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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

The metformin-induced decrease in the number/percentage of CD44+/CD24− MDA-MB-231 cells is dependent on AMPK. (A) MDA-MB-231 cells were treated with recombinant human Sonic hedgehog (rhShh) or the combination of rhShh and metformin; the cells were treated with sterile PBS as a control and the cells were transfected with siCtr or siAMPK#2 in the presence of rhShh and metformin. The CD44+/CD24− subpopulations were examined by flow cytometry. (B) The histogram illustrates the percentage of CD44+/CD24− subpopulations. Error bars represent the means ± SD; **P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group, &&P<0.01 vs. the cells transfected with siCtr in the presence of rhShh and metformin.
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f9-ijmm-36-01-0204: The metformin-induced decrease in the number/percentage of CD44+/CD24− MDA-MB-231 cells is dependent on AMPK. (A) MDA-MB-231 cells were treated with recombinant human Sonic hedgehog (rhShh) or the combination of rhShh and metformin; the cells were treated with sterile PBS as a control and the cells were transfected with siCtr or siAMPK#2 in the presence of rhShh and metformin. The CD44+/CD24− subpopulations were examined by flow cytometry. (B) The histogram illustrates the percentage of CD44+/CD24− subpopulations. Error bars represent the means ± SD; **P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group, &&P<0.01 vs. the cells transfected with siCtr in the presence of rhShh and metformin.

Mentions: We subsequently determined whether AMPK is involved in the metformin-mediated suppression of BCSC stemness through the Shh signaling pathway. In the presence of rhShh and metformin, the siAMPK#2-transfected cells produced a greater number of and larger spheres compared with both the siCtr-transfected cells and the untransfected cells (P<0.01; Fig. 8). In the presence of rhShh and metformin, the siAMPK#2-transfected cells exhibited a significantly greater proportion of CD44+/CD24− stem cell-like cells compared with both the siCtr-transfected cells and untransfected cells (P<0.01; Fig. 9). These results indicate that AMPK is involved in the inhibition of the Shh pathway by metformin.


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)

The metformin-induced decrease in the number/percentage of CD44+/CD24− MDA-MB-231 cells is dependent on AMPK. (A) MDA-MB-231 cells were treated with recombinant human Sonic hedgehog (rhShh) or the combination of rhShh and metformin; the cells were treated with sterile PBS as a control and the cells were transfected with siCtr or siAMPK#2 in the presence of rhShh and metformin. The CD44+/CD24− subpopulations were examined by flow cytometry. (B) The histogram illustrates the percentage of CD44+/CD24− subpopulations. Error bars represent the means ± SD; **P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group, &&P<0.01 vs. the cells transfected with siCtr in the presence of rhShh and metformin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f9-ijmm-36-01-0204: The metformin-induced decrease in the number/percentage of CD44+/CD24− MDA-MB-231 cells is dependent on AMPK. (A) MDA-MB-231 cells were treated with recombinant human Sonic hedgehog (rhShh) or the combination of rhShh and metformin; the cells were treated with sterile PBS as a control and the cells were transfected with siCtr or siAMPK#2 in the presence of rhShh and metformin. The CD44+/CD24− subpopulations were examined by flow cytometry. (B) The histogram illustrates the percentage of CD44+/CD24− subpopulations. Error bars represent the means ± SD; **P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group, &&P<0.01 vs. the cells transfected with siCtr in the presence of rhShh and metformin.
Mentions: We subsequently determined whether AMPK is involved in the metformin-mediated suppression of BCSC stemness through the Shh signaling pathway. In the presence of rhShh and metformin, the siAMPK#2-transfected cells produced a greater number of and larger spheres compared with both the siCtr-transfected cells and the untransfected cells (P<0.01; Fig. 8). In the presence of rhShh and metformin, the siAMPK#2-transfected cells exhibited a significantly greater proportion of CD44+/CD24− stem cell-like cells compared with both the siCtr-transfected cells and untransfected cells (P<0.01; Fig. 9). These results indicate that AMPK is involved in the inhibition of the Shh pathway by metformin.

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