2. Metformin inhibits heme oxygenase-1 expression in cancer cells
2.4. Down-regulation of HO-1 expression by metformin
Metformin strongly suppressed HO-1 expression in HeLa cells (Fig. 9A and B), which lack the endogenous LKB1 required for AMPK activation by metformin (Spicer et al., 2003; Tiainen et al., 1999). Further results indicated that metformin strongly induces AMPK activation in HepG2 cells after 24 h of treatment. However, metformin failed to induce AMPK phosphorylation in HeLa cells (Fig. 12A), which was also reported in a previous study (Vitale-Cross et al., 2012), suggesting that metformin regulates HO-1 expression through AMPK-independent mechanisms. To verify whether AMPK activation plays an indispensable role in the down-regulation of HO- 1 expression by metformin, AMPK expression was knockdown by a small interfering RNA (siRNA). Results indicated that in AMPK-depleted HepG2 cells, metformin treatment also significantly reduced HO-1 protein levels to a similar extent as the control siRNA-transfected cells (Fig. 12B). To confirm these findings, HepG2 cells were transfected with pcDNA 3.1 (control) or a dominant-negative form of AMPK (DN-AMPK) for 48 h, followed by incubation with metformin for 24 h. Transfection with DN-AMPK did not abolish metformin-induced HO-1 inhibition (Fig. 12C). Corroborating results were obtained by pretreating HepG2 cells with the specific AMPK inhibitor
compound C (10 M) for 1 h followed by treatment with 5 mM metformin for an additional 24 h. Compound C did not reverse the suppressive effects of metformin on HO-1 expression (Fig. 12D). Overall, these data suggested that metformin suppresses HO-1 expression in cancer cells by an alternative, AMPK-independent signaling mechanism.
- + Metformin 5 mM -
AMPK
+ HO-1
AMPKa siRNA Control
siRNA B
p-AMPK
-actin
HepG2
1.0 0.2 1.0 0.2
1.0 3.9 1.1 1.0
1.0 0.9 0.2 0.2
+ + -
Metformin 5 mM -
HO-1 D
-actin
HepG2
+ -
Compound C 10 M - +
1.0 0.3 0.2 1.0 A
AMPK
p-AMPK
p-AMPK
-actin
-actin
AMPK HeLa
HepG2
Metformin (mM) - 1 2.5 5 1.0 1.2 2.9 3.6
0.0 0.0 0.0 0.0
- + Metformin 5 mM -
-actin
+ DN-AMPK pcDNA
C
AMPK
HO-1
HepG2
1.0 0.1 1.1 0.1
1.0 0.9 2.3 2.3
Fig. 12. Metformin suppresses HO-1 expression in cancer cells in an AMPK- independent manner. (A) Effects of metformin on AMPK activation in HepG2 and HeLa cells by Western blotting. (B) Effects of metformin and AMPK siRNA on HO-1 expression. HepG2 cells were transfected with AMPK siRNA or control siRNA for 48 h. The transfected cells were treated with 5 mM metformin for 24 h. HO-1 protein expression and phosphorylation of AMPK and AMPK were assayed by Western blotting. (C) Effects of metformin and dominant-negative AMPK (DN-AMPK) on HO-1 expression. HepG2 cells were transfected with DN-AMPK or the pcDNA 3.1 control for 48 h. The transfected cells were treated with 5 mM metformin for 24 h. HO-1 and AMPK expression levels were determined by Western blotting. (D) Effects of metformin and compound C (AMPK inhibitor) on HO-1 expression. HepG2 cells were pretreated for 1 h with 10 M compound C and then the cells were treated for an additional 24 h with 5 mM metformin. HO-1 protein expression was assessed by Western blotting.
