Alain Latil, Centre de Recherche et Développement Pierre Fabre, 3 Av. Hubert Curien, BP 13562, 31035 Toulouse Cedex 1, France. e-mail: email@example.com
What's known on the subject? and What does the study add?
Pervasive inflammatory infiltrates, mainly composed of chronically activated T cells and monocytes/macrophages, have been observed in benign prostatic hyperplasia (BPH). Permixon®, a hexanic lipidosterolic extract of Serenoa repens (hexanic LSESr) used to treat urinary dysfunction in BPH patients, has anti-inflammatory activities. This paper provides new insights into the anti-inflammatory properties of Permixon®. We report that hexanic LSESr inhibits early steps of leukocyte infiltration in vitro by downregulating MCP-1/CCL2 and VCAM-1 expression.
• To investigate the mechanisms by which hexanic lipidosterolic extract of Serenoa repens (hexanic LSESr) may prevent leukocyte infiltration in benign prostatic hyperplasia by studying its impact on monocyte chemoattractant protein 1/chemokine (C-C motif) ligand 2 (MCP-1/CCL2) and vascular cell adhesion molecule 1 (VCAM-1) expression in vitro.
MATERIALS AND METHODS
• After pretreatment with hexanic LSESr, human prostate (epithelial and myofibroblastic) cells and vascular endothelial cells were stimulated with proinflammatory cytokines.
• MCP-1/CCL2 and VCAM-1 mRNA expression was quantified by real-time PCR.
• ELISA kits were used to determine MCP-1/CCL2 levels in culture supernatants and VCAM-1 expression in living cells.
• Hexanic LSESr reduced MCP-1/CCL2 mRNA levels in both epithelial (BPH-1) and myofibroblastic (WPMY-1) prostate cell lines.
• Hexanic LSESr downregulated MCP1/CCL2 secretion by WPMY-1 cells in a concentration-dependent manner, more efficiently than Serenoa repens extracts obtained by supercritical carbon dioxide extraction.
• Hexanic LSESr inhibited tumour-necrosis-factor-α-induced MCP-1/CCL2 secretion by the human vascular endothelial cell line EAhy.926, as well as surface VCAM-1 protein expression, in a concentration-dependent manner.
• Hexanic LSESr impedes key steps of monocyte and T cell attraction and adherence by inhibiting MCP-1/CCL2 and VCAM-1 expression by human prostate and vascular cells in an inflammatory environment.
• These findings provide new insights into the anti-inflammatory effects of the hexanic lipidosterolic extract of Serenoa repens, Permixon®, in benign prostatic hyperplasia.
monocyte chemoattractant protein 1/chemokine (C-C motif) ligand 2
lipidosterolic extract of Serenoa repens
supercritical carbon dioxide
Dulbecco's modified Eagle's medium
fetal bovine serum
vascular cell adhesion molecule 1
intercellular adhesion molecule 1
platelet-endothelial cell adhesion molecule 1
tumour necrosis factor α
BPH affects nearly three in four men by the age of 70 . BPH is a progressive disease that causes lower urinary tract symptoms and carries a risk of acute urinary retention and BPH-related surgery, thus undermining quality of life .
BPH is associated with hyperproliferation of stromal and epithelial prostate cells due to complex cellular alterations affecting proliferation, differentiation, apoptosis and senescence . Androgens and aging are traditionally considered to be the main determinants of prostate enlargement, but a potentially important role of chronic inflammation has emerged in recent years . In particular, mononuclear cell infiltration has been found in surgical specimens  and a strong correlation has been reported between histological inflammation and both the International Prostate Symptom Score and prostate volume. Chronic inflammatory infiltrates are mainly composed of chronically activated T cells and macrophages which produce cytokines that might promote fibromuscular growth in BPH [6,7]. An important role of cytokines in the initiation and progression of BPH is suggested by a recent study of the monocyte chemoattractant protein 1/chemokine (C-C motif) ligand 2 (MCP-1/CCL2), which stimulates monocyte recruitment and activation during inflammation .
