Effect of silibinin on hippo signaling pathway in breast cancer
Amir hosein Nikoukar
,1,* Nasrin motamed
,2 Inas nabhani
1. University of Tehran
2. University of Tehran
3. University of Tehran
In spite of the fact that medical and pharmacological industries have been vastly developed daily, cancer is still the second cause of death worldwide, and it is due to the genomic complexities of cancer. finding the new molecular and cellular processes promoting cancer formation and development, may lead to achieve better strategies in cancer prevention, finding new treatments with lower side effects, and improve the prognosis of patients. hippo signaling pathway is one of the many cellular processes, first discovered in drosophila melanogaster in genetic screening, has an intense role in controlling organ size that deregulate in variety of human malignancies. the core of this pathway includes a kinase cascade that negatively controls the activities of two transcription co-activators yap1/taz, that upregulate in tumorigenesis. hence targeting the components of this pathway can be useful in improving new methods of target therapy and moving further in individualized medicine. one of the major difficulties in cancer treatments is that patients suffering from side effects of conventional therapies, therefore substituent better methods for reducing these effects could be worthy. one of these methods is the traditional use of phytochemicals which have been used in medical practices with fewer side effects. silibinin, the major active constituent of silymarin, a standardized extract of the milk thistle, is a member of phytochemicals that traditionally use in hepatic diseases, and recently proven that could be useful in cancer prevention and treatment. the objective of this study was to investigate and compare the silibinin effect on cell viability of different breast cancer cell lines, and function of hippo signaling pathway under the treatment by silibinin.
breast cancer cell lines mda-mb-231, mda-mb-453, mcf-7, and t47d were cultured in rpmi1640 medium supplemented with 10% fetal bovine serum (fbs) at 37°c in 5% co2 under 90% humidity.
to evaluate the cell viability under different concentration of silibinin, mtt assay was applied. mentioned cell lines were cultured in 96-well plate (12000 cells per/well for mcf-7, 10000 cells per/well for mda-mb-453, 6000 per/well for t47d, and 4500 per /well for mda-mb-231). next day, different concentrations of silibinin (0, 50, 75, 100, 150 and 200 µm) were supplemented to the media for 24, 48 and 72 hours.
rna extraction and real-time pcr
cells cultured in t25 flasks. after reaching to the appropriate confluency, the media replaced with different concentrations of silibinin (50, 75 and 100 µm for mda-mb-231 and mda-mb-453; 75, 100 and 150 for mcf-7 and t47d, according to mtt assay results) for 48h. total rna was extracted from the treated cells, using rnx plus kit (cinnacolon). for cdna synthesis, master mix kit (pishgam biotech) was used. to assess the alternations of yap1, taz (wwtr1), survivin (birc5), and puma (bbc3) transcriptions under the silibinin treatments, the real-time pcr was performed.
according to regulatory role of hippo signaling pathway on yap1/taz protein levels, the immunocytochemistry was applied. after 48h treatment of cells with appropriate concentration of silibinin (75µm for mda-mb-231; 100µm for mcf-7 and mda-mb-453 and 150 µm for t47d according to mtt assay results) immunostaining was performed for each cell line before and after treatment by primary antibodies for yap1/taz. then, samples were visualized by florescent microscope and the protein levels of yap1/taz were evaluated by image j software.
According to mtt assay results, we observed silibinin reduced the cell viability of all cell lines in time and dose dependent manner. after that cells treated with appropriate concentrations of silibinin for 48h. the real-time pcr results showed that silibinin significantly reduced mrna levels of yap1, taz and survivin while significantly increased mrna level of puma in all cell lines. the results from icc showed that silibinin significantly reduced yap1/taz protein levels in all cell lines.
As the other studies, we observed that silibinin has inhibitory effect on breast cancer cell lines. considering the remarkable role of hippo signaling pathway in controlling organs size and its important role in cancer inhibition, and according to effective roles of silibinin in cancer prevention and treatment, in this study we evaluate the effects of silibinin on hippo signaling pathway and demonstrated that silibinin can influences the functions of hippo signaling pathway.
Hippo pathway, silibinin, breast cancer