|Year : 2015 | Volume
| Issue : 1 | Page : 32-35
Visfatin effects on telomerase gene expression in AGS gastric cancer cell line
M Mohammadi1, N Zarghami2, M Hedayati3, S Ghaemmaghami4, RM Yamchi5, M Mohaddes6
1 Department of Clinical Biochemistry, Division of Molecular Medicine, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
2 Department of Cellular and Molecular, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Cellular and Molecular Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Department of Clinical Biochemistry, Division of Molecular Medicine, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz; Cellular and Molecular Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
5 Department of Clinical Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
6 Department of Medical Genetic, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
|Date of Web Publication||3-Feb-2016|
Department of Cellular and Molecular, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran
Source of Support: Tabriz University of Medical Sciences and
Cellular and Molecular Endocrine Research Center, Research
Institute for Endocrine Sciences, Shahid Beheshti University of
Medical Sciences, Conflict of Interest: None
Aim: The aim of this study was to assess visfatin expression and its effect on human telomerase gene expression in AGS gastric cancer cell line. Materials And Methods: In this study, human gastric cancer (AGS) cell line was established as an in vitro model. Reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay was performed to show that visfatin expression in messenger ribonucleic acid (mRNA) and protein level respectively. After stimulating with increasing concentrations of visfatin for times of 24 h and 48 h, cell proliferation was assessed by 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) assay. In order to investigate telomerase gene expression affected by visfatin in AGS cell line, total RNA was extracted and complementary deoxyribonucleic acid was synthesized buy using commercially available kits. Expression of human telomerase reverse transcriptase (hTERT) mRNA was carried out by real-time RT-PCR. Results: After visfatin treatment gastric cell line proliferation was enhanced and was increased the expression of hTERT. Conclusions: The obtained data showed that visfatin induces endogenously gastric cancer cell proliferation and increases telomerase (hTERT) gene expression, as a cancer gene. Based on this study, it is suggested that expression of this adipocytokine protein in real samples could be biomarker for gastric cancer.
Keywords: Gastric cancer, human telomerase reverse transcriptase, nicotinamide phosphoribosyl-transferase, telomerase, visfatin
|How to cite this article:|
Mohammadi M, Zarghami N, Hedayati M, Ghaemmaghami S, Yamchi R M, Mohaddes M. Visfatin effects on telomerase gene expression in AGS gastric cancer cell line. Indian J Cancer 2015;52:32-5
|How to cite this URL:|
Mohammadi M, Zarghami N, Hedayati M, Ghaemmaghami S, Yamchi R M, Mohaddes M. Visfatin effects on telomerase gene expression in AGS gastric cancer cell line. Indian J Cancer [serial online] 2015 [cited 2019 Aug 18];52:32-5. Available from: http://www.indianjcancer.com/text.asp?2015/52/1/32/175567
| » Introduction|| |
Gastric cancer is the fourth common cancer and the second leading cause of cancer death world-wide, with a wide variation in incidence rates across different geographical areas.
Pathogenesis of gastric cancer is complex and not completely understood there are many factors that contribute to gastric carcinogenesis. Several epidemiological studies have investigated the effect of increasing body weight as well as, metabolic syndrome, body mass index and other anthropometric measurements on the risk of gastric cancer.,, Obesity is closely linked to an increase adipose tissue. Adipocytes secrete a number of different factors that are commonly referred to as “adipokines” (adipocyte-derived cytokines). Adipokines are involved in a variety of biological functions, including the regulation of energy balance, glucose homeostasis, lipid metabolism and inflammation. In the last two decades, many researchers have tried to discover the possible role of adipocytokines in the regulation of angiogenesis and tumor growth. In a previous study in gastric cancer has shown that resistin and visfatin increased more than other adipocytokines and may be good biomarkers of gastric cancer.
