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. 2020 Jul 8;177(17):3991–4006. doi: 10.1111/bph.15149

Metformin mitigates gastrointestinal radiotoxicity and radiosensitises P53 mutation colorectal tumours via optimising autophagy

Long Chen 1, Fengying Liao 1, Zhongyong Jiang 1, Chi Zhang 1, Ziwen Wang 1, Peng Luo 1, Qingzhi Jiang 1,2, Jie Wu 1, Qing Wang 1,2, Min Luo 1,3, Xueru Li 4, Yu Leng 4, Le Ma 1, Gufang Shen 1, Zelin Chen 1, Yu Wang 1, Xu Tan 1, Yibo Gan 1, Dengqun Liu 1, Yunsheng Liu 1, Chunmeng Shi 1,✉
PMCID: PMC7429484  PMID: 32472692

Abstract

Background and Purpose

There is an urgent but unmet need for mitigating radiation‐induced intestinal toxicity while radio sensitising tumours for abdominal radiotherapy. We aimed to investigate the effects of metformin on radiation‐induced intestinal toxicity and radiosensitivity of colorectal tumours.

Experimental Approach

Acute and chronic histological injuries of the intestine from mice were used to assess radioprotection and IEC‐6 cell line was used to investigate the mechanisms in vitro. The fractionated abdominal radiation model of HCT116 and HT29 tumour grafts was used to determine the effects on colorectal cancer.

Key Results

Metformin alleviated radiation‐induced acute and chronic intestinal toxicity by optimising mitophagy which was AMPK‐dependent. In addition, our data indicated that metformin increased the radiosensitivity of colorectal tumours with P53 mutation both in vitro and in vivo.

Conclusion and Implications

Metformin may be a radiotherapy adjuvant agent for colorectal cancers especially those carrying P53 mutation. Our findings provide a new strategy for further precise clinical trials for metformin on radiotherapy.

graphic file with name BPH-177-3991-g009.jpg

Abbreviations

CASP3

caspase 3

CC3

cleaved caspase 3

HO1

haem oxygenase 1

KEAP1

Kelch like ECH associated protein 1

MitoSOX

mitochondrial superoxide

MMP

mitochondrial membrane potential

mTOR

mechanistic target of rapamycin kinase

NQO1

NAD(P)H quinone dehydrogenase 1

NRF2

nuclear factor, erythroid 2 like 2

P53

tumour protein P53

P62/SQSTM1

sequestosome 1

TMRM

tetramethylrhodamine, methyl ester

WAI

whole abdominal radiation

What is already known

  • Intestinal radiotoxicity is a major limiting factor in radiation dosage, but no mitigation strategy exists.

  • Clinical trials of metformin as a radiotherapy adjuvant agent for colorectal cancer are in progress.

What does this study add

  • Metformin alleviated radiation‐induced acute and chronic intestinal toxicity in mice.

  • Radiosensitisation by metformin of colorectal tumours is related to the genetic status of P53.

What is the clinical significance

  • Metformin has potential as a radiotherapy adjuvant for colorectal cancers especially those with P53 mutation.

1. INTRODUCTION

Colorectal cancer is the second most common cause of cancer death in the United Kingdom and the third most prevalent cancer in the world (Bray et al., 2018; Massat, Moss, Halloran, & Duffy, 2013). Radiotherapy is an important treatment or adjuvant therapy for colorectal cancers (Miller et al., 2019). Although radiotherapy strategies have been greatly improved, intestinal radiotoxicity is still the major limiting factor during radiotherapy (De Ruysscher et al., 2019; Fransson & Widmark, 2007). In addition, some colorectal tumours are resistant to radiation therapy, such as those with the P53 mutation (Tchelebi, Ashamalla, & Graves, 2014). Thus, there is an urgent but unmet need to find safe and effective treatment to reduce radiation‐induced intestinal toxicity while enhancing radiosensitivity of colorectal tumours (Hauer‐Jensen, Denham, & Andreyev, 2014).

Mitochondria are not only the main source of ROS but also the important targets of ionizing radiation. In addition, radiation‐induced mitochondrial dysfunction can lead to cell death such as apoptosis and cause delayed effects like unstable genomes and inflammation (Banerjee et al., 2016; Kam & Banati, 2013; Szumiel, 2015). Autophagy, a conserved lysosomal degradation process, can reduce ROS by degrading long‐lived proteins in the cytoplasm and damaged organelles such as mitochondria (also called mitophagy) to maintain cell homeostasis (Mizushima, 2007). It is reported that deficiency of autophagy leads to increased ROS in intestinal stem cells, which reduces intestinal regeneration after radiation (Asano et al., 2017). However, the mechanisms of pharmacological regulation of mitophagy during radiation‐induced intestinal injuries are still unknown.

Metformin, a cheap and safe drug widely used for type 2 diabetes (Qaseem, Barry, Humphrey, Forciea, & Clinical Guidelines Committee of the American College of, 2017), has been reported to regulate generation of ROS by acting on the mitochondrial respiratory‐chain complex 1 (Vial, Detaille, & Guigas, 2019). In addition, metformin has been used as a radiotherapy adjuvant agent in clinical trials for colorectal cancer (NCT02473094). Some studies have reported that metformin increases survival and could be an effective radiotherapy adjuvant agent in colorectal cancer, but there was significant heterogeneity between studies (Coyle, Cafferty, Vale, & Langley, 2016; Meng, Song, & Wang, 2017). Therefore, there is an imperative need to study the detail mechanisms and effects of metformin on radiotherapy to implement optimal personalized radiotherapy of colorectal cancer.

In this study, our results indicated that metformin reduced intestinal radiotoxicity by optimising autophagy. In addition, metformin radiosensitised colorectal tumours with P53 mutant. Overall, our results suggested that metformin was a potential candidate as a radiotherapy adjuvant for colorectal cancers especially those with the P53 mutation.

2. METHODS

2.1. Animals

Six‐ to 8‐week‐old male C57BL/6 (RRID:IMSR_CARD:2016) mice purchased from the Laboratory Animal Center of the Army Medical University (AMU) were maintained on an ad libitum diet. All experiments were approved by the Animal Care and Use Committee of the AMU. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath & Lilley, 2015) and with the recommendations made by the British Journal of Pharmacology.

2.2. Whole abdominal radiation and metformin treatment

Whole abdominal radiation (WAI) was used to induce intestinal injuries while protecting the bone marrow. Radiation procedures and metformin administration are detailed below and in the Supporting Information.

2.3. Cell culture

The normal rat small intestine epithelial cell line IEC‐6 (RRID:CVCL_0343) and human colorectal adenocarcinoma cell lines, HCT116 (RRID:CVCL_0291) and HT29 (RRID:CVCL_0320), were purchased from the American Type Culture Collection (ATCC) (Huang et al., 2018). The human colorectal adenocarcinoma cell line HCT116 P53KO (RRID:CVCL_HD97) was generously provided by the Xiangya School of Medicine (China). Cells were maintained in a humidified incubator with 5% CO2 at 37°C, cultured in the recommended medium and confirmed to be free of mycoplasma.

2.4. Isolation of Intestinal crypts

Intestinal epithelial cells were prepared from the jejunum of adult male C57BL/6 mice as described previously (O'Rourke, Ackerman, Dow, & Lowe, 2016). Briefly, the jejunum was dissected from mice after euthanasia, washed with ice‐cold PBS and cut into 2–3 cm segments. The segments were digested in ice‐cold chelator buffer (O'Rourke et al., 2016) for 30 min at 4°C with mild shaking. Villi and crypts in the supernatant were separated using a 70‐μm filter and then the supernatant was centrifuged at the speed of (300 g) for (5 min) to collect crypts.

2.5. Radiation

Mice was exposed to a dose of radiation under anaesthesia using an X‐RAD 160‐225 instrument (Precision X‐Ray, Branford, CT; filter: 2 mm AI; 50 cm, 300 kV/s, 4 mA, 0.9 Gy/min). The whole abdominal of mice was exposed to radiation with a lead shielding and the other parts of the mouse were shielded (Figure S1A). Mice were randomized into groups. The doses and schedules for administration of metformin and radiation are provided in the relevant figure legends. The total body of control mice were shielded with a lead shielding before exposed to radiation.

Cells were seeded and treated before exposed to the chosen dose of radiation or shielded with a lead shielding.

2.6. Metformin treatment

For mice, metformin or PBS was given through gavage at chosen concentrations every day for 7 days before radiation as shown in Figure 1SB. ML385 (30 mg kg−1) was injected intraperitoneally 3 h before metformin treatment. For long‐term treatment metformin, metformin was added to the drinking water (2 mg/ml). For cells, they were treated with metformin (1 mM) or PBS prior to radiation and cells were collected 24 h after radiation. Chloroquine (CQ, 50 μM), ML385 (10 μM) and dorsomorphin (CC, 10 μM) were always incubated with metformin.

