Pathogenesis of lung cancer [25].Intriguingly, HS7 also dosedependently reduced the activatedphosphorylated ERK protein (pERK) in CL10 cells without having considerably affecting the total ERK protein level (Fig. 6a).Lai et al. Chin Med (2017) 12:Web page 7 ofof downstream element HIF1 have been also enhanced accordingly (Fig. 7b). In comparison, HS7 vigorously inhibits these 3 signaling pathways and HIF1 without the need of the feedback activation of pathway components or compensatory activation of parallel circuits induced by the synthetic inhibitors. This result implies that the multitargeting HS7 could exert additional helpful anticancer activity than the single use of every synthetic signaling pathway inhibitor.Fig. 6 Inhibitory effects of HS7 around the ERK and STAT3 signaling pathways in CL10 lung cancer cells just after 72 h of therapy. a HS7 decreased the protein levels of phosphorylated ERK (pERK) inside a dose dependent manner. b HS7 dosedependently decreased both the protein levels of total (STAT3) and phosphorylated STAT3 (pSTAT3). Cell lysates were analyzed by Western blot, applying tubulin or GAPDH as loading controlHS7 induces CDK inhibitors in CL10 cellsThe signal transducer and activator of transcription three (STAT3) is among the 3 main downstream pathways (AKTmTOR, ERK and STAT3) activated by EGFR phosphorylation, which market proliferation and survival of NSCLC cells [9, 26]. Constitutive activation of STAT3 is also a common feature in NSCLC and it might be activated by JAK2 independent of recognized driver mutations [27]. We were interested to investigate if HS7 also inhibits this prosurvival and proliferative signaling in CL10 cells. As expected, HS7 considerably decreased the activatedphosphorylated STAT3 protein (pSTAT3) at dose of 6.25 g mL and pretty much diminished both the pSTAT3 and total STAT3 protein levels when the dose was increased to 12.five gmL (Fig. 6b).HS7 is far more successful than the synthetic inhibitors around the inhibition of signaling pathways in CL10 cellsTo evaluate the effects of HS7 using the synthetic inhibitors of those 3 signaling pathways, we treated CL10 cells with LY294002 (PI3KAKT inhibitor), U0126 (MEKERK inhibitor), AG490 (JAKSTAT3 inhibitor) for 72 h in the doses which substantially reduced the proliferation of CL10 cells (Fig. 7a). At this 72 h treatment time point, LY294002 didn’t show inhibitory impact on the pAKT protein but decreased the pERK protein as U0126 did (Fig. 7b). On the other hand, almost certainly because of the feedback or compensatory activation at this time point, the reduction of pERK by U0126 or LY294002 was also accompanied with induction of pAKT and pSTAT3 (Fig. 7b). The reduction of pSTAT3 by AG490 also was not observed at this time point, nevertheless, the compensatory induction of pAKT was shown (Fig. 7b). Constant with these feedback or compensatory inductions of pAKT by these synthetic inhibitors, the protein levelsIt has been shown that inhibition of AKT and ERK signaling by the common EGFRTKI gefitinib is accompanied with induction of CDK inhibitors for example p15, p21 and p27, and hypophosphorylation of retinoblastoma protein (pRb) [28, 29]. We therefore examined the alterations of those cellcycle regulating components in Acetylcholinesterase Inhibitors Related Products HS7treated cells. As anticipated, treatment with HS7 for 72 h dosedependently increased the protein levels of p15, p21 and p27 (Fig. 8). As shown in the rightdown side of Fig. 9, induction of these CDK inhibitors can avert the pRb inactivation (phosphorylation) by CDK246 and the subsequent release of E2F.