Inhibition of EGFR Suppresses Ethyl Alcohol and Tobacco Cell Effects on Growth of Human Oral Keratinocytes and Human Papillomavirus 16 Entry as a Function of Furin

Abstract

Background: Reported are increased risks for malignant transformation in human oral keratinocytes (HOK) from ethyl alcohol (ETOH), tobacco products or human papilloma virus oncogenic subtype 16 (HPV 16) infections. We examined various HOK cell responses to these factors to show inhibitors of epidermal growth factor receptor (EGFR) also inhibits furin; proprotein convertase (FC) and HPV 16 entry in HOK. Methods: Immortalized HOK by HPV 16 (HPV 16B) or human telomerase (hTERT); primary foreskin keratinocytes (NHFK), primary HOK, buccal keratinocytes (NHBK) and oral SCC-25 were treated with dibenz[a,l]pyrene (DBP), anthraquinone; nitrosamine (NNAL) or ethyl alcohol (ETOH) and acetaldehyde (AA). ETOH was tested for synthesis of malondialdehyde (MDA) and alcohol dehydrogenase expression (ADH). ETOH, and PAH were evaluated by Western immunoblot for oncogene changes, and phosphorylated EGFR expression. Inhibition of EGFR by WZ4002 and Erlotinib and/or carcinogens effect on HPV 16 entry were studied. A green fluorescent pseudovirus (PsV); chloromethylketone (CMK) an inhibitor of furin activity and Western immunoblot of furin cell distribution further characterized HPV 16 entry. Results: ETOH (10 μM) increased expression of phosphorylated EGFR and HPV 16 entry through furin activity, and membrane, nuclear and cytoskeletal accumulations. CMK suppressed HPV 16 entry and blockage of ADH while aldehyde dehydrogenase (ALDH) enhanced HPV 16 entry. Similarly PAH, DBP (4-8 nM), anthraquinone (98 nM) and NNAL (6.9 μM) enhanced HPV 16 entry through furin activity and mem-J. L. Schwartz et al. 91 brane, nuclear and cytoskeletal accumulations. Furthermore, WZ4002 and Erlotinib suppressed expressions of phosphorylated EGFR, FC activity, and HPV 16 entry. ETOH and DBP treatments also enhanced expressions of protease activated receptor-1 (PAR-1), and p21 waf1 while depressed p16 and p27 KIP1 expressions in HOK/HPV 16B cells. Conclusion: EGFR inhibitors are candidates for suppression of alcohol and tobacco effects on EGFR phosphorylated expression; keratinocyte growth, and HPV 16 entry and prevention treatment for HPV related diseases.

Emerging paradigms: unmasking the role of oxidative stress in HPV-induced carcinogenesis

“…Acetaldehyde is considered a ROS that can be formed as a outcome of the metabolism of ethyl alcohol (ethanol) through the action of the ADH enzyme [ 169 , 184 ]. Acetaldehyde, present in premalignant as well as malignant lesions, induces DNA damage by forming bulky adducts and inhibiting DNA repair mechanisms like O6-methylguanine transferase (MGMT) [ 184 , 185 ].…”

“…Acetaldehyde is considered a ROS that can be formed as a outcome of the metabolism of ethyl alcohol (ethanol) through the action of the ADH enzyme [ 169 , 184 ]. Acetaldehyde, present in premalignant as well as malignant lesions, induces DNA damage by forming bulky adducts and inhibiting DNA repair mechanisms like O6-methylguanine transferase (MGMT) [ 184 , 185 ]. It also enhances cellular permeability, causes DNA damage through the generation of bulky adducts like N2-acetaldehyde deoxyguanine, alters the activity of enzyme convertases, and disrupts cellular oxidative balance by binding to glutathione reductase, resulting in lipid peroxidation and the formation of hydroxyl radicals [ 184 ].…”

“…Acetaldehyde, present in premalignant as well as malignant lesions, induces DNA damage by forming bulky adducts and inhibiting DNA repair mechanisms like O6-methylguanine transferase (MGMT) [ 184 , 185 ]. It also enhances cellular permeability, causes DNA damage through the generation of bulky adducts like N2-acetaldehyde deoxyguanine, alters the activity of enzyme convertases, and disrupts cellular oxidative balance by binding to glutathione reductase, resulting in lipid peroxidation and the formation of hydroxyl radicals [ 184 ]. Alcohol dehydrogenase (ADH) first converts ethanol to acetaldehyde, which is then further metabolized by aldehyde dehydrogenases (ALDH) to acetate [ 186 ].…”