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. 2018 Nov 28;23(12):3110.
doi: 10.3390/molecules23123110.

FAK and S6K1 Inhibitor, Neferine, Dually Induces Autophagy and Apoptosis in Human Neuroblastoma Cells

Dinh-Chuong Pham  1 , Yu-Chuan Chang  2 , Shian-Ren Lin  3 , Yuh-Ming Fuh  4 , May-Jywan Tsai  5 , Ching-Feng Weng  6
Affiliations

FAK and S6K1 Inhibitor, Neferine, Dually Induces Autophagy and Apoptosis in Human Neuroblastoma Cells

Dinh-Chuong Pham et al. Molecules. .

Abstract

Human neuroblastoma cancer is the most typical extracranial solid tumor. Yet, new remedial treatment therapies are demanded to overcome its sluggish survival rate. Neferine, isolated from the lotus embryos, inhibits the proliferation of various cancer cells. This study aimed to evaluate the anti-cancer activity of neferine in IMR32 human neuroblastoma cells and to expose the concealable molecular mechanisms. IMR32 cells were treated with different concentrations of neferine, followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to assess cell viability. In an effort to determine the molecular mechanisms in neferine-incubated IMR32 cells, cell cycle arrest, cell migration, and focal adhesion kinase (FAK), the 70-kDa ribosomal S6 kinase 1 (S6K1), poly (ADP-ribose) polymerase (PARP), caspase-3, Beclin-1, and microtubule-associated protein 1A/1B-light chain 3 (LC3) protein expressions were investigated. Neferine strongly disrupted the neuroblastoma cell growth via induction of G2/M phase arrest. Furthermore, neferine provoked autophagy and apoptosis in IMR32 cells, confirmed by p-FAK, and p-S6K1 reduction, LC3-II accumulation, Beclin-1 overexpression, and cleaved caspase-3/PARP improvement. Finally, neferine markedly retarded cell migration of neuroblastoma cancer cells. As a result, our findings for the first time showed an explicit anti-cancer effect of neferine in IMR32 cells, suggesting that neferine might be a potential candidate against human neuroblastoma cells to improve clinical outcomes with further in vivo investigation.

