Targeting Lactate Dehydrogenase A With Catechin Resensitizes SNU620/5FU Gastric Cancer Cells To 5-Fluorouracil Part 2

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2.5. CA and 5Fu Cotreatment Induces Mitochondrial ROS-Dependent Apoptosis

Since LDHA inhibition directly increases the mitochondrial ROS and loss of its membrane potential, subsequently inducing apoptosis[16,29], the factors related to the mitochondrial ROS-mediated apoptosis were examined. CA or 5FU single treatment slightly increased mitochondrial ROSlevel in SNU620/5FU cells when measured by flow cytometry using MitoSOX. However, cotreatment with CA and 5FU significantly enhanced mitochondrial ROS production. This increased ROS production was reversed by Mito-TEMPO, a mitochondria-targeted antioxidant (Figure 5A-C). Growth inhibition following treatment with CA and 5FU was also recovered upon Mito-TEMPO treatment in SNU620/5FU cells (Figure 5D). Further, apoptosis was evaluated via Annexin V-FITC/PI staining in SNU620/5FU cells. Cotreatment with CA and 5FU increased the number of apoptotic cells among SNU620/5FU cells(Figure 6A, B). Nuclear morphologic fragmentation appears during apoptosis, and it can be observed by 4',6-diamidino-2-phenylindole(DAPI)staining [30,31]. DNA fragmentation was found to have significantly increased in the cotreatment group, compared to that in the control group (Figure 6C). Finally, biomarkers of apoptosis including Bcl-2, Bax, Caspase-9, Caspase-3, and PARP were examined. The proapoptotic cascade increased following cotreatment with CA and 5FU in SNU620/5FU cells (Figure 6D). These findings suggest that cotreatment with CA and 5FU induces apoptosis in SNU620/5FU cells.

Figure 5. Cotreatment with catechin(CA)and 5-fluorouracil(5FU)increases the production of mitochondrial ROS:(A)SNU620/5FU cells were treated with the indicated concentrations of CA，5FU（10 μM）， and Mito-TEMPO（20 μM） for 48 h. The mitochondrial ROS of the cells was measured via FACS analysis using MitoSOXTM Red;(B)the bar graph shows the rate of cells that were positive for MitoSOX staining;(C)fluorescence microscopy images (X100)of SNU620/5FU cells stained with MitoSOX were presented;(D) the viabilities of the cells were measured via MTT assay. The results are shown as mean ± SEM.*p<0.01,p<0.001, and ##p<0.001, compared to the respective control. The experiments were independently performed in triplicate.

Figure 6. Increase of apoptosis by catechin(CA)and 5-fluorouracil(5FU) cotreatment; SNU620/5FU cells were treated with CA （10 uM） and/or 5FU （10 μM） for 48 h∶（A） the number of apoptotic cells was analyzed via FACSanalysis using PI-Annexin V staining;(B) the bar graph indicates the percentage of cells in early and late apoptotic phases. The results are shown as mean ± SEM.**p<0.001, compared to the control;(C) the nuclei of the cells were stained with DAPL, and fluorescence microscopic images were taken (X200). The white arrowhead indicates apoptotic cells. Scale bar, 10 um; (D)the expression levels of proteins related to the apoptotic pathway were measured by Western blot analysis. The experiments were independently performed in triplicate.

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3. Discussion

In this study, to characterize and evaluate chemoresistance to 5FU, we used SNU620/5FU cells, which were established by long-term exposure to 5FU with a serial increment of drug concentration [32]. The parental SNU620 cells have been reported to harbor a p53 mutant with a homozygous deletion in exon 5[33]. Based on a DNA microarray, several genes involved in chemoresistance, including thymidylate synthetase, damage-specific DNA binding protein 2, clusterin, and midkine, are elevated in SNU620/5FU cells [32]. Several previous studies have used the SNU620/5FUcell line as a model to evaluate the in vitro efficacy of drugs against resistance to 5FU through anti-mitosis, AMPK activation, and cannabinoid receptor signaling [34-36]. However, the metabolic characteristics and expression levels of metabolism-related genes in the SNU620/5FU cells and their roles in chemoresistance have not been largely investigated.