2.7. Cell toxicity and proliferation

The effect of metformin on CRC cells cell viability was assessed using the Cell Counting Kit‐8 (Dojindo Lab, Tokyo, Japan). Briefly, 5000 cells per well were seeded in 96‐well plates and cultured in the chosen concentrations of metformin for 24h after cell adhesion and then exposed to radiation. Next, 10 μL of Cell Counting Kit‐8 solution was added to each well and incubated for 2 h. The absorbance was measured at 450 nm using a spectrophotometer.

2.8. Histology

The intestine of mice was dissected after euthanasia, then washed in ice cold PBS and fixed in 10% neutral‐buffered formalin for 24 h. Next, tissues were dehydrated using a concentration gradient of alcohol prior to paraffin embedding. Sections of tissues (5 μm) were prepared for staining with haematoxylin and eosin (H&E), Masson's Trichrome and immune‐histochemical (IHC) reagents. For immune‐histochemistry the small intestinal tracts of mice were collected and fixed in 4% formaldehyde, dehydrated, embedded in paraffin, cut into 5 μm‐thick sections and stained with H&E and immune‐histochemical reagents. De‐paraffinised sections were rehydrated and antigen retrieved using 1 M sodium citrate buffer (pH 6.5). Endogenous peroxidase was blocked using 3% H2O2 for 10 min. Furthermore, sections were 100 blocked with 1:200 goat serum in PBS/0.2% Triton for 1 h at room temperature and then incubated with primary antibodies (1:200) at 4 °C overnight, followed by incubation with the appropriate secondary antibody (1:2000) for 1 h. Then nuclei were stained with DAPI. Images were captured using a fluorescent microscope (Olympus BX51).

2.9. Determination of villi height and surviving crypt

Villi height and surviving crypts were objectively analysed and quantified through photographs of H&E stained sections from at least three mice per group using ImageJ 1.37. The villi height was quantified from the crypt villi junction to villi tip and the surviving crypts were defined as containing five or more adjacent chromophilic non‐Paneth cells, at least one Paneth cell and a lumen.

2.10. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labelling (TUNEL) assay

Apoptotic cells were detected in situ using TUNEL staining (Roche) according to the manufacturer's instructions.

2.11. Mitochondrial ROS and mitochondrial membrane potential (MMP) detection

Mitochondrial ROS was measured using mitochondrial superoxide (Thermo Fisher Scientific) and mitochondrial membrane potential was measured using tetramethylrhodamine methyl ester (Thermo Fisher Scientific), both according to the manufacturer's instructions. Briefly, cells were first primed with metformin (1 mM) for 24 h prior to radiation (15 Gy). After 24 h of radiation, cells were washed twice in PBS and loaded with 5 μM mitochondrial superoxide for 10 min or 100 nM tetramethylrhodamine methyl ester for 30 min at 37 °C, followed by washing twice in PBS before being subjected to flow cytometry (Accuri C6, BD Biosciences.

3. CELL DEATH ASSAY

Briefly, cells were first primed with the indicated concentration of metformin for 24 h prior to radiation. After 36 h of radiation, cells were washed twice in PBS and then harvested and stained using the Dead Cell Apoptosis Kit (Thermo Fisher Scientific) at room temperature in the dark for 15 min and then subjected to flow cytometry (Accuri C6, BD Biosciences).

3.1. Immunofluorescence

Cells were cultured on glass coverslips and treated with vehicle or 1 mM metformin for 24 h prior to irradiation (15 Gy). Cells were fixed with 4% neutral paraformaldehyde at specific time points post‐radiation, permeabilised with 0.1% Triton X‐100 and blocked with 1% BSA, then incubated with the primary antibody (1:200) overnight at 4 °C, followed by incubation with appropriate secondary antibody. For experiments that used mitotracker, cells were stained with mitotracker red prior to fixation. Then, nuclei were stained with DAPI. Images were captured using a fluorescent microscope (Olympus BX51).

3.2. Transfection with adenovirus expressing the GFP‐LC3B fusion protein

Cells grown to approximately 70% confluence were transfected with Ad‐GFP‐LC3B (Beyotime, China) according to the manufacturer's instructions. After continued incubation for 24 hours, the cells received subsequent processing.

3.3. Quantitative real‐time PCR analysis

Total RNA was extracted using PureLink® RNA Mini Kit (Thermo Fisher Scientific). cDNA synthesis was performed following the manufacturer's protocol with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Real‐time PCR was performed using an SYBR Green qPCR master mix (Takara) according to the manufacturer's protocol. The primers are listed in supplementary Table 1. All data were normalised to the control using β‐actin as the internal control by ΔCT method.

3.4. Western blotting

Cells or crypt cells were lysed in RIPA Buffer containing protease inhibitors and phosphatase inhibitors (Roche) on ice for 30 min and then centrifuged at 16 000 × g for 15 min at 4 °C to obtain 146 total protein lysates. The Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Fisher Scientific) was used to extract nuclear and cytoplasmic protein. The concentrations were determined using a BCA kit (Thermo Fisher Scientific). The sample was heated with 5X loading buffer (Beyotime) to 100 °C for 5 min. Equal amount of proteins (30μg) from each sample were subjected to SDS‐PAGE (4%‐12%), followed by transfer to a PVDF membrane (Merck Millipore, Darmstadt, Germany). The membranes were blocked with 5% skimmed milk for one to two hours at room temperature and then immunoblotted with primary antibodies (1:1000) overnight at 4 °C and washed with Tris‐buffered saline with Tween (TBST) buffer three times (5 minutes once), followed by incubation with appropriate secondary antibodies (1:2000) at room temperature for 1 hour. After washing with TBST buffer three times (5 minutes once), the intensity of bands was visualised and determined using an enhanced chemiluminescence detection system (Bio‐Rad Laboratories) by an ECL kit (Thermo Scientific, Waltham, USA). β‐Actin was used as the loading control for whole cell lysates and histone H3 was used as the loading control for nuclei. Lysates were sampled from three samples per group. The western blot was conducted and the experimental details provided conform to the British Journal of Pharmacology Guidelines (Alexander et al., 2018).

3.5. Xenotransplantation assays

Five‐ to six‐week‐old athymic nude mice were purchased from Huafukang Bioscience (Beijing, China). A total of 5 × 106 tumour cells in PBS were subcutaneously injected at the abdominal flank region of the athymic nude mice. Palpable tumours were observed after a week. Mice were randomly divided into four groups and then treated daily with metformin (250 mg kg−1) or PBS through gavage. Fractionated WAI (4 Gy/fraction) was administered four times in total; once every four days (Figure 7a). The health status of the mice was observed daily, including diarrhoea after radiation. The xenograft tumour size and body weight were measured every two days using sliding calipers and the volume was estimated using the formula: volume = length × width2 × 0.5. At the end point, the xenografts were photographed and weighed.

3.6. Materials

Metformin (PHR1084), ML385 (SML1833), dorsomorphin previously known as compound C (P5499) and BrdU (5‐bromo‐2'‐deoxyuridine; B5002) were from Sigma‐Aldrich. Cell Counting Kit‐8 was from Dojindo Molecular Technologies (ck04). H&E Staining Kit (ab245880), Trichrome Stain Kit (ab150686) and DAB substrate kit (ab64238) were from Abcam. In Situ Cell Death Detection Kit, POD (11684817910) and Protease Inhibitor Cocktail (5892791001) was from Roche. Mitochondrial superoxide (M36008), tetramethylrhodamine methyl ester (T668), MitoTracker (M7512), Dead Cell Apoptosis Kit (V13242), RevertAid First Strand cDNA Synthesis Kit (K1622), Nuclear and Cytoplasmic Extraction Reagents (78833) and BCA Protein Assay Kit(A53225) were from Thermo 58 Scientific. Adenovirus expressing GFP‐LC3B fusion protein was from Beyotime (C3006). Primary antibodies used for IHC, IF and immunoblot analysis: anti‐mouse BrdU (#5292S), anti‐mouse β‐Actin 60 (#3700S), anti‐mouse LC3B (#83506S), anti‐rabbit Cleaved Caspase3 (#9664s), anti‐rabbit γ‐H2AX (#9718S), anti‐rabbit PARP (#9532S), anti‐rabbit Keap1 (#4678S), anti‐rabbit Histone H3 62 (#4499S), anti‐rabbit AMPKα (#5831S), anti‐rabbit Phospho‐AMPKα (#2535S) and anti‐rabbit Caspase 3 (#9662S) were from Cell Signaling Technologies; anti‐mouse (ab56416), anti‐mouse Parkin (ab56416), anti‐mouse Heme Oxygenase 1(ab13248), anti‐rabbit NQO1(ab34173) and anti‐65 rabbit Tomm20 (ab186735) were from Abcam; anti‐rabbit NRF2 (#16396‐1‐AP ) Pink1 (#23274‐1‐AP ) Parkin (#14060‐1‐AP ) were from Proteintech. Secondary antibody: Anti‐rabbit IgG, HRP‐linked antibody (#7074, CST), anti‐mouse IgG, HRP‐linked antibody (#7076, CST) and Anti‐rabbit IgG 68 Alexa Fluor® 555 Conjugated Antibody (#4413, CST).

