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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 8  |  Page : 237-242

Effect of lenalidomide on the human gastric cancer cell line SGC7901/vincristine Notch signaling


Department of Oncology, Renmin Hospital of Wuhan University, Wuhan 430060, China

Date of Web Publication26-Mar-2018

Correspondence Address:
Wanjun Ding
Department of Oncology, Renmin Hospital of Wuhan University, Wuhan 430060
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.183181

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 > Abstract 


Aim of Study: To examine the function of lenalidomide (LEN) on the human multidrug resistance (MDR)-type gastric cancer line SGC7901/vincristine (VCR) via regulating Notch signaling.
Materials and Methods: Quantitative polymerase chain reaction was used for checking the genes of Notch, DNA methyltransferase (DNMT), RBP-J, Hes1/5, Deltex1, MDR/multidrug resistant protein (MRP); the cell proliferation and cell death were detected by cell counting kit-8 (CCK8) staining, Ki-67 expression, and propidium-iodide staining, and methylated DNA immunoprecipitation assay (MeDIP) was used for checking the 5 mC enrichment, indicating the DNA methylation of the Notch2 gene loci.
Results: LEN reduced the mRNA expression of Notch2 (P < 0.01) and increased the expression of the DNMT3A (P < 0.001) in SGC7901/VCR cell, suggesting the involvement of epigenetic regulation by DNMT3A on Notch2 gene expression. Consistently, Notch2 gene expression showed no obvious change between the LEN treatment and the control when the DNMT3A was knockdown using the interference of shRNA. The modulation of DNA methylation process on gene expression was then confirmed by 5 mC enrichment on Notch2 gene loci after LEN treatment. Furthermore, LEN could suppress the downstream genes in Notch2 signaling including RBP-J (P < 0.05), Hes1 (P < 0.001), and Deltex1 (P < 0.01). Due to the changes of gene expression pattern in Notch pathway, LEN showed a phenotype of cell proliferation suppression using CCK8 staining. Meanwhile, the expression of the genes associated with MDR and MRP was also significantly decreased (MDR, P < 0.01; MRP, P < 0.001) after LEN treatment. Therefore, inhibition of cell proliferation by LEN via Notch2 signaling combined with the MDR/MRP expression modulation contributes to the efficacy of LEN on the gastric cancer cell line SGC7901/VCR.
Conclusion: The data implicate that LEN would be an effective chemical for the therapy of drug-resistant human gastric cancer cell and the gastric cancer patients.

Keywords: DNA methyltransferase 3A, gastric cancer cell, lenalidomide, multidrug resistance/multidrug-resistant protein, Notch2


How to cite this article:
Ding W, Zeng T, Tao W, Ge W, Deng J, Lei H, Xiao Y, Liao F. Effect of lenalidomide on the human gastric cancer cell line SGC7901/vincristine Notch signaling. J Can Res Ther 2018;14, Suppl S1:237-42

How to cite this URL:
Ding W, Zeng T, Tao W, Ge W, Deng J, Lei H, Xiao Y, Liao F. Effect of lenalidomide on the human gastric cancer cell line SGC7901/vincristine Notch signaling. J Can Res Ther [serial online] 2018 [cited 2019 Aug 19];14:237-42. Available from: http://www.cancerjournal.net/text.asp?2018/14/8/237/183181




 > Introduction Top


Recently, the multidrug resistance (MDR) has been the major barrier of the chemotherapy for gastric cancer, which is associated with the efficacy of anti-tumor drugs. Although there are many mechanisms underlying MDR including drug administration, drug metabolism, drug cytotoxicity, DNA repair activation, and cell apoptosis, novel drugs for the effective treatments remain necessary.[1],[2],[3] Here, we investigate the function of lenalidomide (LEN, Revlimid) as the thalidomide analog on the human MDR-type gastric cancer line SGC7901/vincristine (VCR). It has been reported that LEN exhibited efficacy on human multiple myeloma,[4],[5],[6] but no evidence showed that it plays a role in the human gastric cancer.

