Inhibitory Effect of Glucocorticoid Receptor on Colorectal Cancer Growth by Inhibiting Glycolysis Through Autophagy

Materials Express

Inhibitory effect of glucocorticoid receptor on colorectal cancer growth by inhibiting glycolysis through autophagy

Zhiheng Xu1,∗, Kailun Yang2, and Xiaohua Li2 1Gastrointestinal Thyroid Surgery, The First Afﬁliated Hospital of Guangzhou University of Traditional Chinese Medicine, Guangzhou 510405, Guangdong, PR China 2First Clinical Medical Institute, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong, PR China

ABSTRACT Article Glucocorticoid receptor (GR) affects the development and progression of most malignant tumors by regulating autophagy. The GR gene is notIP: expressed 192.168.39.210 in colon On: cancer. Fri, 01 To Oct explore 2021 the05:04:18 role and mechanism of GR in colon cancer, dexamethasone (DXM) wasCopyright: used to stimulate American the Scientific expression Publishers of GR. The expression of the autophagy Delivered by Ingenta markers Beclin 1 (BECN1) and light chain 3 (LC3B) was then detected by qRT-PCR and western blotting. The effects of the differential expression of GR on autophagy, ATP, lactic acid accumulation, and glucose utilization in colon cancer cells were studied. Differential expression of GR affected glycolysis, apoptosis, and migration of colon cancer cells, as determined by flow cytometry and cell viability and migration assays, respectively. The DXM-induced elevation of GR expression significantly promoted the expression of the autophagy-related genes BECN1 and LC3B, and decreased the ATP production, lactic acid accumulation, and glucose uptake in colon cancer cells. These events resulted in the inhibition of colon cancer cell growth, which also involved decreased cell viability and mobility and increased rate of apoptosis. These findings indicate that the GR can promote autophagy and inhibit glycolysis in colon cancer cells, reduce their proliferation and migration, and promote their apoptosis in vitro. Keywords: Colon Cancer, LoVo Cells, Glucocorticoid Receptor, Glycolysis.

1. INTRODUCTION Autophagy-regulated apoptosis is an important way to Colon cancer is a common digestive tract tumor that occurs maintain normal cell function. Autophagy is significantly in the rectum at the junction of the sigmoid colon [1]. different from normal tissues in solid malignant tumor tis- The cancer usually develops in the fifth decade of life. sues, such as endometrial cancer and gastric cancer [4, 5]. Typically there are no obvious characteristics in the early Autophagy also affects the growth and metastasis of colon stage of colon cancer, with symptoms, such as blood in cancer [6]. the stool, developing later in the primary cancer and fol- Glucocorticoids (GCs), also known as adrenal cortex lowing metastasis [2]. At this time, the prognosis of the hormones, are a class of steroid hormones produced by the patient is unfavorable and the mortality is high. Exploring adrenal cortex [7]. GCs have diverse effects in the body. the development mechanism of colon cancer provides a It regulates the metabolism of fat, sugar, and protein, and theoretical basis for the development of clinical drugs [3]. is important in maintaining the balance of the environ- ment inside and outside the body [8]. GCs are widely used ∗Author to whom correspondence should be addressed. in clinical treatment because of their anti-inflammatory Email: [email protected] and anti-viral effects [9]. Autophagy is an important

