Potential mechanism of phytochemical-induced apoptosis in human prostate adenocarcinoma cells: Therapeutic synergy in genistein and beta-lapachone combination treatment.

BACKGROUND: Prostate cancer is the second leading cause of male death in the United States. The incidence increases most rapidly with age, and multiple genetic and epigenetic factors have been implicated in the initiation, progression, and metastasis of the cancer. Nevertheless, scientific knowledge of the molecular mechanisms underlying the disease is still limited; and hence treatment has only been partially successful. The objective of the current studies was to examine the role of caspase 3 (CPP32) and NAD(P)H:quinone oxidoreductase (NQO1) in the signaling of genistein-and beta-lapachone (bLap)-induced apoptosis in human prostate carcinoma cells PC3. RESULTS: Both genistein and bLap produced dose-dependent growth inhibition and treatment-induced apoptosis in PC3. Treatment with caspase 3 inhibitor, DEVD-fmk before exposure to genistein, significantly inhibited caspase 3 expression and treatment-induced apoptosis; implicating CPP32 as the main target in genistein-induced apoptosis in PC3. Contrary to this observation, inhibition of CPP32 did not significantly influence bLap-induced apoptosis; implying that the major target of bLap-induced apoptosis may not be the caspase. Treatment with NQO1 inhibitor, dicoumarol (50 microM), prior to exposure of PC3 to bLap led to significant decrease in bLap toxicity concurrent with significant decrease in treatment-induced apoptosis; thus implicating NQO1 as the major target in beta-lapachone-induced apoptosis in PC3. In addition, the data demonstrated that NQO1 is the major target in bLap-genistein (combination)-induced apoptosis. On the contrary, blocking NQO1 activity did not significantly affect genistein-induced apoptosis; implying that NQO1 pathway may not be the main target for genistein-induced apoptosis in PC3 cells. Furthermore, blocking NQO1 and CPP32 did not confer 100% protection against genistein-induced or bLap-induced apoptosis. CONCLUSION: The data thus demonstrate that both genistein-and bLap-induced apoptosis are mostly but not completely dependent on CPP32 and NQO1 respectively. Other minor alternate death pathways may be involved. This suggests that some death receptor signals do not utilize the caspase CPP32 and/or the NQO1 death pathways in PC3. The demonstrated synergism between genistein and bLap justifies consideration of these phytochemicals in chemotherapeutic strategic planning.

Imaging of luciferase and GFP-transfected human tumours in nude mice.

Studies were performed to compare green fluorescent protein (GFP)-transfected and fi re fl y luciferase (Luc)-transfected MCF-7 human breast tumour cells both in vitro and in vivo. For in vitro studies, cells were serially diluted in 96-well microplates and analysed using a NightOwl LB 981 Molecular Light Imager and a Victor multilabel reader. For in vivo studies, nude mice were injected either intraperitoneally, intravenously or subcutaneously with transfected cells and then imaged using the NightOwl Imager after intraperitoneal injection of d-luciferin for Luc tumours, or excitation at 470 nm for GFP tumours. In vitro imaging studies revealed that both GFP and Luc transfectants were quantifiable. However, the Luc-transfected cells were detectable at a significantly lower concentration compared to GFP transfectants. In vivo studies demonstrated that GFP-transfected tumours were detectable as subcutaneous and intraperitoneal tumours but not as deep tissue lesions, whereas Luc-transfected tumours were detectable as subcutaneous and intraperitoneal tumours and as deep tissue lesions resulting from intraperitoneal or intravenous inoculation. These findings demonstrate that GFP-transfected cells may be useful for imaging studies of superficial tumours where both excitation and emission wavelengths are able to penetrate tissues, whereas luciferase-transfected cells appear superior for imaging studies of primary and metastatic tumours in distant sites and deep tissues.

Determination of chemotherapeutic activity in vivo by luminescent imaging of luciferase-transfected human tumors.

Human DU-145 prostate and MCF-7 breast tumor cell lines were stably transfected with plasmid pcDNA3.1-Luc expressing firefly luciferase. Studies were performed with the transfected cell lines to evaluate luminescent imaging for measuring the efficacy of anti-cancer agents. In vitro experiments demonstrated a dose response of both cell lines to topotecan (Hycamtin) with an IC50 of 0.013 microM for MCF-7 Luc cells and 0.002 microM for DU-145 Luc cells. In vivo imaging experiments were performed using athymic nude mice inoculated i.p. with 5 x 10(6) MCF-7 cells or s.c. with 5 x 10(6) DU-145 cells and then treated with topotecan at 2.5 mg/kg body weight. Tumor progression and regression were monitored for 27 days. Animals inoculated s.c. with DU-145 Luc cells and then treated with topotecan demonstrated significant tumor growth and regression as measured with calipers and luminescent imaging. High correlation was observed between caliper and imaging results. The correlation coefficient was 0.75 for the control untreated group and 0.93 for the topotecan-treated group. Similarly, tumor progression and regression were measurable using luminescent imaging for untreated and topotecan-treated mice inoculated i.p. with MCF-7 Luc cells. These data indicate that luminescent imaging is a useful tool for evaluating anti-cancer drugs in vivo and may prove to be particularly useful for the development of novel agents. Luminescent imaging could also be used to locate and harvest residual tumors in drug-treated animals in order to study mechanisms of drug resistance.