The Notch family genes encode evolutionarily conserved transmembrane receptors that play a key role in cellular differentiation and cell function. The cleavage of transmembrane unit of Notch receptor and its ligand results in Notch intracellular fragment (NIC) release. NIC translocating to nucleus in association with RBP-J regulates the Hes1/5 gene expression. Alternatively, NIC together with Deltex1 suppresses the Jun N-terminal kinase pathway in an RBP-J independent manner.[7],[8],[9] Notch is assumed to decide the cell fate during lymphocytes development.[8] However, the modulation of Notch expression and subsequent cell function by LEN in the gastric cancer MDR cell line remains poorly understood.

It is reported that tumor is associated with the epigenetic changes in cancer cells.[10] DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) regulate mammalian genomes by cytosine methylation, an epigenetic mark that is essential for normal development and primarily occurs at CpG dinucleotides.[11],[12] Most CpG-rich regions (CpG islands) overlap with proximal promoters, where DNA methylation is linked to gene silencing.[13] However, unbiased genome-wide analyses have shown that DNA methylation is widespread across the chromatic portion of mammalian genomes and predominantly takes place in regions outside proximal promoters, including intergenic regions and gene bodies,[14] where tissue- or tumor-specific DNA methylation is frequently localized.[15] The link between DNMT function and Notch expression with the involvement LEN in SGC7901/VCR cells would be observed to elucidate the mechanisms of LEN for human gastric cancer, implicating that LEN would be a potential drug for the tumor.


 > Materials and Methods Top


Cell culture and drug treatment

Gastric cancer cell line SGC7901 and VCR-resistant gastric cancer SGC7901/VCR cells were kindly provided by the Department of Gastroenterology, Xijing Hospital, the Fourth Military Medical University (Xi'an China). The MDR subline SGC7901/VCR was developed by exposing the parental SGC7901 cells to increasing concentrations of the anticancer drug VCR. All cells were cultivated in RMPI-1640 supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a 5% CO2 atmosphere. The medium for SGC7901/VCR cells was further supplemented with VCR (0.25 μmol/L). Before their use in experiments, the SGC7901/VCR cells were cultured in a drug-free medium for 2 weeks. LEN was dissolved in dimethylsulfoxide (DMSO) at 200 mM as a stock solution and stored at −20°C. For in vitro study, dilutions of the stock solutions were made in the culture medium, and filter sterilized; the final concentration of LEN was 2 mM. One percent of DMSO was used as a control without LEN treatment.

Evaluation of proliferation

Cell treated with LEN was cultured for 3 days before they were analyzed for proliferation using cell counting kit-8 (CCK8 kit, DOJINDO, Shanghai, China). Cells were seeded into 96-well plates at densities of 1 × 104 SGC7901/VCR cells per well and incubated in a humidified atmosphere of 5% CO2+ 95% air overnight; then, normal cell medium containing either test compounds or solvents at the desired concentration was added. After 72 h incubation, 10 μL CCK8 (5 g/L in phosphate buffered saline) was added. The plates were incubated for 4 h and the blue dye formed was dissolved in 100 μL DMSO or Me2 SO. The absorbance at 450 nm was recorded using an enzyme-linked immunosorbent assay reader.

Sample preparation and quantitative polymerase chain reaction

Cells used for real-time polymerase chain reaction (PCR) analysis were cultured under the same conditions as those for evaluation of proliferation; however, the culture period was 2 instead of 3 days. Total RNA was extracted from the cells using the RNeasy mini kit (Qiagen, Beijing, China), followed by complementary DNA (cDNA) synthesis using the Superscript III first-strand synthesis kit (Invitrogen, Shanghai, China). Quantitative PCR (qPCR) was performed on Bio-Rad amplifier using the Bio-Rad real-time PCR mix. Data were analyzed using the computed tomography; value normalized to the endogenous reference gene.

