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. 2014 Nov 12;9(11):e111814.
doi: 10.1371/journal.pone.0111814. eCollection 2014.

Potent anti-cancer effect of 3'-hydroxypterostilbene in human colon xenograft tumors

Tzu-Chun Cheng  1 , Ching-Shu Lai  2 , Min-Ching Chung  2 , Nagabhushanam Kalyanam  3 , Muhammed Majeed  3 , Chi-Tang Ho  4 , Yuan-Soon Ho  5 , Min-Hsiung Pan  6
Affiliations

Potent anti-cancer effect of 3'-hydroxypterostilbene in human colon xenograft tumors

Tzu-Chun Cheng et al. PLoS One. .

Abstract

Here we report that 3'-hydroxypterostilbene (HPSB), a natural pterostilbene analogue, was more potent than pterostilbene against the growth of human cancer cells (COLO 205, HCT-116, and HT-29) with measured IC50 values of 9.0, 40.2, and 70.9 µM, respectively. We found that HPSB effectively inhibited the growth of human colon cancer cells by inducing apoptosis and autophagy. Autophagy occurred at an early stage and was observed through the formation of acidic vesicular organelles and microtubule-associated protein 1 light chain 3-II production. At the molecular levels, the results from western blot analysis showed that HPSB significantly down-regulated phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) signalings including decreased the phosphorylation of mammalian target of rapamycin (mTOR). Significant therapeutic effects were demonstrated in vivo by treating nude mice bearing COLO 205 tumor xenografts with HPSB (10 mg/kg i.p.). These inhibitory effects were accompanied by mechanistic down-regulation of the protein levels of cyclooxygenase-2 (COX-2), matrix metallopeptidase-9 (MMP-9), vascular endothelial growth factor (VEGF), and cyclin D1, as well as by the induction of apoptosis in colon tumors. Our findings suggest that HPSB could serve as a novel promising agent for colon cancer treatment.

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Conflict of interest statement

Competing Interests: NK and MM are employees of Sabinsa Corporation. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Chemical structure of pterostilbene and 3′-hydroxypterostilbene.
Figure 2
Figure 2. Pterostilbene and 3′-hydroxypterostilbene induced apoptosis in COLO205 cancer cells.
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 Materials and Methods. (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 Materials and Methods. (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. *P<0.05 indicates statistically significant difference from the pterostilbene-treated group.
Figure 3
Figure 3. Autophagy induction by pterostilbene and 3′-hydroxypterostilbene in COLO 205 cancer cells.
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. *P<0.05 indicates statistically significant difference from the pterostilbene-treated group.
Figure 4
Figure 4. 3′-Hydroxypterostilbene down-regulated mTOR, PI3K/Akt and MAPKs signaling in COLO 205 cancer cells.
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.
Figure 5
Figure 5. Autophagy inhibitor sensitized pterostilbene and 3′-hydroxypterostilbene-induced apoptosis in COLO 205 cancer cells.
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. * P<0.05 and ** P<0.01 indicates statistically significant difference from the pterostilbene-treated group.
Figure 6
Figure 6. Pterostilbene and 3′-hydroxypterostilbene reduced the growth of COLO 205 xenografts in nude mice.
Experimental treatment protocol as described in Materials and Methods. Mice bearing COLO 205 xenografts were i.p. 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. * P<0.05 and ** P<0.01, compared with control group. # P<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.
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This study was supported by the National Science Council NSC 101-2628-B-022-001-MY4, 102-2628-B-002-053-MY3, and NTU-103R7777 (for Dr. Pan) and by the Health and welfare surcharge of tobacco products MOHW103-TD-B-111-01 (for Dr. Ho). Sabinsa Corporation provided support in the form of salaries for authors NK & MM, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.