Volume 110, April 2016, Pages 150–156
Open Access
Abstract
Pumpkin
seeds have been known in folk medicine as remedy for kidney, bladder
and prostate disorders since centuries. Nevertheless, pumpkin research
provides insufficient data to back up traditional beliefs of
ethnomedical practice. The bioactivity of a hydro-ethanolic extract of
pumpkin seeds from the Styrian pumpkin, Cucurbita pepo L. subsp. pepo
var. styriaca, was investigated. As pumpkin seed extracts are
standardized to cucurbitin, this compound was also tested.
Transactivational activity was evaluated for human androgen receptor,
estrogen receptor and progesterone receptor with in vitro yeast
assays. Cell viability tests with prostate cancer cells, breast cancer
cells, colorectal adenocarcinoma cells and a hyperplastic cell line from
benign prostate hyperplasia tissue were performed. As model for
non-hyperplastic cells, effects on cell viability were tested with a
human dermal fibroblast cell line (HDF-5). No transactivational activity
was found for human androgen receptor, estrogen receptor and
progesterone receptor, for both, extract and cucurbitin. A cell growth
inhibition of ~ 40–50% was observed for all cell lines, with the
exception of HDF-5, which showed with ~ 20% much lower cell growth
inhibition. Given the receptor status of some cell lines, a
steroid-hormone receptor independent growth inhibiting effect can be
assumed. The cell growth inhibition for fast growing cells together with
the cell growth inhibition of prostate-, breast- and colon cancer cells
corroborates the ethnomedical use of pumpkin seeds for a treatment of
benign prostate hyperplasia. Moreover, due to the lack of androgenic
activity, pumpkin seed applications can be regarded as safe for the
prostate.
Graphical abstract
Keywords
- Cancer cells;
- Benign prostate hyperplasia;
- Steroid hormone receptors;
- Cell viability;
- Pumpkin
JEL classification
- Obstetrics;
- Gynaecology;
- Urinary-tract disorder
1. Introduction
Pumpkins
are known all over the world and belong to the family Cucurbitaceae,
which includes economically very important species. Aqueous extracts of
pumpkin seeds are used in folk medicine to treat urinary tract
complications. Hydroethanolic pumpkin seed extracts showed promising
activities on stress urinary incontinence, on urination frequency and on
nocturia in clinical trials, which draw the research interest to the
polar part, instead of the more well-known seed oil [1], [2], [3] and [4].
Despite
the widespread use, effects on cell growth and receptor interaction are
not known. Pumpkins are consumed mostly as food, but Cucurbitaceae
representatives are also used in several countries such as Mexico, North
India, China, and in the Caribbean, in ethnomedicinal applications [5], [6], [7], [8] and [9].
In Central Europe, pumpkin seeds are recommended for bladder and
prostate problems and several dietary supplements are commercially
available for this purpose. Pumpkins are also used for prostate problems
in the Caribbean area [7]. In Austria a special variety, the Styrian oil pumpkin, Cucurbita pepo L. subsp. pepo var. styriaca, had been grown for decades [10] and is also used as food supplement.
Nevertheless,
the bioactive properties of pumpkins have been barely investigated. In
addition, pumpkin seeds are often tested in a complex mixture with other
plants and ingredients. For example, Jiang et al. [11] and [12] demonstrated inhibition of prostate cancer in vitro
and in a xenograft model with a polyherbal dietary supplement where
pumpkin seeds are only one of 33 ingredients. This makes an
interpretation of the efficacy of individual extracts and compounds
impossible.
In a
randomized, double-blind, placebo-controlled trial with 47 benign
prostatic hyperplasia patients, international prostate symptom score
(IPSS) was reduced by a 3 months treatment with pumpkin seed oil, but
prostate specific antigen and prostate volume were not reduced [13].
IPSS is a standardized questionnaire that is used to evaluate symptoms
of benign prostate hyperplasia. Another clinical trial with 2245
patients with benign prostate hyperplasia, who received during 3 months
an ethanolic pumpkin seed extract, resulted also in an efficient
improvement of BPH symptoms, especially in early stages, which was
measured with a decreased IPSS [14].
