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Metformin induces an intracellular reductive state that protects oesophageal squamous cell carcinoma cells against cisplatin but not copper-bis(thiosemicarbazones).

Damelin LH, Jivan R, Veale RB, Rousseau AL, Mavri-Damelin D - BMC Cancer (2014)

Bottom Line: Metformin was found to reduce cell proliferation significantly in all OSCC cell lines, with an accumulation of cells in G0/G1 phase of the cell cycle.Our results indicate that a major mechanism of metformin-induced cisplatin resistance results from a significant increase in glycolysis, intracellular NAD(P)H levels with a concomitant increase in reduced intracellular thiols, leading to decreased cisplatin-DNA adduct formation.The glutathione synthesis inhibitor buthionine sulfoximine significantly ablated the protective effect of metformin.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag X3, Johannesburg 2050, South Africa. Demetra.Mavri-Damelin@wits.ac.za.

ABSTRACT

Background: Oesophageal squamous cell carcinoma (OSCC) is a highly aggressive carcinoma with a poor survival rate. One of the most commonly used chemotherapeutic drugs, cisplatin, displays varied and often poor efficacy in vivo. Therefore, alternative, cost-effective and more efficacious treatments are required. Metformin has been previously shown to reduce proliferative rates in various carcinoma cell lines. We report for the first time, the effect of metformin on OSCC cell proliferation and show that it antagonises cisplatin-induced but not copper-bis(thiosemicarbazone)-induced cytotoxicity in OSCC cells.

Methods: Cell proliferation and stage of the cell cycle were quantified by trypan blue counts and flow cytometry, respectively. All cytotoxicity measurements were made using the tetrazolium based MTT assay. Metabolic alterations to cells were determined as follows: glycolysis via a lactate dehydrogenase assay, reducing equivalents by MTT reduction and reduced intracellular thiols by monobromobimane-thiol fluorescence, and glutathione depletion using buthionine sulfoximine. Inductively coupled plasma mass spectrometry was used to quantify cisplatin-DNA adduct formation.

Results: Metformin was found to reduce cell proliferation significantly in all OSCC cell lines, with an accumulation of cells in G0/G1 phase of the cell cycle. However, metformin significantly protected OSCC cells against cisplatin toxicity. Our results indicate that a major mechanism of metformin-induced cisplatin resistance results from a significant increase in glycolysis, intracellular NAD(P)H levels with a concomitant increase in reduced intracellular thiols, leading to decreased cisplatin-DNA adduct formation. The glutathione synthesis inhibitor buthionine sulfoximine significantly ablated the protective effect of metformin. We subsequently show that the copper-bis(thiosemicarbazones), Cu-ATSM and Cu-GTSM, which are trapped in cells under reducing conditions, cause significant OSCC cytotoxicity, both alone and in combination with metformin.

Conclusions: This is the first study showing that metformin can be used to decrease cell proliferation in OSCC cells. However, metformin protects against cisplatin cytotoxicity by inducing a reducing intracellular environment leading to lower cisplatin-DNA adduct formation. As such, we advise that caution be used when administering cisplatin to diabetic patients treated with metformin. Furthermore, we propose a novel combination therapy approach for OSCC that utilises metformin with metformin-compatible cytotoxic agents, such as the copper-bis(thiosemicarbazones), Cu-ATSM and Cu-GTSM.

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Metformin-induced cisplatin resistance is reversed by glutathione depletion in OSCC cells. The glutathione synthesis inhibitor, BSO was used to confirm the involvement of elevated glutathione levels in metformin induced cisplatin resistance in OSCC cells. MTT assays for cytotoxicity were performed as described, with cells treated with cisplatin alone (C), or in the presence 0.4 mM BSO (CB), or metformin and cisplatin (CM), or metformin and cisplatin in the presence of 0.4 mM BSO (CMB). Data is expressed as the percentage difference of LD50 values for each treatment relative to cisplatin alone (n = 3, mean ± SD). Predictably, the inhibition of glutathione synthesis increased cisplatin toxicity as LD50 values for cisplatin-BSO treated cells were significantly lower than cisplatin alone. Importantly, the presence of the inhibitor ablates the protective effect of metformin, with LD50 values for cisplatin-metfomin-BSO treated cells approaching those of cisplatin alone.
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Figure 6: Metformin-induced cisplatin resistance is reversed by glutathione depletion in OSCC cells. The glutathione synthesis inhibitor, BSO was used to confirm the involvement of elevated glutathione levels in metformin induced cisplatin resistance in OSCC cells. MTT assays for cytotoxicity were performed as described, with cells treated with cisplatin alone (C), or in the presence 0.4 mM BSO (CB), or metformin and cisplatin (CM), or metformin and cisplatin in the presence of 0.4 mM BSO (CMB). Data is expressed as the percentage difference of LD50 values for each treatment relative to cisplatin alone (n = 3, mean ± SD). Predictably, the inhibition of glutathione synthesis increased cisplatin toxicity as LD50 values for cisplatin-BSO treated cells were significantly lower than cisplatin alone. Importantly, the presence of the inhibitor ablates the protective effect of metformin, with LD50 values for cisplatin-metfomin-BSO treated cells approaching those of cisplatin alone.

