Open Access

CD38 affects the biological behavior and energy metabolism of nasopharyngeal carcinoma cells

  • Authors:
    • Yanshan Ge
    • Yuehua Long
    • Songshu Xiao
    • Lin Liang
    • Zhengxi He
    • Chunxue Yue
    • Xiong Wei
    • Yanhong Zhou
  • View Affiliations

  • Published online on: December 3, 2018     https://doi.org/10.3892/ijo.2018.4651
  • Pages: 585-599
  • Copyright: © Ge et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Nasopharyngeal carcinoma (NPC) is the most common malignant tumor type in Southern China and South‑East Asia. Cluster of differentiation (CD)38 is highly expressed in the human immune system and participates in the activation of T, natural killer and plasma cells mediated by CD2 and CD3 through synergistic action. CD38 is a type II transmembrane glycoprotein, which was observed to mediate diverse activities, including signal transduction, cell adhesion and cyclic ADP‑ribose synthesis. However, the significance of CD38 in NPC biological behavior and cellular energy metabolism has not been examined. In order to elucidate the effect of CD38 on the biological behavior of NPC cells, stable CD38‑overexpressed NPC cell lines were established. It was demonstrated that CD38 promoted NPC cell proliferation with Cell Counting Kit‑8 and colony formation assays. It was also indicated that CD38 inhibited cell senescence, and promoted cell metastasis. Furthermore, it was determined that CD38 promoted the conversion of cells to the S phase and decreased the content of reactive oxygen species and Ca2+. Additionally, cell metabolism assays demonstrated that CD38 increased the concentration of ATP, lactic acid, cyclic adenosine monophosphate and human ADP/acrp30 concentration in NPC cells. To investigate the possible mechanism, bioinformatics analysis and mass spectrometry technology was used to determine the most notably changing molecule and signaling pathways, and it was determined and verified that CD38 regulated the metabolic‑associated signaling pathways associated with tumor protein 53, hypoxia inducible factor‑1α and sirtuin 1. The present results indicated that CD38 may serve a carcinogenic role in NPC by regulating metabolic‑associated signaling pathways.

Introduction

Nasopharyngeal carcinoma (NPC) refers to malignant tumors that occur on the top and sidewall of the nasopharyngeal cavity (1,2). NPC is one of the most prevalent malignant tumor types in China since 2002-2014 (3,4). Risk factors for NPC include Epstein-Barr virus infection (5), genetic factors, intake of salted food and cigarette smoking (6-9). Female patients accounted for ~30% of cases from 1983-2008 in Hong Kong, indicating that NPC is more common in males (10,11). Currently, radiation therapy is the preferred method used in the treatment of NPC, due to the majority of the NPC cases being poorly differentiated carcinomas (12). The treatment methods of NPC also include surgical treatment and chemotherapy, to improve patient survival rate (13-16).

Nutritional competition among cells can affect the growth, survival and functions. Different cells have different nutrient intake conditions and energy metabolism phenotypes. Therefore, the state of cells is associated with energy metabolism (17). Cluster of differentiation (CD)38 is located on chromosome 4 and was originally observed as a lymphocyte surface antigen. The molecular weight is 45 kDa (18). A number of studies demonstrated that CD38 was a transmembrane protein with receptors, extracellular enzymes and other functions (19-21). CD38 is primarily considered as a Type II membrane protein with a small N-terminal tail protruding into the cell and a catalytically active C-terminus located extracellularly (22). CD38 is not only expressed in T lymphocytes, but also expressed in other immune cells, including plasma cells, natural killer cells, monocytes and dendritic cells (23). CD38 is an important target in the treatment of multiple myeloma (24). In particular, the CD38 targeted therapies exhibit single-agent activity in early-phase clinical trials (25-27).

Although numerous studies have been conducted on CD38, there is relatively limited research regarding the energy metabolism of CD38 in NPC (28). The purpose of the present study was to determine the expression level of CD38 in NPC, investigate the role of CD38 in the biological behavior and cell metabolism of NPC cells, and to investigate the possible mechanisms.

Materials and methods

Cell culture and transfection

Human NPC cells, 5-8F cells, were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were grown at 37°C in an atmosphere containing 5% CO2. Transfection steps were according to the manufacturer's protocols of the lentiviral transfection reagent (Shanghai GenePharma, Co., Ltd., Shanghai, China). Cell transfection was accomplished by using Lipofectamine® 2000, according to the manufacturer's instruction (Invitrogen; Thermo Fisher Scientific, Inc.). Firstly, 2×105 cells were seeded into each well of a 6-well plate 24 h prior to transfection. For each transfection, 2 µg pEGFP-N1-CD38 plasmid or pEGFP-N1 vector plasmid (Sangon Biotech Co., Ltd., Shanghai, China) was transfected into 5-8F cells. The plasmids were diluted with 100 µl RPMI-1640 and 4 µl Lipofectamine 2000 was added into 100 µl RPMI-1640. The two solutions were combined, mixed gently and incubated at room temperature for 30 min. Subsequently, the 200 µl mixture and 200 µl RPMI-1640 were added into each well. The cells were then incubated at 37°C for 24 h, followed by replacing the transfection media with RPMI-1640 with 10% fetal bovine serum. After an additional 48 h culture, the cells were harvested for the following western-blot experiments. The level of CD38 protein expression was detected by western blot analysis, according to the subsequent protocols, and two replicates were conducted for western blot analysis, to confirm whether the stable cell line was constructed successfully. The primary antibody used was mouse anti-CD38 (ImmunoWay Biotechnology Company, Plano, TX, USA; 1:1,000 dilution; cat. no. YM0122) at 4°C overnight. The secondary antibody used was anti-mouse horseradish peroxidase (HRP)-conjugated antibody at 37°C for 1 h (LiankeBio, Hangzhou, China; 1:5,000 dilution; cat. no. GAM007).

Ethical statement

The present study was approved by the Ethics Committee of Hunan Provincial Tumor Hospital (Changsha, China) and conducted in accordance with the Declaration of Helsinki. Prior to the start of the study, all participants provided written informed consent.

