A screening of growth inhibitory activity of Iranian medicinal plants on prostate cancer cell lines

Background: Prostate cancer has been known as one of the most common malignancy in the men and it is therefore very important to prevent and treat this cancer. In this study, the anticancer effects of 20 species of medicinal plants in Iran, especially those grown in Chaharmahal and Bakhtiari province, were investigated on prostate cancer cell lines to identify potential natural alternatives for the development of prostate cancer anticancer drugs. Methods: The plants were gathered from Chaharmahal va Bakhtyari and their aerial parts extracted through maceration method using ethanol 70%. Anti-proliferative activity of extracts on PC-3, DU145 and HDF cell lines was evaluated by MTT assay 48 hours after treatment. Results: Euphorbia szovitsii Fisch. & C.A.Mey. and Achillea wilhelmsii had anti-proliferative activity more than other plants on PC-3. Also IC50s for Urtica dioica, Euphorbia szovitsii Fisch. & C.A.Mey. and Medicago sativa were lower amount among the examined plants on Du-145. Conclusion: According to our result, Euphorbia szovitsii Fisch. & C.A.Mey., U. dioica and Medicago sativa with good anti-proliferative activity can serve as an effective source of natural products to develop new antiprostate cancer drugs.


Introduction
Prostate cancer has been reported with a high incidence of 7.9%, representing the fourth leading cancer in the general population, according to the International Agency for Research on Cancer (IARC) of the World Health Organization released (WHO) in 2014 [1]. In a more recent report, 26% of newly diagnosed cases of cancer in the United States and 9% of men's deaths are related to prostate cancer [2]. In other reports, the incidence rates of prostate cancer are different in different Asian countries, ranging from low incidence (2/100,000 population in Iran) to high incidence (20/100,000 population in the Philippines). In Iran, prostate cancer is one of the most common cancers among men, with the highest prevalence in Tehran (41.2%) and comparatively lower prevalence in other large and industrial provinces (36.8%), and small towns and villages (22.1%) [3][4][5][6][7][8]. These reports suggest that prostate cancer is one of the most common causes of mortality in men, comprising an important health issue, which makes its treatment essential. However, despite the many treatments for prostate cancer, due to drug resistance, several complications of used medications and treatments, the available treatment options have not been able to reduce the survival rates of prostate cancer patients and the survival rates remain far less-than-optimal [9][10][11][12].
Given the treatments that are currently being used to fight prostate cancer and associated complications, drug resistance especially in metastatic prostate cancers, ever-increasing costs of common treatments, and the increasing incidence of prostate cancer in both developing and developed countries, it is necessary to discover newer therapeutic approaches with higher efficacy to reduce the incidence and mortality of prostate cancer. In this regard, it is necessary to find cytotoxic plants against various cancers, especially prostate cancer, which, despite lower side effects, can replace chemotherapy and difficult treatments, and also be used for treatment-resistant cases [13][14][15][16][17]. These plants have high levels of phytochemicals that will have many therapeutic effects [18,19]. Iran especially Chaharmahal and Bakhtiari province is rich in medicinal plants and many medicinal plants are found only in these areas and are native to these regions and the climatic conditions of these areas have caused these plants to contain high concentrations of phytochemical compounds with various therapeutic effects.
Taken together, our aim was to investigate certain species of medicinal plants in Iran, especially those growing in Chaharmahal va Bakhtiari province in Iran, whose effects have not yet been immunosorbent assay (ELISA) reader.
The percentage of inhibition was measured as [1-(optical density of test/ optical density of negative control)] × 100. The IC50 val ue (the concentration with 50% cell inhibition) was calcu lated via the graph of inhibition percentage versus different extract concentrations.

Statistical analysis
The dose-response curves of the plants were fitted by means of the computer program GraphPad Prism 6.0 (GraphPad Software, USA), and IC50 was defined by regression analysis.

Results
In this study, 20 species of Iranian medicinal plants were investigated. The most of the plants were from Lamiaceae family ( Table 1).
Anticancer activity (IC50) of the 20 medicinal plants on DU-145 and PC-3 has been shown in tables 2. Extracts with IC50>300 μg/ml in MTT assay were considered inactive.

