The Role of Autophagy in Usnic Acid-Induced Toxicity in Hepatic Cells

Abstract

The use of usnic acid and usnic acid-containing products is associated with acute liver failure; however, mechanistic studies of hepatotoxicity caused by usnic acid are limited. In this study, we investigated and characterized the possible mechanisms, especially the role of autophagy in usnic acid's toxicity in human HepG2 cells. Usnic acid caused apoptosis as demonstrated by an increased caspase-3/7 activity and an increased subdiploid nucleus formation. Usnic acid-induced autophagy as demonstrated by the conversion of LC3B-I to LC3B-II, degradation of P62, and an increased number of puncta. Inhibition of autophagy by treating cells with autophagy inhibitors (3-methyladenine or chloroquine) or by small interfering RNA against Atg7 aggravated usnic acidinduced apoptosis and decreased cell viability, indicating that autophagy plays a protective role against usnic acid-induced toxicity. Moreover, usnic acid activated the MAPK signaling pathway. Usnic acid-elicited apoptosis was enhanced and autophagy was decreased when JNK was suppressed by a specific inhibitor. Additionally, inhibition of autophagy decreased the activity of JNK. Taken together, our results suggest that usnic acid perturbs various interrelated signaling pathways and that autophagy induction is a defensive mechanism against usnic acid-induced cytotoxicity.

Activation of the Nrf2 signaling pathway in usnic acid-induced toxicity in HepG2 cells

“…Increased accumulation in S phase could be attributed to an increase in cells that are actively replicating DNA; however, this may not be the case for usnic acid treatment. Our previous report showed that usnic acid inhibited cell proliferation (Chen et al 2014a), and our current results show that usnic acid reduces the expression of key cyclin-related proteins that are critical for S phase progression, as indicated in Fig. 2.…”

“…Although different sensitivities were observed in distinct cells, we obtained similar toxicity results with multiple endpoints. Our mechanistic studies in HepG2 cells revealed that various signaling pathways were affected by usnic acid (Chen et al 2014a, 2015). In this study, we continued to use this cell line, not only because the liver is the primary target organ for usnic acid’s toxicity (Guo et al 2008), but also the feasibility and capability of HepG2 cells for genetic modifications (silencing genes of interest in this study) enabled us to elucidate in-depth molecular mechanisms (Chen et al 2014a).…”

“…The sub-diploid DNA peaks and cell cycle distribution of the HepG2 cells were measured using flow cytometry-based cell cycle analysis as described previously (Chen et al 2014a). …”

“…The scramble shRNA sequence does not contain significant homology to known genes and was used as a negative control in silencing experiments. The generation of the lentiviral shRNA vectors and the establishment of the stable HepG2 cell line derivatives were described previously (Chen et al 2014a). In total, two stable cell lines were generated, namely SC (scramble control) and sh-Nrf2.…”

“…Our previous studies demonstrated that usnic acid disturbs cell cycle progression and induces apoptosis in HepG2 cells (Chen et al 2014a). Usnic acid treatment increased ROS generation in both HepG2 cells and mouse primary hepatocytes, which was detected using the cell permeable redox-sensitive fluorescent dyes dihydrodichlorofluorescein diacetate and CellROX Green (Han et al 2004; Sahu et al 2012).…”