Emodin synergistically enhances docetaxel induced apoptosis in androgen-responsive prostate cancer cells Free

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Docetaxel (Taxotere) remains the effective drug for the treatment of various malignancies, including prostate cancer. However, decline in its potency due to emergence of drug resistant tumor cells and overwhelming toxic effects associated with docetaxel treatment warrants investigation for the development of novel combination treatment at reduced dosing without reducing potency. Emodin (6-Methyl-1,3,8-trihydroxyanthraquinone) is an active ingredient from the root of the plant: Rheum palmatum (Rhuharb), with potential antitumor activity. We determined, efficacy of Emodin alone or in combination with Docetaxel on two human prostate cancer cells, LNCaP and PC-3ML. Cytotoxicity of Taxotere (TX), Emodin (E) alone or their combination (E+TX) in LNCaP or PC-3ML cells was evaluated by colorimetric (MTT) assay. Cell-cycle phase fractionation was done by flow cytometry. Apoptotic activity was determined by ELISA. The relative protein levels of PARP, caspase-3, survivin, p27, Total-AKT and phosphorylated AKT were determined by western blotting. Student’s t-test was used for statistical validation of observations. Interaction of combined drug treatment versus single drug treatment was characterized by isoeffect calculation using the Calcusyn program. Both E and TX caused dose dependent cell kill in PC-3ML and LNCaP cells (IC50 for E = 58 μM for PC-3ML and 50 μM for LNCaP cells; IC50 for TX = 35 nM for PC-3ML and 29 nM for LNCaP cells). LNCaP cells were more sensitive to both E and TX. Combined application of E (50 μM) and TX (25 ng) for 48h significantly increased cytotoxicity in LNCaP cells, but not in PC-3ML cells. Isobologram analysis revealed a synergistic interaction between E and TX on LNCaP cell kill (Combination Index value; CI=0.5∼“Synergism”), but an antagonistic interaction on PC-3ML cell kill (CI=1.6∼“Antagonism”). The synergistic enhancement in cell kill in E+TX-treated LNCaP cells was associated with increased apoptosis (3.7 folds), likely due to a significant decrease (p<0.001) in the levels of proteins involved in cell survival and due to inhibition of apoptosis: P-Akt and survivin, respectively. Furthermore, increased apoptosis in LNCaP cells exposed to E+TX was associated with increases in caspase-3 activity (10.3 folds) and PARP cleavage (2 folds). Cell-cycle phase fractionation revealed treatment with E alone in LNCaP cells causes a significant G0-G1 arrest, TX caused G2-M arrest and combined application with E+TX caused further increase in G2-M arrest. Increased G0-G1 cell cycle arrest in LNCaP cell treated with EM is reflected in increased protein levels of p27 (1.5 folds). These results show that, Emodin has a strong potential as a single or as an adjuvant non-toxic anti-tumor agent for co-administration with TX for the treatment of androgen-responsive prostate cancer [Funded by NIH grant 2R01 CA 061038 (BLL)].