Received: 15 December 2022 | Revised: 4 May 2023 | Accepted: 8 June 2023

DOI: 10.1002/mc.23603

RESEARCH ARTICLE

Mitochondria‐targeted metformin (mitomet) inhibits lung

cancer in cellular models and in mice by enhancing the
generation of reactive oxygen species

Mohammad Shameem1 | Alireza Jian Bagherpoor1 | Ali Nakhi1,2 |

Peter Dosa1,2 | Gunda Georg1,2 | Fekadu Kassie1,3

1
Masonic Cancer Center, University of
Minnesota, Minneapolis, Minnesota, USA Abstract
2
Department of Medicinal Chemistry, Institute Lung cancer is the leading cause of cancer‐related mortality in the United States.
for Therapeutics Discovery and Development,
University of Minnesota, Minneapolis,
Although some epidemiological studies have shown an inverse relationship between
Minnesota, USA the use of metformin, a widely used antidiabetic drug, and the incidence of lung
3
College of Veterinary Medicine, University of cancer, the real benefits of the drug are unclear as the efficacy is low and the
Minnesota, Saint Paul, Minnesota, USA
outcomes are quite heterogeneous. To develop a more potent form of metformin,
Correspondence we synthesized mitochondria‐targeted metformin (mitomet) and tested its efficacy in
Fekadu Kassie, Masonic Cancer Center,
in vitro and in vivo models of lung cancer. Mitomet was cytotoxic to transformed
University of Minnesota, Minneapolis, MN
55455, USA. bronchial cells and several non‐small cell lung cancer (NSCLC) cell lines but relatively
Email: kassi012@umn.edu
safe to normal bronchial cells, and these effects were mediated mainly via induction
Funding information of mitochondrial reactive oxygen species. Studies using isogenic A549 cells showed
Masonic Cancer Center and College of that mitomet was selectively toxic to those cells deficient in the tumor suppressor
Veterinary Medicine, University of Minnesota;
Masonic Cancer Center, University of gene LKB1, which is widely mutated in NSCLC. Mitomet also significantly reduced
Minnesota the multiplicity and size of lung tumors induced by a tobacco smoke carcinogen in
mice. Overall, our findings showed that mitomet, which was about 1000 and 100
times more potent than metformin, in killing NSCLC cells and reducing the
multiplicity and size of lung tumors in mice, respectively, is a promising candidate
for the chemoprevention and treatment of lung cancer, in particular against
LKB1‐deficient lung cancers which are known to be highly aggressive.

KEYWORDS
4‐(Methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK), lung tumor, metformin,
mitochondria‐targeted metformin (mitomet), non‐small cell lung cancer (NSCLC),
reactive oxygen species (ROS)

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medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
© 2023 The Authors. Molecular Carcinogenesis published by Wiley Periodicals LLC.

Molecular Carcinogenesis. 2023;1–11. wileyonlinelibrary.com/journal/mc | 1

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2 | SHAMEEM ET AL.

1 | INTRODUCTION cells, deficient or proficient in LKB1, revealed that mitomet induced