Discussion
According to the results of studies and also the report of IARC of the WHO in 2014 [6], despite extensive studies on the discovery of anticancer drugs, the incidence and prevalence of various cancers remain high and in many cases, drug resistance leads to lack of appropriate response; therefore, it is essential to conduct further studies and discover drugs with more potent effects that can serve as alternatives to chemical drugs and reduce the side effects of current medications and therapies. Phytotherapy is one of the approaches that have been used to treat various cancers in recent years [13][14][15][16][17]. In this regard, the present study was carried out to screen medicinal plant species in Iran, especially Chaharmahal and Bakhtiari province, on prostate cancer cell lines. Of the stud- So far, the preventive and anticancer effects of many medicinal plants, as well as the effects of their derivatives on different cell lines, have been studied. In a review (2015), the effects of 34 Iranian medicinal plants tested for their anticancer effects on different cell lines and with animals and human subjects were reported [23]. U. dioica, which has been investigated for its effect on prostate cancer cell lines including LNCaP and hPCPs, is one of the plants in Iran whose effects on some prostate cancer cell lines have been studied [24]. In our study, the effect of this plant studied on prostate cancer cell lines but they are used as anticancer agents according to public beliefs and Iranian traditional medicine books, or whose anti-inflammatory and antioxidant effects were confirmed in studies so that they may be used to produce more efficient and novel drugs to treat prostate cancer.

Plant material
The plants were collected

Preparation of extracts
The herbal samples were cleaned, shade dried in, pulverized to powder in a mechanical grinder and mac erated in ethanol (70%) at room temperature for 72 hours. In the next step, the hydro alcoholic extracts were concentrated by a rotary evaporator under re duced pressure. Samples were dissolved in DMSO % 0.1 (dimethyl sulfoxide, Sigma) [20][21][22][23]. Finally, extracts were diluted in RPMI 1640 at con centration of 5 mg/ml.

Cell lines and culture medium
The following cancer cell lines were used for this study: PC-3 and DU145 (prostate cancer cell lines) and HDF (Human Dermal Fibroblasts) as non-cancer cell line. Cells were obtained from National Cell Bank of Iran (Pasteur Institute, Tehran, Iran).

Antiproliferative assay
3-[4, 5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay (MTT assay) was used for evaluating cell viability. The cells (PC-3, DU145, and HDF) cells were seeded in 96-well plates and incubated at 37°C. After 24 h of incubation, when cells reached more than 80-90% confluence, the medium was removed and the cells were treated with fresh medium containing various concentrations of plant extracts to be tested (10 µg/ ml-1 mg/ml). After 48 h, the supernatant liquids were eliminated and a medium including MTT solution (0.5 mg/ml) was added to the wells which were incubated for 4 h. In the next step, supplements were eliminated, and the formazan crystals were dissolved in DMSO. The absorbance of the plates were determined at 570 nm with a reference wavelength of 630 nm in an enzyme linked on the other two cell lines of prostate cancer, i.e. PC-3 and DU-145, was investigated. A study (2014) showed that the cytotoxic effect of aqueous U. dioica extract on LNCaP cell line is mediated by apoptosis and oxidative stress [25]. Aqueous U. dioica extract results in significant inhibition of adenosine deaminase (ADA) activity in prostate tissue [26].
Medicago sativa, as one of the other plants in our study, has good anti-proliferative activity on DU-145 cell line. In the previous study, anticancer effect of M. sativa on multidrug-resistant tumor cells lines has been reported. M. sativa could induce apoptosis in these cells lines [27]. Also some of isolated compounds from M. sativa have been indicated to have antitumor activity against different cancer cell lines such as leukemia, cervix and breast cancer [28,29]. The results of present study and other studies that have examined the effect of U. dioica and M. sativa on cancers suggest them as effective medicinal plants in the pre-vention and treatment of cancers.
Thymus vulgaris, Taverniera spartea, Camellia sinensis, Ferula gummosa, Allium sativum, Curcuma longa and Zingiber officinale are other species in Iran, which are similar to the studied plants studied in the current work in terms of plant families or chemical compounds, and their anticancer effects on prostate cancer have also been investigated [30][31][32][33][34][35]. However, according to the available results, none of the plant species in Iran have yet been studied for their effects on prostate cancer cell lines (DU-145 and PC-3).
In the present study, Euphorbia szovitsii Fisch. & C.A.Mey, that is a species of the Euphorbia genus, was found to have better antitumoural effects on both prostate cancer cell lines than other plants under study. The plants from the Euphorbia genus have traditionally been used to treat inflammation and tumors [36,37]. Besides that, cytotoxic and immunosuppressive effects have

Conclusion
According

Conflicts of interest statement
The authors declare no conflict of interest.