Patient samples

A total of 20 NPC specimens and 17 non-tumor NPC epithelial tissues were collected from the Cancer Hospital of Hunan Province, Central South University (Changsha, China). The mean age of the patients was 45±5.5 years, including 28 males and 9 females. Biopsy samples were collected from March 17, 2017 to May 17, 2017. The non-cancerous nasopharyngeal tissues were collected from independent patients with chronic inflammation of nasopharyngeal mucosa or polyps.

Immunohistochemistry

For immunohistochemistry, paraffin-embedded specimens were sliced to 4-µm thickness and fixed with 4% paraformaldehyde for 24 h at 4°C. Immunohistochemistry was conducted using the peroxidase anti-peroxidase technique (29) following a microwave antigen retrieval procedure, which included incubating twice in 100% analytical reagent (AR) ethanol for 5 min, followed by 95% AR ethanol for 5 min, 75% AR ethanol for 5 min, 50% AR ethanol for 5 min and then in distilled water for 5 min at room temperature, and then washed in 1X PBS for 3 min twice at room temperature. Antibody for CD38 was purchased from Immunoway Biotechnology Company. The antibody against CD38 (cat. no. YM0122; Immunoway Biotechnology Company; 1:1,000 dilution) was overlaid on NPC and non-tumor nasopharyngeal epithelial tissues sections and incubated overnight at 4°C. Anti-mouse HRP-conjugated secondary antibody (dilution 1:5,000; cat. no. GAM007; LiankeBio) was performed at room temperature for 30 min. Color reaction was developed with 3,3'-diaminobenzidine tetrachloride chromogen solution. All slides were counterstained with hematoxylin for 3 min at room temperature.

Sections were blindly evaluated by two investigators (Department of Pathology, the First Xiangya Hospital, Central South University, Changsha, China) in an effort to provide a consensus on staining patterns by optical microscopy at ×40 and ×200 magnification (Olympus Corporation, Tokyo, Japan). CD38 staining was assessed according to the methods described by Hara and Okayasu (30) with minor modifications. Each case was rated according to a score that added a scale of intensity of staining to the area of staining. At least 10 high-power fields were selected randomly, and >1×103 cells were counted for each section. The intensity of staining was graded on the following scale: 0, no staining; 1+, mild staining; 2+, moderate staining; and 3+, intense staining. The area of staining was evaluated as follows: 0, no staining of cells in any microscopic fields; 1+, <30% of tissue stained positive; 2+, 30-60% stained positive; and 3+, >60% stained positive. The minimum score when calculated was 0 and the maximum was 6. A combined staining score of ≤2 was considered to be negative staining (low staining), a score of 3-4 was considered to be moderate staining, whereas, a score of 5-6 was considered to be strong staining. An optimal cut-off level was identified as follows: A staining index score of 0-2 was used to define tumors with negative expression and 3-7 indicated positive expression of these two proteins. Agreement between the two evaluators was 95%, and all scoring discrepancies were resolved through discussion between the two evaluators.

Cell proliferation and colony formation assay

The cells were counted and seeded in 96-well plates in a total amount of 100 µl RPMI-1640 supplemented with 10% fetal bovine serum at 1×103 cells/well. In accordance with each 100 µl RPMI-1540 supplemented with 10% fetal bovine serum by adding 10 µl Cell Counting Kit-8 (CCK-8) detection reagent (Shanghai Qibao Xintai Biotechnology Co., Ltd., Shanghai, China; cat. no. C008-3), and incubated at 37°C for 1-7 days. Absorbance was measured at a wavelength of 450 nm with a microplate reader (Beckman Coulter, Inc., Brea, CA, USA). For the colony formation assay, the cells were digested and then were counted at ×100 magnification with a light microscope. A total of 8×102 cells were planted in each well of a 6-well plate, cultured in a CO2 incubator for 14 days at 37°C. Cells were fixed with 4% paraformaldehyde solution for 15 min at room temperature and stained with 0.1% crystal violet solution for 20 min at room temperature, and the number of clones was counted at ×40 magnification with a light microscope.

Scratch assay and Transwell

The cells (2.5×105) were seeded in 6-well plates and allowed to grow at 37°C in an atmosphere containing 5% CO2 for 24 h. Subsequently, the cells were scratched with a 10 µl pipette tip and an image was captured with a light microscope every 12 h at ×100 magnification. The RPMI-1640 was changed every 12 h at 37°C and images were captured in situ until the cell scratch healed. For the Transwell assay, cell counts were performed following digestion of the cells with 0.25% 1X Trypsin EDTA (Gibco; Thermo Fisher Scientific, Inc.). Transwell chambers were used to hold inserts containing cultured cells (Transwell chamber; 8-mm pore size; Costar, High Wycombe, UK). A total of 2×104 cells were seeded in each chamber, serum-free RPMI-1640 was added to the upper chamber, and RPMI-1640 culture medium containing 600 µl 10% fetal bovine serum was added to the lower chamber. When cells were visible in 24 plates for 48 h at 37°C, the culturing was stopped and the cells were washed 3 times with PBS. Cells were fixed with 4% paraformaldehyde solution for 10 min and room temperature and stained with 0.1% crystal violet solution for 10 min at room temperature. The cells were imaged with a light microscope at ×400 magnification.

β-galactosidase staining

The detection of cellular senescence was performed using a Senescence-associated β-Galactosidase Staining kit (Beyotime Institute of Biotechnology, Haimen, China), according to the manufacturer's protocol. In a 6-well plate (1.2×106), RPMI-1640 supplemented with 10% fetal bovine serum was aspirated and the cells were washed once with PBS, and then 1 ml β-galactosidase staining fixative from a Senescence-associated β-Galactosidase Staining kit was added for 15 min at room temperature. Subsequently, the β-galactosidase staining fixative was aspirated, the cells were washed 3 times with PBS for 3 min each time. Following this, PBS was removed and 1 ml stain solution was added to each well. Finally, the cells were incubated overnight at 37°C. The staining was observed with a light microscope at ×200 magnification, the β-galactosidase positive cells (blue) were considered senescent.

