A Study On The Role Of Crh Related Peptides In Migration & Apoptosis Of Cancer Cells And Relevant Mechanisms Via Regulating PLA2

Corticotropin-Releasing Hormone(CRH) is a 41 amino acids polypeptide which was originally discovered in central nervous system(CNS). Lately, several peptides which have similar sequence with CRH were isolated from CNS and peripheral tissues. These similar peptides are Urocortin(Ucn), Ucn2 and Ucn3, which collectively known as CRH-related peptides along with CRH. CRH-related peptides act through two CRH receptors, CRHR1 and CRHR2. A large number of researchs found that CRH-related peptides and CRH receptors exist in tumor tissues and affect tumor’s development in a paracrine and autocrine manner. There are evidences showing that CRH-related peptides play an important role in the progress of tumor cells’ apoptosis and migration, but the specific mechanisms are far from clear.PartⅠ Effects of Ucn on the migration of hepatic cancer cellsUcn is a member of CRH-related peptides, which has been reported to play a role in many biological processes, including inflammation and cancer development. Growing evidences shows that phospholipase A2(PLA2) enzymes also participate in inflammation and tumor’s development. Our previous work showed that Ucn differently adjusted cytosolic PLA2(c PLA2)and Ca2 +-independent PLA2(i PLA2). Some studies have shown that these two PLA2 take part in tumor’s migration. Thepresent study investigated how Ucn affected hepatorna carcinoma cell’s migration through regulating c PLA2 and i PLA2’s expression. Firstly, we studied the effects of Ucn on PLA2’s expression and Hep G2 cells’ migration using western blotting, wound healing and transwell assay. The results have shown that Ucn markedly suppressed i PLA2’s expression and Hep G2’s migration at 24 h, which can be attenuated by Anti-30(CRHR2’s inhibitor)/BEL(i PLA2’s inhibitor)/si RNA-i PLA2.On the contrary, Ucn2 significantly promoted c PLA2’s expression and Hep G2 cells’ migration at 36 h, which can be abolished by Anta(CRHR1’s inhibitor),PYR(c PLA2’s inhibitor)or si RNA-c PLA2. Unlike Hep G2 cells which express both CRH receptors themselves, SMMC-7721 cells hardly express these two CRH receptors. So we transfected CRHR1 or CRHR2 to SMMC-7721 cells steadily to observe the effects of Ucn. We found that Ucn obviously advanced SMMC-7721-R1 cells’ migration and the expression of c PLA2. However,Ucn obviously restrained SMMC-7721-R2 cells’ migration and the expression of i PLA2. Pretreatment of SMMC-7721-R1 cells with PYR blocked cell migration while BEL enhanced SMMC-7721-R2 cells’ migration. Moreover,we also found that NF-κB’s inhibitor PDTC attenuated the Ucn induced c PLA2 increasing as well, which indicated that NF-κB participated in c PLA2’s regulation. These results suggest that Ucn plays dual roles in the hepatoma carcinoma cells’ migration. Ucn suppress cells’ migration through down-regulating c PLA2 and promoted cells’ migration via up-regulating i PLA2.Part Ⅱ effects of CRH-related peptides on the apoptosis of prostate cancer cellsA growing number of researchs show that CRH-related peptides affect tumor’s development, such as proliferation, apoptosis and angiogenesis. Previously, wediscovered that CRHR mediated apoptosis of mouse prostate cancer cell line RM-1 through altering the ratio of Bcl-2:Bax, mitochondrial membrane potential, and caspase-9 activity. Ucn2 underwent opposite effects to CRH in RM-1. In the present study, we investigated the role of cytosolic PLA2(c PLA2)in CRH/ Ucn2 induced changing of apoptosis and the ratio of Bcl-2:Bax. Knockdown of c PLA2 attenuated CRH(CRHR1 agonist)-induced apoptosis and the decrease of Bcl-2:Bax ratio. When it comes to Ucn2, deficiency of c PLA2 aggravated Ucn2-inhibited apoptosis and the increase of Bcl-2:Bax ratio. Silencing of IL-1β markedly inhibited CRH-induced c PLA2 expression increase, whereas the decrease of c PLA2 expression induced by Ucn2 wasn’t consistent with either TNF-α or IL-1β. We also found that CRH time-dependently increased c PLA2 phosphorylation through activated CRHR1. In addition, In HEK293 cells transfected with CRHR1 or CRHR2, we found that CRH or Ucn2 up- or down-regulated c PLA2 expression of HEK293-R1 or HEK293-R2 cells respectively. Taken together,the above results indicated that CRH-related peptides promoted or restrained apoptosis via up- or down-regulating c PLA2 expression. Moreover, IL-1βwas involved in CRH induced increase in c PLA2 expression.