|Year : 2019 | Volume
| Issue : 1 | Page : 38-41
Downregulation of DBN1 is related to vincristine resistance in colon cancer cells
Zhongmin Han, Hemei Huang, Tao Zhang
Department of Medical Technology, Zhengzhou Railway Vocational and Technical College, Zhengzhou, Henan 450052, P.R. China
|Date of Web Publication||13-Mar-2019|
Prof. Zhongmin Han
Department of Medical Technology, Zhengzhou Railway Vocational and Technical College, Zhengzhou, Henan 450052
Source of Support: None, Conflict of Interest: None
Objective: This study was aimed to investigate the relationship between the expression of drebrin (DBN1) gene and resistance in colon cancer to reveal the mechanism of tumor drug resistance and provide a basis for the reversal of this drug resistance in tumor cells.
Materials and Methods: The human colon carcinoma cell line HCT-8 was used, and vincristine (VCR)-resistant colon cancer cell line HCT-8/V was established by gradually increasing the concentration of VCR. Polymerase chain reaction (PCR) primers were designed for DBN1 gene. The DBN1 differential expression in colon cancer sensitive and resistant cell lines was detected by fluorescence quantitative PCR. Western blot analysis was used to study DBN1 expression in the resistant cells further.
Results: VCR resistance of HCT-8/V cell line was established. Quantitative PCR and Western blot results showed that DBN1 expression in the resistant cell line was significantly lower, the difference being statistically significant (P < 0.05).
Conclusion: Low DBN1 gene expression may be associated with colon cancer cell resistance to VCR.
Keywords: Colon cancer, DBN1, drug resistance, HCT-8, vincristine
|How to cite this article:|
Han Z, Huang H, Zhang T. Downregulation of DBN1 is related to vincristine resistance in colon cancer cells. J Can Res Ther 2019;15:38-41
| > Introduction|| |
Colon cancer is a common gastrointestinal cancer and is very harmful to human life and health. Over the recent years, its prevalence has increased. Chemotherapy is an important comprehensive treatment for colon cancer., Cancer chemotherapy includes long-term use of multiple high-dose drug combinations. Cells become resistant to drugs with long-term use, and it has become a problem in the treatment of colon cancer and other malignant tumors. It has been found that 90% of the cancer deaths are related to cancer drug resistance. Tumor resistance mechanisms involve molecules at multiple cellular levels, such as changes in drug targets, repair of damaged cells, activation or suppression of cell death signaling pathways, and epigenetic changes (such as gene mutation, deletion, gene amplification, DNA methylation abnormality, and microRNA posttranscriptional regulation).,,
Vincristine (VCR) is the most commonly used chemotherapeutic drug for the treatment of colon cancer. VCR is a cell cycle-specific drug that can bind to tubulin, inhibit assembly of microtubule structures, and arrest mitosis in metaphase.
Drebrin (DBN1), an actin-binding protein not previously known to be involved in colorectal cancer metastasis, was found to be overexpressed in HCT-116 metastatic derivative E1 and was further validated immunohistochemically in patient tissues. Mitra et al. demonstrated that DBN1 exhibits a highly significant differential expression in recurrent tumors, and as a multivariate marker, DBN1 exhibits potential clinical utility in risk stratification of Stage I to III nonsmall cell lung cancer patients.
In this study, we verified the expression of DBN1 by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis and investigated its role in modulating VCR resistance, which may offer a new candidate target for gene therapy for VCR-resistant ovarian cancer.
| > Materials and Methods|| |
Cells and culture
Human colon cancer HCT-8 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in Dulbecco's modified Eagle's medium (Santa Clara, State of California) containing 10% fetal calf serum, 100 μg/mL penicillin, and 100 μg/mL streptomycin, at 37°C in a CO2 incubator. The cells were subcultured every 2–3 days, after treatment with 0.02% ethylenediaminetetraacetic acid and 0.1% trypsin.
Establishment of vincristine-resistant colon cancer cell line
VCR concentration was gradually increased to establish VCR-resistant cancer cell line HCT-8/V. The sensitive HCT-8 cells were cultured in a medium containing VCR with an initial concentration of 5 ng/mL, which was gradually increased to 10, 100, 1000, and 2000 ng/mL. After acquiring resistant cells for a concentration of VCR, cloning by limiting dilution was performed on the resistant cells, which were then used for the next concentration and screening. Finally, HCT-8 cells were cultured in a medium with 2000 ng/mL of VCR, after more than twenty passages. The cells had become resistant to VCR 1 week before the experiment.
RNA extraction and quantitative real-time reverse transcription-polymerase chain reaction
Cells in the exponential growth phase were collected. Total RNA was isolated from these cells in Petri dish More Detailses using the RNeasy kit (QIAGEN China Co., Ltd. Shanghai, China.) and first-strand complementary DNA was synthesized as previously described. PCR was performed on the Mx 3000P real-time PCR system (Stratagene, La Jolla, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as internal control.
