Antiproliferative and apoptotic effects of silibinin in rat prostate cancer cells.
BACKGROUND
The tremendous impact of prostate cancer (PCA) on the US male population has led to an increased attention on its prevention and on therapeutic intervention. Short-term models are needed to quickly screen the efficacy of promising agents against PCA. We have established recently several rat PCA cell lines from primary PCA in rats induced by a MNU-testosterone protocol, but their usefulness as a model for screening PCA preventive and therapeutic agents remains to be established. With the rationale that agents found effective in these cells could be promising for efficacy testing in long-term in vivo experiments, e.g., with MNU-testosterone-induced PCA in rats, the major goal of our study was to assess the antiproliferative and apoptotic efficacy in rat PCA cell lines of silibinin, a major active flavonoid component of silymarin, which consists of a group of flavonoid antioxidants occurring in milk thistle (Silybum marianum).


METHODS
Three rat PCA cell lines, namely H-7, I-8, and I-26, were treated with silibinin or silymarin, a crude silibinin-containing preparation, at various doses for varying lengths of time. Cell growth and viability studies were carried out by using hemocytometer and Trypan blue dye exclusion methods. Cell cycle distribution studies were conducted by using PI staining and flow cytometry analysis, and DNA synthesis was assessed by bromodeoxyuridine incorporation. Apoptotic cell death was assessed as DNA damage by using an enzyme-linked immunosorbent assay method and by annexin V and PI staining followed by flow cytometry analysis.


RESULTS
Silibinin resulted in a significant growth inhibition and reduction in cell viability in each cell line studied in both a dose- and a time-dependent manner. Silibinin treatment of H-7 and I-8 cells at 100 microM dose for 12 and 24 hr resulted in a G1 arrest but caused S phase arrest after a 48-hr treatment period in each cell line studied. Similar silibinin treatment of I-26 cells resulted in a slight S phase arrest at all time points studied. Consistent with these findings, silibinin showed a strong inhibition of DNA synthesis. Silibinin also induced a substantial apoptotic death in each cell line studied. Similar to silibinin, silymarin induced growth inhibition and reduced viability in a dose- and time-dependent manner.


CONCLUSIONS
This study demonstrates that silibinin as well as silymarin induce growth inhibition and apoptosis in rat PCA cells. These results form a strong rationale for PCA prevention and therapeutic intervention studies with silibinin and silymarin in animal models, such as the MNU-testosterone rat PCA model, to establish their efficacy and to further define their mechanisms of action under in vivo conditions.
Tyagi
Alpana
A
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Bhatia
Neehar
N
Condon
Mark S
MS
Bosland
Maarten C
MC
Agarwal
Chapla
C
Agarwal
Rajesh
R
D000818:Animals; D017304:Annexin A5 / Q000737:chemistry; D017209:Apoptosis / Q000187:drug effects*; D001973:Bromodeoxyuridine / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D004396:Coloring Agents / Q000737:chemistry; D004249:DNA Damage / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D008297:Male; D011419:Propidium / Q000737:chemistry; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D051381:Rats; D000077385:Silybin; D012838:Silymarin / Q000494:pharmacology*; D014343:Trypan Blue / Q000737:chemistry; D014407:Tumor Cells, Cultured
D017304:Annexin A5; D004396:Coloring Agents; D012838:Silymarin; D011419:Propidium; D000077385:Silybin; D001973:Bromodeoxyuridine; D014343:Trypan Blue