J Breast Cancer.  2011 Mar;14(1):8-13. 10.4048/jbc.2011.14.1.8.

Silibinin Enhances Ultraviolet B-Induced Apoptosis in MCF-7 Human Breast Cancer Cells

• Noh EM
• Yi MS
• Youn HJ
• Lee BK
• Lee YR
• Han JH
• Yu HN
• Kim JS
• Jung SH

• KMID: 2175636
• DOI: http://doi.org/10.4048/jbc.2011.14.1.8

Abstract

PURPOSE
Chemotherapies for breast cancer generally have strong cellular cytotoxicity and severe side effects. Thus, significant emphasis has been placed on combinations of naturally occurring chemopreventive agents. Silibinin is a major bioactive flavonolignan extracted from milk thistle with chemopreventive activity in various organs including the skin, prostate, and breast. However, the mechanism underlying the inhibitory action of silibinin in breast cancer has not been completely elucidated. Therefore, we investigated the effect of silibinin in MCF-7 human breast cancer cells and determined whether silibinin enhances ultraviolet (UV) B-induced apoptosis.
METHODS
The effects of silibinin on MCF-7 cell viability were determined using the MTT assay. The effect of silibinin on PARP cleavage, as the hallmark of apoptotic cell death, and p53 protein expression in MCF-7 cells was analyzed using Western blot. The effect of silibinin on UVB-induced apoptosis in MCF-7 cells was analyzed by flow cytometry.
RESULTS
A dose- and time-dependent reduction in viability was observed in MCF-7 cells treated with silibinin. Silibinin strongly induced apoptotic cell death in MCF-7 cells, and induction of apoptosis was associated with increased p53 expression. Moreover, silibinin enhanced UVB-induced apoptosis in MCF-7 cells.
CONCLUSION
Silibinin induced a loss of cell viability and apoptotic cell death in MCF-7 cells. Furthermore, the combination of silibinin and UVB resulted in an additive effect on apoptosis in MCF-7 cells. These results suggest that silibinin might be an important supplemental agent for treating patients with breast cancer.

Keyword

Apoptosis; MCF-7; Silibinin; p53; Ultraviolet B

MeSH Terms

Apoptosis
Blotting, Western
Breast
Breast Neoplasms
Cell Death
Cell Survival
Humans
MCF-7 Cells
Milk Thistle
Prostate
Silymarin
Skin
Silymarin

Figure

• 

  Figure 1 Structure of silibinin.

• 

  Figure 2 Effects of silibinin on MCF-7 cell viability. (A) Cells were cultured in 96-well plates until 90% confluence and 200 µM silibinin was then added for 24, 36, 48, and 72 hr. The MTT assay was used to detect cell viability. The optical density (O.D.) value of the control was regarded as 100%. Data points are the means±SEs of more than three experiments (p<0.005). (B) Cells were cultured in 96-well plates until 90% confluence and various concentrations of silibinin (0-200 µM) were added for 72 hr. The MTT assay was used to detect cell viability. The O.D. value of the control was regarded as 100%. Data points are the means±SEs of more than three experiments (p<0.005).

• 

  Figure 3 Effect of silibinin on PARP cleavage in MCF-7 cells. MCF-7 cells (5×106 cells) were treated with various concentrations of silibinin (0-200 µM) for 72 hr, and whole cell extracts were used for Western blot analysis of PARP. β-actin was used as the loading control.

• 

  Figure 4 Effect of silibinin on p53 and PTEN expression in MCF-7 cells. MCF-7 cells (5×106 cells) were treated with various concentrations of silibinin (0-200 µM) for 72 hr. Cells were lysed with lysis buffer and the amounts of p53/PTEN and β-actin as a loading control were measured by Western blot (A). The relative density of the electrophoretic band was obtained with a LAS-1000 analyzer (Fujifilm, Japan) (B). Data are means±SEs of four separate experiments (p<0.005).

• 

  Figure 5 Effect of silibinin and ultraviolet (UV) B on MCF-7 cell viability. MCF-7 cells were treated with 200 µM silibinin and UVB alone or in combination for 72 hr. The MTT assay was used to detect cell viability. The optical density value of the control was regarded as 100%. Data points are the means±SEs of more than three experiments. *p<0.001 (Student's t-test).

• 

  Figure 6 Effect of silibinin on ultraviolet (UV) B-induced apoptosis in MCF-7 cells. MCF-7 cells were stimulated with UVB (25 mJ/cm2) and silibinin 200 µM for 16 hr. The cells were stained with FITC-conjugated annexin V and propidium iodide and then analyzed by flow cytometry.

Reference

1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin. 2009. 59:225–249.

2. Lee SO, Jeong YJ, Im HG, Kim CH, Chang YC, Lee IS. Silibinin suppresses PMA-induced MMP-9 expression by blocking the AP-1 activation via MAPK signaling pathways in MCF-7 human breast carcinoma cells. Biochem Biophys Res Commun. 2007. 354:165–171.

3. Tyagi AK, Agarwal C, Chan DC, Agarwal R. Synergistic anti-cancer effects of silibinin with conventional cytotoxic agents doxorubicin, cisplatin and carboplatin against human breast carcinoma MCF-7 and MDA-MB 468 cells. Oncol Rep. 2004. 11:493–499.

4. Li L, Gao Y, Zhang L, Zeng J, He D, Sun Y. Silibinin inhibits cell growth and induces apoptosis by caspase activation, down-regulating survivin and blocking EGFR-ERK activation in renal cell carcinoma. Cancer Lett. 2008. 272:61–69.

5. Kaur M, Agarwal R. Silymarin and epithelial cancer chemoprevention: how close we are to bedside? Toxicol Appl Pharmacol. 2007. 224:350–359.