Flow cytometry of cell cycle and apoptosis

Cells were harvested with 0.5% trypsin and centrifuged at 1,000 × g for 5 min at room temperature. Cell cycle analysis was performed using a cell cycle staining kit (LiankeBio; cat. no. CCS012), cells were fixed in ice-cold 70% ethanol overnight at 4°C. Following being washed thrice with cold PBS, cells were suspended in 500 µl PBS, and 10 µl RNase A was added for 5 min at room temperature. Subsequently, 10 µl propidium iodide (PI; LiankeBio; cat. no. CCS012) was added into the cell resuspension solution, and incubated for 30 min at 4°C. The cells were finally washed twice with PBS prior to analysis. As for apoptotic analysis, it was performed using a Hoechst 33342/PI Apoptosis Assay kit (Shanghai BestBio Biotechnology, Shanghai, China), according to the manufacturer's protocols. The samples were washed twice with ice-cold PBS and suspended in 500 µl staining buffer, and then incubated with 5 µl Hoechst 33342 and 5 µl PI in the dark for 20 min at 4°C. The cells were finally washed twice with PBS prior to analysis. Analysis was performed on a MoFlo™ XDP High-Performance Cell Sorter and the data were analyzed with Summit v5.2 Software (both from Beckman Coulter, Inc.).

Flow cytometry analysis of ROS and Ca2+ concentration

Cells were digested with 0.25% 1X Trypsin EDTA and cultured in RPMI-1640, and 5 µl 2,7-dichlorofluorescin diacetate (DCFH-DA) from a ROS kit (Applygen Technologies, Inc., Beijing, China) was added to the test tube. The blank group contained no reagents, whereas 5 µl DCFH-DA and 5 µl reactive oxygen donor was added to the positive control group and incubated in a constant temperature water bath at 37°C for 20 min. Subsequently, cells were washed with 1X PBS, and then resuspended in 500 µl 1X PBS. ROS was detected with a flow cytometer (Beckman Coulter, Inc.). For the Ca2+ concentration, following digestion of the cells with 0.25% 1X Tryspin EDTA, the cells were resuspended with the culture medium. A total of 1 µl Fluorescein-2 AM Ca2+ fluorescent probe from a Ca2+ analysis kit (S1056; Beyotime Institute of Biotechnology) was added to each tube of the sample to be tested. Subsequently, 1 µl dimethyl sulfoxide solution was added into the cell suspension of the control group, and then the tube was inverted and mixed thoroughly every 3-5 min to allow full contact between the cells and the Ca2+ fluorescence probe. The cells were then washed 2-3 times with 1X PBS and the cells were resuspended by adding 500 µl 1X PBS. The Ca2+ concentration was detected with a flow cytometer (Beckman Coulter, Inc.). The data were analyzed with Summit v5.2 Software (Beckman Coulter, Inc.).

ATP and lactic acid (HL) detection

The detection of ATP content was performed using an ATP content test kit (S0026; Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Relative light unit values were determined using a Luminometer. A standard curve was drawn and the ATP concentration of the sample to be tested was calculated. The measured concentration of ATP is divided by the concentration of the protein and the unit is finally converted to nmol/mg protein. The detection of HL content was performed using a HL content test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer's protocol.

Cyclic AMP (cAMP) and adiponectin (ADP/acrp30) tests

A cAMP test kit (cat. no. E-EL-0056c; Elabscience, Wuhan, China) was used according to the manufacturer's protocols. The optical density (OD) of each well was measured immediately with a microplate reader at a wavelength of 450 nm. A standard curve was drawn and the solubility was calculated from the curve. An ADP/acrp30 test kit (cat. no. E-EL-H0004c; Elabscience) was used according to the manufacturer's protocol. The OD of each well was measured immediately with a microplate reader at a wavelength of 450 nm. A standard curve was drawn and the concentration was calculated from the curve.

NADP+/NADPH assay

For the extraction of NADP+, 1 ml acidic extract (Nanjing Jiancheng Bioengineering Institute) was added to 5×106 cells, and then the cells were sonicated for 1 min, boiled 5 min at 100°C, cooled in an ice bath at 4°C for 5 min and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was extracted and an equal volume of alkaline extract (Nanjing Jiancheng Bioengineering Institute) was added to neutralize the cells. The cells were then mixed well at 10,000 × g for 10 min at 4°C. Subsequently, reagent one to seven reagents (Nanjing Jiancheng Bioengineering Institute) was added and mixed well. Absorbance was measured at a wavelength of 570 nm and an optical path of 1 cm. Following this, the NADP+ content was calculated according to the formula: NADP+ = 4.57 × (ΔA-0.062)/Cpr, ΔA = A2-A1, where Cpr represents the concentration of protein, A1 represents the absorbance of the control group and A2 represents the absorbance of the experimental group. For the extraction of NADPH, 1 ml alkaline extract was added to 5×106 cells, and then the cells were sonicated for 1 min, boiled for 5 min at 100°C, cooled in an ice bath at 4°C for 5 min and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was extracted and an equal volume of acidic extract was added to neutralize the cells. The cells were then mixed well at 10,000 × g for 10 min at 4°C. Subsequently, reagent one to seven reagents was added and mixed well. Absorbance was measured at a wavelength of 570 nm and an optical path of 1 cm. Following this, the NADPH content was calculated according to the formula: NADPH = 7.2 × (ΔA-0.072)/Cpr, ΔA = A2-A1, where Cpr represents the concentration of protein, A1 represents the absorbance of the control group and A2 represents the absorbance of the experimental group.

Bioinformatics analysis

The NPC-associated dataset GSE13597 (31) based on GPL96 (HG-U133A) Affymetrix Human Genome U133A Array (32) was downloaded from the Gene Expression Omnibus (GEO) database (www.ncbi.nlm.nih.giv/geo/), including 25 NPC and 3 normal samples. The Empirical Bayes method (33) was used to select significant differentially expressed genes (DEGs) based on the limma (http://www.biocon-ductor.org/packages/release/bioc/html/limma.html) package of Bioconductor v3.6 (http://www.bioconductor.org/biocLite.R). In the volcano map, the DEGs with a fold change of >2 were selected. Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.kegg.jp/) enrichment analysis of DEGs was performed based on R software v3.4.1 (https://www.r-project.org/). Finally, Cytoscape software v3.6.0 (https://cytoscape.org/) was used to build protein-protein interaction (PPI) networks and module analyzes to determine the key genes.