stronger cytotoxicity in LKB1‐deficient cells as compared to the
Although cancer chemoprevention, the use of natural or synthetic effect in LKB1‐proficient cells. Also, in a mouse model of lung cancer,
agents to prevent, reverse or suppress cancer development, has been both metformin and mitomet significantly reduced the multiplicity
found to be effective in populations at high risk for prostate, colon, and size of lung tumors but mitomet was about 100 times more
and breast cancers,1–3 at present, there is no clinically proven potent than metformin. These effects were paralleled by the
chemopreventive agent against lung cancer. Therefore, there is a depletion of GSH, which suggests that induction of oxidative stress
critical unmet need for the development of novel lung cancer is responsible, at least in part, for the anti‐lung tumor activities of
chemopreventive agents with proven safety and efficacy. Several metformin and mitomet. Overall, the present study clearly demon-
observational studies reported the beneficial effects of metformin, a strated that mitomet is a highly promising agent for the prevention
drug used for the treatment of type 2 diabetes mellitus (DM), on the and treatment of lung cancer.
survival of lung cancer patients with DM.4–6 Although these
observations led to the repurposing of metformin for the prevention
and treatment of lung cancer,7–9 the real benefit of metformin for 2 | M A T E R I A L S AN D M E T H O D S
lung cancer patients remains unclear due to the high heterogeneity
among the outcomes of both epidemiological and clinical 2.1 | Cell lines and chemicals
studies.7,10–12
One potential reason for these inconsistent and weak anti‐lung Immortalized bronchial epithelial cell line 2B (BEAS‐2B) and its
cancer effects of metformin could be the poor uptake of the drug by malignant (1170) derivative were provided by Dr. Klein‐Szanto (Fox
lung cancer cells. The organic cation transporter OCT1, which is Chase Cancer Center). Cell line 1170 was developed from BEAS‐2B
responsible for metformin uptake by cancer cells, exhibits polymor- cells explanted along with beeswax pellets containing cigarette
phisms, under‐expression, and inactivating mutations in cancer smoke condensate into rat tracheas that had been denuded of
13–15
cells, thereby reducing the ability of metformin to reach active bronchial epithelium and further transplanted into the dorsal
intracellular concentrations. Moreover, even in normal tissues, OCT1 subcutaneous tissues of nude mice. NSCLC cell lines H522, H2030,
is highly expressed in liver tissue but minimally expressed in lungs and H1299, H2009, H838, A549, and H1975 were purchased from
other tissues.16 ATCC. All NSCLC cell lines were tested for mycoplasma infection and
The principal mechanism for the anticancer effects of metformin is authenticated by short tandem repeat method at MD Anderson's Cell
the inhibition of complex I of the mitochondrial respiratory chain,17,18 Line Core Facility in November 2019 and cultured in RPMI 1640
which suggests that enhancing the accumulation of metformin at the medium supplemented with 10% fetal bovine serum in 5% CO2
mitochondria could increase the anticancer effects of the drug. In fact, incubator at 37°C. A549 cells expressing LKB1 were established as
previous reports showed that tagging of metformin to triphenylpho- follows. LKB1 coding sequences (Sino Biological) were PCR amplified
sphonium (TPP+), a mitochondrial homing moiety with high lipophilicity and cloned into the piggyBac transposon vector system (containing a
group, enhanced the uptake, retention, and antiproliferative effects of CMV promoter and a NEO selection cassette) via gateway cloning
19,20
the drug in pancreatic cancer cells without affecting normal cells. method. Subsequently, LKB1 mutant A549 cells were transfected
The greater uptake of the drug by cancer cells, relative to normal cells, with WT LKB1‐piggyBac expression vector and transposase plasmid
was ascribed to the higher mitochondrial membrane potential in tumor using lipofectamine 3000 reagent (Invitrogen). After 3 days of
cells than in normal cells.21 transfection, single‐cell clones resistant to G‐418 (200 μg/mL) were
In the present study, we synthesized mitochondria‐targeted selected and LKB1 expression was confirmed by western blot
22
metformin (mitomet) as described previously and assessed the analysis.
differential cytotoxicity of mitomet and its parent drug metformin Metformin was purchased from Sigma‐Aldrich and mitomet was
towards normal (BEAS‐2B), transformed bronchial cells (1170) and synthesized from metformin according to established methods.22
non‐small cell lung cancer (NSCLC) cells. Moreover, we examined the Chemical structures of metformin and mitomet are depicted in
potential lung cancer chemopreventive effects of mitomet using a Figure 1. The tobacco smoke carcinogen 4‐(methylnitrosamino)−1‐(3‐
mouse model of lung cancer. Both metformin and mitomet‐induced pyridyl)−1‐butanone (NNK) was purchased from Toronto Research
dose‐dependent cytotoxic effects in 1170 and NSCLC cells but not in Chemicals.
BEAS‐2B cells. However, the concentrations of mitomet required to
induce these effects were about three orders of magnitude (1000
times) lower than the concentrations of metformin. The cytotoxic 2.2 | Cell viability assay
effects of metformin and mitomet in NSCLC cells were mediated via
both apoptosis and ferroptosis, and probably ascribed to enhanced The effects of metformin and mitomet on the viability of BEAS‐2B,
formation of mitochondrial reactive oxygen species (mROS) and the 1170, and NSCLC cells were determined by methylthiazole tetrazo-
resulting depletion of glutathione (GSH). Studies in isogenic A549 lium (MTT; Biotium) assay as described previously.23
 10982744, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/mc.23603 by Egyptian National Sti. Network (Enstinet), Wiley Online Library on [21/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SHAMEEM ET AL. | 3

F I G U R E 1 Metformin and mitomet inhibit NSCLC cell growth. (A) Chemical structures of metformin and mitomet. (B) Effects of metformin
and mitomet on the viability of BEAS‐2B and 1170 cells. Cells were exposed to different concentrations of metformin and mitomet for 72 h, cell
viability was determined by MTT assay, and the percentage of cell viability was presented as a bar graph. Experiments were performed in
triplicate and data were presented as mean ± standard error of the mean (SEM). Statistical significance between vehicle and treatment groups at
each concentration was determined by one‐way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. (C) LKB1 expression in different lung cancer cell
lines as determined by Western immunoblotting. (D) Effects of metformin and mitomet on the viability of different NSCLC cells. Cells were
treated with metformin and mitomet at different concentrations for 72 h and then subjected to MTT assay. The results were presented as a bar
graph. Experiments were performed in triplicate and data were presented as mean ± SEM. Statistical significance between vehicle and treatment
groups at each concentration was determined by one‐way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. (E) Effect of metformn and mitomet on
colony formation. NSCLC cells were treated with different concentrations of metformin and mitomet for 10 days and subsequently stained with
crystal violet, and images were captured by using iBright CL 1500 (Invitrogen). NSCLC, non‐small cell lung cancer.