Keywords: FAK/S6K1; apoptosis; autophagy; human neuroblastoma cells; neferine.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Neferine suppresses cell proliferation in human neuroblastoma cells. (A,B) IMR32 cells were treated with 1, 10, 20, and 30 μM of neferine or 20, 50, 100, and 400 μM of TMZ for 24 h; (C,D) Normal human astrocytes (NHA) were exposed to the indicated doses of neferine and TMZ for 24 h. Cell viability was analyzed by MTT assay, and the surviving cells were determined and presented as a percentage of the non-treated cells. Data are presented as mean ± standard deviation (SD) in three independent experiments. * p < 0.05, *** p < 0.001 as compared with the non-treated control.
Figure 1
Figure 1
Neferine suppresses cell proliferation in human neuroblastoma cells. (A,B) IMR32 cells were treated with 1, 10, 20, and 30 μM of neferine or 20, 50, 100, and 400 μM of TMZ for 24 h; (C,D) Normal human astrocytes (NHA) were exposed to the indicated doses of neferine and TMZ for 24 h. Cell viability was analyzed by MTT assay, and the surviving cells were determined and presented as a percentage of the non-treated cells. Data are presented as mean ± standard deviation (SD) in three independent experiments. * p < 0.05, *** p < 0.001 as compared with the non-treated control.
Figure 2
Figure 2
Neferine induces G2/M cell cycle arrest in human neuroblastoma cells. (A,B) IMR32 cells were cultured at the described concentrations of neferine (A) or TMZ (B) for 24 h, and then flow cytometry analysis was used to determine the cell cycle distributions; (C,D) The quantification of cell cycle arrest in neferine-treated or TMZ-treated IMR32 cells was shown. Data are expressed as mean ± SD in three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 as compared with the non-treated control.
Figure 2
Figure 2
Neferine induces G2/M cell cycle arrest in human neuroblastoma cells. (A,B) IMR32 cells were cultured at the described concentrations of neferine (A) or TMZ (B) for 24 h, and then flow cytometry analysis was used to determine the cell cycle distributions; (C,D) The quantification of cell cycle arrest in neferine-treated or TMZ-treated IMR32 cells was shown. Data are expressed as mean ± SD in three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 as compared with the non-treated control.
Figure 3
Figure 3
Neferine inhibits FAK, p-FAK, S6K1, and p-S6K1 protein levels in human neuroblastoma cells. (A,B) IMR32 cells were incubated with the desired concentrations of neferine (A) or TMZ (B) for 24 h. Then, all cells were harvested and lysed for Western blot analysis; (C,D) Changes in the levels of FAK, p-FAK, S6K1, and p-S6K1 proteins after being normalized to the levels of beta actin were presented. Data are shown as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 as compared with the non-treated control.
Figure 3
Figure 3
Neferine inhibits FAK, p-FAK, S6K1, and p-S6K1 protein levels in human neuroblastoma cells. (A,B) IMR32 cells were incubated with the desired concentrations of neferine (A) or TMZ (B) for 24 h. Then, all cells were harvested and lysed for Western blot analysis; (C,D) Changes in the levels of FAK, p-FAK, S6K1, and p-S6K1 proteins after being normalized to the levels of beta actin were presented. Data are shown as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 as compared with the non-treated control.
Figure 4
Figure 4
Neferine triggers autophagy in human neuroblastoma cells. (A,B) IMR32 cells were exposed to the indicated concentrations of neferine (A) or TMZ (B) for 24 h. Then, autophagy-related proteins, including Beclin-1 and LC3, were assessed using Western blot analysis; (C,D) Changes in the levels of Beclin-1, LC3-I, and LC3-II after being normalized to the levels of beta actin were shown. The data are expressed as mean ± SD in three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 as compared with the non-treated control.
Figure 4
Figure 4
Neferine triggers autophagy in human neuroblastoma cells. (A,B) IMR32 cells were exposed to the indicated concentrations of neferine (A) or TMZ (B) for 24 h. Then, autophagy-related proteins, including Beclin-1 and LC3, were assessed using Western blot analysis; (C,D) Changes in the levels of Beclin-1, LC3-I, and LC3-II after being normalized to the levels of beta actin were shown. The data are expressed as mean ± SD in three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 as compared with the non-treated control.
Figure 5
Figure 5
Neferine induces apoptosis in human neuroblastoma cells. (A,B) IMR32 cells were incubated at the described concentrations of neferine (A) or TMZ (B) for 24 h. Then, apoptosis-related proteins, including cleaved Caspase-3 and PARP, were determined using Western blot analysis; (C,D) Changes in the levels of cleaved Caspase-3 and PARP after being normalized to the levels of beta actin were displayed. Data are shown as mean ± SD in three independent experiments. * p < 0.05, ** p < 0.01 as compared with the non-treated control.
Figure 6
Figure 6
Neferine inhibits migration in human neuroblastoma cells. (A,B) IMR32 cell monolayer was scratched and treated with neferine (A) or TMZ (B) in time-dependent manner for wound-healing migration assay. Images were accessed by inverted microscope; (C,D) The quantification of cell migration in wound healing assay after treatment with neferine (C) or TMZ (D) were measured. Data are shown as percentages of the recovered scratch area relative to non-treated control. Results are presented as mean ± SD in three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared with the non-treated control.
Figure 6
Figure 6
Neferine inhibits migration in human neuroblastoma cells. (A,B) IMR32 cell monolayer was scratched and treated with neferine (A) or TMZ (B) in time-dependent manner for wound-healing migration assay. Images were accessed by inverted microscope; (C,D) The quantification of cell migration in wound healing assay after treatment with neferine (C) or TMZ (D) were measured. Data are shown as percentages of the recovered scratch area relative to non-treated control. Results are presented as mean ± SD in three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared with the non-treated control.
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