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Metformin exerts anticancer effects through the inhibition of the Sonic hedgehog signaling pathway in breast cancer.

Fan C, Wang Y, Liu Z, Sun Y, Wang X, Wei G, Wei J - Int. J. Mol. Med. (2015)

Bottom Line: Its anticancer effects, which are mediated by the activation of AMP-activated protein kinase (AMPK), have become notable.The aim of the present study was to elucidate the role of the Shh pathway in mediating the anticancer effects of metformin and the correlation between AMPK and the Shh pathway.Furthermore, the small interfering RNA (siRNA)‑mediated downregulation of AMPK reversed the inhibitory effects of metformin on rhShh‑induced Gli-1 expression and stemness.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemotherapy, Cancer Center, Qilu Hospital, Shandong University, Jinan, Shandong 250012, P.R. China.

ABSTRACT
Metformin, a widely prescribed antidiabetic drug, has previously been shown to lower the risk of certain types of cancer, including that of breast cancer, and to improve prognosis. Its anticancer effects, which are mediated by the activation of AMP-activated protein kinase (AMPK), have become notable. The Sonic hedgehog (Shh) signaling pathway is involved in changes in mammary ducts and malignant transformation. The aim of the present study was to elucidate the role of the Shh pathway in mediating the anticancer effects of metformin and the correlation between AMPK and the Shh pathway. We investigated the effectiveness of metformin in inhibiting the proliferation, migration, invasion and stemness of breast cancer cells in vitro using RNA extraction and reverse transcription‑polymerase chain reaction (RT-PCR), western blot analysis, cell proliferation assay, scratch-wound assay (cell migration assay), cell invasion assay, mammosphere culture and flow cytometry. In in vivo experiments, a tumor xenograft model was used to detect the effects of metformin on cancer cell proliferation. The results revealed that the treatment of breast cancer cells with metformin led to the inhibition of the Shh signaling pathway. Importantly, metformin inhibited recombinant human Shh (rhShh)‑induced cell migration, invasion, and stemness, and impaired cell proliferation both in vitro and in vivo. Furthermore, the small interfering RNA (siRNA)‑mediated downregulation of AMPK reversed the inhibitory effects of metformin on rhShh‑induced Gli-1 expression and stemness. Our findings identified a role of the Shh signaling pathway in the anticancer effects of metformin in breast cancer. Furthermore, we revealed that the metformin-mediated inhibition of the Shh signaling pathway may be dependent on AMPK.

No MeSH data available.


Related in: MedlinePlus

Metformin inhibits rhShh-induced sphere formation and reduces the number of CD44+/CD24− MDA-MB-231 cells. (A) Representative images of MDA-MB-231 cells in the presence of recombinant human Sonic hedgehog (rhShh), metformin and their combination. (B) The number of spheres was counted under a microscope. Data represent the means ± SD of 3 separate experiments (**P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group) (C) Cells were treated with rhShh, metformin and their combination. After 4 days, the cells were stained with anti-CD44-APC and anti-CD24-PE antibodies, and the CD44+/CD24− subpopulations were examined by flow cytometry. (D) Histogram illustrates the percentages of the CD44+/CD24− subpopulations. All data represent the means ± SD of 3 separate experiments (**P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group).
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f6-ijmm-36-01-0204: Metformin inhibits rhShh-induced sphere formation and reduces the number of CD44+/CD24− MDA-MB-231 cells. (A) Representative images of MDA-MB-231 cells in the presence of recombinant human Sonic hedgehog (rhShh), metformin and their combination. (B) The number of spheres was counted under a microscope. Data represent the means ± SD of 3 separate experiments (**P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group) (C) Cells were treated with rhShh, metformin and their combination. After 4 days, the cells were stained with anti-CD44-APC and anti-CD24-PE antibodies, and the CD44+/CD24− subpopulations were examined by flow cytometry. (D) Histogram illustrates the percentages of the CD44+/CD24− subpopulations. All data represent the means ± SD of 3 separate experiments (**P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group).

Mentions: To determine whether metformin inhibits the stemness of breast cancer cells through the suppression of the Shh signaling pathway, the MDA-MB-231 cells were cultured at a very low density (1 cell/µl) in 96-well plates containing serum-free medium with rhShh (1 µg/ml) alone, metformin (3 mM) alone, or a combination of both for 7 days. The cells treated with rhShh produced a greater number of and larger spheres compared with control cells (Fig. 6A and B). Treatment with metformin significantly reduced the number and size of these spheres compared with the control cells, and also inhibited the rhShh-induced development of a greater number of and larger spheres (P<0.01; Fig. 6A and B).


