Fig 1.
Growth inhibition of KML001 in prostate cell lines.
Cells treated with various concentrations of KML001 were incubated for 72 h. The Alamar blue assay was done in triplicate. Mean values are given, the value of control being 100%. PC3 (●, thick solid line), DU145 (▲, thin solid line), and LNCaP (▼, thin solid line).
Table 1.
IC50 values of KML001 in prostate cancer cells.
Fig 2.
Structural features observed by electron microscope (10000× and 5000×) in PC3, DU145, and LNCaP prostate cancer cells treated with KML001 for 24 and 48 h.
Fig 3.
Induction of apoptosis by KML001 in prostate cancer cells.
(A) FACS analysis of annexin V/PI staining. Results show early apoptosis, defined as annexin V-positive and PI-negative cells, and late apoptosis, defined as annexin V-positive and PI-positive cells. Results were expressed as means ± SD of three independent experiments. (B) Western blot analysis of the time- and dose-dependent cleavage of PARP and activation of procaspase-3.
Fig 4.
Induction of autophagy by KML001 in prostate cancer cells.
(A) Western blot analysis of the time- and dose-dependent conversion of LC3-I to-II. (B) Inhibition by 3-MA of KML001-induced conversion of LC3 in prostate cancer cells. (C) Cells were exposed to 10 μM (PC3), 5 μM (DU145), or 2 μM (LNCaP) KML001 in the presence or absence of 1 mM 3-MA for 72 h. Results were expressed as means ± SD of three independent experiments. * p < 0.05 by one-way ANOVA.
Fig 5.
Regulation of autophagy and apoptosis by ROS.
All 3 prostate cancer cells were treated with the indicated concentration of KML001 in the absence or presence of 5 mM NAC for 24 h. (A) KML001 induces dose-dependent ROS (blue) accumulation. Cells were stained with DCFH-DA and washed with PBS. More than three fields in each cell were observed by fluorescence microscope (200×), and representative images are shown. (B) NAC inhibition of KML001-induced conversion of LC and caspase activation in prostate cancer cells. (C) Cells were exposed to 10 μM (PC3), 5 μM (DU145), or 2 μM (LNCaP) KML001 in the presence or absence of 1 mM NAC for 72 h. Results were expressed as means ± SD of three independent experiments. * p < 0.05 by one-way ANOVA.
Fig 6.
KML001 treatment has (A) a growth inhibitory effect on DU145 prostate cancer cells in mice, and (B) no effect on body weight of mice.
Vehicle and KML001 group mice orally received saline and KML001 (2.5 or 10 mg/kg/d) for 4 weeks, respectively. * p < 0.05 vs. vehicle.
Fig 7.
KML001 treatment has (A) anti-proliferative effect, (B) apoptotic effect, and (C) autophagic effect on DU145 prostate cancer cells in mice.
Three fields in each mice were observed by fluorescence (200×) and bright field microscopes (200×), respectively. Representative images of each treatment group were shown. Vehicle and KML001 group mice orally received saline and KML001 (2.5 or 10 mg/kg/d) for 4 weeks, respectively. * p < 0.05 vs. vehicle.