br Formerly we synthesized a new coumarin derivative with ph
Formerly, we synthesized a new coumarin derivative with phe-nylsulfonylfuroxan, a NO donor (compound 1) (Liu et al., 2014). We proved that the IC50 of compound 1 was 0.26 μM for NSCLC A549 cells, and this hybrid simutaneously triggered caspase-dependent apoptosis and cytoprotective autophagy in NSCLC Dorsomorphin (Wang et al., 2018). To further enhance the anti-lung cancer eﬀect, we synthesized a novel seco-B-ring derivative of coumarin with 4-fluorobenzyl fragment at 3-position of coumarin skeleton (compound 6), which had a better se-lectivity against malignant cells (Guo et al., 2018). This study was aim at revealing the potential of compound 6 for apoptosis and autophagy induction in NSCLC cells as well as the underlying mechanism involved in this process.
In the present study, we first observed that compound 6 displayed
the enhanced capacities to inhibit cell growth and alter morphological features of NSCLC cells compared with the lead compound 1, and its IC50 for A549 cells was only 53 nM. Simultaneously, compound 6 could induce apoptosis, as evidenced by flow cytometry and western blot analysis. The process of apoptosis begins with the release of cytochrome c derived from mitochondrial disruption, which then causes self-clea-vage and activation of caspase-9. Caspase-3, as eﬀector caspases, is downstream of the activator caspases and acts to catalyze proteolytic cleavage of multiple key proteins such as PARP to initiate a caspase cascade, eventually leading to apoptosis (McArthur and Kile, 2018). Our experiments proved that the cleavage of caspase-9 was notably increased after compound 6 exposure, which subsequently activated caspase-3 and then in turn targeted on PARP. The pan-caspase inhibitor z-VAD-fmk could save the cells and reduce the activities of caspase-3 and caspase-9 as well as the cleavage of PARP. Therefore, we conclude that compound 6-induced apoptosis was caspase-dependent in A549
Fig. 6. . Inhibition eﬀect of compound 6 on A549 cells is concerned with PI3K/Akt/mTOR signaling pathway. (A and B) A549 cells were dose- and time- dependently exposed to compound 6. (A) The expressions of mTOR, p-mTOR, p-P70S6K and p-4EBP1 were analyzed by western blotting. (B) The protein level of p-Akt was also analyzed by western blotting. (C) After treated with 3-MA or LY294002, A549 cells were exposed to 50 nM compound 6 in the absence or presence of 50 nM PFOA for 48 h. Cell viability was measured by MTT assay and the data were represented as mean ± SD (**P < 0.01, ***P < 0.001). (D and E) A549 cells were pretreated with LY294002, and then exposed to compound 6 in combination with or without 50 nM PFOA for 48 h. (D) Western blotting was employed to evaluate the expressions of caspase-3, cleaved-caspase 3 and cleaved-caspase 9. (E) The expressions of p-mTOR and p-Akt were also assessed by western blotting. Relative expression of corresponding protein were quantified by Image J software and presented in bar charts (*P < 0.05).
Recently, it has been reported that coumarin and its derivatives can bring about autophagy in prostate cancer, breast cancer, and neuro-blastoma cells (Ren et al., 2016; Suparji et al., 2016; Yao et al., 2016). We observed the double-membrane autophagosomes by electron mi-croscopy in compound 6-treated cells. The occurrence of autophagy was further confirmed by detecting the specially labeled autophagic va-cuoles, the conversion of LC3-I to LC3-II, and the formation of autop-hagic flux. These outcomes suggest that compound 6 can induce
autophagy in cancer cells like its lead compound 1.
The role of autophagy in the chemotherapy of cancer cells is con-cerned by many researchers (Bhat et al., 2018; White et al., 2015). Despite the need to consider diﬀerent cell types and stimuli, most stu-dies show that cytoprotective autophagy preferentially appears in cancer treatment. We have proved that the lead compound 1 induced cytoprotective autophagy that rescued NSCLC cells from cell death (Wang et al., 2018). To elucidate the character of autophagy in com-pound 6-treated NSCLC cells, two kinds of autophagy inhibitors