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. 2022 Jun;13(3):1423-1432.
doi: 10.21037/jgo-22-298.

Effects of 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione on autophagy and the PI3K/AKT/mTOR signaling pathway in human cholangiocarcinoma QBC939 cells

Yongfei He #  1 , Yanghong Li #  2 , Tianyi Liang  1 , Shutian Mo  1 , Yuan Liao  1 , Zijun Chen  1 , Shuqi Zhao  1 , Qingfu Ran  3 , Chuangye Han  1   4 , Pham Thi Thai Hoa  5
Affiliations

Effects of 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione on autophagy and the PI3K/AKT/mTOR signaling pathway in human cholangiocarcinoma QBC939 cells

Yongfei He et al. J Gastrointest Oncol. 2022 Jun.

Abstract

Background: 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) has been reported to have good antitumor effects. The aim of this study was to investigate whether DMDD induces apoptosis and autophagy in human cholangiocarcinoma (CCA) QBC939 cells and determine its effect on the PI3K/AKT/mTOR signaling pathway.

Methods: QBC939 cells were cultured in vitro and changes in cell viability were detected by the Cell Counting Kit (CCK8) assay after treatment with different concentrations of DMDD for 24, 48, and 72 h. The cells were divided into control and DMDD-treated groups (treated concentrations were 10, 15, and 20 µM/L), and the cell cycle, apoptosis, and autophagic vesicles were assessed. The expression levels of PI3K, AKT, mTOR, microtubule-associated protein 1 light chain 3 beta (LC3-II)/I, Beclin-1, and P62 were detected by Western blot. A xenograft mouse model was constructed to detect the effect of DMDD on CCA.

Results: The experimental results showed that DMDD was able to inhibit proliferation, migration, and invasion and induce cell cycle arrest and autophagy of QBC939 cells. In addition, DMDD decreased the protein expression of PI3K, AKT, and mTOR and increased the expression of LC3-II/I, Beclin-1, and P62. In mice, DMDD was able to inhibit the growth of tumors.

Conclusions: DMDD inhibits CCA cell viability and induces cell cycle arrest and autophagy by a mechanism that may be related to the downregulation of the PI3K/AKT/mTOR signaling pathway.

Keywords: 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD); PI3K/AKT/mTOR; autophagy; cholangiocarcinoma (CCA).

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-298/coif). The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Effect of DMDD on the proliferation of QBC939 cells. DMDD, 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione.
Figure 2
Figure 2
Effect of DMDD on the migration and invasion of bile duct cancer cells. (A,B) Effect of different concentrations of DMDD on the migration ability of the QBC939 cell line. (C,D) Effect of different concentrations of DMDD on the invasion ability of the QBC939 cell line. (A) Cell migration was observed by scanning electron microscopy. (C) Stained with crystal violet. Compared with the control group, *, P<0.05; ***, P<0.001. DMDD, 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione; CK, control; L, DMDD L group; M, DMDD M group; H, DMDD H group.
Figure 3
Figure 3
Effect of DMDD on the cell cycle of cholangiocarcinoma cells. (A,B) The effect of different concentrations of DMDD on the cell cycle of QBC939 cells. Compared with the control group, *, P<0.05; ***, P<0.001. DMDD, 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione; CK, control; L, DMDD L group; M, DMDD M group; H, DMDD H group; G1, DNA synthesis; S, DNA synthesis; G2, DNA synthesis.
Figure 4
Figure 4
Effect of DMDD on autophagic vesicles in cholangiocarcinoma cells. (A,B) Effect of different concentrations of DMDD on autophagic vesicles in the QBC939 cell line. Monodansylcadaverine stains the cells. Compared with the control group, **, P<0.01; ***, P<0.001. DMDD, 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione; CK, control; L, DMDD L group; M, DMDD M group; H, DMDD H group.
Figure 5
Figure 5
Effect of DMDD on the PI3K/AKT/mTOR signaling pathway in cholangiocarcinoma cells. (A) Western blot analysis. (B-G) Expression analysis of PI3K, AKT, mTOR, Beclin-1, P62, and LC3-II/I proteins. Compared with the control group, *, P<0.05; **, P<0.01; ***, P<0.001. DMDD, 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione; CK, control; L, DMDD L group; M, DMDD M group; H, DMDD H group.
Figure 6
Figure 6
DMDD inhibits tumor growth in vivo. (A) Tumor images. (B) Tumor volume curve after treatment. DMDD, 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione; CK, control; L, DMDD L group; M, DMDD M group; H, DMDD H group.
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