Cytotoxic Effect of Saffron Stigma Aqueous Extract on Human Prostate Cancer and Mouse Fibroblast Cell Lines Hassan Ahmadnia1*, Jalil Tavakkol Afshari2, Jamshid Tabeshpour3, Mehdi Younesi Rostami4, Ehsan Mansourian5, Alireza Akhavan Rezayat5, Azam Brook2 Purpose: Several lines of experimental evidence have shown that saffron has anticarcinogenic effects. This study aimed at evaluating the possible anticancer effect of saffron stigma aqueous extract on human prostate cancer (PC3) and mouse fibroblast cells (L929) as non-cancerous control cells. Materials and Methods: Saffron stigma aqueous extract at concentrations of 100, 200, 400, 600, 800, 1600 and 3200 μg/mL were prepared. PC3 and L929 cells were incubated with different concentrations of saffron extracts in different time intervals (24, 48, 72, 96 and 144 hours). MTT assay was used for each cell line to investigate the cytotoxic effect of saffron. Morphological alterations were also observed under light inverted microscope. Results: In fibroblast cell line after 24 hours, Saffron extract did not affect significantly the normal cells and they were intact in morphologic view. After 96 hours in the cells with highest concentration (1600 μg/mL), cell death and cellular form changes as well as severe granulation was observed. In prostate cell line after 24 hours, the only changes were observed in cells with the concentration of 1600 μg/mL. The cells were granulated and the form of the cells were spherule. After 72 hours, in group with the concentration of 1600 μg/mL, severe granulation was observed and the cell count decreased and some cells were dead. Conclusion: Saffron aqueous extract has an in vitro inhibitory effect on the proliferation of human prostate cell and mouse L929 cells which is dose-dependent. Keywords: Saffron aqueous extract; L929 cells; PC3 cells; anticancer effect; MTT assay; cytotoxic effect INTRODUCTION The most prevalent visceral malignant neoplasm in men, prostate cancer is responsible for one third of all such cancers.(1) According to American Cancer Society, 220,000 Americans have been diagnosed with prostate cancer, annually.(2) Different kinds of treat- ments have been introduced for prostate cancer such as cabazitaxel, docetaxel and mitoxantrone.(3) But, unfor- tunately, a variety of adverse effects following synthet- ic medicines are observed in the clinical settings.(3) To minimize such adverse effects, many researchers have been persuaded to search for herbal therapies with the least adverse effects such as Adiantum venusutum(4), Abelmoschus moschatus(5) and Aspidosperma tomen- tosum.(6) Herbal medicine is a new alternative therapy in cancer patients.(7) Consumption of certain botanicals could be associated with reduced cancer incidence. One of these botanical agents is Crocus sativus L., commonly known as saffron. It is a stemless herb of the Iridaceae family that is widely cultivated in Iran and other countries. In the world, 205 tons of saffron are produced every year, 1Urology, Endoscopic & Minimally Invasive Surgery Research Center, Ghaem Hospital, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 2Immunology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran 3Faculty of Pharmacy, Damghan Bransh, Islamic Azad University, Damghan, Iran. 4Department of Urology, Imam Hospital, Mazandaran University of Medical Sciences, Sari, Iran. 5Department of Urology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. *Correspondence: Urology, Endoscopic & Minimally Invasive Surgery Research Center, Ghaem Hospital, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. E-mail: ahmadniah@mums.ac.ir Received June 2020 & Accepted November 2020 while Iran has an important role in this production as the grand producer (80 percent of total). Khorasan prov- ince, sited at north east of Iran, accounts for 137 tons of the above-mentioned totals.(8-10) Mostly used as a spice, flavoring agent, food coloring and herbal medicine, saffron is produced from dried red stigma with a small portion of the yellowish stamina attached.(9) The stigmas of saffron has been demonstrat- ed to have several components including carotenoids, crocin, crocetin, picrocrocin, anthocyanin, lycopene, monoterpene aldehydes and safranal as well as proteins, sugars, vitamins, flavonoids, amino acids and minerals (Figure 1).(11-15) Anticancer(9), anti-inflammatory(16), an- tidepressive(17), antioxidant(18) and antibacterial(19) activ- ities of saffron have been established by many research- ers as its pharmacological effects. Since 1990, research has been focused on anti-carcinogenic activity of saf- fron.(7, 8) Saffron and its main components have shown anti-tumor and anti-carcinogenic activities both in vitro and in vivo.