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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

AMP-activated protein kinase (AMPK)α1 is involved in the inhibitory effects of metformin on the expression of Gli-1. (A) MDA-MB-231 cells were transfected with siAMPK#1 (PRKAA1-RNAi-24250-1), siAMPK#2 (PRKAA1-RNAi-24251-1), siAMPK#3 (PRKAA1-RNAi-24252-1), or siCtr, and 48 h after transfection, the cells were lysed and examined by western blot analysis; β-actin was used for loading control. (B) Untransfected MDA-MB-231 cells were treated with recombinant human Sonic hedgehog (rhShh) or the combination of rhShh and metformin, and the control group was treated with PBS. MDA-MB-231 cells were transfected with siCtr or siAMPK#2 in the presence of rhShh and metformin. Gli-1 protein levels were examined by western blot analysis. β-actin levels were measured as a loading control.
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f7-ijmm-36-01-0204: AMP-activated protein kinase (AMPK)α1 is involved in the inhibitory effects of metformin on the expression of Gli-1. (A) MDA-MB-231 cells were transfected with siAMPK#1 (PRKAA1-RNAi-24250-1), siAMPK#2 (PRKAA1-RNAi-24251-1), siAMPK#3 (PRKAA1-RNAi-24252-1), or siCtr, and 48 h after transfection, the cells were lysed and examined by western blot analysis; β-actin was used for loading control. (B) Untransfected MDA-MB-231 cells were treated with recombinant human Sonic hedgehog (rhShh) or the combination of rhShh and metformin, and the control group was treated with PBS. MDA-MB-231 cells were transfected with siCtr or siAMPK#2 in the presence of rhShh and metformin. Gli-1 protein levels were examined by western blot analysis. β-actin levels were measured as a loading control.

Mentions: Previous studies have found that AMPK plays a critical role in facilitating the anticancer effects of metformin (9,10). Therefore, we then investigated whether the metformin-mediated inhibition of the Shh signaling pathway is dependent on AMPK. To inhibit AMPK expression, siRNA was used. Fig. 7A shows that the 3 sequences of AMPK siRNA (siAMPK#1, siAMPK#2 and siAMPK#3) effectively depleted AMPK expression at the protein level in the transfected MDA-MB-231 cells compared with cells transfected with siCtr. The sequence siAMPK#2-transfected cells were selected to examine the role of AMPK in the metformin-mediated inhibition of the Shh signaling pathway. Western blot analysis revealed a significant increase in Gli-1 expression following the combined treatment of MDA-MB-231 cells with rhShh and metformin, and transfection with siAMPK#2 compared with the cells transfected with siCtr or in the untransfected cells (Fig. 7B).


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)

AMP-activated protein kinase (AMPK)α1 is involved in the inhibitory effects of metformin on the expression of Gli-1. (A) MDA-MB-231 cells were transfected with siAMPK#1 (PRKAA1-RNAi-24250-1), siAMPK#2 (PRKAA1-RNAi-24251-1), siAMPK#3 (PRKAA1-RNAi-24252-1), or siCtr, and 48 h after transfection, the cells were lysed and examined by western blot analysis; β-actin was used for loading control. (B) Untransfected MDA-MB-231 cells were treated with recombinant human Sonic hedgehog (rhShh) or the combination of rhShh and metformin, and the control group was treated with PBS. MDA-MB-231 cells were transfected with siCtr or siAMPK#2 in the presence of rhShh and metformin. Gli-1 protein levels were examined by western blot analysis. β-actin levels were measured as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7-ijmm-36-01-0204: AMP-activated protein kinase (AMPK)α1 is involved in the inhibitory effects of metformin on the expression of Gli-1. (A) MDA-MB-231 cells were transfected with siAMPK#1 (PRKAA1-RNAi-24250-1), siAMPK#2 (PRKAA1-RNAi-24251-1), siAMPK#3 (PRKAA1-RNAi-24252-1), or siCtr, and 48 h after transfection, the cells were lysed and examined by western blot analysis; β-actin was used for loading control. (B) Untransfected MDA-MB-231 cells were treated with recombinant human Sonic hedgehog (rhShh) or the combination of rhShh and metformin, and the control group was treated with PBS. MDA-MB-231 cells were transfected with siCtr or siAMPK#2 in the presence of rhShh and metformin. Gli-1 protein levels were examined by western blot analysis. β-actin levels were measured as a loading control.
Mentions: Previous studies have found that AMPK plays a critical role in facilitating the anticancer effects of metformin (9,10). Therefore, we then investigated whether the metformin-mediated inhibition of the Shh signaling pathway is dependent on AMPK. To inhibit AMPK expression, siRNA was used. Fig. 7A shows that the 3 sequences of AMPK siRNA (siAMPK#1, siAMPK#2 and siAMPK#3) effectively depleted AMPK expression at the protein level in the transfected MDA-MB-231 cells compared with cells transfected with siCtr. The sequence siAMPK#2-transfected cells were selected to examine the role of AMPK in the metformin-mediated inhibition of the Shh signaling pathway. Western blot analysis revealed a significant increase in Gli-1 expression following the combined treatment of MDA-MB-231 cells with rhShh and metformin, and transfection with siAMPK#2 compared with the cells transfected with siCtr or in the untransfected cells (Fig. 7B).

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