It has recently been found that the expression of the transglutaminase 3 (TGM3) is significantly reduced in esophageal cancer (EC). However, the potential of TGM3 as a useful biomarker or as a molecular target for EC diagnosis and treatment is unclear. The aim of the present study was to explore the role of the TGM3 in EC. The expression level of TGM3 were measured by real-time polymerase chain reaction and western blot analysis in EC cell lines, including SKGT-4, KYSE-510, OE33, OE21 and the normal esophageal epithelial cell line HEEC; fifty-eight pairs of tissues samples were also measured. Through exogenous expression of TGM3 in EC cells it was found that the expression of TGM3 is closely associated with tumor cell growth and apoptosis. TGM3 was found downregulated in EC and closely associated with tumor proliferation and migration. In addition, overexpression of TGM3 apparently induces EC cell proliferation, migration, invasion and promotes cell apoptosis
Esophageal cancer (EC) is the seventh most common malignant tumor in the world, the mortality of which is ranked sixth among the malignant tumors worldwide (
The transglutaminase 3 (TGM 3) enzyme is an enzyme with the ability to catalyze the irreversible cross-linking of peptide-bound glutamine residues either with peptide-bound lysines or with primary amines (
The nuclear factor kappa B (NF-κB) transcription factor family is composed of p50, p52, RelA/p65, c-rel and Rel B. The homodimers and heterodimers of these molecules are sequestered in the cytoplasm in an inactive form by the inhibitor of kappa B (IκB). Upon stimulation, the IκB kinase complex (IKK) phosphorylates the κB inhibitor, which then releases NF-κB and allows its phosphorylation, nuclear translocation, binding and subsequent activation of target genes involved in the regulation of cell proliferation, survival, angiogenesis and metastasis (
In the present study, we confirmed that the expression levels of TGM3 are downregulated in EC cell lines compared with normal primary esophageal epithelial cells, and tissue specimens compared with paired adjacent normal tissues, by means of RT-PCR and western blotting. We further evaluated the effect of ectopic TGM3 expression in the SKGT-4, KYSE-510, OE33 and OE21 cell lines. We provide the first evidence that the ectopic expression of TGM3 in EC cell lines inhibits cell proliferation and migration and induces apoptosis in cancer cells
Fifty-eight pairs of primary EC tissue samples and adjacent non-cancerous tissues (located >3 cm away from the tumor) were obtained from the Department of Oncology, The Second Affiliated Hospital, Zhengzhou University (Zhengzhou, China) between June 2014 and December 2015. All subjects were diagnosed and confirmed by a pathological evaluation. None of the subjects received any biotherapy or chemotherapy treatment before recruitment to this study. The study was approved by the Institutional Ethics Committee of the Second Affiliated Hospital, Zhengzhou University and all of the patients provided written informed consent in accordance with the institutional guidelines.
The EC cell lines SKGT-4 and KYSE-510 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). OE33 and OE21 were obtained from the European Collection of Animal Cell Cultures (ECACC; Porton Down, Salisbury, UK). All EC cell lines were grown in RPMI-1640 medium supplemented with 10% filtered fetal calf serum, 100 units/ml penicillin, 100
Bay11-7082, a specific inhibitor of NF-κB signaling, was purchased from CsA (Biomol, Plymouth Meeting, PA) and diluted in culture medium to obtain the desired concentration. Recombinant human TGM3 was obtained from Abcam Inc. (Cambridge, MA, USA), and the rabbit anti-TGM3 antibody was from Adipo Bioscience Inc. (Santa Clara, CA, USA). EC cells were transfected with recombinant human TGM3 (10
Total RNA was isolated using the TRIzol reagent (Invitrogen Life Technologies). The concentration of RNA were determined using a NanoDrop ND-1000 instrument (Thermo Fisher Scientific, Waltham, MA, USA), and aliquots of the samples were stored at −80°C. For reverse transcription, cDNA was synthesized by using a reverse transcription kit (Promega, Madison, WI, USA). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed (StepOne; Applied Biosystems, Foster City, CA, USA) using SYBR-Green (Takara, Shiga, Japan). All PCR experiments were performed in triplicate. The TGM3 primer sequences are as follows: sense: 5′-TCAACTGGCAGACGGCCTTCA-3′ and antisense 5′-GTACCGTCCTATGGGTGCGCT-3′.
Total protein was extracted using RIPA lysis buffer and the concentrations in different samples were determined using the BCA kit (Beyotime Institute of Biotechnology, Haimen, China). An equivalent amount of protein (30
Cells were seeded in 96-well plates at 6×103 cells/well and the surviving fractions were determined at 0, 24, 48, 72 and 96 h using the MTT assay, as previously described (
EC cells were grown to confluence in 12-well plastic dishes and treated with recombinant human TGM3 protein or a scramble peptide. Then, 24 h after transfection, linear scratch wounds (in triplicate) were created on the confluent cell monolayers using a 200
For the invasion assays, 24 h after transfection, 1×105 cells in serum-free media were seeded in Transwell migration chambers. The upper chamber of the Transwell inserts was coated with Matrigel (Sigma-Aldrich, St. Louis, MO, USA). Medium containing 20% FBS was added to the lower chamber. After 24 h, the non-invading cells were removed with cotton wool. Invasive cells located on the lower surface of the chamber were stained with May-Grunwald-Giemsa stain (Sigma-Aldrich) and counted using a microscope (Olympus Corp., Tokyo, Japan). From these images, the number of invasive cells was counted. Experiments were independently repeated three times.
