Study of the Effect of Silymarin on Viability of Breast Cancer Cell Lines

Background: Breast cancer is the most prevalent cancer and results in 14% of cancer-related deaths among women worldwide. The aim of this study is to investigate the anticancer effects of Silymarin on two breast cancer cell lines (BT-474, SK-BR-3). Methods and Material: Two breast cancer cell lines—SK-BR-3 and BT-474—were incubated for 24 hours in standard conditions before adding 100, 200, 400, 800, 1600 μM Silymarin to each well. Alamar blue was then added to the wells after 24, 48 and 72 hours of incubation and cell viability was determined using fluorescence reader to detect the optical density. Results were analyzed using generalized estimating equations (GEE) method in STATA 12.0. Results: we demonstrated the Silybum marianum inhibition of two-cell lines SK-BR-3 and BT-474 growth at different concentrations after 24, 48 and 72 hours. Silymarin increased cell death in both cell lines. Conclusion: Silymarin can be combined with other anti-neoplastic agents to obtain better results. Corresponding author. S. Hajighasemlou et al.


Introduction
Silybum marianum (milk thistle) is a plant of the Asteraceae family that grows naturally in some parts of Europe, Asia (including Iran) and the United States [1].Silymarin is a complex mixture of polyphenolic molecules, including seven closely related flavonolignans (silybin A, silybin B, isosilybin A, isosilybin B, silychristin, isosilychristin, silydianin) and one flavonoid (taxifolin); the active ingredient is Silibinin which has long been used in traditional medicine and shows antioxidant, anti-inflammatory and anti-cancer properties and can induce apoptosis in some cells [2] [3].Anti-neoplastic properties of Silymarin have been demonstrated in cancers of prostate [4]- [6], Ovaries, lung, skin, bladder and breast [7]- [11].
Although the exact mechanisms involved in antineoplastic effects of silymarin in breast cancer have not been identified, possible underlying explanations include induction of G1 arrest and apoptosis through inhibiting cyclin-dependent kinases activity and epidermal growth factor receptor signaling, and increasing Cip1/p21 and p27 [9] [12]- [16].
Breast cancer is a major health problem more commonly seen in the developed countries.Breast cancer is the leading cause of death in women 40 -59 years and more than a million new cases are detected annually [17] [18].In Iran breast cancer is the commonest cancer among women comprising 21.4% of all cancers among females.Studies indicate that breast cancer presents about one decade earlier in Iranian women than in developed countries [19]- [21].
In present study, we examine the in vitro effect of different Silymarin concentrations on two breast cancer cell lines: SK-BR-3 and BT-474 by determining cell viability after 24, 48 and 72 hours of incubation with alamar blue using fluorescent reader.

Materials and Methods
Silymarin powder (Sigma) solution in Dimethyl Sulfoxide (DMSO, Sigma) was prepared as stock at the concentration of 30,000 µL/mL and kept at −20˚C.During the test required concentrations using phosphate buffered saline (PBS) were prepared from this stock solution.Cell culture medium (Gibco®) consisted of RPMI1640 and DMEM, trypsin, antibiotics, fresh bovine serum (FBS) and anti-mycoplasma.Alamar blue fluorescence reagent from Invitrogen and cell lines SK-BR-3 and BT-474 were purchased from Cell Bank of Iran, Pasteur Institute.Cells were cultured at standard conditions (temperature of 37˚C, humidity of 95% and 5%CO 2 gas pressure) and the culture was replaced at 48 and 72-hour intervals.
A suspension of 25,000 cells were added to each well and incubated for 24 hours at 37˚C to stick to the bottom of the wells.10 wells were assigned for each concentration (100, 200, and 400, 800 and 1600 mM) and 10 wells without Silymarin as our controls.The DMSO concentration in the controls wells was adjusted to be the same as the test wells.
After incubation the wells for 24, 48 and 72 hours, 25 µl Alamar blue was added to each well and the plates were incubated for an additional 3 hours.Absorbance was measured at 530 and 590 nm using a fluorescence reader.