(7-9, 20) As far as we know, the possible effects of saffron on prostate cancer has not been evaluated and reported. UROLOGICAL ONCOLOGY Urology Journal/Vol 18 No.6/ November-December 2021/ pp. 633-638. [DOI: 10.22037/uj.v16i7.6331] Therefore, this study aimed to evaluate the in vitro cytotoxic effects of saffron stigma aqueous extract on prostate cell line and non-neoplastic fibroblast cells of mouse as a normal cell line. MATERIALS AND METHODS Preparation of Saffron Extract Saffron harvested from saffron farms of a city of Kho- rasan province (Ghaen), was used in this experiment. 15 g of ground petal stigma was mixed with 400 mL of distilled water in a Soxhlet extractor for 18 h, to prepare saffron aqueous extract. Then, to be sterilized, it was concentrated to 100 mL with a rotatory evaporator in low pressure and filtered through a 0.2-mm filter. The obtained solution was kept at 4°C to 8°C. Different con- centrations of saffron extract (100, 200, 400, 600, 800, 1600 and 3200 μg/mL) were prepared immediately and refrigerated before the experiments. Urological Oncology 634 Figure 1. Molecular structures of the most important carotenoid secondary metabolites of saffron. Figure 2. After 48 hours of incubation with the concentration of 400 μg/mL, PC3 cells were granulated and spheroid (left picture) in comparison with the cells which were not incubated with saffron extract, as a control group (right picture). The absorbance of the dye was read at Ex: 488 nm, Em: 585 nm. Cytotoxic Effect of Saffron Stigma Aqueous Extract-Ahmadnia et al. Morphologic Observation of Cell Lines Human prostate carcinoma cells (PC3) and mouse fi- broblast cell lines (L929) were bought from the Nation- al Cell Bank of Iran (Pasture Institute, Tehran, Iran). Both of the cell lines were cultured and passaged. Tryp- an blue test was used to determine cell viability. Equal parts of 0.4% trypan blue dye to the cell suspension were added to obtain a 1 to 2 dilution and weremixed by pipetting up and down. The incubation time was less than three minutes at room temperature. The percentage of viable cells were calculated by dividing the number of viable cells by the number of total cells and multiply- ing by 100 or % viable cells. Six-well plates were used for both cell lines. In each well, 5 × 105 PC3 cells or 2 × 105 L929 cells were cultivated in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich, St Louis, Missouri, USA) with 10% fetal calf serum (Gibco, Paisley, UK). The media were supplemented with 100 IU/mL strep- tomycin and 100 IU/mL penicillin (Jaberebn-e-Hayan, Cytotoxic Effect of Saffron Stigma Aqueous Extract-Ahmadnia et al. Figure 3. Viability percent for PC3 and L929 cells after 24, 48, 72, 96 and 144 h incubation with different concentrations of saffron extract. Figure 4. After 72 h incubation, the PC3cells incubated with 400 μg/mL of saffron extract were granulated and spheroid with reduction in cell counts (left picture) in comparison with the cells which were not incubated with saffron extract as a control group (right picture). The absorbance of the dye was read at Ex: 488 nm, Em: 585 nm. Vol 18 No 6 November-December 2021 635 Tehran, Iran). The cells were incubated at 37°C in a hu- midified 5% CO 2 atmosphere for 24 h. Then, the cells were exposed to saffron extract as follows: The media (2 mL capacity) were replaced with fresh media and plates were incubated with different concentrations of saffron extract (100, 200, 400, 600, 800, 1600 and 3200 μg/ mL) in the situation aforementioned for 24, 48, 72, 96 and 144 h, and the cells were observed by light inverted microscope for morphological alterations. Each extract concentration was observed 3 times to check their reli- ability. The viability of the cells was determined to be higher than 95% during the experiment. Quantitative Assessment Surviving cells (percent) compared to the controls = (optical density of treated cells in each well × 100)/ (mean optical density of control ) The absorbance of trypan blue dye was read using fluo- rescence microplate reader (Ex: 488 nm, Em: 585 nm). Statistical Analysis Data are presented as mean ± SD. Statistical analyses were performed in Graph pad Prism software version 8.0. RESULTS Effect of saffron on L929 cell viability and morphological alterations Incubation of L929 cells with different concentrations of saffron extract for 24 h, did not significantly affect the cells and they were intact in morphological view. In addition, there were no changes in number, cytoplasm, and nucleus of the cells. Incubation of the cells with 1600 μg/mL of saffron extract for 48 and 72 hours, showed a slight granulation of the cells while no chang- es in number, cytoplasm, and nucleus of the cells were observed. After 96 and 144 hours, at the concentration of 800 μg/mL, the cells were