Lidocaine induces apoptosis in head and neck squamous cell carcinoma through activation of bitter taste receptor T2R14

Highlights

• •Lidocaine activates T2R14 to increase intracellular Ca²⁺ in HNSCC cells
• •Lidocaine decreases cell viability, depolarizes mitochondria, and leads to ROS
• •T2R14 activation with lidocaine induces apoptosis and inhibits the UPS via ROS
• •Lidocaine may be more effective in HPV+ HNSCCs with high TAS2R14 expression

Summary

Head and neck squamous cell carcinomas (HNSCCs) have high mortality and significant treatment-related morbidity. It is vital to discover effective, minimally invasive therapies that improve survival and quality of life. Bitter taste receptors (T2Rs) are expressed in HNSCCs, and T2R activation can induce apoptosis. Lidocaine is a local anesthetic that also activates bitter taste receptor 14 (T2R14). Lidocaine has some anti-cancer effects, but the mechanisms are unclear. Here, we find that lidocaine causes intracellular Ca²⁺ mobilization through activation of T2R14 in HNSCC cells. T2R14 activation with lidocaine depolarizes mitochondria, inhibits proliferation, and induces apoptosis. Concomitant with mitochondrial Ca²⁺ influx, ROS production causes T2R14-dependent accumulation of poly-ubiquitinated proteins, suggesting that proteasome inhibition contributes to T2R14-induced apoptosis. Lidocaine may have therapeutic potential in HNSCCs as a topical gel or intratumor injection. In addition, we find that HPV-associated (HPV+) HNSCCs are associated with increased TAS2R14 expression. Lidocaine treatment may benefit these patients, warranting future clinical studies.

Introduction

Head and neck squamous cell carcinomas (HNSCCs) arise in the mucosa of oral and nasal cavities, larynx, and pharynx, resulting from exposure to environmental carcinogens and/or human papillomavirus (HPV). HNSCCs account for ∼900,000 new cancer diagnoses per year, with a 5-year mortality rate of ∼50%. This high mortality is partly due to frequent late-stage diagnosis, lack of preventive screening, and high rates of metastasis. Surgery, radiation, chemotherapy, and occasionally immunotherapy are possible treatments. However, patients often suffer from side effect burden negatively impacting their quality of life (QoL). Long-term survivors can be left with physical deformities, chronic pain, tracheostomy or feeding tube dependence, and inability to orally communicate, among other impacts. It is vital to develop new targeted therapies to effectively treat HNSCC while maintaining QoL.

A majority of HNSCCs are localized to the oral cavity and oropharynx. A main function of this region is taste perception. Humans can perceive five tastes: sour, salty, sweet, umami, and bitter. Sweet, umami, and bitter tastes are perceived through the activation of specialized G-protein-coupled receptors (GPCRs). GPCR taste receptors are classified into two groups: taste family 1 (T1Rs; sweet and umami) and taste family 2 (T2Rs; bitter) receptors. While there are only 3 T1R isoforms, there are 25 T2R isoforms in humans, which protect against ingestion of bitter tasting toxins. Canonical T2R signaling causes an intracellular calcium (Ca²⁺) increase and cyclic adenosine monophosphate (cAMP) decrease. This Ca²⁺ response is frequently through activation of PLCβ2 via Gβγ subunits of the G-protein heterotrimer, which produces inositol triphosphate (IP₃) to release Ca²⁺ from the endoplasmic reticulum (ER).

Beyond their role in bitter taste perception, T2Rs are involved in innate immunity, thyroid function, cardiac physiology, and other biologic processes. Furthermore, T2Rs have been studied in cancers, including ovarian, breast, and HNSCC. Of the 25 T2Rs, T2R14 is one of the most well studied. We show that some bitter agonists that activate various T2Rs, including T2R14, cause an increase in nuclear and mitochondrial Ca²⁺ that leads to apoptosis in squamous airway epithelial and HNSCC cells. The underlying mechanisms of T2R-induced apoptosis are largely unknown, despite our studies and others linking T2Rs to apoptosis and suggesting they may have therapeutic potential.

Lidocaine is commonly used as a topical or injectable local anesthetic to block voltage-gated sodium (Na_v) channels to inhibit pain signals from sensory neurons. Some studies suggest that lidocaine may also have anticancer effects and decrease the rates of metastasis in some malignancies. It remains a mystery if lidocaine exerts these effects through Na_v channel inhibition or another mechanism. Interestingly, lidocaine is bitter and activates heterologously expressed T2R14. From this, we hypothesized that lidocaine may have pro-apoptotic effects in HNSCCs and possibly other cancers through T2R14.

