Fig. 5: β2-AR and EGFR pathways regulate the ROS metabolism NRF-2 mediated.

Evaluation of the ROS metabolism in cancer cells directly obtained from the HNSCC-bearing mice. a Western Blot analysis of Nrf-2 expression in both cytoplasmic and nuclear protein extracts. ICI treatment reduced the Nrf-2 nuclear translocation, in a more evident way if in combination with CTX. b RT-PCR for the HO-1, NQO-1, GCLC, and G6PD gene expression level analysis. The β2-AR blockade diminishes the gene expression level of HO-1, with a synergistic effect in combination with CTX, which is able to induce a milder effect by itself. The MEK 1/2 inhibition did not replicate this effect. No significant effects have been observed about the NQO-1 expression. The expression of GCLC and G6PDH is significantly reduced only after treatment with ICI in combination with CTX. c Immunohistochemistry assay on mice tongues engrafted with UMSCC 103 for the HO-1 and NQO1 detection. The ICI and CTX treatments sensibly reduced the expression of HO1, which increases if we combine the drugs. The expression of NQO1 was affected only in mice subjected to the combination of ICI plus CTX. d Cell-ROX assay for the evaluation of the oxidative stress in UMSCC 103 induced by our drugs. With both flow cytometer and e fluorescent microscopy analysis, we observed an increased level of oxidation after treatment with the β2-AR inhibitor, as well as with CTX and U0126. The same drugs are even more effective in combination, but this increased effect is counteracted by the KI696. (*P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001 vs. CTR).