Medical management is often preferred for BPH, owing to the potential complications of surgery. The hexanic lipidosterolic extract of Serenoa repens (hexanic LSESr), Permixon®, has been prescribed for BPH for more than 25 years in Europe and is one of the most widely used phytotherapeutic agents in this setting. Permixon® inhibits 5α-reductase activity , reduces prostate cell proliferation and enhances their apoptosis [10,11], and also displays anti-inflammatory activities . The aim of the present study was to obtain new insights into the anti-inflammatory properties of this hexanic LSESr by evaluating its effects on early steps of leukocyte infiltration, using cultured human prostate and vascular cell lines.
MATERIALS AND METHODS
We tested four LSESr batches obtained by hexanic extraction (Permixon®) and two batches obtained by supercritical carbon dioxide (SC-CO2) extraction at Pierre Fabre Plantes et Industrie (Gaillac, France). Batches of Serenoa repens extracts were freshly prepared for each experiment by dissolution in ethanol (80 mg/mL) and further dilution to final concentrations of 10–56 µg/mL. The maximum final concentration of ethanol in the culture medium was <0.1%.
Curcumin (CAS Number 458-37-7) and quercetin (CAS Number 117-39-5) were purchased from Sigma-Aldrich (L’Isle d’Abeau, France).
Human prostate epithelial (BPH-1), myofibroblastic (WPMY-1) and cancer (PC3) cell lines were obtained from the German Resource Centre for Biological Material, the European Collection of Cell Cultures and the American Type Culture Collection, respectively.
The human endothelial cell line EAhy.926 was obtained from Dr E. Dejana (Mario Negri Institute, Milan, Italy). This cell line is the fusion product of human umbilical vein endothelial cells and the non-endothelial lung carcinoma cell line A549 .
WPMY-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mm l-glutamine (Sigma). BPH-1 cells were cultured in RPMI 1640 medium supplemented with 20% FBS, 20 ng/mL testosterone, 5 µg/mL transferrin, 5 ng/mL sodium selenite and 5 µg/mL insulin (Sigma). WPMY-1 and BPH-1 culture media were changed twice a week at a split ratio of 1:3, and the cells were used at passages 20–25. EAhy.926 cells were cultured in DMEM supplemented with 10% FBS, gentamicin (50 µg/mL), fungizone (2.5 µg/mL) and 2% HAT (hypoxanthine/aminopterine/thymidine). EAhy.926 cells were subcultured weekly at a split ratio of 1:10 and were used up to passage 20.
Total RNA was isolated from prostate cell lines by using the RNAble reagent and Qiagen RNeasy mini-preps according to the manufacturers’ instructions (Eurobio and Qiagen, Courtaboeuf, France). The quantity and purity of extracted RNA were assessed with a NanoDrop ND 1000 spectrophotometer (Labtech International, Paris, France). First-strand cDNA synthesis was performed with 1 µg of total RNA and Superscript II reverse transcriptase (Life Technologies, Courtaboeuf, France) in a final volume of 20 µL.
Real-time qPCR was performed with the fluorescent double-stranded DNA-binding dye SYBR Green. Primers were chosen with the assistance of the computer programs Oligo-4.0 (National Biosciences, Plymouth, MN, USA) and Primer Express v2.0 (Applied Biosystems, Life Technology, Foster City, CA, USA). Searches of the dbEST, htgs and nr (the non-redundant set of the GenBank, EMBL and DDBJ database sequences) databases confirmed the total gene specificity of the nucleotide sequences chosen as primers and the absence of DNA polymorphisms. Dissociation curves showed that each primer set yielded a single product, which was purified and sequenced to confirm its specificity. To avoid amplification of contaminating genomic DNA, one of the two primers was placed at the junction between two exons. For each primer pair, we included no-template and no-reverse-transcriptase controls, which produced negligible signals (usually Ct > 40), suggesting that primer-dimer formation and genomic DNA contamination effects were negligible.
