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Glucose starvation induces resistance to metformin through the elevation of mitochondrial multidrug resistance protein 1

Metformin, a drug for type 2 diabetes mellitus, has shown therapeutic effects for various cancers. However, it had no beneficial effects on the survival rate of human malignant mesothelioma (HMM) patients. The present study was performed to elucidate the underlying mechanism of metformin resistance in HMM cells. Glucose‐starved HMM cells had enhanced resistance to metformin, demonstrated by decreased apoptosis and autophagy and increased cell survival. These cells showed abnormalities in mitochondria, such as decreased ATP synthesis, morphological elongation, altered mitochondrial permeability transition pore and hyperpolarization of mitochondrial membrane potential (MMP). Intriguingly, Mdr1 was significantly upregulated in mitochondria but not in cell membrane. The upregulated mitochondrial Mdr1 was reversed by treatment with carbonyl cyanide m‐chlorophenyl hydrazone, an MMP depolarization inducer. Furthermore, apoptosis and autophagy were increased in multidrug resistance protein 1 knockout HMM cells cultured under glucose starvation with metformin treatment. The data suggest that mitochondrial Mdr1 plays a critical role in the chemoresistance to metformin in HMM cells, which could be a potential target for improving its therapeutic efficacy.

The effects and mechanism of glucose starvation in the context of chemoresistance of cancer cells are largely unknown. Glucose is metabolized mainly in mitochondria, which is essential for generating energy for cell proliferation. Glucose starvation disturbs mitochondrial function and changes the cellular phenotype. 6,[14][15][16] Under glucose-deficient conditions, autophagy provides energy to sustain cellular metabolism and considered a hindrance to cancer therapy that disturbs cellular physiology. 17,18 In contrast, excessive autophagy contributes to cell death, which is considered to be a target for effective cancer therapy. 19 HMM is an aggressive tumor that is closely associated with exposure to asbestos fibers approximately 20-40 years prior to tumorigenesis. HMM patients present poor therapeutic response due to traditional anticancer drug resistance, such as cisplatin and pemetrexed. 20 Metformin, which is commonly used for the management of type II diabetes mellitus, has been suggested as an alternative anticancer drug. 21,22 Recent studies have shown the potential therapeutic value of metformin in HMM cells, which is exerted through the inhibition of mTOR and cell cycle arrest. 21 However, metformin treatment causes resistance in human breast cancer cells, although the precise mechanism has not been elucidated. 23 Metformin treatment has shown no evidence to relieve HMM patients, which may be due to the tumor microenvironment. 24 Expanding our understanding of the mechanism of drug resistance is urgently needed to improve the prognosis in cancer patients and to develop effective therapeutic strategies. The present study was performed to determine the impact of glucose deficiency on the development of resistance to metformin and the underlying mechanism in HMM cells. The results of the present study suggest that glucose starvation enhances drug resistance in HMM cells via mitochondrial Mdr1 elevation. Identification of the mechanism associated with enhanced resistance to metformin will be valuable for improving therapeutic efficacy in cancer patients.

| Cell lines and culture conditions
The HMM cell lines MS1 and NIH-513 (H513) were kindly provided by Dr Jablons (University of California San Francisco, San Francisco, CA, USA) and Dr R. Kratzke (University of Minnesota, Minnesota, MN, USA), respectively. Met-5A benign transformed mesothelial cells were purchased from the ATCC (Manassas, VA, USA). These mesothelial cell lines were cultured as described previously, 25 and various concentrations of glucose (0, 1, 5 and 10 mmol/L) were added to the medium.

| Cell proliferation and viability assay
Cell viability was evaluated by using an MTT assay (Sigma-Aldrich, St. Louis, MO, USA). Briefly, MTT solution (MTT dissolved in PBS, 5 mg/mL) was added to each well and incubated for 1 hour at 37°C. Subsequently, each well was treated with 100 mL of a solution which contained DMSO (Sigma-Aldrich) and 2-propanol (Millipore, Billerica, MA, USA) at a 9:1 ratio and absorbance was measured at a wavelength of 570 nm using a microplate reader (BioTek Epoch, Izasa, Barcelona, Spain). For the cell proliferation assay, 5 × 10 4 cells were seeded in 6-well plates and incubated in conditioned medium with glucose concentrations ranging from 0 to 10 mmol/L for 3 days.
The surviving cells on each day were counted manually under a light microscope.

| Apoptosis assay
The apoptosis assay was performed using an EzWay Annexin

| Western blotting assay
The cells were lysed using EzRIPA buffer (ATTO, Tokyo, Japan). The  and Lamin A (Santa Cruz Biotechnology, #sc-6214) were used as endogenous control markers for the cytoplasm, mitochondria and nucleus, respectively.