3.7. Data and statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). Equalization, randomization and blinding were used for each group in both in vivo and in vitro experiments. In addition, the group size in this study was set as at least five to ensure the implementation of statistical analysis and no data points were excluded from the statistical analysis in any test. All group sizes represent the numbers of experimental independent biological repeats and statistical analysis was performed using these independent values.

To reduce variation, the data of real‐time PCR, mitochondrial ROS and mitochondrial membrane potential were normalized to fold mean of the controls. Each value was divided by the mean of the control values and expressed in the form of “fold change.” For these results, the y‐axis in the figures was labelled with “fold mean of the controls.”

Kaplan–Meier statistics were used to analyse survival in mice. All data are presented as means ± SDs. Statistical analyses were applied using the unpaired two‐tailed Student's t‐test and one‐way ANOVA with GraphPad Prism 7.04 (RRID:SCR_002798) statistical software (GraphPad, San Diego, USA). In multigroup studies with parametric variables, post hoc tests were conducted only if F in ANOVA (or equivalent) achieved the necessary level of statistical significance (P < 0.05) and there was no significant variance inhomogeneity. The threshold for statistical significance was set at the level of P being 0.05 and in all cases, P < 0.05 was considered statistically significant. In addition, asterisks denote statistical significance (ns, non‐significance; *P < 0.05). Additional materials and methods are available in the online supplementary methods.

3.8. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018) and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander et al., 2019).

4. RESULTS

4.1. Metformin improves survival and reduces body weight loss in mice after whole abdominal radiation

Whole abdominal radiation was used to cause intestinal injuries while protecting the bone marrow (Figure S1A). First, half‐lethal dosage (10 Gy), lethal dosage (12 Gy), high dosage (15 Gy) of whole abdominal radiation, medium dose (250 mg·kg−1) and high dose (500 mg·kg−1) of metformin were used to investigate effects of metformin in mice. Interestingly, our results showed that metformin reduced or delayed mortality after different dosage of whole abdominal radiation (Figures 1a and S1C, E). Mice treated with PBS all died after 12‐Gy whole abdominal radiation. However, the survival rates of the mice treated with different dose of metformin were 40% (250 mg·kg−1) and 30% (500 mg·kg−1). In addition, mice treated with metformin lost less body weight after whole abdominal radiation (Figures 1b and S1D, F). To further evaluate the effects of metformin on radiation‐induced intestinal damage, samples were collected at 0 h, 4 h, 3.5 days and 5 days (Figure S1B) because they are important time points for evaluating apoptosis and regeneration of crypts after radiation (Wei et al., 2016). Interestingly, HE staining revealed the crypt‐villus architecture and survival crypts in the metformin‐group were also well protected compared with the PBS‐group (Figure 1c–e). Chronic radiation enteritis often occurs after abdominal radiation and is associated with significant and ongoing mortality (Sher & Bauer, 1990). We then evaluated the long‐term effects of metformin on whole abdominal radiation‐induced chronic damage in mice. Sixteen mice in the metformin‐treated group survived more than 30 days after whole abdominal radiation and only one of them died within 12 months, while only five of nine of mice in the PBS‐group survived (Figure 1f). Interestingly, mice treated with metformin appeared to be larger and heavier compared with mice in the PBS‐group (Figures 1g and S1G). Furthermore, the thinner intestinal walls (Figure S1H) and the shorter intestines (Figures 1h and S1I) were observed in mice treated with PBS. HE staining showed that mice in the PBS‐group had lower villus density and length and had fewer crypt numbers (Figure 1i). Trichrome staining showed more collagen deposition in the PBS‐group in the intestinal submucosa and lamina propria (Figure 1i). These results indicated that metformin alleviated acute and chronic radiation‐induced intestinal damage in mice.

FIGURE 1.

FIGURE 1

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Metformin protected mice from intestinal radiotoxicity. (a) Kaplan–Meier survival analysis of mice treated with 250 (Met250) or 500 (Met500) mg·kg−1 of metformin or PBS after 12‐Gy whole abdominal radiation (WAI). P < 0.05 for Met250 + IR or Met250 + IR, both compared with PBS + IR group. n = 10. (b) Body weight over time post different doses of radiation. n = 10. (c) HE‐stained sections of mice intestine. Scale bars: 500 μm (top) and 100 μm (bottom). (d) Villi height at 96 h after WAI. n = 5. (e) Average number of surviving crypts per section 3.5 days after WAI, n = 5. (f) Kaplan–Meier survival analysis of mice after 12‐Gy WAI for Met versus PBS, by log‐rank test. n = 10. (g) Body weight of surviving mice after 12 months. n = 10. (h) Length of the intestine from surviving mice 12 months after WAI. n = 5. (i) HE and trichrome stains of intestine from surviving mice 12 months after WAI. Scale bars: 100 μm. (*P < 0.05)

4.2. Metformin reduces radiation‐induced loss of crypt cells

Survival and regeneration of crypts promote recovery of intestinal injuries after radiation and 5‐bromo‐2'‐deoxyuridine (BrdU) was one of the markers for regenerative crypts (Wei et al., 2016). At the time points (3.5 and 5 days) for regeneration of crypts, more BrdU positive cells in crypts were observed in the metformin‐group compared with the PBS‐group, while there was no significant difference at 0 day (Figure 2a,b). Ionizing radiation causes DNA damage and apoptosis in crypt cells, which induced detrimental effects on intestinal regeneration. Usually, DNA damage was usually evaluated by γH2AX at 4 h after radiation (Turinetto & Giachino, 2015). The crypts of mice in the metformin‐group showed a lower level of γH2AX staining than that in the PBS‐group after whole abdominal radiation at 4 h time point (Figure 2c). To identify the role of apoptosis, we performed HE, TUNEL and Cleaved caspase 3 (CC3) staining of intestine sections at 4 h after whole abdominal radiation. Our data showed that more apoptotic cells of crypts were found in the PBS‐group than that in the metformin‐group (Figure 2d–g). Then, we determined the apoptosis‐related protein level of CC3, caspase 3 (CASP3) and PARP from crypt cells; metformin noticeably reduced the level of apoptosis‐related protein (Figure 2h). Altogether, these results indicated that metformin reduced radiation‐induced apoptosis in crypt cells.

FIGURE 2.

FIGURE 2

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Metformin reduced radiation‐induced loss of crypt cells. BrdU (a) and γH2AX (c) in a cross section at indicated time points. Red, BrdU or γH2AX; blue, DAPI. Scale bar: 20 μm. (b) Average number of regenerated crypts per circumference after whole abdominal radiation (WAI). (d) Representative apoptosis in crypts with HE‐stained. The arrows indicated the apoptotic crypts cells. (e) Representative images of TUNEL immunofluorescence in the crypts. Green, TUNEL; blue, DAPI. Scale bar: 20 μm. (f) Representative images of intestinal sections stained with cleaved caspase 3 (C‐CASP3). Red, C‐CASP3; blue, DAPI. Scale bar: 20 μm. (g) Quantitation of γH2AX+, apoptosis+, TUNEL+ and cleaved caspase 3+ crypt cells. (h) Expression of PARP, caspase 3, and cleaved caspase 3(C‐CASP3) from crypts analysed by western blotting. For subparts (b) and (g) (*P < 0.05), n = 5

4.3. Metformin enhances mitophagy and reduces apoptosis after radiation in vitro

IEC‐6, a rat cell line derived from intestinal epithelium, was used as in vitro model system to investigate the mechanisms of metformin. Consistent with in vivo results, metformin reduced apoptosis and the level of apoptosis‐related protein including cleaved caspase 3 and PARP after radiation (Figure 3a–c). Ionizing radiation has been reported to increase mitochondrial oxidative stress which plays a key role in radiation‐induced apoptosis (Taneja, Tjalkens, Philbert, & Rehemtulla, 2001). Consistent with previous report (Yamamori et al., 2012), mitochondrial membrane potential (MMP) and mitochondria‐derived RO, mitochondrial superoxide, increased 24 h after radiation, whereas metformin effectively reduced those in IEC‐6 cells (Figure 3d,e). Mitophagy eliminates damaged mitochondria to reduce cellular oxidative stress, reduce ROS, maintain normal cell homeostasis and resist apoptosis (Ashrafi & Schwarz, 2013; Hu, Wang, Huang, Zhao, & Wang, 2016; Larson‐Casey, Deshane, Ryan, Thannickal, & Carter, 2016; Zhang et al., 2019). Firstly, we analysed autophagy‐related proteins. Our results showed that LC3 II increased slightly after radiation, but metformin treatment greatly increased LC3 II protein regardless of radiation. Chloroquine further confirmed that the increased LC3 II is due to enhanced autophagy rather than lysosomal dysfunction (Figure 3f). Moreover, metformin increased the level of mitophagy‐related proteins including ubiquitin‐binding protein p62, Pink1 and Parkin (Figures 3g and S2A). This was also confirmed through the results of immunofluorescence staining of LC3II (Figure 3h). The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology.

