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. 2023 Jul 24;12(4):702–708. doi: 10.1093/toxres/tfad060

Metformin and Aspirin: Anticancer effects on A549 and PC3 cancer cells and the mechanisms of action

Farzaneh Motafeghi 1,2,✉, Romina Shahsavari 3, Parham Mortazavi 4, Aysan Babaei 5, Pouria Samadi Mojaveri 6, Omid Abed Khojasteh 7, Mohammad Shokrzadeh 8
PMCID: PMC10470367  PMID: 37663811

Abstract

 

Metformin exerts its anticancer effect through two mechanisms, directly affecting the tumor and indirectly reducing systemic insulin levels. The anticancer effects of aspirin occur by inhibiting Cyclooxygenase (COX)-2. COX-2 is absent in many cell types under normal conditions and increases under pathological conditions such as cancer. This study aims to investigate the effect of metformin and aspirin and their combination of them on A549 and PC3 cell lines. Metformin and aspirin were investigated separately and in combination on two cancer cell lines, A549 and PC3. The examined groups include the negative control of untreated cells and the positive control of cisplatin and drugs at concentrations of 15, 10, and 20 μg/ mL to investigate the mechanism of oxidative stress factors (reactive oxygen species, lipid peroxidation, Glutathione (GSH)) and apoptosis (lactate dehydrogenase). The results showed that aspirin, metformin, and their combination could affect cancer cell growth by damaging mitochondria, releasing reactive oxygen species, and activating the oxidative stress pathway. Also, these two drugs show the activation of the apoptotic pathway in cancer cells by increasing the lactate dehydrogenase factor and releasing it from the cells. By disrupting the balance of oxidants and antioxidants in the cell, metformin and aspirin cause an increase in the level of reactive oxygen species and a decrease in the level of glutathione reserves, followed by an increase in the level of lipid peroxidation and a decrease in cell viability. Unlike common chemotherapy drugs, these drugs have no known severe side effects; Therefore, in the not-so-distant future, these drugs can also be used as anticancer drugs.

Highlights

  • Metformin and aspirin, commonly used drugs for diabetes and inflammation, inhibit the growth of cancer cell lines, A549 and PC3.

  • Metformin and aspirin, either separately or in combination, can potentially impede cancer cell growth by disrupting mitochondrial function, inducing the release of reactive oxygen species (ROS), and activating oxidative stress pathways.

  • Furthermore, these drugs can trigger apoptosis, a programmed cell death mechanism, in cancer cells by increasing lactate dehydrogenase (LDH) levels and facilitating its release from the cells.

Keywords: metformin, aspirin, LDH, apoptosis, anticancer

Introduction

Noncommunicable diseases are the leading cause of mortality worldwide.1 In the 21st century, cancer is anticipated to be the primary cause of death and the most significant obstacle to extending life expectancy in all countries. Cancer incidence and mortality are on the rise globally. The causes of cancer incidence are complex, but they reflect aging and population growth and changes in the prevalence and distribution of key cancer risk factors, many of which are associated with social and economic development.2,3

Over 120 million patients worldwide are prescribed Metformin (Met) for the treatment of type 2 diabetes mellitus (TDM2).4 Metformin classically acts as an insulin-sensitizing agent.5,6 It has an anti-inflammatory effect,7 anti-apoptotic, anticancer, liver protector, heart protector,8 eye protector,9 kidney protector,10 and antioxidant.11

Metformin exerts its direct (insulin-independent) anticancer effect by inhibiting complex I of the electron transport chain (ETC) in the mitochondria. As a result, it increases the activation of adenosine monophosphate-activated protein kinase (AMPK) and causes energy stress12 AMPK. It acts as an energy-signaling protein while maintaining homeostasis through the ratio of AMP to ATP; metformin directly inhibits the mitochondrial respiratory chain, reducing ATP production and thereby increasing the AMP-to-ATP ratio.13