Mentions: To confirm that increased thiol levels can protect OSCC cells against cisplatin, cells were treated with the cell permeable thiol derivative, N-acetyl cysteine (NAC) (10 mM) prior to cisplatin exposure [35]. Predictably, all OSCC cell lines were significantly protected against cisplatin cytotoxicity by NAC pre-treatment (Figure 5). Therefore, our hypothesis, that a metformin-dependent increase in intracellular thiols is primarily responsible for the observed protection against cisplatin, seemed highly plausible. Since glutathione is the major thiol species within the cells, we confirmed its role in metformin-induced cisplatin resistance using the glutathione synthase inhibitor, BSO [32], to deplete intracellular glutathione pools. Cells were treated with metformin in the presence of BSO, prior to cisplatin exposure. Glutathione depletion by BSO almost completely reversed the protective effect of metformin for all OSCC cell lines, confirming the role of reduced-glutathione in metformin-induced cisplatin resistance (Figure 6). We also observed that BSO increased cisplatin cytotoxicity, with lower LD50 values, and this was anticipated as decreased intracellular glutathione levels would result in less cisplatin-thiol sequestration and an increase in cisplatin-DNA adduct formation.


Metformin induces an intracellular reductive state that protects oesophageal squamous cell carcinoma cells against cisplatin but not copper-bis(thiosemicarbazones).

Damelin LH, Jivan R, Veale RB, Rousseau AL, Mavri-Damelin D - BMC Cancer (2014)

Metformin-induced cisplatin resistance is reversed by glutathione depletion in OSCC cells. The glutathione synthesis inhibitor, BSO was used to confirm the involvement of elevated glutathione levels in metformin induced cisplatin resistance in OSCC cells. MTT assays for cytotoxicity were performed as described, with cells treated with cisplatin alone (C), or in the presence 0.4 mM BSO (CB), or metformin and cisplatin (CM), or metformin and cisplatin in the presence of 0.4 mM BSO (CMB). Data is expressed as the percentage difference of LD50 values for each treatment relative to cisplatin alone (n = 3, mean ± SD). Predictably, the inhibition of glutathione synthesis increased cisplatin toxicity as LD50 values for cisplatin-BSO treated cells were significantly lower than cisplatin alone. Importantly, the presence of the inhibitor ablates the protective effect of metformin, with LD50 values for cisplatin-metfomin-BSO treated cells approaching those of cisplatin alone.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4020916&req=5

