2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione mediates the effect of ROS-enhanced PI3K/Akt/mTOR pathway on autophagy in breast cancer

Abstract

Several studies have suggested a potential antitumor effect of 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD). To further understand the mechanism of action of this compound, we investigated its effect on the phosphatidylinositol-3-kinase (PI3K)/serine-threonine kinase (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. We show that DMDD application significantly inhibited the proliferation of breast cancer cell lines MDA-MB-231 and ER-α positive MCF-7. Furthermore, DMDD application resulted in increased intracellular reactive oxygen species (ROS) levels, apoptosis and autophagy, whereas it downregulated the expression of PI3K, Akt and mTOR mRNA and proteins, and increased the expression of LC3II/I and p62 proteins. In a mouse breast cancer xenograft model, DMDD inhibited tumor growth. Expression analyses suggest that ROS levels were higher in DMDD treated tumor tissues, whereas immunohistochemical analyses suggest that apoptotic cells were more prevalent in the DMDD treated group compared to the control group. Taken together, our results suggest that the molecular mechanism of action of DMDD may involve the enhancement of breast cancer autophagy through the PI3K/Akt/mTOR signaling pathway by mediating ROS expression.

Keywords: 2‐dodecyl‐6‐methoxycyclohexa‐2,5‐diene‐1,4‐dione; PI3K/Akt/mTOR signaling pathway; ROS; autophagy; breast cancer.

Fig. 1

2‐dpdecyl‐6‐methoxycyclohexa‐2,5‐diene‐1,4‐dione (DMDD) chemical constitution.

Fig. 2

(A, C) Cell viability was analyzed by a cell counting kit (CCK)‐8 assay. The cells were incubated with DMDD at different concentrations for 24/48 h. (B, D) Cell viability was analyzed by a CCK‐8 assay. The cells were incubated with PTX at different concentrations for 24/48 h. (E, F) Cell proliferation ability was detected by a colony formation assay. The cells were incubated with 1 μmol·L⁻¹ PTX or DMDD at different concentrations for 24 h. The assay results were expressed as the relative value of the experimental group/control group. (G, H) Cell migration ability was detected by a transwell assay. The cells were incubated with 1 μmol·L⁻¹ PTX or DMDD at different concentrations for 24 h (scale bar = 100 μm). The assay results were expressed as the relative value of the experimental group/control group. DMDD, 2‐dpdecyl‐6‐methoxycyclohexa‐2,5‐diene‐1,4‐dione; PTX, paclitaxel; I, control; II, PTX; III, DMDD‐L; IV, DMDD‐M; V, DMDD‐H. All data are presented as the mean ± SD from three biologically independent experiments, one‐way ANOVA analysis was adopted for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, vs. Control).

Fig. 3

(A, C) Cell invasion ability was detected by transwells assay. The cells were incubated with 1 μmol·L⁻¹ PTX or DMDD at different concentrations for 24 h (scale bar = 100 μm). The assay results were expressed as the relative value of the experimental group/control group. (C, D) Cell cycle was detected by flow cytometry. The cells were incubated with 1 μmol·L⁻¹ PTX or DMDD at different concentrations for 24 h. DMDD, 2‐dpdecyl‐6‐methoxycyclohexa‐2,5‐diene‐1,4‐dione; PTX, paclitaxel; I, control; II, PTX; III, DMDD‐L; IV, DMDD‐M; V, DMDD‐H. All data are presented as the mean ± SD from three biologically independent experiments, one‐way ANOVA analysis was adopted for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, vs. Control).

Fig. 4

(A, B) Cell apoptosis level was detected by flow cytometry. The cells were incubated with 1 μmol·L⁻¹ PTX or DMDD at different concentrations for 24 h. (C, D) ROS levels in cells was detected by flow cytometry. The cells were incubated with 1 μmol·L⁻¹ PTX or DMDD at different concentrations for 24 h. The assay results were expressed as the relative value of the experimental group/control group. (E, F) The level of autophagy was detected by a Monodansyla damantanamine‐autophagy detection kit. The cells were incubated with 1 μmol·L⁻¹ PTX or DMDD at different concentrations for 24 h. DMDD, 2‐dpdecyl‐6‐methoxycyclohexa‐2,5‐diene‐1,4‐dione; PTX, paclitaxel; I, control; II, PTX; III, DMDD‐L; IV, DMDD‐M; V, DMDD‐H. All data are presented as the mean ± SD from three biologically independent experiments, one‐way ANOVA analysis was adopted for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, vs. Control).

Fig. 5

DMDD promoted the proliferation of MCF‐7 and MDA‐MB231 cells through PI3K/Akt/mTOR signaling pathway. (A–D) The protein levels of mTOR, PI3K, Akt, LC3I and LC3II in MCF‐7 and MDA‐MB231 cells were determined using Western blotting. The amount of each product was normalized to the housekeeping gene β‐Actin. The cells were incubated with 1 μmol·L⁻¹ PTX or DMDD at different concentrations for 24 h. (E) mTOR, PI3K and Akt mRNA expression in MCF‐7 and MDA‐MB231 cells. The amount of each product was normalized to the housekeeping gene GAPDH. The cells were incubated with 1 μmol·L⁻¹ PTX or DMDD at different concentrations for 24 h. DMDD, 2‐dpdecyl‐6‐methoxycyclohexa‐2,5‐diene‐1,4‐dione; PTX, paclitaxel; I, control; II, PTX; III, DMDD‐L; IV, DMDD‐M; V, DMDD‐H. All data are presented as the mean ± SD from three biologically independent experiments, one‐way ANOVA analysis was adopted for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, vs. Control).

Fig. 6

DMDD inhibits tumor growth in breast cancer mice. (A) Construction of mouse breast cancer xenografts and drug intervention timeline. (B) Mouse tumors, from left to right: model group, paclitaxel treatment group, DMDD low, medium and high groups. (C) The tumor growth curve of mouse breast cancer xenografts from day 5 to the day 20; the vertical axis is the tumor volume and the horizontal axis is the growth time. (D) Akt, PI3K and mTOR mRNA expression in mouse tumor tissue. The amount of each product was normalized to the housekeeping gene GAPDH. (E) ROS level in mouse tumor tissue. The assay results were expressed as the relative value of the experimental group/control group. (F, G) HE staining, Tunel staining and immunohistochemistry in mouse tumor tissue; yellow and red arrows refer to the damaged and apoptotic tissues. Scale bar = 100 μm. Paclitaxel, DMDD, 2‐dpdecyl‐6‐methoxycyclohexa‐2,5‐diene‐1,4‐dione. I, model; II, PTX; III, DMDD‐L; IV, DMDD‐M; V, DMDD‐H. All data are presented as the mean ± SD from three biologically independent experiments, one‐way ANOVA analysis was adopted for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, vs. model).