| Quantitative real-time PCR
Total RNA was extracted using TRIzol LS Reagent (Ambion, Austin, TX, USA) and quantitation was performed using a BioTek Epoch Microplate Spectrophotometer (Izasa, Barcelona, Spain). A total of 1000 ng of RNA was subjected to cDNA synthesis using a QuantiTect Reverse Transcription Kit (Enzynomics, Seoul, South Korea). An SYBR Green RT-PCR Kit (Enzynomics) was used for gene expression analysis. The primer sequences are presented in Table S1.
The relative changes in gene expression levels were normalized to 18S rRNA and calculated using the ΔΔC t method. 26

| Autophagy detection
The autophagy activity was assessed using a Cyto-ID Autophagy detection kit (Enzo Life Sciences, Farmingdale, NY, USA). Briefly, the Cyto-ID Autophagy Detection Reagent was added to the cell pellet, and incubated for 30 minutes at 37°C protected from light and analyzed using flow cytometry (Becton Dickinson).

| Transmission electron microscopy
Cell pellets were immersed in Karnovsky's solution (2% glutaraldehyde, 0.05 mol/L cacodylate, 2% paraformaldehyde and distilled water) and incubated overnight. 27 After washing with 0.05 mol/L sodium cacodylate buffer, the cells were subjected to post-fixation using 2% osmium tetroxide for 2 hours, followed by washing in distilled water. For fixation, 0.5% uranyl acetate was added, and the cells were then washed with ethanol. Propylene oxide was added to the pellet for transition. For infiltration, the cells were incubated in propylene oxide and Spurr's resin mixed at a 1:1 ratio for 2 hours at room temperature. For solidification, the solution was replaced with fresh Spurr's resin and incubated at 70°C overnight. After thin sectioning using an ultramicrotome (MT-X; RMC, Tucson, AZ, USA), the intracellular organelles morphology was examined using a JEM 1010 transmission electron microscope (JEOL, Tokyo, Japan).

| Assessment of mitochondrial function
The cellular level of ATP was measured using the ATP Colorimetric/ Fluorometric Assay Kit (BioVision, Milpitas, CA, USA), according to the manufacturer's recommendations. Briefly, a mixture of ATP assay buffer, probe, converter and developer was added to the cell lysate obtained from 1 × 10 6 cells. In addition, the resulting absorbance was measured at a wavelength of 570 nm using a microplate reader (BioTek Epoch) and calculated using a standard curve.
Mitochondrial membrane potential was evaluated using Using the depolarization baseline with red/green ratio decreased by CCCP treatment, the MMP data were normalized.

| Production of knockout cells using the clustered regulated interspaced short palindromic repeats/Cas9 technique
Human malignant mesothelioma cells were transfected with 2 μg of MDR1 CRISPR/Cas9 KO plasmids containing a GFP-coding region and either control or MDR1 (Table S1; Santa Cruz Biotechnology) using the HiPerFect Transfection Reagent (Qiagen, Hilden, Germany) following the manufacturer's recommendations. GFP-positive cells were selectively collected by using a BD Aria III cell sorter (BD Biosciences Clontech, Palo Alto, CA, USA) 3 days post-transfection. The knockout efficiency for the target gene was verified by real-time RT-PCR for MDR1.

| Statistical analysis
The experiments described above were performed independently at least 3 times. Data were expressed as the mean ± SD. GraphPad Prism Software (GraphPad Software, San Diego, CA, USA) was used for all graphs and statistical analysis. Tukey's pairwise comparison and one-way ANOVA were applied for comparisons between groups. Statistical significance was accepted at P < 0.05.

| Survived human malignant mesothelioma cells under glucose-starved conditions desensitized against to metformin treatment
To HMM cells precultured in medium with 5 or 10 mmol/L glucose exhibited a significant reduction of cell viability compared to 0 or 1 mmol/L glucose ( Figure 1D,E). These data revealed that adapted HMM cells to glucose-deficient conditions acquired greater resistance to metformin than did cells cultured under glucose abundant conditions. The expression of LC3B, p-AMPK and AMPK was increased by either glucose starvation or metformin treatment in HMM cells ( Figure 2D). However, that was decreased in the treatment of glucose-starved HMM cells with metformin. In addition, mTOR expression was decreased by either glucose starvation or metformin treatment in MS1.

| MDR1 is significantly elevated in mitochondria of glucose-starved HMM cells by treatment with metformin
The treatment of glucose-starved HMM cells with metformin increased the mRNA expression of MDR1 and ABCG2 ( Figure 3A,B). In particular, MDR1 mRNA expression in glucose-starved cells with metformin Previous research reported that MDR1 is increased in hepatocellular carcinoma cells with mitochondrial defects. 28 Moreover, dependent on glucose content decreasing and metformin treatment, the mRNA expression of MDR1 and PINK1 showed positive correlation ( Figure S2). The Mdr1 fluorescence in glucose-starved MS1 cells with metformin treatment was significantly increased and exhibited colocalization with MitoTracker ( Figure 3E). However, the Mdr1 expression in HMM cells cultured in complete medium was unchanged by metformin treatment. In addition, mitotracker expression increasing which was a mitochondria damage indicator was detected in HMM cells cultured under glucose starvation and metformin treatment compared to those HMM cells cultured in complete medium ( Figure S3).

