Cytotoxic effects of the cigarette smoke extract of heated tobacco products on human oral squamous cell carcinoma: the role of reactive oxygen species and CaMKK2

Background The increasing prevalence of heated tobacco products (HTPs) has heightened concerns regarding their potential health risks. Previous studies have demonstrated the toxicity of cigarette smoke extract (CSE) from traditional tobacco's mainstream smoke, even after the removal of nicotine and tar. Our study aimed to investigate the cytotoxicity of CSE derived from HTPs and traditional tobacco, with a particular focus on the role of reactive oxygen species (ROS) and intracellular Ca.sup.2+. Methods A human oral squamous cell carcinoma (OSCC) cell line, HSC-3 was utilized. To prepare CSE, aerosols from HTPs (IQOS) and traditional tobacco products (1R6F reference cigarette) were collected into cell culture media. A cell viability assay, apoptosis assay, western blotting, and Fluo-4 assay were conducted. Changes in ROS levels were measured using electron spin resonance spectroscopy and the high-sensitivity 2ʹ,7ʹ-dichlorofluorescein diacetate assay. We performed a knockdown of calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) by shRNA lentivirus in OSCC cells. Results CSE from both HTPs and traditional tobacco exhibited cytotoxic effects in OSCC cells. Exposure to CSE from both sources led to an increase in intracellular Ca.sup.2+ concentration and induced p38 phosphorylation. Additionally, these extracts prompted cell apoptosis and heightened ROS levels. N-acetylcysteine (NAC) mitigated the cytotoxic effects and p38 phosphorylation. Furthermore, the knockdown of CaMKK2 in HSC-3 cells reduced cytotoxicity, ROS production, and p38 phosphorylation in response to CSE. Conclusion Our findings suggest that the CSE from both HTPs and traditional tobacco induce cytotoxicity. This toxicity is mediated by ROS, which are regulated through Ca.sup.2+ signaling and CaMKK2 pathways. Graphical Abstract Keywords: Heated tobacco products (HTPs), Calcium, Oral cancer, Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), Reactive oxygen species (ROS)
Author(s): Nagao Kagemichi[sup.1,2], Masanari Umemura[sup.1], Soichiro Ishikawa[sup.1,3], Yu Iida[sup.1,2], Shota Takayasu[sup.1], Akane Nagasako[sup.1], Rina Nakakaji[sup.1,3], Taisuke Akimoto[sup.2], Makoto Ohtake[sup.2], Takahiro Horinouchi[sup.4], Tetsuya Yamamoto[sup.2] and Yoshihiro Ishikawa[sup.1] Background Tobacco is responsible [...]