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Effects of metformin on retinoblastoma growth in vitro and in vivo.

Brodowska K, Theodoropoulou S, Meyer Zu Hörste M, Paschalis EI, Takeuchi K, Scott G, Ramsey DJ, Kiernan E, Hoang M, Cichy J, Miller JW, Gragoudas ES, Vavvas DG - Int. J. Oncol. (2014)

Bottom Line: Recent studies suggest that the anti-diabetic drug metformin may reduce the risk of cancer and have anti-proliferative effects for some but not all cancers.This was associated with activation of AMPK, inhibition of the mTOR pathways and autophagy marker LC3B.This suggests that the potential beneficial effects of metformin seen in epidemiological studies may be limited to specific tumor types or be related to indirect effects/mechanisms not observed under acute laboratory conditions.

View Article: PubMed Central - PubMed

Affiliation: Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA.

ABSTRACT
Recent studies suggest that the anti-diabetic drug metformin may reduce the risk of cancer and have anti-proliferative effects for some but not all cancers. In this study, we examined the effects of metformin on human retinoblastoma cell proliferation in vitro and in vivo. Two different human retinoblastoma cell lines (Y79, WERI) were treated with metformin in vitro and xenografts of Y79 cells were established in nu/nu immune-deficient mice and used to assess the effects of pharmacological levels of metformin in vivo. Metformin inhibited proliferation of the retinoblastoma cells in vitro. Similar to other studies, high concentrations of metformin (mM) blocked the cell cycle in G0‑G1, indicated by a strong decrease of G1 cyclins, especially cyclin D, cyclin-dependent kinases (4 and 6), and flow cytometry assessment of the cell cycle. This was associated with activation of AMPK, inhibition of the mTOR pathways and autophagy marker LC3B. However, metformin failed to suppress growth of xenografted tumors of Y79 human retinoblastoma cells in nu/nu mice, even when treated with a maximally tolerated dose level achieved in human patients. In conclusion, suprapharmacological levels (mM) of metformin, well above those tolerated in vivo, inhibited the proliferation of retinoblastoma cells in vitro. However, physiological levels of metformin, such as seen in the clinical setting, did not affect the growth of retinoblastoma cells in vitro or in vivo. This suggests that the potential beneficial effects of metformin seen in epidemiological studies may be limited to specific tumor types or be related to indirect effects/mechanisms not observed under acute laboratory conditions.

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Metformin at higher mM levels increases the doubling time and causes cell death of retinoblastoma cells. (A and B) Retinoblastoma cell lines WERI and Y79 were treated with 1.25 and 5 mM of metformin for 48 h. Trypan blue exclusion test was performed on days 3, 6 and 9; metformin at mM levels caused proliferation inhibition; doubling time increased proportionally to metformin dose. The results are the average of three independent experiments. (C–F) The retinoblastoma cell lines Y79 and WERI were treated for 48 h with 5 mM of metformin, and cell viability and death was measured by calcein AM and DAPI staining using FACS; comparing to control, mM levels of metformin cause increased cell death and decreased viability (**p<0.01 for WERI and ***p<0.001 for Y79). The data are representative of three independent experiments (n=12).
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f2-ijo-45-06-2311: Metformin at higher mM levels increases the doubling time and causes cell death of retinoblastoma cells. (A and B) Retinoblastoma cell lines WERI and Y79 were treated with 1.25 and 5 mM of metformin for 48 h. Trypan blue exclusion test was performed on days 3, 6 and 9; metformin at mM levels caused proliferation inhibition; doubling time increased proportionally to metformin dose. The results are the average of three independent experiments. (C–F) The retinoblastoma cell lines Y79 and WERI were treated for 48 h with 5 mM of metformin, and cell viability and death was measured by calcein AM and DAPI staining using FACS; comparing to control, mM levels of metformin cause increased cell death and decreased viability (**p<0.01 for WERI and ***p<0.001 for Y79). The data are representative of three independent experiments (n=12).

Mentions: In order to determine whether metformin affects human retinoblastoma cell viability and proliferation, we analyzed the effect of the drug on two human retinoblastoma cell lines: WERI and Y79. Cells were treated with various concentrations of metformin (12 μM up to 10 mM) and the viability was assed by the MTT assay. Increasing doses of metformin led to a corresponding reduction in cell viability but at doses in the mM range of concentrations (Fig. 1A and B). Reduced viability was not observed at μM concentrations (Fig. 1C and D). Assessment of cell growth and doubling time by trypan blue exclusion showed decreased growth rates in the presence of mM levels of metformin. Doubling time increased from 2.2 to 5.1 days for the Y79 cell line and from 3 to 5.4 days for the WERI cell line (Fig. 2A and B). Metformin treatment at 5 mM also increased the proportion of non-viable cells and decreased the proportion of viable cells (Fig. 2D and F) as judged by calcein AM/DAPI flow cytometry when compared to control (Fig. 2C and E).


