Autophagy Plays a Critical Role in Insulin Resistance- Mediated Chemoresistance in Hepatocellular Carcinoma Cells by Regulating the ER Stress

Abstract

The high mortality of hepatocellular carcinoma (HCC) patients is associated with several independent risk factors including type 2 diabetes mellitus (T2DM) and insulin resistance (IR), which could be caused by various pathological processes such as tumorigenesis and inflammation in the liver. In previous report, we declared that IR contributes to multidrug resistance in HCC by activation of endoplasmic reticulum (ER) stress. Here, our study revealed that the enhanced autophagy induced by IR significantly prompts the chemotherapeutic drug resistance in hepatoma cells, which was validated by stimulation and inhibition of the autophagy respectively. A potential reason is that autophagy acts as a regulator of ER stress in the IR-mediated chemoresistance in HCC. In conclusion, autophagy facilitates the HCC survival in chemotherapeutic drug treatment by maintaining the homeostasis in the ER indicating the regulatory role of autophagy in ER stress contributes to IR-mediated chemoresistance in hepatocellular carcinoma cells. Collectively, these data implied inhibition of autophagy is a potential treatment of inherent IR-mediated chemoresistance in HCC.

Keywords: Autophagy; Chemoresistance; Endoplasmic reticulum; Hepatocellular carcinoma; Insulin resistance.

Figure 1

Analysis of autophagy activation in HepG2/IR cells. (A) Ultrastructure detected by transmission electron microscopy (TEM) (original magnification, ×20,000). The fluorescence intensity of MDC was observed by fluorescence light microscopy (FLM) (original magnification, ×1,000). (B) HepG2 cells were treated with 0.5 μmol/L insulin for 72 hr to induce HepG2/IR cells, and then treated with 10mmol/L PH for 24 hours to reverse IR. Cell lysates were used for autophagy markers detection by immunoblotting. The quantitative data are the Beclin-1/β-actin, LC3-II/LC3-I, P62/β-actin. All experiments were repeated three times, and data were presented as mean ± SD of triplicate experiments. ** P<0.01 vs HepG2 cells.

Figure 2

Autophagic enhancement promotes IR-mediated chemotherapeutic drug resistance in hepatoma cells. (A-D) Autophagy markers were detected by immunoblotting at 48 hr following treatment with 16mg/L DDP with or without pretreated by 2 μmol/L RAPA for 4 hr in both HepG2 and HepG2/IR cells. The quantitative data are the Beclin-1/β-actin (B), LC3-II/LC3-I (C) and P62/β-actin (D). (E-F). HepG2 and HepG2/IR cells were exposed to 16mg/L DDP for 48 hr with or without pretreated by 2μmol/L RAPA for 4 hr and then harvested. Apoptotic cell rates were detected with Annexin V-FITC/PI double staining assay and then acquired by flow cytometry. The apoptotic cell rate is shown (F). (G-I) The analysis of cleaved Caspase-3 and Bcl-2 at 48 hr following treatment with 16mg/L DDP with or without pretreated by 2μmol/L RAPA for 4 hr by Western blot. The quantitative data are the Bcl-2/β-actin (H) and Cleaved Caspase-3/β-actin (I). (J) FLM, original magnification×1,000. (K) TEM, original magnification×2,000. All experiments were repeated three times, and data were presented as mean ± SD of triplicate experiments. * P<0.1, ** P<0.01, vs control group of HepG2 cells; △ P<0.1, △△ P<0.01, vs control group of HepG2/IR cells; # P<0.1, ## P<0.01. vs HepG2 cells in the same treatment group.

Figure 3

Autophagy inhibition impaired IR-mediated chemotherapeutic drug resistance in hepatoma cells. (A-D) Autophagy markers were detected by immunoblotting at 48 hr following treatment with 16mg/L DDP with or without pretreated by 2 mmol/L 3-MA for 4 hr in both HepG2 and HepG2/IR cells. The quantitative data are the Beclin-1/β-actin (B), LC3-II/LC3-I (C) and P62/β-actin (D). (E-F) HepG2 and HepG2/IR cells were exposed to 16mg/L DDP for 48 h with or without pretreated by 2mmol/L 3-MA for 4 hr and then harvested. Apoptotic cell rates were detected with Annexin V-FITC/PI double staining assay followed by flow cytometry. The apoptotic cell rate is shown (F). (G-I) The analysis of cleaved Caspase-3 and Bcl-2 at 48 hr following treatment with 16mg/L DDP with or without pretreated by 2mmol/L 3-MA for 4 hr by Western blot. The quantitative data are the Bcl-2/β-actin (H) and Cleaved Caspase-3/β-actin (I). (J) FLM, original magnification×1,000. (K) TEM, original magnification×2,000. All experiments were repeated three times, and data were presented as mean ± SD of triplicate experiments. * P<0.1, ** P<0.01, vs control group of HepG2 cells. △ P<0.1, △△ P<0.01, vs control group of HepG2/IR cells. # P<0.1, ## P<0.01 vs HepG2 cells in the same treatment group.

Figure 4

Autophagy regulates the ER stress response in HepG2/IR cells. (A) FLM, original magnification×1,000 and TEM, original magnification×2,000. (B) Western blot analysis of GRP78, XBP-1, PERK and p-PERK following 4 hr treatment by 2μmol/L RAPA or 2mmol/L 3-MA. The quantitative data are the GRP78/β-actin, XBP-1/β-actin and p-PERK/PERK. All experiments were repeated three times, and data were presented as mean ± SD of triplicate experiments. * P<0.1, ** P<0.01, vs control group of HepG2 cells. △ P<0.1, △△ P<0.01, vs control group of HepG2/IR cells. # P<0.1, ## P<0.01 vs HepG2 cells in the same treatment group.