Liquid chromatography-mass spectrometry (LC-MS)/MS analysis

A total of 5×105 cells were washed twice with PBS, and then 150 µl radioimmunoprecipitation assay (RIPA) buffer (CWBIO, Beijing, China) was added. Following being lysed at 4°C for 1 h, the cells were disrupted by sonication for 10 min. Subsequently, the cells were centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was collected. Following this, the Coomassie Brilliant Blue method (34) was used for determination of protein concentration. Subsequently, the proteins were separated by western blot analysis. SDS-PAGE was stained with Coomassie Brilliant Blue on a shaker for 1 h at room temperature. The Coomassie blue-stained protein strip was cut into 1 mm3 size pellets, collected in Eppendorf® (EP) tubes, and washed three times with ultrapure water. Subsequently, 0.5 ml decolorizing solution without gelatinous particles was added, decolorized at 37°C and the decolorizing solution was replaced once, until it became colorless and transparent, and was washed 3 times with 25 mM NH4HCO3. Additionally, 0.5 ml 100% ACN was added, the cells were dried at 37°C for 5 min, and then 250 µl 25 mM dithiothreitol (DTT) and 25 mM NH4HCO3 was added. Following this, the cells were incubated in a water bath at 56°C for 1 h, and then the supernatant was aspirated. Immediately after cooling to room temperature for 3 min, 250 µl protein protection solution (55 mM IAA and 25 mM NH4HCO3) was added. The reaction was allowed to stand at room temperature for 30 min in the dark, and the supernatant was aspirated. Subsequently, 250 µl 25 mM DTT and 25 mM NH4HCO3 was added and allowed to react at room temperature for 30 min to remove excess IAA. Subsequently, washed 3 times with 0.5 ml 25 mM NH4HCO3. Following this, the cells were dehydrated twice with 0.5 ml 100% ACN, dried at 37°C for 5 min and then washed once with 0.5 ml 25 mM NH4HCO3. After, the cells were dehydrated 2 times with 0.5 ml 100% ACN, dried at 37°C for 5 min, and then 0.5 ml trypsin was added and incubated for 30 min at 4°C. Following this, 0.5 ml 25 mM NH4HCO3 was added and reacted at 37°C for 18 h. Subsequently, the protein was centrifuged at 10,000 × g for 5 min at 4°C, the enzymatic solution was aspirated into a new EP tube and 100 µl extract (0.1% TFA and 70% ACN) was added to the original EP tube. The tube was then incubated in a 37°C constant temperature water bath for 30 min, and shaken at room temperature for 15 min with an oscillator, underwent ultrasonication for 15 min, and the extract and the enzymatic solution were combined in a new EP tube. The ZipTip C18 column was desalted and concentrated to ~3 µl in a freeze concentrator, and then 20 µl 0.1% FA was added. Subsequently, Nano-LC LTQ Orbitrap ETD Mass Spectrometry was performed.

Western blot analysis

Total cellular protein was extracted with RIPA buffer, and then the bicinchoninic assay method was used for determination of protein concentration (Thermo Fisher Scientific, Inc.). A total of 50 µg sample protein volume mixed with 5X loading buffer (cat. no. WB2001; Suzhou New Secco Biotechnology Co., Ltd., Suzhou, China) was used and boiled in water for 5 min at 100°C following loading. Proteins were separated in 10% SDS-PAGE gels and electroblotted onto nitrocellulose membranes (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA). The membranes were blocked using Tris-buffered Tween-20 (25 mM Tris-HCl, 150 mM NaCl, pH 7.5 and 0.05% Tween-20) containing 5% non-fat milk at 4°C overnight followed by overnight incubation at 4°C with primary antibodies: Mouse anti-CD38 (Immunoway Biotechnology Company; 1:1,000 dilution; cat. no. YM0122); mouse anti-cyclin dependent kinase 4 (CDK4; Wuhan Boster Biological Technology, Ltd., Wuhan, China; 1:500 dilution; cat. no. BM1572), rabbit anti-c-Jun N-terminal kinase 2 (Immunoway Biotechnology Company; 1:1,000 dilution; cat. no. YM3721), rabbit anti-B-cell lymphoma-2 (Bcl-2; Immunoway Biotechnology Company; 1:1,000 dilution; cat. no. YT0427), rabbit anti-signal transducer and activator of transcription 1 (STAT1; Immunoway Biotechnology Company; 1:1,000 dilution; cat. no. YT6124), rabbit anti-extracellular signal-regulated kinase (ERK; Immunoway Biotechnology Company; 1:500 dilution; cat. no. YT1622), mouse antitumor protein 53 (TP53; Wuhan Boster Biological Technology, Ltd.; 1:500 dilution; cat. no. BM0101), rabbit anti-Ezrin (Immunoway Biotechnology Company; 1:1,000 dilution; cat. no. YT1651), rabbit anti-cyclin D1 (Wuhan Boster Biological Technology, Ltd.; 1:500 dilution; cat. no. BM4272), rabbit anti-integrin subunit α5 (ITGA5; Wuhan Boster Biological Technology, Ltd.; 1:500 dilution; cat. no. BA1641-2), rabbit anti-AKT1 (Wuhan Boster Biological Technology, Ltd.; 1:500 dilution; cat. no. A00024), rabbit anti-GAPDH (Wuhan Boster Biological Technology, Ltd.; 1:1,000 dilution; cat. no. A00227), rabbit anti-cluster of differentiation 24 (CD24; Immunoway Biotechnology Company; 1:1,000 dilution; cat. no. YT0741), rabbit anti-glucose transporter 1 (GLUT1; Immunoway Biotechnology Company; 1:1,000 dilution; cat. no. YT1928), rabbit anti-GLUT4 (Immunoway Biotechnology Company; 1:1,000 dilution; cat. no. YT5523), rabbit antitumor protein TP53 induced glycolysis regulatory phosphatase (Immunoway Biotechnology Company; 1:1,000 dilution; cat. no. YN2999), rabbit anti-succinate dehydrogenase complex flavoprotein subunit A (SDHA; Immunoway Biotechnology Company; 1:2,000 dilution; cat. no. YT4226), rabbit anti-dihydrolipoamide dehydrogenase (DLD; Wuhan Boster Biological Technology, Ltd.; 1:500 dilution; cat. no. PB0608), rabbit anti-ribonucleotide reductase regulatory subunit M2 (RRM2; Wuhan Boster Biological Technology, Ltd.; 1:300 dilution; cat. no. BM5490), rabbit anti-ATP synthase peripheral stalk subunit D (ATP5H; Wuhan Boster Biological Technology, Ltd.; 1:500 dilution; cat. no. PB0281), rabbit anti-ATP synthase F1 (ATP5F1; Wuhan Boster Biological Technology, Ltd.; 1:1,000 dilution; cat. no. A10633-1), rabbit anti-hypoxia inducible factor (HIF)-1α (Wuhan Boster Biological Technology, Ltd.; 1:500 dilution; cat. no. BA0912-2), rabbit anti-sirtuin (SIRT1; Immunoway Biotechnology Company; 1:1,000 dilution; cat. no. YT4302), rabbit anti-SIRT2 (Immunoway Biotechnology Company; 1:1,000 dilution; cat. no. YT4303) and mouse anti-SIRT6 (Immunoway Biotechnology Company; 1:500 dilution; cat. no. YM1274). The membranes were incubated with anti-rabbit or anti-mouse HRP-conjugated secondary antibody at 37°C for 1 h (dilution 1:5,000; cat. nos. GAR0072 and GAM007, respectively; LiankeBio). Specific signals were visualized using an enhanced chemiluminescent detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Rabbit anti-β-tubulin was used as a loading control. The controls used were mouse anti-β-tubulin (Immunoway Biotechnology Company; 1:5,000 dilution; cat. no. YM3030) and rabbit anti-β-tubulin (Affinity Biosciences. Cincinnati, OH, USA; 1:5,000 dilution; cat. no. AF7011)

