3. Metformin induces microRNA-34a to down-regulate Sirt1/Pgc-1/Nrf2
3.6. Up-regulation of DR5 expression by metformin sensitizes wild-type p53 cancer cells to TRAIL-induced apoptosis
Previous studies demonstrated that Sirt1 inhibition sensitized cancer cells to TRAIL-induced apoptosis through induction CHOP and DR5 expression (Kim et al., 2013) and Nrf2 expression has been associated with TRAIL resistance in cancer cells (Arlt et al., 2009). It was hypothesized that the down-regulation of Sirt1 and Nrf2 expression by metformin would up- regulate CHOP, DR5 expression and sensitize TRAIL-resistant MCF-7 cells to TRAIL-induced apoptosis. MCF-7 cells were treated with 1 mM metformin for 24 and 48 h, followed by qRT-PCR assessment of CHOP and DR5 mRNA levels. Metformin significantly induced CHOP and DR5 mRNA levels (Fig. 23A and B). Western blot analysis indicated that metformin induced DR5 protein levels after 24 and 48 h of treatment (Fig. 23C). To elucidate role of Sirt1 in the regulation of DR5 expression, MCF-7 cells were transfected with siRNA Sirt1, and the cells were harvested for analysis of DR5 protein expression by Western blotting. As shown in Fig. 23D, the deletion of Sirt1 clearly induced DR5 protein expression. Notably, when wild-type p53 MCF-7 cells were pre-treated with metformin 1 mM for 24 h, followed by treatment with TRAIL 25 ng/mL for an additional 48 h, metformin clearly potentiated TRAIL-mediated cell death (Fig. 24A).
Interestingly, pre-treatment with even a low concentration of metformin
significantly enhanced TRAIL-mediated survival inhibition in MCF-7 cancer cells (Fig. 24B). To investigate whether the death caused by metformin and TRAIL treatment was related to apoptosis, PARP protein expression was analyzed as a marker of apoptosis. MCF-7 cells were pretreated with 1 mM metformin for 24 h and then treated with 25 ng/mL TRAIL for an additional 24 h. PARP protein levels in cell lysates were analyzed by Western blotting.
Pretreatment with metformin potentially enhanced TRAIL-induced cleaved PARP protein levels (Fig. 24C). To confirm the up-regulation DR5 expression by metformin relates to TRAIL sensitization. Knockdown of DR5 by using siRNA significantly restored cell viability in response to metformin and TRAIL treatment (Fig. 24D). Moreover, the knockdown of DR5 also suppressed the metformin-mediated sensitization to TRAIL-induced apoptosis (Fig. 24E). However, metformin did not increase TRAIL sensitivity in p53-mutated MDA-MB-231 cells (Fig. 24F). These results indicate that metformin sensitizes wild-type p53 cancer cells to TRAIL- induced apoptosis through DR5 up-regulation.
Overall, metformin induced p53/miR-34a expression to down-regulate Sirt1/Pgc-1/Nrf2 pathway leads to increased susceptibility of wild-type p53 cancer cells to oxidative stress and TRAIL-induced apoptosis (Fig. 25).
Fig. 23. Effects of metformin on CHOP and DR5 expression in breast cancer cells. Metformin induces CHOP (A) and DR5 (B) mRNA expression in MCF-7 cells after 24 and 48 h of treatment. CHOP and DR5 mRNA levels were analyzed by qRT-PCR. All experiments were performed in triplicate.
Bars represent mean ± SD; *P < 0.05 vs. control. (C) Metformin induced DR5 protein expression in MCF-7 cells after 24 and 48 h of treatment. DR5 and -actin protein levels in cell lysates were analyzed by Western blotting.
(D) Sirt1 depletion induced DR5 protein levels in cancer cells. MCF-7 cells were transfected with Sirt1 or control siRNA for 48 h. Cell lysates were subjected to Western blotting using antibodies specific for Sirt1, DR5 and -
actin.