2.5. Reduction of HO-1 contributes to anti-proliferative effects of metformin in cancer cells
Previous study has shown that metformin (3–10 mM) significantly decreased proliferation of multidrug-resistant MCF-7/adr cells (Kim et al., 2011). In the present study, the hypothesis that the anti-proliferative effects of metformin in cancer cells are partially mediated by HO-1 inhibition is tested. Therefore, HepG2, A549 and HeLa cells were treated with increasing doses of metformin (1–10 mM) and the specific HO-1 inhibitor ZnPPIX (5 M) for 48 h. 5-Bromo-2’-deoxyuridine (BrdU) incorporation assay was performed to determine the BrdU incorporation during DNA synthesis of these drug- treated cells as described in the Materials and methods. Metformin and ZnPPIX significantly decreased proliferation in HepG2 and A549 cells after 48 h of treatment (Fig. 13A and B). Meanwhile, metformin treatment did not significantly reduce proliferation of HeLa cells (Fig. 13C). To overcome the resistance to metformin of HeLa cells, it was supposed that metformin pretreatment could enhance the anti-proliferative effects of the anti-cancer drug paclitaxel. To test this hypothesis, HeLa cells were pretreated with 5 mM metformin for 24 h followed by 100 nM paclitaxel for an additional 48 h. Fig. 13D indicates that metformin pretreatment sensitizes HeLa cells to the anti-proliferative effects of paclitaxel compared to the effects of treatment with paclitaxel only. Finally, to further confirm whether HO-1
suppression is responsible for anti-proliferation effect of metformin, HepG2 cells were transfected with an HO-1 expression vector or pCMV6-Neo empty vector, and then the cells were treated with metformin. The elevation of HO-1 levels in transfected cells was confirmed by Western blot analysis, and the overexpressed level of HO-1 protein was also significantly reduced by metformin treatment (Fig. 14A). HO-1 induction by transfection with HO-1 expression vector significantly attenuated metformin-induced inhibition of cell growth (Fig. 14B). These results suggest that metformin inhibits cancer cell proliferation or sensitizes cancer cells to anti-cancer drugs by suppressing HO-1 expression.
Taken together, metformin inhibited Raf/ERK signaling to down-regulate Nrf2 and HO-1 expression resulted in suppressing cell proliferation and increasing sensitivity of cancer cells to therapeutic drugs (Fig. 15).
0 20 40 60 80 100
BrdU incorporation (% of control)
* *
* *
A549
Metformin (mM) - 1 2.5 5 10 - ZnPPIX (M) - - - - - 5 B
0 20 40 60 80 100
BrdU incorporation (% of control) *
* *
HepG2
Metformin (mM)- 1 2.5 5 10 - ZnPPIX (M)- - - - - 5 A
0 20 40 60 80 100
BrdU incorporation (% of control) *
Hela
Metformin (mM) - 1 2.5 5 10 - ZnPPIX (M) - - - - - 5 C
0 20 40 60 80 100
BrdU incorporation (% of control)
*
*
# Hela
Metformin (mM) - 5 - 5
Paclitaxel (nM) - - 100 100 D
Fig. 13. Effects of metformin on proliferation of cancer cells. Effects of metformin and a specific HO-1 inhibitor on the proliferation of (A) HepG2, (B) A549 and (C) HeLa cell lines. Cells were treated with increasing doses of metformin (1–10 mM) or the specific HO-1 inhibitor ZnPPIX (5 M) for 48 h. (D) Metformin pretreatment enhanced the anti-proliferative effects of paclitaxel in HeLa cells. HeLa cells were pretreated with 5 mM metformin for 24 h followed by 100 nM paclitaxel for an additional 48 h. The BrdU incorporation assay was performed as described in the Materials and methods. All experiments were performed in quadruplicate. Bars represent mean ± SD. *P < 0.05 vs. control; #P < 0.05 vs. cells treated with paclitaxel alone.
Fig. 14. Role of HO-1 suppression in anti-proliferative effect of metformin in cancer cells. (A) Metformin suppresses HO-1 overexpression by gene transfer. HepG2 cells were transfected with a mammalian expression plasmid vector containing the full length cDNA of human HO-1 (HO-1 expression vector) or pCMV6 vector for 24 h, and then the cells were treated with metformin for an additional 24 h. Cell lysates were subjected to Western blotting using antibodies specific for HO-1 and -actin. (B) HO-1 overexpression restores anti-proliferative effect of metformin in cancer cells.
HepG2 cells were transfected with an HO-1 expression vector or pCMV6 vector for 24 h, and then the cells were treated with metformin for an additional 48 h. The BrdU incorporation assay was performed as described in the Materials and methods. All experiments were performed in quadruplicate. *P < 0.05 vs the control; #P < 0.05 vs. cells treated with metformin alone.
HO-1 A
-actin
HepG2
1.0 2.7 1.8 0.1
Metformin 10 mM - - + +
pCMV6 vector + - - +
HO-1 expression vector - + + -
HepG2
Metformin 10 mM - - + + B
pCMV6 vector + - - +
HO-1 expression vector - + + -
0 20 40 60 80 100
BrdU incorporation (% of control)
*
*
#
Fig. 15. Proposed signaling pathways underlying the effects of metformin on down-regulation of HO-1 expression in cancer cells. Ɵ means an inhibitory effect.
Nrf2 + 1 Nrf2 MEK1/2
ERK1/2
Nrf2 transcription (Autoregulation) PD98059
Cytosol
Nucleus
Raf
Metformin
Nrf2 Keap1
ARE‐like binding site
+ 1
HO-1 transcription
ARE binding site
mRNA Chemoresistance Radioresistance Tumorigenicity
protein
HO-1