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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 decreases Sonic hedgehog (Shh), Smo, Patched (Ptc) and Gli-1 expression in MDA-MB-231 cells. MDA-MB-231 cells were treated with metformin at concentrations of 0, 1, 3 or 9 mM for 12 h or with 3 mM of metformin for 0, 3, 6 and 12 h. (A and E) The mRNA levels of Shh, Smo, Ptc and Gli-1 were measured by RT-PCR; GAPDH served as a control. (C and G) The protein levels of Shh, Smo, Ptc and Gli-1 were measured by western blot analysis; β-actin levels were measured as a loading control. Histograms illustrate the (B and F) mRNA levels relative to those of GAPDH and (D and H) protein expression relative to that of β-actin. All data are presented as the means ± SD of 3 independent experiments. *P<0.05 vs. the control group; **P<0.01 vs. the control group.
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f1-ijmm-36-01-0204: Metformin decreases Sonic hedgehog (Shh), Smo, Patched (Ptc) and Gli-1 expression in MDA-MB-231 cells. MDA-MB-231 cells were treated with metformin at concentrations of 0, 1, 3 or 9 mM for 12 h or with 3 mM of metformin for 0, 3, 6 and 12 h. (A and E) The mRNA levels of Shh, Smo, Ptc and Gli-1 were measured by RT-PCR; GAPDH served as a control. (C and G) The protein levels of Shh, Smo, Ptc and Gli-1 were measured by western blot analysis; β-actin levels were measured as a loading control. Histograms illustrate the (B and F) mRNA levels relative to those of GAPDH and (D and H) protein expression relative to that of β-actin. All data are presented as the means ± SD of 3 independent experiments. *P<0.05 vs. the control group; **P<0.01 vs. the control group.

Mentions: To determine whether metformin inhibits the Shh signaling pathway in breast cancer cells, RT-PCR was employed to measure the Shh, Smo, Ptc and Gli-1 mRNA levels following treatment with metformin. The MDA-MB-231 cells were treated with various concentrations of metformin (0–9 mmol/l), as previously described (31), for 12 h or with 3 mM metformin for different periods of time (0–12 h). As shown in Fig. 1A and E, the mRNA expression levels of Shh, Smo, Ptc and Gli-1 decreased in a dose- and time-dependent manner following treatment with metformin. The changes in Shh, Smo, Ptc and Gli-1 protein expression observed in these cells following incubation with various concentrations of metformin for 12 h or with 3 mM metformin for different periods of time (0–12 h) were then determined by western blot analysis. Metformin also decreased the protein levels in a dose- and time-dependent manner (Fig. 1C and G). Treatment with metformin also suppressed Shh, Smo, Ptc and Gli-1 protein expression in the MCF-7 and BT-549 cells, as shown in Fig. 2.


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 decreases Sonic hedgehog (Shh), Smo, Patched (Ptc) and Gli-1 expression in MDA-MB-231 cells. MDA-MB-231 cells were treated with metformin at concentrations of 0, 1, 3 or 9 mM for 12 h or with 3 mM of metformin for 0, 3, 6 and 12 h. (A and E) The mRNA levels of Shh, Smo, Ptc and Gli-1 were measured by RT-PCR; GAPDH served as a control. (C and G) The protein levels of Shh, Smo, Ptc and Gli-1 were measured by western blot analysis; β-actin levels were measured as a loading control. Histograms illustrate the (B and F) mRNA levels relative to those of GAPDH and (D and H) protein expression relative to that of β-actin. All data are presented as the means ± SD of 3 independent experiments. *P<0.05 vs. the control group; **P<0.01 vs. the control group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-ijmm-36-01-0204: Metformin decreases Sonic hedgehog (Shh), Smo, Patched (Ptc) and Gli-1 expression in MDA-MB-231 cells. MDA-MB-231 cells were treated with metformin at concentrations of 0, 1, 3 or 9 mM for 12 h or with 3 mM of metformin for 0, 3, 6 and 12 h. (A and E) The mRNA levels of Shh, Smo, Ptc and Gli-1 were measured by RT-PCR; GAPDH served as a control. (C and G) The protein levels of Shh, Smo, Ptc and Gli-1 were measured by western blot analysis; β-actin levels were measured as a loading control. Histograms illustrate the (B and F) mRNA levels relative to those of GAPDH and (D and H) protein expression relative to that of β-actin. All data are presented as the means ± SD of 3 independent experiments. *P<0.05 vs. the control group; **P<0.01 vs. the control group.
Mentions: To determine whether metformin inhibits the Shh signaling pathway in breast cancer cells, RT-PCR was employed to measure the Shh, Smo, Ptc and Gli-1 mRNA levels following treatment with metformin. The MDA-MB-231 cells were treated with various concentrations of metformin (0–9 mmol/l), as previously described (31), for 12 h or with 3 mM metformin for different periods of time (0–12 h). As shown in Fig. 1A and E, the mRNA expression levels of Shh, Smo, Ptc and Gli-1 decreased in a dose- and time-dependent manner following treatment with metformin. The changes in Shh, Smo, Ptc and Gli-1 protein expression observed in these cells following incubation with various concentrations of metformin for 12 h or with 3 mM metformin for different periods of time (0–12 h) were then determined by western blot analysis. Metformin also decreased the protein levels in a dose- and time-dependent manner (Fig. 1C and G). Treatment with metformin also suppressed Shh, Smo, Ptc and Gli-1 protein expression in the MCF-7 and BT-549 cells, as shown in Fig. 2.

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