Visfatin is also known as pre-B cell enhancing factor, a growth factor for early B cell proliferation  and it is the secretory form of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme of mammalian nicotinamide adenine dinucleotide (NAD) biosynthesis. NAD+ is central to many cellular processes and cancer cells have a high rate of NAD+ turnover compared with normal cells. Several studies have shown the role of visfatin in different cancer.,,,
Recent studies have shown that possible role of visfatin in the regulation of angiogenesis and tumor growth. In prostate cancer cells, exogenous visfatin increased proliferate. In breast cancer cells, transcription of human visfatin genes is regulated by hypoxia inducible factor-1, a key factor in a malignant tumor progression. About visfatin in gastric cancer, recent study was performed by Nakajima et al., was found to correlate significantly with stage progression, perhaps encouraging its use as a biomarker for gastric cancer progression. There are many factors that contribute to gastric carcinogenesis.,,,,,, Currently, telomerase has been a major focus., Telomerase activation is associated with an early stage of stomach carcinogenesis.
Telomerase is composed of human telomerase ribonucleic acid (RNA) component, human telomerase-associated protein and human telomerase catalytic subunit human telomerase reverse transcriptase (hTERT), have been identified.,, Recent studies have demonstrated a close correlation between telomerase activity and hTERT expression., It has been shown that enhanced expression of hTERT is a direct determinant of telomerase activity in cancers. Therefore, numerous studies have focused on the cancer-specific regulation of hTERT and its application of tumor diagnosis and treatment. Based on these studies, we postulate that visfatin may up regulate the expression of hTERT and hTERT may mediate the function of visfatin in tumor growth and progression.
The relationship between high visfatin levels and gastric cancer risk is unclear and the molecular basis for such a link remains poorly understood. In the present study, we attempted to investigate whether high serum level of visfatin in gastric cancer patients is good biomarker and could be used as a potential diagnostic and prognostic tool. For this purpose, at the first step, we should investigate endogenous expression or only exogenous form of visfatin has the effect on cancer progression then we aimed to evaluate the effect of visfatin on the cell viability in AGS cells, also to evaluate the expression of hTERT in response to visfatin.
| » Materials and Methods|| |
Cell lines and culture conditions
Human gastric adenocarcinoma consisting of mucus secreting epithelial cells and was obtained from were purchased from the institute pastor cell bank in Iran. The cells were incubated in Ham's F-12 (Sigma-Aldrich/USA) culture medium containing 100 ng/ml of penicillin, 100 ng/ml of streptomycin, 15 mM Hepes, 1.2 g/L sodium bicarbonate and 10% fetal bovine serum (Biochorom/Belin). The cell monolayer in a 25 cm 2 flask was sub-cultured at 1:5 ratios every 3 days by treatment with 0.1% trypsin and 0.03% ethylenediaminetetraacetic acid. The flask was maintained in the incubator at 37°C in a humidified atmosphere with 5% CO2.
Cell proliferation assay
Growing cells (7 × 103 cells/well) were seeded in 96-well plates after 24 h incubation at 37°C, incubated with various concentrations of recombinant human visfatin (0, 5, 10, 50, 100, 200 ng/ml) for 12 h, 24 h and 48 h. Cell number and viability were determined using a hemocytometer after staining with trypan blue. Cell proliferation was analyzed using the XTT assay kit ( BIOTIUM, Inc, USA.) according to the manufacturer's instructions. Briefly, add 50 μL of the activated XTT solution (Mix 25 μL activation reagent with 5 mL XTT solution to derive activated XTT solution) to each well and were incubated for 4 h. Measure the absorbance of the samples with a spectrophotometer (enzyme-linked immunosorbent assay [ELISA] reader) at a wavelength of 470 nm wavelength and a reference wavelength of 630 nm.
Total RNA extraction
Before RNA extraction AGS cells were cut out by harvesting medium for each flask. Adherent cells were washed twice with phosphate buffered saline and trypsinized; the cell pellets were collected by centrifugation at 1000 g for 10 min at 4°C. Total RNA was extracted from each cell culture flask using the guanidine isothiocyanate based RNA Xplus solution (Sinna Gen INC, IRI) according to the manufacturer protocol. Briefly 1 ml of RNX plus reagent in a clean RNase-free tube and was incubated for 5 min at room temperature. After incubation, 200 μl chloroform was added, shaken rigorously for 15 s and incubated for another 5 min. The mixture was centrifuged at 12000 g for 15 min. The aqueous phase was transferred to a clean RNase-free tube. The total RNA was precipitated by adding 0.5 ml isopropyl alcohol and incubating for 15 min at room temperature. The pellet including total RNA was washed using 75% ethanol and centrifuged at 7500 g for 8 min. After drying the ethanol, the RNA pellet was dissolved in TE buffer. The amount of extracted RNA was quantified by measuring the absorbance at 260 nm. The purity of the RNA was checked by measuring the ratio of the absorbance at 260 and 280 nm. The absence of degradation of the RNA was confirmed by RNA electrophoresis on a 1.5% agarose gel containing ethidium bromide.