Effects of metformin on retinoblastoma growth in vitro and in vivo.

Brodowska K, Theodoropoulou S, Meyer Zu Hörste M, Paschalis EI, Takeuchi K, Scott G, Ramsey DJ, Kiernan E, Hoang M, Cichy J, Miller JW, Gragoudas ES, Vavvas DG - Int. J. Oncol. (2014)

Metformin at higher mM levels increases the doubling time and causes cell death of retinoblastoma cells. (A and B) Retinoblastoma cell lines WERI and Y79 were treated with 1.25 and 5 mM of metformin for 48 h. Trypan blue exclusion test was performed on days 3, 6 and 9; metformin at mM levels caused proliferation inhibition; doubling time increased proportionally to metformin dose. The results are the average of three independent experiments. (C–F) The retinoblastoma cell lines Y79 and WERI were treated for 48 h with 5 mM of metformin, and cell viability and death was measured by calcein AM and DAPI staining using FACS; comparing to control, mM levels of metformin cause increased cell death and decreased viability (**p<0.01 for WERI and ***p<0.001 for Y79). The data are representative of three independent experiments (n=12).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215581&req=5

f2-ijo-45-06-2311: Metformin at higher mM levels increases the doubling time and causes cell death of retinoblastoma cells. (A and B) Retinoblastoma cell lines WERI and Y79 were treated with 1.25 and 5 mM of metformin for 48 h. Trypan blue exclusion test was performed on days 3, 6 and 9; metformin at mM levels caused proliferation inhibition; doubling time increased proportionally to metformin dose. The results are the average of three independent experiments. (C–F) The retinoblastoma cell lines Y79 and WERI were treated for 48 h with 5 mM of metformin, and cell viability and death was measured by calcein AM and DAPI staining using FACS; comparing to control, mM levels of metformin cause increased cell death and decreased viability (**p<0.01 for WERI and ***p<0.001 for Y79). The data are representative of three independent experiments (n=12).
Mentions: In order to determine whether metformin affects human retinoblastoma cell viability and proliferation, we analyzed the effect of the drug on two human retinoblastoma cell lines: WERI and Y79. Cells were treated with various concentrations of metformin (12 μM up to 10 mM) and the viability was assed by the MTT assay. Increasing doses of metformin led to a corresponding reduction in cell viability but at doses in the mM range of concentrations (Fig. 1A and B). Reduced viability was not observed at μM concentrations (Fig. 1C and D). Assessment of cell growth and doubling time by trypan blue exclusion showed decreased growth rates in the presence of mM levels of metformin. Doubling time increased from 2.2 to 5.1 days for the Y79 cell line and from 3 to 5.4 days for the WERI cell line (Fig. 2A and B). Metformin treatment at 5 mM also increased the proportion of non-viable cells and decreased the proportion of viable cells (Fig. 2D and F) as judged by calcein AM/DAPI flow cytometry when compared to control (Fig. 2C and E).

Bottom Line: Recent studies suggest that the anti-diabetic drug metformin may reduce the risk of cancer and have anti-proliferative effects for some but not all cancers.This was associated with activation of AMPK, inhibition of the mTOR pathways and autophagy marker LC3B.This suggests that the potential beneficial effects of metformin seen in epidemiological studies may be limited to specific tumor types or be related to indirect effects/mechanisms not observed under acute laboratory conditions.

View Article: PubMed Central - PubMed

Affiliation: Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA.

ABSTRACT
Recent studies suggest that the anti-diabetic drug metformin may reduce the risk of cancer and have anti-proliferative effects for some but not all cancers. In this study, we examined the effects of metformin on human retinoblastoma cell proliferation in vitro and in vivo. Two different human retinoblastoma cell lines (Y79, WERI) were treated with metformin in vitro and xenografts of Y79 cells were established in nu/nu immune-deficient mice and used to assess the effects of pharmacological levels of metformin in vivo. Metformin inhibited proliferation of the retinoblastoma cells in vitro. Similar to other studies, high concentrations of metformin (mM) blocked the cell cycle in G0‑G1, indicated by a strong decrease of G1 cyclins, especially cyclin D, cyclin-dependent kinases (4 and 6), and flow cytometry assessment of the cell cycle. This was associated with activation of AMPK, inhibition of the mTOR pathways and autophagy marker LC3B. However, metformin failed to suppress growth of xenografted tumors of Y79 human retinoblastoma cells in nu/nu mice, even when treated with a maximally tolerated dose level achieved in human patients. In conclusion, suprapharmacological levels (mM) of metformin, well above those tolerated in vivo, inhibited the proliferation of retinoblastoma cells in vitro. However, physiological levels of metformin, such as seen in the clinical setting, did not affect the growth of retinoblastoma cells in vitro or in vivo. This suggests that the potential beneficial effects of metformin seen in epidemiological studies may be limited to specific tumor types or be related to indirect effects/mechanisms not observed under acute laboratory conditions.

Show MeSH
Related in: MedlinePlus