Effects of Petasitin as Natural Extract on Proliferation and Pathological Changes of Pediatric Neuroblastoma SK-N-SH Cells

Abstract

This research was developed to investigate the impact and mechanisms of petasitin as natural extract on the proliferation and pathological changes of pediatric neuroblastoma SK-N-SH cells. The sample cells were selected as experimental materials and randomly rolled into a control (Ctrl), a low-dose (LD), a medium-dose (MD), and a high-dose (HD) group, which were subjected to regular culture, 0.5, 1.5, and 5 μM/L petasitin for 12 hours, respectively. Meanwhile, the cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry (FCT) was employed to assess the cycle progression and apoptosis of SK-N-SH cells, while Hoechst 33258 fluorescent staining was applied to observe the nuclear changes. Meanwhile, the real-time quantitative PCR (RT-qPCR) was applied to measure the mRNA levels of PI3K, PTEN, AKT1, and mTOR genes in SK-N-SH cells. Western blot was conducted to determine the protein expression levels (PELs) of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR in SK-N-SH cells. The results revealed that after treatment for 48 and 72 hours, the optical density (OD) values in the petasitin treatment groups were lower and exhibited great differences to those in the Ctrl group (P < 0.05), while inhibition rates (IRs) were higher (P < 0.05). Furthermore, the petasitin treatment groups exhibited an obvious increase in the G1/G0 cell cycle ratio (CCR) and a great decrease in S and G2/M phase CCR (P < 0.05). Moreover, the apoptotic rates in the petasitin treatment groups were much higher at different time points, showing observable differences with P < 0.05. Besides, the Hoechst 33258 staining positivity rates (PRs) of SK-N-SH cell nuclei in the petasitin treatment groups were higher and presented great differences with P < 0.05 to those in the Ctrl group. In addition, the cells in petasitin treatment groups exhibited greatly downshifted mRNA levels of PI3K and AKT1 and obviously elevated PTEN (all P < 0.05). PELs of PI3K, AKT1, and mTOR exhibited no great differences (P > 0.05), but phosphorylation levels of p-PI3K, p-Akt, and p-mTOR were decreased in the petasitin treatment groups, presenting great differences with P < 0.05. Together, these results suggested that petasitin exerted a suppressive role in proliferation and promote the apoptosis of pediatric SK-N-SH cells by adjusting PI3K-Akt-mTOR signaling pathway (SPW).