FIGURE 3.

FIGURE 3

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Metformin protected IEC‐6 cells from radiation in vitro. For subparts (a) to (g), cells were seeded in 6‐well plates and treated with indicated treatment before exposed to 15‐Gy radiation or shielded with a lead shielding. (a) Annexin V‐PI was used to label apoptotic cells through flow cytometry 36 h after 15‐Gy radiation. (b) Quantitation of apoptosis rates. (c) Expression of PARP, caspase 3, and cleaved caspase 3 (CCASP3) from IEC‐6 cells analysed by western blotting. Tetramethylrhodamine methyl ester (TMRM) and mitochondrial superoxide (MitoSOX) were used to quantify the mitochondrial membrane potential (d) and mitochondria‐derived ROS (e) 24 h after 15‐Gy radiation. (f) Expression of autophagy‐related LC3‐I and LC3‐II from IEC‐6 cells 24 h after 15‐Gy radiation. (g) Expression of Parkin, Pink1, Tomm20 and P62 from IEC‐6 cells 24 h after 15‐Gy radiation analysed by western blotting. (h) Intracellular distribution of LC3 and mitochondria in IEC‐6 cells treated with indicated treatments 24 h after 15‐Gy radiation examined by confocal microscopy. For subparts (b), (d), and (e) (*P < 0.05), n = 5

4.4. Metformin promotes mitophagy through activation of AMPK‐NRF2

Mitophagy mediated by Pink1/Parkin is dependent on p62/SQSTM1, a key selective autophagy receptor and deletion of p62 impairs mitophagy in mice (Geisler et al., 2010; Lamark, Svenning, & Johansen, 2017; Nguyen et al., 2019). It has been reported that NRF2 induces p62 expression at the transcriptional level to promote mitophagy. Next, we examined whether metformin affects the expression of NRF2. As we predicted, metformin activated NRF2 and its downstream targets including haem oxygenase 1 (HO1) and NAD(P)H quinone dehydrogenase 1 (NQO1; Figures 4a,b and S3A, B). We found that metformin caused noticeable induction of NRF2 expression on protein level instead of mRNA level (Figure S3C). Therefore, metformin might elevate NRF2 by promoting its stability. kelch like ECH‐associated protein 1 (KEAP1) was reported to promote degradation of NRF2 to inhibit activation of NRF2 (Wang et al., 2019). Western blot showed that metformin decreased expression of KEAP1 (Figure 4c). In addition, metformin increased the phosphorylation level of AMPK as previously reported. To identify whether the reduced KEAP1 was related to AMPK, dorsomorphin a specific AMPK inhibitor, was used (Zhou et al., 2001). Our results showed that dorsomorphin rescued reduced KEAP1 and reversed activation of NRF2 by metformin. Taken together, our data demonstrated that metformin might activate NRF2 by autophagic degradation of KEAP1, which was AMPK‐dependent. To further investigate the role of NRF2 on mitophagy of metformin, a specific NRF2 inhibitor (ML385) was used to block NRF2 (Liu et al., 2018; Singh et al., 2016). Our results showed that ML385 effectively inhibited the activation of NRF2 and mitophagy, as evidenced by the results of western blot and immunofluorescence staining (Figures 4d,e and S3D).

FIGURE 4.

FIGURE 4

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Metformin promoted mitophagy through activation of AMPK‐NRF2. (a) NRF2, NQO1, and HO1 expression levels in whole cell lysates from IEC‐6 cells 24 h after 15‐Gy radiation. (b) NRF2 expression levels in nuclei from IEC‐6 cells 24 h after 15‐Gy radiation. (c) NRF2, KEAP1, AMPK, and p‐AMPK expression levels in whole cell lysates from IEC‐6 cells 24 h after 15‐Gy radiation. Dorsomorphin (CC) was used as an AMPK inhibitor. (d) NRF2, NQO1, HO1, Pink1 and P62 expression levels in whole cell lysates from IEC‐6 cells with the indicated treatments 24 h after 15‐Gy radiation. Dorsomorphin (CC) was used as a NRF2 inhibitor. (e) Intracellular distribution of LC3 and mitochondria in IEC‐6 cells 24 h after 15‐Gy radiation examined through confocal microscopy

4.5. Activation of NRF2 is essential for radioprotection of metformin

Then we investigated whether NRF2 played a key role for radioprotection of metformin. In vitro, inhibition of NRF2 by ML385 abolished the effects of metformin on mitochondrial‐derived ROS and apoptosis (Figures 5a–c and S4A). In vivo, ML385 effectively blocked the accumulation of NRF2 in the nucleus of crypt cells in mice treated with metformin (Figure 5d) and eliminated the radioprotective effects of metformin after 12Gy whole abdominal radiation. Mild diarrhoea was observed in the mice treated with metformin alone, while mice in the other groups developed very severe diarrhoea and weak melena after 12‐Gy whole abdominal radiation. Moreover, the beneficial effects of metformin on survival and weight loss in mice were also eliminated by ML385 (Figure 5e,f). HE staining also showed that ML385 inhibited the protective effects of metformin on the fracturing of small intestine villi and crypts after whole abdominal radiation. Compared with mice in the metformin‐group, mice treated with ML385 and metformin had fewer residual crypts and shorter villi (Figure 5g–i). TUNEL staining showed that ML385 also significantly reduced the anti‐apoptotic effects conferred by metformin (Figures 5j and S4B). Collectively, these data indicated that metformin protected mice from radiation‐induced injuries by regulating mitophagy which is AMPK‐NRF2 dependent.

FIGURE 5.

FIGURE 5

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Activation of NRF2 is essential for radioprotection of metformin. (a) Relative value of the mitochondria‐derived ROS and apoptosis rates (b) were detected in IEC‐6 cells 24 h after 15‐Gy radiation, both compared with PBS group. (c) PARP, caspase 3 and cleaved caspase 3 expression levels from IEC‐6 cells 24 h after 15‐Gy radiation. (d) Images (top) of stained by NRF2 immunofluorescence in mice crypts. Red, NRF2; blue, DAPI. Scale bar: 20 μm. Corresponding NRF2 expression levels (bottom) in crypts from mice 24 h after ML385 treatment. (e) Kaplan–Meier survival analysis of mice after 12‐Gy whole abdominal radiation (WAI ; n = 10). P < 0.05 for Met + IR versus PBS + IR. (f) Body weight of mice over time after 12‐Gy WAI. (g) HE‐stained sections of mice intestine after 12‐Gy WAI. Scale bars: 500 μm. (h) Average number of surviving crypts per section at the indicated time after 12‐Gy WAI. (i) Villi height at the indicated time after WAI. (j) TUNEL+ crypt cells at 4 h after WAI. For subparts (a), (b), (h), (j) and (i), n = 5 (*P < 0.05)

4.6. Metformin enhances the radiosensitivity of colorectal tumour cells with P53 mutation in vitro

One of the important aspects of radiotherapy adjuvants is their effects on tumour radiosensitivity. To address this feature of metformin, we studied its effects on colorectal tumours. To choose reasonable dosage for metformin and radiation, we first examined the effects of different doses of radiation and metformin on the growth viability of colorectal tumour cells including HT29 and HCT116. As determined by our data (Figure S5A–c), 6‐Gy radiation and 500‐mM metformin were used as optimal doses to complete the follow‐up experiments. Interestingly, our results showed that metformin enhanced the radiosensitivity of HT29 but not HCT116 including cell viability and apoptosis (Figures 6a–d and S5D). Then we examined the protein levels related to apoptosis and autophagy, which contribute to radiosensitivity of tumours (Chaurasia et al., 2019). Our results showed that metformin promoted autophagy in HCT116 rather than HT29 cells, but increased apoptosis was observed in the HT29 cells (Figures 6e,f and S5D). Previous studies have reported that HCT116 (expressing a wild type P53 protein) and HT29 (expressing a mutation P53 protein) cells have different P53 genetic background (Cordani et al., 2016). To verify whether the opposite effects of metformin were related to P53 status in the two colorectal tumour cells, we explored the effects of metformin on HCT P53KO cell line. Interestingly, metformin enhanced the radiosensitivity of HCT116 P53KO tumour cells, including increased apoptosis and reduced autophagy as observed in HT29 (Figures 6g,h and S5D). Collectively, these results demonstrated that metformin might radiosensitise colorectal tumour cells with P53 mutation.

FIGURE 6.