The indirect anticancer effect (insulin-dependent) is carried out by inhibiting gluconeogenesis in the liver. The activation of AMPK inhibits the transcription of gluconeogenesis genes, which reduces blood glucose and insulin levels and increases glucose absorption in muscles. Insulin has mitogenic effects, as numerous tumor cells, including those of breast, prostate, and colon cancer, exhibit high levels of insulin receptors. Reducing circulating insulin decreases the phosphoinositide-3-kinase (PI3K) axis, which is involved in growth, proliferation, and differentiation. Unlike the direct pathway, the indirect pathway does not require metformin accumulation in tumor cells.12

Aspirin or salicylate is usually prescribed for people with coronary artery disease. However, studies show evidence that aspirin can also play a role in cancer prevention by slowing the spread of cancer and its metastasis.14 The anti-inflammatory properties of aspirin can mediate the antitumor effect. The anti-inflammatory effect of aspirin works by inhibiting Cyclooxygenase (COX)I/COX2 and modulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) or signal transducer and activator of transcription 3 (STAT3) pathway. In addition, aspirin has been shown to activate the AMPK pathway. The combination of metformin and aspirin is thought to significantly inhibit the phosphorylation of mTOR and STAT3 to induce apoptosis.15

A case-control study on 700 patients with colorectal cancer showed for the first time the effect of aspirin on cancer.16 Multiple cellular mechanisms that could plausibly mediate this advantage have been identified, and concentrations as low as one mmol of aspirin salt can induce apoptosis in human tumor cell lines.16

Lactate dehydrogenase (LDH) is an enzyme primarily present in cells. When there is cellular damage, LDH is released into the bloodstream, and its levels can be used to indicate tissue damage. In cancer diagnosis, serum LDH levels are important due to the tissue destruction caused by tumor growth. Elevated serum LDH levels are usually observed in hematopoietic malignancies such as Hodgkin’s lymphoma17 and non-Hodgkin’s lymphoma (NHL). The removal of the primary tumor through surgery substantially decreases serum LDH levels within the first week after surgery. Tumor metastasis can lead to increased LDH levels, suggesting its potential as a diagnostic marker for cancer.

Furthermore, LDH is a prognostic factor in various malignancies. LDHA has been found to be A significant survival indicator for patients with aggressive lymphoma and is included as one of the risk factors in the International Prognostic Index (IPI). A retrospective analysis by Jin et al. revealed that elevated pre-treatment serum LDH levels were associated with poor survival rates in patients with metastatic nasopharyngeal carcinoma. Similarly, patients with elevated post-treatment serum LDH levels also exhibited significantly lower survival rates than those with normal levels. The specific role of serum LDHA in diagnosing and predicting prognosis requires further investigation, given no clinical differences in its subtypes.18

This study aims to investigate the anticancer effect of metformin and aspirin and their combination of them through oxidative stress pathways on A549 and PC3 cell lines.

Materials and methods

Material and reagents

The ELISA kit was purchased from Abcam, the USA, to measure LDH. 2,5-diphenyl-2H-tetrazolium bromide (MTT) reagent, thiobarbituric acid (TBA), DCF-DA, and Ellman's reagent 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich.

Cell lines and cell culture

A549 and PC-3 human lung and prostate cancer cell lines were acquired from the Pasteur Institute of Iran. The cell suspensions were grown in a Dulbecco's Modified Eagle's Medium culture medium containing 10% FBS and 1% antibiotics to achieve logarithmic growth. When the primary culture was nearly confluent, trypsin-ethylene diamine tetra acetic acid (EDTA) was added to the flask and incubated for 5 min to separate the cells completely from the flask. Then, 100 μL of cell suspension was added to each well in 96-well plates, and the plates were incubated for 24 h to evaluate the oxidative stress and cell survival tests.19

