Focal adhesion kinase silencing augments docetaxel-mediated apoptosis in ovarian cancer cells

Abstract

Objective: Docetaxel causes cell death through induction of apoptosis; however, cell death characteristics for docetaxel have not yet been fully elucidated. We examined the role of focal adhesion kinase (FAK) cleavage in docetaxel-mediated apoptosis.

Methods: FAK degradation after treatment with docetaxel was determined in both taxane-sensitive (HeyA8 and SKOV3) and taxane-resistant (HeyA8-MDR and SKOV3-TR) ovarian cancer cell lines by Western blot analysis. Cell growth was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. FAK-targeting small interfering RNA (siRNA) was used to decrease FAK expression. Apoptosis and caspase activity were determined using commercially available kits.

Results: SKOV3 and HeyA8 cell lines were both sensitive to docetaxel (IC50 levels, 1-6.2 nmol/L), whereas the SKOV3-TR and HeyA8-MDR cells were resistant (IC50>or=250 nmol/L for both). Docetaxel induced high rates of apoptosis in SKOV3 and HeyA8 cells (84% and 66% apoptosis, respectively) but minimal apoptosis (5-8%) in SKOV3-TR and HeyA8-MDR cells. Similarly, FAK was cleaved in SKOV3 and HeyA8 cells in response to docetaxel treatment but unchanged in the resistant cells. Caspase-3 and caspase-8 activity also increased significantly in docetaxel-treated SKOV3 and HeyA8 cells but not in the taxane-resistant cells. DEVD-fmk (caspase-3 blocker) was able to block both FAK cleavage and apoptosis mediated by docetaxel in SKOV3 and HeyA8 cells. FAK siRNA transfection resulted in 70% to 90% decrease in FAK levels in all cell lines within 72 hours. FAK silencing augmented docetaxel-mediated growth inhibition (5- to 8-fold increase) and apoptosis in both of the taxane-sensitive and taxane-resistant cell lines.

Conclusions: Docetaxel induces FAK cleavage, mediated through activation of caspase-3, in taxane-sensitive ovarian cancer cells but not in taxane-resistant cells. The absence of FAK degradation may contribute to cell survival in taxane-resistant cells. FAK silencing promotes the in vitro efficacy of docetaxel in both taxane-sensitive and taxane-resistant cell lines and may serve as a novel therapeutic approach.

Fig. 1

Effect of docetaxel on ovarian cancer cell growth: (A) HeyA8 and HeyA8-MDR or (B) SKOV3 or SKOV3-TR cells were plated in 96-well plates and subsequently incubated with increasing concentrations of docetaxel for 96 hours, and cell viability was determined. Points, mean of three independent experiments; bars, SE.

Fig. 2

Effect of docetaxel on caspase-3 (A), caspase-8 (B), and caspase-9 (C) activity. Ovarian cancer cells were treated with docetaxel for 24 hours followed by fluorometric profiling of caspase activity using a commercially available kit. Columns, means of three independent experiments; bars, SE.

Fig. 3

Docetaxel treatment results in FAK cleavage. The taxane-sensitive (A) and taxane-resistant (B) SKOV3 cells were cultured in the presence of docetaxel for 48 or 72 hours. Western blot analysis for FAK in SKOV3 and SKOV3-TR cells in the presence or absence of the caspase-3 inhibitor DEVD-fmk.

Fig. 4

Effect of FAK silencing on docetaxel-sensitivity in ovarian cancer cell lines. A, Western blot analysis of SKOV3 whole-cell lysates probed with anti-FAK and anti-actin monoclonal antibodies. Bottom, densitometry results. Control siRNA did not significantly affect FAK expression compared with untreated cells whereas FAK-specific siRNA resulted in >90% suppression of FAK expression by 72 hours. Effects of docetaxel on (B) SKOV3, (C) HeyA8, (D) SKOV3-TR, and (E) HeyA8-MDR growth were tested alone or in combination with FAK siRNA or control siRNA. Points, means of three independent experiments; bars, SE.

Fig. 5

FAK gene silencing potentiates docetaxel-induced caspase-3 activity in (A) SKOV3 and HeyA8 and (B) SKOV3-TR and HeyA8-MDR cells. Columns, means of three experiments; bars, SE. *, P < 0.01; **, P < 0.001.

Fig. 6

Effects of docetaxel on (A) SKOV3 and SKOV3-TR or (B) HeyA8 and HeyA8-MDR ovarian cancer cells. The percentage of apoptosis was determined by terminal deoxynucleotidyl transferase – mediated nick end labeling. Cells were treated with or without IC₉₀ concentration of docetaxel for the taxane-sensitive cell lines. Points, means of three different experiments; bars, SE. Effect of docetaxel with or without the caspase-3 inhibitor (DEVD-fmk) on (C) SKOV3 and SKOV3-TR and (D) HeyA8 and HeyA8-MDR ovarian cancer cell apoptosis. Columns, means of three independents experiments; bars, SE.

Fig. 7

Docetaxel-mediated apoptosis with or without FAK siRNA in (A) SKOV3 and HeyA8 and (B) SKOV3-TR and HeyA8-MDR ovarian cancer cells. Columns, means of three independent experiments; bars, SE. Doc, docetaxel.