FIGURE 6

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Metformin enhances radiosensitivity of colorectal tumour cells with P53 mutation in vitro. Cell viability of HCT116 (a) and HT29 (b) tumour cells were measured by the Cell Counting Kit‐8 assay at 24 h after 15‐Gy radiation. Apoptosis rates of HCT116 (c) and HT29 (d) cells treated with indicated treatments were detected by flow cytometry of annexin V‐PI marked cells 36 h after 15‐Gy radiation. (e–g) PARP, cleaved caspase 3, P62 and LC3 expression levels in whole cell lysates from indicated tumour cells 24 h after 15‐Gy radiation. (h) Apoptosis rates of HCT116 P53KO cells treated with indicated treatments were detected by flow cytometry of annexin V‐PI marked cells 36 h after 15‐Gy radiation. For subparts (a–d) and (h), N = 5 (*P < 0.05)

4.7. Metformin enhances radiosensitivity of tumours with P53 mutation in vivo

HT29 and HCT116 tumour grafts in nude mice were used to determine the effects of metformin on tumours in vivo. In this study, fractionated radiotherapy was used to evaluate the radiosensitivity of metformin on colorectal tumours (Figure 7a). During the whole experiment, our results showed that metformin inhabited the growth of HT29 tumour but not the HCT116 tumours with fractionated radiotherapy (Figure 7b,c). At the endpoint, the tumour size and tumour weight were inhibited by combination of metformin and radiotherapy compared with combination of PBS and radiotherapy. In addition, mice treated with metformin had higher survival rate, milder gastrointestinal toxicity symptoms such as diarrhoea and less loss of body weight during radiotherapy (Figure 7h,i). These results suggested that metformin enhanced radiosensitivity of tumours with P53 mutation in vivo and simultaneously reduced radiation‐induced gastrointestinal toxicity.

FIGURE 7.

FIGURE 7

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Metformin enhances radiosensitivity of tumours with P53 mutation in vivo. (a) Schematic diagram of metformin (250 mg·kg−1) and fractionated whole abdominal radiation (WAI; 4 Gy/fraction) was given for four times. HCT116 (b) and HT29 (c) tumours growth curves. (d–g) The dissected xenografts of HCT116 and HT29 tumours were photographed and weighed at the endpoint. (h) Kaplan–Meier survival analysis of mice post‐fractionated WAI. (i) Body weight of mice over time post‐fractionated WAI. For the HCT116 tumour graft endpoint, group PBS (n = 10), group Met (n = 10), group PBS + IR (n = 9), group Met + IR (N = 10); for the HT29 tumour graft endpoint, group PBS (n = 8), group Met (n = 6), group PBS + IR (n = 8), group Met + IR (n = 7). For all subparts, * P < 0.05

5. DISCUSSION

Screening new radiotherapy adjuvant agent currently faces two major challenges. One is that many compounds are difficult to reach the effective concentration in vivo or clinical potential toxicity limits their use and the other is that they simultaneously protect the tumour (Hauer‐Jensen et al., 2014). Metformin, a cheap and safe drug widely used for type 2 diabetes, enters cells requiring an organic cation transporter (OCT) (Vancura, Bu, Bhagwat, Zeng, & Vancurova, 2018), which is highly expressed in the intestine and tumours, such as colorectal cancer and prostate cancer (He & Wondisford, 2015), which means that metformin could be used at mM concentration range in the intestine as a safe dosage (de Mey et al., 2018). In addition, some cohort studies have reported that metformin prevents colorectal cancer and protects radiation‐induced haematopoietic injuries and pulmonary fibrosis (Wang et al., 2017; Xu et al., 2015). In this study, we demonstrated that metformin protected the intestinal crypts from radiation, thereby alleviating intestinal injuries and its protective effects attributed to the activation of mitophagy, which is AMPK‐NRF2 dependent. AMPK, the energy sensor, activated by metformin through multiple pathways, is an important target for radiosensitivity of tumours (Sanli et al., 2010; Sanli, Steinberg, Singh, & Tsakiridis, 2014). Autophagy is required for intestinal regeneration after radiation and plays a key role in regulating intestinal inflammation (Asano et al., 2017; Pott, Kabat, & Maloy, 2018). However, the effects of pharmacological regulation of autophagy on intestinal radiation‐induced injuries are still unknown. Here, our data supported that optimising autophagy may be a meaningful strategy for preventing radiation‐induced intestinal damage, although we cannot rule out other radioprotective mechanisms of metformin such as microbiota. Interestingly, intestinal microbiota contributes to intestinal radiotoxicity (Ciorba et al., 2012; Ferreira, Muls, Dearnaley, & Andreyev, 2014; Riehl et al., 2018) and metformin is reported to alter intestinal microbiota by regulating metabolism (Bauer et al., 2018; Greenhill, 2019).

Some studies have reported that metformin increases the radiosensitivity of colorectal tumours, but there was still a great heterogeneity between studies (Coyle et al., 2016; de Mey et al., 2018; Muaddi, Chowdhury, Vellanki, Zamiara, & Koritzinsky, 2013; Zannella et al., 2013), suggesting that metformin may not be beneficial for radiotherapy in all colorectal tumours. P53 status was reported to regulate the reactivity of colorectal cancer cells to metformin (Abu El Maaty, Strassburger, Qaiser, Dabiri, & Wolfl, 2017; Buzzai et al., 2007) and metformin can radiosensitize P53 mutant tumour cells through DNA damage repair pathway. Interestingly, our data showed that metformin might promote apoptosis but not protective autophagy to radiosensitize colorectal tumours with P53 mutation. Previous studies reported that wild type P53 triggers activation of AMPK signalling and inhibition of the mTOR to induce autophagy while Mutant P53 can inhibit autophagy through multiple pathways including blockage of AMPK signalling (Cordani, Butera, Pacchiana, & Donadelli, 2017; Morselli et al., 2008). Our results indicated that the deficiency of the AMPK/P53 axis may partially explain radiosensitization of metformin in P53 mutant colorectal tumours. However, we cannot completely rule out other effects of metformin such as DNA methylation which has been reported to play an important role in radiation‐induced oxidative stress (Miousse, Kutanzi, & Koturbash, 2017) and metformin regulated DNA methylation via multiple ways (Cuyas et al., 2018; Sabit, Abdel‐Ghany, Said, Mostafa, & El‐Zawahry, 2018; Zhong et al., 2017). Nevertheless, our results supported that regulation of AMPK‐induced autophagy may be an effective strategy for radiosensitizing colorectal tumours. Therefore, pharmacological AMPK activator such as metformin may be effective radiotherapy adjuvants for colorectal cancer especially those with P53 mutation.

6. CONCLUSION

In summary (as shown in the Figure 8), we found that metformin reduced acute and chronic radiation‐induced intestinal toxicity and metformin might increase the radiosensitivity of colorectal tumours with P53 mutation. Metformin may be potential radiotherapy adjuvant agent for colorectal cancers especially those carrying the mutation P53.

FIGURE 8.

FIGURE 8

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Proposed model of metformin as radiotherapy adjuvant for P53 mutation colorectal tumours via optimising autophagy. In normal intestinal epithelial cells, metformin promotes AMPK‐dependent autophagic degradation of Keap1, then Nrf2 moves to the nucleus and reduces mtROS by transcriptionally activating mitophagy, thereby protecting radiation‐induced intestinal injuries. However, in P53 mutation colorectal tumours, protective autophagy is inhibited by mutant p53 protein, resulting in radiosensitive effects

AUTHOR CONTRIBUTIONS

C.S and L.C. designed and performed the experiments. F.L. and L.C. analysed the data and drafted and wrote the manuscript with input from all co‐authors. C.S. conceived and supervised the study. Z.J., C.Z., Z.W., P.L., Q.Z.J., J.W., Q.W., M.L., X.L., Y.L., L.M. and G.S. took part in the IHC experiments and analysis of corresponding results. Z.C., Y.W., X.T., Y.G., D.L. and Y.L. analysed and interpreted data from experiments. All authors discussed the results and commented on the manuscript.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design and Analysis, Immunoblotting and Immunochemistry and Animal Experimentation, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.

ETHICAL APPROVAL

The Ethics Committee of the Animal Center of the Army Medical University approved this study.

