Figure 1.
High ER-α36 expression sensitizes breast cancer cells to pterostilbene treatment.
a. ER-α36 mRNA levels were measured in Mb231, Mb231/Si36, MCF-7, and MCF-7/ER36 cells using RT-PCR. b. The relative mRNA expressions are presented as the ratio of the OD of the corresponding mRNA bands compared to the OD of the β-actin bands. c. Western blots of ER-α36 and ER-α66 proteins in Mb231, Mb231/Si36, MCF-7, and MCF-7/ER36 cells using β-actin levels as the loading control. d. MTT assays measuring the cell proliferation of the two paired cells after control and pterostilbene treatments for 72 h. The relative cell numbers were normalized to those of their respective pterostilbene untreated controls (set as 100%). (Bars, SE. *p<0.05, **p<0.01).
Figure 2.
High ER-α36 expression enhances pterostilbene-induced apoptosis in breast cancer cells.
a. Apoptosis induction in Mb231, Mb231/Si36, MCF-7, and MCF-7/ER36 cells treated with 30 µM pterostilbene for 72 h was analyzed through flow cytometry (X-Axis: Annexin V; Y-Axis: Propidium iodide, PI). The apoptosis rates are presented as both early and late apoptotic cells compared to total cells. b. Statistical analysis of FACS in (a). c. Caspase-3 expression in the two paired cells after control and 30 µM pterostilbene treatment for 72 h. (Means ± SE, **p<0.01).
Figure 3.
Pterostilbene inhibits the ERK1/2 and Akt activation via ER-α36 in breast cancer cells.
a. Mb231, Mb231/Si36, MCF-7, and MCF-7/ER36 cells were treated with the indicated concentrations of pterostilbene for 72 h. b Mb231, Mb231/Si36, MCF-7, and MCF-7/ER36 cells were treated with 30 µM pterostilbene for the indicated hours. Changes in p-ERK1/2 and p-Akt protein levels were analyzed through western blotting. The total ERK1/2 and Akt proteins were used as loading controls.
Figure 4.
ER-α36 knockdown reduces the sensitivity of the xenograft tumors of ER-α36-positive breast cancer to pterostilbene.
Mb231/Si36 and Mb231 cells were injected into the left and right breast pad, respectively of 5 to 6 weeks old nude mice. When the tumors reached about 50 mm3 size, the mice were randomly assigned into two groups (n = 3): vehicle control (Veh) and pterostilbene treatment (+Pter). a. Tumor volumes were measured once every two days. Red arrows indicate the timed of pterostilbene administration. b. Tumor weights were calculated and are shown as a plot with the median and whiskers from minimum to maximum. c. The representative images of tumors in each group are shown. d. Paraffin-embedded tissue sections of the above tumors were subjected to H&E staining (X 40, left panels) and immunohistochemical (IHC) staining using the antibody against ER-α36 (X 100, right panels). (**p<0.01, NS: no significance).