Abstract
BackgroundTo analyze the expression at basal level of inflammation-related cytokines and chemokines and the activation status of the NF-κB pathway, together with the proliferation and apoptosis indexes in two widely used in vitro tumor models, the androgen-dependent human Prostate Cancer (PC) cell line LNCaP and the androgen-independent PC3 , and in primary cultures of human PC cells. To assess in these models and primary cultures, the effects of Serenoa repens (LSESr, Permixon®) on proliferation/apoptosis ratio, inflammation-related genes expression and NF-κB pathway activation.MethodsThe expression of IL-6, CCL-5, CCL-2, COX-1, COX-2, iNOS inflammation-related genes has been evaluated at the mRNA level in two in vitro human PC models (LNCaP and PC3 cell lines) and in 40 independent human prostatic primary cultures obtained from PC patients undergoing radical prostatectomy. Tissue fragments were collected from both PC lesions and normal hyperplastic tissue counterparts for each case. All cultures were treated with two different amounts of Permixon® (44 and 88 μg/ml) for different time points (16, 24, 48 and 72 hours), depending on the cell type and the assay; the expression of inflammation-related genes, cell growth (proliferation/apoptosis ratio) and NF-κB activation has been analyzed in treated and untreated cells by means of semi-quantitative RNA-PCR, cell proliferation and immunofluorescence respectively.ResultsWe detected a significant reduction (p <0.001) in PC and normal cells proliferation due to Permixon ® treatment. This result was related to an increase of the apoptotic activity showed by an increase in the number of anti-caspase-3 fluorescent cells. Almost all the inflammation-related genes (IL-6, CCL-5, CCL-2, COX-2 and iNOS) were expressed at the basal level in in vitro cultured cells and primary cultures and down-regulated by Permixon® treatment. This treatment interfered with NF-kB activation, detecting by the translocation of more than 30% of NF-κB p65 subunit to the nucleus.ConclusionsThe present study confirms the expression of inflammatory pattern in PC. We showed the effect of Permixon® on down-regulation of inflammatory-related genes in cell lines and in primary cultures. The inhibitory effect of Permixon® on cell growth could be partly associated to the down-regulation of inflammatory-related genes and to the activation of NF-κB pathway in prostate tissue.
Highlights
Evidences in literature suggest that prostatic inflammation is involved in the pathogenesis and progression of prostatic diseases, such as benign prostatic hyperplasia (BPH) and prostate cancer (PC)
Human samples were screened by means of Reverse transcriptase polymerase chain reaction (RT-PCR) for the expression of CD45 and CK5/CK8 markers to assess the epithelial origin of cultured cells; only CD45 negative/CK5-CK8 positive cultures have been used for Sequences 5’-3’
This study shows for first time that primary human Prostate Cancer (PC) cells are influenced in growth and apoptosis pathways by natural compounds such as LSESr (Permixon), which down-regulates the inflammation pattern
Summary
Evidences in literature suggest that prostatic inflammation is involved in the pathogenesis and progression of prostatic diseases, such as benign prostatic hyperplasia (BPH) and prostate cancer (PC). The aim of our study is to analyze and to compare the expression of the inflammatory pathways in human PC cell lines, such as androgen-dependent LNCap and androgenindependent PC3 cell lines and in primary cultures of human prostate adenocarcinoma cells On these different settings, we evaluated the effect of LSESr (Permixon) on different inflammatory factors, analyzing whether its potential anti-inflammatory activity affects proliferation and apoptosis. To analyze the expression at basal level of inflammation-related cytokines and chemokines and the activation status of the NF-κB pathway, together with the proliferation and apoptosis indexes in two widely used in vitro tumor models, the androgen-dependent human Prostate Cancer (PC) cell line LNCaP and the androgenindependent PC3 , and in primary cultures of human PC cells. To assess in these models and primary cultures, the effects of Serenoa repens (LSESr, PermixonW) on proliferation/apoptosis ratio, inflammation-related genes expression and NF-κB pathway activation
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