Inflammation and Invasion in Oral Squamous Cell Carcinoma Cells Exposed to Electronic Cigarette Vapor Extract

Abstract

Electronic cigarettes (eCig) represent a new avenue of tobacco exposure that involves heating oil-based liquids and the delivery of aerosolized flavors with or without nicotine, yet little is known about their overall health impact. The oral cavity is an anatomic gateway for exposure that can be compromised by activating myriad of signaling networks. Oral squamous cell carcinoma (OSSC) is a common malignancy affecting 30,000 people in the United States each year. Our objective was to determine the impact of eCig and nicotine on gingival OSSC invasion and their secretion of pro-inflammatory molecules. Gingiva-derived Ca9-22 cells and tongue-derived Cal27 cells were exposed to eCig vapor extract (EVE) generated from Red Hot or Green Apple (Apple) flavored eCig solution +/- nicotine for 6 hours. Isolation of protein lysates and collection conditioned media was done after treatment. Real-time cellular invasion was assessed using a RTCA DP instrument. Protein expression was determined using western blot. Compared to controls, we observed: elevated NF-kB, TNF-α, ERK, JNK, MMP-13 and cell invasion by Ca9-22 treated with Apple EVE; increased TNF-α and JNK by Ca9-22 treated with Red Hot EVE; and increased TNF-α and JNK by Cal27 cells treated with both Apple and Red Hot EVE. We conclude that eCig flavoring and nicotine orchestrated differential cell invasion and inflammatory effects. This study provides an important initial step in dissecting mechanisms of cancerous invasion and molecular avenues employed by OSCC.

Keywords: OSCC; eCIG; gingiva; inflammation; invasion.

Figure 1

Expression of NF-kB, pERK, and pJNK in Ca9-22 cells exposed to EVE. ELISA was performed to determine NF-kB released into the cell medium. ERK and JNK were determine by western blot. Released of NF-kB in the media was increased only with Apple EVE and nicotine EVE in treated Ca9-22 cells as compared to media only treated controls (A). A representative western blot results for ERK and JNK us shown in (B) ERK protein was activated in cells treated with Apple EVE and Nicotine and significantly decreased in cells exposed to Red Hot EVE with nicotine (C). JNK protein was activated by both flavors in Ca9-22 cells when nicotine was present (D). Data are shown with *p ≤ 0.05.

Figure 2

| Expression of NF-kB (A), pERK (B), and pJNK (C) in Cal27 cells exposed to EVE. ELISA was performed to determine NF-kB released into the cell medium. ERK and JNK were determine by western blot. Released NF-kB was decreased with both flavors and nicotine in these cells (A) as compared to media only treated controls cells. A representative western blot results for ERK and JNK is shown in (B) Activation of ERK and JUNK proteins protein was decreased after exposure to both flavoring with nicotine except for induction of pJNK by Red Hot (C, D). Data are shown with *p ≤ 0.05.

Figure 3

Expression of TNF-α in OSCCs exposed to EVE. ELISA was performed to determine TNF-α released into the cell medium. Released TNF-α levels were increased in conditioned media from Ca9-22 (A) and Cal27 (B) exposed to both EVE flavors plus nicotine. Data are shown with *p ≤ 0.05.

Figure 4

Ca9-22 invasion with Green Apple or Red Hot EVE. A representative diagram of readout cells over time invasion is shown in (A). At 24 hours of culture, Green Apple EVE did not affect Ca9-22 invasion with or without nicotine (B, C). Treatment with Red Hot EVE decreased Ca9-22 invasion (D) and Red Hot with nicotine enhanced Ca9-22 invasion (E) when compared to c untreated controls. Data are shown with *p ≤ 0.05.

Figure 5

Cal27 invasion with Green Apple or Red Hot EVE. A representative diagram of readout cells over time invasion is shown in (A). Green Apple EVE did not affect Cal27 invasion in cells with or without nicotine (B, C). Red Hot EVE reduced Cal27 invasion in cells with and without nicotine (D, E) as compared to untreated controls. Data are shown with *p ≤ 0.05.

Figure 6

MMP-9 and MMP-13 during Red Hot EVE treatment of Ca9-22 cells. A representative Ca9-22 MMP9 and 13 western blot picture is shown in (A) Western blot analysis showed decreased MMP-9 when Ca9-22 cells were treated with Red Hot EVE in the presence or absence of nicotine (B). MMP-13 was increased in cells exposed to Red Hot EVE with nicotine (C) when compared to untreated control cells. Data are shown with *p ≤ 0.05.

Figure 7

MMP-9 and MMP-13 during Red Hot EVE treatment of Cal27 cells. A representative Cal27 MMP9 and 13 western blot picture is shown in (A) Western blot analysis showed that MMP-9 did not change when cells were treated with Red Hot EVE with or without nicotine (B) when compared to untreated control cells. MMP-13 was decreased with Red Hot EVE treatment alone (C). Data are shown with *p ≤ 0.05.