| Dysfunctional mitochondria induced by glucose starvation and/or metformin treatment
To further examine the mitochondria integrity, the expression of Pink1, a mitochondrial damage marker, was measured. The

| MDR1 regulates chemoresistance under glucose-starved conditions
To evaluate whether MDR1 is directly mediated metformin resistance in glucose-starved HMM cells, MDR1 was knocked out using the CRISPR/CAS9 system. The control cells ( Figure 5A). The viability of the MDR1 KO HMM cells was similar to those of the parental cells. In contrast to the parental HMM cells, the treatment of glucose-starved MDR1 KO HMM cells with metformin induced a significant reduction of viability ( Figure 5B).
Apoptosis and autophagy assays were performed to assess cell death pathways that were regulated by MDR1. Compared to those of the control, increased apoptosis was found in the glucose-starved MDR1 KO HMM cells by metformin treatment ( Figure 5C). The autophagic response was increased in metformin treatment against glucosestarved MDR1 KO cells, while this response was decreased in the control cells ( Figure 5D). However, a consistent level of autophagy was exhibited in MDR1 KO HMM cells cultured under glucose starvation or metformin treatment compared to those of the parental cells.

| D ISCUSS I ON
Despite efforts in anticancer drug development, cancer mortality remains high due to the therapeutic resistance of cancer cells.
During progression to the malignant stage, cancer cells survive in an adverse microenvironment and develop drug resistance. [8][9][10] This study was performed to elucidate the mechanism of enhanced metformin resistance in glucose-starved HMM cells. HMM cells used a distinct metabolism to generate energy compared to normal mesothelial cells and glucose starvation promoted more severe metabolic alterations. 30 resistance. The viability of HMM cells cultured under glucose starvation with metformin was unchanged and apoptosis was decreased compared to those of cells cultured in complete medium.
Excessively activated autophagy induces cell death during cancer therapy. 18  The de-phosphorylation of Ser 656 on dynamin-related protein 1, a mitochondrial fission marker, induced mitochondrial elongation and prompted an anti-apoptotic response. 36 The mPTP penetrated through the mitochondrial inner and outer membranes, which maintain the calcium homeostasis. Excessive calcium was accumulated in damaged mitochondria through mPTP overactivation, which disturbs homeostasis and leads to cell death. 37 In line with this, glucose starvation or metformin treatment in- Metformin combined with serum in medium obtains a hydrophobic feature that could be a substrate of MDR1. 44,45 Multidrug resistance has been attributed to ABC transporters that encode p-glycoprotein, which regulates drug resistance via the efflux of hydrophobic substrates from the intracellular membrane. 46 Furthermore, MDR1 interacts with the membrane as a fluid mosaic model located in lipid bilayer membranes and activate binding with ATP, which contributes to the efflux of the accumulated drug and small molecules to the extracellular space. 47 The MDR1 is expressed not only in the plasma membrane, but also in mitochondria and nucleus. 48 Moreover, p-glycoprotein is highly expressed to protect attenuated mitochondria and promotes ion and chemical homeostasis. 49,50 Mitochondrial Mdr1 has a distinct role as a unidirectional pump that sequesters drug infiltrated mitochondria and is subsequently degraded by lysosome to prevent drug distribution. 48  The role of MDR1 was verified by knockout using the CRISPR/ Cas9 system. In contrast to the parental cells, apoptosis and autophagy were increased and viability was decreased in the treatment of glucose-starved MDR1 KO HMM cells with metformin. ATP is required for autophagy activation. However, the ATP amount was insufficient in glucose-starved HMM cells with metformin because that energy was wasted in MDR1 overexpression. Therefore, an inadequate ATP amount could not facilitate autophagy. However, further research is necessary on the relationship between autophagy and MDR1. In addition, the expression level of ABCG2 mRNA was increased in glucose-starved HMM cells with metformin treatment. However, the extent of ABCG2 upregulation was less dramatic than that of MDR1 in our study. The role of ABCG2 in metformin resistance and the underlying mechanism warrants further study.
The present study demonstrated that overexpressed Mdr1 in defected mitochondria rescued glucose-starved HMM cells from metformin treatment. Although MDR1 is important in chemoresistance, the mechanism of its expression and localization are not yet clear in HMM cells. Therefore, tumor recurrence and chemoresistance in malignant mesothelioma patients could be suppressed by Mdr1 repression through MMP reduction, which promote the anticancer efficacy.

CO N FLI C T S O F I NTE R E S T
All authors declare no potential of conflicts of interest.