The following primers were used: MCP-1/CCL2, 5′-AGTCTCTGCCGCCCTTCTGTG-3′ (forward) and 5′-CATCTGGCTGAGCGAGCCC-3′ (reverse); CCR2A, 5′-AGTTGGAAGTGTGTGATCTGTGGG-3′ (forward) and 5′- CAGCGATGGAGCGTATACTGTGAT-3′ (reverse); CCR2B, 5′-TCTACAGGGAGACAGTGGATGGAG-3′ (forward) and 5′- ATAAACCAGCCGAGACTTCCTGCT-3′ (reverse); vascular cell adhesion molecule 1 (VCAM-1), 5′-GGAAAAAGGAATCCAGGTGGAGA-3′ (forward) and 5′-ACACTTGACTGTGATCGGCTTCC-3′ (reverse); intercellular adhesion molecule 1 (ICAM-1), 5′-CCTTCCTCACCGTGTACTGGACTC-3′ (forward) and 5′-CTGGCAGCGTAGGGTAAGGTTCT-3′ (reverse); platelet-endothelial cell adhesion molecule 1 (PECAM-1), 5′-TGATGCCGTGGAAAGCAGATACT-3′ (forward) and 5′-GTTCTTCTCGGAACATGGATGTCC-3′ (reverse); PPIA, 5′-GAGCACTGGAGAGAAAGGATTTGGTT-3′ (forward) and 5′-CGTGTGAAGTCACCACCCTGACA-3′ (reverse); GUSB, 5′-GAACGGGAGGTGATCCTGCC-3′ (forward) and 5′-GACCCCATTCACCCACACGA-3′ (reverse); RPLP0, 5′-GGCGACCTGGAAGTCCAACT-3′ (forward) and 5′-CCATCAGCACCACAGCCTTC-3′ (reverse).
All qPCR reactions were performed with an ABI Prism 7900 Sequence Detection System (Applied Biosystems) and the SYBR® Green PCR Core Reagents kit (Life Technology). The thermal cycling conditions comprised an initial denaturation step at 95 °C for 10 min and 40 cycles at 95 °C for 15 s and 65 °C for 1 min.
To control for differences in the amount of starting material, the data were normalized to the geometric mean of a set of three ‘housekeeping’ genes (PPIA, GUSB and RPLP0), expression levels of which have been empirically shown not to change as a function of treatment, using geNorm  and NormFinder . The value of the target gene was subsequently normalized such that the control value was 1.
MCP-1/CCL2 protein levels were determined with ELISA kits. Cells were seeded in six-well culture plates and treated with increasing concentrations of LSESr or vehicle for 24 h before adding proinflammatory cytokines (10 ng/mL) for another 5 h. Cytokine concentrations and time point treatment were chosen according to the Penna et al.  study. Cell viability was not affected by the selected cytokine concentrations at any of the time points, as shown by LDH and ATPlite assays (data not shown).
Supernatants were harvested and MCP-1/CCL2 levels were measured with the MCP-1/CCL2 Quantikine ELISA kit according to the manufacturer's recommendations (R&D Systems Europe Ltd, Lille, France). Each experiment was repeated at least three times.