2.3 | Colony formation assay 2.5 | Annexin V/propidium iodide apoptosis assay

Colony formation assay was performed as described previously.24 NSCLC To determine the apoptotic effects of metformin and mitomet in
cells were seeded into six‐well plates and grown overnight. Cells were NSCLC cells, each cell line was treated with different concentrations
treated with different concentrations of metformin and mitomet and of the drugs for 72 h. Subsequently, 1 × 106 cells were washed twice
grown for 10 days. After that, cells were washed with 1× phosphate‐ with cold PBS and stained with 5 μL Annexin V‐fluorescein
buffered saline (PBS) and stained with crystal violet. Finally, cells were isothiocyanate and 5 μL PI (BD Pharmingen) for 15 min at room
washed with tap water gently and air dried at room temperature. temperature in the dark. The proportion of apoptotic cells was
determined by BD LSRII flow cytometer.

2.4 | Detection of cytosolic and mitochondrial ROS

levels using flow cytometry 2.6 | Cell cycle analysis

After 24 h of exposure to metformin or mitomet, NSCLC cells were NSCLC cells were seeded in six‐well plates, incubated overnight,
stained with 2′,7′dichlorodihydrofluorescein diacetate (CH2DCF‐DA; synchronized by using double thymidine block, and exposed to
Invitrogen) or MitoSOX Red Mitochondrial Superoxide Indicator different concentrations of metformin and mitomet for different time
(Thermo Fisher Scientific) for the detection of cytosolic or mitochon- periods. Thereafter, cells were collected, washed in 1× PBS, fixed
drial ROS, respectively. A working concentration of 10 µM H2DCF‐ with ice‐cold 70% ethanol, and stored at −20°C. Fixed cells were
DA or 5 µM mitoSOX Red was used, and cells were incubated at rinsed again with cold PBS, re‐suspended in PBS containing 50 μg/mL
37°C for 30 min. After washing off excess dye, cells were trypsinized propidium iodide (PI) and 100 μg/mL DNase‐free RNase, and
and analyzed by flow cytometry. analyzed by BD LSRII flow cytometer.
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4 | SHAMEEM ET AL.

2.7 | Western blot analysis 2.10 | Statistical analyses

For the preparation of lysates from cell cultures, DMSO‐ or drug‐ Results from MTT, mROS, and apoptosis assays were reported as
treated cells were incubated in 1× RIPA buffer with protease and mean ± standard error of the mean (SEM) of triplicate determinations.
phosphatase inhibitors (Pierce) for 20 min on ice. Subsequently, cell Between groups, comparisons were performed using one‐way ANOVA.
lysates were centrifuged at 14,000g for 15 min at 4°C, and the One‐way ANOVA and two‐way ANOVA were used to analyze
supernatants were collected, aliquoted, and stored at −80°C. differences in the multiplicity of total lung tumors and the size of lung
Western immunoblotting analysis of the different proteins was tumors, respectively, between control and treatment groups. Two‐sided
performed, as we reported previously.23 For each protein, at least p values ≤ 0.05 were considered statistically significant and all analyses
three Western blot assays were carried out. were conducted using GraphPad Prism 9 software (GraphPad).

