Endocrine Abstracts

Introduction: The biological rhythm pattern is synchronized through circadian oscillation of cortisol and melatonin release. Increased cortisol levels and circadian rhythm disruption act as an oncogenic factor in endometrial cancer through among others- dysregulation of cell proliferation/apoptosis and invasion.

Aim: To investigate, whether there is an oscillatory expression of the clock genes, MT1 and GR expression in human endometrial carcinoma cells. To explore whether glucocorticoids and melatonin can affect the expression of these genes and further to evaluate whether dexamethasone and melatonin affect the viability and invasiveness of Ishikawa cells.

Material and methods: Ishikawa cells were cultured and serum starved for 16h. Following starvation, cells were serum shocked and maintained in DCC-supplemented medium in the presence of dexamethasone (10^-7M), melatonin (10^-7M,10^-8M) and GR antagonist RU486(10^-5M), alone or co-incubated with dexamethasone and melatonin for 54h. Cell viability and cell invasion were evaluated by MTS and scratch assay, respectively. The mRNA levels of circadian clock genes: CLOCK, BMAL1, CRY-1, PER-2, ROR-α, REV-ERBb, glucocorticoid receptor-alpha and melatonin receptor were measured by qPCR.

Results: Dexamethasone induced cell invasiveness of Ishikawa cells was inversed by 10% in the presence of melatonin at 10^-8M for 54h. Co-incubation of dexamethasone-treated cells with melatonin (10^-7M,10^-8M) reduced the Ishikawa cell viability as compared to cells incubated with dexamethasone alone(10^-7M). For the first time, we showed that following synchronization with serum shock, Ishikawa cells expressed Bmal-1, Clock, Per-2, Cry1 in an oscillatory manner with a peak observed every approximately 36h. Interestingly, MT-1 and GRα also exhibited almost the same oscillaroty expression pattern. Incubation of Ishikawa cells with dexamethasone at concentration 10^-7did not affect the oscillatory pattern of Clock, Per-2, Cry, while it decreased the amplitude of Cry-1 expression at 18h of incubation. However, dexamethasone disrupted the pattern of Bmal-1 expression, mainly by increasing the frequency of oscillations; this effect was reversed by co-incubation with RU-486 implying a GR-mediated effect. Notably, melatonin at concentration of 10^-8M reversed the disruption of Bmal-1 expression pattern, without changing the GRα expression. Long-term incubation with melatonin alone at both concentrations did not affect significantly the oscillatory pattern, however at the highest concentration appeared to increase the amplitude of the oscillation in Bmal-1, Clock, Per-2, Cry-1 expression (approximately by 27%, 127%,83% and 73% respectively) with more robust effect at 18h of incubation (first peak).

Conclusion: Melatonin ameliorates the glucocorticoid-induced invasiveness of human endometrial cancer cells possibly through reversing the glucocorticoid-induced disruption of circadian clock system. Further studies need to confirm the causal link.