Simultaneous activation and blockade of autophagy to fight hepatocellular carcinoma

Abstract

ABSTRACTAutophagy is considered a target for cancer treatment, although few compounds manipulating this process have been added to the anticancer arsenal in humans. Pharmacological manipulation of autophagy has therefore been considered in the treatment and chemosensitization of hepatocellular carcinoma (HCC), a heterogeneous malignancy that remains difficult to treat (limited impact of genomic discoveries for the implementation of personalized precision medicine). We analyzed the autophagy marker proteins p62 and LC3 in paired tumor and adjacent cirrhotic non-tumor tissues of human HCC. We show strong variability in p62 and LC3-II levels between tumor parts of different HCC patients and between tumor and non-tumor HCC in the same patient, suggesting heterogeneity in autophagy flux. This diversity in flux led us to consider a non-personalized method of autophagy targeting, combining simultaneous activation and blockade of autophagy, which could, in theory, benefit a substantial number of HCC patients, irrespective of tumor autophagic flux. We show that the combination of sodium butyrate (NaB, autophagy inducer) and chloroquine (CQ, autophagy blocker) has a marked and synergistic cytotoxic effect in vitro on all human liver cancer cell lines studied, compared with the cellular effect of each product separately, and with no deleterious effect on normal hepatocytes in culture. Cancer cell death was associated with accumulation of autophagosomes, induction of lysosome membrane permeabilization and increased oxidative stress. Our results suggest that simultaneous activation and blockade of autophagy may be a valuable approach against HCC, and that microbiota-derived products improve the sensitivity of HCC cells to antitumor agents.Abbreviations AV: annexin V; CI: combination index; CTSB: Cathepsin B; CTSD: Cathepsin D; CTSF: Cathepsin F; CQ: chloroquine; DEN: N-diethylnitrosamine; DMEM: Dulbecco’s modified eagle medium; FBS: fetal bovine serum; FSC: forward scatter; GNS: N-acetylglucosamine-6-sulfatase; HCC: hepatocellular carcinoma; HDACi: histone deacetylase inhibitor; HCQ: hydroxychloroquine; LMP: lysosomal membrane permeabilization; LAMP1: lysosome-associated membrane protein; LIPA: Lysosomal acid lipase; LSR: Lysosomal staining cells; MAP1LC3A: microtubule associated protein 1 light chain 3 alpha; NaB: sodium butyrate; NASH: non-alcoholic steatohepatitis; NRF2: nuclear factor erythroid 2–related factor 2; PI: propidium iodide; PMSF: phenylmethanesulfonyl fluoride; ROS: reactive oxygen species; SCARB2: Scavenger receptor class B member 2; SQSTM1/p62: sequestosome 1; SMPD1: Sphingomyelin phosphodiesterase 1; SSC: side scatter; TFEB: transcription factor EB.

Keywords

• Liver cancer
• cholangiocarcinoma
• microbial metabolite
• butyrate
• chloroquine
• therapy