Growth inhibitory and apoptotic effects of linarin, linarin acetate and acacetin on human prostate carcinoma cells

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This study was carried out to assess the anticancer efficacy of linarin (LN), linarin acetate (LA) and acacetin (AC), the flavonoid compounds with same flavone ring structure but different substitution, against human prostate cancer (PCA) cells. LN was isolated and purified from Chrysanthemum zawadaskii var, Latilobum (Compositae); LA was synthesized from LN; and acacetin was obtained commercially, which is abundant in Cirsium rhinoceros Nakai (Compositae). LNCaP and DU145 PCA cells were treated with LN, LA and AC in the range of 5-100 μM doses for 24-72 h, and cell growth and death was determined by cell counting. Cell cycle progression and apoptosis were analyzed by saponin/propidium iodide (PI) and annexin V/PI staining followed by flow cytometry analysis, respectively. Higher doses of LN were effective in inhibiting growth of DU145 cells whereas LA did not show any growth inhibition. Interestingly, LN showed a significant cell growth inhibition at 24 h of treatment in LNCaP cells, which decreased progressively with an increase in treatment time. Conversely, LA followed a reverse trend showing a time-dependent increase in LNCaP cell growth inhibition when compared with LN. Both these compounds were also effective in causing PCA cell death. LN caused up to 5-7-fold increase in cell death at 24 h of treatment in both cell lines that decreased with the time; however, LA enhanced cell death by 2-3-fold with the increase in treatment time. Further, we observed that LN-induced cell death was associated with apoptosis induction in both cell lines; however, LA caused a moderate increase in apoptotic cell death only in DU145 cells. The third compound, AC showed a strong and both time- as well as –dose-dependent cell growth inhibition, which was more prominent in LNCaP cells (43-77%) as compared to DU145 cells (39-49%). Similarly, AC also increased cell death, which was associated with a strong and significant increase in apoptosis (up to 5 fold in LNCaP cells and up to 3 fold in DU145 cells). In cell cycle analysis, LN and LA did not show any profound effect on cell cycle arrest in both the cell lines; only a moderate increase in G1 arrest by LA in LNCaP cells at 24-72 h of treatment, and a slight increase in S phase arrest by LA in DU145 cells at 72 h of treatment were observed. Conversely, AC showed a strong effect on cell cycle arrest in both the cell lines. In LNCaP cells, lower doses (5-50 μM) of AC showed G2-M arrest whereas 100 μM dose resulted in a strong G1 arrest following 24 h treatment. At higher treatment times (48 and 72 h), lower doses of AC showed G1 arrest whereas its higher doses (50-100 μM) caused G2-M arrest. In DU145 cells, however, AC showed an increase in G1 arrest at 5-50 μM doses, and G2-M arrest at 50-100 μM doses after 24 h of treatment; higher treatment times showed only G2-M arrest. The findings in the present study reveal the structural determinants in anticancer efficacy of these three novel flavonoids against PCA cells.