Here, we demonstrated that SNU620/5FU cells have advanced glycolytic phenotypes, including elevated lactate production and the expression of enzymes related to the conversion of pyruvate to lactate, such as LDHA. The phosphorylation of PDHA1, representing the reduced PDH activity by elevated PDK2 and PDK3, also increased in SNU620/5FU-resistant cells, compared with that in SNU620 parent cells. However, OCR was not significantly decreased in resistant cells. A possible explanation for this discrepancy is the nutrient plasticity of cancer cells for the TCA cycle, that is, amino acids or fatty acid-derived acetyl-CoA, not only glucose-derived acetyl-CoA, could supply substrates to the TCA cycle [37].In correlation with this, 5FU-resistant gastric cancer cells promote stemness via mitochondrial fatty acid oxidation [38]. In addition, 5FU-resistance has been related to increased mitochondrial mass and activity, including the expression of electron transport chain (ETC) enzymes and oxygen consumption [39,40]. Thus, we focused on the modulation of LDHA to resensitize the 5FU-resistance. Based on our results, the inhibition of LDHA activity with oxamate or CA successfully suppressed the growth of resistant SNU620/5FU cells and resensitized them to 5FU treatment. These findings showed a good correlation with those of previous studies, which reported that genetic or pharmacological inhibition of LDHA successfully reduced resistance to chemotherapy, including 5FU [19,41,42]. Thus, we assumed that the inhibition of LDHA might be sufficient to suppress the resistance against 5FU in SNU620/5FU cells.

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Several synthetic molecules, such as oxamate, FX11, PSTMB, and GNE-140 have been established as small-molecule LDHA inhibitors [11,43]. Among these, FX11 has been reported as a sensitizer to chemotherapy in resistant tumor cells [44]. However, although many LDHA inhibitors are under scrutiny for approval as novel anticancer drugs, none of them have been approved yet. Thus, more drug-like candidates are required to generate new LDHA inhibitors [45]. Natural compounds have been regarded as potential resources for anticancer drugs, particularly in overcoming chemoresistance as a combination therapy [46,47]. Therefore, targeting cancer metabolism, especially glucose metabolism with plant-derived natural products is an emerging research trend for the development of novel cancer therapeutics [48].

Further, several reports have suggested that natural products, such as gossypol, galloflavin, crocetin, machine A, and EGCG, are potent LDHA inhibitors [43,49,50]. Although among these, gossypol has the most potent inhibitory action on LDHA (IC50=9.8 uM), it also inhibits the activity of LDHB, which converts lactate to pyruvate [51]. Our results demonstrated that EGCG, an established LDHA inhibitor and a sensitizer to 5FU chemotherapy [22-25], exert inhibitory effects on both LDHA and LDHBactivities. Among the natural product-derived LDHA inhibitors showing LDHA-specific inhibitory activities, such as machine A(IC50 =84 μM）， crocetin （IC50 =54.9 μM）， and CA （IC50 =40.69 μM）【49，50】， CA showed the best suppressive effect on LDHA activity with respect to the ICsn value. Although the chemical structures of CA and EGCG are very similar except for the additional gallic acid moiety, their inhibitory selectivities were not the same. In addition, despite CA being the simplest compound, compared with its derivatives, it showed the best inhibitory action on both lactate production and LDHA activity. Thus, an extensive structure-activity relationship study should be conducted to demonstrate CA as a potential novel backbone for the development of specific and potent LDHA inhibitors.

EGCG has been known that a competitive inhibitor of NADPH. In addition, EGCG inhibits the NADPH oxidase translocation and reproduction [52-54]. However, according to the molecular modeling results, CA and EGCG are structurally located in different places (Figure 3A, B).In addition, they did not show the correlation in the specificity in the inhibition of LDHA and LDHB activity, which use the NADH and NAD+ as a cofactor, respectively. From these data, we assumed that CA has no direct correlation with EGCG in the mechanism of competing with NAD(P)H. By the way, inhibition of LDHA by several inhibitors, such as FX11, GSK 2837808A, NCI-737, and NCI-006, reduced the NADt production and increased the NADH accumulation, thereby decreasing the ratio of NAD+/NADH[16,17,55]. Apoptosis induced by p53/NAD-dependent DNA damage pathway is also raised by LDHA inhibition using siRNA or chemical inhibitor, NH-2 [56]. The GCN2-ATF4 signaling pathway was also reported as another mechanism responsible for apoptotic cell death induced by the LDHA inhibitor, GSK 2837808A [17]. Since the NAD/NADH ratio plays a key role in redox homeostasis and cell proliferation [5758], the CA as an LDHA inhibitor might affect the 5FU-resistant cancer cells through modulation of the NAD+/NADH ratio.