2.8 | Comparative anti‐lung tumor effects of 3 | RESULTS

metformin and mitomet in A/J mice
3.1 | Mitomet‐induced differential cytotoxicity
The mouse tumor bioassay was performed following the guidance of in transformed bronchial cells
the National Research Council's Guide for the Care and Use of
Laboratory Animals and the Institutional Animal Care and Use To determine if metformin and mitomet preferentially kill trans-
Committee. Briefly, 6‐week‐old female A/J mice (Jackson Laboratory) formed cells, we compared the cytotoxicity of the drugs towards
were randomized into five groups (10 mice for group 1 and 15 mice normal bronchial cells (BEAS‐2B) and transformed bronchial cells
each for the remaining groups) and received physiological saline (1170) using the MTT assay. As depicted in Figure 1B, exposure of
solution as vehicle control (group 1) or two doses of NNK at an interval BEAS‐2B and 1170 cells to relatively low concentrations of
16
of a week (groups 2–5). Metformin (group 3, 200 mg/kg) or mitomet metformin and mitomet for 72 h induced differential toxicity toward
(1 mg/kg for group 4; 2 mg/kg for group 5) were given to the mice transformed 1170 cells. These effects were particularly remarkable
three times a week (Mondays, Wednesdays, and Fridays) by with mitomet in which the highest concentration of the drug
intraperitoneal injection for a total of 16 weeks (from 4 weeks after (1.25 µM) reduced the viability of 1170 cells by 72%, compared to
the second dose of NNK, at which time airway and alveolar epithelial a 26% reduction in BEAS‐2B cells.
hyperplasia are formed,25 until termination of the study at Week 20).
The dose of metformin used in this study is believed to be relevant to
humans since mice that received 200 mg/kg of metformin have been 3.2 | Comparative cytotoxic and antiproliferative
shown to achieve serum concentrations consistent with steady‐state effects of metformin and mitomet in LKB1‐proficient
values that have been reported in patients.16 Although no human and LKB1‐deficient NSCLC cell lines
studies have been carried out with humans, given that mitomet
exhibited a 1000‐fold higher potency than metformin, it would achieve Mutations or under‐expression of the tumor suppressor gene LKB1 has
a therapeutically effective plasma concentration in humans. Body been shown to be associated with a marked increase in ROS levels and
weights were measured throughout the study. At the end of Week 20, sensitivity to ROS‐induced cell death.26,27 To determine the role of
mice were euthanized with an overdose of carbon dioxide and the LKB1 expression in metformin/mitomet‐induced cytotoxicity in NSCLC
lungs were harvested, and the number and size of tumors on the cells, three LKB1‐proficient NSCLC cell lines (H522, H1299, and H1975;
surface of the lung were determined under a dissecting microscope. Figure 1C) and three LKB1‐deficient cell lines (A549, H2030, and H838;
Lungs from five mice/group were immediately kept in liquid nitrogen Figure 1C) were exposed to different concentrations of the two drugs
and stored at −80°C for subsequent downstream studies. for 72 h and cytotoxicity determined by MTT assay. As depicted in
Figure 1D and Supporting Information: Figure 1, LKB1‐deficient A549
and H2030 cells as well as LKB1‐proficient H1975 cells were relatively
2.9 | Detection of intracellular GSH more sensitive to the cytotoxic effects of mitomet than LKB1‐deficient
H838 and LKB1‐proficient H522 and H1299 cells. On the other hand,
GSH was measured by Glutathione Colorimetric Detection Kit among metformin‐treated NSCLC cells, LKB1‐deficient A549 cells and
(Invitrogen) in either total cell extracts or lung tumor extracts. Total LKB1‐proficient H1299 cells showed the highest sensitivity, whereas
GSH and GSSG (oxidized GSH) were measured following the LKB1‐deficient H2030 cells were the least sensitive cells. Interestingly,
recommendation of the manufacturer. Total GSH content was mitomet was about 1000 times more potent than metformin, regardless
calculated from a standard curve created from a 250‐µM GSH of LKB1 expression, in killing NSCLC cells.
standard supplied by the manufacturer. The Free GSH concentration To investigate if the effect of metformin or mitomet on cell
in samples was calculated by subtracting the GSSG content from the proliferation is influenced by LKB1 expression status, the colony‐
total GSH. forming efficiency of metformin‐ or mitomet‐treated LKB1‐proficient
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SHAMEEM ET AL. | 5

and LKB1‐deficient NSCLC cells was assessed (Figure 1E). The data metformin resulted in a significant G1 cell cycle arrest only at early
revealed that, as in the MTT assay, the colony‐forming efficiency of time point (24 h), whereas mitomet induced G1 cell cycle arrest at all
A549, H2030, and H1975 cells was completely abolished upon time points (24, 36, and 48 h; Figure 2A). In 1975 cells treated with
treatment with 0.625 µM or higher of mitomet, whereas H1299 cells metformin and mitomet, significant G1 arrest was observed at 36 and
were the most resistant. In metformin‐treated cells, LKB1‐deficient 48 h, respectively (Figure 2B).
cells (A549, H2030, and H838) were more sensitive than LKB1‐ To assess the mechanism through which metformin and mitomet
proficient cells (H1975, H1299 and H522). Overall, although the data induce cytotoxicity in NSCLC cells, A549, H2030, H522, and H1975
do not clearly indicate that LKB1 expression is indispensable for the cells were exposed to different concentrations of the drugs, and the
cytotoxicity or antiproliferative effects of metformin and mitomet in proportion of apoptotic cells was determined by flow cytometry. As
NSCLC cells, both drugs inhibited cell viability and colony formation depicted in Figure 2C,D and Supporting Information: Figure 2, compared
efficiency in a concentration‐dependent manner. to the vehicle control (DMSO), both metformin and mitomet
significantly increased the percentage of apoptotic cells and, with the
exception of A549 cells, all three cell lines treated with mitomet
3.3 | Effects of metformin and mitomet on cell exhibited a higher proportion of apoptotic cells as compared to
cycle and cell death of NSCLC cells metformin‐treated cells (Figure 2C). In contrast to our expectation,
LKB1‐proficient cells (H1975 and H522) exhibited a higher percentage
To determine if metformin and mitomet inhibit cell proliferation of apoptotic cells compared to LKB1‐deficient A549 and H2030 cells, in
through induction of cell cycle arrest, A549 and H1975 cells were particular in mitomet‐treated cells. The lower percentage of apoptosis
exposed to metformin and mitomet for specified time periods, and induced by metformin and mitomet in A549 cells, compared to what
cell cycle distribution was evaluated by flow cytometry. In A549 cells, would be expected on the basis of cytotoxicity assays, suggests that

