d-Limonene sensitizes docetaxel-induced cytotoxicity in human prostate cancer cells: Generation of reactive oxygen species and induction of apoptosis

<b>Background:</b><i> </i> Clinical trials have shown that docetaxel combined with other novel agents can improve the survival of androgen-independent prostate cancer patients.<i> d</i> -Limonene, a non-nutrient dietary component, has been found to inhibit various cancer cell growths without toxicity. We sought to characterize whether a non-toxic dose of <i> d</i> -limonene may enhance tumor response to docetaxel in an <i> in vitro</i> model of metastatic prostate cancer. <b> Materials and Methods: </b> Human prostate carcinoma DU-145 and normal prostate epithelial PZ-HPV-7 cells were treated with various concentrations of <i> d</i> -limonene, docetaxel or a combination of both, and cell viability was determined by MTT assay. Intracellular reactive oxygen species (ROS), reduced glutathione (GSH) and caspase activity were measured. Apoptosis and apoptosis-related proteins were studied by enzyme-linked immunosorbent assay and Western blotting, respectively. <b> Results:</b> <i> d</i> -Limonene and docetaxel in combination significantly enhanced the cytotoxicity to DU-145 cells than PZ-HPV-7 cells. Exposure of DU-145 cells to a combined <i> d</i> -limonene and docetaxel resulted in higher ROS generation, depletion of GSH, accompanied by increased caspase activity than docetaxel alone. It also triggered a series of effects involving cytochrome <i> c</i> , cleavages of caspase-9, 3 and poly (ADP-ribose) polymerase, and a shift in Bad:Bcl-xL ratio in favor of apoptosis. Apoptotic effect was significantly blocked on pretreatment with <i> N</i> -acetylcystein, indicating that antitumor effect is initiated by ROS generation, and caspase cascades contribute to the cell death. <b> Conclusion:</b> Our results show, for the first time, that <i> d</i> -limonene enhanced the antitumor effect of docetaxel against prostate cancer cells without being toxic to normal prostate epithelial cells. The combined beneficial effect could be through the modulation of proteins involved in mitochondrial pathway of apoptosis. <i> d</i> -Limonene could be used as a potent non-toxic agent to improve the treatment outcome of hormone-refractory prostate cancer with docetaxel

AMR-Me inhibits PI3K/Akt signaling in hormone-dependent MCF-7 breast cancer cells and inactivates NF-κB in hormone-independent MDA-MB-231 cells

AMR-Me, a C-28 methylester derivative of triterpenoid compound Amooranin isolated from Amoora rohituka stem bark and the plant has been reported to possess multitude of medicinal properties. Our previous studies have shown that AMR-Me can induce apoptosis through mitochondrial apoptotic and MAPK signaling pathways by regulating the expression of apoptosis related genes in human breast cancer MCF-7 cells. However, the molecular mechanism of AMR-Me induced apoptotic cell death remains unclear. Our results showed that AMR-Me dose-dependently inhibited the proliferation of MCF-7 and MDA-MB-231 cells under serum-free conditions supplemented with 1 nM estrogen (E2) with an IC50 value of 0.15 µM, 0.45 µM, respectively. AMR-Me had minimal effects on human normal breast epithelial MCF-10A + ras and MCF-10A cells with IC50 value of 6 and 6.5 µM, respectively. AMR-Me downregulated PI3K p85, Akt1, and p-Akt in an ERα-independent manner in MCF-7 cells and no change in expression levels of PI3K p85 and Akt were observed in MDA-MB-231 cells treated under similar conditions. The PI3K inhibitor LY294002 suppressed Akt activation similar to AMR-Me and potentiated AMR-Me induced apoptosis in MCF-7 cells. EMSA revealed that AMR-Me inhibited nuclear factor-kappaB (NF-κB) DNA binding activity in MDA-MB-231 cells in a time-dependent manner and abrogated EGF induced NF-κB activation. From these studies we conclude that AMR-Me decreased ERα expression and effectively inhibited Akt phosphorylation in MCF-7 cells and inactivate constitutive nuclear NF-κB and its regulated proteins in MDA-MB-231 cells. Due to this multifactorial effect in hormone-dependent and independent breast cancer cells AMR-Me deserves attention for use in breast cancer prevention and therap