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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 recombinant human Sonic hedgehog (rhShh)-induced tumor growth in the in vivo model of breast cancer. (A) Representative bioluminesence images of the control and the treatment groups: control (ctr; untreated), rhShh (intratumoral injections of rhShh, 1 mg/kg body weight), Met (oral gavage of metformin, 100 mg/kg body weight), Met + rhShh (a combination of both drugs). (B) Chart representing the average photon counts from the control and the treatment groups. The bars indicate the means ± SD of triplicate samples, **P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group. (C) The expression levels of Gli-1 in the tumors from the xenograft model were measured by immunohistochemistry. Gli-1 immunostaining was regarded as positive with brown granules exhibited in the cytoplasm of a cell. Final magnification, ×40. Scale bar, 50 µm.
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f4-ijmm-36-01-0204: Metformin inhibits recombinant human Sonic hedgehog (rhShh)-induced tumor growth in the in vivo model of breast cancer. (A) Representative bioluminesence images of the control and the treatment groups: control (ctr; untreated), rhShh (intratumoral injections of rhShh, 1 mg/kg body weight), Met (oral gavage of metformin, 100 mg/kg body weight), Met + rhShh (a combination of both drugs). (B) Chart representing the average photon counts from the control and the treatment groups. The bars indicate the means ± SD of triplicate samples, **P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group. (C) The expression levels of Gli-1 in the tumors from the xenograft model were measured by immunohistochemistry. Gli-1 immunostaining was regarded as positive with brown granules exhibited in the cytoplasm of a cell. Final magnification, ×40. Scale bar, 50 µm.

Mentions: To examine the effects of metformin on rhShh-induced tumor growth in vivo, a total of 1.0×106 MDA-MB-231 cells expressing GFP were implanted into the mammary fat pads of BALB/c-nu mice. We then used IVIS to measure tumor growth in order to improve the quality of the quantitative results. An analysis of the bioluminescence imaging data indicated that the rhShh treatment group generated significantly larger tumors than the control group. However, the administration of metformin significantly inhibited tumor growth (Fig. 4A). Furthermore, the mice in the combined treatment group presented with tumors much smaller than those of the mice in the rhShh treatment group (Fig. 4A). An approximately 2.3-fold difference in the average signal intensity was observed between the rhShh treatment group and the combination treatment group (P<0.01; Fig. 4B).


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 recombinant human Sonic hedgehog (rhShh)-induced tumor growth in the in vivo model of breast cancer. (A) Representative bioluminesence images of the control and the treatment groups: control (ctr; untreated), rhShh (intratumoral injections of rhShh, 1 mg/kg body weight), Met (oral gavage of metformin, 100 mg/kg body weight), Met + rhShh (a combination of both drugs). (B) Chart representing the average photon counts from the control and the treatment groups. The bars indicate the means ± SD of triplicate samples, **P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group. (C) The expression levels of Gli-1 in the tumors from the xenograft model were measured by immunohistochemistry. Gli-1 immunostaining was regarded as positive with brown granules exhibited in the cytoplasm of a cell. Final magnification, ×40. Scale bar, 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4-ijmm-36-01-0204: Metformin inhibits recombinant human Sonic hedgehog (rhShh)-induced tumor growth in the in vivo model of breast cancer. (A) Representative bioluminesence images of the control and the treatment groups: control (ctr; untreated), rhShh (intratumoral injections of rhShh, 1 mg/kg body weight), Met (oral gavage of metformin, 100 mg/kg body weight), Met + rhShh (a combination of both drugs). (B) Chart representing the average photon counts from the control and the treatment groups. The bars indicate the means ± SD of triplicate samples, **P<0.01 vs. the Ctr group, ##P<0.01 vs. the rhShh group. (C) The expression levels of Gli-1 in the tumors from the xenograft model were measured by immunohistochemistry. Gli-1 immunostaining was regarded as positive with brown granules exhibited in the cytoplasm of a cell. Final magnification, ×40. Scale bar, 50 µm.
Mentions: To examine the effects of metformin on rhShh-induced tumor growth in vivo, a total of 1.0×106 MDA-MB-231 cells expressing GFP were implanted into the mammary fat pads of BALB/c-nu mice. We then used IVIS to measure tumor growth in order to improve the quality of the quantitative results. An analysis of the bioluminescence imaging data indicated that the rhShh treatment group generated significantly larger tumors than the control group. However, the administration of metformin significantly inhibited tumor growth (Fig. 4A). Furthermore, the mice in the combined treatment group presented with tumors much smaller than those of the mice in the rhShh treatment group (Fig. 4A). An approximately 2.3-fold difference in the average signal intensity was observed between the rhShh treatment group and the combination treatment group (P<0.01; Fig. 4B).

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