Figure 1.
Nab-paclitaxel inhibits human gastric cancer cells proliferation.
(A) Total cell extracts of gastric cancer cells were analyzed by immunoblotting for expression of stathmin and phospho-stathmin. (B–D) Human gastric cancer cells SNU16(B), NCI-N87 (C) and AGS (D) were plated on 96-well plates and treated with 1 nM to 1000 nM concentrations of nab-paclitaxel, 5-fluorouracil, oxaliplatin and epirubicin. After 72 hours, 10 μl WST-1 reagent was added in each well and incubated for 2 additional hours. The absorbance at 450 nm was measured using a microplate reader. The resulting number of viable cells was calculated by measuring absorbance of color produced in each well. Data are the mean ± SD of triplicate determinations.
Figure 2.
Effects of nab-paclitaxel on cell cycle progression, expression of phospho-stathmin and mitotic cell death in vitro.
(A) SNU16 cells were cultured for 24 hours and then treated with 100 nM nab-paclitaxel for 12 hours. Cell cycle analysis was performed by flow cytometry. Results shown are representative of two independent experiments. (B–D) Expression of phospho-stathmin and mitotic cell death was evaluated in SNU16 (B), NCI -N87 (C), and AGS (C) cells. Cultured cells were treated with nab-paclitaxel (10 μM) and showed increased mitotic arrest configuration and phospho-stathmin expression by immunocytochemical staining. Mitotic arrests were detected in the presence of phospho-stathmin expression. (D) A sub-confluent monolayer of human gastric cancer cells AGS, NCI-N87 and SNU16 was treated with nab-paclitaxel (10 μM) for 3, 6 and 9 hours and analyzed by immunoblotting for phospho-stathmin and total stathmin. (E) Human gastric cancer cell were treated with nab-paclitaxel (10 μM), oxaliplatin (10 μM), and epirubicin (10 μM) for 16 hours and analyzed for cleaved PARP-1 and caspase-3. Data are representative of two independent experiments with similar results.
Figure 3.
Nab-paclitaxel inhibits growth of established local tumor.
(A) SCID mice were subcutaneously injected with SNU16 cells (20×106) and treated with nab-paclitaxel, oxaliplatin and epirubicin for 2 weeks. (B) SCID mice were subcutaneously injected with NCI-N87 cells (10×106) and treated with nab-paclitaxel for 2 weeks. Relative tumor volume and tumor weight on the final day were assessed. Data are representative of mean values ± standard deviation from 6–8 mice per group. Symbol * represents significant difference (p<0.05) and symbol ** represents significant differences (p<0.001) compared to vehicle controls; ns = no significant difference.
Figure 4.
Effects of nab-paclitaxel treatment on expression of phospho-stathmin in vivo.
SCID mice were subcutaneously injected SNU16 cells (20×106) or NCI-N87 cells (10×106) and treated with nab-paclitaxel for 2 weeks. Intratumoral expression of phospho-stathmin was measured in SNU16 (A) or NCI-N87 (B) tumor tissue by immunostaining tissue sections for phospho-stathmin (ser38) antigen and photographed under a fluorescent microscope. Tumor lysates were prepared from tumor tissue samples obtained from SNU16 (A) or NCI-N87 (B) tumor bearing SCID mice after nab-paclitaxel therapy and then analyzed by immunoblotting for phospho-stathmin and total stathmin.
Figure 5.
Effects of nab-paclitaxel treatment on cell proliferative and apoptotic activity in vivo.
SCID mice were subcutaneously injected SNU16 cells (20×106) and treated with nab-paclitaxel for 2 weeks. (A) Intratumoral proliferation was measured by immunostaining tissue sections with Ki67 nuclear antigen followed by fluorescent microscope photography. Ki67-positive cells were counted in five high power fields per sample. Fold change in proliferative index was standardized to controls (set to 1) and other samples are compared relative to this sample. (B) Intratumoral apoptosis was measured by staining tumor tissue section with the TUNEL procedure and subsequent fluorescent microscope photography. TUNEL-positive apoptotic cells were counted in five high power fields per sample. For both immunostaining experiments, each group had at least three samples counted and the data are expressed as the mean ± standard deviation. Symbol * represents significant difference compared to vehicle group (p<0.05).
Figure 6.
Effects of nab-paclitaxel therapy on the overall survival of mice.
(A) Antitumor effect of nab-paclitaxel compared with oxaliplatin. SNU cells (40×106) were injected intraperitoneally into SCID mice, followed by treatment after 2 weeks with nab-paclitaxel (10 mg/kg, 2 times a week) and oxaliplatin (5 mg/kg, 2 times a week) for 2 weeks. (B) Antitumor effect of nab-paclitaxel compared with oxaliplatin and docetaxel. SNU cells (40×106) were injected intraperitoneally into SCID mice, followed by treatment after 2 weeks with nab-paclitaxel (10 mg/kg, 2 times a week), oxaliplatin (5 mg/kg, 2 times a week), or docetaxel (3 mg/kg, 2 times a week) for 2 weeks. The curve represents the animal survival time from the beginning of therapy. Symbol * represents significant difference compared with vehicle control (p<0.05), symbol § represents significant differences compared with oxaliplatin (p<0.05), and symbol # represents significant differences compared with docetaxel (p<0.05).