CA induced cell death in both parental SNU620 and 5FU-resistant SNU620/5FU cells. However, CA-induced cell death was LDHA dependent through shRNA depletion in SNU620/5FU cells. In addition, the cotreatment of CA and 5FU showed additional anticancer effects on 5FU-resistant cells. As previously reported [16,59], inhibition of LDHA increases the ROS and consequently suppressed the growth of cancer cells. When LDHA was inhibited, the energy metabolism was converted from glycolysis to the TCA cycle and mitochondrial oxidative phosphorylation [60]. Thus, cancer cells make more ROS production, resulting in the damage of mitochondrial membrane and mitochondrial-induced apoptosis [61,62].In good agreement with previous studies, CA induces apoptotic cell death through a mitochondrial ROS-dependent pathway. Increased ROS-defense ability and reduced apoptotic signals are common properties of chemoresistant cancer cells, especially in 5FU-resistant cells 3,63]. Further, alterations in cancer metabolism, particularly in glucose metabolism, also lead to resistance to chemotherapy through the alteration of cellular activities, such as aberrant DNA repair, enhanced autophagy, reduced apoptosis, defense against ROS, and increased secretion of exosomes [9,64,651. Thus, targeting glycolytic enzymes, including LDHA, with CA might be an alternative strategy for overcoming chemoresistance, especially in 5FU-resistant gastric cancer. Generally, rapidly dividing malignant tumors is highly sensitive to DNA synthesis inhibitors including 5FU, compared to normal tissue. However, some cancer cells can develop resistance to the treatment through several mechanisms, as previously described [34-36]. The cotreatment of bioactive compounds and conventional chemotherapy has a higher effect, compared to a single compound, on slowing the development of resistance |66. Therefore, the biological effects of specific phytochemicals with proven cytotoxic effects ad-ministered with conventional chemotherapy to target a wider range of signaling pathways in cancer cells, including cancer metabolism and mitochondrial functions, should be superior to single compounds in cancer management since they may delay the development of resistance [67]. In this study, the combination of CA and 5FU showed a higher inhibition on cell viability, compared to that of a single treatment. Moreover, we demonstrated that the inhibition of LDHA activity and subsequent mitochondrial ROS-mediate apoptosis might be the mechanism underlying the sensitizing effect of CA on 5FU-resistant cells (Figure 7).

In addition to the potency and specificity of LDHA inhibition, CA is safer than previously established natural product-derived LDH inhibitors. Moreover, CA is a well-known chemical ingredient of green tea, and its safety and pharmacodynamic properties have been confirmed in previous studies [68-70]. However, a precise toxicity assessment of the coadministration of CA and 5FU has not yet been conducted. In addition, the in vivo efficacy of CA in 5FU-resistant cancer cells was not examined in this study. However, previous studies report on the in vivo anticancer efficacy of CA and its derivatives to overcome chemoresistance, including the 5FU-resistance in human gastric and colon cancer [71-77]. In this study, we focused on the LDHA inhibition as a major molecular mechanism of CA of overcoming the resistance to 5FU. Therefore, to develop CA as a novel adjuvant for chemoresistant cancer cells, the in vivo efficacy and safety of CA and 5FU cotreatment should be evaluated through extensive animal studies, including xenograft models and good laboratory toxicity assessments.