F I G U R E 2 Metformin and mitomet suppress cell proliferation and survival in NSCLC cells. (A, B) Metformin and mitomet induce cell cycle
arrest in A549 (A) and H1975 (B) cells. Cells were synchronized by a double thymidine block, treated with metformin (5 mM) or mitomet (5 μM)
for different time intervals, stained with propidium iodide, and cell cycle distribution was analyzed by flow cytometer. Representative histograms
and bar graphs showing the mean ± SEM of the percentage of accumulated cells from two experiments are presented. Statistical significance
between vehicle and treatment groups was determined by two‐way ANOVA followed by Bonferroni post‐tests. *p < 0.05, **p < 0.01,
***p < 0.001. (C, D) Metformin and mitomet induce apoptosis in A549 (C) and H1975 cells (D). A549 and H1975 cells were treated with
metformin (2.5, 5, and 10 mM) or mitomet (2.5, 5, and 10 μM) in a complete culture medium for 72 h. Apoptosis was measured by Annexin‐V/PI
staining followed by flow cytometry. Data are presented as representative scatter plots and bar graphs showing the means ± SEM of the
percentage of early and late apoptotic cells from triplicate experiments. Statistical significance between vehicle and treatment groups was
determined by one‐way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. (E) Representative images showing the effects of ferrostatin‐1, glutathione,
and the caspase inhibitor Z‐VAD‐FMK on the cytotoxicity of mitomet in A549 and H1975 cells. NSCLC, non‐small cell lung cancer; PI, propidium
iodide. [Color figure can be viewed at wileyonlinelibrary.com]
 10982744, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/mc.23603 by Egyptian National Sti. Network (Enstinet), Wiley Online Library on [21/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
6 | SHAMEEM ET AL.

other forms of cell death could be involved. To examine if ferroptosis, an sensitive to therapeutic strategies that further increase the production
iron‐dependent form of non‐apoptotic cell death caused by excessive of ROS. Before assessing the effects of metformin and mitomet on
peroxidation of membrane phospholipids and production of ROS,28 mROS, we analyzed the background level of mROS in LKB1‐deficient
contributes to metformin‐ and mitomet‐induced cytotoxicity in NSCLC (A549, H2030) and LKB1‐proficient (H1975 and H522) cells. These
cells, A549 and H1975 cells were pretreated with ferrostatin‐1, a studies showed that LKB1‐deficient cells exhibited significantly higher
selective inhibitor of ferroptosis, or GSH, a potent inhibitor of levels of mROS as compared to LKB1‐proficient cells (Figure 3A). Upon
28
ferroptosis, and then exposed to metformin or mitomet for 72 h. As treatment with metformin or mitomet, mROS levels significantly
depicted in Figure 2E, both ferrostatin‐1 and GSH markedly abrogated increased, compared to the level in control cells, and the effects of
mitomet‐induced cytotoxicity in A549 cells, but not in H1975 cells, mitomet were stronger than that of except in H2030 cells (Figure 3B
whereas the pan‐caspase inhibitor z‐VAD‐fmk impacted cytotoxicity in and Supporting Information: Figure 3). Unlike mROS, cytosolic ROS,
neither of the cell lines. analyzed using the redox‐sensitive dye dichlorodihydrofluorescein
diacetate (H2DCF‐DA), did not show significant changes in any of
metformin or mitomet‐treated NSCLC cell lines (data not shown).
3.4 | Metformin and mitomet‐induced mROS In line with the enhanced generation of mROS, levels of GSH, the
major intracellular thiol for antioxidant defense against ROS, were
Owing to their abundant antioxidant systems, cancer cells thrive on markedly reduced in metformin‐ and mitomet‐treated NSCLC cells
levels of ROS that are moderately higher than those in their normal (Figure 3C). One major pathway through which cancer cells combat
29
counterparts. However, this feature renders cancer cells more oxidative stress and restore redox balance is induction of the NRF2

F I G U R E 3 Metformin and Mitomet induce mitochondrial ROS (mROS) in NSCLC cells. Basal mROS (A) and metformin‐or mitomet‐induced
ROS (B) in NSCLC cells. Untreated or drug‐treated A549 and H1975 cells were stained with MitoSOX Red Mitochondrial Superoxide and
analyzed by flow cytometry. Data are presented as representative histogram and bar graphs showing the percentage change in mROS mean
fluorescence intensity (MFI) ± SEM compared to the control group in three experiments. Statistical significance between vehicle and treatment
groups was determined by one‐way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Measurement of intracellular GSH in metformin‐ and
mitomet‐treated A549 and H1975 cells. Cells were treated with different concentrations of metformin or mitomet for 8 h and the amount of
free GSH quantified following the manufacturer's protocol. Data are presented as mean ± SEM of free GSH from three experiments relative to
control. Statistical significance between control and treatment groups was determined by one‐way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.
(D) Modulation by metformin and mitomet of the expression of antioxidant and apoptosis‐related proteins in A549 and H1975 cells as
determined by Western immunoblotting. The assay was performed as described in the Materials and Methods section. [Color figure can be
viewed at wileyonlinelibrary.com]
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SHAMEEM ET AL. | 7