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way to maintain homeostasis. During autophagy, damaged key role in tumor function and survival [20]. Autophagy organelles or cytoplasmic components are encapsulated by is one of the intrinsic functions affected by PD-L1 lig- autophagosomes of bilayer membranes and transported to ands. PD-1 is involved in T cell inhibition of glucose lysosomes for their degradation and reuse [10]. GCs can uptake and glycolysis by blocking CD28-mediated nutri- induce whitening of brown fat, a process that is regulated ent metabolism [21]. Due to nutritional starvation, glucose by autophagy [11]. depletion causes autophagy via the AMP-activated protein Glycolysis is the process in which glucose is converted kinase (AMPK) and mammalian rapamycin target protein to energy in the body. The three irreversible steps of (mTORC1) pathways [22]. In this study, dexamethasone glycolysis process are catalyzed by hexokinase, phospho- (DXM) was used to stimulate the expression of GR, to fructokinase, and pyruvate kinase, with the phosphofruc- explore the role and mechanism of GR in colon cancer. tokinase is the rate-limiting enzyme of glycolysis [12–14]. Under normal physiological conditions, low concentrations 2. MATERIALS AND METHODS of blood glucose stimulate the phosphorylation of pyru- 2.1. Promoter Analysis of the Autophagy-Related vate kinase in the liver, reducing its activity and reducing Factors Beclin 1 and LC3B the utilization of glucose in the liver [15]. Cortisol exerts Bioinformatics technology was used to mine data from its biological effects primarily by binding to the gluco- public databases to predict gene interactions and their corticoid receptor (GR) [16, 17]. Adrenocorticotropic hor- effects on cellular biology. In this study, the public mone inhibits glycolysis and reduces glucose utilization databases NCBI and ENSEMBL were used to query the by down-regulating the activity of phosphofructokinase in information of the target genes Beclin 1 (BECN1) and the liver [18]. Light chain 3B (LC3B). The Berkeley Drosophila Genome Autophagy can inhibit tumor cell growth and also pro- Project (BDGP), FPROM, TSSG, and TSSW online sites mote tumor survival. In the past decade, many targeted were used to predict the promoter regions and sequences anti-cancer therapies have been discovered and proven to of the promoter region. The AliBaba 2.1, GPMiner, and be effective in the treatment of hematological and solid PROMO online sites were used to predict the binding tumors [19]. The programmed death receptor-1 (PD-1)/ sites of the nuclear transcription factor GR at the promoter regions of the BECN1 and LC3B genes. programmed death ligand-1 signalingIP: 192.168.39.210 pathway plays On: a Fri, 01 Oct 2021 05:04:18 Copyright: American Scientific Publishers Delivered by Ingenta Table I. Bioinformatics analysis of promoter regions.

Mean Websites BECN1 LC3B Article Prediction of PubMed https://www.ncbi.nlm.nih.gov/gene/8678 https://www.ncbi.nlm.nih.gov/gene/81631 promoters ENSEMBL (1) http://asia.ensembl.org/Homo_sapiens/ (1) http://asia.ensembl.org/Homo_sapiens/ Location/View?db=core;g=ENSG00000126581; Location/View?db=core;g=ENSG00000140941; r=17:42810134-42833350 r=16:87383953-87404779 (2) (2) http://asia.ensembl.org/Homo_sapiens/ http://asia.ensembl.org/Homo_sapiens/ Location/View?db=core;g=ENSG00000126581; Location/View?db=core;g=ENSG00000140941; r=17:42822800-42825000 r=16:87380800-87394801 BDGP http://www.fruitfly.org/cgi-bin/seq_tools/ http://www.fruitfly.org/cgi-bin/seq_tools/ promoter.pl promoter.pl FPROM http://www.softberry.com/cgi-bin/programs/ http://www.softberry.com/cgi-bin/ promoter/fprom.pl programs/promoter/fprom.pl TSSG http://www.softberry.com/cgi-bin/programs/ http://www.softberry.com/cgi-bin/ promoter/tssg.pl programs/promoter/tssg.pl TSSW http://www.softberry.com/cgi-bin/programs/ http://www.softberry.com/cgi-bin/ promoter/tssw.pl programs/promoter/tssw.pl Prediction of AliBaba 2.1 http://gene-regulation.com/cgi-bin/pub/ http://gene-regulation.com/cgi-bin/pub/ transcription programs/alibaba2/webbaba2.cgi programs/ binding factors alibaba2/getmat.cgi?seg=2.1.1.1& file=seq_122.out&left=986&right=995&seq= seq_122#focus GPMiner http://gpminer.mbc.nctu.edu.tw/ http://gpminer.mbc.nctu.edu.tw/ show_prediction/show.php?OS=human&ID= show_prediction/show.php?OS=human&ID= 20190906_090545&scale=3&GC_window= 20190906_092407&scale=3&GC_window= 15&TFBS_core=1.00&TFBS_matrix=0.95& 15&TFBS_core=1.00&TFBS_matrix=0.95& OR_zscore=5&OR_number=20&OR_occur=2 OR_zscore=5&OR_number=20&OR_occur=2 &stability_window=15& &stability_window=15& miRNA_MFE=&miRNA_score= miRNA_MFE=&miRNA_score= PROMO http://alggen.lsi.upc.es/cgi-bin/promo_v3/ http://alggen.lsi.upc.es/cgi-bin/promo_v3/ promo/promoinit.cgi?dirDB=TF_8.3 promo/promoinit.cgi?dirDB=TF_8.3