Supporting information

Figure S1. (A)Schematic diagram demonstrating WAI exposure field for C57BL/6 mice. Only a 30 mm abdominal area of the mice containing the intestine was irradiated (irradiation field), thus avoiding hematopoietic injuries. (B) Schematic diagram of metformin, radiation treatment and specimen collection. Mice were given metformin daily for one week before receiving radiation. Samples were collected at four time points: 0, 4h, 3.5d and 5d. (C)Kaplan–Meier survival analysis of mice treated with different concentrations of metformin or PBS after 10Gy WAI(n=10). P<0.05 for Met250+IR, P <0.05 forMet500+IR, both compared with PBS+IR group. (D) Body weight of mice treated with different concentrations of metformin or PBS at times post 10Gy WAI. (E)Kaplan–Meier survival analysis of mice treated with different concentrations of metformin or PBS after 15Gy WAI(n=10). P=0.017 for Met250+IR, p=0.033 forMet500+IR, both compared with PBS+IR group. (F) Body weight of mice treated with different concentrations of metformin or PBS at times post 15Gy WAI. (G) Surviving mice from the metformin (right), PBS (middle) treated with WAI and an age matched control (left). Fur color turned white in the irradiated area. (H)Representative picture from the necropsy of a mouse 12 months after WAI. (I) Representative picture of intestine from surviving mice after 12 months. For Fig C ‐ F, n=10.

Figure S2. (A) Relative expression level of autophagy‐related genes: Beclin1, LC3, Pink1, Parkin, P62 24h after 15Gy radiation. For Fig A, n=5. (*, P<0.05).

Figure S3. Relative expression level of NRF2 pathway‐related genes 24h after 15Gy radiation: HO1(A), NQO1(B) and NRF2(C). (D) Relative expression level of NQO1, HO1 and P62 with the indicated treatments 24h after 15Gy radiation. For Fig A ‐ D, n=5. (*, P<0.05).

Figure S4. (A) Induction of apoptosis was detected by flow cytometry of annexin V‐PI marked cells 36h after 15Gy radiation. (B) Representative images of TUNEL immunofluorescence in the crypts. Green, TUNEL; blue, DAPI. Scale bar: 20 μm. For Fig A and B, n=5. (*, P<0.05).

Figure S5. (A) Effects of different dosage of radiation on cell viability of colorectal tumour cells. (B) Effects of different concentration of metformin on cell viability of HCT116 colorectal tumour cells. (C) Effects of different concentration of metformin on cell viability of HT29 colorectal tumour cells. (D and E) Annexin V‐PI was used to label apoptotic cells through flow cytometry. For Fig A ‐ E, n=5. (*, P<0.05).

Table S1. Sequences of the primers used for qRT‐PCR

Click here for additional data file. (1.5MB, pdf)

ACKNOWLEDGEMENTS

This work was supported by the National Key Research and Development Program (2016YFC1000805), University Innovation Team Building Program of Chongqing (CXTDG201602020) and Intramural Research Project Grants (AWS17J007 and 2018‐JCJQ‐ZQ‐001).

Chen L, Liao F, Jiang Z, et al. Metformin mitigates gastrointestinal radiotoxicity and radiosensitises P53 mutation colorectal tumours via optimising autophagy. Br J Pharmacol. 2020;177:3991–4006. 10.1111/bph.15149

Long Chen and Fengying Liao contributed equally to this work.

