Apoptosis Cell Death Effect of Scrophularia Variegata on Breast Cancer Cells via Mitochondrial Intrinsic Pathway

© 2015 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2015, 5(3), 443-446 doi: 10.15171/apb.2015.060 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
Breast cancer is one of the leading causes of death among women in the world.At the present, using of natural compounds such as medicinal plants in cancer therapy has aroused general because of its minimal side effect, safety and efficiency. 1,2Scrophularia variegata is an Iranian medicinal plant and which is used for various inflammatory disorders in traditional medicine.][5][6][7][8][9][10] In the present study we investigated the cytotoxic effect and induction of apoptosis of S. variegata in the MCF-7 human breast cancer cell line.

Plant material and preparation
The plant was collected from Taleqan region (Alborz province) in May 2010, in Iran.A voucher specimen was deposited in the herbarium of the Institute of Medicinal Plants (IMP).Aerial parts of the plant were dried, powdered (100 g) and macerated with a 90% ethanol solution for 3 days with three changes of the solution.The resulting extract was filtered and evaporated under vacuum into a dried powder.

Phytochemical, anti-oxidant and total flavonoid assay
We analyzed the chemical components of extract by thin layer chromatocheraphy (TLC).In addition, the antioxidant capacity of the plant extract by the DPPH (2, 2-diphenylpicrylhydrazyl) test and also total flavonoid content were estimated by aluminum chloride colorimetric assay as described previously. 3

Cell culture and cell viability assay with MTT test
The MCF-7 human breast cancer cell line and normal human fibroblast cells (L929) were prepared from the National Cell Bank of Iran (NCBI) and maintained by culturing in RPMI 1640 medium (Sigma, St Louis, USA) supplemented with 10% heat-inactivated fetal calf serum (Gibco, USA).The cell viability was assayed by MTT (3-(4, 5-dimethylthiazoyl)-2, 5-diphenyltetrazolium bromide) as previously described. 3

Cell apoptosis and cell cycle assay
Detection of apoptosis was evaluated with an Annexin V-FITC apoptosis Kit (Invitrogen, USA) according to the manufacturer's protocol.Moreover, the cell cycle distribution was measured by PI staining as previously described. 3

DNA fragmentation analysis and measurement of caspase activity
To confirm breast cancer apoptosis, we evaluated the fragmented DNA from MCF-7 cells by gel electrophoresis as previously described. 3In addition, caspase-3 and caspase-9 activity were assessed according to the manufacturer's instruction of the caspase colorimetric assay kit (R&D systems).

Statistical analysis
Data represented as mean±standard deviation.Statistical analyses were carried out by one-way analysis of variance (ANOVA) and a post-hoc Bonferroni's test to express the difference among the groups.All analyses performed using SPSS software16.Data considered statistically significant at P<0.05.

Antioxidant activity and total phenolic compounds
The results showed that the extract had the strong antioxidant and free radical scavenging capacities.These results are shown in Tables 1and 2.

Cytotoxicity effect of S. variagata extract on MCF-7 tumor cell
In this study normal human fibroblast cells (L929) were used as normal cells compared with MCF-7 human breast cancer cell.In a preliminary experiment on L929, the results indicated that extract up to 200 µg/ ml did not any significant toxicity for 48 h (Data not shown).On the other hand, a successful antitumor drug should kill cancer cells without causing excessive side effects to normal cells that this ideal situation is achievable by apoptosis induction in cancer cells.As shown Figure 1, the extract significantly (p<0.05)inhibited MCF-7 cell growth in dose and time dependent manner.So, the results indicate that the extract can induce a cytotoxicity effect in MCF-7 human breast cancer cell line.

Effect of S. variagata extract in MCF-7 apoptosis and cell cycle arrest
Apoptosis is a normal physiologic process that plays an important role in homeostasis and growth of the normal and cancer cells also dysregulation of apoptosis is usually considered as a major cancer property. 11,12n the present study, to determine whether the toxicity effect of S. variagata extract on MCF-7 cells was associated to apoptotic cell death, the apoptosis induction were measured by annexin V-FITC/PI staining.As shown in Figure 2, our findings indicated that the amount of apoptotic cells increased with increasing concentration of S. variagata extract.These results indicated that the cytotoxic effects of S. variagata extract could be mediated by the induction of apoptosis via mitochondrial intrinsic pathway in MCF-7 cells.Moreover, some studies showed that other genus of scrophularia such as S. floribunda and S. striata extract induced apoptosis in tumor cells through induction of cell cycle arrest. 3,13Our findings demonstrated that S. variagata extract can induce a G2/M phase cell cycle arrest in MCF-7 cells in a dose-dependent manner.

Effects of S. variagata extract on caspases induction and DNA fragmentation
To confirm the effects of extract on the induction of apoptosis in the breast cancer cell line, the extract was examined for the appearance of DNA ladder and induction of caspases in treated cells.Our results demonstrated that increasing of the caspase-3 and caspase-9 activity and internucleosomal DNA fragmentation was dose dependently apparent in the cells indicating that the extract can cause apoptosis in the MCF-7 cells (Figure 3 A, B).Therefore, the findings in this study showed that the Scrophularia variagata could be as an anticancer agent against breast cancer by cell growth inhibition and apoptosis induction.

Conclusion
Our in vitro study demonstrated that S. variagata has cytotoxic activity on MCF-7 breast cancer cell line.The ability of it medicinal plant to induce apoptosis through G2/M phase cell cycle arrest and an increase in caspases activity on human breast cancer cell line candidate it for further studies as a potential natural anti-cancer agent.

Figure 1 .
Figure 1.Cytotoxicity effect of S. variegata on MCF-7 tumor cell line.The results showed that the extract significantly (*P<0.05)inhibited MCF-7 cell growth in dose and time dependent manner compared with non-treated (control group) after 48 h.The results shown are representative of three independent experiments.

| 445 Figure 2 .
Figure 2. Effect of S. variegata extract on the inducing of apoptosis and cell cycle arrest.Apoptosis inducing effect of S. variegata extract on MCF-7 human breast cancer cell line evaluated by Annexin V-FITC (AV)/PI method.The results shown are representative of three independent experiments.

Table 1 .
Antioxidant capacity and total phenolic compounds of S. variagata extract