Limits...
Metformin inhibition of neuroblastoma cell proliferation is differently modulated by cell differentiation induced by retinoic acid or overexpression of NDM29 non-coding RNA.

Costa D, Gigoni A, Würth R, Cancedda R, Florio T, Pagano A - Cancer Cell Int. (2014)

Bottom Line: In this study we report the antiproliferative effect of metformin treatment in a high risk neuroblastoma cell model, focusing on possible effects associated to different levels of differentiation and/or tumor initiating potential.We found that metformin significantly inhibits the proliferation of NB cells, an effect that correlates with the inhibition of Akt, while AMPK activity resulted unchanged.Notably, metformin effects were modulated in a different ways by differentiating stimuli, being abolished after retinoic acid treatment but potentiated by overexpression of NDM29.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Medicine (DIMES), University of Genova, Genova, Italy ; IRCCS-AOU San Martino-IST, Genova, Italy.

ABSTRACT

Background: Metformin is a widely used oral hypoglycemizing agent recently proposed as potential anti-cancer drug. In this study we report the antiproliferative effect of metformin treatment in a high risk neuroblastoma cell model, focusing on possible effects associated to different levels of differentiation and/or tumor initiating potential.

Methods: Antiproliferative and cytotoxic effects of metformin were tested in human SKNBE2 and SH-SY5Y neuroblastoma cell lines and in SKNBE2 cells in which differentiation is induced by retinoic acid treatment or stable overexpression of NDM29 non-coding RNA, both conditions characterized by a neuron-like differentiated phenotype.

Results: We found that metformin significantly inhibits the proliferation of NB cells, an effect that correlates with the inhibition of Akt, while AMPK activity resulted unchanged. Notably, metformin effects were modulated in a different ways by differentiating stimuli, being abolished after retinoic acid treatment but potentiated by overexpression of NDM29.

Conclusion: These data suggest the efficacy of metformin as neuroblastoma anticancer agent, and support the requirement of further studies on the possible role of the differentiation status on the antiproliferative effects of this drug.

No MeSH data available.


Related in: MedlinePlus

Metformin reduces cell proliferation and viability of Neuroblastoma SKNBE2 cells. A) Cell Index curves reporting SKNBE2 neuroblastoma cell proliferation, untreated (CTR) or treated with metformin (Met), as resulting by Xelligence RTCA DP analysis. Histograms highlight Cell Index values after 24 and 48 hrs. B) Cell counting assay of SKNBE2 cells untreated (CTR) or treated with metformin (Met). Y-axis is referred to cell number x 106. C) [3H]-thymidine incorporation assay of SKNBE2 cells, untreated (CTR) or treated with metformin (Met). Y-axis is referred to cpm x 103. D) Cell viability analysis of SKNBE2 cells untreated (CTR) or treated with metformin (Met) as determined by ATPlite assay. Y-axis is referred to luminescence x 105. E) Cell death analysis by FACS analysis for sytox blue staining. The percentage of cell death is reported as Met/CTR ratio. F) Western blot analysis of caspase-3 cleavage. Equal loading of proteins was ensured by normalization for pro-caspase 3 and α-tubulin expression. Cleaved Caspase 3 was not detected. G) Western blot analysis of Akt phosphorylation/activation. 1 = Complete Medium (C.M., RPMI 10%), 2 = C.M. + 20 mM Met for 5 min, 3 = C.M. + 20 mM Met for 15 min, 4 = C.M. + 20 mM Met for 30 min, 5 = C.M. + 20 mM Met for 60 min. Equal loading of proteins was ensured by normalization for total Akt and α-tubulin expression. Densitometric analysis of phospho-Akt/total Akt ratio is also reported. (*p < 0.05; **p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4128937&req=5