Cell viability test

The cell lines were exposed for 48 h to different concentrations (10, 15, and 20 μg/mL) of metformin and aspirin and their combination. After that, 10 μl of MTT solution was added to each well and incubated for 4 h. The absorption rate was determined by BioTek ELx800 microplate reader at 630 nm.20

Measurement of intracellular glutathione (GSH)

In order to remove cellular proteins, a combination of TCA and EDTA 10% has been used. After centrifugation for 15 min, add DTNB reagent to the supernatant solution and incubate for 15 min until the color reaction is done and its absorbance was read with a spectrophotometer at 412 nm.21

Measurement of lipid peroxidation

To check the amount of Malondialdehyde (MDA) produced, phosphoric acid and TBA reagent were added to the cell suspension and incubated for 30 min in a Bain-Marie at 100 °C. After the incubation, the microtubes were transferred to an ice container to complete the reaction. Then, 500 ml of n-butanol were added and centrifuged to obtain the lipid part, and the supernatant was separated to measure absorption with a spectrophotometer at 532 nm.22,23

ROS assessment

The DCF-DA reagent is used to evaluate free radicals in mitochondria. Consequently, the reagent was added to the cell suspension, which was then incubated for 15 min at 4 ° C. The absorbance at 485–530 nm was measured using a fluorescence microplate reader.24,25

LDH test

Cells were treated in 24-well culture vessels with different doses of metformin and aspirin (10, 15, 20 μg/ml) for 48 h. ELISA kits(Abcam, the USA.) were used to determine the level of LDH enzyme activity in the supernatant solution and cell-lysed sediment.26

Statistical analysis

The software Prism, version 8, was used to conduct statistical analyses. Using a one-way analysis of variance followed by Tukey’s test, the differences between groups were determined (P < .05).

Results

Metformin and aspirin-induced mitochondrial dysfunction and release LDH from A549 and PC-3 cell lines

The study investigated the effects of metformin and aspirin on two types of cancer cells, A549 (lung cancer) and PC-3 (prostate cancer), with a focus on growth inhibition, mitochondrial dysfunction, reactive oxygen species (ROS) production, MDA production, GSH reserves, and LDH leakage.

The MTT test results indicated that both drugs inhibited the growth of cancer cells. The combination treatment resulted in more significant growth inhibition than either drug alone. At the highest concentration of the combined treatment, the growth of lung cancer cells was inhibited by 63% and prostate cancer cells by 72% (Fig. 1).

Fig. 1.

Fig. 1

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A549 and PC3 cell line was exposed to the metformin and aspirin and combination of them in different concentrations, to evaluation of cell viability. **P < .01, ***P < .001, ****P < .0001 compared with the cisplatin group.

Exposure to metformin and aspirin caused damage to the mitochondria of the cancer cells, which led to an increase in ROS production and release. This excess ROS production disrupted normal cellular processes and contributed to the inhibition of cancer cell growth. The rate of ROS release was significantly elevated in cells that received the highest concentration of the two drugs, with a 56% increase for lung cancer cells and a 62% increase for prostate cancer cells (Fig. 2).

Fig. 2.

Fig. 2

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A549 and PC3 cell line was exposed to the metformin and aspirin and combination of them in different concentrations, to evaluation of intercellular ROS. **P < .01, ****P < .0001 compared with the cisplatin group.

The excessive production of ROS also caused reactive oxygen radicals to migrate to lipid membranes, leading to their destruction and the release of MDA. The study found that metformin and aspirin caused the production of MDA at a concentration of 20 μg/ml in the group receiving the combined treatment. The increase in MDA production was significant, with a 64% increase in lung cancer and a 72% increase in prostate cancer (Fig. 3).

Fig. 3.

Fig. 3

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A549 and PC3 cell line was exposed to the metformin and aspirin and combination of them in different concentrations, to evaluation of lipid proxidation. *P < .05, **P < .01, ***P < .001, ****P < .0001 compared with the cisplatin group.