0 2 4 6 8 10 12 14 16
Relative CHOP mRNA to 18S rRNA (fold of control)
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*
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Metformin (mM) - 1 - 1
24 h 48 h
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Relative DR5 mRNA to 18S rRNA (fold of control)
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Metformin (mM) - 1 - 1
24 h 48 h
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-actin DR5
Metformin (mM) - 1 - 1
24 h 48 h
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Control siRNA siRNA Sirt1
-actin DR5 D
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- +
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Control Metformin 1 mM TRAIL 25 ng/mL
Metformin 1 mM + TRAIL 25 ng/mL
Metformin (mM) Cleaved
PARP
-actin Full-length
PARP
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TRAIL (ng/mL) - - 25 25 -
C
1.0 1.3 2.5 7.2
Metformin (mM) 0.02 0.1 0.5 1 5 B
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Cell viability (% of control)
Metformin alone Metformin + TRAIL 25 ng/mL
*
* *
* *
* *
*
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# MCF-7
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Cell viability (% of control)
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*
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Control siRNA - - - -
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Control siRNA Cleaved
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1.0 4.5 1.7 1.0
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Metformin 1 mM + + -
TRAIL 25 ng/mL - + + - -
Metformin (mM) 0.02 0.1 0.5 1 5 F
-
0 20 40 60 80 100
Cell viability (% of control)
Metform in alone Metform in + TRAIL 25 ng/m L
* * * * * *
MDA-MB-231
Fig. 24. Metformin sensitizes wild-type p53 cancer cells to TRAIL-induced apoptosis. (A) Metformin sensitized wild-type p53 MCF-7 breast cancer cells to TRAIL-induced cell death. MCF-7 cells were pre-treated with 1 mM metformin for 24 h, followed by stimulating to 25 ng/mL TRAIL for an additional 48 h. After treatment, cellular morphology images were captured.
(B) The effect of metformin on TRAIL-induced survival inhibition in MCF-7 cells as measured by MTT assay. All experiments were performed in quadruplicate. Bars represent mean ± SD; *P < 0.05 vs. control. #P < 0.05 vs. cells treated with TRAIL and metformin alone. (C) Metformin enhanced TRAIL-induced apoptosis in MCF-7 breast cancer cells. MCF-7 cells were pre-treated with 1 mM metformin for 24 h and then treated with 25 ng/mL TRAIL for an additional 24 h. PARP and -actin protein levels in cell lysates were analyzed by Western blotting. (D) Effects of knockdown of DR5 expression on the suppression of cell viability caused by pre-treatment with metformin and followed by TRAIL. MCF-7 cells were transfected with DR5 siRNA or a non-specific control siRNA for 24 h. The siRNA-transfected cells were pre-treated with metformin for 24 h, and then with TRAIL for an additional 48 h. Cell viability was measured by MTT assay. All experiments were performed in quadruplicate. Bars represent mean ± SD. *P < 0.05 vs.
control; #P < 0.05 vs. cells treated with metformin or TRAIL alone; ¥P <
0.05 vs. cells treated with both metformin and TRAIL. (E) Effects of knockdown of DR5 expression on the metformin-mediated sensitization to TRAIL-induced apoptosis. MCF-7 cells were transfected with DR5 siRNA or a non-specific control siRNA for 48 h. The siRNA-transfected cells were pre-treated with metformin for 24 h, and then with TRAIL for an additional 12 h. DR5, PARP and -actin protein levels in cell lysates were analyzed by Western blotting. (F) Effect of metformin on TRAIL-induced survival inhibition in p53-mutated MDA-MB-231 cells as measured by MTT assay.
All experiments were performed in quadruplicate. Bars represent mean ± SD; *P < 0.05 vs. control.
Fig. 25. Proposed signaling pathways underlying the effects of metformin on Sirt1/Pgc-1/Nrf2 pathway leads to increased susceptibility of cancer cells to oxidative stress and therapeutic agents.
p53Ac
Metformin
miR-34a
3’-UTR AAA
Sirt1 mRNA Sirt1
deacetylation
Pgc-1 PPAR Nrf2
CHOP CHOP DR5
Nrf2
DR5 DR5 DR5 TRAIL sensitization
SOD2HO-1 HO-1 Nrf2
Oxidative stress sensitization
SOD2 CHOP