VCAM-1 protein expression by the human umbilical vein endothelial cell line EAhy.926 was measured with a cell-based ELISA. Confluent EAhy.926 cells in 96-well plates were pretreated for 2 h with increasing concentrations of hexanic LSESr or vehicle and then stimulated with tumour necrosis factor α (TNF-α) (300 U/mL, R&D Systems) for another 24 h. The cell monolayers were washed twice and incubated for 1 h at 37 °C with 100 µL of primary mouse anti-human antibodies against VCAM-1 (CD106), ICAM-1 or PECAM-1 (Beckman Coulter Immunotech, Marseille, France). The monolayers were then washed twice and incubated for 1 h at 37 °C with 100 µL of a horseradish-peroxidase-conjugated anti-mouse antibody (GE Healthcare, Saclay, France). VCAM-1, ICAM-1 and PECAM-1 were quantified with a colorimetric method (at 450 nm) using phosphate-citrate buffer with urea hydrogen tablets and o-phenylenediamine as substrates. ELISA values were determined after 20 min of incubation at room temperature and were expressed as OD450nm (target protein) minus OD450nm (non-specific binding).
Results are expressed as mean ±sd. anova and the Tukey test were used for multiple comparisons, and statistical significance was inferred at P < 0.05. SigmaStat 3.5 software was used for all analyses.
EFFECT OF HEXANIC LSESR ON MCP-1/CCL2, VCAM-1, ICAM-1 AND PECAM-1 MRNA LEVELS IN PROSTATE CELL LINES
Basal MCP-1/CCL2 mRNA expression was approximately 10-fold higher in WPMY-1 myofibroblastic cells than in BPH-1 epithelial cells (data not shown). MCP-1/CCL2 mRNA expression fell significantly in both cell lines after hexanic extract treatment (−55% and −72% in BPH-1 and WPMY-1 cells, respectively) (Fig. 1A).
The cytokine cocktail (interferon-γ/interleukin-17/TNF-α[IFN-γ/IL-17/TNF-α]) induced respective 80-fold and 120-fold increases in MCP-1/CCL2 mRNA in BPH-1 and WPMY-1 cells. This effect was attenuated by hexanic extract (40 µg/mL) in both cell lines (Fig. 1B), with an approximate 50% reduction in WPMY-1 cells.
Hexanic extract treatment of WPMY-1 cells stimulated with the proinflammatory cocktail reduced VCAM-1 mRNA levels but had no effect on either ICAM-1 or PECAM-1 mRNA levels (Fig. 1C).
HEXANIC LSESR INHIBITION OF MCP-1/CCL2 PROTEIN EXPRESSION IN WPMY-1 CELLS
MCP-1/CCL2 protein levels in WPMY-1 cell supernatants were strongly enhanced by the proinflammatory cocktail (from 139 ± 10 pg/mL to 1711 ± 65 pg/mL). Hexanic extract reduced MCP-1/CCL2 protein secretion in a concentration-dependent manner, by up to 50% at 40 µg/mL. WPMY-1 cells exposed to curcumin and quercetin (at 3.7 and 30 µg/mL, respectively) released significantly less MCP-1/CCL2 than vehicle-treated cells (Fig. 2A).
Basal MCP-1/CCL2 protein secretion by WPMY-1 cells was approximately halved by hexanic extract treatment (40 µg/mL) (Fig. 2B).
EFFECT OF SIX BATCHES OF SERENOA REPENS HEXANIC AND SC-CO2 EXTRACTS ON MCP-1/CCL2 PROTEIN EXPRESSION BY WPMY-1 CELLS
In proinflammatory conditions, the four batches obtained by hexanic extraction (SR870, SR876, SR877 and SR900) induced similar maximal inhibition of MCP-1/CCL2 protein expression (P < 0.001). The two SC-CO2 extracts (SR194 and SR906) did not significantly inhibit MCP-1/CCL2 protein expression (Fig. 3).
HEXANIC LSESR INHIBITION OF VCAM-1 AND MCP-1/CCL2 PROTEIN EXPRESSION BY EAHY.926 CELLS
Activation of EAhy.926 cells with TNF-α (300 U/mL) increased the surface expression of VCAM-1 (Fig. 4A) and ICAM-1 but did not affect PECAM-1 expression. TNF-α induced a 20-fold increase in VCAM-1 expression by EAhy.926 cells compared with vehicle-treated cells; pretreatment with hexanic extract for 2 h reduced cytokine-induced VCAM-1 expression in a concentration-dependent manner, by more than 60% at 56 µg/mL (P < 0.001) (Fig. 4A,B), but had no effect on either ICAM-1 or PECAM-1 expression (Fig. 4B).