The production of ROS and MDA resulted in a severe decrease in antioxidant reserves, including GSH. The study found that metformin and aspirin caused mitochondrial damage, which led to a reduction in GSH reserves. The decrease in GSH reserves was most pronounced in the group receiving the highest concentration of the combined treatment, where they decreased by 50% (Fig. 4).

Fig. 4.

Fig. 4

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A549 and PC3 cell line was exposed to the metformin and aspirin and combination of them in different concentrations, to evaluation of GSH. *P < .05, **P < .01,***P < .001 ****P < .0001 compared with the cisplatin group.

The study also investigated the effect of metformin and aspirin on LDH leakage in cancer cells. LDH is an enzyme released from damaged cells and can be used as a marker for cellular damage. The results showed that both drugs significantly affected the leakage of LDH in cancer cells, indicating further cellular damage caused by exposure to the drugs (Fig. 5).

Fig. 5.

Fig. 5

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A549 and PC3 cell line was exposed to the metformin and aspirin and combination of them in different concentrations, to evaluation of LDH. **P < .01, ****P < .0001 compared with the cisplatin group.

Overall, the findings suggest that metformin and aspirin induce mitochondrial dysfunction and oxidative stress in cancer cells, leading to cellular damage and inhibition of cancer cell growth. The combination treatment resulted in more significant growth inhibition than either drug alone, indicating a potential synergistic effect. The study highlights the importance of understanding the mechanisms underlying the effects of these drugs on cancer cells and their potential use in combination therapy.

Discussion

Oxidative stress is an imbalance between the state of oxidants and antioxidants in the body.27 When this balance is lost, many intracellular molecules, including lipids, proteins, DNA and RNA,, are changed and damaged.28 The cell damage caused by these ROS is dependent on their intracellular concentration and the ratio of ROS to endogenous antioxidant species. These reactive species induce DNA fissures and disrupt DNA repair.29 Extensive experimental evidence indicates the role of free radicals in the initiation, promotion, and progression of cancer.30,31 ROS, whether from endogenous or exogenous sources, if not detoxified by antioxidants, can increase cell oxidative stress. Mitochondria, the primary source of intracellular ROS through producing superoxide radicals during normal oxidative phosphorylation, causes tumor induction, which increases with cell age.31,32

ROS produced from mitochondrial respiration has been reported to be higher in neoplastic cells than normal cells.33 Cancer cells with high metabolism need high concentrations of ROS to maintain their proliferation rate.28 These tumor cells are in hypoxic conditions because the neoplasm has more blood supply. With hypoxia, transcription factors of hypoxia-inducing factors are activated, related to cell proliferation and angiogenesis with tumor progression.27 Also, the free radical can react by abstracting a hydrogen atom in phospholipids (polyunsaturated fatty acids), which leads to lipid peroxyl radicals and lipid hydroperoxides (lipid peroxidation).34,35 Lipid peroxidation can lead to genotoxic forms, including malondialdehyde and 4-hydroxy-2-alkenal, which can interact and mutate genomic DNA. Release occurs after the onset of lipid peroxidation caused by ROS.35

Metformin is a hypoglycemic drug that is widely prescribed for the treatment of type II diabetes and reduces blood glucose levels through various metabolic changes,36 and helps to overcome insulin resistance in T2DM patients. Currently, the pharmacological importance of metformin for protection against several diseases and malignancies is recognized. On a cellular basis, the anticancer effect of metformin may be exerted by blocking cell proliferation or inducing apoptosis.37

In the study conducted by Park et al. in 2019 regarding the effects of inducing oxidative stress in hepatocellular carcinoma cells, metformin showed an effect on glucose consumption, lactate production, ROS production, and their role in the apoptosis of H4IIE liver cells. Without interfering with glycolysis, it caused an increase in apoptosis caused by oxidative stress in cancer cells, which is consistent with our study in doses of 10, 15, and 20 μg/ml of metformin on A549 and PC3 cancer lines.37

