The results obtained using various BPH cultures grown in the presence of serum from a man treated with PA corroborate the results previously reported for the direct application of PA on mouse10
or human4, 5
prostatic fibroblasts. The main criticism of PA pharmacology and clinical studies is that the precise compound responsible for the effect of PA is unknown and, therefore, it has never been demonstrated that the PA levels in the serum and prostate after oral absorption are as high as those used in in vitro
or animal studies. To address this issue, we used the serum of a man before and after the ingested PA. This study is the first to demonstrate that the serum of a man treated with PA could induce a response in prostatic cells with relevant modification of the transcriptome and inhibition of prostatic cell growth. Nonetheless, because the experiments were performed with the serum of only one man, which is a limitation of this study, our results cannot be generalized because there may be variability in the absorption or metabolism of the drug among individuals.
The effect on prostatic cells is supported by many aspects of our study. The inhibitory effect on cell growth when using various models of prostatic cell growth reinforce the validity of these findings, especially the results obtained when using fresh prostatic cells from five different men and when using 3D organotypic cultures, which more closely resemble the in vivo
conditions than the regular cell line cultures.4, 9
Fresh prostatic cells in these models were more likely to proliferate in the presence of normal serum than immortalized cells were, which is in line with earlier reports.4
Primary and organotypic cultures may, therefore, be interesting tools to analyse the effects of different drugs on prostate cell proliferation when cultured with human serum. PA did not have any observed effect on the PNT2 prostatic epithelial cells. There was little proliferative effect observed for both types of serum used; longer culture times may be necessary to see an effect. The effect on WPMY cell proliferation was observed only for the highest serum concentrations. PA was previously observed to have a similar effect on PMF and Madin–Darby canine kidney epithelial cells exposed to similar concentrations of PA.4
Therefore, it seems likely that PA is mainly active on myofibroblast growth rather than BPH epithelial cell growth, corroborating previous findings obtained using rats.10
This result may also indicate that human serum is not a suitable culture medium for prostatic epithelial cells, again highlighting the close relationship between the epithelium and the stroma during prostate growth.11
The effect of PA on cancerous cell lines has also been reported, and treatment with PA is associated with a reduced incidence of prostate cancer in TRAMP mice,6
indicating a global effect on pathological prostate growth. Our results also suggest that gene expression is modified in response to exposure to PA, supporting the hypothesis that sufficient drug levels were present in the serum to induce an effect. Additional experiments, such as measuring individual RNA and protein levels and repeating the assays using sera from multiple individuals, would be required to confirm each individual candidate. Nonetheless, this gene analysis has identified relevant candidate genes in the context of the reduction of cell growth.
The effect on prostatic cells is also supported by the transcriptome analysis because major modifications were observed in pathways previously reported to be involved in BPH.12
We observed an upregulation of genes related to tumour suppressor functions (KRIT1
) and a downregulation of genes involved in tumour progression, including genes related to inflammation and oxidative stress. It has been hypothesized that KRIT1
is a tumour suppressor gene,13
can phosphorylate p53, which is a tumour suppressor gene involved in many cancers.14
Downregulation of oncogenes was also observed. RASEF is a member of the RAS oncogene family and is involved in various aspects of intracellular membrane transport, including exocytosis.15
Little is known about this gene, but it has been reported that the expression of RASEF is increased in familial cutaneous malignant melanoma,16
thereby suggesting a potential oncogenic role.
Other genes may also be linked to tumour development. MALAT-1 expression was shown to be significantly correlated with the risk of metastasis in early-stage non-small-cell lung cancer.17
C1orf181 was reported to be a breast cancer antigen.18
ASNS was found to be associated with pancreatic and gastric cancer and is associated with cell cycle progression.19, 20
KLC1 has been reported to be involved in kidney and breast tumours that are associated with the VHL tumour suppressor protein and in the synthesis and maintenance of primary cilia.21
Inflammatory mechanisms and responses to stress were also affected by the treated serum as illustrated by the overexpression of the SMG1
gene and the downregulation of the TPST1
genes, which have been shown to downregulate the production of reactive oxygen species.22
Of reactive oxygen species, H2
has been reported to be a second messenger for various physiologically active agents, such as TGF-beta and epidermal growth factor, both of which are involved in BPH processes.
Tyrosine kinase genes have been reported to be involved in BPH and prostate cancer processes.12
ZEB2, which was shown to be upregulated, plays a crucial role in the regulation of TGF-beta signalling.23
This effect may be related to β-sitosterol, which was able to significantly induce the expression and secretion of TGF-beta1 in primary cultures of BPH.24
The overexpression of the first receptor of the fibroblast growth factor receptor family (FGFR1), a tyrosine kinase receptor, highlights the frequent impact of fibroblast growth factor family members on BPH.8, 11, 25
Nonetheless, the overexpression of FGFR1 in the general context of reducing proliferation is unexpected, and additional data are required for the accurate interpretation of this result because the overexpression of FGFR1 has been reported to be associated with the proliferation of both benign and cancerous prostate lesions.
Altogether, the results of this transcriptome analysis suggest the drug had an effect on prostatic cells, especially on pathways usually involved in BPH.12
Nonetheless, no firm conclusion could be made regarding the different genes that were up- or downregulated because PCR was not used to confirm these results. Here again, the aim was to show that there is an effect on prostatic cells after the absorption of PA and not to elucidate the underlying mechanisms. Another aim was to describe an original method to investigate the effect of various phytotherapeutic drugs of unknown composition on prostatic cell growth that could be used to determine the underlying mechanisms.
The oral intake of PA could result in sufficient serum levels of active substances to induce a transcriptome modification and the inhibition of prostatic myofibroblast growth. Nonetheless, our results should not be construed as justifying the medical usefulness of PA but as a proof of concept that the oral absorption of PA could provide sufficient concentrations of active substances to the prostate.