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2.2. Cell Culture In vitro culture of LoVo human colon cancer cells was performed using Gib’s F-12K medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 Cin at atmosphere of 5% CO2. For the culture, LoVo cells stored at liquid nitrogen temperature were rapidly resus- citated at 37 C and centrifuged at 1000 rpm for 10 min. The cell pellet was resuspended in 2 mL of medium and transferred to a 15 mL centrifuge tube. Eight milliliters of medium were added and the suspensions were cultured in a fresh culture flask. Transfer to a 15 mL centrifuge tube and add 8 mL of medium to dilute. Routine culture in a new culture flask; after the cell adherence rate is above 80%, discard the medium, incubate the cells with 0.25% trypsin for 3 min in the incubator, resuspend the cells in 12 mL medium and re-suspend the cells in a new one. Routine culture in culture flasks. Fig. 2. Bioinformatics analysis of the BECN1 promoter region. The AliBaba 2.1 online website predicted that GR binds to the 1389–1398 bp promoter region of BECN1. 2.3. Enzyme-Linked Immunosorbent Assay (ELISA) GR expression levels were measured using an ELISA kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the supplied protocol. A spectrophotometer was used to determine the absorbance of each well at 450 nm, and Article

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Fig. 1. Bioinformatics analysis of the BECN1 promoter region. The Fig. 3. Bioinformatics analysis related of the LC3B promoter region. AliBaba 2.1 online website predicted that GR binds to the 211–220 bp The AliBaba 2.1 online website predicted that GR binds to the 986– and 1337–1346 bp promoter region of BECN1. 995 bp and 1083–1092 bp promoter region of LC3B.

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changes in the samples were monitored using an EVO 18 scanning electron microscope (Carl Zeiss Microscopy, GmbH, Oberkochen, Germany) at a high pressure of 10.0 kV. The sample was coated with a thinner gold ﬁlm prior to inspection [23, 24].

2.5. qRT-PCR RT-PCR was used to detect mRNA expression. The total RNA extracted by nano-magnetic beads was retrieved using the MagBeads Total RNA Extraction Kit (TIAN- GEN, Beijing, China). cDNA was synthesized by reverse transcription [25, 26] by pre-denaturation at 95 Cfor 15 min, deformation at 95 C for 5 s, and annealing at 60 C for 30 s for a total of 45 cycles. The speciﬁcity of the primers was determined by the dissolution curve of the PCR products, and the relative expression amount of mRNA was calculated as 2−Ct ∗ 100% [27, 28].

2.6. Western Blot Analysis Proteins were examined by western blotting by using mon- oclonal antibodies against GR, BECN1, and LC3B proteins (each 1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Glyceraldehyde 3-phosphate dehy- drogenase (GAPDH, 1:5000; Sigma-Aldrich, St. Louis, MO, USA) served as a loading control. The cells were IP: 192.168.39.210 On: Fri,incubated 01 Oct 2021 with 05:04:18 horseradish peroxidase (HRP)-labeled sec- Copyright: American Scientificondary antibodyPublishers (1:1000, Santa Cruz Biotechnology) for Delivered by1hat25 IngentaC. Quantiﬁcation of band density was performed using the Odyssey infrared imaging system (LICOR Bio- Fig. 4. science, Lincoln, NB, USA). Article Bioinformatics analysis related of the LC3B promoter region. The AliBaba 2.1 online website predicted that GR binds to the 1876– 1885 bp and 2001–2010 bp promoter region of LC3B. 2.7. Cell Glycolysis Cells were treated with the ATP Fluorescence Assay Kit the contents of each well were calculated using a standard (Roche Applied Science, Basel, Switzerland) according to curve. the operating manual. The ﬂuorescence value at 570 nm was measured with a microplate reader to calculate intra- 2.4. Scanning Electron Microscopy (SEM) cellular ATP levels. The intracellular L +-lactate level

Samples for SEM and a 100 ng/mL solution of Fe3O4 was measured using a lactate assay kit. The cells were sus- nanoparticles in ethanol were prepared. Morphological pended in the assay buffer containing lactate and incubated

Fig. 5. Elevation of glucocorticoid receptor (GR) expression by DXM. (A) The expression GR in different groups, compared with that in HCoEpiC cells. (B) The dose-response curve of DXM in LoVo cells in vitro. ∗∗∗P<0.001 in HCoEpiC cells; # < 0.05 in LoVo cells; ###P<0.001.