REFERENCES

  1. Abu El Maaty, M. A. , Strassburger, W. , Qaiser, T. , Dabiri, Y. , & Wolfl, S. (2017). Differences in p53 status significantly influence the cellular response and cell survival to 1,25‐dihydroxyvitamin D3‐metformin cotreatment in colorectal cancer cells. Molecular Carcinogenesis, 56, 2486–2498. 10.1002/mc.22696 [DOI] [PubMed] [Google Scholar]
  2. Alexander, S.P.H. , Roberts, R.E. , Broughton, B.R.S. , Sobey, S.G. , George, C.H. , Stanford, S,C. … Ahluwalia, A. (2018). Goals and practicalities of immunoblotting and immunohistochemistry: A guide for submission to the British Journal of Pharmacology, Br J Pharmacol 175, 407–411. 10.1111/bph.14112 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alexander, S. P. H. , Kelly, E. , Mathie, A. , Peters, J. A. , Veale, E. L. , Armstrong, J. F. , … Southan, C. (2019). The concise guide to pharmacology 2019/20: Introduction and other protein targets. British Journal of Pharmacology, 176(Suppl 1), S1–S20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Asano, J. , Sato, T. , Ichinose, S. , Kajita, M. , Onai, N. , Shimizu, S. , & Ohteki, T. (2017). Intrinsic autophagy is required for the maintenance of intestinal stem cells and for irradiation‐induced intestinal regeneration. Cell Reports, 20, 1050–1060. 10.1016/j.celrep.2017.07.019 [DOI] [PubMed] [Google Scholar]
  5. Ashrafi, G. , & Schwarz, T. L. (2013). The pathways of mitophagy for quality control and clearance of mitochondria. Cell Death and Differentiation, 20, 31–42. 10.1038/cdd.2012.81 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Banerjee, S. , Aykin‐Burns, N. , Krager, K. J. , Shah, S. K. , Melnyk, S. B. , Hauer‐Jensen, M. , & Pawar, S. A. (2016). Loss of C/EBPδ enhances IR‐induced cell death by promoting oxidative stress and mitochondrial dysfunction. Free Radical Biology & Medicine, 99, 296–307. 10.1016/j.freeradbiomed.2016.08.022 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bauer, P. V. , Duca, F. A. , Waise, T. M. Z. , Rasmussen, B. A. , Abraham, M. A. , Dranse, H. J. , … Lam, T. K. (2018). Metformin alters upper small intestinal microbiota that impact a glucose‐SGLT1‐sensing glucoregulatory pathway. Cell Metabolism, 27(101–117), e105. [DOI] [PubMed] [Google Scholar]
  8. Bray, F. , Ferlay, J. , Soerjomataram, I. , Siegel, R. L. , Torre, L. A. , & Jemal, A. (2018). Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians, 68, 394–424. 10.3322/caac.21492 [DOI] [PubMed] [Google Scholar]
  9. Buzzai, M. , Jones, R. G. , Amaravadi, R. K. , Lum, J. J. , DeBerardinis, R. J. , Zhao, F. , … Thompson, C. B. (2007). Systemic treatment with the antidiabetic drug metformin selectively impairs p53‐deficient tumor cell growth. Cancer Research, 67, 6745–6752. 10.1158/0008-5472.CAN-06-4447 [DOI] [PubMed] [Google Scholar]
  10. Chaurasia, M. , Gupta, S. , Das, A. , Dwarakanath, B. S. , Simonsen, A. , & Sharma, K. (2019). Radiation induces EIF2AK3/PERK and ERN1/IRE1 mediated pro‐survival autophagy. Autophagy, 15, 1391–1406. 10.1080/15548627.2019.1582973 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ciorba, M. A. , Riehl, T. E. , Rao, M. S. , Moon, C. , Ee, X. , Nava, G. M. , … Stenson, W. F. (2012). Lactobacillus probiotic protects intestinal epithelium from radiation injury in a TLR‐2/cyclo‐oxygenase‐2‐dependent manner. Gut, 61, 829–838. 10.1136/gutjnl-2011-300367 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cordani, M. , Butera, G. , Pacchiana, R. , & Donadelli, M. (2017). Molecular interplay between mutant p53 proteins and autophagy in cancer cells. Biochimica et Biophysica Acta. Reviews on Cancer, 1867, 19–28. 10.1016/j.bbcan.2016.11.003 [DOI] [PubMed] [Google Scholar]
  13. Cordani, M. , Oppici, E. , Dando, I. , Butturini, E. , Dalla Pozza, E. , Nadal‐Serrano, M. , … Donadelli, M. (2016). Mutant p53 proteins counteract autophagic mechanism sensitizing cancer cells to mTOR inhibition. Molecular Oncology, 10, 1008–1029. 10.1016/j.molonc.2016.04.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Coyle, C. , Cafferty, F. H. , Vale, C. , & Langley, R. E. (2016). Metformin as an adjuvant treatment for cancer: A systematic review and meta‐analysis. Annals of Oncology, 27, 2184–2195. 10.1093/annonc/mdw410 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Curtis, M. J. , Alexander, S. , Cirino, G. , Docherty, J. R. , George, C. H. , Giembycz, M. A. , … Ahluwalia, A. (2018). Experimental design and analysis and their reporting II: Updated and simplified guidance for authors and peer reviewers. British Journal of Pharmacology, 175, 987–993. 10.1111/bph.14153 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cuyas, E. , Fernandez‐Arroyo, S. , Verdura, S. , Garcia, R. A. , Stursa, J. , Werner, L. , … Neuzil, J. (2018). Metformin regulates global DNA methylation via mitochondrial one‐carbon metabolism. Oncogene, 37, 963–970. 10.1038/onc.2017.367 [DOI] [PubMed] [Google Scholar]
  17. De Ruysscher, D. , Niedermann, G. , Burnet, N. G. , Siva, S. , Lee, A. W. M. , & Hegi‐Johnson, F. (2019). Radiotherapy toxicity. Nature Reviews Disease Primers, 5, 13. 10.1038/s41572-019-0064-5 [DOI] [PubMed] [Google Scholar]
  18. Ferreira, M. R. , Muls, A. , Dearnaley, D. P. , & Andreyev, H. J. (2014). Microbiota and radiation‐induced bowel toxicity: Lessons from inflammatory bowel disease for the radiation oncologist. The Lancet Oncology, 15, e139–e147. 10.1016/S1470-2045(13)70504-7 [DOI] [PubMed] [Google Scholar]
  19. Fransson, P. , & Widmark, A. (2007). 15‐year prospective follow‐up of patient‐reported outcomes of late bowel toxicity after external beam radiotherapy for localized prostate cancer. A comparison with age‐matched controls. Acta Oncologica, 46, 517–524. 10.1080/02841860601113596 [DOI] [PubMed] [Google Scholar]
  20. Geisler, S. , Holmstrom, K. M. , Skujat, D. , Fiesel, F. C. , Rothfuss, O. C. , Kahle, P. J. , & Springer, W. (2010). PINK1/Parkin‐mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nature Cell Biology, 12, 119–131. 10.1038/ncb2012 [DOI] [PubMed] [Google Scholar]
  21. Greenhill, C. (2019). Effects of metformin mediated by gut microbiota. Nature Reviews. Endocrinology, 15, 2 10.1038/s41574-018-0133-y [DOI] [PubMed] [Google Scholar]
  22. Harding, S. D. , Sharman, J. L. , Faccenda, E. , Southan, C. , Pawson, A. J. , Ireland, S. , … NC‐IUPHAR . (2018). The IUPHAR/BPS guide to pharmacology in 2018: Updates and expansion to encompass the new guide to immunopharmacology. Nucleic Acids Research, 46, D1091–D1106. 10.1093/nar/gkx1121 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hauer‐Jensen, M. , Denham, J. W. , & Andreyev, H. J. (2014). Radiation enteropathy—Pathogenesis, treatment and prevention. Nature Reviews. Gastroenterology & Hepatology, 11, 470–479. 10.1038/nrgastro.2014.46 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. He, L. , & Wondisford, F. E. (2015). Metformin action: Concentrations matter. Cell Metabolism, 21, 159–162. 10.1016/j.cmet.2015.01.003 [DOI] [PubMed] [Google Scholar]
  25. Hu, L. , Wang, H. , Huang, L. , Zhao, Y. , & Wang, J. (2016). The protective roles of ROS‐mediated mitophagy on (125)I seeds radiation induced cell death in HCT116 cells. Oxidative Medicine and Cellular Longevity, 2016, 9460462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Huang, Y. , Zhou, J. , Luo, S. , Wang, Y. , He, J. , Luo, P. , … Shi, C. (2018). Identification of a fluorescent small‐molecule enhancer for therapeutic autophagy in colorectal cancer by targeting mitochondrial protein translocase TIM44. Gut, 67, 307–319. 10.1136/gutjnl-2016-311909 [DOI] [PubMed] [Google Scholar]
  27. Kam, W. W. , & Banati, R. B. (2013). Effects of ionizing radiation on mitochondria. Free Radical Biology & Medicine, 65, 607–619. 10.1016/j.freeradbiomed.2013.07.024 [DOI] [PubMed] [Google Scholar]
  28. Kilkenny, C. , Browne, W. , Cuthill, I. C. , Emerson, M. , & Altman, D. G. (2010). Animal research: Reporting in vivo experiments: the ARRIVE guidelines. British Journal of Pharmacology, 160, 1577–1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lamark, T. , Svenning, S. , & Johansen, T. (2017). Regulation of selective autophagy: The p62/SQSTM1 paradigm. Essays in Biochemistry, 61, 609–624. 10.1042/EBC20170035 [DOI] [PubMed] [Google Scholar]
  30. Larson‐Casey, J. L. , Deshane, J. S. , Ryan, A. J. , Thannickal, V. J. , & Carter, A. B. (2016). Macrophage Akt1 kinase‐mediated mitophagy modulates apoptosis resistance and pulmonary fibrosis. Immunity, 44, 582–596. 10.1016/j.immuni.2016.01.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liu, X. , Zhu, Q. , Zhang, M. , Yin, T. , Xu, R. , Xiao, W. , … Lu, G. (2018). Isoliquiritigenin ameliorates acute pancreatitis in mice via inhibition of oxidative stress and modulation of the Nrf2/HO‐1 pathway. Oxidative Medicine and Cellular Longevity, 2018, 7161592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Massat, N. J. , Moss, S. M. , Halloran, S. P. , & Duffy, S. W. (2013). Screening and primary prevention of colorectal cancer: A review of sex‐specific and site‐specific differences. Journal of Medical Screening, 20, 125–148. 10.1177/0969141313501292 [DOI] [PubMed] [Google Scholar]
  33. McGrath, J. C. , & Lilley, E. (2015). Implementing guidelines on reporting research using animals (ARRIVE etc.): New requirements for publication in BJP . British Journal of Pharmacology, 172, 3189–3193. 10.1111/bph.12955 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Meng, F. , Song, L. , & Wang, W. (2017). Metformin improves overall survival of colorectal cancer patients with diabetes: A meta‐analysis. Journal Diabetes Research, 2017, 5063239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. de Mey, S. , Jiang, H. , Corbet, C. , Wang, H. , Dufait, I. , Law, K. , … de Ridder, M. (2018). Antidiabetic biguanides radiosensitize hypoxic colorectal cancer cells through a decrease in oxygen consumption. Frontiers in Pharmacology, 9, 1073. 10.3389/fphar.2018.01073 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Miller, K. D. , Nogueira, L. , Mariotto, A. B. , Rowland, J. H. , Yabroff, K. R. , Alfano, C. M. , … Siegel, R. L. (2019). Cancer treatment and survivorship statistics, 2019. CA: A Cancer Journal for Clinicians, 69, 363–385. 10.3322/caac.21565 [DOI] [PubMed] [Google Scholar]
  37. Miousse, I. R. , Kutanzi, K. R. , & Koturbash, I. (2017). Effects of ionizing radiation on DNA methylation: From experimental biology to clinical applications. International Journal of Radiation Biology, 93, 457–469. 10.1080/09553002.2017.1287454 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mizushima, N. (2007). Autophagy: Process and function. Genes & Development, 21, 2861–2873. 10.1101/gad.1599207 [DOI] [PubMed] [Google Scholar]
  39. Morselli, E. , Tasdemir, E. , Maiuri, M. C. , Galluzzi, L. , Kepp, O. , Criollo, A. , … Kroemer, G. (2008). Mutant p53 protein localized in the cytoplasm inhibits autophagy. Cell Cycle, 7, 3056–3061. 10.4161/cc.7.19.6751 [DOI] [PubMed] [Google Scholar]
  40. Muaddi, H. , Chowdhury, S. , Vellanki, R. , Zamiara, P. , & Koritzinsky, M. (2013). Contributions of AMPK and p53 dependent signaling to radiation response in the presence of metformin. Radiotherapy and Oncology, 108, 446–450. 10.1016/j.radonc.2013.06.014 [DOI] [PubMed] [Google Scholar]
  41. Nguyen, T. D. , Shaid, S. , Vakhrusheva, O. , Koschade, S. E. , Klann, K. , Tholken, M. , … Haberbosch, I. (2019). Loss of the selective autophagy receptor p62 impairs murine myeloid leukemia progression and mitophagy. Blood, 133, 168–179. 10.1182/blood-2018-02-833475 [DOI] [PubMed] [Google Scholar]
  42. O'Rourke, K. P. , Ackerman, S. , Dow, L. E. , & Lowe, S. W. (2016). Isolation, culture, and maintenance of mouse intestinal stem cells. Bio‐Protocol, 6, e1733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pott, J. , Kabat, A. M. , & Maloy, K. J. (2018). Intestinal epithelial cell autophagy is required to protect against TNF‐induced apoptosis during chronic colitis in mice. Cell Host & Microbe, 23(191–202), e194. [DOI] [PubMed] [Google Scholar]
  44. Qaseem, A. , Barry, M. J. , Humphrey, L. L. , Forciea, M. A. , & Clinical Guidelines Committee of the American College of P . (2017). Oral pharmacologic treatment of type 2 diabetes mellitus: A clinical practice guideline update from the American College of Physicians. Annals of Internal Medicine, 166, 279–290. [DOI] [PubMed] [Google Scholar]
  45. Riehl, T. E. , Alvarado, D. , Ee, X. , Zuckerman, A. , Foster, L. , Kapoor, V. , … Stenson, W. F. (2018). Lactobacillus rhamnosus GG protects the intestinal epithelium from radiation injury through release of lipoteichoic acid, macrophage activation and the migration of mesenchymal stem cells. Gut, 68(6), 1003–1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sabit, H. , Abdel‐Ghany, S. E. , Said, O. A. , Mostafa, M. A. , & El‐Zawahry, M. (2018). Metformin reshapes the methylation profile in breast and colorectal cancer cells. Asian Pacific Journal of Cancer Prevention, 19, 2991–2999. 10.22034/APJCP.2018.19.10.2991 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sanli, T. , Rashid, A. , Liu, C. , Harding, S. , Bristow, R. G. , Cutz, J. C. , … Tsakiridis, T. (2010). Ionizing radiation activates AMP‐activated kinase (AMPK): A target for radiosensitization of human cancer cells. International Journal of Radiation Oncology, Biology, Physics, 78, 221–229. 10.1016/j.ijrobp.2010.03.005 [DOI] [PubMed] [Google Scholar]
  48. Sanli, T. , Steinberg, G. R. , Singh, G. , & Tsakiridis, T. (2014). AMP‐activated protein kinase (AMPK) beyond metabolism: A novel genomic stress sensor participating in the DNA damage response pathway. Cancer Biology & Therapy, 15, 156–169. 10.4161/cbt.26726 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sher, M. E. , & Bauer, J. (1990). Radiation‐induced enteropathy. The American Journal of Gastroenterology, 85, 121–128. [PubMed] [Google Scholar]
  50. Singh, A. , Venkannagari, S. , Oh, K. H. , Zhang, Y. Q. , Rohde, J. M. , Liu, L. , … Biswal, S. (2016). Small molecule inhibitor of NRF2 selectively intervenes therapeutic resistance in KEAP1‐deficient NSCLC tumors. ACS Chemical Biology, 11, 3214–3225. 10.1021/acschembio.6b00651 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Szumiel, I. (2015). Ionizing radiation‐induced oxidative stress, epigenetic changes and genomic instability: The pivotal role of mitochondria. International Journal of Radiation Biology, 91, 1–12. 10.3109/09553002.2014.934929 [DOI] [PubMed] [Google Scholar]
  52. Taneja, N. , Tjalkens, R. , Philbert, M. A. , & Rehemtulla, A. (2001). Irradiation of mitochondria initiates apoptosis in a cell free system. Oncogene, 20, 167–177. 10.1038/sj.onc.1204054 [DOI] [PubMed] [Google Scholar]
  53. Tchelebi, L. , Ashamalla, H. , & Graves, P. R. (2014). Mutant p53 and the response to chemotherapy and radiation. Sub‐Cellular Biochemistry, 85, 133–159. 10.1007/978-94-017-9211-0_8 [DOI] [PubMed] [Google Scholar]
  54. Turinetto, V. , & Giachino, C. (2015). Multiple facets of histone variant H2AX: A DNA double‐strand‐break marker with several biological functions. Nucleic Acids Research, 43, 2489–2498. 10.1093/nar/gkv061 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Vancura, A. , Bu, P. , Bhagwat, M. , Zeng, J. , & Vancurova, I. (2018). Metformin as an anticancer agent. Trends in Pharmacological Sciences, 39, 867–878. 10.1016/j.tips.2018.07.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Vial, G. , Detaille, D. , & Guigas, B. (2019). Role of mitochondria in the mechanism(s) of action of metformin. Front Endocrinol (Lausanne), 10, 294. 10.3389/fendo.2019.00294 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wang, J. , Wang, Y. , Han, J. , Mei, H. , Yu, D. , Ding, Q. , … Lin, Z. (2017). Metformin attenuates radiation‐induced pulmonary fibrosis in a murine model. Radiation Research, 188, 105–113. 10.1667/RR14708.1 [DOI] [PubMed] [Google Scholar]
  58. Wang, Z. , Chen, Z. , Jiang, Z. , Luo, P. , Liu, L. , Huang, Y. , … Shi, C. (2019). Cordycepin prevents radiation ulcer by inhibiting cell senescence via NRF2 and AMPK in rodents. Nature Communications, 10, 2538. 10.1038/s41467-019-10386-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wei, L. , Leibowitz, B. J. , Wang, X. , Epperly, M. , Greenberger, J. , Zhang, L. , & Yu, J. (2016). Inhibition of CDK4/6 protects against radiation‐induced intestinal injury in mice. The Journal of Clinical Investigation, 126, 4076–4087. 10.1172/JCI88410 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Xu, G. , Wu, H. , Zhang, J. , Li, D. , Wang, Y. , Wang, Y. , … Meng, A. (2015). Metformin ameliorates ionizing irradiation‐induced long‐term hematopoietic stem cell injury in mice. Free Radical Biology & Medicine, 87, 15–25. 10.1016/j.freeradbiomed.2015.05.045 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Yamamori, T. , Yasui, H. , Yamazumi, M. , Wada, Y. , Nakamura, Y. , Nakamura, H. , & Inanami, O. (2012). Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint. Free Radical Biology & Medicine, 53, 260–270. 10.1016/j.freeradbiomed.2012.04.033 [DOI] [PubMed] [Google Scholar]
  62. Zannella, V. E. , Dal Pra, A. , Muaddi, H. , McKee, T. D. , Stapleton, S. , Sykes, J. , … Wouters, B. G. (2013). Reprogramming metabolism with metformin improves tumor oxygenation and radiotherapy response. Clinical Cancer Research, 19, 6741–6750. 10.1158/1078-0432.CCR-13-1787 [DOI] [PubMed] [Google Scholar]
  63. Zhang, Y. , Wang, Y. , Xu, J. , Tian, F. , Hu, S. , Chen, Y. , & Fu, Z. (2019). Melatonin attenuates myocardial ischemia‐reperfusion injury via improving mitochondrial fusion/mitophagy and activating the AMPK‐OPA1 signaling pathways. Journal of Pineal Research, 66, e12542 10.1111/jpi.12542 [DOI] [PubMed] [Google Scholar]
  64. Zhong, T. , Men, Y. , Lu, L. , Geng, T. , Zhou, J. , Mitsuhashi, A. , … Huang, Y. (2017). Metformin alters DNA methylation genome‐wide via the H19/SAHH axis. Oncogene, 36, 2345–2354. 10.1038/onc.2016.391 [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zhou, G. , Myers, R. , Li, Y. , Chen, Y. , Shen, X. , Fenyk‐Melody, J. , … Moller, D. E. (2001). Role of AMP‐activated protein kinase in mechanism of metformin action. The Journal of Clinical Investigation, 108, 1167–1174. 10.1172/JCI13505 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. (A)Schematic diagram demonstrating WAI exposure field for C57BL/6 mice. Only a 30 mm abdominal area of the mice containing the intestine was irradiated (irradiation field), thus avoiding hematopoietic injuries. (B) Schematic diagram of metformin, radiation treatment and specimen collection. Mice were given metformin daily for one week before receiving radiation. Samples were collected at four time points: 0, 4h, 3.5d and 5d. (C)Kaplan–Meier survival analysis of mice treated with different concentrations of metformin or PBS after 10Gy WAI(n=10). P<0.05 for Met250+IR, P <0.05 forMet500+IR, both compared with PBS+IR group. (D) Body weight of mice treated with different concentrations of metformin or PBS at times post 10Gy WAI. (E)Kaplan–Meier survival analysis of mice treated with different concentrations of metformin or PBS after 15Gy WAI(n=10). P=0.017 for Met250+IR, p=0.033 forMet500+IR, both compared with PBS+IR group. (F) Body weight of mice treated with different concentrations of metformin or PBS at times post 15Gy WAI. (G) Surviving mice from the metformin (right), PBS (middle) treated with WAI and an age matched control (left). Fur color turned white in the irradiated area. (H)Representative picture from the necropsy of a mouse 12 months after WAI. (I) Representative picture of intestine from surviving mice after 12 months. For Fig C ‐ F, n=10.

