Immunohistochemical expression of autophagosome markers LC3 and p62 in preneoplastic liver foci in high fat diet-fed rats

Abstract

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive deposition of droplets in hepatocytes. Patients with NAFLD can be at risk for nonalcoholic steatohepatitis, which can lead to hepatocellular carcinoma. Autophagy is a cellular pathway that is crucial for survival and homeostasis, and which protects against pathophysiological changes like obesity and cancer. We determined the expression of autophagy markers in preneoplastic hepatic lesions and the effects of an autophagy repressor chloroquine (CQ) or inducer amiodarone (AM) in a steatosis-related hepatocarcinogenesis model. Male F344 rats were fed a control diet or high fat diet (HFD), and subjected to initiation and promotion steps with N-nitrosodiethylamine injection at week 0 and a partial hepatectomy at week 3. Several HFD-fed rats were administered 0.1% CQ and 0.5% AM in their drinking water during week 2 and 8. CQ and AM did not improve HFD-induced obesity. AM, but not CQ, significantly decreased the number of glutathione S-transferase placental form-positive preneoplastic liver foci in the liver. Autophagosome markers LC3 and the LC3-binding protein p62 were heterogeneously expressed in the preneoplastic foci. CQ might inhibit autophagy by significantly increased p62/LC3 ratio, while AM might have a potential of inducing autophagy by showing an increased gene expression of the autophagy regulator, Atg5. These results suggest that preneoplastic lesions express autophagosome markers and that AM might decrease steatosis-related early hepatocarcinogenesis by potentially inducing autophagy in HFD-fed rats, while inhibition of autophagy by CQ did not alter the hepatocarcinogenesis. However, an immunohistochemical trial revealed a technical limitation in detecting autophagosome markers because there were variations in each preneoplastic lesion.

Metronidazole enhances steatosis-related early-stage hepatocarcinogenesis in high fat diet-fed rats through DNA double-strand breaks and modulation of autophagy

“…In NASH patients and HFD-fed mice, microtubule-associated protein 1A/1B light chain 3 (LC3), the autophagosome marker, and p62, the autophagic ux marker that binds to LC3, were highly expressed in hepatic samples (González-Rodríguez et al 2014;Zhang et al, 2016). We also con rmed the speci c expression of both autophagosome markers in preneoplastic hepatic foci in HFD-fed rats (Masuda et al 2019). In addition to steatosis, autophagy also plays a role in DNA damage response (DDR).…”

Hydroxysafflor yellow�A induces autophagy in human liver cancer cells by regulating Beclin�1 and ERK expression

“…A number of studies have reported that autophagy can inhibit tumorigenesis (13)(14)(15); however, when tumors form under oxygen-deficient conditions, autophagy is able to promote tumor growth and survival (16). Additionally, >30 autophagy-associated proteins have been identified, including light chain 3 (LC3), which was the first autophagosome protein marker to be discovered, the Beclin 1 complex and p62 (17)(18)(19). The expression of the Beclin 1 complex is closely associated with the occurrence and development of various tumors (18) and p62 is an autophagic substrate protein (19).…”

“…Additionally, >30 autophagy-associated proteins have been identified, including light chain 3 (LC3), which was the first autophagosome protein marker to be discovered, the Beclin 1 complex and p62 (17)(18)(19). The expression of the Beclin 1 complex is closely associated with the occurrence and development of various tumors (18) and p62 is an autophagic substrate protein (19). Therefore, the three aforementioned proteins were selected as target proteins in the present study.…”

Flutamide exacerbates steatosis and promotes early hepatocarcinogenesis in high-fat diet-fed non-obese steatotic rats: Insights from clustering analysis of mitophagy regulators AMBRA1 and LC3

“…In a previous study conducted in our laboratory, FL was observed to enhance precancerous lesions in a medium-term liver carcinogenesis rat model; however, the underlying mechanism remains unclear 27 . In this study, we hypothesized that FL induces mitochondrial injury followed by mitophagy in an HFD-mediated early hepatocarcinogenesis model [28][29][30][31] . This model, based on the medium-term hepatocarcinogenesis assay 32 , served as a valuable tool for understanding the augmentation of autophagy in precancerous lesions facilitated by fatty liver conditions [28][29][30][31] .…”