Figure 1: Metformin reduces cell proliferation and viability of Neuroblastoma SKNBE2 cells. A) Cell Index curves reporting SKNBE2 neuroblastoma cell proliferation, untreated (CTR) or treated with metformin (Met), as resulting by Xelligence RTCA DP analysis. Histograms highlight Cell Index values after 24 and 48 hrs. B) Cell counting assay of SKNBE2 cells untreated (CTR) or treated with metformin (Met). Y-axis is referred to cell number x 106. C) [3H]-thymidine incorporation assay of SKNBE2 cells, untreated (CTR) or treated with metformin (Met). Y-axis is referred to cpm x 103. D) Cell viability analysis of SKNBE2 cells untreated (CTR) or treated with metformin (Met) as determined by ATPlite assay. Y-axis is referred to luminescence x 105. E) Cell death analysis by FACS analysis for sytox blue staining. The percentage of cell death is reported as Met/CTR ratio. F) Western blot analysis of caspase-3 cleavage. Equal loading of proteins was ensured by normalization for pro-caspase 3 and α-tubulin expression. Cleaved Caspase 3 was not detected. G) Western blot analysis of Akt phosphorylation/activation. 1 = Complete Medium (C.M., RPMI 10%), 2 = C.M. + 20 mM Met for 5 min, 3 = C.M. + 20 mM Met for 15 min, 4 = C.M. + 20 mM Met for 30 min, 5 = C.M. + 20 mM Met for 60 min. Equal loading of proteins was ensured by normalization for total Akt and α-tubulin expression. Densitometric analysis of phospho-Akt/total Akt ratio is also reported. (*p < 0.05; **p < 0.01).

Mentions: In order to investigate the effect of metformin on NB, SKNBE2 cells were treated for 12–72 hrs with metformin (20 mM) and proliferation rate analyzed using multiple experimental approaches. First, we measured cell proliferation using the xCELLigence RTCA DP system. SKNBE2 growth curves showed a time-dependent decrease in the proliferation rate of metformin-treated cells, resulting in a statistically significant difference after 48 hrs (as evidenced by the calculated Cell Index, see Methods), and lasting up to the end of the experimental observation (72 hrs) (Figure 1A).


Metformin inhibition of neuroblastoma cell proliferation is differently modulated by cell differentiation induced by retinoic acid or overexpression of NDM29 non-coding RNA.

Costa D, Gigoni A, Würth R, Cancedda R, Florio T, Pagano A - Cancer Cell Int. (2014)

Metformin reduces cell proliferation and viability of Neuroblastoma SKNBE2 cells. A) Cell Index curves reporting SKNBE2 neuroblastoma cell proliferation, untreated (CTR) or treated with metformin (Met), as resulting by Xelligence RTCA DP analysis. Histograms highlight Cell Index values after 24 and 48 hrs. B) Cell counting assay of SKNBE2 cells untreated (CTR) or treated with metformin (Met). Y-axis is referred to cell number x 106. C) [3H]-thymidine incorporation assay of SKNBE2 cells, untreated (CTR) or treated with metformin (Met). Y-axis is referred to cpm x 103. D) Cell viability analysis of SKNBE2 cells untreated (CTR) or treated with metformin (Met) as determined by ATPlite assay. Y-axis is referred to luminescence x 105. E) Cell death analysis by FACS analysis for sytox blue staining. The percentage of cell death is reported as Met/CTR ratio. F) Western blot analysis of caspase-3 cleavage. Equal loading of proteins was ensured by normalization for pro-caspase 3 and α-tubulin expression. Cleaved Caspase 3 was not detected. G) Western blot analysis of Akt phosphorylation/activation. 1 = Complete Medium (C.M., RPMI 10%), 2 = C.M. + 20 mM Met for 5 min, 3 = C.M. + 20 mM Met for 15 min, 4 = C.M. + 20 mM Met for 30 min, 5 = C.M. + 20 mM Met for 60 min. Equal loading of proteins was ensured by normalization for total Akt and α-tubulin expression. Densitometric analysis of phospho-Akt/total Akt ratio is also reported. (*p < 0.05; **p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4128937&req=5