Two main reasons for increased cellular ROS production in cancer treatment are mitochondrial ROS production and inhibition of the cellular antioxidant systemArsenic trioxide, which is approved for the treatment of leukemia, reduces the membrane potential of mitochondria and inhibits complexes I and II, disrupts the mitochondrial ETC and electron leakage, resulting in increased ROS production. Many chemotherapy drugs, such as anthracycline doxorubicin also target mitochondria and induce cellular ROS production. Inhibition of the antioxidant system is a major contributor to the increased production of ROS by cells during chemotherapy, including low molecular mass antioxidants such as GSH and ascorbic acid, enzymes that regenerate reduced forms of antioxidants, and interacting enzymes, such as peroxidases, catalases, and superoxide dismutase (SOD),.38

According to studies, ROS inhibitor compounds disrupt the cytotoxic activity of metformin. For this purpose, a potent ROS-inhibiting antioxidant, such as NAC, was used with metformin. NAC suppressed metformin-induced glucose consumption and significantly interfered with increased ROS production and decreased cell viability due to metformin. These results clearly show that metformin-induced cytotoxicity is mediated through ROS production and oxidative stress, ultimately leading to apoptosis of H4IIE cells.37

ZHEN-YUAN GAO1 et al. studied two breast cancer cell lines, MDA-MB-231 and MDA-MB-435, treated with metformin at different concentrations (1.25, 2.5, 5, 10, or 20 mmol/l) for 24. They were treated for 48 or 72 h. It showed that metformin is a partial inhibitor of complex 1 of the mitochondrial ETC and causes the abnormal flow of electrons to oxygen and causes the accumulation of ROS in the mitochondrial matrix. High levels of ROS can induce apoptosis and senescence in tumor cells. Metformin’s effectiveness against cancer cells is linked to the production of ROS.39

COX is the most important and well-defined molecular target for ASA and NSAIDs. Nevertheless, the chemopreventive effects of NSAIDs on certain cancer cells, including colon cancer cells, are partially independent of their capacity to impede COX activity. The findings of Ajay Goel et al. suggest alternative mechanisms to inhibition of prostaglandin synthesis, such as mitochondrial respiratory dysfunction, increased regulation of pro-apoptotic proteins and increased oxidative stress.40

Huizhu Gan et al’.s studies investigate cell viability and colony formation in lung cancer cell lines A549 and H1299 treated cells with saline or various aspirin concentrations. Increasing aspirin concentrations (2.5 and 5.0 mM) led to a significant decrease in cell viability. The colonization assay of the present study revealed that aspirin treatment made A549 cells incapable of malignancy development.41

Haider Raza et al. studied the effect of aspirin in doses of 5 and 10 μg/mL for 24 and 48 h on HepG2 human hepatoma cells. They showed that the toxicity caused by ASA was due to the increase in the production of ROS, the decrease in GSH reserves, and the increase in oxidative stress, with which Mitochondrial dysfunction is associated.41

Pflaum et al’.s studies showed that although the two cell lines MCF-7 and MDA-MB-231 presented some response characteristics, metformin generally caused more oxidative stress and prevented the cells from adapting to doxorubicin treatment. Metformin is a potential candidate for future trials to prevent or reverse acquired chemoresistance in patients undergoing doxorubicin-based therapy, according to these findings.17,42,43

According to research by Sung et al., compared with control groups, people who used aspirin alone showed a significant reduction in overall cancer risk.

Similarly, those who used metformin alone had a reduced overall cancer risk(HR .79, 95% CI .71–.88). Patients receiving both aspirin and metformin demonstrated the greatest reduction in overall cancer risk (HR .53, 95% CI .45–.63).

