A Study On The Circadian Rhythms Of Well-differentiated Cancer Cell Lines Cultured In Vitro And Its Correlation With Invasion And Metastasis

Objectives To study the circadian rhythms in three well-differentiated cancer cell lines Eca-109,CNE-1and SW480 and to investigate the relationship between circadian rhythms and cancer invasion and metastasis.Methods(1) These three cell lines were cultured in vitro. (2)Per-1and Clock mRNAs were assayed by RT-PCR.(3) MMP-9 and COX-2 mRNAs were assayed by RT-PCR.(4) The expression of MMP-9 protein products were analyzed by immunofluorescence staining and flow cytometry.(5)Apoptosis of these cancer cell lines at different time points were measured by DAPI.Results(1) The expression of clock genes Per-1 and Clock showed phase oscillation(P＜0.05) in these three cancer cell lines,which was obvious in the well- differentiated esophageal cancer cell line Eca-109(P＜0.01).(2)The results of RT-PCR showed that MMP-9 and COX-2 genes displayed almost the same phase oscillations as Per-1 and Clock genes in these three cancer cell lines.(3) MMP-9 protein in these three cell lines which were assayed by flow cytometry showed similar phase fluctuation. (4) The apoptosis at the time point which expressed highest levels with Per-1 and Clock genes in cell lines Eca-109,CNE-1 and SW480 were significantly lower than that in the lowest expression levels.Conclusions(1) Well-differentiated cancer cells which were cultured in vitro showed the phase oscillation in their expressions of clock genes Per-1 and Clock.The highest expression levels and lowest expression levels of these two genes in different types of cancer cells were at different time points.Among them,the expression of Per-1and Clock genes of Eca-109 cell line displayed circadian rhythms.(2)The Eca-109 cell line,therefore,may have the potential to be a useful tool for future investigation of circadian rhythm in cancer.(3)To our knowledge, this is the first report describing the expression of invasion and metastasis genes MMP-9 and COX-2 in these three cancer cell lines showed phase fluctuations,especially,Eca-109 cell line which displays circadian rhythm.(4) These results indicate that the phase fluctuation of MMP-9 and COX-2 gene expressiom is similar to the essentially biological fluctuations,which suggests that cancer biological behavior,to some extent,is still affected by its own biological rhythm.