Methylated DNA immunoprecipitation

Purified genomic DNA prepared from cultured cells was digested overnight with proteinase K for methylated DNA immunoprecipitation (MeDIP). Digested DNA (5 μg) was used for a standard MeDIP assay as described.[16] In brief, DNA was immunoprecipitated using 10 μg of monoclonal antibody against 5-methylcytidine (Eurogentec, Liège, Belgium) in 300 μL IP buffer (10 mM sodium phosphate [pH 7.0], 140 mM NaCl, 0.05% Triton X-100) for 5 h at 4°C and then washed three times with 800 μL IP buffer. Immunoprecipitated DNA was recovered with proteinase K-digestion, followed by column-based purification (DNA Wizard, Promega, Fitchburg, WI, USA). Recovered DNA fractions were diluted and measured using qPCR method.

shRNA and transient gene transfection

DNMT3A shRNAs were purchased from OriGene Technologies (Rockville, MD, USA). They were used for knocking down DNMT3A expression [17] using lipofectamine 2000 (Invitrogen) and treated with 0.5 μg/mL of puromycin for 48 h. The total RNA from cells was subjected to reverse transcription-PCR analysis of DNMT3A expression.

Statistics

Results are mean ± standard deviation. Statistical differences were determined using a two-tailed paired Student's t-test in SPSS Statistics Software 17.0. P < 0.05 was considered statistically significant.


 > Results Top


To determine the effect of LEN on the SGC7901/VCR Notch signaling genes expression, we checked the mRNA expression of Notch subtype genes by qPCR, and it was found that LEN could reduce Notch2 mRNA expression but have no significant effect on the Notch1/Notch3 transcription, and Notch4 mRNA expression was not detected on SGC7901/VCR cells [Figure 1]a; furthermore, the DNA methylation regulator genes DNMT1, DNMT3A, and DNMT3A expression were measured and only DNMT3A expression was dramatically promoted by LEN, suggesting LEN could mediate Notch2 expression via the DNMT3A [Figure 1]b.
Figure 1: Lenalidomide regulated the Notch2 gene expression and increased the expression of DNA methyltransferase 3A. (a and b) Notch family gene expression (a) and DNA methyltransferase family gene expression (b) after lenalidomide treatment at a concentration of 2 mM in the SGC7901/vincristine cell line were detected by quantitative polymerase chain reaction. Dimethylsulfoxide treatment was a control without lenalidomide. Student's t-test, **P < 0.01, ***P < 0.001. Each experiment was performed in triplicate

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To further prove this hypothesis, we constructed the shRNA of DNMT3A. The efficacy of shRNA in SGC7901/VCR cells was verified by qPCR [Figure 2]a, consistently the in vitro depletion of DNMT3A abrogates the effect of LEN on the Notch2 mRNA expression, indicating that DNMT3A was the activator by LEN for Notch2 transcription [Figure 2]b. To investigate the underlying mechanism of LEN-DNMT3A signaling to Notch2, the methylation level of Notch2 loci was detected using MeDIP-PCR, and [Figure 2]c shows that LEN increased the methylation level of Notch2 loci, suggesting an epigenetic modification in Notch gene loci by LEN in the gastric cancer cell line.
Figure 2: Knockdown of DNA methyltransferase 3A suppressed the lenalidomide mediated Notch2 downregulation and lenalidomide modulated the 5 mC enrichment of Notch2. (a) The efficacy of DNA methyltransferase 3A shRNA. DNA methyltransferase 3A mRNA expression was measured after DNA methyltransferase 3A shRNA or control shRNA treatment. (b) Notch mRNA expression after DNA methyltransferase 3A or control shRNA treatment in the presence or absence of lenalidomide. (c) Methylated DNA immunoprecipitation assay determined the 5 mC enrichment of Notch2 in the presence or absence of lenalidomide. Student's t- test, **P < 0.01, ***P < 0.001. Each experiment was performed in triplicate

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As Notch2 would affect series of downstream genes, for example, RBP-J, Hes1/5, some critical genes among them were detected by qPCR. As shown in [Figure 3]a, [Figure 3]b, and [Figure 3]d, these genes were consistently decreased in the presence of LEN, but Hes5 exhibited no significant difference [Figure 3]c.
Figure 3: Lenalidomide regulated the downstream signaling of Notch2. (a-d) SGC7901/vincristine cells were treated with or without 2 mM lenalidomide. Levels of RBP-J (a), Hes1 (b), Hes5 (c) and Deltex1 (d) mRNA expression were detected by quantitative polymerase chain reaction. Dimethylsulfoxide treatment was a control without lenalidomide. Student's t-test, *P< 0.05, **P < 0.01, ***P < 0.001. Each experiment was performed in triplicate

