Silibinin’s effect on total EGFR, Erb-b2, and Erb-b3 expression in human colon cancer

Abstract

14124 Purpose: Flavonoid, silibinin, has been shown to have strong chemotherapeutic activity in prostate, breast, bladder, skin, ovarian, and colon cancer. It has no documented LD50 and has been proven safe in multiple animal models and human trials. Cell cycle arrest and inhibition of protein levels of various cyclins are commonly reported in silibinin’s mechanism of action. We investigated silibinin’s activity on upstream cell-signaling targets, EGFR, Erb-b2, Erb-b3, Akt, Erk1/2, and mTOR, using three human colon cancer cells lines: Fet, Geo, HCT 116. Methods: The MTT cell-viability assay and FACS analysis were performed after silibinin treatment to substantiate its effect on proliferation and cell-cycle. Western blots for EGFR, Erb-b2, Erb-b3, Erk1/2, Akt, and mTOR were completed and quantified with densitometry. Standardization of protein loading was confirmed with cytoskeletal and house-keeping proteins, B-actin and GAPDH. Means were contrasted using analysis of variance with Dunnet’s correction for multiple testing. All statistical testing was two-sided with a significance level of 5%. Results: The MTT assay revealed a dose-dependent inhibitory effect. Treatment of FET and GEO at 75 micrograms/ml resulted in 50% inhibition of cell-viability (IC-50) at 72 hrs. An IC50 dose of 40 ug/ml was established for HCT116. FACS analysis demonstrated statistically significant cell-cycle arrest in all cell lines. G2-M phase arrests in Fet and Geo cell lines (p<0.001) and a G1 arrest in HCT116 (p=0.005) were noted. Western blots showed decreased protein level expressions of total and phospho-EGFR, Erb-b2, Erb-b3, Akt, Erk1/2, and mTOR. Conclusions: Inhibition of protein expression levels of the Erb-b family of receptor tyrosine kinases plays a role in the mechanism of silibinin’s action. Strong inhibition of protein levels of total EGFR, Erb-b2, and Erb-b3 was noted as well as downstream decreases in total Erk1/2, Akt, and mTOR. Given silibinin’s inhibition of cancer proliferation and its safety profile, it is a promising compound requiring further mechanistic study, and applications in cancer therapy should be pursued given its effects on the Erb-b class of receptor tyrosine kinases and intermediary proteins, Erk1/2, Akt, and mTOR. No significant financial relationships to disclose.