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2.8. Cell Viability LoVo and HCoEpiC cells were recovered at the loga- rithmic phase of growth and suspended in medium. The cell density was adjusted to 5 × 105 cells/mL and 100-L aliquots were added to wells of a 96-well plate. Three groups were set up with seven wells each, and the other wells contained cell-free medium. The plate was incubated for 12 hours at 37 Cina5%CO2 atmosphere. One group of LoVo cells was added to medium containing DXM and cultured for 24 hours. Then, 20 Lof 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bro- Fig. 6. Morphology and size of Fe O nanoparticles, as determined 3 4 mide (MTT) solution (5 mg/mL) was added to each well, by SEM. and the cells were incubated for 4 h. Then, 150 L of dimethyl sulfoxide was added for 10 min and the for 30 min. The amount of lactic acid produced was absorbance due to the formazan in solution was deter- quantiﬁed by measuring the absorbance at 570 nm using mined by spectrometry at 490 nm. a model 680 microplate reader (Bio-Rad, Hercules, CA, USA). For the glucose uptake assay, LoVo cells that had 2.9. Cell Migration been treated with DXM for 24 h were continuously cul- LoVo and HCoEpiC cells were diluted to 106 cells/mL in tured for 2 h in a medium containing 25 mM glucose. The medium, and 500 L of each cell suspension was added collection medium was used for a glucose oxidase assay to wells of 6-well plates. After 24 h of routine culture, using a commercial kit as described by the manufacturer. the cells were crossed with 200 L of vertical cells, and The glucose concentration was measured at a wavelength the old medium was discarded. The cells were washed of 450 nm. The glucose content of the experimental group three times with phosphate buffered saline. LoVo cells Article minus the glucose content of the untreated group repre- were added to serum-free medium containing DXM or sented the cell glucose reuptake value.IP: 192.168.39.210 On: Fri,drug- 01 Oct and 2021 serum-free 05:04:18 medium, and conventional culturing Copyright: American Scientific Publishers Delivered by Ingenta AB

Fig. 7. Effects of DXM-induced elevation of GR expression on the protein expression of autophagy markers in LoVo cells. (A)–(D) GR, BECN1, and LC3B protein expression detected by western blotting. ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001 versus HcoEpic group; #P<0.05, ##P<0.01 versus LoVo cells.

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was continued. After 24 h, the cells were observed by 3. RESULTS AND DISCUSSION microscopy. 3.1. GR Binds to Autophagy-Related Gene Promoter to Regulate Its Expression 2.10. Cell Apoptosis The findings of the bioinformatics analysis of promoter According to the instructions of Annexin V-FITC/PI apop- regions using the various website databases are shown in tosis detection kit (Beyotime, Shanghai, China), apopto- Table I. The autophagy-related factor BECN1 was located sis was detected. After 5 ∗ 105 cells/mL were mixed with in the c42824316-42810132 region of human chromo- 200 L of binding buffer, 10 L of annexin V-FITC and some 17. The AliBaba 2.1 online website predicted that 5 L of PI were added to incubate in dark for 15 min GR binds to the 211–220 bp, 1337–1346 bp, and 1389– respectively, and apoptosis was observed under fluores- 1398 bp stretches of the promoter region of BECN1 cence microscope. (Figs. 1 and 2). LC3B is located in the 87392336– 87404774 region of human chromosome 16. AliBaba 2.1 2.11. H&E Staining predicted four sites on the LC3B promoter for the binding The cells were fixed with 95% ethanol for 20 min, of the transcription factor GR: the 986–995 bp, 1083–1092 washed twice with PBS, soaked for 2 min with hema- bp, 1876–1885 bp, and 2001–2010 bp regions (Figs. 3 and 4). toxylin dye solution. After washing the excess dye in the cells with tap water, dye with eosin for 1 min, 3.2. DXM Promotes GR Expression and wash with tap water. After natural air drying, the To verify the GR expression-promoting effect of DXM, cells were sealed with neutral gum and observed under LoVo cells were treated with different concentrations of microscope. DXM and the expression of GR was detected by ELISA. At a concentration of 50 M, DXM promoted GR expres-

