Pinus mugo Essential Oil Impairs STAT3 Activation through Oxidative Stress and Induces Apoptosis in Prostate Cancer Cells

Abstract

Essential oils (EOs) and their components have been reported to possess anticancer properties and to increase the sensitivity of cancer cells to chemotherapy. The aim of this work was to select EOs able to downregulate STAT3 signaling using Western blot and RT-PCR analyses. The molecular mechanism of anti-STAT3 activity was evaluated through spectrophotometric and fluorometric analyses, and the biological effect of STAT3 inhibition was analyzed by flow cytometry and wound healing assay. Herein, Pinus mugo EO (PMEO) is identified as an inhibitor of constitutive STAT3 phosphorylation in human prostate cancer cells, DU145. The down-modulation of the STAT3 signaling cascade decreased the expression of anti-proliferative as well as anti-apoptotic genes and proteins, leading to the inhibition of cell migration and apoptotic cell death. PMEO treatment induced a rapid drop in glutathione (GSH) levels and an increase in reactive oxygen species (ROS) concentration, resulting in mild oxidative stress. Pretreatment of cells with N-acetyl-cysteine (NAC), a cell-permeable ROS scavenger, reverted the inhibitory action of PMEO on STAT3 phosphorylation. Moreover, combination therapy revealed that PMEO treatment displayed synergism with cisplatin in inducing the cytotoxic effect. Overall, our data highlight the importance of STAT3 signaling in PMEO cytotoxic activity, as well as the possibility of developing adjuvant therapy or sensitizing cancer cells to conventional chemotherapy.

Keywords: STAT3; apoptosis; essential oil; oxidative stress.

Figure 1

EOs inhibit constitutive tyrosine-phosphorylated STAT3 with strong potency. DU145 cells were treated with the indicated concentrations of EOs for 1 h, and total protein extracts were analyzed by Western blot with pTyr⁷⁰⁵STAT3 antibody and with anti-STAT3 antibody after membrane stripping. β-Actin is shown as the internal loading control. The data shown are representative of four independent experiments. Pinus mugo essential oil (PMEO), Lavandula angustifolia essential oil (LAEO), Pinus sylvestris essential oil (PSEO) and Cupressus sempervirens essential oil (CSEO).

Figure 2

EOs induce cytotoxicity in DU145 cell line. DU145 cells were treated with increasing concentrations of EOs belonging to the strong cluster for 24 and 48 h, and cell viability was analyzed by WST-8 assay. (a) The graphs report the % viability of DU145 cells after EO treatment. The results represent the mean ± SEM value of six independent experiments. (b) EO doses required to affect 50% cell viability (IC50) are reported in the graph. (c) The graphs report the % viability of human fibroblast cells after EO treatment.

Figure 3

Chemical structure of main PMEO components.

Figure 4

Pinus mugo EO modulates constitutive STAT3 signaling. (a) DU145 cells were treated with 50 μg/mL PMEO for the indicated time points, and total protein extracts were analyzed by Western blot with pTyr⁷⁰⁵STAT3 antibody and with anti-STAT3 antibody after membrane stripping. β-Actin is shown as the internal loading control. The data shown are representative of four independent experiments. (b) DU145 cells were exposed to 50 μg/mL PMEO for 24 h, and total RNA was analyzed by real-time PCR assay. The data were normalized against SDHA RNA, and the levels of mRNA are expressed as the value relative to untreated cells. Each bar represents the mean ± SD of four independent experiments performed in triplicate. p < 0.0001 (***); p < 0.001 (**); p < 0.01(*). (c) DU145 cells were treated with the indicated concentrations of PMEO for 24 h, and total protein extracts were analyzed by Western blot using antibodies specific for Bcl2, MCL1, Cyclin D1, Survivin, XIAP and COX2 proteins. β-Actin is shown as the internal loading control. The data shown are representative of four independent experiments.

Figure 5

Pinus mugo EO modulates IL6-induced STAT3 activation. LnCAP cells were treated with the indicated concentrations of PMEO for 45 min and then with 20 ng/mL IL-6. Total protein extracts were analyzed by Western blot using anti-pTyr⁷⁰⁵STAT3 antibody and anti-STAT3 antibody after membrane stripping. β-Actin is shown as the internal loading control. The data shown are representative of four independent experiments.

Figure 6

Pinus mugo EO induces oxidative stress and modulates STAT3 tyrosine phosphorylation. (a) DU145 cells loaded with H₂DCF-DA were treated with 50 and 75 μg/mL PMEO for 30 min or 1 h, and then the fluorescence was analyzed for ROS production. ROS levels are expressed as the value relative to untreated cells. The data are presented as means ± SEM of four independent experiments. p < 0.0001 (***); p < 0.01(*). (b) DU145 cells were treated with 50 µg/mL PMEO for the indicated time points, and GSH levels were spectrophotometrically analyzed by DTNB. GSH levels are expressed as the value relative to untreated cells. Data are presented as means ± SEM of five independent experiments. (c) DU145 cells were pretreated with 10 mM NAC for 1 h and then treated with the indicated concentrations of PMEO for 1 h more. Total protein extracts were analyzed by Western blot with pTyr⁷⁰⁵STAT3 antibody and with anti-STAT3 antibody after membrane stripping. β-Actin is shown as the internal loading control. The data shown are representative of four independent experiments.

Figure 7

Pinus mugo EO induces apoptosis in DU145 cells. (a) DU145 cells were treated with 50 and 75 μg/mL PMEO for 24 h, stained with Annexin V/PI and analyzed by flow cytometry for apoptosis detection. (b) DU145 cells were treated with 50 μg/mL PMEO for the indicated time points, and (c) with indicated doses for 24 h, and total protein extracts were analyzed by Western blot for the expression of cleaved caspase-3 and PARP1. PARP1 antibody recognizes both intact PARP (116 kDa) and the cleaved fragment (89 kDa). Β-Actin was used as the internal loading control. The data shown are representative of four independent experiments.

Figure 8

Pinus mugo EO inhibits cell migration in DU145 cells. (a) DU145 cells were treated with 25 and 50 μg/mL PMEO for the indicated time points. (b) % Wound closure was calculated using ImageJ/Fiji software (https://imagej.net/Fiji). (c) DU145 cells were treated with 25, 50 and 75 μg/mL PMEO for 24 h, and total protein extracts were analyzed by western blot using anti-ZEB1, anti-TWIST-1 and anti-Vimentin antibodies. Β-Actin was used as the internal loading control. The data shown are representative of four independent experiments.

Figure 9

Fa–CI plot of interaction between Pinus mugo EO and cisplatin. PMEO plus cisplatin proved to be synergistic in DU145 cells.