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Inhibitory effects of metformin at low concentration on epithelial-mesenchymal transition of CD44(+)CD117(+) ovarian cancer stem cells.

Zhang R, Zhang P, Wang H, Hou D, Li W, Xiao G, Li C - Stem Cell Res Ther (2015)

Bottom Line: Low dose of metformin reduced survivin expression in CSCs.Metformin was also associated with a reduction of snail2, twist, and vimentin in CD44(+)CD117(+) ovarian CSCs in vivo.Our results implicate that metformin at low dose inhibits selectively CD44(+)CD117(+) ovarian CSCs through inhibition of EMT and potentiates the effect of cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Gynecology, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200092, China. rrzhangcn@126.com.

ABSTRACT

Background: Although metformin, a first-line drug for treating diabetes, may play an important role in inhibition of epithelial ovarian cancer cell growth and cancer stem cells (CSCs), metformin at low dose showed less effect on the proliferation of ovarian cancer cells. In this study, we evaluated the effect of metformin at low dose on ovarian CSCs in order to understand the molecular mechanisms underlying.

Methods: The inhibitory effects of metformin at los dose on proliferation and population of ovarian cancer cells including SKOV3 and A2780 were assessed by cell proliferation assay and flow cytometry. Quantitative real-time PCR assay on expression of Bcl-2, Survivin and Bax was performed to determine the effect of metformin at low dose on epithelial-mesenchymal transition (EMT) of cancer cells and CSCs. Tumor sphere formation assay was also performed to evaluate the effect of metformin on spheres forming ability of CSCs. The therapeutic efficacy and the anti-CSC effects of metformin at low dose were investigated by using both SKOV3 cells and primary tumor xenografts. In addition, the CSC frequency and EMT in tumor xenograft models were also assessed by flow cytometry and quantitative real-time PCR.

Results: Metformin at low dose did not affect the proliferation of ovarian cancer cells. However, it inhibited population of CD44(+)CD117(+) selectively, neither CD133(+) nor ALDH(+) cells. It suppressed expression of snail2, twist and vimentin significantly in cancer cells and CD44(+)CD117(+) CSCs in vitro. Low dose of metformin reduced survivin expression in CSCs. Low concentrations of metformin inhibited the secondary and the tertiary tumor sphere formation, decreased SKOV3 and primary ovarian tumor xenograft growth, enhanced the anticancer effect of cisplatin, and lowered the proportion of CD44(+)CD117(+) CSCs in the xenograft tissue. Metformin was also associated with a reduction of snail2, twist, and vimentin in CD44(+)CD117(+) ovarian CSCs in vivo.

Conclusions: Our results implicate that metformin at low dose inhibits selectively CD44(+)CD117(+) ovarian CSCs through inhibition of EMT and potentiates the effect of cisplatin.

No MeSH data available.


Related in: MedlinePlus

Low concentrations of metformin reduced EMT of ovarian cancer cells and the CD44+CD117+ CSC population. SKOV3 cells were incubated with 0.1 mM metformin for 72 hours. The CD44+CD117+ cells were isolated by flow cytometry. Total RNA was isolated and mRNA was quantitated by real-time RT-PCR. mRNA expression of snail1, snail2, twist, vimentin, and E-cadherin was analyzed. Data show that treatment with 0.1 mM metformin reduced snail2, twist, and vimentin, but not snail1expression level, and upregulated the E-cadherin expression level (a). Bands detected with antibodies indicate the immune reactivity for snail1, snail2, twist, vimentin, E-cadherin, and β-actin: 72 hours of treatment with 0.1 nM metformin significantly downregulated snail2, twist, and vimentin, but not snail1, and upregulated E-cadherin as shown by western blotting (b). Total RNA was isolated from CD44+CD117+ cells and the mRNA was quantitated by real-time RT-PCR. Metformin at 0.1 mM significantly reduced snail2, twist, and vimentin, but not snail1 expression level, and upregulated the E-cadherin expression level (c). * Significant difference compared with control (P < 0.05)
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Fig3: Low concentrations of metformin reduced EMT of ovarian cancer cells and the CD44+CD117+ CSC population. SKOV3 cells were incubated with 0.1 mM metformin for 72 hours. The CD44+CD117+ cells were isolated by flow cytometry. Total RNA was isolated and mRNA was quantitated by real-time RT-PCR. mRNA expression of snail1, snail2, twist, vimentin, and E-cadherin was analyzed. Data show that treatment with 0.1 mM metformin reduced snail2, twist, and vimentin, but not snail1expression level, and upregulated the E-cadherin expression level (a). Bands detected with antibodies indicate the immune reactivity for snail1, snail2, twist, vimentin, E-cadherin, and β-actin: 72 hours of treatment with 0.1 nM metformin significantly downregulated snail2, twist, and vimentin, but not snail1, and upregulated E-cadherin as shown by western blotting (b). Total RNA was isolated from CD44+CD117+ cells and the mRNA was quantitated by real-time RT-PCR. Metformin at 0.1 mM significantly reduced snail2, twist, and vimentin, but not snail1 expression level, and upregulated the E-cadherin expression level (c). * Significant difference compared with control (P < 0.05)