2.12. Statistical Analysis sion (Fig. 5(A)). The half-maximal response value (EC50) GraphPad 8.0 software (GraphPad, La Jolla, CA, was 27.688 mM (Fig. 5(B)). LoVo cells were subsequently USA) was used to analyze the data. All experiments treated with DXM at 27.688 mM. were repeated three times and the data are expressed as mean ± standard deviation.IP: ANOVA 192.168.39.210 was used On: to Fri,3.3. 01 Oct Morphological 2021 05:04:18 Observation of Fe3O4 by SEM determine whether marked differencesCopyright: existed American among ScientificMorphology Publishers of the Fe3O4 nanoparticles is very impor- the experimental groups. P<0.05 was regardedDelivered as bytant Ingenta for the efﬁcient extraction of nucleic acids. SEM of signiﬁcant. the nano-magnetic beads using a narrow electron beam Article AB

Fig. 8. Effects of DXM-induced elevation of GR expression on the mRNA expression of autophagy markers in LoVo cells. (A)-(C) GR, BECN1, and LC3B mRNA expression detected by qRT-PCR. ∗P<0.05 in HCoEpiC cells, ∗∗P<0.01, ∗∗∗P<0.001; #P<0.05 in LoVo cells.

368 Mater. Express, Vol. 10, 2020 Inhibitory effect of glucocorticoid receptor on colorectal cancer growth Materials Express Xu et al. revealed a uniform circular shape (diameter approximately LC3B (P<0.05 and P<0.05, respectively) in LoVo cells 400 nm) and uniform distribution of magnetic beads (Figs. 7 and 8). (Fig. 6). 3.5. GR Activation Inhibits Glycolysis 3.4. Activation of GR Promotes Autophagy in The ATP level, lactic acid accumulation, and glucose con- Colon Cancer Cells sumption were determined in LoVo colon cancer cells after To further verify whether GR binds to the BECN1 and DXM intervention (Fig. 9). Compared with normal cells, LC3B promoters to regulate their expression, the GR significant increases were evident in the colon cancer cells agonist DXM (27.688 mM, 24 h) was used to treat for the ATP levels (P<0.01), glucose uptake (P<0.01), LoVo human colon cancer cells; HCoEpiC human normal and lactic acid accumulation (P<0.05). DXM interven- colonic epithelial cells were used as the untreated con- tion resulted in decreases in glycolysis, ATP (P<0.001), trols, and drug-free LoVo cells were used as the experi- glucose uptake (P<0.05), and lactic acid accumulation mental controls. The mRNA and protein expression of GR, (P<0.001). BECN1, and LC3B were detected by qRT-PCR and western blotting, respectively. Protein and mRNA expression 3.6. Activation of GR Promotes Apoptosis in of GR in the LoVo colon cancer cells was downregu- Colon Cancer Cells lated compared with the HCoEpiC normal cells (P< Flow cytometry revealed significantly lower rate of apop- 0.001 and P<0.05, respectively). Significant decreases tosis of colon cancer cells compared to that of normal were also evident for the protein and mRNA expres- cells. DXM treatment significantly promoted apoptosis in sion of the autophagy-related genes BECN1 (P<0.05 LoVo cells (Fig. 10). and P<0.01, respectively) and LC3B (P<0.01 and P<0.001, respectively). Compared with the experimental 3.7. Activation of GR Inhibits Colon Cancer group, DXM treatment significantly promoted protein and Cell Growth and Metastasis mRNA expression of GR (P<0.05 and P<0.05, respec- DXM significantly inhibited the growth of malignant Article tively), BECN1 (P<0.01 and P<0.05, respectively), and tumors. Colon cancer cells proliferated significantly IP: 192.168.39.210 On: Fri, 01 Oct 2021 05:04:18 Copyright: American Scientific Publishers ABDelivered by Ingenta

Fig. 9. Effect of DXM-induced elevation of GR expression on glycolysis of LoVo cells. (A) DXM activates GR to promote ATP production in LoVo cells. (B) The effect of DXM-induced elevation of GR expression on glucose uptake in LoVo cells. (C) DXM-induced elevation of GR expression inhibits lactate accumulation in LoVo cells. ∗∗P<0.01 in HCoEpiC cells, ∗∗∗P<0.001; #P<0.05 in LoVo cells, ###P<0.001.