Figure S2. (A) Relative expression level of autophagy‐related genes: Beclin1, LC3, Pink1, Parkin, P62 24h after 15Gy radiation. For Fig A, n=5. (*, P<0.05).

Figure S3. Relative expression level of NRF2 pathway‐related genes 24h after 15Gy radiation: HO1(A), NQO1(B) and NRF2(C). (D) Relative expression level of NQO1, HO1 and P62 with the indicated treatments 24h after 15Gy radiation. For Fig A ‐ D, n=5. (*, P<0.05).

Figure S4. (A) Induction of apoptosis was detected by flow cytometry of annexin V‐PI marked cells 36h after 15Gy radiation. (B) Representative images of TUNEL immunofluorescence in the crypts. Green, TUNEL; blue, DAPI. Scale bar: 20 μm. For Fig A and B, n=5. (*, P<0.05).

Figure S5. (A) Effects of different dosage of radiation on cell viability of colorectal tumour cells. (B) Effects of different concentration of metformin on cell viability of HCT116 colorectal tumour cells. (C) Effects of different concentration of metformin on cell viability of HT29 colorectal tumour cells. (D and E) Annexin V‐PI was used to label apoptotic cells through flow cytometry. For Fig A ‐ E, n=5. (*, P<0.05).

Table S1. Sequences of the primers used for qRT‐PCR

Click here for additional data file. (1.5MB, pdf)

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