4. Materials and Methods

4.1.Materials

Antibodies against poly(ADP-ribose)polymerase, caspase-3, and caspase-9 were purchased from Cell Signaling Technology(Cell Signaling Technology, Danvers, MA, USA). Antibodies against LDHA, glyceraldehyde 3-phosphate dehydrogenase(GAPDH), and PDHA1 were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and those against phosphor-PDHA1 and PDK3 were purchased from Abcam (Abcam, Cambridge, MA, USA). Further, antibodies against PDK2 and PDK4 were purchased from Signalway Antibody (Signalway Antibody, Dallas, TX, USA), those against PDK1 was obtained from Enzo Life Sciences (Enzo Life Sciences, Farmingdale, NY, USA), and those against B-cell lymphoma-2(Bcl-2) and Bcl-2-associated X protein were purchased from Novus Biologicals (Novus Biologicals, Littleton, CO, USA). MitoSOX was purchased from Invitrogen (Invitrogen, Carlsbad, CA, USA). Chemicals and reagents, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT),4',6-diamidino-2-phenylindole, oxamate, nicotinamide adenine dinucleotide, CA, and CA derivatives (EC GC, EGC, and EGCG) were purchased from Merck(Merck, Darmstadt, Germany).β-nicotinamide adenine dinucleotide(oxidized form)was purchased from Tokyo Chemical Industry (Tokyo Chemical Industry, Tokyo, Japan).

4.2.Cell Culture

Human gastric cancer SNU620, SNU620/5FU, and AGS, pancreatic cancer Panic-1 and MIA PaCa-2, and colon cancer LS174T and RKO cells were obtained from the Korean Cell Line Bank(KCLB, Seoul, Korea).SNU620, SNU620/5FU, AGS, and LS174T cells were cultured in Roswell Park Memorial Institute medium 1640 (RPMI-1640)(Welgene, Daegu, Korea), supplemented with 10% heat-inactivated fetal bovine serum (FBS)(Gibco, New York, NY, USA)and 1%penicillin/streptomycin (Invitrogen), and the Panc-1, MIA PaCa-2, and RKO cells were cultured in Dulbecco's modified Eagle's medium (Welgene) containing 10% heat-inactivated FBS and 1%penicillin/streptomycin. All cells were cultured in a humidified CO, incubator at 37 °C, and 5% CO2.

4.3. Cell Viability Assay

The cytotoxicity levels of CA and 5FU in SNU620 and SNU620/5FUcells were measured using an MTT assay. Cells were cultured in 24-well plates(2× 104 cells/well) with the indicated concentrations of CA and 5FU for the stated day. MTT solution (2.0 mg/mL)was then added to each well, followed by 3-4 h of incubation at 37°C and 5% CO, in a cell culture incubator. The culture medium was subsequently removed, and the absorbance of formazan crystals was made from live cells. DMSO was added to dissolve the formazan crystals, and it was measured at 540 nm using a Spectramax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).

4.4. Extracellular Acidification Rate(ECAR) and Oxygen Consumption Rate(OCR)

ECAR and OCR, indicating the cellular rates of glycolysis and oxidative phosphorylation, respectively, were monitored with the Seahorse XF analyzer(Agilent Technologies, Santa Clara, CA, USA), as described previously [78,79]. Briefly, 60,000 SNU620 or SNU620/5FU cells per well were seeded in Seahorse XF six-well plates in RPMI medium supplemented with 1% penicillin/streptomycin. Following a 30 min incubation, the medium from each well was replaced with 80 uL of the prewarmed serum-free medium with5 mM oxamate, a standard LDH inhibitor [11]. Cells were then incubated at 37℃C for 24 h. After incubation， the medium from each well was replaced with 180 μL of prewarmed XFbase medium (containing 10 mM glucose,2 mM glutamine, and 1 mM sodium pyruvate; pH 7.4) to measure ECAR and OCR. Results were analyzed using the Wave 2.6.0.31 software (Agilent Technologies).

4.5.Quantitatioe Reverse transcription-polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted using the RiboEx Total RNA Extraction Kit(GeneAll Biotechnology, Seoul, Korea), and cDNA was synthesized using a reverse transcriptase kit (Promega, Madison， WI， USA）. In reverse transcription， 1 μg of total RNA was used， and the total amount of cDNA synthesized is 20 μL. Each kit was used according to the manufacturer's instructions. Quantitative PCR was performed using a StepOneTM Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA), with the Real HelixgPCR Kit (NanoHelix, Daejeon, Korea), for 40 cycles consisting of 15 s at 95℃Cand 1 min at 60°C. Relative mRNA levels were normalized to the levels of 18S ribosomal RNA, which served as an endogenous control. Sequences of the primers used for qRT-PCR are listed in Table 1.

