Synergistic Effect between Human Papillomavirus 18 and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone on Malignant Transformation of Immortalized SHEE Cells

Abstract

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific nitrosamine (TSNA) that induces malignant tumors in rodents. High-risk human papillomavirus (hr-HPV) infection is an important cause of several human cancers. Epidemiological evidence has shown that HPV cooperatively induces carcinogenesis with tobacco smoke. In the present study, the synergistic carcinogenesis of NNK and HPV18 was investigated. Immortalized human esophageal epithelial SHEE cells containing the HPV18 E6E7 gene were constructed by lentiviral transfection. SHEE-E6E7 cells were exposed to NNK along with SHEE-V cells without HPV18 E6E7 as a negative control. The cooperation of NNK and HPV was examined by wound-healing, transwell, and colony-forming assays. The results showed that NNK exposure promoted the migration, invasion, and proliferation abilities of both SHEE-E6E7 and SHEE-V cells; however, the changes in these phenotypic features were remarkably stronger in SHEE-E6E7 cells than those in SHEE-V cells. Our findings indicate that NNK promotes malignant transformation of human esophageal epithelial cells and suggest a synergistic carcinogenesis with the HPV18 E6E7 oncogene. As reported previously, the formation of pyridyloxybutylated DNA adducts is a crucial step in NNK-mediated carcinogenesis. In order to clarify the influence of HPV on the formation of NNK-induced DNA adducts, the amounts of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts were determined using high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry. We observed that the levels of HPB-releasing adducts in SHEE-E6E7 cells were significantly higher (p < 0.01) than those of SHEE-V cells, which was in line with results of the phenotypic assays. In conclusion, this study provides direct evidence that NNK and HPV18 exhibit a synergistic effect on formation of DNA adducts, resulting in malignant transformation of esophageal epithelial cells. Such knowledge on the interaction between infection and smoking habits in the development of cancers informs cancer-prevention strategies. Further studies to delineate the molecular mechanism and to identify specific intervention targets are worthwhile.

Chemopreventive Role of Apigenin against the Synergistic Carcinogenesis of Human Papillomavirus and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

“…However, few evidences have been provided for the DNA alkylation by NNK in HPV-infected cells. In the present and our previous studies [21], we consistently observed that the level of total POB-DNA adducts in HPV-positive cells was significantly higher than that in HPV-negative cells, suggesting synergistic carcinogenesis of HPV and NNK. Moktar et al [45] reported that single-and double-strand DNA breaks induced by cigarette smoke condensate were dose-dependent and persistent in HPV-transformed human ectocervical cells, which gave support to our results.…”

“…Immortalized human esophageal epithelial cells (SHEE) were purchased from the BeNa Culture Collection (BNCC) Biotechnology Company (Beijing, China) and were transfected with the HPV-18 E6E7 gene (SHEE-E6E7) or with empty vector (SHEE-V) by lentiviral transfection described in our previous study [21]. Cells were cultured using Dulbecco's Modified Eagle Medium (DMEM, Hyclone, South Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, DAKEWE, Shenzhen, China) and 1% penicillin and streptomycin (Hyclone, South Logan, UT, USA).…”

“…After pretreated with AP for 24 h, SHEE cells were exposed to NNK in a freshly prepared medium containing 1% S9 rat liver microsome for another 24 h. Then the collected cells were subjected to DNA isolation as described in our previous study [21]. The obtained DNA was dissolved in deionized water containing 30 nM [D 4 ] HPB as the internal standard, followed by acidic hydrolysis with HCl at 80 • C for 3 h to release HPB.…”

“…In recent decades, a number of epidemiological evidences indicate that the synergistic effect between tobacco smoke and hr-HPV shows associations with the occurrence and development of cervical cancer, as well as head and neck cancers [16][17][18][19][20]. Our recent investigation reported that NNK and HPV-18 synergistically promote malignant transformation and DNA damage of human esophageal epithelial cells (SHEE) [21]. Therefore, it is necessary to elucidate the mechanism and explore possible chemoprevention of the synergism between NNK and hr-HPV.…”

Infection with Human Papillomavirus 18 Promotes Alkylating Agent-Induced Malignant Transformation in a Human Esophageal Cell Line

“…71 The latest findings indicated that NNK and HPV18 exhibit a synergistic effect on malignant transformation of esophageal epithelial cells. 72 This evidence suggested that HPV is not the independent risk factor of EC, combined with the fact of high nitrosamine exposure in Huai'an. 73 HPV18 infection Het-1A stable transfected cell model was established, and then Het-1A-HPV18 cells were exposed to MNNG in our study.…”