Reverse transcription polymerase chain reaction
First-strand complementary deoxyribonucleic acid (cDNA) was generated using the First-strand cDNA synthesis Kit (Fermentas), with random hexamer, according to the manufacturer's protocol. An aliquot of the RT reaction was amplified under conditions (1 min at 94°C, 1 min at 60°C and 2 min at 72°C) in a total volume of 50 μl. In parallel, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as a housekeeping gene, PCR was performed to control for the RNA input in the RT-PCRThe following primers were used: Human visfatin forward sequence 5'-TGCCTTCGGTTCTGGTGGAGGTT-3‘; human visfatin reverse sequence 5'-ACAA AATTCCCTG CTGGCGTCCT-3‘; and housekeeping gene GAPDH the following primers were used: Forward sequence 5'-CAAGGTCATCCATGACAACTTTG-3‘, GAPDH reverse sequence 5'-GT CCACCA CCCTGTTG CTGTAG-3‘. Reaction products were separated on polyacrylamide gel.
Visfatin expression and secretion was detected by a human visfatin ELISA using a competitive enzyme immunoassay technique (RayBiotech, Australia), from cell lysate and cell supernatant (after 24 and 48 h without serum) according to the manufacturer's instructions. Briefly, cell lysate was harvested from culture flask of AGS cell also supernatants were harvested from serum free culture flask of AGS after 24 and 48 h. Standards, controls and samples were assayed at a wavelength of 450 nm for wavelength correction. A range of visfatin dilutions was used to generate a standard curve to determine visfatin concentration in the sample supernatant.
Quantitative real-time RT-PCR
Levels of hTERT RNA molecules were determined by quantitative real-time RT-PCR technique using the Syber Green-I (Roche, Germany) by the Rotor-Gene ™ 6000 system (Corbett Research, Australia) according to the manufacturer's instructions. After cDNA synthesis, specific primers: Visfatin human forward primer 5‘-CCGCCTGAGCTGTACTTTGT-3‘, visfatin human reverse primer 5‘-CAGGTGAGCCACGAACTGT-3‘, used to amplify hTERT messenger RNA (mRNA). Alternative spliced variants of hTERT mRNA were not measured because they do not reconstitute telomerase activity., The GAPDH mRNA measured as the internal control by specific primers (forward sequence 5‘-CAAGGTCATCCATGACAACTTTG-3‘, GAPDH reverse sequence 5‘-GTCCACCACCCT GTTGCTGTAG-3‘). The program for real-time PCR reaction was as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s and extension at 72°C for 30 s. Finally, amplicons were assessed by melting curve analysis of 70-95°C. The quantity of PCR product generated from amplification of the hTERT gene was standardized using the quantity of GAPDH product for each sample to obtain a relative level of gene expression. After quantitation, results were analyzed by 2–ΔΔCt method. All data were derived from at least three independent experiments and statistical analyses were performed using independent t-test. Values were presented as mean ± standard error of the mean (SEM) P < 0.05.
The results were expressed as mean ± SEM and analyzed using the SPSS 15. Student's t-test was used for comparisons between two groups. P < 0.05 was considered to be statistically significant.
| » Results|| |
Visfatin expression and secretion
To determine the expression of visfatin in gastric cancer AGS cell line in mRNA level, RT-PCR was performed that were positively detected in cell line. To confirm of this result, we performed ELISA based on competitive enzyme immunoassay in cell lysate sample and cell free serum supernatant after 24 and 48 h. Results showed visfatin positively express and secrete in all samples. In cell lysate, visfatin concentration was higher than cell supernatants, but this differentiation was not significant. Also in 48 h visfatin concentration was higher than 24 h, but differentiation was not significant.
