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Program Number: B041
Session Name: Poster Session

Cigarette Smoke Condensate Attenuates T2R-Mediated Apoptosis in Head and Neck Squamous Cell Carcinoma

Kyle Polen, BS¹; Sarah Sywanycz, BS²; Lily Huang, BS¹; Brianna L Hill, PhD²; Zoey A Miller, BS²; Robert J Lee, PhD²; Ryan M Carey, MD²; ¹Perelman School of Medicine, University of Pennsylvania; ²Department of Otorhinolaryngology-Head & Neck Surgery, University of Pennsylvania Health System

Smoking tobacco has been shown to play a role in the development of head and neck squamous cell carcinomas (HNSCCs), yet up to 50% of patients diagnosed with smoking-related cancers continue to smoke despite counseling. Smoking-related HNSCCs exhibit more aggressive behavior, and continuing to smoke post-diagnosis is associated with worse survival and locoregional control. Smoking has been shown to diminish bitter taste perception, which is interesting in the context of HNSCC due to recent evidence that bitter taste receptors (T2Rs) are also expressed in HNSCC cells, and tumor T2R4 and T2R14 expression levels are associated with survival outcomes. Canonical T2R signaling in taste buds occurs through Gα-mediated cAMP decrease, Gα-activation of phospholipase C, and downstream Ca²⁺ release which leads to “normal” taste perception, but in HNSCC cells, this signaling results in downstream mitochondrial depolarization, reduced proliferation, and apoptosis via caspase-3/7 activation. We hypothesize that cigarette smoke condensate (CSC) exposure may decrease HNSCC cell T2R expression and reduce responses (e.g. Ca²⁺, apoptosis) to T2R agonists.

HNSCC cell lines UM-SCC-47, FaDU, and RPMI 2650, which have varying endogenous expression levels of the 25 members of the T2R receptor family, were used for experimental work. Cells were incubated for 24 and 48 hours in the presence or absence of 25 µg/mL CSC (“smoked” vs “control”). Following exposure, cell responses to T2R agonists denatonium benzoate (15 mM; agonist for T2R4, 8, 10, 13, 39, 43, 46, & 47) and flufenamic acid (500 uM; agonist for T2R14) were assessed with live cell Ca²⁺ imaging (Fluo-4-AM), qPCR normalized to UBC housekeeping gene, and cell viability and apoptosis assays (CellEvent and Crystal Violet). All figures used the following annotations: P < 0.05 (*) and P < 0.01 (**).

qPCR analysis of smoked SCC-47 cells showed significantly lower relative expression of TAS2R4 and TAS2R14 (genes encoding T2R4 and T2R14) compared to control cells (Fig 1). Live cell Ca²⁺ imaging showed that smoked SCC-47 cells exhibit a significantly blunted Ca²⁺ response to denatonium stimulation at 24 and 48 hours and flufenamic acid at 48 hours compared to controls. Smoked FaDu and RPMI 2650 cells showed decreased Ca²⁺ responses to flufenamic acid treatment but no differences to denatonium stimulation at 24 hours compared to controls (Fig 2). An apoptosis assay detecting caspase-3/7 activity showed that 24-hour and 48-hour smoked SCC-47 cells underwent significantly reduced denatonium-induced cell apoptosis compared to controls. Viability assays demonstrated similar results with reduced denatonium-mediated cell death in smoked SCC-47 cells (Fig 3).

HNSCC cigarette smoke exposure reduced TAS2R4 and TAS2R14 gene expression and decreased calcium and apoptosis responses to T2R agonists. As T2Rs continue to be investigated alongside the development of directed therapies, patients’ exposure to cigarette smoke may be an important consideration in understanding tumor behavior and responsiveness to T2R treatment or endogenous ligands.

 

 

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