Pterostilbene inhibits lung squamous cell carcinoma growth in vitro and in vivo by inducing S phase arrest and apoptosis

Abstract

Natural dietary components have become the subject of an increasing amount of interest due to the side effects of anticancer treatment. Pterostilbene, an analog of resveratrol, is primarily found in grapes, and has been suggested to exert antioxidant and anticancer effects in different tumor types. The present study aimed to investigate the antitumor effects and molecular mechanisms of pterostilbene in the human lung squamous cell carcinoma (SqCC) cell line, H520. The results of the present study indicate that pterostilbene significantly reduced cell viability and induced S phase arrest, and that treatment with pterostilbene was associated with the downregulation of cyclin A and cyclin E, as with the upregulation of p21 and p27 expression in H520 cells. In the apoptosis analysis, pterostilbene induced S phase accumulation and the activation of caspase-3, -8 and -9 in H520 cells, potentially through the activation of extrinsic and intrinsic apoptotic pathways. Additionally, the in vivo study demonstrated that pterostilbene effectively inhibited lung SqCC growth in a H520 ×enograft model. Given the in vitro and in vivo antitumor effects of pterostilbene demonstrated in the present study, pterostilbene may serve a novel and effective therapeutic agent to for patients with SqCC.

Keywords: apoptosis; cell cycle; lung squamous cell carcinoma; pterostilbene.

Figure 1.

Effect of pterostilbene on the viability of H520 and H226 cells by an MTT assay. (A) Cells were incubated with increasing concentrations of pterostilbene in culture medium for 24 and 48 h. Cell viability was then assessed by an MTT assay. Data presented are the mean ± SD of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. 0.1% DMSO-treated group. (B) Representative bright-field images of cell morphology (magnification, ×100).

Figure 2.

Effects of pterostilbene on cell cycle distribution and apoptosis in H520 cells. (A) Cell cycle analysis of pterostilbene-treated cells. Cells were plated as described in the Materials and methods section with 50, 25 and 12.5 µM pterostilbene for 48 h. Cell cycle distributions were then determined by staining with a PI solution and flow cytometry analysis. Data are representative of three independent experimental repeats. (B) Flow cytometric analysis of Annexin V staining to quantify pterostilbene-induced apoptosis in H520 cells. The dot plots show the results of the treatment of H520 cells with pterostilbene for 48 h. Data are presented as the mean ± SD of triplicate experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control. PI, propidium iodide.

Figure 3.

Effects of pterostilbene on S phase-associated regulatory proteins. The expression of S phase regulatory proteins in pterostilbene-treated H520 cells was examined by western blotting. GAPDH was used as the loading control. The fold change in the band intensity compared with that of the vehicle-treated cells is denoted under the western blots. Data are representative of three independent experiments.

Figure 4.

Effects of pterostilbene on the activity of caspase-3, −8, and −9 in H520 cells. The cells were treated with 50, 25 and 12.5 µM pterostilbene for 48 h and harvested and the caspase activity was analyzed by flow cytometry. Data are representative of three independent experiments. ***P<0.001 vs. 0.1% DMSO-treated group.

Figure 5.

Effects of pterostilbene on the mitochondrial potential and cytochrome c release in H520 cells. Cells were treated with 50, 25 and 12.5 µM pterostilbene for 48 h. (A) Cells were stained with fluorescent JC-1 dye, and the mean JC-1 fluorescence intensity was analyzed by flow cytometry. Gate P6 was used to determine the JC-1 green percentage. (B) Cytosolic lysates were prepared, and the expression of cytochrome c in pterostilbene-treated H520 cells was examined by western blot analysis. (C) Total lysates were prepared, and the expression of Bax and Bcl-2 proteins in pterostilbene-treated H520 cells was examined by western blotting. The fold change in the band intensity compared with that of the vehicle-treated mice is denoted. Data are representative of three independent experimental repeats. *P<0.05 and ***P<0.001 vs. 0.1% DMSO-treated group.

Figure 6.

Effects of pterostilbene on H520 ×enograft growth in nude mice. H520 cells (1×10⁷) were inoculated under the skin of nude mice and tumors were permitted to grow to ~10 mm³. Subsequently, pterostilbene (50 mg/kg) or vehicle control was administered intraperitoneally three times weekly until day 38 (n=6). (A) Tumor size was measured at 3–5 day intervals. *P<0.05, **P<0.01 and ***P<0.001 vs. vehicle control group (10% DMSO + 90% glyceryl trioctanoate). (B) Representative tumor images and (C) tumor weights of each group on day 38 are presented. *P<0.05 vehicle control group (10% DMSO + 90% glyceryl trioctanoate). (D) Body weight and (E) organ weight of the heart, lung, liver, kidney and spleen of pterostilbene-treated and vehicle control-treated mice. NS, not significant.