Figure 1: Metformin reduces cell proliferation and viability of Neuroblastoma SKNBE2 cells. A) Cell Index curves reporting SKNBE2 neuroblastoma cell proliferation, untreated (CTR) or treated with metformin (Met), as resulting by Xelligence RTCA DP analysis. Histograms highlight Cell Index values after 24 and 48 hrs. B) Cell counting assay of SKNBE2 cells untreated (CTR) or treated with metformin (Met). Y-axis is referred to cell number x 106. C) [3H]-thymidine incorporation assay of SKNBE2 cells, untreated (CTR) or treated with metformin (Met). Y-axis is referred to cpm x 103. D) Cell viability analysis of SKNBE2 cells untreated (CTR) or treated with metformin (Met) as determined by ATPlite assay. Y-axis is referred to luminescence x 105. E) Cell death analysis by FACS analysis for sytox blue staining. The percentage of cell death is reported as Met/CTR ratio. F) Western blot analysis of caspase-3 cleavage. Equal loading of proteins was ensured by normalization for pro-caspase 3 and α-tubulin expression. Cleaved Caspase 3 was not detected. G) Western blot analysis of Akt phosphorylation/activation. 1 = Complete Medium (C.M., RPMI 10%), 2 = C.M. + 20 mM Met for 5 min, 3 = C.M. + 20 mM Met for 15 min, 4 = C.M. + 20 mM Met for 30 min, 5 = C.M. + 20 mM Met for 60 min. Equal loading of proteins was ensured by normalization for total Akt and α-tubulin expression. Densitometric analysis of phospho-Akt/total Akt ratio is also reported. (*p < 0.05; **p < 0.01).
Mentions: In order to investigate the effect of metformin on NB, SKNBE2 cells were treated for 12–72 hrs with metformin (20 mM) and proliferation rate analyzed using multiple experimental approaches. First, we measured cell proliferation using the xCELLigence RTCA DP system. SKNBE2 growth curves showed a time-dependent decrease in the proliferation rate of metformin-treated cells, resulting in a statistically significant difference after 48 hrs (as evidenced by the calculated Cell Index, see Methods), and lasting up to the end of the experimental observation (72 hrs) (Figure 1A).

Bottom Line: In this study we report the antiproliferative effect of metformin treatment in a high risk neuroblastoma cell model, focusing on possible effects associated to different levels of differentiation and/or tumor initiating potential.We found that metformin significantly inhibits the proliferation of NB cells, an effect that correlates with the inhibition of Akt, while AMPK activity resulted unchanged.Notably, metformin effects were modulated in a different ways by differentiating stimuli, being abolished after retinoic acid treatment but potentiated by overexpression of NDM29.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Medicine (DIMES), University of Genova, Genova, Italy ; IRCCS-AOU San Martino-IST, Genova, Italy.

ABSTRACT

Background: Metformin is a widely used oral hypoglycemizing agent recently proposed as potential anti-cancer drug. In this study we report the antiproliferative effect of metformin treatment in a high risk neuroblastoma cell model, focusing on possible effects associated to different levels of differentiation and/or tumor initiating potential.

Methods: Antiproliferative and cytotoxic effects of metformin were tested in human SKNBE2 and SH-SY5Y neuroblastoma cell lines and in SKNBE2 cells in which differentiation is induced by retinoic acid treatment or stable overexpression of NDM29 non-coding RNA, both conditions characterized by a neuron-like differentiated phenotype.

Results: We found that metformin significantly inhibits the proliferation of NB cells, an effect that correlates with the inhibition of Akt, while AMPK activity resulted unchanged. Notably, metformin effects were modulated in a different ways by differentiating stimuli, being abolished after retinoic acid treatment but potentiated by overexpression of NDM29.

Conclusion: These data suggest the efficacy of metformin as neuroblastoma anticancer agent, and support the requirement of further studies on the possible role of the differentiation status on the antiproliferative effects of this drug.

No MeSH data available.


Related in: MedlinePlus