Autophagy inhibitor sensitized pterostilbene and 3′-hydroxypterostilbene-induced apoptosis in COLO 205 cancer cells.

<p>Cells were pretreated with 25 µM CQ for 1 h before treatment with 50 µM of pterostilbene or 3′-hydroxypterostilbene for 24 h. (A) Cell viability was determined by MTT assay. (B, C) Sub-G1 cell population (%) was analyzed and quantification after PI staining followed by flow cytometry. Data were presented as mean ±SD of triplicate experiments. ^*<i>P</i><0.05 and ^**<i>P</i><0.01 indicates statistically significant difference from the pterostilbene-treated group.</p

Pterostilbene and 3′-hydroxypterostilbene induced apoptosis in COLO205 cancer cells.

<p>Cells were treated with various concentrations (5, 10, 25 and 50 µM) of pterostilbene or 3′-hydroxypterostilbene for 24 h. (A) Determination of sub-G1 cells in COLO 205 cells by flow cytometry after PI staining as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111814#s2" target="_blank">Materials and Methods</a>. (B, C) After treatment, total cell lysates were prepared from COLO 205 cells and the cleavage of PARP, DFF-45, pro-caspase 8 and pro-caspase 9 were analyzed by Western blotting. (D) Kinetics of caspase activation in COLO 205 cells. Cells were treated with 25 and 50 µM of pterostilbene or 3′-hydroxypterostilbene for 24 h. Caspase activities were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111814#s2" target="_blank">Materials and Methods</a>. (E) Cells were treated with 50 µM of pterostilbene or 3′-hydroxypterostilbene for 15 min. Mitochondrial membrane potential and ROS production were stained with DiOC6 (40 nM) and DCFH-DA (20 µM) and measured by flow cytometry. The values are expressed as means ±SE of triplicate tests. *<i>P</i><0.05 indicates statistically significant difference from the pterostilbene-treated group.</p

Autophagy induction by pterostilbene and 3′-hydroxypterostilbene in COLO 205 cancer cells.

<p>Cells were treated with 50 µM pterostilbene or 3′-hydroxypterostilbene for 24 h and stained with acridine orange. (A) Green and red fluorescence in acridine orange-stained cells were observed under fluorescence microscope. (B, C) Detection and quantification of autophagy in COLO 205 cells. Cells were treated with 25 and 50 µM of pterostilbene or 3′-hydroxypterostilbene for 24 h and stained with acridine orange. The measurement of green and red fluorescence in acridine orange-stained cells was performed using flow cytometry. (D) Cell lysates were prepared after 24 h treatment and the protein expression of LC3 I/II were analyzed by Western blotting. Data were presented as mean ±SD of triplicate experiments. *<i>P</i><0.05 indicates statistically significant difference from the pterostilbene-treated group.</p

Effects of pterostilbene and 3′-hydroxypterostilbene administration on the body weight and organ weight in a COLO 205 xenograft model^a.

a<p>COLO 205 cells were injected into 3–4 week old BALB/c nude mice (5×10⁶ cells per mouse). After tumors grew to about 100–200 mm³, mice were <i>i.p.</i> treated with pterostilbene or 3′-hydroxypterostilbene for 15 days. All mice of each group were sacrificed by CO₂ asphyxiation at the end of experiment. Comparisons were analyzed using ANOVA followed by Fisher's least significant difference test.</p><p>Effects of pterostilbene and 3′-hydroxypterostilbene administration on the body weight and organ weight in a COLO 205 xenograft model^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111814#nt102" target="_blank">a</a>}.</p

Pterostilbene and 3′-hydroxypterostilbene reduced the growth of COLO 205 xenografts in nude mice.

<p>Experimental treatment protocol as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111814#s2" target="_blank">Materials and Methods</a>. Mice bearing COLO 205 xenografts were <i>i.p.</i> injection with pterostilbene or 3′-hydroxypterostilbene for 15 days where control group was received corn oil. (A) Photograph of xenograft tumors developed in each group is shown at the end of day15. (B) Average tumor volumes were recorded during the treatment and (C) average tumor weights were measured at the end of experiment. Six samples were analyzed in each group, and values represent the mean ±SD. ^*<i>P</i><0.05 and ^**<i>P</i><0.01, compared with control group. ^#<i>P</i><0.05, compared with pterostilbene-treated group. (D) Total proteins of xenograft tumors in each group were extracted for western blot analysis. COX-2, MMP-9, VEGF, PCNA, cyclin D1 protein expression and cleaved caspase-3 were detected by using specific antibodies. Similar results were obtained in three independent experiments.</p

3′-Hydroxypterostilbene down-regulated mTOR, PI3K/Akt and MAPKs signaling in COLO 205 cancer cells.

<p>COLO 205 cells were treated with 50 µM 3′-hydroxypterostilbene at different times. Cell lyates were prepared and the protein levels of (A) p-mTOR, p-P70S6K, (B) p-PI3K, p-Akt and (C) p-ERK1/2, p-JNK1/2, p-p38 were analyzed by Western blotting analysis. All analyses were representative of at least three independent experiments. The values under each lane indicate relative density of the band normalized to β-actin using a densitometer.</p

Chemical structure of pterostilbene and 3′-hydroxypterostilbene.

<p>Chemical structure of pterostilbene and 3′-hydroxypterostilbene.</p