In another set of experiments, TNF-α (300 U/mL) induced a 21-fold increase in MCP-1/CCL2 expression (up to 23 000 ng/mL) by EAhy.926 cells after 24 h; hexanic extract pretreatment for 2 h before cytokine challenge significantly attenuated this stimulation, but only at the highest concentration tested (56 µg/mL, −74%) (data not shown).
MCP-1/CCL2 is a low-molecular-weight monomeric polypeptide whose primary function is to promote monocyte and macrophage migration to sites of inflammation . MCP-1/CCL2 is involved in monocyte infiltration in inflammatory diseases such as rheumatoid arthritis and also in inflammatory responses to tumours.
Given the lack of an in vitro model of BPH-related inflammatory processes, we used a cocktail of proinflammatory cytokines (IFN-γ/IL-17/TNF-α), known to be secreted by prostate-infiltrating CD4+ cells in BPH , to create an inflammatory environment. CD4+ cells infiltrating human prostate tissue also produce MCP-1/CCL2.
We found that hexanic LSESr inhibited MCP-1/CCL2 mRNA expression in human epithelial and myofibroblastic prostate cell lines in both basal and inflammatory conditions, suggesting the involvement of the same pathway. Basal MCP-1/CCL2 mRNA expression was approximately 10-fold higher in myofibroblastic WPMY-1 cells than in BPH-1 epithelial cells, in keeping with a prostatic stromal cell origin of this chemokine; indeed, MCP-1/CCL2 transcripts are expressed primarily by stromal smooth muscle cells . As our results suggested that the myofibroblastic cell line WPMY-1 was a more accurate cellular model of MCP-1/CCL2 expression in BPH, we used these cells to investigate the effects of hexanic LSESr.
When WPMY-1 cells cultured in an inflammatory environment were exposed to hexanic extract, MCP-1/CCL2 protein secretion fell in a concentration-dependent manner. Hexanic LSESr also inhibited basal MCP-1/CCL2 protein secretion. Both curcumin, the active polyphenolic compound of powdered tumeric (Curcuma longa) root, and the bioflavonoid quercetin, used as positive controls, inhibited MCP-1/CCL2, as previously described [20,21].
In addition to its critical role in monocyte recruitment and activation, MCP-1/CCL2 (i) regulates memory T cell and natural killer cell migration and infiltration, (ii) suppresses apoptosis of T cells and promotes their survival and (iii) rescues fully functional cells from the apoptotic programme, possibly promoting their migration towards new sites of inflammation where growth factors are available .
It is noteworthy that only batches obtained by hexanic extraction, contrary to SC-CO2 extracts, reduced MCP-1/CCL2 protein expression, underlining the importance of the extraction procedure used to prepare phytotherapeutic products. Our data confirm the reproducibility of hexanic LSESr batches used in Permixon®, as previously reported by Scaglione et al. , in terms of their effect on 5α-reductase activity.
In response to MCP-1/CCL2 stimulation, monocytes/macrophages migrate across the endothelial cell layer. VCAM-1 overexpression participates in the early steps of leukocyte extravasation by favouring the rolling adherence of monocytes and T cells on vessel walls, as well as acting in concert with chemotactic proteins such as MCP-1/CCL2 to promote leukocyte infiltration of various tissues [24,25].
