Effect Of PGE2-EP4 In COX-2 Signal Pathway On Biological Characteristics Of Oral Squamous Cell Carcinoma And Mechanisms

Oral Squamous Cell Carcinoma(OSCC)is one of the most common malignancies in the world,and its incidence is increasing annually.In the past 20 years,despite the impact of developing technology on the treatment of OSCC.However,the five-year survival rate of OSCC has not improved significantly,so recent studies have focused on the pathogenesis,diagnosis,and treatment of OSCC.In recent years,tumor microenvironment and malignant tumor,especially cyclooxygenase(COX)pathway in mediating the relationship between inflammation and cancer have remained an important ongoing concern for decades.Studies have documented that the high expression of COX-2 in OSCC is associated with the occurrence,invasion and chemotherapeutic drug resistance.On the other hand,Nonsteroidal Anti-inflammatory drugs(NSAIDs)can inhibit the development of malignant tumors,but its long-term use will produce many side effects such as gastric ulcer and myocardial infarction,limiting the clinical application of these drugs.Prostaglandin E2(PGE2)is the downstream products of the COX-2 pathway.Studies have shown that PGE2 can promote the proliferation and invasion of various malignant tumors,the mechanism may be to inhibit the local immune response.However,PGE2 and its receptor EP4 on OSCC biological characteristics of the impact and mechanism is not yet fully understood.For this reason,the possible mechanism of PGE2 induced the proliferation and apoptosis of OSCC cells were studied by exogenous addition of PGE2 and corresponding antagonists to find a potential therapeutic target for the treatment of OSCC.(1)Cell counting kit 8(CCK8)was performed to detect the effect of exogenous PGE2 on the proliferation of Tca8113 cells.It was found that PGE2 could significantly promote cell proliferation in a dose-dependent,and the maximal was obtained at 1.0?mol/L after 48 h.(2)The mechanism of PGE2 promoting the proliferation of Tca8113 cells was studied by Western blotting.We found that exogenous addition of PGE2 could activate the phosphorylation of ERK,while the effect was nearly abolished by U0126,an MEK inhibitor.In addition,we found that U0126 also antagonized the proliferation of Tca8113 cells by CCK8.The above results indicated that PGE2 could promote the proliferation of Tca8113 cells by activating ERK phosphorylation.(3)Four types of EP receptors were all expressed in Tca8113 cells by Western blotting.(4)To determine the type of EP receptor bound to PGE2,EP1,EP2 and EP4 receptor antagonists was investigated.The results showed that EP4 antagonist,L-161982,antagonized the proliferation of Tca8113 cells in a time and dose-dependent manner.Moreover,we observed that L-161982 inhibited the phosphorylation of ERK induced by PGE2.Thus PGE2 induced the proliferation of Tca8113 cells via the phosphorylation of ERK was confirmed.(5)Next,the Further investigation is warranted which EP receptor can mediate PGE 2 induced the proliferation of Tca8113 cells,and three types of EP receptor agonists were studied.The results showed that only the agonist of EP4,PGE 1-OH,could rescue from celecoxib-induced proliferation arrest in Tca8113 cells,and PGE 1-OH also induced the proliferation of Tca8113 cells in a concentration-dependent manner.Furthermore,we found that PGE 1-OH promoted cell proliferation by activating ERK phosphorylation,while U0126 could abolish this phenomenon.Take together;these results suggest that PGE2 may promote cell proliferation by binding to the EP4 receptor.(6)Flow cytometry analysis showed that L-161982 could induce the apoptosis of Tca8113 cells and arrest cell cycle at s phase in a concentration-dependent manner.Western blotting analysis revealed that L-161982 might induce apoptosis down-regulate the level of Bcl-2 protein and up-regulate the level of Bax protein,and arrest the cell cycle by up-regulating P21 and down-regulating of CDK2 and CyclinA2.In summary,this study found that PGE2 could induce cell proliferation via ERK phosphorylation by EP4 receptor and the EP4 antagonist could affect the apoptosis and cell cycle of OSCC cells,so EP4 receptor may be a potential target for treatment of OSCC.