Synergistic Carcinogenesis of HPV18 and MNNG in Het-1A Cells through p62-KEAP1-NRF2 and PI3K/AKT/mTOR Pathway

Abstract

N-methyl-N´-nitro-N-nitrosoguanidine is a clear carcinogen, increasing evidence that indicates an etiological role of human papillomavirus in esophageal carcinoma. Studies have reported the synergistic effect on environmental carcinogens and viruses in recent years. On the basis of establishing the malignant transformation model of Het-1A cells induced by synergistic of HPV18 and MNNG, this study was to explore the synergistic carcinogenesis of MNNG and HPV. Our research indicated that HPV&MNNG led to a significant increase in the protein-expression levels of c-Myc, cyclinD1, BCL-2, BAX, E-cadherin, N-cadherin, mTOR, LC3II, and p62, with concomitant decreases in p21 and LC3I. HPV18 and MNNG induced accumulation of p62 and its interaction with KEAP1, which promoted NRF2 nuclear translocation. p62 loss prevents growth and increases autophagy of malignant cells by activating KEAP1/NRF2-dependent antioxidative response. In addition, PI3K and p-AKT were stimulated by HPV&MNNG, and PI3K/AKT/mTOR is positively associated with cell proliferation, migration, invasion, and autophagy during malignant transformation. Taken together, MNNG&HPV regulates autophagy and further accelerates cell appreciation by activating the p62/KEAP1/NRF2 and PI3K/AKT/mTOR pathway. MNNG&HPV may improve Het-1A cell autophagy to contribute to excessive cell proliferation, reduced apoptosis, and protection from oxidative damage, thus accelerating the process of cell malignant transformation and leading to cancerous cells.

Figure 1

Protein expression of Het-1A during malignant transformation process (V+: HPV&MNNG group; V-: HPV group; N+: MNNG group; N-: control group; P10: 10^th passage; P20: 20^th passage; P35: 35^th passage).

Figure 2

p62 loss prevents growth of Het-1A-E6E7-MNNG-35 cells. (a) p62 mRNA and protein expression in 35^th Het-1A-E6E7-MNNG cell after silencing of p62. (b, c) Effect of p62 loss on cell cycle and apoptosis was evaluated by the flow cytometry assay. ^∗P < 0.05. (d) Effect of p62 loss on cell function protein.

Figure 3

p62 loss increases autophagy in malignant Het-1A cells. (a) Autophagy-related protein expression in Het-1A-HPV and Het-1A-NC cell with MNNG exposure. Het-1A-HPV and Het-1A-NC cells were treated by MNNG at concentrations of 0, 2, 4, and 8 μmol/L for 24 hours. (b) p62 mRNA and protein expression in Het-1A-HPV after silencing of p62. (c) Autophagy-related protein expression in Het-1A-HPV after silencing of p62. Het-1A-HPV-si-p62 and Het-1A-HPV-si-NC were treated with MNNG at concentrations of 0, 2, 4, and 8 μmol/L for 24 hours.

Figure 4

p62 promotes malignant Het-1A cell autophagy by activating KEAP1/NRF2-dependent antioxidative response. (a) mRNA expression of HO-1, NQO-1, and NRF2 in cells at 35^th passage (V+: HPV&MNNG group; V-: HPV group; N+: MNNG group; N-: control group). (b) Expression of KEAP1 protein and nuclear protein NRF2 in cells at 35^th passage (V+: HPV&MNNG group; V-: HPV group; N+: MNNG group; N-: control group). (c) Oxidative damage of the 35^th Het-1A-HPV-MNNG cells after silencing of p62 (green fluorescence was GFP; red fluorescence was the silver nanocluster at the location of the free radical aggregation shown by the excitation at 580 nm). (d) mRNA expression of SOD-1, SOD-2, HO-1, NQO-1, and NRF2 in the 35^th Het-1A-HPV-MNNG cells after silencing of p62. (e) Expression of KEAP1 protein and nuclear protein NRF2 in the 35^th Het-1A-HPV-MNNG cells after silencing of p62. (f) Interaction between p62 and KEAP1.

Figure 5

HPV&MNNG activates autophagy of Het-1A cells by the PI3K/AKT/mTOR pathway. (a) Expression of PI3K and p-AKT in cells at 10^th passage, 20^th passage, and 35^th passage (V+: HPV&MNNG group; V-: HPV group; N+: MNNG group; N-: control group). (b) Cell activity of 35^th Het-1A-E6E7-MNNG was detected by CCK8 after treatment with LY294002. (c) Protein expression of PI3K/AKT/mTOR pathway after treatment with LY294002. (d) Expression of autophagy-related proteins after treatment with LY294002. (e, f) Het-1A-E6E7-MNNG cell cycle and apoptosis were evaluated by the flow cytometry assay after inhibition of PI3K signal; ^∗P < 0.05. (g) Expression of cell cycle and apoptosis-related proteins after treatment with LY294002.