Capsaicin Targets tNOX (ENOX2) to Inhibit G1 Cyclin/CDK Complex, as Assessed by the Cellular Thermal Shift Assay (CETSA)

Abstract

Capsaicin (8-methyl-N-vanillyl-6-noneamide), which is an active component in red chili peppers, is used as a chemopreventive agent that shows favorable cytotoxicity against cancer cells. Accumulating evidence indicates that capsaicin preferentially inhibits a tumor-associated NADH oxidase (tNOX, ENOX2) that is ubiquitously expressed in cancer but not in non-transformed cells. This attenuates cancer cell growth by inducing apoptosis. The capsaicin-mediated inhibition of tNOX was recently shown to prolong the cell cycle. However, the molecular events underlying this regulation have not yet been investigated. In the present study, we used a cellular thermal shift assay (CETSA) to detect “target engagement” of capsaicin and its consequent impact on cell cycle progression. Our results indicated that capsaicin engaged with tNOX and triggered the proteasomal degradation of tNOX, which leads to the inhibition of NAD+-dependent SIRT1 deacetylase. Ultimately, the acetylation levels of c-Myc and p53 were enhanced, which suppressed the activation of G1 cyclin/Cyclin-dependent kinase complexes and triggered cell cycle arrest in cancer cells. The results obtained when tNOX was overexpressed in non-cancer cells validated its importance in cell cycle progression. These findings provide the first molecular insights into the regulatory role of tNOX and the anti-proliferative property of capsaicin in regulating the cell cycle of bladder cancer cells.

A superior loading control for the cellular thermal shift assay

“…Moreover, both APP-CTFs (α- and β) behaved similarly upon temperature exposure while APP-βCTF levels were not influenced by the presence of a binding ligand either. Other commonly used loading controls, vinculin, β-actin, GAPDH, HSP70 and SOD-1, all exhibited temperature sensitivities in our study, as previously reported 7 – 11 . By means of example, we validated the use of APP-αCTF as a CETSA loading control for the kinase, MEK (mitogen-activated protein kinase kinase), in the presence of two inhibitors using the wild-type HEK293 cell line.…”

“…Traditional qWB loading controls have been utilised previously but are ill-suited for this unintended purpose. Vinculin, β-actin, GAPDH, α-tubulin and even heat-shock chaperone 70 (HSP70) decrease in concentration as temperature increases 7 – 11 . Superoxide dismutase-1 (SOD-1), a promising candidate owing to its reported stable range of 35–95 °C 12 – 14 , was found to transition through different unfolded states with increasing temperature 13 , 15 .…”

Unveiling the Molecular Mechanisms Driving the Capsaicin-Induced Immunomodulatory Effects on PD-L1 Expression in Bladder and Renal Cancer Cell Lines

“…In BC, CPS triggers the proteasomal degradation of tNOX, leading to the inhibition of NAD+-dependent SIRT1 deacetylase and the enhancement of c-Myc and p53 that suppresses G1 cyclin-dependent kinase activation and triggers cell cycle arrest [ 14 ]. Moreover, by downregulating SIRT1, CPS enhances the acetylation of cortactin and β-catenin to decrease MMP-2 and MMP-9 activation and impair cell migration in BC cells [ 15 , 16 ]. CPS suppresses cell proliferation, and induces cell cycle arrest and reactive oxygen species (ROS) production in BC cells, through FOXO3a-mediated pathways [ 17 , 18 ].…”

Current strategies and technologies for finding drug targets of active components from traditional Chinese medicine

“…Atikul et al used the CETSA technology to verify the binding of capsaicin to the cell cycle-related enzyme tNOX, and found that capsaicin can bind to tNOX in T24 bladder cancer cells. This combination inhibits the production of NAD+ and down-regulates tNOX, thereby inhibiting sirtuin-1 (SIRT1) deacetylase activity [93]. Ultimately, decreased SIRT1 deacetylase activity will enhance the acetylation of c-Myc and p53, leading to cell cycle arrest, thereby inhibiting the growth of cancer cells.…”