Progesterone Effects On Primary Cutaneous Malignant Melanoma Growth And The Expression Of Anti-inflammatory Molecules

Objective1.To evaluate the prognoses of newly diagnosed primary cutaneous malignant melanoma(CMM) in Hubai area,including relationship between clinical stage and gender.2.To study progesterone receptors (PR) expression in primary CMM,and PR effects onmalignant melanoma growth,malignant transformation and immune inhibitoryphenotype.3.To study progesterone effects on malignant melanoma cell lines growth,apoptosis,theexpression regulation of anti-inflammatory molecules (immunosuppressive moleculesIL-10,TGF-β1 and HLA-G),and the signaling pathway related to it in vitro.Methods1.Among patients with primary CMM recorded in the area in the last 20 years,acollection of complete clinical and pathological data was reviewed,and the prognoseswere assessed.2.PR,proliferative cell nuclear antigen (PCNA),Bcl-2 and human leukocyte antigen -G(HLA-G) expression in a series of 40 specimens from 35 patients with CMM wereevaluated by immunohistochemistry,and the correlations between theimmunohistochemistry findings and clinicohistopathology date were also analyzed. 3.PR protein expression was identified by Indirect Immunofluorescence Assay in culturedmalignant melanoma cell lines A375 and A875 in vitro.4.Cultured malignant melanoma cell lines A375 and A875 were treated with a serialconcentration of progesterone,its antagonist RU486,MEK1/2inhibitor U0126 or AKtinhibitor LY294002.5.MTT assay was employed to determine the growth regulatory effects of progesterone,and Flow Cytometry Analysis was used to determine apoptosis rate.6.The level of IL-10,TGF-β1 and HLA-G mRNA and protein expression weredetermined by RT-PCR and ELISA or Western blot Assay.7.Activated MPAK,PI3K/Akt pathway was evaluated by the level of ERK1/2 and Aktphosphorylation,respectively,with Western blot Assay.Results1.The prognosis assessments of primary cutaneous malignant melanoma in Hubai area.1) Among 43 cases newly diagnosed primary CMM in Hubai area,27 (62.8%)wereclinical stageâ… -â…¡tumors and 16 (37.2%) stageâ…¢-â…£,sex ratio F/M 2.91:1,the meanage 57.9±1.92 years old,the mean diagnosis delay 1.74±0.2 years,mean Breslowthickness 1.66±0.19mm,ulceration 27.9%,radial growth pattern 20.9% and verticalgrowth pattern 79.1%,non-brisk inflammatory infiltrate 58% and brisk 23.3%.2) The relation between clinical staging and other prognostic variables:no significantdeference of clinical staging was observed between males and females (x²=0.430,P=0.512),advanced clinical stage was significantly correlated with Breslow thicknessmore than 1mm (x²=14.149,P=0.001),and tumors with vertical growth pattern(x²=6.475,P= 0.009) .3) In last two decades,there were significant shorter delay in diagnosis in the second10-years than in the first (p=0.017),and no significant differences of other variables.2.Expression and the role of progesterone receptor in primary CMM1) PR expression was detected in 25.7% (9/35) of CMM,no PR expression was observedin nevi,the difference is significant (Fisher's exact tests,P＜0.01 ) . 2) There is a slight increasing of PR expression in female,aged less than 55 years,diagnosis delay longer than 1 years,ulceration and non-acral histopathology subtype,but not reaching statistic significance(P=0.416,0.416,0.129,0.416,0.416,respectively).3) PR expression was significantly inversely correlated with PCNA expression(r=0.012,P=0.946),and not with Bcl-2.4) PR and immunosuppressive molecule human leukocyte antigen-G were detected insome cases,but significant correlation was not obtained (r=0.012,P=0.946),HLA-Gexpression was significantly correlated with Bcl-2(r=0.440,P=0.008).3.The effects of progesterone on growth regulation in malignant melanoma cells linesA375 and A875 in vitro.1) Both A375 and A875 are progesterone receptor-negative.2) Lower concentration progesterone enhanced both two cell lines proliferation,but thisgrowth regulation effect reversed when incubated with 1×10^-7M and higherconcentration progesterone.3) The growth regulatory effect can be inhibited by MAPK inhibitor U0126,but can notbe abolished by progesterone antagonist RU486.4) High concentration (≥1×10^-7M) progesterone promoted A375 and A875 apoptosis in adose-dependented manner.5) The level of ERK1/2 phosphorylation increased by lower concentration (1×10^-9M)progesterone,but reduced by high concentration (1×10^-6M) progesterone.4.Progesterone effects on immune inhibitory ligands expression in melanoma cells invitro.1 ) TGF-β1 and IL-10 mRNA and protein can be detected in both A375 and A875 cellsunder serum-free culture conditions as measured by RT-PCR and ELISA.2) High concentration progesterone(≥1×10^-7M) enhanced IL-10 expression both in A375and A875 cells,but did not alter TGF-β1 expression,and not induce HLA-Gexpression.3) LY294002 abrogated progesterone effects on IL-10 expression regulation,but not progesterone antagonist RU486.4) High concentration progesterone(≥1×10^-7M) increased the level of Aktphosphoryiation in A375 cells.Conclusions1.The newly diagnosis cutaneous primary malignant melanoma in this area had apredilection for males,and were more likely to have advanced stages of disease atpresentation.Advanced clinical stage was associated with Breslow thickness andvertical growth pattern.2.Progesterone receptors expressed in some of primary cutaneous malignant melanoma,and PR expression associated with tumor growth and malignant transformation.3.Progesterone exhibit growth regulation on malignant melanoma cell throughnon-genomic mechanism.4.Progesterone stimulates PI3K-dependent signal pathway leading to the up-regulation ofIL-10 gene expression in melanoma cells.