Results
Results of the effect of Silybum marianum were evaluated on two cell lines BT-474 and SK-BR-3 are summarized in the Figure 1 and Figure 2.
Time trends of cells were examined using generalized estimating equations (GEE) that account for correlation between samples.For BT-474 cell line we found significant difference between samples of different incubation periods (P-value < 0.001).We then compared the control group with different silymarin concentrations of different incubation periods (one to three days).Table 1 shows the significance difference between the control and silymarin groups.This difference remains significant but declines after Day 1.For SK-BR-3 cell line we also found a significant interaction between incubation period (days) and different concentrations, which suggests a varying degree of silymarin effect at the same concentration but in different incubation periods; we could not however detect any pattern for such decline.

Discussion
Anti-growth and anti-tumor effects of Silybum marianum have been noted in many cancers over recent years.This study was the first to employ Alamar blue in order to detect cell viability which is more sensitive than the conventional MTT assay [22].
In the present study, cell viability was significantly reduced in comparison to the controls.This effect increased over the time course from 24 -72 hours, and also from lower to higher concentrations of Silymarin from 0 -1600 µM.Our data are in consistent with that of Gharagozloo and colleagues showed that Silybum marianum has an inhibitory effect on HepG2 cell line growth [23].It would be compatible with the research by Singh who has studied the effect of Silybum marianum on human endothelial cell line and has showed that this drug is able to inhibit cell proliferation [24], also compatible with the research by Li and colleagues that examined the effect of Silybum marianum on cell Anip973 line (lung cancer), and showed that Silybum marianum inhibited cell proliferation and activates apoptosis via the mitochondrial pathway [25].Sigh and colleagues in other study, the effect of Silybum marianum on Hairless SKH-1 mice, demonstrated that it has a strong protective effect against photocarcinogen and inhibitory effects on inflammatory responses and angiogenesis [26].Similarly, Rajamanickam and colleagues examined the effect of Silybum marianum on colorectal cancer in a mouse model of APC (min/+) and showed that it had anticancer effects in this model of cancer that is consistent with the findings from this study [27].The results would be compatible with the results of the study by Kim and colleagues in 2011, showed that Silymarin could reduce the ligand-induced EGFR and metalloproteinase 9 (MMP-9) in both cell lines SK-BR-3 and BT-474 [28].Our results are compatible with research by Provinciali that showed antitumor effects of the silybin-phosphatidylcholine complex (IdB 1016) on the development of mammary tumors in HER-2/neu transgenic mice [29].
Here we suggest that a similar mechanism is involved as has been proposed for silymarin effect on prostate cancer: G1 phase cell cycle arrest most probably through inhibition of cyclin-dependent kinases (CDK) activity and epidermal growth factor receptor (erbB2) signaling [9] [12]- [14].
As showed above in vitro and in vivo studies and the results of the present study, all confirmed antioxidant and anticancer properties of these drugs.The highest concentration of Silymarin used in this study was the 1600 μM; higher doses are suggested to achieve the optimum dose.The highest level of cell apoptosis observed in the present study at 72-hour incubation proposed that long-term incubation of cells with the drug may increase efficiency of its action on the cancer cells.Both cell lines studied here were Her2 positive, a similar study on Her2 negative cell lines would be valuable.

Conclusion
We demonstrated the Silybum marianum inhibition of two cell lines SK-BR-3 and BT-474 growth at different concentrations, and increased cell death in both cell lines.Silymarin can be combined with other anti-neoplastic agents to obtain better results.

Figure 1 .
Figure 1.Silymarin effect on the BT-474 cell line after 24, 48 and 72 hours of incubation: Cell viability was significantly reduced compared to control for all concentrations at 24 and 48 hours and for concentrations of ≥400 µM at 72 hours.

Figure 2 .
Figure 2. The effect of Silymarin on the SK-BR-3 cell line after 24, 48 and 72 hours: Cell viability differed significantly at all concentrations only after 72 hours; the difference was not significant at any concentration after 24 or 48 hours of incubation.

Table 1 .
Comparisons of different concentrations with control group.