Beside those studies, only a few in vivo experiments were conducted, but mostly with pumpkin seed oil; these studies show a benevolent impact on prostate via inhibition of testosterone-induced hyperplasia [15] and [16].
Another
possibility to prevent an immense growth of hyperplastic tissue could
be obtained by cytotoxic effects. Cytotoxic activity was reported for
fig-leaf gourd (Cucurbita ficifolia), a Cucurbitaceae species that is common in Mexico [17]. This was also reported for compounds such as cucurbitacins, moschatin and cucurmosin from other Cucurbitaceae species [18], [19], [20] and [21].
Nevertheless,
these studies present only a small glimpse in the potential of
Cucurbitaceae as therapeutic and do not explain satisfactorily
traditional ethnomedical applications. More studies are needed to
corroborate ethnopharmacological records or to dismiss them. In this
study, we tested two effects of pumpkin seed extract and cucurbitin, (3R)-3-aminopyrrolidine-3-carboxylic
acid, that is used to standardize this extract. In the first place the
transactivation of human androgen receptor, estrogen receptor α and
progesterone receptor has been tested, and secondly the impact on cell
viability of several cancer cell lines, a hyperplastic cell line and a
normal fibroblastic cell line has been evaluated. Experiments with an
androgen-sensitive and an androgen-insensitive prostate cancer cell line
were performed to test if possible effects are mediated by an
androgen-dependent route. Experiments were performed in at least three
independent assays and within these tests in at least duplicates.
2. Materials and methods
2.1. Chemicals and media
Dimethyl sulfoxide (DMSO), 17β-estradiol (E2), β-naphthoflavone, 5α-dihydrotestosterone (5α-DHT), N-lauroylsarcosine (sodium salt), disodium hydrogen phosphate dihydrate (Na2HPO4 ∗ 2 H2O), sodium dihydrogen phosphate dihydrate (NaH2PO4 ∗ 2 H2O), potassium chloride (KCl), magnesium sulfate heptahydrate (MgSO4 ∗ 7 H2O), sodium carbonate (Na2CO3), copper(II) sulfate pentahydrate (CuSO4 ∗ 5 H2O) and o-nitrophenyl-β-galactopyranoside
(ONPG) were purchased from Sigma Aldrich (St. Louis, MO, USA), Fluka
(Buch, Switzerland), and Merck (Darmstadt, Germany). Cucurbitin was
purchased from Phytolab (Vestenbergsgreuth, Germany).
For
yeast media preparation, yeast nitrogen base was purchased from Difco
(Franklin Lakes, NJ), amino acids from Serva Feinbiochemica (Heidelberg,
Germany), and dropout medium without tryptophan from Sigma Aldrich.
Cell culture media and reagents were purchased from Biochrom (Berlin,
Germany), Sigma-Aldrich or Invitrogen (Lofer, Austria).
Cell
culture reagents were purchased from Biochrom (Berlin, Germany),
Sigma-Aldrich (St. Louis, MO, USA) or Invitrogen (Lofer, Austria).
Sodium selenite, human transferrin, methylthiazolyldiphenyl-tetrazolium
bromide (MTT), staurosporine and insulin (solution from bovine pancreas)
was purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.2. Extracts
The
pumpkin seed extracts were kindly provided by APOMEDICA Pharmazeutische
Produkte GmbH (Graz, Austria). The crude extract is a hydroethanolic
extract (60%) of Cucurbita pepo L. ssp. pepo var. styriaca semen with a drug:extract ratio of 15–25:1. The content of cucurbitin measured by GC was 0.41% (m/m).
A second batch of the extract was granulated on maltodextrin (30%) as carrier.
The
crude extract was diluted with distilled water (sterile filtered,
0.22 μm filters from Millipore (Millipore Ireland Ltd.) or with DMSO in
dilutions 1:2 (KRD2 or KRW2), 1:5 (KRD5 or KRW5) and 1:10 (KRD10 or
KRW10). The extract granulate was extracted with DMSO or water in
concentrations of 500 mg/ml (KGD500 or KGW500), 100 mg/ml (KGD100 or
KGW100), 50 mg/ml (KGD50 or KGW50), 20 mg/ml (KGD20 or KGW20) and
10 mg/ml (KGD10 or KGW10).
