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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 suppresses the rhShh-induced cell migration and invasion. (A) Representative images of the wounds at 0 and 48 h in the presence of recombinant human Sonic hedgehog (rhShh), metformin or their combination. (B) Histogram illustrates the relative wound width at 0 and 48 h. The migration distance of each cell was measured using Adobe Photoshop. (C) Matrigel invasion assay. Cells were seeded into Matrigel-coated invasion chambers and were treated with rhShh, metformin or their combination for 24 h. Representative images of stained and invaded cells are shown. Magnification, ×10. (D) The number of invaded cells was quantified by counting the cells from 5 random fields. 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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f5-ijmm-36-01-0204: Metformin suppresses the rhShh-induced cell migration and invasion. (A) Representative images of the wounds at 0 and 48 h in the presence of recombinant human Sonic hedgehog (rhShh), metformin or their combination. (B) Histogram illustrates the relative wound width at 0 and 48 h. The migration distance of each cell was measured using Adobe Photoshop. (C) Matrigel invasion assay. Cells were seeded into Matrigel-coated invasion chambers and were treated with rhShh, metformin or their combination for 24 h. Representative images of stained and invaded cells are shown. Magnification, ×10. (D) The number of invaded cells was quantified by counting the cells from 5 random fields. 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: We then investigated the effects of metformin on the migration potential of the MDA-MB-231 cells using the scratch-wound assay (for cell migration). The cells were seeded in 6-well plates, grown to confluence, and scratched using a 200-µl pipette tip to create a wound. The cells were then incubated for 48 h in the presence of rhShh (1 µg/ml), metformin (3 mM), or a combination of both. Phase-contrast images were obtained at the 0- and 48-h time points. When compared with the control group, the rhShh-treated MDA-MB-231 cells displayed a higher rate of migration (P<0.01), and the leading edges along the scraped area had almost integrated at 48 h (Fig. 5A and B). By contrast, treatment with metformin resulted in a significant decrease in cellular migration compared with the control group, and combination treatment with rhShh and metformin led to a marked inhibition of the rhShh-induced wound gap closure (P<0.01; Fig. 5A and B). A Transwell invasion assay was also performed to analyze the effects of treatment with rhShh (1 µg/ml), metformin (3 mM), or their combination on the invasive capacity of the cells. The number of rhShh-treated cells that had migrated across both the Matrigel and the insert was 3-fold higher than that of the control group, while the number of migratory metformin-treated cells was approximately 70% that of the control group (P<0.01; Fig. 5C and D). The cells administered the combined treatment showed a markedly reduced invasive capacity compared with those treated with rhShh alone (Fig. 5C and D). These results suggest that metformin impairs the effects of rhShh in promoting cell invasion.


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 suppresses the rhShh-induced cell migration and invasion. (A) Representative images of the wounds at 0 and 48 h in the presence of recombinant human Sonic hedgehog (rhShh), metformin or their combination. (B) Histogram illustrates the relative wound width at 0 and 48 h. The migration distance of each cell was measured using Adobe Photoshop. (C) Matrigel invasion assay. Cells were seeded into Matrigel-coated invasion chambers and were treated with rhShh, metformin or their combination for 24 h. Representative images of stained and invaded cells are shown. Magnification, ×10. (D) The number of invaded cells was quantified by counting the cells from 5 random fields. 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

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

f5-ijmm-36-01-0204: Metformin suppresses the rhShh-induced cell migration and invasion. (A) Representative images of the wounds at 0 and 48 h in the presence of recombinant human Sonic hedgehog (rhShh), metformin or their combination. (B) Histogram illustrates the relative wound width at 0 and 48 h. The migration distance of each cell was measured using Adobe Photoshop. (C) Matrigel invasion assay. Cells were seeded into Matrigel-coated invasion chambers and were treated with rhShh, metformin or their combination for 24 h. Representative images of stained and invaded cells are shown. Magnification, ×10. (D) The number of invaded cells was quantified by counting the cells from 5 random fields. 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: We then investigated the effects of metformin on the migration potential of the MDA-MB-231 cells using the scratch-wound assay (for cell migration). The cells were seeded in 6-well plates, grown to confluence, and scratched using a 200-µl pipette tip to create a wound. The cells were then incubated for 48 h in the presence of rhShh (1 µg/ml), metformin (3 mM), or a combination of both. Phase-contrast images were obtained at the 0- and 48-h time points. When compared with the control group, the rhShh-treated MDA-MB-231 cells displayed a higher rate of migration (P<0.01), and the leading edges along the scraped area had almost integrated at 48 h (Fig. 5A and B). By contrast, treatment with metformin resulted in a significant decrease in cellular migration compared with the control group, and combination treatment with rhShh and metformin led to a marked inhibition of the rhShh-induced wound gap closure (P<0.01; Fig. 5A and B). A Transwell invasion assay was also performed to analyze the effects of treatment with rhShh (1 µg/ml), metformin (3 mM), or their combination on the invasive capacity of the cells. The number of rhShh-treated cells that had migrated across both the Matrigel and the insert was 3-fold higher than that of the control group, while the number of migratory metformin-treated cells was approximately 70% that of the control group (P<0.01; Fig. 5C and D). The cells administered the combined treatment showed a markedly reduced invasive capacity compared with those treated with rhShh alone (Fig. 5C and D). These results suggest that metformin impairs the effects of rhShh in promoting cell invasion.

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