Effect Of CRH Family Peptide On Apoptosis And Migration Of Tumor Cells And Its Mechanism

Corticotropin-releasing hormone(C RH) family are composed of C RH, Urocortin1(Ucn1), Urocortin2(Ucn2) and Urocortin3(Ucn3). They are widely distributed in the central nervous system and the periphery system. CRH family act through two transmembrane receptors, corticotropin-releasing hormone receptor type 1(CRHR1) and corticotropin-releasing hormone receptor type 2(C RHR2). Corticotropin-releasing hormone receptors(CRHRs) are G protein-coupled receptors that may play different roles in some tissues.In the present study, we investigated the role of CRHRs in apoptosis of prostate cancer cells, and it was found to be dependent on the effects of cytosolic calcium-dependent phospholipase A2(c PLA2). The experiments were carried out with mouse prostate cancer cells(RM-1 cells). The apoptosis rate of RM-1 cells stimulated by C RH or Ucn2 was detected by Flow cytometry after Annexin V–FITC/PI double staining. The results showed that CRH induced apoptosis(9.76%) compared with the control(3.92%), while Ucn2 repressed apoptosis(8.18%) compared with treatment with TG alone(14.84%).Researchers found that abnormal expression of c PLA2 is related to the occurrence and development of various tumors. To determine whether c PLA2 expression was involved in CRHRs-mediated apoptosis, c PLA2 m RNA was silenced with si RNA, then the apoptosis rate was detected. Results showed that silencing of c PLA2 attenuated CRH- induced apoptosis(5.31% vs. 9.76%) and strengthened Ucn2-inhibited apoptosis induced by TG(4.59% vs. 8.18%), indicating that c PLA2 was an apoptotic inducer in CRH/Ucn2 treatment. Silencing of c PLA2 also attenuated the Bcl-2/Bax ratio inhibited by CRH, whereas silencing of c PLA2 aggravated the Bcl-2/Bax ratio induced by Ucn2.Moreover, proinflammatory mediators such as IL1β & TNFα may enhance c PLA2 expression. In this part, the results indicated that CRH increased c PLA2 expression in a time and concentration-dependent manner mainly through interleukin 1β(IL1β) upregulation,while Ucn2 decreased c PLA2 expression through neither tumor necrosis factor α nor IL1β. CRH also suppressed decay of c PLA2 m RNA but Ucn2 merely suppressed its production. Overexpression of CRHR1 or CRHR2 in HEK293 cells correspondingly upregulated or downregulated c PLA2 expression after CRH or Ucn2 stimulation, respectively.In a word, the observation demonstrated that c PLA2 participates in CRHR1-induced apoptosis and C RHR2- inhibited apoptosis in RM-1 cells.Corticortropin-releasing hormone(CRH) family are multifunctional endocrine- factors and containing CRH, Urocortin1(Ucn1), Urocortin2(Ucn2), Urocortin3(Ucn3). They regulate proliferation, apoptosis and migration of various types of cancer cells through membrane receptors, corticortropin-releasing hormone type 1(C RHR1) and corticortropin-releasing hormone type 2(C RHR2). The role of CRH family in migration of cancer cells is not clear.Most researches indicate that abnormal transforming growth factor β1(TGFβ1)signals are related to the occurrence and development of cancers. In the early stage, TGFβ1 could inhibit cell proliferation and induce cell apoptosis. Whereas in the terminal stage, TGFβ1 is generally recognized as an inducible factor of Epithelial-Mesenchymal Transition(EMT) through induction of transcriptional factors such as Snail1 & Slug and hence inhibition of calcium epithelial mucin(E-cadherin) expression. E-cadherin is involved in forming adherin junctions between adjacent loops of membrane, and downregulation of E-cadherin is considered as a mark of EMT. Deregulation of TGFβ1 signals promotes aggressive metastatic properties in late-stage cancers. Modern epidemiological survey also showed that more than 90% of breast cancer patients ultimately died of tumor metastasis.The purpose of this study is to investigate the mechanism of Ucn1 effects on TGFβ1-induced Snail1 & Slug in breast cancers. The experiments were carried out with human breast cancer cell lines(MCF-7 cells & MDA-MB-231 cells). TGFβ1-Smad pathway is a canonic signal, in which Smad2/3 is the most important signal transducted factor. The results showed that Ucn1 inhibited Smad2/3 activation, while silencing of Smad7 abrogated its effects. It was further demonstrated that phosphorylated Smad2/3 translocated to the nuclear while Ucn1 inhibited activated Smad2/3 nuclear translocation, leading to downregulation Snail1 & Slug, the EMT promoters.Ucn1 has equal affinity for both CRHR1 and C RHR2. To find out whether these two C RH receptors play the similar role, Antalarmin(CRHR1 antagonist) and Antisauvagine-30(C RHR2 antagonist) were further used to abrogate the effects of Ucn1 on TGFβ1 signaling. The results indicated that both CRHR1 and CRHR2 participated in Ucn1-inhibited TGFβ1 signaling.This study demonstrated that Ucn1 as a potential mediator inhibited oncogenic signaling by TGFβ1 via activation of both CRHR1 and CRHR2.