4.6.Wester" Blot Analysis

The cells were washed with 1× PBS, and total proteins were extracted from cells using RIPA buffer and 1% NP-40 lysis buffer containing protease inhibitor cocktail tablets (Roche, Basel, Switzerland). Each proteins concentrations were measured using the Bio-Rad protein assay. Equal amounts of protein were fractionated from each sample through 8-15% SDS-PAGE, and then the proteins were transferred to nitrocellulose membranes(GE Healthcare, Munich, Germany)via electrophoresis. The membranes were blocked at room temperature (20-25°C) for 1 h using 5% nonfat dry milk and incubated with primary antibodies at 4°C overnight. Subsequently, these membranes were washed three times with 1× Tris-buffered saline for 10 min. Specific bands of proteins were measured with a chemiluminescence imaging system (ImageQuant LAS 4000; GE Healthcare).The expression of proteins was adjusted by GAPDH. 4.7.Lactate Production Assay

Lactate production was measured in the culture media of SNU620, SNU620/5FU, AGS, Panc-1, MIA PaCa-2, LS174T, and RKO cells. These cells were incubated for 1d at 37°C, and the culture media were subsequently replaced with phenol red-free medium, followed by incubation for 1 h at 37°C. The medium of each cell was then evaluated using a commercial lactate fluorometric assay kit(BioVision, Milpitas, CA, USA).

4.8. LDHA and LDHB Activity Assays

To detect LDHA activity, the indicated concentrations of CA were incubated for 20 min in a buffer containing 2 mM pyruvate， 20 μM NADH， and 20 mM HEPES-K+（pH7.2）. For LDHBactivity, a buffer containing 1 M Tris-HCl(pH8.0),25 mM NAD+, and 2 M sodium L-lactate was used. Briefly, 10 nanograms of each of the purified recombinant LDHA and LDHB proteins were used for the in vitro LDHA and LDHB activity assays. One microgram of total protein from cell lysates was used for intracellular LDHA and LDHB activity assays as an enzyme source. Fluorescence of NADH at an excitation wavelength of 340 nm and an emission wavelength of 460 nm was detected using a spectrofluorometer (Spectramax M2; Molecular Devices), as previously described [80]. LDHA activity was measured by the decreased amount of NADH, whereas LDHIB activity was evaluated by measuring the amount of NADH converted from NAD+.

4.9.Protein-Small Molecule Interaction

The interaction between protein and small molecules was predicted using the Pyrex program. The LDHA (PDB ID:1l10) molecules and the 2D structures of CA and EGCG obtained from the NCBI PubChem compound database were used in Pyrex. The ID of CA was 9064 and that of EGCG was 65064. The relative distribution of the surface charge was shown with the acidic region in red, the basic region in blue, and the neutral region in white. Hydrogen bonds in LDHA complexes with CA or EGCG, respectively, were shown as black dotted lines. Sequences were obtained from UniProt (https://www.uniprot.org, accessed on 9 November 2020)with accession numbers P00338(LDHA).

4.10.Transfection of Short Hairpin RNA (shRNA)

The pLKO.1 mock-vector and shRNA targeting LDHA vector were used, as previously described [50]. Cells were seeded at six-well plates (2 × 10° cells/well) and incubated overnight. SNU620/5FU cells were transfected using polyethyleneimine (PED)(Polyplus-transfection,llkirch, France), gene, and PEI ratio is 1:3. Then, transfected cells were treated with 1 ug/mL puromycin for 1 week. The control cell line was generated following infection with a scrambled plasmid.

4.11.LDHA Overexpression

The plasmid pDEST27-LDHA was constructed by subcloning of LDHA cDNA (purchased from Korea Human Gene Bank, Daejeon, Korea) into pDEST27(Invitrogen) vectors. Two sets of subcloned for LDHA were conducted. For reconstruction of the LDHA, the SNU620/5FU-shLDHA cells were transfected with pDEST27-LDHA and empty pDEST27 plasmid. Briefly, the cells were cultured up to 70% confluency. Then, the cells were treated with a mixture including 3ug of DNA and Lipofectamine 2000 (Invitrogen) for 48 h. After incubation, the cells were selected with 200 ug/mL G418 for 1 week. Then, to confirm the efficacy of transfection, we performed the Western blot assay.

