DG-8d, a novel diosgenin derivative, decreases the proliferation and induces the apoptosis of A549 cells by inhibiting the PI3k/Akt signaling pathway.Lung neoplasm has a relatively poor prognosis, and the clinical efficacy of targeted medicine remains unsatisfactory.  In our previous study, we showed that a novel diosgenin derivative 8d (DG-8d), which contained 5-(3-pyridyl)-1,3,4-thiadiazole moiety, had significant cytotoxic activity on human tumor cells, especially the A549 cells.  However, the underlying mechanism of DG-8d was unknown.  In this study, the pharmacological effect of DG-8d on the A549 cells was inspected.


 The findings proved that DG-8d could inhibit cell growth and induce apoptosis.  The effect of DG-8d on the proliferation and apoptosis in the A549 cells were improved with LY294002 (PI3K inhibitor).  Moreover, the effect of DG-8d on apoptosis was further confirmed by AO-EB dye, mitochondrial depolarization and accrued intracellular ROS.  Gene and protein detection showed that DG-8d or DG-8d combined with LY294002 could down-regulate signaling molecules of Bcl-2, PI3k, p-Akt, p-FoxO3a and up-regulate signaling molecules of Bax snd Bim.  DG-8d could inhibit the proliferation and induce the apoptosis of the A549 cells, which maybe mainly because of the suppression of the PI3k/Akt pathways.  Finally, we believe that DG-8d can be developed as a possible agent for carcinoma therapy.
DG-8d
Cell viability and apoptosis were detected by CCK-8 assays, morphological changes and quantitative analysis of flow cytometry. 
CCK-8 assays
Cell viability and apoptosis were detected by CCK-8 assays, morphological changes and quantitative analysis of flow cytometry. 
flow cytometry
Levels of gene and protein expression of apoptosis-related and PI3k/Akt pathway were evaluated by qRT-PCR, immunostaining and Western blot analysis.



qRT-PCR
Levels of gene and protein expression of apoptosis-related and PI3k/Akt pathway were evaluated by qRT-PCR, immunostaining and Western blot analysis.



immunostaining
Levels of gene and protein expression of apoptosis-related and PI3k/Akt pathway were evaluated by qRT-PCR, immunostaining and Western blot analysis.



Western blot analysis
The effect of DG-8d on the proliferation and apoptosis in the A549 cells were improved with LY294002 (PI3K inhibitor). 
LY294002 (PI3K inhibitor)
Moreover, the effect of DG-8d on apoptosis was further confirmed by AO-EB dye, mitochondrial depolarization and accrued intracellular ROS. 
AO-EB dye
Moreover, the effect of DG-8d on apoptosis was further confirmed by AO-EB dye, mitochondrial depolarization and accrued intracellular ROS. 
mitochondrial depolarization
Moreover, the effect of DG-8d on apoptosis was further confirmed by AO-EB dye, mitochondrial depolarization and accrued intracellular ROS. 
intracellular ROS