Involvement of Cdk5/p25 in Digoxin-triggered Prostate Cancer Cell Apoptosis

Abstract

Cardiac digitalis has been considered to be a treatment for breast cancer. Our previous study indicates that digoxin, one member in digitalis, decreases the proliferation of prostate cancer cells, but the mechanisms remain unclear. In the present study, Ca 2؉ proved to be an important factor in digoxin-triggered prostate cancer cell death. Because cyclin-dependent kinase (Cdk)5 and p35 cleavage (p25 formation) have been reported to be targets of intracellular Ca 2؉ , and subsequently correlated to apoptosis, we not only demonstrated first that Cdk5, p35, and p25 proteins were all expressed in prostate cancer cells (including lymph node carcinoma of the prostate (LNCaP) and DU-145 cells), but also showed where p25 formation and Cdk5 kinase activity were affected by treatment with digoxin. The inhibitor of p35 cleavage (calpeptin) was used to reduce p25 formation, and the result suggested that p25 accumulation might be the major cause of digoxin-triggered LNCaP cell death. Butyrolactone-I and roscovitine, two Cdk5 kinase inhibitors, were also found to prevent digoxin-triggered LNCaP cell death. In addition, treatment of siRNA-Cdk5 diminished digoxin-triggered cell death, as compared with the treatments of siRNA-Cdk1 or siRNA-Cdk2, which implies the specific involvement of Cdk5 in digoxin-triggered cell death. Caspase inhibitor set and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay were used to demonstrate that digoxin-triggered LNCaP cell apoptosis through Cdk5 activation. These results suggest that Cdk5/p35 and p25 are novel players in digoxin-triggered prostate cancer cell apoptosis and, therefore, become potential therapeutic targets.Prostate cancer is a common malignancy and age-related cancer in men (1). Although the cancer marker prostate-specific antigen helps to identify prostate cancer, the lethality is still very high. Traditionally, before prostate cancer becomes androgen-independent, the therapy is gonadectomy. Once prostate cancer has become androgen-independent, definitive radio-or chemotherapies are used.Digoxin, a purified digitalis preparation derived from the leaves of Digitalis lanata (foxglove), plays a vital role in the therapy of congestive heart failure (2) and is the most widely used digitalis glycoside. It has direct and indirect cardiovascular actions for treating heart failure (3). Epidemiological studies have shown the anti-cancer effects of digitalis (4). Breast cancer patients treated with digitalis have been found to have a lower death rate than non-treated patients (4). Five years after mastectomy, cancer recurrence rates in control patients who were not given digitalis increased by 9.6-fold, as compared with patients who were treated with digitalis (5). Our previous study demonstrated that digoxin is able to induce the rise of intracellular Ca 2ϩ and the toxicity of prostate cancer cells, but detailed mechanisms still remain unknown (6).Cdk5 is a unique member of a small serine/threonine cyclindependent kinase (cdk) 1 family. Although most Cdks are invol...

Regulation of Androgen Receptor and Prostate Cancer Growth by Cyclin-dependent Kinase 5

“…In prostate cancer cells we discovered that digoxin-triggered Cdk5 overactivation may lead to apoptosis (24). This is the first report demonstrating the role of Cdk5 in prostate cancer cells.…”

“…Lysates were analyzed by direct immunoblotting (20 -35 g/lane) or blotting after immunoprecipitation (0.5-1 mg/immunoprecipitation) using methods modified from those previously described (12,21,23,24). Immunoprecipitates were collected by binding to 25-40 l of the ExactaCruz beads (Santa Cruz).…”

“…The ExactaCruz beads with target proteins were incubated at 30°C for 30 min in kinase reaction buffer containing 10 g of substrates (histone H1, Upstate Biotechnology; homemade human recombinant AR), 10 l of magnesium/ATP mixture (Upstate Biotechnology), 5 l of 5ϫ assay dilution buffer (Upstate Biotechnology; 100 mM MOPS (pH 7.2), 125 mM ␤-glycerophosphate, 25 mM EGTA, 5 mM Na 3 VO 4 , and 5 mM dithiothreitol), and 1-3 Ci of [ 32 P]ATP in a final volume of 25 l. The reaction was terminated by adding 7 l of 5ϫ SDS sample buffer and boiling for 3 min. The samples were separated by 10% SDS-polyacrylamide gel electrophoresis and visualized on the x-ray film (Fujifilm) (24,26).…”

Enhancing the anticancer properties of cardiac glycosides by neoglycorandomization

“…With respect to other implicated cellular players, the nonlethal cardenolide concentrations that inhibit breast cancer cell proliferation also activate Src kinase, stimulate the interaction between Na ϩ ͞K ϩ -ATPase, the activated Src kinase, and epidermal growth factor (EGFR), and lead to the activation of extracellular signal-regulated kinases 1 and 2 (ERK1͞2) and subsequent cell cycle arrest caused by increased levels of p21 Cip1 (35). Cardiac glycosides have also been demonstrated to initiate apoptosis via the classical caspase-dependent pathways in malignant T lymphoblasts (36) and prostate cancer cells (37). In the latter, cardiac glycosides also inhibit testosterone production in vivo (38).…”

Digoxin Effectively Inhibited Cell Growth and Induced Senescence in Cervical Cancer Cell Line

“…It exerts its therapeutic potential directly and indirectly on the cardiovascular system (3). Recently, it has been proved that some cardiac glycosides such as digoxin have both therapeutic and suppressive potential in cancer therapy (4).…”