Dichloroacetate induces protective autophagy in LoVo cells: involvement of cathepsin D/thioredoxin-like protein 1 and Akt-mTOR-mediated signaling

Abstract

Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK), and recently it has been shown as a promising nontoxic antineoplastic agent. In this study, we demonstrated that DCA could induce autophagy in LoVo cells, which were confirmed by the formation of autophagosomes, appearance of punctate patterns of LC3 immunoreactivity and activation of autophagy associated proteins. Moreover, autophagy inhibition by 3-methyladenine (3-MA) or Atg7 siRNA treatment can significantly enhance DCA-induced apoptosis. To determine the underlying mechanism of DCA-induced autophagy, target identification using drug affinity responsive target stability (DARTS) coupled with ESI-Q-TOF MS/MS analysis were utilized to profile differentially expressed proteins between control and DCA-treated LoVo cells. As a result, Cathepsin D (CTSD) and thioredoxin-like protein 1 (TXNL1) were identified with significant alterations compared with control. Further study indicated that DCA treatment significantly promoted abnormal reactive oxygen species (ROS) production. On the other hand, DCA-triggered autophagy could be attenuated by N-acetyl cysteine (NAC), a ROS inhibitor. Finally, we demonstrated that the Akt-mTOR signaling pathway, a major negative regulator of autophagy, was suppressed by DCA treatment. To our knowledge, it was the first study to show that DCA induced protective autophagy in LoVo cells, and the potential mechanisms were involved in ROS imbalance and Akt-mTOR signaling pathway suppression.

Figure 1

DCA induced autophagosome formation in LoVo cells. (a) Representative TEM depicting ultrastructures of LoVo cells treated with 29 mM DCA for 48 h. The percentage of the cells with autophagosomes was analyzed from at least 100 randomly chosen TEM fields. (b) The punctate patterns of LC3 staining was observed in the cytoplasm of the LoVo cells in response to 29 mM DCA treatment for 24 h, whereas LC3-GFP could not be seen in the control. Notably, a significant increase in both the percentage of the cells with punctate patterns of LC3 and the average number of punctate patterns of LC3 per cell was observed in LoVo cells when the treatment time was prolonged to 48 h. (c) LC3-II protein was detectable in LoVo cells following DCA treatment for 24 h, whereas the expression of LC3-II was undetectable in the control. (d) Western blot assay of Beclin 1, Atg7 and Atg12-Atg5 conjugate using lysates from LoVo cells treated with PBS (control) or 29 mM DCA for 48 h. β-Actin was used as a loading control. All data are representative of three independent experiments. *P<0.05; **P<0.01

Figure 2

Inhibition of autophagy augmented DCA-induced proliferation inhibition and apoptosis in LoVo cells. (A) Representative images of transmission electron microscopy (upper) and LC3 immunofluorescence staining (lower). (B) The percentage of the cells with autophagosomes was markedly decreased when treated with 3-MA plus DCA compared with DCA alone (P<0.05). (C) The LC3 dots per cell were significantly decreased in 3-MA plus DCA-treated LoVo cells compared with treatment with DCA alone (P<0.01). (D) DCA-triggered apoptosis was markedly enhanced in the presence of 3-MA compared with treatment with DCA alone, 3-MA alone and PBS control (P<0.01). a, PBS control; b, DCA; c, 3-MA; d, DCA plus 3-MA. (E, F) Cell viability and cell number were also significantly reduced when treated with DCA plus 3-MA compared with DCA alone and 3-MA alone (P<0.05). (G, H) Cell number and viability of DCA-treated LoVo cells were also significantly reduced when silencing Atg7 by siRNA (P<0.01). Western blot was conducted to evaluate interference efficiency. *P<0.05; **P<0.01

Figure 3

Identification of molecular targets of DCA using DARTS method. (a) After treatment with DCA (29 mM DCA), LoVo cell lysates were subjected to thermolysin digestion and Coomassie staining. On MS analysis, two differential protein bands were identified to be CTSD and TXNL1. (b) The protein levels of CTSD and TXNL1 were examined by western blotting in LoVo cells following DCA (29 mM) treatment for 48 h. β-Actin was used as a loading control. **P<0.01

Figure 4

ROS mediated DCA-induced autophagy in LoVo cells. (a) Cellular ROS generation was assayed by DCFH-DA staining after treated with PBS, 1.2 mM DCA, 6 mM DCA and 30 mM DCA for 48 h in LoVo cells. (b, c) The levels of both SOD and CAT activity did not change significantly when treated with different concentrations of DCA for 48 h (P>0.05). (d) Cellular ROS generation was reduced by treatment with a combination of 30 mM DCA and 10 mM NAC for 48 h compared with DCA treatment alone (P<0.05). (e, f) The percentage of the cells with autophagosomes, and the LC3 dots per cell were markedly reduced in DCA plus NAC-treated LoVo cells compared with DCA alone (P<0.05). (g) Representative picture of transmission electron microscopy (upper) and the punctate patterns of LC3 staining (lower). Cells were treated with DCA (30 mM) or NAC (10 mM) for 48 h. (h) Western blots assay of autophagy-related proteins. β-Actin was used as a loading control. The data are representative of three independent experiments. *P<0.05; **P<0.01

Figure 5

DCA inhibited the Akt-mTOR signaling pathway in LoVo cells. Cell lysates from LoVo cells either treated with PBS (control) or 29 mM DCA for 48 h were subjected to western blot analysis. β-Actin was used as a loading control