Mentions: To investigate the effect of metformin at low dose (0.1 mM) on the EMT of SKOV3 cells, SKOV3 cells were treated with metformin at 0.1 mM for 72 hours and expression of EMT components, including snail1, snail2, twist, vimentin, and E-cadherin, was then analyzed by quantitative real-time PCR. We observed that metformin at 0.1 mM showed no effect on snail1 expression, a 2.3-fold decrease on snail2, a 2.4-fold decrease on twist, a 3.4-fold decrease on vimentin, and a 1.9-fold increase on E-cadherin (Fig. 3a). Western blotting assay showed that metformin increased significantly the expression of E-cadherin and decreased snail2, twist, and vimentin, which were consistent with the quantitative RT-PCR results (Fig. 3b). To determine expression of the EMT biomarkers, flow cytometry was used to isolate CD44+CD117+ cells. Data from quantitative real-time PCR showed that metformin at 0.1 mM caused no change in snail1 expression, but a 5.9-fold decrease for snail2, a 5.6-fold decrease for twist, a 7.7-fold decrease for vimentin, and a 2.32-fold increase for E-cadherin (Fig. 3c).Fig. 3


Inhibitory effects of metformin at low concentration on epithelial-mesenchymal transition of CD44(+)CD117(+) ovarian cancer stem cells.

Zhang R, Zhang P, Wang H, Hou D, Li W, Xiao G, Li C - Stem Cell Res Ther (2015)

Low concentrations of metformin reduced EMT of ovarian cancer cells and the CD44+CD117+ CSC population. SKOV3 cells were incubated with 0.1 mM metformin for 72 hours. The CD44+CD117+ cells were isolated by flow cytometry. Total RNA was isolated and mRNA was quantitated by real-time RT-PCR. mRNA expression of snail1, snail2, twist, vimentin, and E-cadherin was analyzed. Data show that treatment with 0.1 mM metformin reduced snail2, twist, and vimentin, but not snail1expression level, and upregulated the E-cadherin expression level (a). Bands detected with antibodies indicate the immune reactivity for snail1, snail2, twist, vimentin, E-cadherin, and β-actin: 72 hours of treatment with 0.1 nM metformin significantly downregulated snail2, twist, and vimentin, but not snail1, and upregulated E-cadherin as shown by western blotting (b). Total RNA was isolated from CD44+CD117+ cells and the mRNA was quantitated by real-time RT-PCR. Metformin at 0.1 mM significantly reduced snail2, twist, and vimentin, but not snail1 expression level, and upregulated the E-cadherin expression level (c). * Significant difference compared with control (P < 0.05)
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Fig3: Low concentrations of metformin reduced EMT of ovarian cancer cells and the CD44+CD117+ CSC population. SKOV3 cells were incubated with 0.1 mM metformin for 72 hours. The CD44+CD117+ cells were isolated by flow cytometry. Total RNA was isolated and mRNA was quantitated by real-time RT-PCR. mRNA expression of snail1, snail2, twist, vimentin, and E-cadherin was analyzed. Data show that treatment with 0.1 mM metformin reduced snail2, twist, and vimentin, but not snail1expression level, and upregulated the E-cadherin expression level (a). Bands detected with antibodies indicate the immune reactivity for snail1, snail2, twist, vimentin, E-cadherin, and β-actin: 72 hours of treatment with 0.1 nM metformin significantly downregulated snail2, twist, and vimentin, but not snail1, and upregulated E-cadherin as shown by western blotting (b). Total RNA was isolated from CD44+CD117+ cells and the mRNA was quantitated by real-time RT-PCR. Metformin at 0.1 mM significantly reduced snail2, twist, and vimentin, but not snail1 expression level, and upregulated the E-cadherin expression level (c). * Significant difference compared with control (P < 0.05)
Mentions: To investigate the effect of metformin at low dose (0.1 mM) on the EMT of SKOV3 cells, SKOV3 cells were treated with metformin at 0.1 mM for 72 hours and expression of EMT components, including snail1, snail2, twist, vimentin, and E-cadherin, was then analyzed by quantitative real-time PCR. We observed that metformin at 0.1 mM showed no effect on snail1 expression, a 2.3-fold decrease on snail2, a 2.4-fold decrease on twist, a 3.4-fold decrease on vimentin, and a 1.9-fold increase on E-cadherin (Fig. 3a). Western blotting assay showed that metformin increased significantly the expression of E-cadherin and decreased snail2, twist, and vimentin, which were consistent with the quantitative RT-PCR results (Fig. 3b). To determine expression of the EMT biomarkers, flow cytometry was used to isolate CD44+CD117+ cells. Data from quantitative real-time PCR showed that metformin at 0.1 mM caused no change in snail1 expression, but a 5.9-fold decrease for snail2, a 5.6-fold decrease for twist, a 7.7-fold decrease for vimentin, and a 2.32-fold increase for E-cadherin (Fig. 3c).Fig. 3

