg chow), MLT and 9cRA daily (Mel = 9cRA), and MLT alternating every other day with 9cRA (Mel/RA). All treatments were initiated one day following NMU administration, and the animals were palpated for tumor development each week. Rats receiving 9cRA developed fewer mammary tumors (26%) compared to diluent-treated control animals (55%). Although we have previously reported that MLT suppresses NMU-induced mammary tumor formation (1 l), no difference in tumor incidence was seen between MLT-treated animals (60%) and control animals (55%) in this particular study. In animals receiving 9cRA and MLT every other day (Mel/RA), tumor incidence was further suppressed (to 20%) past that seen with 9cRA alone. A significant decrease in tumor incidence to only 5% (one animals with one tumor) was seen with the daily administration of MLT (late afternoon injections) and 9cRA. In addition, the latency to onset of tumor formation was significantly lengthened from 18 to 21 or 22 weeks in both groups receiving the combination of MLT and 9cRA over controls. Thus, it appears that the in vitro suppressive effects of MLT and RA on breast tumor cell growth is supported by the data from these in vivo pre-clinical studies. Although the most efficacious regimen for

Figure 5. RAR transactivation assay of MCF-7 cells treated with MLT and/or atRA. Cells were grown in serum-free medium for 3 days, and treated with either all-tranretionic acid (atRA) [25 nM] or MLT (1 nM), or treated simultaneously with MLT and atRA, or pre-treated with MLT for the times indicated (5 min, 30 min, or 1 h) followed by atRA. Cells were incubated for 18 h following the final treatment to allow for expression of the reporter gene. Control cells were treated with ethanol (0.01%). Statistical analysis compares luciferase expression for each treatment versus (*) control or versus (**) atRA. These data represent one run in triplicate. p < 0.05.

Figure 5. RAR transactivation assay of MCF-7 cells treated with MLT and/or atRA. Cells were grown in serum-free medium for 3 days, and treated with either all-tranretionic acid (atRA) [25 nM] or MLT (1 nM), or treated simultaneously with MLT and atRA, or pre-treated with MLT for the times indicated (5 min, 30 min, or 1 h) followed by atRA. Cells were incubated for 18 h following the final treatment to allow for expression of the reporter gene. Control cells were treated with ethanol (0.01%). Statistical analysis compares luciferase expression for each treatment versus (*) control or versus (**) atRA. These data represent one run in triplicate. p < 0.05.

0 10 20 AI Treatments

Weeks Post-Carcinogen

Figure 6. Incidence of NMU-induced mammary tumors in rats treated with 9cRA, MLT or a combination of MLT and 9c RA. N-nitroso-N-methylurea (50 mg/kg body weight) was administered i.p. to Sprague-Dawley rats at 50 days of age. Treatment with diluent (saline with 0.01% ethanol), MLT (500 ^g/day), 9cRA (RA) [30 mg/kg/chow], MLT and 9cRA on alternating days (Mel/RA), or MLT and 9cRA every day (Mel + RA) were initiated on Day 1 following NMU administration, and continued through Week 22 of the experiment. MLT was administered s.c. daily at 3 p.m. Twenty rats were in each treatment group and were housed under long photoperiod conditions (12 h light/12 h dark). This graph also depicts latency to tumor onset. * p < 0.01.

suppressing tumor formation was the daily administration of both MLT and 9cRA, which almost completely prevented tumor formation, the animals receiving MLT and 9cRA every other day, and thus, only half the total retinoid dosage of those animals treated daily with 9cRA, also showed a lower tumor incidence than those treated daily with 9cRA alone. These data, however, must be interpreted with care because the decrease in tumor incidence from 26% to 20% is not statistically significant. From these studies it appears as though the addition of MLT sensitizes the animals to the antitumor effects of 9cRA, so that the dosage of 9cRA can be cut in half while maintaining an equivalent or even greater tumor suppressive effect. Thus, the additive or synergistic interaction between these two signaling pathways could allow non-toxic doses of retinoids to be used as a chemotherapeutic agent, thus providing a more effective, less toxic treatment regimen for breast cancer.

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