Figure 6: Metformin-induced cisplatin resistance is reversed by glutathione depletion in OSCC cells. The glutathione synthesis inhibitor, BSO was used to confirm the involvement of elevated glutathione levels in metformin induced cisplatin resistance in OSCC cells. MTT assays for cytotoxicity were performed as described, with cells treated with cisplatin alone (C), or in the presence 0.4 mM BSO (CB), or metformin and cisplatin (CM), or metformin and cisplatin in the presence of 0.4 mM BSO (CMB). Data is expressed as the percentage difference of LD50 values for each treatment relative to cisplatin alone (n = 3, mean ± SD). Predictably, the inhibition of glutathione synthesis increased cisplatin toxicity as LD50 values for cisplatin-BSO treated cells were significantly lower than cisplatin alone. Importantly, the presence of the inhibitor ablates the protective effect of metformin, with LD50 values for cisplatin-metfomin-BSO treated cells approaching those of cisplatin alone.
Mentions: To confirm that increased thiol levels can protect OSCC cells against cisplatin, cells were treated with the cell permeable thiol derivative, N-acetyl cysteine (NAC) (10 mM) prior to cisplatin exposure [35]. Predictably, all OSCC cell lines were significantly protected against cisplatin cytotoxicity by NAC pre-treatment (Figure 5). Therefore, our hypothesis, that a metformin-dependent increase in intracellular thiols is primarily responsible for the observed protection against cisplatin, seemed highly plausible. Since glutathione is the major thiol species within the cells, we confirmed its role in metformin-induced cisplatin resistance using the glutathione synthase inhibitor, BSO [32], to deplete intracellular glutathione pools. Cells were treated with metformin in the presence of BSO, prior to cisplatin exposure. Glutathione depletion by BSO almost completely reversed the protective effect of metformin for all OSCC cell lines, confirming the role of reduced-glutathione in metformin-induced cisplatin resistance (Figure 6). We also observed that BSO increased cisplatin cytotoxicity, with lower LD50 values, and this was anticipated as decreased intracellular glutathione levels would result in less cisplatin-thiol sequestration and an increase in cisplatin-DNA adduct formation.

Bottom Line: Metformin was found to reduce cell proliferation significantly in all OSCC cell lines, with an accumulation of cells in G0/G1 phase of the cell cycle.Our results indicate that a major mechanism of metformin-induced cisplatin resistance results from a significant increase in glycolysis, intracellular NAD(P)H levels with a concomitant increase in reduced intracellular thiols, leading to decreased cisplatin-DNA adduct formation.The glutathione synthesis inhibitor buthionine sulfoximine significantly ablated the protective effect of metformin.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag X3, Johannesburg 2050, South Africa. Demetra.Mavri-Damelin@wits.ac.za.

ABSTRACT

Background: Oesophageal squamous cell carcinoma (OSCC) is a highly aggressive carcinoma with a poor survival rate. One of the most commonly used chemotherapeutic drugs, cisplatin, displays varied and often poor efficacy in vivo. Therefore, alternative, cost-effective and more efficacious treatments are required. Metformin has been previously shown to reduce proliferative rates in various carcinoma cell lines. We report for the first time, the effect of metformin on OSCC cell proliferation and show that it antagonises cisplatin-induced but not copper-bis(thiosemicarbazone)-induced cytotoxicity in OSCC cells.

Methods: Cell proliferation and stage of the cell cycle were quantified by trypan blue counts and flow cytometry, respectively. All cytotoxicity measurements were made using the tetrazolium based MTT assay. Metabolic alterations to cells were determined as follows: glycolysis via a lactate dehydrogenase assay, reducing equivalents by MTT reduction and reduced intracellular thiols by monobromobimane-thiol fluorescence, and glutathione depletion using buthionine sulfoximine. Inductively coupled plasma mass spectrometry was used to quantify cisplatin-DNA adduct formation.

Results: Metformin was found to reduce cell proliferation significantly in all OSCC cell lines, with an accumulation of cells in G0/G1 phase of the cell cycle. However, metformin significantly protected OSCC cells against cisplatin toxicity. Our results indicate that a major mechanism of metformin-induced cisplatin resistance results from a significant increase in glycolysis, intracellular NAD(P)H levels with a concomitant increase in reduced intracellular thiols, leading to decreased cisplatin-DNA adduct formation. The glutathione synthesis inhibitor buthionine sulfoximine significantly ablated the protective effect of metformin. We subsequently show that the copper-bis(thiosemicarbazones), Cu-ATSM and Cu-GTSM, which are trapped in cells under reducing conditions, cause significant OSCC cytotoxicity, both alone and in combination with metformin.

Conclusions: This is the first study showing that metformin can be used to decrease cell proliferation in OSCC cells. However, metformin protects against cisplatin cytotoxicity by inducing a reducing intracellular environment leading to lower cisplatin-DNA adduct formation. As such, we advise that caution be used when administering cisplatin to diabetic patients treated with metformin. Furthermore, we propose a novel combination therapy approach for OSCC that utilises metformin with metformin-compatible cytotoxic agents, such as the copper-bis(thiosemicarbazones), Cu-ATSM and Cu-GTSM.

Show MeSH
Related in: MedlinePlus