br All these results suggested
All these results suggested that compound 6-induced cytotoxic eﬀect and apoptosis were very likely enhanced by PI3K inhibition.
Inactivation of PI3K/Akt/mTOR signaling pathway after compound 6 exposure in NSCLC cells
Frequently, the PI3K/Akt/mTOR pathway is hyperactivated in di-verse human cancers, which may result in tumorigenesis and drug re-sistance. To assess the assignment of PI3K/Akt/mTOR signaling pathway in compound 6-treated NSCLC cells, we primarily examined the level of phosphorylated mTOR by western blotting and found that the phosphorylation of mTOR-S2448 declined in a dose- and time-dependent manner (Fig. 6A). Subsequently, we found that the expres-sions of the upstream protein p-Akt as well as two downstream sub-strates p-p70S6K and p-4E-BP1 were all eﬃciently suppressed after compound 6 exposure (Fig. 6A and B). Thus, compound 6 could
downregulate PI3K/Akt/mTOR signaling pathway and the synthesis of related protein in some degree.
As a further verification, perfluorooctanoic Malonyl Coenzyme A (PFOA), an mTOR activator, was applied to reverse the inhibitory activity of compound 6 on PI3K/Akt/mTOR pathway. As expected, compound 6-induced cell viability reduction and apoptosis were visibly improved by PFOA compared with compound 6-treated alone (Fig. 6C and D). Western blotting revealed that the phosphorylation of Akt and mTOR-S2448 both increased after pretreatment with PFOA (Fig. 6E). In addition, the inhibition of cell viability and apoptosis induced by compound 6 combined with 3-MA or LY294002 were also reversed by PFOA (Fig. 6C and D). Fig. 6E further illustrated that 3-MA or LY294002 synergisti-cally enhanced the anti-tumor activity by acting as inhibitors of PI3K pathway indeed.
In summary, the PI3K/Akt/mTOR signaling pathway was in-activated after compound 6 exposure in NSCLC cells.
Natural bioactive compounds have become an essential source of innovative anti-cancer drugs due to their novel structure, less adverse reactions and some special therapeutic eﬀects (Rejhova et al., 2018). Coumarin, a class of natural compounds found in a variety of plants, is beneficial to chemoprevention and human health (Song et al., 2015). A number of compounds containing the coumarin moiety exhibit diverse
Fig. 5. PI3K signaling pathway inhibitors promote the cytotoxicity and apoptosis induced by compound 6 in A549 cells. (A–D) A549 cells were exposed to 50 nM compound 6 in combination with or without 2 mM 3-MA or 10 μM LY294002 for 48 h. (A) The protein levels of LC3-I/II were evaluated by western blotting. (B) MTT assay was applied to detect the cell viability. The data were represented as mean ± SD (**P < 0.01, ***P < 0.001). (C) Cell apoptosis was analyzed by flow cytometry after Annexin-V and PI double-staining for 15 min. The percentage of Annexin V-positive cells was presented in bar charts as mean ± SD (*P < 0.05). (D) Western blotting was employed to evaluate the protein levels of PARP, cleaved-PARP, cleaved-caspase 3 and cleaved-caspase 9. (E–F) A549 cells were treated with the indicated concentrations of compound 6 and LY294002 alone as well as in combination with constant ratio for 48 h. (E) Cell viability was measured by MTT assay (IC50 of LY294002 = 19.76 μM). *P < 0.05, ***P < 0.001, statistically significant diﬀerence between compound 6 and combination treatment. The data was cal-culated by CompuSyn software. (F) Combination index plot (left) and isobologram (right), in which CI values <1, =1 and >1 indicate synergism, additive eﬀect and antagonism, respectively.
and eﬀective biological or pharmaceutical activities, which raises considerable research interest in their synthesis from last few decades (Li et al., 2015, 2016).