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Fig. 10. Effect of altered GR expression on apoptosis of LoVo cells. (A) and (B), The apoptosis results of HCoEpiC detected by flow cytometry; (C) and (D), the apoptosis results of LoVo cells detected by flow cytometry; (E) and (F), the apoptosis results of DXM detected by flow cytometry.

compared with normal cells (P<0.001), while DXM 3.8. Morphological Changes of Colon Cancer signiﬁcantly decreased the proliferation of LoVo cells (P< Cells After GR Activation 0.001) (Fig. 11(A)). The cell scratch assay was used After H & E staining, cell bodies were observed under microscope. Under normal conditions, HCoEpiC cells are to detect the effect of DXM activation on the migra- endothelial like cells with spindle shape. LoVo cells were tion of LoVo cells. GR expression was inversely pro- densely distributed in spindle shape, with a large number portional to cell migration ability, and was signiﬁcantly of cells and a full body. After DXM treatment, LoVo cells different from that of untreated LoVo cells (P<0.001, were sparsely distributed, mainly in spindle shape, the cells Fig. 11(B)). were oval in shape, the antennae were not obvious, and the

370 Mater. Express, Vol. 10, 2020 Inhibitory effect of glucocorticoid receptor on colorectal cancer growth Materials Express Xu et al.

Fig. 11. Effect of GR expression on proliferation and migration of LoVo cells. (A) The effect of differential GR expression on LoVo cell viability, as detected by MTT assay. (B) The effect of differential GR expression on LoVo cell migration, as detected by the cell scratch assay. ∗∗∗P<0.001 in HCoEpiC cells; ###P<0.001 in LoVo cells. cell body became smaller, the number of cells decreased The LC3B gene is indispensable in the formation of (Fig. 12). autophagosome membranes. he contents of the model is positively correlated with the degree of autophagy, so it is 3.9. Discussion often used to measure autophagy activity [31]. Presently, to GR is a nuclear transcription factor. Its role and the mech- further verify whether GR binds to the BECN1 and LC3B anism underlying its involvement in neuropathic pain is promoters to regulate the expression of both genes, colon controversial. Herein, DXM was used to stimulate the cancer cells were treated with the GR agonist DXM. Pro- expression of GR, and the expression of the autophagy tein and mRNA expression of GR, BECN1, and LC3B in Article markers BECN1 and LC3B were detected by qRT-PCR colon cancer cells were downregulated compared to that in and western blotting. To assess theIP:effect 192.168.39.210 of the differen- On: Fri,normal 01 Oct cells. 2021 DXM 05:04:18 treatment significantly promoted pro- tial expression of GR on the autophagyCopyright: of colon American cancer Scientifictein and mRNAPublishers expression of GR, BECN1, and LC3B in Delivered by Ingenta cells, the levels of ATP, glucose, and lactic acid accumu- LoVo cells. lation were determined. The effects of GR on the glycoly- The degree of glycolysis is related to cell type and sis, apoptosis, proliferation, and migration of colon cancer growth status. Some tumor cells synthesize ATP by gly- cells were determined using an intake assay, flow cytome- colytic hydrolysis to account for 60% of the total [32]. try, MTT assay, and cell scratch assay, respectively. Glycolysis is a relatively inefficient process of produc- GCs are steroid hormones produced by the adrenal cor- tivity [33]. One molecule of glucose produces 38 ATP tex [7] exert their biological functions via their action with molecules through the aerobic metabolic pathway, while the GR [29]. To verify the GR-promoting effect of DXM, one molecule of glucose produces two molecular ATPs LoVo cells were treated with different concentrations of through the glycolytic pathway [34]. Therefore, even if DXM and the expression of GR was detected by ELISA. mitochondrial oxidative phosphorylation is slightly sup- DXM (50 M) promoted GR expression. pressed, a significant increase in glycolysis is needed to Among the many known autophagy markers, BECN1 provide enough energy. In the present study, ATP levels, stimulates autophagy and inhibits tumor cell growth and glucose uptake, lactic acid accumulation and glycolysis can induce death by regulating autophagy levels [30]. increased in the presence of DXM.

ABC

0.5 µm 0.5 µm 0.5 µm

Fig. 12. HE staining results of cells in each group. (A), Morphology of HCoEpiC cells under microscope; (B), morphology of LoVo cells under microscope; (C), morphology of LoVO cells in DXM group under microscope.

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Received: 7 December 2019. Accepted: 8 December 2019. Article

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