Bottom Line: Low dose of metformin reduced survivin expression in CSCs.Metformin was also associated with a reduction of snail2, twist, and vimentin in CD44(+)CD117(+) ovarian CSCs in vivo.Our results implicate that metformin at low dose inhibits selectively CD44(+)CD117(+) ovarian CSCs through inhibition of EMT and potentiates the effect of cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Gynecology, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200092, China. rrzhangcn@126.com.

ABSTRACT

Background: Although metformin, a first-line drug for treating diabetes, may play an important role in inhibition of epithelial ovarian cancer cell growth and cancer stem cells (CSCs), metformin at low dose showed less effect on the proliferation of ovarian cancer cells. In this study, we evaluated the effect of metformin at low dose on ovarian CSCs in order to understand the molecular mechanisms underlying.

Methods: The inhibitory effects of metformin at los dose on proliferation and population of ovarian cancer cells including SKOV3 and A2780 were assessed by cell proliferation assay and flow cytometry. Quantitative real-time PCR assay on expression of Bcl-2, Survivin and Bax was performed to determine the effect of metformin at low dose on epithelial-mesenchymal transition (EMT) of cancer cells and CSCs. Tumor sphere formation assay was also performed to evaluate the effect of metformin on spheres forming ability of CSCs. The therapeutic efficacy and the anti-CSC effects of metformin at low dose were investigated by using both SKOV3 cells and primary tumor xenografts. In addition, the CSC frequency and EMT in tumor xenograft models were also assessed by flow cytometry and quantitative real-time PCR.

Results: Metformin at low dose did not affect the proliferation of ovarian cancer cells. However, it inhibited population of CD44(+)CD117(+) selectively, neither CD133(+) nor ALDH(+) cells. It suppressed expression of snail2, twist and vimentin significantly in cancer cells and CD44(+)CD117(+) CSCs in vitro. Low dose of metformin reduced survivin expression in CSCs. Low concentrations of metformin inhibited the secondary and the tertiary tumor sphere formation, decreased SKOV3 and primary ovarian tumor xenograft growth, enhanced the anticancer effect of cisplatin, and lowered the proportion of CD44(+)CD117(+) CSCs in the xenograft tissue. Metformin was also associated with a reduction of snail2, twist, and vimentin in CD44(+)CD117(+) ovarian CSCs in vivo.

Conclusions: Our results implicate that metformin at low dose inhibits selectively CD44(+)CD117(+) ovarian CSCs through inhibition of EMT and potentiates the effect of cisplatin.

No MeSH data available.


Related in: MedlinePlus