antioxidant system.30 In line with this, metformin‐ or mitomet‐treated NRF2 and its downstream effectors xCT and GPX4 but induced PARP
A549 and H1975 cells exhibited overexpression of NRF2 and GSH cleavage in H2009 cells (Figure 4A). Upon transfection of the cells
peroxidase 4 (GPX4), an antioxidant enzyme that converts lipid with LKB1‐specific siRNA, the level of PARP cleavage was increased
hydroperoxides into nontoxic lipid alcohols to prevent cell death and the effects of mitomet on NRF2, xCT, and GPX4 were
(Figure 3D). The level of xCT, the cystine–glutamate antiporter which attenuated. Contrary to the findings in H2009 cells, in H1299 cells,
mediates the uptake of extracellular cysteine for GSH biosynthesis the level of PARP cleavage in LKB1‐deficient cells was similar to that
and antioxidant defense, was also increased in metformin‐ and of LKB1‐proficient cells and LKB1 silencing did not clearly modulate
mitomet‐treated H11975 cells. In spite of the overexpression of mitomet‐induced alterations in the expression of NRF2, xCT, and
these antioxidant proteins, metformin and mitomet still induced GPX4, indicating that consequences of LKB1 silencing differ
cleavage of PARP, a marker for apoptotic cell death, in A549 and according to the cell line.
H1975 cells, in particular in mitomet‐treated H1975 cells. Overall, To further assess the role of LKB1 in modulating the anticancer
these results suggest that the cytotoxicity of metformin and mitomet effects of mitomet, we developed A549 cells stably expressing LKB1
could be ascribed, at least in part, to the induction of mROS. and compared mitomet‐induced cytotoxicity and level of apoptosis
and mROS generation in LKB1‐deficient and LKB1‐proficient
isogenic A549 cells. Unlike LKB1‐deficient A549 cells, A549 cells
3.5 | Effects of silencing or overexpression of stably expressing LKB1 did not exhibit PARP cleavage (Figure 4B) OR
LKB1 in modulating the phenotypes of NSCLC cells reduction in cell viability (Figure 4C) upon exposure to mitomet.
These results were further corroborated in flow cytometry‐based
To determine the role of LKB1 in regulating redox homeostasis and apoptosis assays in which mitomet induced significant and
thereby modulating the cytotoxicity of mitomet in NSCLC cells, concentration‐dependent apoptotic effects only in LKB1‐deficient
H1299 (LKB1 wild type, p53 null, and Kras wild type) and H2009 A549 cells (Figure 4D). In line with the differential cytotoxicity of
(LKB1 wild type, p53 mutant, and Kras mutant) NSCLC cells were mitomet in LKB1‐deficient A549 cells, comparison of mROS levels in
transfected with siRNA specific to LKB1 or control siRNA, treated LKB1‐deficient and LKB1‐proficient isogenic A549 cells demon-
with mitomet and modulation of NRF2 and other oxidative stress‐ strated a significant and concentration‐dependent increase of mROS
related proteins as well as PARP cleavage were assessed. Similar to levels in the former cells (Figure 4E). Overall, these results clearly
the results in A549 and H1975 cells, mitomet increased levels of showed that LKB1‐deficiency markedly enhances the sensitivity of

F I G U R E 4 Effects of LKB1 silencing on the phenotypes of NSCLC cells. (A) LKB1 silencing by a specific siRNA modulated the expression of
antioxidant proteins and apoptosis in H2009 but not in H1299 cells. The level of the proteins was determined by the Western immunoblotting
assay as described in Section 2. (B). Effects of LKB1 expression on mitomet‐induced PARP cleavage in isogenic A549 cells as determined by
Western immunoblotting. The assay was performed as described in Section 2. (C) Representative images showing differential cytotoxic effects
of metformin and mitomet in LKB1‐deficient A549 cells. (D) Effects of LKB1 expression on mitomet‐induced apoptosis in isogenic A549 cells.
The assay was performed as described in Figure 2. Data are presented as representative scatter plots and bar graphs showing the means ± SEM
of the percentage of early and late apoptotic cells from triplicate experiments. Statistical significance between vehicle and treatment groups was
determined by one‐way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. (E) Effects of LKB1 expression on mitomet‐induced mROS formation in
isogenic A549 cells. The assay was performed as described in Figure 3. Data are presented as representative histogram and bar graphs showing
the percentage change in mROS mean fluorescence intensity (MFI) ± SEM compared to the control group in three experiments. Statistical
significance between control and treatment groups was determined by one‐way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. [Color figure can be
viewed at wileyonlinelibrary.com]
 10982744, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/mc.23603 by Egyptian National Sti. Network (Enstinet), Wiley Online Library on [21/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
8 | SHAMEEM ET AL.

