3. Metformin induces microRNA-34a to down-regulate Sirt1/Pgc-1/Nrf2
3.3. Down-regulation of Sirt1 by metformin inhibits Nrf2 expression
We performed qRT-PCR to verify that metformin significantly reduced Nrf2 mRNA levels in MCF-7 cells after 24 h of treatment (Fig. 19B). To confirm that Sirt1 positively regulates Nrf2 expression, MCF-7 cells were transfected with Sirt1 siRNA for 48 h. Knockdown of Sirt1 notably reduced Nrf2 protein levels. Additionally, MCF-7 cells were transfected with a Sirt1 expression vector, which remarkably induced Nrf2 protein levels (Fig. 19C). These results suggest that down-regulation of Nrf2 expression by metformin was mediated through a reduction of Sirt1 in cancer cells.
Next experiments were performed to investigate effects of metformin on the expression of cytoprotective HO-1 and anti-oxidative SOD2 enzymes that were regulated by Nrf2 (Cherry et al., 2014). Metformin reduced HO-1 and SOD2 protein levels after 24 h of treatment in MCF-7 cells (Fig. 19D).
However, metformin up-regulated catalase protein levels in the same condition of treatment (Fig. 19D). Because the expression of SOD2, catalase and HO-1 plays an important role for controlling balance of ROS and cellular protection against oxidative stress, intracellular ROS production was determined in metformin or H2O2-stimulated MCF-7 breast cancer cells. As showed in Fig. 20A, metformin (1–5 mM) did not affect to basal ROS, while ROS production was 5-fold increased significantly by stimulating with H2O2
(100 M) compared with the control after 1 h of treatment. Metformin also did not change ROS generation after 24 h of treatment (data not shown).
Interestingly, pre-treatment with metformin for 24 h significantly enhanced H2O2-induced cell cytotoxicity and apoptosis in MCF-7 breast cancer cells (Fig. 20B, C, D and E). However, pre-treatment with metformin did not enhance H2O2-induced cell cytotoxicity in p53-mutated MDA-MB-231 cells (Fig. 20F and G). These results indicated that metformin increases susceptibility of wild-type p53 cancer cells to oxidative stress.
-actin Nrf2
Metformin (mM) - 1 2.5 5
1.0 0.5 0.2 0.1
Nrf2
-actin
Metformin (mM) - 5 - 5
48 h 72 h
1.0 0.4 1.0 0.1
24 h A
Metformin (mM) - 1 2.5 5
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Relative Nrf2 mRNA to 18S rRNA (fold of control)
*
* *
B
Sirt1
Control siRNA siRNA Sirt1
-actin Nrf2
pCMV6 vector Sirt1 expression vector
+ - - -
- + - -
- - + -
- - - +
1.0 0.2 1.0 3.1
1.0 0.5 1.0 2.0
C
Metformin (mM) - 1 2.5 5 HO-1
1.0 0.9 0.6 0.4
-actin SOD2 Catalase
1.0 0.5 0.5 0.4
1.0 1.5 1.8 2.0
D
Fig. 19. Metformin-mediated down-regulation of Sirt1 inhibits Nrf2 expression in MCF-7 breast cancer cells. (A) Effects of metformin on Nrf2 protein levels in MCF-7 cells after 24 h of treatment (upper panel) and after 48 and 72 h of treatment (lower panel). Nrf2 and -actin protein levels in cell lysates were assayed by Western blotting. (B) Effects of metformin on Nrf2 mRNA expression. qRT-PCR demonstrated the effect of metformin on Nrf2 mRNA levels in MCF-7 cells after 24 h of treatment. All experiments were performed in triplicate. Bars indicate mean ± SD; *P < 0.05 vs. control. (C) Role of Sirt1 in regulating Nrf2 expression in cancer cells. MCF-7 cells were transfected with Sirt1 siRNA, a Sirt1 expression, control siRNA, or pCMV6 vector for 48 h. Cell lysates were subjected to Western blotting using antibodies specific for Sirt1, Nrf2 and -actin. (D) Effects of metformin on HO-1, SOD2 and catalase expression in MCF-7 cells. HO-1, SOD2, catalase and -actin protein levels in cell lysates were assayed by Western blotting.
B Control Metformin 5 mM
Metformin 5 mM + H2O2100 M H2O2100 M
0 1 5 6
ROS production (relative to control)
*
Metformin (mM)
H2O2(M) - 1 2.5 5
- - - -
- 100 A
Metformin (mM) Cleaved
PARP
-actin Full-length
PARP
5 - 5
H2O2(M) - - 100 100 -
1.0 1.0 0.9 2.6
E
Metformin (mM) 0.5 1 5
C
-
0 2 0 4 0 6 0 8 0 1 0 0
Cell viability (% of control)
M e tfo rm in a lo n e M e tfo rm in + H 2O 2 100 M
*
*
*
*
* *
#
#
#
MCF-7
Metformin (mM) 0.5 1 5
D
-
1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0
LDH leakage (% of control)
M e tfo rm in alo n e M e tfo rm in + H 2O 2 100 M
*
*
*
*
# #
MCF-7 #
Metformin (mM) 0.5 1 5
F
-
0 2 0 4 0 6 0 8 0 1 0 0
Cell viability (% of control)
M e tfo rm in a lo n e M e tfo rm in + H 2O 2 100 M
MDA-MB-231
Metformin (mM) 0.5 1 5
G
-
0 2 0 4 0 6 0 8 0 10 0 12 0
LDH leakage (% of control)
M etfo rm in alo ne M etfo rm in + H 2O 2 100 M
MDA-MB-231
Fig. 20. Effects of metformin on intracellular ROS production, H2O2-induced cytotoxicity and apoptosis. (A) Effects of metformin and H2O2 on intracellular ROS production in MCF-7 cells. Intracellular ROS production was determined using the fluorescent probe H2DCFDA. Bars represent mean
± SD. *P < 0.05 vs. control, n=6. (B and C) Effects of metformin on H2O2- induced survival inhibition in wild-type p53 MCF-7 cells as measured by MTT assay. (D) Effects of metformin on H2O2-induced cytotoxicity in wild- type p53 MCF-7 cells as measured by LDH assay. All experiments were performed in quadruplicate. Bars represent mean ± SD; *P < 0.05 vs. control.
#P < 0.05 vs. cells treated with H2O2 and metformin alone. (E) Effects of metformin on H2O2-induced apoptosis. MCF-7 cells were pre-treated with 5 mM metformin for 24 h, followed by stimulating to 100 M H2O2 for an additional 12 h. PARP and -actin in cell lysates were analyzed by Western blotting. (F) Effects of metformin and H2O2 on cell viability of p53-mutated MDA-MB-231 cells as measured by MTT assay. (G) Effects of metformin and H2O2 on cytotoxicity of p53-mutated MDA-MB-231 cells as measured by LDH assay. All experiments were performed in quadruplicate. Bars represent mean ± SD.