Figure 1.
Long term treatment of nicotine alters mRNA levels of epithelial to mesenchymal transition expression in head and neck squamous cell carcinoma cell lines UMSCC10B and HN-1.
A) Snail, B) Twist, C) Vimentin were all induced corresponding to an increased EMT phenotype. D) Western blot showing increased EMT expression in Snail and decreased E-cadherin in UMSCC10B and HN1 following long-term exposure to nicotine. E) Nicotine-treated HN-1 cells (ii) had a more mesenchymal morphology compared to non-treated cells (i). F) Effect of nicotine on expression of EMT markers in normal oral keratinocytes. G) EMT is confirmed by immunofluorescence, with nicotine treated cells displaying higher expression of the mesenchymal marker vimentin. All images were photographed under 40X. *p<0.05, **p<0.01.
Figure 2.
Long term treatment of nicotine increases mRNA expression of stem cell genes in head and neck squamous cell carcinoma cell lines UMSCC10B and HN-1.
A) CD44, B) Bmi1, C) Oct4, D) Nanog. E) Effect of nicotine on expression of stem cell markers in normal oral keratinocytes. *p<0.05, **p<0.01.
Figure 3.
Increased cell survival, sphere formation and tumor size after exposure to nicotine.
A) Nicotine enhanced the ability of 10B and HN-1 cells to form colonies under low serum conditoins, suggesting increased survival capacity. B) Photos on left are representative images of spheres formed by nicotine-treated or control HN-1 cells. Graph on right indicates the significant increase in number of spheres formed by nicotine-treated HN-1 cells compared to untreated cells. C) Representative photos comparing tumor sizes between 1 mM-nicotine-treated HN-1 cells and untreated HN-1 cells. D) Relative expression of the multipotency regulator INHBA between nicotine-treated cells and control cells. *p<0.05, **p<0.01.
Figure 4.
Long-term nicotine exposure increases invasiveness of 10B and HN-1.
A) Bar graph indicating the results of a matrigel invasion assay. B) Representative wells of invaded 10B cells. Permutations include control, control with addition of 0.1 mM nicotine during the invasion assay, cells treated long term in 0.1 mM nicotine without addition of nicotine during assay, cells treated long term in 0.1 mM nicotine with addition of 0.1 mM nicotine during assay. C) Representative wells of invaded HN-1 cells, with same permutations as above. *p<0.05, **p<0.01, ***p<0.001.
Figure 5.
Long-term nicotine exposure increases size of tumors formed in nude mice.
A) Fluorescent whole-body imaging of nude mice each injected with 500 k of either control or 1 mM-nicotine-treated HN-1 cells overexpressing RFP. B) Photographs and weights of tumors dissected from mice 47 days post-injection.
Figure 6.
Long-term nicotine exposure increases tumorigenicity.
A) Fluorescent whole-body imaging of nude mice each injected with 5 k of either control or 1 mM-nicotine-treated HN-1 cells overexpressing RFP. B) Photographs and weights of tumors dissected from mice 54 days post-injection.
Figure 7.
MicroRNA profile of nicotine-treated vs non-treated cells.
A) Log expression values are computed based on fold expression from the median value within each row. Genes were excluded based on the criteria of having at least 20% of expression values showing a 1.5 fold increase or decrease relative to the median. Downregulated genes that clustered with miR-101 and upregulated genes that clustered with miR-9 were magnified within the figure. B) qPCR validation of miR-9 and miR-101 expression.
Figure 8.
Overexpression of E-cadherin decreases expression of (A) Oct-4, (B) Nanog, (C) Snail, and (D) Twist, indicating a reversal in phenotype induced by long term nicotine treatment.
Expression values are compared against an empty vector control. E) indicates the overexpression level of e-cadherin.
Figure 9.
Effect of nicotine withdrawal and subsequent reintroduction on long term nicotine-treated cells.
Bar graphs showing expression of (A) Snail, (B) Vimentin, (C) BMI1, (D) CD44, (E) Oct-4, (F), Nanog under various conditions.