NSCLC cells to mitomet. However, co‐existing genetic alterations gain during the later stages of the bioassay (Figure 5B). Upon analysis
might dampen these effects in some NSCLC cells. of lung tumors on the surface of the lung, we observed that the
vehicle control group treated with combinations of DMSO and corn
oil exhibited 27.9 ± 6.2 lung tumors/mouse, whereas the groups
3.6 | Mitomet reduced the multiplicity and size treated with metformin or the higher dose of mitomet (2 mg/kg) had
of NNK‐induced lung tumors in mice 16.2 ± 4.8 and 15.6 ± 7.3 lung tumors/mouse, corresponding to a
significant reduction of tumor multiplicity by 42%, and 44%,
Our studies in normal and transformed bronchial cells, which are respectively (Figure 5C). Mice treated with the lower dose of
commonly used as in vitro models for lung cancer chemoprevention mitomet (1 mg/kg) had 22.3 ± 8.0 lung tumors/mouse, which corre-
studies,31 showed that mitomet induced significant and differential sponds to a non‐significant reduction by 22% compared to the
cytotoxic effects in transformed (1170) bronchial cells. To determine vehicle control group. Stratification of the lung tumors into different
whether similar effects are induced in an in vivo model of lung cancer, size classes indicated that metformin and the higher dose of mitomet
we assessed the comparative efficacy and potency of metformin and significantly reduced the multiplicity of tumors with a size of ≥1 mm
mitomet to inhibit the multiplicity and size of lung tumors induced by (Figure 5D). Assessment of GSH levels in lung tumors from the
the tobacco smoke carcinogen NNK in A/J mice. The NNK‐A/J control group versus metformin and mitomet groups showed that, as
mouse model of lung cancer is widely used to identify and develop in the in vitro studies, both metformin and mitomet significantly
anti‐lung cancer agents.32 The experimental design for the mouse reduced GSH (Figure 5E). Overall, the mouse lung tumor bioassay
tumor bioassay is shown in Figure 5A. showed that mitomet was as effective as metformin in reducing the
Measurement of the body weight of the mice indicated that multiplicity and size of NNK‐induced lung tumors, probably via
neither metformin nor mitomet reduced body weight gain signifi- induction of oxidative stress, at a dose level 100‐fold lower than that
cantly, although metformin‐treated mice exhibited lower body weight of metformin.

F I G U R E 5 Mitomet was more potent than metformin in reducing the multiplicity and size of NNK‐induced lung tumors in mice. (A) Study
design of the mouse lung tumor bioassay. (B) Safety of metformin and mitomet as demonstrated by the absence of adverse effects on body
weight gain. (C, D) Effects of metformin and mitomet on the multiplicity (C) and size (D) of NNK‐induced lung tumors. Differences in the total
number of tumors between vehicle and treatment groups were determined by one‐way ANOVA, whereas differences in the size of the tumors
were analyzed by two‐way ANOVA. **p < 0.01, ***p < 0.001. (E) Effects of metformin and mitomet on GSH levels in lung tumors. GSH levels
were determined as described in Figure 3. Data are presented as mean ± SEM of free GSH from three experiments relative to control. Statistical
significance between vehicle and treatment groups was determined by one‐way ANOVA. ***p < 0.001. [Color figure can be viewed at
wileyonlinelibrary.com]
 10982744, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/mc.23603 by Egyptian National Sti. Network (Enstinet), Wiley Online Library on [21/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SHAMEEM ET AL. | 9