Hexanic LSESr inhibited both MCP-1/CCL2 protein secretion and cytokine-induced VCAM-1 (CD106) expression by cultured human vascular endothelial cells, in a concentration-dependent manner. Expression of VCAM-1, which is involved in T cell and monocyte adhesion, was also selectively inhibited. Upon activation by TNF-α, endothelial cells overexpress ICAM-1, a molecule involved in neutrophil adhesion. Interestingly, hexanic LSESr did not affect the expression of ICAM-1 or that of PECAM-1, a constitutive protein of intercellular junctions, highlighting the specificity of its effects. Likewise, hexanic LSESr inhibited WPMY-1 prostate cell mRNA expression of VCAM-1 but not that of ICAM-1 or PECAM-1.
T cells and monocytes/macrophages are two major cell types involved in BPH inflammation . The present results, although obtained in cell culture models, support a favourable effect of hexanic LSESr on BPH-related inflammation. Hexanic LSESr may attenuate inflammation by blocking crucial steps of leukocyte adhesion and migration, by inhibiting MCP-1/CCL2 production by prostate stroma cells and by reducing MCP-1/CCL2 and VCAM-1 expression by vascular endothelial cells.
As stromal cells have important paracrine functions in epithelial cell homeostasis, alterations in the stroma can modify stromal–epithelial interactions and BPH progression . Inflammatory cells in the prostate microenvironment can promote epithelial cell proliferation, a feature of BPH.
In this study, the enhancement of PC3 cell proliferation by WPMY-1 cell conditioned medium was attenuated when the latter cells were treated with hexanic extract for 24 h, unlike BPH-1 the proliferation of which remained unchanged (data not shown). Our qPCR data indicate that PC3 cells express both known MCP-1/CCL2 receptor variants (CCR2A and CCR2B) , contrary to BPH-1 cells (data not shown). Hence, although we cannot exclude a direct action of hexanic LSESr on PC3 cell proliferation, hexanic extract might inhibit the proliferation of prostate epithelial cells by downregulating MCP-1/CCL2 production by stromal cells. A direct stimulatory effect of MCP-1/CCL2 on prostate epithelial cells has recently been described . The possible value of MCP-1/CCL2 circulating levels as a surrogate marker of prostatic inflammation and BPH progression is worth investigating.
Testosterone inhibits proinflammatory cytokine production by isolated peripheral blood monocytes/macrophages , and testosterone levels fall with aging. Thus, leukocytes attracted to aging prostate tissue might promote abnormal cell proliferation .
The precise cellular mechanisms underlying the inhibition of MCP-1/CCL2 and VCAM-1 expression by hexanic LSESr remain to be clarified. The possible impact of hexanic LSESr on leukocyte diapedesis and nuclear factor κB activation is now being investigated, along with the effect of specific components of hexanic LSESr. Regarding the ‘active fraction(s)’ in hexanic LSESr, which contain several types of lipids but also other specific chemical entities, experiments are ongoing to test their effects on MCP-1/CCL2 and VCAM-1 expression.
Whatever the underlying mechanism, MCP-1/CCL2 downregulation would tend to slow the course of BPH. Indeed, systemic administration of CCL2-neutralizing antibodies to mice has been shown to significantly slow prostate growth and to attenuate CD68+ macrophage infiltration in vivo, effects accompanied by a significant decrease in microvascular density . A role of CCL2-mediated macrophage infiltration in BPH has been suggested by Fujita et al. .
In conclusion, inflammation appears to play a pivotal role in the pathogenesis of BPH and thus represents a key target for anti-BPH drugs. Several lines of evidence indicate that blood mononuclear cells are involved in the inflammatory process. By inhibiting MCP-1/CCL2 and VCAM-1 expression, hexanic LSESr may impede the attraction, adherence and extravasation of monocytes and T cells in the prostatic microenvironment.
This work highlights for the first time the direct action of Permixon® on the expression of key proinflammatory genes and provides new insights into the anti-inflammatory mechanism of this pharmaceutical product.
The authors thank Claire Issac, Jérôme Rouquet and Thierry Taillandier for their excellent technical assistance.
CONFLICT OF INTEREST
None declared. Source of funding: Institut de Recherche Pierre Fabre.