Statistical analysis

The association between the expression of CD38 and clinicopathological characteristics in NPC were tested using a χ2 test. The differences between the groups were analyzed using an unpaired Student's t-test when there were only two groups. All statistical analyses were performed using SPSS software (SPSS, Inc., Chicago, IL, USA). Some charts were produced with GraphPad Prism v5.01 (GraphPad Software, Inc., La Jolla, CA, USA). The error bars represent the mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference.

Results

CD38 is highly expressed in NPC

To detect the protein expression levels of the CD38 molecular in NPC and non-tumor nasopharyngeal epithelial tissues, 20 NPC and 17 non-tumor nasopharyngeal epithelial tissues were collected. Immunohistochemistry was performed to detect the expression of CD38. The results demonstrated that 7 samples were negative, 3 samples were weak positive, 5 samples were positive and the other 5 samples were strong positive in NPC tissues. In the control group, 14 samples were negative and 3 samples were weak positive. Of the samples, 65.0% (13/20) had a positive expression of CD38 in NPC tissues and 17.6% (3/17) had a positive expression in the adjacent non-cancerous tissues. Whereas, negative expression was exhibited in 35.0% (7/20) and 82.4% (14/17) of NPC and the adjacent non-cancerous tissues, respectively (P<0.01). The present results demonstrated that CD38 was highly expressed in NPC (Fig. 1A and Table I).

Table I

The difference in cluster of differentiation 38 expression between NPC and non-tumor nasopharyngeal epithelial tissues by immunohistochemistry.

Table I

The difference in cluster of differentiation 38 expression between NPC and non-tumor nasopharyngeal epithelial tissues by immunohistochemistry.

SamplesNegative (−)Weak positive (+)Positive (++)Strong positive (+++)χ2P-value
NPC7 (35%)3 (15%)5 (25%)5 (25%)12.170.0068
Non-tumor nasopharyngeal epithelial tissues14 (82.35%)3 (17.65%)00

[i] NPC, nasopharyngeal carcinoma.

CD38 promotes the proliferation of NPC cells

The immunohistochemistry results indicated that CD38 was highly expressed in NPC. Combined with our previous results, CD38 is a putative functional marker of side population (SP) cells in human NPC cell lines (35). It was speculated that CD38 served an important role in the pathogenesis of NPC. Thus, CD38-overexpressed-5-8F stable cell lines (5-8F/CD38; Fig. 1B) were successfully constructed. There are no difference between the 2 5-8F/CD38 columns in part B, and there are no difference between the 2 5-8F/Vector columns in part B. There is only one clone, and WB made 2 replicates. To clarify the effect of CD38 on the biological behavior of NPC cells, the effect of CD38 on the proliferation of 5-8F cells was examined using a CCK-8 assay. The results demonstrated that CD38 promoted the proliferation of 5-8F cells (Fig. 1C). To further confirm that CD38 promotes the proliferation of NPC cells, a colony formation assay was performed. The results indicated that the high expression of CD38 clone number is increased, compared with the control group (Fig. 1D and E). These results demonstrate that CD38 promoted the proliferation of NPC cells.

CD38 not only promotes the migration of NPC cells, but also reduces the cell senescence of NPC cells

The migration of tumor cells serves an important role in tumor development (36,37). In order to understand the impact of CD38 on the migration of tumor cells, a cell wound scratch assay was used to observe the cell scratch healing at different time points. It was determined that 5-8F/CD38 cells had an increased healing ability, compared with 5-8F/Vector cells (Fig. 2A and B). To further validate these results, a Transwell assay was performed. Similarly, it was determined that 5-8F/CD38 cells had increased migration, compared with 5-8F/Vector cells. The experiments demonstrated that CD38 promoted the migration of NPC cells (Fig. 2C and D).