4 | DISC US SION chain by mitomet 20

could lead to an increase in AMP:ATP and
subsequent activation of AMPK, a master sensor of cellular energy
In the present study, we have shown that mitomet is a promising crisis,36 which has been shown to be regulated by liver kinase B1
candidate for the prevention and treatment of lung cancer, as (LKB1) or calcium/calmodulin‐dependent protein kinase β (CaMKKβ)
demonstrated by its efficacy to reduce the multiplicity and size of and exhibits anticancer effects via inhibition of the mTOR signaling
NNK‐induced lung tumors in mice and its selective cytotoxicity to pathway and G1 cell cycle arrest.37,38
NSCLC cells without affecting normal bronchial cells and, probably Liver kinase B1 (LKB1) is mutationally inactivated in about 20%
via enhancement of oxidative stress. Moreover, mitomet exhibited of NSCLCs and this alteration has been found to be an Achilles heel
1000 and 100 times higher potency in cell line models and mouse for the survival of cancer cells, as LKB1 deficiency increases ROS
lung tumor bioassay, respectively, as compared to the parent drug levels and enhances the sensitivity of these cells to ROS‐generating
metformin. Interestingly, although mitomet did not exhibit differen- anticancer drugs.26 However, in the present study, the sensitivity of
tial cytotoxicity towards Kras/LKB1 co‐mutant cell lines as compared LKB1‐deficient and LKB1‐proficient NSCLC cell lines toward mitomet
to Kras‐mutant/LKB1‐wild type or Kras‐wild/LKB1‐wild type NSCLC was not clearly different. Moreover, whereas silencing of LKB1 in
cell lines, studies in isogenic A549 cells (Kras mutant/LKB1‐deficient H2009 cells (LKB1 wild type, Kras mutant, and p53 wild type)
vs. Kras mutant/LKB1‐proficient cells) indicated a higher level of suppressed the expression of NRF2, xCT, GPX4, and SOD2 and
cytotoxicity, mROS generation and apoptosis in parental A549 cells enhanced apoptosis as evidenced by increased PARP cleavage, no
derived from Kras and LKB1 co‐mutated lung cancer, a highly such effects were observed in H1299 cells (LKB1 wild type, Kras wild
26
aggressive and metastatic lung cancer against which no effective type, and p53 null), suggesting that additional unique mutations
treatment is available. The higher potency of mitomet, as compared contained in LKB1‐deficient NSCLC cells might play a role in
to metformin, is ascribed to an alkyl substituent containing a TPP+ modulating the sensitivity of these cells to mitomet. On the other
moiety, which allows metformin to accumulate in the mitochondria hand, studies in isogenic A549 cells (parental LKB1‐deficient A549
and inhibit mitochondrial complex I activity and induce a stronger cells and LKB1 expressing A549 cells) showed that parental LKB1‐
inhibition of mitochondrial complex I activity, the principal mecha- deficient A549 cells were clearly more sensitive than their LKB1‐
nism for the anticancer effects of metformin.20 In line with this, the proficient counterparts to mitomet as demonstrated by enhanced
IC50 values of mitomet to inhibit mitochondrial complex I activity in generation of mROS and increased cytotoxicity and apoptosis. Our
pancreatic cancer cells were found to be 2750 times lower than that observations with isogenic A549 cells are consistent with the findings
20
of metformin. of Shackelford et al.26 in which phenformin, a more potent analog of
Cancer cells exhibit a higher level of ROS than normal cells metformin, differentially induced apoptosis in isogenic derivatives of
due to hypermetabolism, oncogene activation, and antioxidant several NSCLC cell lines lacking functional LKB1. On the other hand,
33
imbalance. While moderate levels of ROS mediate cell signaling ROS stress induced by treatment with H2O2 caused greater levels of
pathways involved in cell proliferation and survival, excess ROS result apoptosis in cells re‐expressing LKB1 compared to their LKB1‐
in damage to cellular structures, including lipid membranes, protein, deficient counterparts39 and LKB1 re‐expression showed an
and nucleic acid, thereby leading to cell death. Therefore, anticancer increased radio‐sensitivity, whereas knockdown of LKB1 increased
therapies that induce oxidative stress by increasing ROS and/or radio‐resistance of LKB1‐proficient cells by suppressing radiation‐
inhibiting antioxidant processes have received significant attention. derived ROS generation in a NRF2‐dependent manner.40 The reasons
The observed anticancer activities of mitomet could be ascribed for these discrepancies are unclear.
mainly to drug‐induced oxidative stress resulting from the inhibition Although the major objective of our study was to assess the role of
of mitochondrial respiratory complex I and subsequent increase in LKB1 expression in the apoptotic and antiproliferative effects of
ROS generation.19,20 In support of this, pretreatment of A549 cells mitomet in NSCLC cells, it is impossible to exclude the impact of p53
with ferrostatin‐1, a specific inhibitor of ferroptosis which is an iron‐ in these effects. However, the proliferation of mitomet‐treated p53
dependent oxidative cell death caused by ROS, and GSH, the most wild‐type A549 cells was not different from the effects in p53 mutant
potent weapon against oxidative stress, markedly reduced mitomet‐ H2030 and H1975 cells, whereas the apoptotic effects of mitomet were
induced cytotoxicity. ROS induces apoptosis by increasing outer even markedly higher in H1975 cells than in A549 cells, suggesting that
mitochondrial membrane permeabilization leading to the release into P53 did not appear to influence the anticancer effects of mitomet. On
the cytosol of proapoptotic proteins such as cytochrome c, the other hand, we observed that mitomet‐induced ferroptosis‐
apoptosis‐inducing factor, and endonuclease G and ultimately to mediated cell death, a nonapoptotic form of cell death, in p53 wild‐
apoptotic cell death by both caspase‐dependent and ‐independent type A549 cells, but not in p53 mutant H2030 or H1975 cells, which
mechanisms.34 On the other hand, ROS‐mediated ferroptosis results could be associated with p53‐mediated transcriptional suppression of
from a build‐up of lipid peroxides in cellular membranes under iron‐ SLC7A11, a component of the cystine/glutamate antiporter, and the
and ROS‐rich conditions eventually damage the membranes and resulting inhibition of cystine uptake and sensitization of cells to
leading to ferroptotic cell death.35 However, other mechanisms may ferroptosis.41 These results indicate that, in addition to cell‐cycle arrest
also be involved in the anti‐lung cancer activities of mitomet. For and apoptosis, other activities such as metabolic regulation could play a
instance, inhibition of complex I of the mitochondrial respiratory role in p53‐mediated tumor suppression.
 10982744, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/mc.23603 by Egyptian National Sti. Network (Enstinet), Wiley Online Library on [21/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
10 | SHAMEEM ET AL.

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