Research Article: Genome protective effect of metformin as revealed by reduced level of constitutive DNA damage signaling

Date Published: October 28, 2011

Publisher: Impact Journals LLC

Author(s): H. Dorota Halicka, Hong Zhao, Jiangwei Li, Frank Traganos, Sufang Zhang, Marietta Lee, Zbigniew Darzynkiewicz.

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Abstract

We have shown before that constitutive DNA damage signaling represented by H2AX-Ser139 phosphorylation and ATM activation in untreated normal and tumor cells is a reporter of the persistent DNA replication stress induced by endogenous oxidants, the by-products of aerobic respiration. In the present study we observed that exposure of normal mitogenically stimulated lymphocytes or tumor cell lines A549, TK6 and A431 to metformin, the specific activator of 5’AMP-activated protein kinase (AMPK) and an inhibitor of mTOR signaling, resulted in attenuation of constitutive H2AX phosphorylation and ATM activation. The effects were metformin-concentration dependent and seen even at the pharmacologically pertinent 0.1 mM drug concentration. The data also show that intracellular levels of endogenous reactive oxidants able to oxidize 2′,7′-dihydro-dichlorofluorescein diacetate was reduced in metformin-treated cells. Since persistent constitutive DNA replication stress, particularly when paralleled by mTOR signaling, is considered to be the major cause of aging, the present findings are consistent with the notion that metformin, by reducing both DNA replication stress and mTOR-signaling, slows down aging and/or cell senescence processes.

Partial Text

In live cells, DNA is continuously being damaged by reactive oxygen species (ROS), the by-products of aerobic respiration in mitochondria [1-6]. Exogenous oxidants originating from environmental pollutants [7],phagocyte-oxidative burst [8-10], and even iatrogenic factors [11], additionally contribute to DNA damage. Such DNA damage involves oxidation of the constituent DNA bases, particularly of guanine by formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxo8dG), base ring fragmentation, modification of deoxyglucose, crosslinking of DNA and protein, and induction of DNA double strand breaks (DSBs) [12, 13]. Another important injurious effect of endogenous and exogenous oxidants is peroxidation of lipids in cell membranes [14].

The effect of metformin was tested on the level of constitutive expression of γH2AX and Ser1981-phoshorylated ATM in human lung adenocarcinoma A549 cells. The cells were grown attached on slides and the expression of these phospho-proteins was measured by laser scanning cytometry (LSC) [57]. The data provide clear evidence that expression of γH2AX in A549 cells growing in the presence of metformin for 48 h was reduced (Figure 1). The reduction was apparent at 1 mM, and was progressively more pronounced following exposure to 5 and 20 mM concentrations of metformin.

The present data demonstrate that exposure of either normal, mitogenically activated lymphocytes, or tumor cell lines (A549, TK6) to metformin leads to a decrease in the level of constitutive phosphorylation of H2AX on Ser139 and constitutive activation of ATM. The observed decrease was evident even at a concentration as low as 0.1 mM metformin (Figures 3 and 4). Pharmacokinetic data indicate that this concentration of metformin is of pharmacological relevance [60].Since the level of constitutive expression of γH2AX and ATM-S1981P to a large extent reports DNA damage signaling in response to DNA damage by endogenous oxidants generated during aerobic respiration [39-45, 58]. the present findings would be consistent with a notion that metformin exerts protective effect on nuclear DNA against oxidative damage. These findings are consistent with the observation that exposure of cells to metformin lowered the extent of reactive oxidants that were able to oxidize the H2DCF-DA substrate (Figure 5). They are also in accordance with numerous studies in which a decrease in the level of ROS in cells treated with metformin has been observed [55, 61-65]. It appears that the mechanisms activated by metformin for neutralizing ROS such as upregulation of the antioxidant thioredoxin [55], and/or suppression of NAD(P)H oxidase activity [61] may prevail over the ROS-generating inhibitory effect on mitochondrial respiratory complex I or catabolic processes activated by AMPK [66, 67].

 

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