(+)-Usnic Acid Induces ROS-dependent Apoptosis Via Nrf2 Expression In Lung Squamous Cell Carcinoma

Objective: Patients with lung squamous cell carcinoma(LUSC)have poor response to current clinical chemotherapy drugs and targeted drugs.And it is urgent to study effective adjuvant therapy drugs and related combined drug strategies for LUSC.(+)-Usnic acid,an important biologically active metabolite of lichens,has been shown to have high anticancer activity at low doses.However,there have been no reports regarding the effect of(+)-usnic acid on LUSC cells.This study found that(+)-usnic acid reduced viability in LUSC cells and can induce reactive oxygen species(ROS)accumulation by inhibiting the expression of Nrf2(Nuclear factor erythroid 2-related factor 2),thereby significantly inducing apoptosis in LUSC cells.Meanwhile,our studies showed that combined treatment of(+)-usnic acid and paclitaxel synergistically suppressed LUSC cells.This study provides experimental basis for choosing effective adjuvant drugs for clinical anti-LUSC.Experimental data supports the further development of(+)-usnic acid and its combined treatment regimen with paclitaxel.Methods:(1)The MTT assay were used to detect the effects of(+)-usnic acid and the combination of(+)-usnic acid and paclitaxel on the viability of LUSC cells(H520 and Calu-1 cells).(2)Flow cytometry analysis were used to detect the effects on apoptosis and reactive oxygen species(ROS)stimulated by(+)-usnic acid in LUSC cells(H520 and Calu-1 cells).(3)Western blotting was used to detect the effect of(+)-usnic acid on the expression of Nrf2 and pathway-related proteins.(4)Real-Time RTPCR(RT-PCR)were used to detect m RNA expression of Nrf2、HO-1 and NQO-1 in LUSC cells(H520 and Calu-1 cells)by(+)-usnic acid in a dosedependent manner.Results:(1)(+)-Usnic Acid suppresses viability and induces apoptosis in LUSC cells via cellular ROS accumulation.(2)Interference of Nrf2 expression contributes to(+)-Usnic Acid-induced ROS production and apoptosis in LUSC cells.(3)PI3K(Phosphatidylinositol 3-kinases)is an intracellular phosphatidylinositol kinase,the serine/threonine specific protein kinase AKT is also known as PKB(protein kinase B).After the body is stimulated,it can generate phospholipids and combine with PI3 K,produce interaction,PI3K/AKT signaling pathway plays an important role in cell growth.This research shows.(+)-Usnic Acid can inhibit the PI3K/AKT signal activity in H520 and Calu-1 cells.When the PI3K/AKT signal is blocked,(+)-usnic Acid have no additional effects on Nrf2 Expression.PI3K/AKT Signaling Mediates(+)-Usnic Acid-inhibited Nrf2 expression.(4)(+)-Usnic Acid enhances aclitaxel cytotoxicity.Conclusion:(+)-Usnic acid induces ROS accumulation and cell apoptosis,as well as enhances the antitumor efficacy of paclitaxel in LUSC cells by interfering with Nrf2 expression via inhibition of the PI3K/AKT pathway.Therefore,(+)-usnic acid may turn out as promising anticancer candidates for adjuvant treatment of LUSC.