The proliferative and differentiation abilities of cells gradually decline over time as the process of change progresses. Senescence also occurs in the tumor process (38,39). Therefore, whether CD38 affects the senescence of NPC cells was further investigated by β-galactosidase staining. The results demonstrated that the number of β-galactosidase-positive 5-8F/CD38 cells was reduced, compared with 5-8F/Vector cells. Additionally, it was demonstrated that CD38 overexpression reduced cellular senescence in NPC cells (Fig. 2E and F).

CD38 affects the cycle distribution of NPC cells

The effect of CD38 overexpression on the cell cycle of NPC cells was further examined. Additionally, the cell cycle distribution of 5-8F/CD38 and 5-8F/Vector cells was investigated with flow cytometry. The results demonstrated that 32.24% of 5-8F/CD38 cells were within the G0/G1 phase and 67.76% of 5-8F/CD38 cells were within the S/G2 phase. However, 41.69% of 5-8F/Vector cells were within the G0/G1 phase and 58.32% of 5-8F/Vector cells were within the S/G2 phase. Compared with the control group, the proportion of 5-8F/CD38 cells in the S phase was significantly increased. This demonstrated that CD38 promoted S phase DNA replication and promoted the proliferation of NPC cells (Fig. 3A and B).

CD38 suppresses the cell apoptosis of NPC cells

Apoptosis was detected using a Hoechst 33342/PI double staining assay and established 5-8F/CD38 cells were analyzed with flow cytometry. Concentrated chromatin in Hoechst 33342-stained apoptotic cells is more pronounced than in normal chromatin, and PI can only pass through dead cells and late apoptotic cells. Therefore, dyeing patterns distinguish normal, apoptotic and dead cell populations (40). As depicted in Fig. 3C and D, the percentage of apoptotic 5-8F/CD38 cells was reduced, compared with 5-8F/Vector cells. The results indicated that CD38 inhibited cell apoptosis, which echoes the results in Fig. 2E.

CD38 regulates the intracellular Ca2+ concentration and ROS of NPC cells

In order to investigate the mechanism of CD38 inhibited apoptosis of NPC cells, the intracellular Ca2+ concentration was detected by Fluorescein-2 AM, a Ca2+ ion fluorescent probe. The result was depicted in Fig. 3E. Compared with the 5-8F/Vector cells, 5-8F/CD38 cells had reduced fluorescence intensity. The results indicated that 5-8F/CD38 decreased intracellular Ca2+ concentration. Subsequently, DCFH-DA, a ROS detection probe, was used to further analyze the effect of CD38 overexpression on ROS production in NPC cells with flow cytometry. The results demonstrated that CD38 overexpression resulted in decreased levels of ROS in NPC cells (Fig. 3E)

CD38 affects the ATP concentration of NPC cells

In order to clarify the effect of CD38 on the ATP concentration of NPC cells, an ATP assay was used to detect the ATP concentration of 5-8F cells. The results demonstrated that the concentration of ATP in 5-8F/CD38 cells was significantly increased, compared with 5-8F/Vector cells (Fig. 4A). Simultaneously, the results indicated that CD38 overexpression significantly increased HL levels of 5-8F cells, compared with 5-8F/Vector cells (Fig. 4B).

CD38 increases the intracellular cAMP concentration of NPC cells

Cell proliferation and differentiation are two basic characteristics of cells. Cell proliferation and differentiation is a simultaneous and contradictory biological process. cAMP serves an important role in the regulation of these contradictions (41,42). To clarify how CD38 affects the concentration of cAMP in NPC cells, cAMP was tested and it was determined that the concentration of cAMP in 5-8F/CD38 cells was 60.25±3.23 ng/ml, and the 5-8F/Vector intracellular cAMP concentration was 53.48±2.49 ng/ml. Therefore, the cAMP concentration of 5-8F/CD38 cells was significantly increased, compared with 5-8F/Vector cells (Fig. 4C).

CD38 reduces the intracellular NADP+/NADPH concentration of NPC cells

NADP+/NADPH is an important indicator of energy metabolism. NADP+/NADPH ratio is not only one of the major markers of cellular redox status, but also serves an important regulatory role in the pentose phosphate pathway and antioxidant metabolism (43). Therefore, whether CD38 affected NADP+/NADPH in NPC cells was further investigated. The results demonstrated that NADP+/NADPH significantly decreased in NPC cells following CD38 overexpression, compared with 5-8F/Vector cells (Fig. 4D).

CD38 affects the human ADP/acrp30 concentration of NPC cells

Human ADP/acrp30, which activates the signaling pathway associated with cAMP-protein kinase A, has an inhibitory function in activation of nuclear factor-κB and intercellular adhesion molecule, as well as vascular endothelial cells (44). In order to clarify the effect of CD38 on the ADP/acrp30 concentration in NPC cells, an ADP/acrp30 kit was used to detect the concentration of ADP/acrp30 in 5-8F/CD38 and 5-8F/Vector cells. The concentration of ADP/acrp30 in 5-8F/CD38 cells was 0.89±0.10 ng/ml and the concentration of ADP/acrp30 in the 5-8F/Vector cells was 0.76±0.04 ng/ml (P>0.05). The results remonstrated that the concentration of ADP/acrp30 in 5-8F/CD38 cells was increased, compared with 5-8F/Vector cells, but the difference was not statistically significant (Fig. 5A).

Bioinformatics analysis of high-throughput data

To determine the most notably changing molecule and signaling pathway, the scientific information retrieval from the GEO database was used to download the GSE13597, which contained 28 samples, including 25 NPC samples and 3 control samples. Analysis of the results determined a total of 910 DEGs, such as RRM2, ζ chain of T-cell receptor-associated protein kinase 70 (ZAP70), TP53 and SDHA, including 210 significantly downregulated DEGs and 266 significantly upregulated DEGs (fold change >2; Fig. 5B). Subsequently, KEGG enrichment and PPI analysis of significantly DEGs was performed (Fig. 5C-E). KEGG pathway analysis determined five significantly enriched pathways: DEGs enriched in cell cycle, DNA replication, TP53 signaling pathway, pathway in cancer and small cell lung cancer. PPI analysis demonstrated interactions between upregulated DEGs and interactions between downregulated DEGs, including, gene CDK4 and minichromosome maintenance complex component 6 interacting with each other in upregulated DEGs, and ZAP70 and protein tyrosine phosphatase, non-receptor type 22 interacting with each other in downregulated DEGs.