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To determine the phenotype of cell function by LEN, the CCK8 staining data showed the suppression of LEN on the gastric cancer cell proliferation [Figure 4]a; meanwhile, it was supported by the Ki-67 expression using qPCR [Figure 4]b. In addition, we measured the cell death of LEN-treated cells, and there was no obvious difference, suggesting the limited cytotoxic to the cell [Figure 4]c. In addition, the LEN showed the effect on genes associated with the MDR in human gastric cancer, including the MDR genes and multidrug resistant protein (MRP), which were decreased in LEN-treated cells [Figure 4]d.
Figure 4: Lenalidomide exhibited function on the cell proliferation. (a) Cell proliferation (% of control) after 3 days incubation with dimethylsulfoxide or lenalidomide using cell counting kit-8 staining. (b) Ki-67 gene expression was detected by quantitative polymerase chain reaction on the day 3. (c) The percentage of cell death measured as propidium iodide-positive cell (%) was checked by flow cytometry. (d) mRNA expressions of multidrug resistance and multidrug resistant protein were detected by quantitative polymerase chain reaction. Student's t-test, *P< 0.05. Each experiment was performed in triplicate

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 > Discussion Top


This study reveals that LEN exhibited a proliferation inhibitory effect on SGC7901/VCR cells, resulting from the Notch2 gene. Furthermore, the treatment of LEN enhances DNMT3A in the human gastric cancer cell. It was reported that DNMT3A was responsible for the suppressed expression of retinoic acid receptor-beta2 in esophageal cancer cells,[17] but there is no report about this gene on gastric cancer. Our findings indicate that LEN epigenetically silencing Notch2 gene and the signaling via enhancing DNMT3A expression, resulting in the cell proliferation suppression phenotype. In fact, Notch inhibitors have been used for the cancer treatment.[18] LEN showed its effect on the genes with a concentration of 2 mM, which is similar to the reported dose in human myeloma H929 cell line in vitro;[19] however, here, the mechanism is different from myeloma, in which ubiquitously expressed E3 ligase protein cereblon is an identified target of LEN.[19],[20] Exposure of gastric cell to LEN interferes the Notch2 signaling which plays a role in cell proliferation, differentiation, and apoptosis. The Notch pathway is an evolutionarily conserved signal transduction system. Notch transduces signals through receptor-ligand interactions; thus, it is potentially involved in the aberrant regulation in tumor cells. Recent studies have shown that in many tumor cell lines, Notch genes were abnormally activated and aberrant Notch signaling was correlated with regulation of tumor cells, for example, Notch1 expression was increased in colon tumor tissue and promoted epithelial to mesenchymal transition.[21] However, our findings showed the exogenous LEN could directly or indirectly suppress the transcription of the specific Notch2 gene without affecting Notch1 or Notch3/4. To further elucidate the mechanism of gene expression, the DNA methyltransferases were analyzed and DNMT3A was found highly expressed after LEN treatment, implicating the silence of the gene by DNA methylation, which is the major epigenetic change involved in LEN treatment. MeDIP assay confirms that LEN could increase the 5 mC marker in Notch2 gene loci compared with DMSO control. Furthermore, LEN also reduced the downstream critical genes compared with the DMSO control; the results confirm the effect of LEN on Notch pathway. The proliferation phenotype was checked by CCK8 assay as well as Ki-67 expression. Considering MDR is an important factor that contributes to the success or failure of chemotherapy in various types of cancer including gastric cancer and patients often show resistance to a number of cytotoxic drugs,[22],[23] we also examined the effect of LEN as single agent on the expression of two common MDR genes, the MDR, encoding the P-glycoprotein,[24] and the MRP gene, which encodes the MRP. LEN reduced the expression of both MDR and MRP.


 > Conclusion Top


LEN showed a negative effect on the Notch2 gene in SGC7901/VCR cell due to the DNA methylation process by DNMT3A; series of Notch2 downstream genes was also regulated by LEN treatment resulting in the proliferation suppression phenotype. Meanwhile, LEN could reduce the MDR gene expression of MDR and MRP. Taken together, our data demonstrated that LEN would be useful in clinical gastric cancer therapy for its inhibition on cell proliferation and the effect on MDR.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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