6. Singh RP, Agarwal R. Prostate cancer chemoprevention by silibinin: bench to bedside. Mol Carcinog. 2006. 45:436–442.

7. Wellington K, Jarvis B. Silymarin: a review of its clinical properties in the management of hepatic disorders. BioDrugs. 2001. 15:465–489.

8. Singh RP, Dhanalakshmi S, Tyagi AK, Chan DC, Agarwal C, Agarwal R. Dietary feeding of silibinin inhibits advance human prostate carcinoma growth in athymic nude mice and increases plasma insulin-like growth factor-binding protein-3 levels. Cancer Res. 2002. 62:3063–3069.

9. Katiyar SK, Korman NJ, Mukhtar H, Agarwal R. Protective effects of silymarin against photocarcinogenesis in a mouse skin model. J Natl Cancer Inst. 1997. 89:556–566.

10. Kohno H, Tanaka T, Kawabata K, Hirose Y, Sugie S, Tsuda H, et al. Silymarin, a naturally occurring polyphenolic antioxidant flavonoid, inhibits azoxymethane-induced colon carcinogenesis in male F344 rats. Int J Cancer. 2002. 101:461–468.

11. Flaig T, Agarwal R, Su L, Harrison GS, Gustafson DL, Glode LM. A phase I study of silibinin in hormone refractory prostate cancer. 2005. 23:In : 2005 ASCO Annual Meeting; Abstract #4698.

12. Kim S, Choi JH, Lim HI, Lee SK, Kim WW, Kim JS, et al. Silibinin prevents TPA-induced MMP-9 expression and VEGF secretion by inactivation of the Raf/MEK/ERK pathway in MCF-7 human breast cancer cells. Phytomedicine. 2009. 16:573–580.

13. Yarosh DB, Boumakis S, Brown AB, Canning MT, Galvin JW, Both DM, et al. Measurement of UVB-induced DNA damage and its consequences in models of immunosuppression. Methods. 2002. 28:55–62.

14. Kulms D, Schwarz T. Molecular mechanisms of UV-induced apoptosis. Photodermatol Photoimmunol Photomed. 2000. 16:195–201.

15. John EM, Schwartz GG, Dreon DM, Koo J. Vitamin D and breast cancer risk: the NHANES I Epidemiologic follow-up study, 1971-1975 to 1992. National Health and Nutrition Examination Survey. Cancer Epidemiol Biomarkers Prev. 1999. 8:399–406.

16. Ferguson HA, Marietta PM, Van Den Berg CL. UV-induced apoptosis is mediated independent of caspase-9 in MCF-7 cells: a model for cytochrome c resistance. J Biol Chem. 2003. 278:45793–45800.

17. Dhanalakshmi S, Mallikarjuna GU, Singh RP, Agarwal R. Dual efficacy of silibinin in protecting or enhancing ultraviolet B radiation-caused apoptosis in HaCaT human immortalized keratinocytes. Carcinogenesis. 2004. 25:99–106.

18. Vogel G, Trost W, Braatz R, Odenthal KP, Brüsewitz G, Antweiler H, et al. Pharmacodynamics, site and mechanism of action of silymarin, the antihepatoxic principle from Silybum mar. (L) Gaertn. 1. Acute toxicology or tolerance, general and specific (liver-) pharmacology. Arzneimittelforschung. 1975. 25:82–89.

19. Mereish KA, Bunner DL, Ragland DR, Creasia DA. Protection against microcystin-LR-induced hepatotoxicity by Silymarin: biochemistry, histopathology, and lethality. Pharm Res. 1991. 8:273–277.

20. Katiyar SK, Roy AM, Baliga MS. Silymarin induces apoptosis primarily through a p53-dependent pathway involving Bcl-2/Bax, cytochrome c release, and caspase activation. Mol Cancer Ther. 2005. 4:207–216.

21. Bhatia N, Zhao J, Wolf DM, Agarwal R. Inhibition of human carcinoma cell growth and DNA synthesis by silibinin, an active constituent of milk thistle: comparison with silymarin. Cancer Lett. 1999. 147:77–84.

22. Ellis RE, Yuan JY, Horvitz HR. Mechanisms and functions of cell death. Annu Rev Cell Biol. 1991. 7:663–698.

23. Raff MC, Barres BA, Burne JF, Coles HS, Ishizaki Y, Jacobson MD. Programmed cell death and the control of cell survival: lessons from the nervous system. Science. 1993. 262:695–700.

24. Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis. 2000. 21:485–495.

25. Finlay CA, Hinds PW, Levine AJ. The p53 proto-oncogene can act as a suppressor of transformation. Cell. 1989. 57:1083–1093.

26. Dhanalakshmi S, Agarwal C, Singh RP, Agarwal R. Silibinin up-regulates DNA-protein kinase-dependent p53 activation to enhance UVB-induced apoptosis in mouse epithelial JB6 cells. J Biol Chem. 2005. 280:20375–20383.

27. Gu M, Dhanalakshmi S, Mohan S, Singh RP, Agarwal R. Silibinin inhibits ultraviolet B radiation-induced mitogenic and survival signaling, and associated biological responses in SKH-1 mouse skin. Carcinogenesis. 2005. 26:1404–1413.

28. Tonks NK, Myers MP. Structural assets of a tumor suppressor. Science. 1999. 286:2096–2097.

29. Mohan S, Dhanalakshmi S, Mallikarjuna GU, Singh RP, Agarwal R. Silibinin modulates UVB-induced apoptosis via mitochondrial proteins, caspases activation, and mitogen-activated protein kinase signaling in human epidermoid carcinoma A431 cells. Biochem Biophys Res Commun. 2004. 320:183–189.