CD38 affects the biological behavior and energy metabolism of NPC cells by downregulating TP53, and upregulating HIF-1α, SIRT1 and SDHA

Based on the previous results, western blot analysis was selected for validation of DEGs associated with cellular energy metabolism, cell proliferation and apoptosis. It was determined that CD38 overexpression yielded unexpected results. Upregulation of cell proliferation-associated genes CDK4, mitogen-activated protein kinase 9 and Cyclin D1 progressed the cell cycle from the G1 phase to the S phase resulting in increased cell proliferation. Although GADPH was a DEG that was upregulated, following CD38 overexpression, western blot analysis demonstrated that GAPDH was downregulated. Subsequently, western blot analysis of DEGs associated with cell apoptosis was performed. It was determined Bcl-2 and Ezrin expression was upregulated and TP53 expression was downregulated following CD38 overexpression. This implied that 5-8F/CD38 inhibited the cell apoptosis, consistent with the results depicted in Fig. 3E. Simultaneously, the DEGs ERK, STAT1, ITGA5, CD24 (45,46) and AKT1, which serve important regulatory roles in cell growth, cell activation and cell signaling (47), were identified with western blot analysis. The results demonstrated that ERK was downregulated, and STAT1, ITGA5, CD24 and AKT1 were upregulated (Fig. 6A).

HIF-1α induces glycolysis by binding to the DNA binding site on the target gene, increasing the glycolysis process. It promotes anaerobic metabolism and facilitates glycolysis of tumor cells under hypoxic conditions. SIRT1, SIRT2 and SIRT6 molecules belong to the NAD+ dependent III histone deacetylase sirtuin family (48,49). Therefore, in order to investigate the possible mechanism of CD38 on the metabolism of 5-8F cells, the expression of HIF-1α, SIRT1, SIRT2 and SIRT6 in 5-8F/CD38 and 5-8F/Vector cells was detected with western blot analysis. The results of LC-MS/MS identified 329 DEGs in 5-8F/CD38 cells, including 52 molecules associated with energy metabolism, such as SDHA, ATP5PD, ATP5F1, RRM2 and DLD, which were upregulated. The results demonstrated that CD38 overexpression upregulated the expression of SDHA and downregulated the expression of DLD, but had no significant effect on the expression of ATP5PD, ATP5F1 and RRM2 (Fig. 6B).

In conclusion, CD38 affects the biological behavior and energy metabolism of NPC cells by regulating the expression of TP53, HIF-1α, SIRT1, SDHA and other genes.

Discussion

CD38 is a cell surface enzyme that serves an important role in various physiological processes, including immune response, inflammation, cancer and metabolic diseases (50-52). CD38 is a multifunctional molecule, and has a number of complex and unique biological characteristics and functions, including: i) Receptor characteristics (53,54); ii) enzyme activity function (55); iii) adhesion molecule properties (56); iv) cell activation and cytokine production (57,58); and v) vector function (59). Previously, CD38 molecules have been demonstrated to be involved in the regulation of a number of physiological and pathological processes, including signal transduction (60), synthesis of cyclic ADP-ribose (61) and the cell cycle (62). In multiple myeloma, CD38 is an important therapeutic target for multiple myeloma (24). Our previous study also determined that CD38 was highly expressed in the SP of NPC and that CD38 may be involved in the regulation of the proliferation and invasion of SP cells by interacting with ZAP70 (36).

In the present study, the results demonstrated that CD38 was highly expressed in NPC tissues, compared with non-tumor nasopharyngeal epithelial tissues. It was speculated that there may be associations between CD38 expression and the clinical characteristics of patients; however, sufficient samples have no been collected yet. In future experiments, sufficient information and samples will be collected in this regard. CD38 promoted the proliferation of 5-8F cells and significantly increased the clonogenic capacity of 5-8F cells. Flow cytometry demonstrated that CD38 promoted S phase DNA replication of NPC cells and promoted the proliferation of NPC cells. CD38 overexpression served an inhibitory role in the apoptosis of 5-8F cells. Abnormal changes in the process of apoptosis causes a variety of diseases (63,64), for example, T-cell resistance against apoptosis contributes to inappropriate T-cell accumulation and the perpetuation of the chronic inflammatory process in inflammatory bowel disease with potential tumorigenic effect. Excessive apoptosis causes atrophy, and reduced apoptosis results in abnormal cell proliferation and tumorigenesis (65). A number of studies demonstrated that in the mouse model, CD38 regulated microglia activation, and the lack of CD38 slowed the process of glioma, reduced tumor expansion and prolonged the life of tumor-bearing mice (57,66,67). The lack of CD38 is associated with increased cell death and reduced expression of metalloproteinase-12 in the tumor mass (57). In a mouse esophageal cancer model, CD38-overexpressing myeloid-derived suppressor cells (MDSCs) were determined to be less mature than MDSCs with low CD38 expression. Furthermore, high CD38 expression in MDSCs caused an increased inhibitory effect on activated T cells, and it promoted increased tumor growth (68). Liao et al (53) determined that CD38 was highly expressed in cervical cancer tissues and affects the expression of phosphoinositide 3-kinase (PI3K), AKT, mouse double minute 2 proto-oncogene and TP53 in cervical cancer cells, indicating that CD38 serves a role in regulating the PI3K/AKT signaling pathway in cervical cancer. It has been reported that knockout of CD38 causes increased cell apoptosis in hairy cell leukemia (HCL), and inhibits the adhesion of HCL cells to monolayer epithelial cells, destroying their ability to form tumors (69). Overexpression of CD38 promoted the proliferation of NPC cells and inhibited the apoptosis in NPC cells. However, a number of studies demonstrated that CD38 exerted its antitumor effect in malignant tumors (69,70). Chini et al (71) determined that CD38 was the major consumption enzyme of NAD. Overexpression of CD38 resulted in the arrest of pancreatic cancer cells and the increase of cell senescence. Additionally, CD38 serves an important role by inhibiting nicotinamide phophoribosyltransferase.