4.12.Apoptosis Analysis

Apoptotic cells were detected using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA), according to the manufacturer's instruction. Briefly, cells were seeded at six-well plates(2×10° cells/well) and treated with indicated concentrations of CA and 5FU for 2 days. After 2 days of treatments, the cell was washed with 1× PBS. The cells were suspended in 500 μL of binding buffer and treated with 5 uL of annexin V-FITC and 5 μL of propidium iodide （Sigma-Aldrich， St. Louis， MO， USA）， followed by incubation for 15 min at room temperature in the dark. Fluorescence intensities were examined using a BD FACS CANTO ⅡI (BD Biosciences).

4.13. Mitochondrial Reactive Oxygen Species (ROS) Detection Assay

Mitochondrial ROS production was detected using a MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fisher Scientific). The cells were seeded at six-well plates （2× 10° cells/well） and Mito-TEMPO（20 μM; Sigma-Aldrich） was pretreated for 1 h before drug treatment. The cells were resuspended in 1 mL of 1× phosphate-buffered saline （PBS）， after which 5 μM of MitoSOXwas added.The cells were then incubated for 10 min at 37 °C. Fluorescence intensity was analyzed using a BD FACS CANTO II (BD Biosciences). Fluorescence image was detected by fluorescence microscope (magnification, 100×)(Axioimager M1 microscope, Carl Zeiss, Oberkochen, Germany).

4.14.DAPI Staining of Nucleus

SNU620/5FU was seeded in a 24-well plate (5× 104 cells/well) and treated with the indicated concentrations of CA and 5FU for 48 h. After washing with 1× PBS, cells were resuspended in 1-mL 1× PBS. The cells were then stained with4ug/mL DAPI for 30 min at room temperature and examined under a fluorescence microscope (magnification, 200×)(Carl Zeiss).

4.15. Statistical Analysis

The results of cell viability, lactate production, ECAR, OCR, qRT-PCR, LDHA, LDHB activities, apoptosis, and mitochondrial ROS were indicated relative to control values and expressed as mean ±standard error of the mean of three independent experiments. Differences above the mean value of each group were analyzed by Student's t-test, whereas differences between groups were analyzed by one-way analysis of variance with Tukey's post hoc test using GraphPad Prism (Version5.0, GraphPad Software, San Diego, CA, USA).

5. Conclusions

Taken together, resistant gastric cancer SNU620/5FU cells have glycolytic phenotypes, including elevated lactate production and higher LDHA expression than those in parental SNU620 cells. Restricting glycolysis with CA, as an LDHA-specific inhibitor, sensitizes SNU620/5FU cells to 5FU. Additionally, cotreatment with CA and 5FU increased mitochondrial ROS and apoptotic cell death in 5FU-resistant cells. Our findings suggest that CA may be a promising candidate for the development of an adjuvant drug that reduces resistance to 5FU-based chemotherapy by restricting LDHA activity.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22105406/s1, Figure S1:Effect of 5FUon the viability of SNU620/5FU clls, Figure S2: The growth of SNU620 and SNU620/5FU cells measured by cell counting, Figure S3:Lactate production levels of SNU620 and SNU620/5FUcells, Figure S4: Expressions of PDK isotypes in SNU620 and SNU620/5FU cells, Figure S5: Effect of oxamate on the viabilities of SNU620 and SNU620/5FU cells, Figure S6: Effects of EGCG on LDHA and LDHBactivities, Figure S7:Effects of catechin (CA)on the expression levels of p-PDHA1, PDHA1, and PDKs proteins, Figure S8:Isothermal titration calorimetry (ITC) analysis of the LDHA and CA, Figure S9:LDHA expression in SNU620/5FU cells transfected with shLDHA, Figure S10: Measurement of glycolytic phenotypes and cell viability in various cancer cells, Figure S11: The raw data of Western blot assay.

This article is extracted from Int. J. Mol. Sci. 2021, 22, 5406. https://doi.org/10.3390/ijms22105406 https://www.mdpi.com/journal/ijms