Western blot analysis
Cellular proteins were extracted with Cell Lysis Buffer (Danfoss, Massachusetts, China) containing 1 mM phenylmethylsulfonyl fluoride. Equal amounts of protein were fractionated with 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and exposed to antibodies against DBN1 (B1310; Santa Clara, State of California) and β-actin (AC-74; Sigma-Aldrich, USA). Blot intensity was quantified by ImageJ software (Synatom Research, Princeton, New Jersey, USA) (NIH) after normalization with corresponding loading control.
All the data were analyzed with SPSS 13.0 (SPSS company, Chicago, Illinois, USA). Data are expressed as mean ± standard deviation. One-way ANOVA and Student's t-test were used for statistical analysis, with P < 0.05 reflecting a statistically significant difference.
| > Results|| |
Establishment of resistant cell lines
The VCR-resistant cell line was established by gradually increasing the concentration of the drug. After screening, the cell line was designated as HCT-8/V. HCT-8/V cells grew well in medium with 2000 ng/mL of VCR. Varying concentrations of VCR were used to treat the colon cancer cell lines HCT-8 and HCT-8/V, and their IC50 values were 13.56 and 165.49 μg/mL, respectively.
Quantitative real-time reverse transcription-polymerase chain reaction
Total RNA from HCT-8/V cells was extracted. Using the designed PCR primers, the DBN1 expression in these cells was detected by quantitative RT-PCR. The results showed that the amplification curves of DBN1 and internal reference GAPDH were suitable [Figure 1]a and [Figure 1]b. The relative expression of DBN1 in sensitive HCT-8 cells was significantly higher (1.76 times) than that in HCT-8/V cells (1.003 ± 0.08090 and 0.5700 ± 0.05033, respectively), and the difference was statistically significant [P < 0.05, [Figure 1]c and [Figure 1]d].
|Figure 1: DBN1 expression from HCT-8 and HCT-8/V was detected by quantitative reverse transcription-polymerase chain reaction. Beta-actin is shown as a loading control. Melt curve of DBN1 (a) and glyceraldehyde 3-phosphate dehydrogenase (b); (c) melt curve of DBN1; (d) analysis of DBN1 expression|
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Western blot analysis
Total cellular protein from HCT-8 and HCT-8/V was extracted, and β-actin was used as internal control. After calibration of the target protein with internal reference, the relative expression of the target protein was obtained. The results showed that the DBN1 expression in HCT-8/V cells was less than that in the sensitive HCT-8 cells [Figure 2]. The expression level of DBN1 in the sensitive HCT-8 cells was 2.56 times more than that in the resistant cells, the difference being statistically significant (P < 0.05).
|Figure 2: DBN1 expression from HCT-8 and HCT-8/V was detected by Western blotting. Beta-actin is shown as a loading control|
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| > Discussion|| |
VCR has been widely used in the clinical treatment of leukemia, lung cancer, and other malignancies, but tumor cells gradually become VCR resistant in the process.,,, VCR resistance mechanism is complex involving genes, such as insulin-like growth factor binding protein 7, multidrug resistance protein 1, long noncoding RNA, and microRNA.,,, To explore the mechanism of VCR resistance in colon cancer cells, the study established a VCR-resistant cell line by gradually increasing the drug concentration and designated it as HCT-8/V. The cell line was resistant to 2000 ng/mL VCR concentration, and HCT-8 cells were 12.7 times more sensitive to this VCR concentration than HCT-8/V.
After being treated with vinca alkaloids, the tumor cells become resistant by changing the site of action of the drug, for example, through tubulin mutation or by changing the sequence of the cytoskeleton, overexpression of drug pumps to reduce the concentration of the drug in the cells, and inhibiting apoptosis signal transduction to reduce apoptotic effect.
DBN1 encodes for the actin-binding protein drebrin, which is an F-actin side-binding protein that remodels actin to facilitate the change of filopodia into dendritic spines during synaptogenesis in developing neurons. It has been revealed that DBN1 expression is a target of p38 mitogen-activated protein kinase signaling during myogenesis and that drebrin promotes myoblast differentiation. DBN1 may play a key role in mouse brain development and may regulate differentiation of neural stem cells by filamentous actin. DBN1 was found to be overexpressed in HCT-116 metastatic derivative cells, and it exhibits potential clinical utility in risk stratification of Stage I to III nonsmall cell lung cancer patients., In this study, we found that cell cycles of HCT-8 and HCT-8/V had no significant changes, but the expression of DBN1 significantly reduced in HCT-8/V cells.
The experimental results showed that the resistant cell line of colon cancer has reduced the expression of DBN1, which may be related to changes in the drug-target sites, but in-depth features and mechanisms need to be further investigated.
| > Conclusion|| |
DBN1 gene expression is down-regulated in VCR-resistance colon cancer cells. Low DBN1 gene expression may be associated with colon cancer cell resistance to VCR.
Financial support and sponsorship
This study was supported by Key Project of Universities and Colleges in Henan, China (Grant No.15A310033).
Conflicts of interest
There are no conflicts of interest.
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