Cyclin D1 serves an important role in cell cycle progression and is one of the key molecules that regulate the G1 restriction point in cell cycle progression (72-74). There is evidence that Cyclin D1 protein expression is vital for normal cell cycle progression (72). Abnormal expression of Cyclin D1 may disrupt cell cycle regulation, resulting in increased genomic instability and tumor induction (75,76). The aforementioned results are similar to those in the present study, confirming the role of Cyclin D1 and CDK molecules in the biological behavior of 5-8F/CD38 cells.

In prokaryotes, cAMP may directly activate RNA polymerase in order to promote its transcription, through the 6-factor phosphorylation level of the enzyme, to promote the transcription level of RNA. Recent studies demonstrated that the role of cAMP in eukaryotic cells was determined to be associated with regulation of transcription factors (77,78), for example, gene expression patterns define key transcriptional events in cell-cycle regulation by cAMP and protein kinase A. cAMP has the effect of inhibiting its cell division and promoting cell differentiation (41,42). cAMP also has a dual effect on cell proliferation, which promotes cell proliferation at early G0 or G1, and is inhibited at late G1 (42). The present study determined that the cAMP concentration of 5-8F/CD38 cells was increased, compared with 5-8F/Vector cells, indicating that CD38 affects the concentration of cAMP in NPC cells. Human ADP/acrp30 can increase the survival of the majority of endothelial progenitor cells in vitro, and inhibit the apoptotic process. The role of human ADP/acrp30 is primarily focused on endothelial cells affecting angiogenesis (79). A previous study also determined that human ADP/acrp30 had the effect of inhibiting mature macrophages, including the ability to inhibit the phagocytosis of mature macrophages (80,81). The present results demonstrated that CD38 overexpression had no significant effect on 5-8F cell intracellular concentrations of ADP/acrp30 in NPC.

HIF-1α regulates the transcriptional activity and expression of different target genes in the body, participates in invasion, metastasis and the production of blood vessels (82). The over-expression of HIF-1α is associated with the poor prognosis of a number of malignant tumors types (83-85), for example, HIF-1α is critical for the inhibitory effect of oleanolic acid on rectal cancer cell proliferation. The present results demonstrated that CD38 overexpression resulted in the upregulation of HIF-1α. This indicated that CD38 may participate in the proliferation and cell metabolism of NPC cells by affecting the activity of HIF-1α. HIF-1α can also participate in tumor angiogenesis through the regulation of vascular endothelial growth factor expression and promoting tumor cell metastasis (48,86).

In the present study, it was demonstrated that CD38 overexpression inhibited cell apoptosis and promoted cell proliferation in NPC cells. It was determined that CD38 may promote cell proliferation through inhibiting TP53, causing the upregulation of CDK4 and Cyclin D1 protein expression, thus promoting the transition from G1 to S phases. It was indicated that CD38 inhibition of apoptosis may be through inhibition of TP53, and also that Bcl-2 protein expression increased. It was demonstrated that CD38 regulated cellular energy metabolism by upregulating SIRT1, SDHA and HIF-1α protein expression (Fig. 7). In conclusion, the present results indicated that CD38 may serve its carcinogenic effect on NPC by regulating the metabolic-associated signaling pathways.

Funding

The present study was supported by National Natural Sciences Foundation of China (grant nos. 81672685, 81402270 and 81672993), the 111 Project (grant no. 111-2-12), the Natural Science Foundation of Xinjiang Autonomous Region (grant no. 2017D01C122), and the Planned Project of Key Subject Construction of the third Xiangya Hospital, Central South University.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

YZ designed the study. YG and YL conducted the experiments. SX, LL, ZH, CY and XW analyzed the data. The manuscript was drafted by YG and YZ. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Written informed consent was obtained from each patient and experimental protocols were approved by the Institutional Review Board of the Hunan Provincal Tumor Hospitals (Changsha, China).

Patient consent for publication

All participants provided written informed consent for publication.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

Not applicable.

Abbreviations:

NPC

nasopharyngeal carcinoma

HL

lactic acid

cAMP

cyclic adenosine monophosphate

GEO

Gene Expression Omnibus

KEGG

Kyoto Encyclopedia of Genes and Genomes

PPI

protein-protein interaction

TP53

tumor protein p53

CDK

cyclin-dependent kinase

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February-2019
Volume 54 Issue 2

Print ISSN: 1019-6439
Online ISSN:1791-2423

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Copy and paste a formatted citation
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Spandidos Publications style
Ge Y, Long Y, Xiao S, Liang L, He Z, Yue C, Wei X and Zhou Y: CD38 affects the biological behavior and energy metabolism of nasopharyngeal carcinoma cells. Int J Oncol 54: 585-599, 2019
APA
Ge, Y., Long, Y., Xiao, S., Liang, L., He, Z., Yue, C. ... Zhou, Y. (2019). CD38 affects the biological behavior and energy metabolism of nasopharyngeal carcinoma cells. International Journal of Oncology, 54, 585-599. https://doi.org/10.3892/ijo.2018.4651
MLA
Ge, Y., Long, Y., Xiao, S., Liang, L., He, Z., Yue, C., Wei, X., Zhou, Y."CD38 affects the biological behavior and energy metabolism of nasopharyngeal carcinoma cells". International Journal of Oncology 54.2 (2019): 585-599.
Chicago
Ge, Y., Long, Y., Xiao, S., Liang, L., He, Z., Yue, C., Wei, X., Zhou, Y."CD38 affects the biological behavior and energy metabolism of nasopharyngeal carcinoma cells". International Journal of Oncology 54, no. 2 (2019): 585-599. https://doi.org/10.3892/ijo.2018.4651