Metformin significantly reduces the risk of lung, esophagus, and bladder cancer. As a result, using aspirin or metformin alone shows a similar decrease in the overall cancer rate, but the most significant decrease was in the simultaneous use of drugs.44

Reproductive viability is one of the crucial characteristics of metastatic and invasive cancer cells. According to the results of a study on the two cell lines MCF-7 and SK-BR-3 in the use of metformin (2.5 and 5 mM) and aspirin (10 mM) and the MDA-MB-231 cell line with aspirin (5 and 10 mM) have reduced colony formation. This effect was increased by combining both drugs, which shows that metformin plus aspirin specifically targets triple breast cancer cell lines (130).

LDH serves as a diagnostic and therapeutic monitoring marker in the case of Wilms’ tumor. In the majority of tumor tissues and even precancerous lesions, total LDH activity is elevated, although this is not always the case. In cancers such as lung and stomach, the total LDH activity does not vary substantially. Necrotic processes within the tumor tissue have been hypothesized to influence the elevated LDH activity. On the other hand, it is known that malignant tissues contain a significantly larger number of connective tissue components than normal tissues, which, according to some authors, may be the cause of falsely low results.45

Overall, the findings suggest that metformin and aspirin induce mitochondrial dysfunction and oxidative stress in cancer cells, leading to cellular damage and inhibition of cancer cell growth. The combination treatment resulted in greater growth inhibition than either drug alone, indicating a potential synergistic effect. The study highlights the importance of understanding the mechanisms underlying the effects of these drugs on cancer cells and their potential use in combination therapy. Our results are in line with other studies.

Conclusion

Based on the results of this study, metformin, and aspirin, in addition to being two widely used drugs in the treatment of diabetes and inflammation, with multiple methods and effects on metabolism and cellular mechanisms, also have antitumor properties; One of the mechanisms discussed here is the induction of oxidative stress in lung and prostate cancer cells. By disrupting the balance of oxidants and antioxidants in the cell, metformin and aspirin cause an increase in the level of ROS and a decrease in the level of glutathione reserves, followed by an increase in the level of lipid oxidation and a decrease in cell viability. Unlike common chemotherapy drugs, these drugs have no known severe side effects; Therefore, in the not-so-distant future, these drugs can also be used as anticancer drugs.

Authors’ contributions

Mphammad Shokrzadeh: he did not cooperate in the project, he was only the mentor of the project.

Farzaneh Motafeghi: contributed to conception and study design and management, analysis of data, writing, and drafting the manuscript.

Parham Mortazavi, Pouria SamadiMojaveri, Omid Abed Khojasteh: checking and correcting grammar and plagiarism and editing the manuscript.

Romina Shahsavari and Aysan Babaei: contributed to in vitro study.

Funding

The Research Council of Mazandaran University of Medical Sciences provided funding for this investigation (IR.MAZUMS.REC.1401.219).

Ethical approval

This paper contains no studies with human participants or animals performed by authors.

All authors read and approved the final manuscript.

Conflict of Interest statement

None declared.

Data and Materials Availability

The authors confirm that the data supporting the findings of this study are accessible.

Supplementary Material

Highlights_MET_and_ASA_tfad060
highlights_met_and_asa_tfad060.docx (14.4KB, docx)

Contributor Information

Farzaneh Motafeghi, Reproductive Endocrinology Research Center, Research Institute for Endocrine Sciences and Metabolism, Shahid Beheshti University of Medical Sciences, Tehran 19839-63113, Iran; Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari 4847193698, Iran.

Romina Shahsavari, Department of Pharmacology and Toxicology, Student Research Committee, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari 4847193698, Iran.

Parham Mortazavi, Isfahan Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan 81746-73461, Iran.

Aysan Babaei, Department of Pharmacology and Toxicology, Student Research Committee, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari 4847193698, Iran.

Pouria Samadi Mojaveri, Faculty of medicine, Mazandaran University of Medical Sciences, Sari 4847193698, Iran.

Omid Abed Khojasteh, Department of Clinical Pharmacy, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari 4847193698, Iran.

Mohammad Shokrzadeh, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari 4847193698, Iran.

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