The organotypic raft assay was performed in the absence of TNF. cells that express HPV16 E6 and E7. Chronic TNF exposure successfully induced the malignant growth and stemness phenotype in the E6-expressing cells but not in the control and E7-expressing cells. We further demonstrated that HPV16 E6 played a key role in TNF-induced cancer stemness suppression of the stemness-inhibiting microRNAs miR-203 and miR-200c. Overexpression of miR-203 and miR-200c suppressed cancer stemness in TNF-treated HPV16-immortalized cells. Overall, our study suggests that chronic inflammation promotes cancer stemness in HPV-infected cells, thereby promoting HPV-associated oral carcinogenesis. a Notch-dependent pathway.31 Furthermore, recent studies have demonstrated that the proinflammatory cytokines TGF and TNF generate CSCs in human cancer.32C34 In the present study, we investigated the effect of chronic inflammation on HPV-associated oral carcinogenesis by treating HPV-immortalized and non-tumourigenic human oral keratinocytes with TNF for extended periods and studied the phenotypic and molecular biological changes. Results Chronic TNF exposure induces calcium resistance in HPV-immortalized cells but not in non-HPV-immortalized cells. Two immortalized oral keratinocyte cell lines (HPV16-immortalized HOK-16B and hTERT-immortalized OKF6/tert) were used Lorcaserin in this study. Keratinocytes normally proliferate in low-Ca2+ (0.15?mmolL?1) keratinocyte growth medium (KGM) but not in high-Ca2+ (1.5?mmolL?1) DMEM containing 10% serum. Proliferation capacity at the physiological calcium level (1.5?mmolL?1), also known as calcium resistance, Lorcaserin is a transformed phenotype of keratinocytes.35 To investigate the effect of inflammation on HPV-associated carcinogenesis, we first examined the effect of short-term proinflammatory cytokine TNF exposure Mouse monoclonal to CD15.DW3 reacts with CD15 (3-FAL ), a 220 kDa carbohydrate structure, also called X-hapten. CD15 is expressed on greater than 95% of granulocytes including neutrophils and eosinophils and to a varying degree on monodytes, but not on lymphocytes or basophils. CD15 antigen is important for direct carbohydrate-carbohydrate interaction and plays a role in mediating phagocytosis, bactericidal activity and chemotaxis (2C10 days) on the proliferation of HPV-positive HOK-16B and HPV-negative OKF6/tert cells in low-Ca2+ medium (Fig. ?(Fig.1a).1a). The short-term TNF exposure had no significant effect on cell growth. Interestingly, after Lorcaserin 4 months of exposure to TNF, HOK-16B cells showed enhanced proliferation capacity in the high-Ca2+ medium and no signs of keratinocyte differentiation and cell death; they were named 16B/TNF (Fig. ?(Fig.1b).1b). However, after the same period of exposure, OKF6/tert cells failed to show enhanced proliferation capacity in the high-Ca2+ medium and were named OKF/TNF (Fig. ?(Fig.1b).1b). Moreover, high Ca2+ markedly increased the expression of differentiation markers, i.e., keratin 1 (KRT1), KRT10, and involucrin (INV), in HOK-16B but not in 16B/TNF cells Lorcaserin (Fig. ?(Fig.1c).1c). Our data indicate that chronic TNF treatment resulted in calcium resistance and a significant reduction in the differentiation potential of the HPV-positive HOK-16B cells. Since TNF is known to affect HPV viral gene expression,24 we measured Lorcaserin the expression levels of E6 and E7 in HOK-16B and 16B/TNF cells (Fig. ?(Fig.1d).1d). E6 and E7 expression levels were not altered by TNF in the HPV16-immortalized oral keratinocytes. Collectively, our findings suggest that the acquired calcium resistance of 16B/TNF cells is independent of the overexpression of E6/E7 by TNF in HPV16-immortalized oral keratinocytes. Open in a separate window Fig. 1 Chronic TNF exposure induces calcium resistance in HPV-immortalized oral keratinocytes.a HPV16-immortalized HOK-16B and hTERT-immortalized OKF6/tert cells were exposed to TNF (5?ngmL?1) in low-Ca2+ (0.15?mmolL?1) keratinocyte growth medium (KGM) for the indicated days, and the cell numbers were counted. b HOK-16B and OKF6/tert cells were exposed to TNF (5?ngmL?1) for 4 months in low-Ca2+ medium to generate 16B/TNF and OKF/TNF cells, respectively. Then, the cell proliferation capacity in high-Ca2+ (1.5?mmolL?1) DMEM containing 10% serum was determined by cell counting. Cells were seeded at a density of 2??104 cells and counted after the indicated incubation period. Passage-matched controls, HOK-16B and OKF6/tert cells, were used for comparison with 16B/TNF and OKF/TNF cells, respectively. c The effect of high Ca2+ on the expression of differentiation markers was determined by qPCR using HOK-16B and 16B/TNF cells. The cells were cultured in low- or high-Ca2+ medium for 2 days and harvested for the assay. *test. d.
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