Metformin exerts anticancer effects through the inhibition of the Sonic hedgehog signaling pathway in breast cancer.

Fan C, Wang Y, Liu Z, Sun Y, Wang X, Wei G, Wei J - Int. J. Mol. Med. (2015)

Metformin inhibits rhShh-induced sphere formation and reduces the number of CD44+/CD24− MDA-MB-231 cells. (A) Representative images of MDA-MB-231 cells in the presence of recombinant human Sonic hedgehog (rhShh), metformin and their combination. (B) The number of spheres was counted under a microscope. Data represent the means ± SD of 3 separate experiments (**P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group) (C) Cells were treated with rhShh, metformin and their combination. After 4 days, the cells were stained with anti-CD44-APC and anti-CD24-PE antibodies, and the CD44+/CD24− subpopulations were examined by flow cytometry. (D) Histogram illustrates the percentages of the CD44+/CD24− subpopulations. All data represent the means ± SD of 3 separate experiments (**P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4494591&req=5

f6-ijmm-36-01-0204: Metformin inhibits rhShh-induced sphere formation and reduces the number of CD44+/CD24− MDA-MB-231 cells. (A) Representative images of MDA-MB-231 cells in the presence of recombinant human Sonic hedgehog (rhShh), metformin and their combination. (B) The number of spheres was counted under a microscope. Data represent the means ± SD of 3 separate experiments (**P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group) (C) Cells were treated with rhShh, metformin and their combination. After 4 days, the cells were stained with anti-CD44-APC and anti-CD24-PE antibodies, and the CD44+/CD24− subpopulations were examined by flow cytometry. (D) Histogram illustrates the percentages of the CD44+/CD24− subpopulations. All data represent the means ± SD of 3 separate experiments (**P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group).
Mentions: To determine whether metformin inhibits the stemness of breast cancer cells through the suppression of the Shh signaling pathway, the MDA-MB-231 cells were cultured at a very low density (1 cell/µl) in 96-well plates containing serum-free medium with rhShh (1 µg/ml) alone, metformin (3 mM) alone, or a combination of both for 7 days. The cells treated with rhShh produced a greater number of and larger spheres compared with control cells (Fig. 6A and B). Treatment with metformin significantly reduced the number and size of these spheres compared with the control cells, and also inhibited the rhShh-induced development of a greater number of and larger spheres (P<0.01; Fig. 6A and B).

Bottom Line: Its anticancer effects, which are mediated by the activation of AMP-activated protein kinase (AMPK), have become notable.The aim of the present study was to elucidate the role of the Shh pathway in mediating the anticancer effects of metformin and the correlation between AMPK and the Shh pathway.Furthermore, the small interfering RNA (siRNA)‑mediated downregulation of AMPK reversed the inhibitory effects of metformin on rhShh‑induced Gli-1 expression and stemness.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemotherapy, Cancer Center, Qilu Hospital, Shandong University, Jinan, Shandong 250012, P.R. China.

ABSTRACT
Metformin, a widely prescribed antidiabetic drug, has previously been shown to lower the risk of certain types of cancer, including that of breast cancer, and to improve prognosis. Its anticancer effects, which are mediated by the activation of AMP-activated protein kinase (AMPK), have become notable. The Sonic hedgehog (Shh) signaling pathway is involved in changes in mammary ducts and malignant transformation. The aim of the present study was to elucidate the role of the Shh pathway in mediating the anticancer effects of metformin and the correlation between AMPK and the Shh pathway. We investigated the effectiveness of metformin in inhibiting the proliferation, migration, invasion and stemness of breast cancer cells in vitro using RNA extraction and reverse transcription‑polymerase chain reaction (RT-PCR), western blot analysis, cell proliferation assay, scratch-wound assay (cell migration assay), cell invasion assay, mammosphere culture and flow cytometry. In in vivo experiments, a tumor xenograft model was used to detect the effects of metformin on cancer cell proliferation. The results revealed that the treatment of breast cancer cells with metformin led to the inhibition of the Shh signaling pathway. Importantly, metformin inhibited recombinant human Shh (rhShh)‑induced cell migration, invasion, and stemness, and impaired cell proliferation both in vitro and in vivo. Furthermore, the small interfering RNA (siRNA)‑mediated downregulation of AMPK reversed the inhibitory effects of metformin on rhShh‑induced Gli-1 expression and stemness. Our findings identified a role of the Shh signaling pathway in the anticancer effects of metformin in breast cancer. Furthermore, we revealed that the metformin-mediated inhibition of the Shh signaling pathway may be dependent on AMPK.

No MeSH data available.


Related in: MedlinePlus