Apoptosis was assayed by an Annexin V apoptosis detection kit (BD Biosciences, San Diego, CA, USA). Following transfection for 72 h, the cells were collected and detected using an Annexin V fluorescein isothiocyanate kit (FITC) according to the manufacturer's instructions. In brief, cells transfected with the recombinant human vasostatin-2 or vasostatin-2 antibody were resuspended in 100 ml binding buffer, at a density of 1×106 cells/ml, then incubated with Annexin V-FITC and PI for 15 min. The cells were analyzed with Beckman CXP software on a FC-500 flow cytometer (Beckman Coulter, Pasadena, CA, USA) within 1 h of cell collection.
The levels of p65 concentrations in culture medium were determined by ELISA (Tanjin Biotechnology Co., Shanghai, China) following the manufacturer's instructions. The absorbance was assessed at 450 nm using a 680XR microplate reader (Bio-Rad Laboratories).
The differences were analyzed using Student's t-test for two groups and examined by the χ2 test. A P-value <0.05 was considered to indicate statistically significant differences. Data were processed as mean ± standard deviation (SD). All statistical analyses were performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA).
In order to confirm the potential roles of TGM3 in EC, RT-PCR was performed to investigate the mRNA levels of TGM3 in 58 paired EC specimens compared to the corresponding adjacent non-cancerous tissues (NCTs) (
It is well-known that cell proliferation is a key event in the formation and development of cellular oncogenesis. In order to investigate the role of TGM3 in EC cell proliferation, TGM3 was overexpressed or repressed in SKGT-4 cells and KYSE-510 cells by transfection with recombinant human TGM3 or TGM3 antibody, qRT-PCR (
One of the hallmarks of cancer is its ability to evade apoptosis (
To analyze the role of TGM3 in cell migration and invasion, which are the key determinants of malignant progression and metastasis, scratch assays and Transwell assays were performed in the SKGT-4 and KYSE-510 cell lines. Cells treated with recombinant human TGM3 were distinctively less migratory and invasive; however, cells treated with the TGM3 antibody were distinctively more migratory and invasive than untreated cells at 48 h after scratching (
The NF-κB signaling pathway has been demonstrated to play an important role in cellular energy homeostasis and reported to be extensively involved in cell proliferation, migration and differentiation (
To further delineate the effect of NF-κB on TGM3 modulated EC cell growth and apoptosis, the NF-κB signaling inhibitor Bay11-7082 (20 mol/l) was used. After a series of functional restoration assays, we found that the decrease in SKGT-4 cell proliferation (
A variety of treatments is currently available for EC. The choice of treatment is crucial, recurrent or distant metastases as the limitations of conventional treatments, therefore, it is necessary to search for novel approaches. Recently, several research groups have reported certain genes and signaling molecules that are potentially involved in EC initiation and progression (
Further studies identified that TGM3 promotes activation of the NF-κB signaling pathway. Recently, abundant studies have confirmed the important roles of NF-κB signaling in cancer development and progression, including proliferation, survival, angiogenesis and metastasis (
In conclusion, this study showed that TGM3 expression is low in EC tissues and cell lines, this result is consistent with the previous studies. We also found that in EC cell lines overexpression of TGM3 inhibits cell proliferation and induces apoptosis by activation of NF-κB signaling pathway. As the TGM3 expression level is associated with EC cell growth process, promotion of TGM3 expression in EC tissues may inhibit tumor cell amplification. Therefore, the present study may provide a therapeutic strategy for patients with EC.
The present study is supported by grants from the Key Scientific Research Project of Henan Province (14A310017) and the Key Project of Henan Provincial Department of Science and Technology (142102310311).
TGM3 expression is frequently downregulated in EC tissues and cell lines. The expression of TGM3 in EC tissues and their matched adjacent non-cancerous tissues was determined using (A) RT-PCR and (B) western blot analysis. The expression level of TGM3 in four EC cell lines and the normal human esophageal epithelial cell line HEEC were assessed by (C) RT-PCR and (D) western blot analysis. Data are representative of three experiments. Error bars represent mean ± SD. *P<0.05 and **P<0.01, differences from their respective controls, respectively.
TGM3 protein affects EC cell proliferation
TGM3 affects EC cells apoptosis
TGM3 protein affects EC cell migration and invasion
Overexpression of TGM3 triggered activation of the NF-κB signaling pathway. (A) After pretreatment with recombinant human TGM3 (Re TGM3) or TGM3 antibody (Anti TGM3), the p65 concentration in the culture medium of SKGT-4 cells was assessed by ELISA. (B) The protein expression of p65 was analyzed by western blotting. Also, the p65 concentration and protein expression in SKGT-4 cells were analyzed. Data represent means ± SD from three independent experiments. *P<0.05, **P<0.01 differences from their respective controls, respectively.
TGM3 regulates EC cell development by targeting NF-κB signaling pathway. (A) The MTT assay shows that overexpression of TGM3 increases the effect of TGM3 in the regulation of proliferation. (B) Inhibition of the NF-κB signaling pathway rescued cell migration induced by TGM3 in EC cells. (C) Disruption of the NF-κB signaling pathway promoted apoptosis induced by TGM3 in EC cells. Data represent mean ± SD. *P<0.01, differences vs. the normal control; #P<0.01, differences vs. the TGM3 scramble.