PFKFB3Control Of Tongue Cancer By Responding To Circadian Clock Outputs

Backgrouds and objectives:Growing evidences have indicated that circadian disruption can distribute to increase cancer risk. Shift work, jet lag and the delay exposure to light at night have been reported that could induce abnormal circadian rhythms, which has been associated with increasing risk of tumorigenesis and improving progress of carcinoma. On the other hand, circadian rhythm has been reported as the independent factor to predict the development of cancer. As a result, chronotherapy has been considered as a nice therapy which can be applied in cancer. However, there is limited research on the relationship between oral cancer and circadian disruption.Recently, PFKFB3has been indicated as the strongest enzyme gene in the family (PFKFB1-4) whose transcription production is iPFK2. iPFK2can improve activation of Fur-2,6-P2which can catalyze the function of PFK1. PFK1is the key enzyme to contributing to improving the progress of glycolysis. In the1927, there is a classical mechanism about energy metabolism of cancer cells defined by Otto Warburg, which named Warburg’s effect. The effect describes that neoplastic cells maintain approve ATP which supports all the physiology of cancer cells.3PO has been approved as an effect inhibitor to inhibit the expression of PFKFB3. All the evidences above indicate that PFKFB3plays essential role in energy metabolism of neoplastic cells. However there is nearly no research forces on the relevance of circadian disruption and PFKFB3function in the occurring and progress of neoplastic cells. If we can find the relationship between the rhythm of PFKFB3and oral cancer, it will give us a new overview for the treatment of oral cancer. Materials and Methods:(1) One human tongue carcinoma cell line (SCC9) was commercially obtained from ATCC The cells were cultured and subcultured. After enough cells were obtained, SCC9s were synchronized and harvested at6different time points during24hours (every4hs). RNA was obtained to reverse into cDNA. RT-PCR was used to analyze the rhythms of PFKFB3, CLOCK, Bmall, Perl and Per2.(2) After the sequence of the promoter of PFKFB3was got, the positions of all the E-boxes were searched by software. The plasmids with gene of CLOCK, mutant CLOCK, Bmall, or PFKFB3were transfected into SCC9s and luciferase reporter assay was chosen to analyze the expression of PFKFB3on different time point (ZT7and ZT19).(3) In vitro,3PO was chosen as a treatment to induced SCC9(DMSO was chosen as control) on different time point (ZT7and ZT19). Flow cytometry, RT-PCR and CCK-8assay were used to analyze the cell cycle, apoptosis and proliferation of SCC9.Results:(1) There was obvious24-hour oscillation of PFKFB3, which is coincident with the expression of CLOCK gene. Both of them increased in early phase during the24hours and reached the peak at around ZT5to ZT9, as same as decreased in late phase and reached the lowest point at around ZT17to ZT21.The rhythm of Bmall was inhibited all the time, without showing any rhythm. Perl and Per2both showed two peaks during24hours.(2) There are totally33E-boxes on the promoter of PFKFB3, with a strict bite position of Bmall/CLOCK. After transfection, CLOCK could obviously improve the transcription of PFKFB3. This improvement could be inhibited signally by the co-transfection of Bmall with CLOCK. However only the Bmall gene could not change the level of the PFKFB3transcription.(3) As the CCK8assay showed, the proliferation was obviously inhibited at ZT7. However there was no effect at ZT19between the inhibitor group and blank control group. The RT-PCR results also confirmed that the transcriptions of proliferation genes CCND-1and TERT decreased signally at ZT7, compared with ZT19. By running RT-PCR, at the same time, we found that the transcriptions of apoptosis genes Cospase3, Cospase7, Bcl-2and Bax all were inhibited obviously at ZT7. However there was no signal change at ZT19. Cell cycle of cells was no difference between ZT7and ZT19.Conclusions:Depended on the results, we have confirmed that the expression of PFKFB3was higher than the normal epithelial tissue of tongue, which also showed the24hours rhythm. The rhythm was highly possible directly controlled by CLOCK/Bmall. On the other hand, we have definite that ZT7was the best time point on which the3PO can inhibit the expression of PFKFB3and the growth of cancer.