Here, we show that lidocaine induces Ca²⁺ responses in HNSCC cells through T2R14. Lidocaine decreases HNSCC cell viability, depolarizes the mitochondrial membrane potential (MMP), elevates mitochondrial reactive oxygen species (ROS), and induces apoptosis. Apoptosis is blocked by specific T2R14 antagonists. We also observe that lidocaine inhibits the proteasome in a T2R14-dependent manner, revealing a new mechanism of T2R agonist-evoked apoptosis and a novel link between GPCR signaling and proteasome inhibition. Our data identify the mechanism of action for the apoptotic effects of lidocaine in cancer cells and suggest that lidocaine may be repurposed as a therapeutic for HNSCCs, as either a topical gel or intratumor injection. In addition, we uncover that HPV-associated (HPV+) HNSCCs are associated with increased expression of TAS2R14, the gene encoding T2R14. HPV+ tumors may thus benefit most from T2R14 activation with lidocaine as a treatment.

Results

Lidocaine stimulation causes an intracellular Ca²⁺ response in HNSCC cells

T2R14 is one of 25 T2Rs strongly expressed in oral and extraoral tissues. Lidocaine activation of T2R14 was reported in heterologous expression. We hypothesized that lidocaine activates T2R14 in HNSCC cells. We screened lidocaine against all 25 T2Rs and found significant activation of only T2R14 (Figure S1). To understand if lidocaine activates endogenous T2R14, Ca²⁺ responses were recorded in living HNSCC cells loaded with Fluo-4. Dose-dependent Ca²⁺ responses were observed in SCC47 oral cancer cells with 0–20 mM lidocaine (Figures 1A, 1B, and 1E ). Lidocaine caused higher Ca²⁺ responses than equimolar denatonium benzoate (Figures 1C and 1D), a structurally similar non-T2R14 bitter agonist that induces apoptosis in HNSCC cells. Denatonium benzoate is considered one of the most bitter tasting compounds due to its activation of eight T2Rs (T2R4, 8, 10, 13, 39, 43, 46, and 47). Lidocaine also induced higher Ca²⁺ responses than other T2R agonists at their respective maximum concentrations in aqueous solution, including T2R14 agonists thujone and flufenamic acid as well as purinergic receptor agonist ATP in SCC47, FaDu, and RPMI2650 HNSCCs (Figures 1F–1H). Lidocaine activated Ca²⁺ responses in SCC4 and SCC90 oral cancer cells comparable with FaDu and SCC47 cells (Figures 1I–1K). Unlike lidocaine, the closely related anesthetic procaine, not known to activate T2R14, did not induce Ca²⁺ responses at equivalent concentrations (Figure 1L). Thus, the Ca²⁺ response to lidocaine is not due to Na_v channel inhibition.

Ca²⁺-linked GPCRs, including T2Rs, most often induce intracellular Ca²⁺ responses primarily originating from the ER IP₃ receptor (IP₃R) Ca²⁺ release, with a sustained Ca²⁺ response requiring store-operated Ca²⁺ entry. Stimulation with lidocaine (1–10 mM) in SCC4 and SCC47 cells dose dependently reduced Ca²⁺ responses with secondary ATP (100 μM) stimulation. ATP activates intracellular Ca²⁺ release via purinergic GPCRs. As lidocaine concentration increased, the post-ATP Ca²⁺ response decreased, suggesting depletion of ER Ca²⁺ stores by lidocaine (Figures S2A–S2F). These data suggest that lidocaine and ATP release Ca²⁺ from the same intracellular stores.

We further tested if the lidocaine Ca²⁺ response was from mobilization of intracellular Ca²⁺ stores or extracellular Ca²⁺ influx from plasma membrane Ca²⁺ channels. We recorded Fluo-4 responses in HBSS with or without extracellular Ca²⁺. Cells stimulated with lidocaine in HBSS without Ca²⁺ had a similar initial Ca²⁺ peak as cells stimulated with extracellular Ca²⁺ (Figures 2A and 2B ). However, there was a faster return to baseline fluorescence in cells stimulated in HBSS without Ca²⁺ (Figure 2A). In addition, Na⁺/Ca²⁺ exchanger inhibitor kb-r7943 had no effect on lidocaine-induced Ca²⁺ (Figure S3A). This indicates initial mobilization from intracellular Ca²⁺ stores rather than extracellular stores…