Visfatin proliferate AGS cell line
After XTT assay was performed in different doses and times, results was shown visfatin proliferate AGS cells and in concentration of 5 ng/ml visfatin had maximum cell viability and cell count was higher than concentration treatment (P < 0.05) [Figure 1].
|Figure 1: Effect of visfatin on cell proliferation in AGS cells. AGS cells were plated in 96-well plates and treated with visfatin at the indicated concentrations (5, 10, 50, 100, and 200 ng/ml), followed by cell viability testing using the XTT assay and live cell counting. (a) 12 h, (b) 24 h,and (c) 48 h after visfatin treatment. All data were expressed as the mean ± standard error of the mean of at least six wells per group. *P < 0.05; **P < 0.01 for visfatin versus control|
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Visfatin up regulate expression of hTERT mRNA
Since telomerase activity is tightly associated with the expression of hTERT, the catalytic reverse transcriptase of telomerase, we evaluated the levels of hTERT mRNA in AGS cells by real-time RT-PCR [Figure 2]. In agreement with the effect on telomerase activity, visfatin also enhanced the expression of hTERT mRNA in a time-dependent fashion [Figure 2]. The levels of hTERT mRNA were increased 1.6-fold (P < 0.05) with 5 ng/ml of visfatin in 24 h, but in 6 and 12 h after visfatin treatment, hTERT level expression was of no significant.
|Figure 2: Effect of visfatin on human telomerase reverse transcriptase, gene expression. AGS cells were plated in T-25 fl asks and incubated for 24 h in the presence of visfatin (5 ng/ml) to examine hTERT gene expression patterns. Total ribonucleic acid was extracted from cells and analyzed by real-time polymerase chain reaction. Expression hTERT messenger RNA species was significantly increased by treatment of AGS cells with visfatin. The data were expressed as mean ± standard error of the mean (n = 6). *P < 0.05; **P < 0.01 for visfatin versus control|
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| » Discussion|| |
Recent studies have aimed to understanding of the influence of adipocyte-derived adipocytokines on tumorgenesis in gastric. Based on previous analyses of the adipokines alterations in gastric cancer, increase of visfatin and resistin higher than other adipokines, we hypothesized in addition, expression adipose tissue and other source there would be endogenous expression of visfatin in gastric cells and progression of gastric cancer.
Our study demonstrates that visfatin express endogenous in gastric cancer cell line AGS and increase the level in cancer cells as well as serum concentration of visfatin in patients. In fact, in mammals, visfatin has two different forms: Intracellular has been named iNampt and extracellular Nampt has been named eNampt.
iNampt involved in the NAD + salvage pathway of NAD synthesis. NAD is an essential cofactor found in all cells and plays a vital role in energy metabolism, serving as a cofactor of histone deacetylase sirtuins, regulates cell death through poly (ADP-ribose) polymerase 1, thus iNampt important in cellular processes. eNampt, has also been named visfatin, as it was found released from adipocytes mostly in the form of serum Nampt, likely functions as a cytokine in circulation.
Telomerase activation is a critical step for human carcinogenesis through the maintenance of telomeres, but the activation mechanism during carcinogenesis remains unclear. Transcriptional regulation of the hTERT gene is the major mechanism for cancer-specific activation of telomerase and a number of factors have been identified to directly or indirectly regulate the hTERT promoter, including cellular transcriptional activators such as nuclear factor kappa beta, myeloid zinc fnger protein-2 c-Myc, specificity protein 1, hypoxia inducible factor-1, activator protein-2, estrogen receptor, And, etc., Recently, several studies suggest that the function of hTERT is not limited to the maintenance of telomeres. Lee et al. reported that hTERT promoted cellular survival independent of telomerase activity. Interestingly, hTERT regulates the expression of cyclin D1, as an important cell cycle protein and vascular endothelial growth factor, as a key angiogenic factor.
In this study, we showed that visfatin has the effect on hTERT expression and activates telomerase thus hTERT may mediate the function of visfatin in tumor growth and progression.
| » Conclusions|| |
In conclusion, we found that visfatin was overexpressed in established gastric cancer cells and induces endogenously gastric cancer cell proliferation and increases telomerase (hTERT) gene expression, as a cancer gene. There for visfatin could be a good biomarker also for as much as, visfatin is caused hTERT increase expression, blocking visfatin signaling and limiting visfatin secretion may be valuable for the treatment of gastric cancer with elevated visfatin levels.
| » Acknowledgment|| |
This study was supported by Tabriz University of Medical Sciences and Cellular and Molecular Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences. We are grateful to all individuals who participated in this project.
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