larger and granulated while at the concentration of 1600 μg/mL, cell death, cellular form changes and severe granulation were no- ticed (Figure 2). Effect of saffron on PC3 Cell viability and morphological alterations PC3 cells were granulated and the form of the cells were spherule when they were exposed to 1600 μg/mL of saffron extract for 24 hours. After 48 and 72 hours of treatment (400, 600 and 800 μg/mL), the cells were granulated and spheroid, while at the concentration of 1600 μg/mL, severe granulation was observed and the cells count decreased and some cells were dead (Fig- ures 2,3,4). The half maximal inhibitory concentration (IC50) is a measure of the potency of a substance in inhibiting a specific biological or biochemical function which was calculated for PC3 cells to be 400 to 800 μg/mL. Granulated and dead cells were noticed at the concen- tration of 1600 μg/mL, after 144 h of treatment (Figure 5). DISCUSSION Prostate cancer is a commonly diagnosed cancer in men, and dietary chemoprevention was considered due Urological Oncology 636 Figure 5. After 144 h, the PC3 cells incubated with 400 μg/mL of saffron extract were granulated and spheroid with reduction in cell counts, presenting the cellular death (left picture) in comparison with the cells which were not incubated with saffron extract as control group (right picture). The absorbance of the dye was read at Ex: 488 nm, Em: 585 nm. Cytotoxic Effect of Saffron Stigma Aqueous Extract-Ahmadnia et al. to its slow growth rate and its long incubation period. Different herbal extracts has shown noticeable benefits such as pomegranate, green tea in chemoprevention of prostate cancer.(21,22) Several effects were recognized for saffron such as anti-carcinogenic, decreasing blood pressure, and con- trolling tonic-clonic and absence seizures.(10) Saffron can cure coronary heart disease and hepatitis, and pro- mote immunity.(23) There are several reports demon- strating the anti-carcinogenic effects of saffron extract, either inhibition of new tumors formation or shrinkage of existing tumors. The anticancer activity of saffron against a wide spectrum of tumors, such as leukemia, transitional cell carcinoma, ovarian carcinoma, colon adenocarcinoma, rhabdomyosarcoma, papilloma, squa- mous cell carcinoma, and soft tissue sarcoma have been demonstrated.(7,9,10,15,20,23-26) The inhibitory effects of Saf- fron aqueous extract on the growth of both TCC 5637 and normal L929 cell lines have been reported(10) as we observed in our research. Moreover, increasing saffron concentrations results in a reduction in the cell survival percent of healthy L929 cells. Higher saffron concen- trations seem to reduce the cell survival rate of healthy cells up to 50% in longer incubation time. Salomi et al. reported anti-promoting and non-muta- genic activity of saffron extract.(7) Protective effect of crocin against adverse consequence of hepatocarcino- genic materials has been demonstrated.(15) In addition, different in vitro studies have reported the protective effects of crocin such as inhibition of intracellular nu- cleic acid synthesis(11) and inhibition of proliferation of promyelocytic leukemia cells in a dose-dependent man- ner.(12, 20) Saffron extract in combination of vitamin E has been shown to protect rats against cisplatin toxicity. (15) A significant inhibitory effect of ethanolic extract of saffron on the colony formation and intracellular DNA and RNA synthesis of Hela cells (cervix epitheloid car- cinoma cells) was reported by Abdullaev and Frenkel. (26) Growth delay of papilloma, decreased incidence of squamous cell carcinoma and soft tissue sarcoma in mice treated by saffron, have been reported.(20) Although saffron extract has well-documented antitu- mor effects, the cellular mechanisms responsible for these effects remain ill-defined. Different hypotheses have been suggested for the antitumor mechanism of saffron such as the inhibitory effect on DNA and RNA synthesis without any effect on protein synthesis and the inhibitory effect on free radicals which is thought to be due to carotenoid component of saffron.(10,20) It is suggested that saffron (dimethyl-crocetin) disrupts DNA-protein interactions e.g. topoisomerases II, which is essential for cellular DNA synthesis.(20) The anti-tu- mor components of saffron were reviewed by some re- searchers. Crocin isolated from saffron inhibits PC12 (rat’s pheochromocytoma cell line) cell growth with increased synthesis of glutathione. The mechanism be- hind its antigrowth effect could be the possible decrease in tumor necrosis factor-alpha levels.(27) CONCLUSIONS This research demonstrates in vitro cytotoxic effects of saffron on human prostate cell lines. 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