Understanding the Pro‐Apoptotic Mechanisms of Bitter Local Anesthetics in Head and Neck Squamous Cell Carcinoma

Within head and neck squamous cell carcinomas (HNSCCs), oral and oropharyngeal squamous cell carcinomas (SCCs) affect ~34,000 people in the US each year. Patients face a 50% 5-year survival rate and overall decline in quality of life due to morbidities of current treatments. Novel targeted therapies are needed for SCCs to prolong survival and decrease off-target effects of treatment. Due to the oral localization of SCCs, bitter taste receptor (T2R) GPCRs have sparked interest as potential therapeutic targets. We showed that upregulated T2R expression may be associated with higher overall survival in HNSCC and may serve as a predictive biomarker. T2R activation by bitter agonists induces apoptosis in HNSCC cells. Here, we show that bitter local anesthetics have apoptotic effects in HNSCC cells: SCC47, SCC4, and FaDu. Lidocaine (10 mM; a concentration within the clinically used range) activates T2R calcium responses that are inhibited by pertussis toxin, an inhibitor of Gi/o - coupled GPCRs, and by suramin, an inhibitor of Ga . This was distinct from the structurally-related T2R agonist denatonium, which activates calcium responses inhibited by Gq inhibitor YM-254890 but not pertussis toxin. Lidocaine induces initial calcium release from intracellular reservoirs sustained by calcium influx. HNSCC cells have a larger calcium response with lidocaine than with T2R agonists denatonium, thujone, procaine, and caffeine. Lidocaine exhausts cells and inhibits subsequent secondary purinergic calcium responses. Using biosensors localized to the ER or mitochondria, we found that ER calcium is depleted upon lidocaine stimulation while mitochondrial calcium is increased. Using lysosomal K+/ H+ proton gradient inhibitor nigericin, we observed that lidocaine also causes calcium depletion of lysosomal stores. Consistent with ER depletion, lidocaine stimulates ER stress, upregulating XBP-1. Lidocaine reduces NADH metabolism via XTT assay, indicative of reduced cellular health. Lidocaine also reduces mitochondrial potential as measured by JC-1 dye and simultaneously promotes apoptosis, with cleavage of both caspase-3 and -7, measured by Western and live cell imaging. Interestingly, lidocaine upregulates both cleaved and full-length caspase-3 and -7 proteins, even in the presence of cycloheximide. The mRNA products of both caspase proteins are not upregulated. This suggests a possible inhibition of protein degradation involved in lidocaine-induced apoptosis. Induction of pro-apoptotic protein BAX also occurs upon lidocaine stimulation. Lidocaine also induced cell death in Matrigel culture. Together, our data show that lidocaine should be viewed as more than a local anesthetic within HNSCC surgical settings. Lidocaine or other T2R agonists may aid in eradication of residual cancer cells and extend time between initial surgery and reoccurrence/metastasis as an alternative or complementary therapy. With their promising effects on oral and oropharyngeal SCC cells and accessibility of the oral cavity to topically applied lidocaine gels, further work is warranted to understand the effects of bitter local anesthetics and T2R signaling in HNSCC and normal surrounding epithelia.