Effects of telomerase inhibitor on epigenetic chromatin modification enzymes in malignancies
1 | INTRODUCTION
Telomeres are guanine‐rich 5′‐TTAGGG‐3′ repeats that provide DNA stability, which consist of about 10 to 15 kilobytes (kb) of DNA at the ends of human chromosomes. Telomeric repeats on the chromosome are extended with an enzyme called telomerase. Telomerase is a DNA polymerase enzyme that is a ribonucleoprotein construct, which contains both RNA and protein.1 Telomerase consists of two essential subunits: a highly conserved catalytic sub- unit (telomerase reverse transcriptase [TERT]) and an RNA component (telomerase RNA component [TERC]), both of which are required for telomerase activity.2 The level of telomerase enzyme is very low in healthy cells, but this enzyme is overexpressed in 80% to 95% of human cancers.
A special functional complex telomerase regulates several cellular mechanisms such as cell proliferation, gene expression, apoptosis, and cellular aging, and are also associated with human diseases such as cancer initiation, tumor maintaining, and therapy resistance.5
2‐[(E)‐3‐Naphtalen‐2‐yl‐but‐2‐enoylamino]‐benzoic acid (BIBR1532) is a selective small‐molecule inhibitor of telomerase enzyme, and it suppresses the human TERT (hTERT) active site. BIBR1532 exhibits a noncompetitive inhibition pattern and is highly specific as a synthetic agent.6
There are four types of leukemia: acute myeloid leukemia, chronic myeloid leukemia (CML), acute lymphocytic leukemia, and chronic lymphocytic leu- kemia. Most types of cancer such as the lung and breast spread to bone marrow from the primary tumor origin, but this situation is different in leukemia. All leukemia subtypes have different prognosis, eg, chronic leukemia patients can live for many years, as the disease does not show itself immediately, and the treatment is more difficult than acute leukemia.7
According to the American Cancer Society’s reports, about 15% of all leukemia new cases are CML in the United States as for 2017.8
Most of the CML cases have a reciprocal translocation of t(9;22)(q34;q11), which is termed as Philadelphia chromosome. As a result of this translocation, the BCR‐ABL1 fusion gene and oncoproteins are formed. ABL1 gene located on chromosome 9 has a tyrosine kinase activity, and it translocates to the BCR gene found on chromosome 22. As a result, this gene shows excessive tyrosine kinase activity.9 K‐562 is a CML cell line originated from the bone marrow of human, and these cells show suspension cell culture properties.
Eighty percentage of the malignant brain tumors are gliomas. Gliomas are aggressive, invasive, and neurolo- gical destructive subtype of primary brain tumors. Gliomas are classified according to both histological properties and the grades of malignancies. Three conventional subtypes of gliomas are astrocytomas, oligodendrogliomas, and ependymomas. According to the grade of malignancy, they are classified as the grade II and III astrocytic tumors, the grade II and III oligodendrogliomas, the grade IV glioblastomas, pediatric diffuse gliomas.10 Telomerase expression is noticed in high levels mostly in malignant gliomas.11
According to Ferrandon et al12 the imetelstat, the telomerase inhibitor, in vivo and in vitro, reduces tumor mass, suppresses proliferation, and increases sensitivity to radiotherapy. Telomeres and telomerase enzyme have been studied in many hematologic malignancies, especially in CML. CML patients have been shown to have a slightly shorter telomeric length than healthy controls. Previous studies suggest that CML patients have a slightly shorter telomeric length than healthy controls, and there is a statistically significant correlation between aging and telomeric shortening.13 We hypothesized that telomerase inhibitors would have cytotoxic, apoptotic, and gene expressions effects on both cells.
2 | MATERIALS AND METHODS
2.1 | Cell culture
Human CML cell line K‐562 (cat no. CCL‐243) and human glioma cell line U87MG (cat no. HTB‐14) were obtained from ATCC. K‐562 and U87MG cells were cultured in 25 and 75‐cm2 cell culture flasks in RPMI‐1640 (Bio Ind, USA) medium and Dulbecco modified Eagle medium (Bio Ind), respectively. Then, the cells were incubated in a cell culture incubator (Thermo Electron Corporation’s Class 100, USA) at 37°C, 95% humidity, and 5% CO2. Cell viability and proliferation controls were performed with trypan blue dye exclusion test.
2.2 | Chemical agents
BIBR1532, provided by ApexBio Technology (USA), was dissolved in 1 mL dimethyl sulfoxide (Merck Millipore, USA) to adjust the stock concentration to 10 mM. The BIBR1532 stock solution was diluted to prepare working solutions. To determine the cytotoxic values of the BIBR1532 inhibitor in the U87MG and K‐562 cell lines, the experiments were performed at the dose range of 3.125 to 100 μM.
2.3 | Cell proliferation assays
The optimum concentrations for each cell types during the 72‐hour incubation period were determined using the WST‐1 Cell Proliferation Assay Kit (Roche). K‐562 and U87MG cells were incubated at a density of 1.95 × 103 to 1 × 106 cells/mL in 1 mL of RPMI‐1640 and Dulbecco modified Eagle medium in 96‐well plates, respectively. WST‐1 solution was added, 10 μL/well, as specified in the user guide. The quantitative value of formazan dye transformation was measured using Multiskan FC (Ther- mo Fisher Scientific, USA) microplate reader at 450‐ and 620‐nm wavelengths. Optimal cell concentrations were calculated using GraphPad Prism 5.0 (GraphPad Software, USA) software at the 72nd hour.
2.4 | Cytotoxicity assays
The cytotoxic activity of BIBR1532 in K‐562 and U87MG cells were determined using the WST‐1 Cell Proliferation Assay Kit (Roche). Cells were incubated at the concen- tration of 1.25 × 105 cells/mL, and BIBR1532 was added to cells in a dose range of 3.125 to 100 μM in 96‐well plates for 24‐, 48‐, and 72‐hour incubation periods. The quantitative value of formazan dye transformation was measured using Multiskan FC (Thermo Fisher Scientific) microplate reader at 450‐ and 620‐nm wavelengths. BIBR1532‐free cell groups were used as the control groups for each cell line. IC50 doses of BIBR1532 on K‐562 and U87MG cells were calculated using CalcuSyn software (Biosoft, USA) in a time‐dependent and dose‐ dependent manner.
2.5 | Apoptosis assays
The Annexin V‐FITC Detection Kit (BD Pharmingen, USA) and flow cytometry (BD Accuri C6, USA) were used for apoptosis analyses. An apoptosis assay was performed to observe the apoptotic effect of the untreated control groups compared with the BIBR1532‐treated cell lines. Phosphatidylserine translocates to the outside of the cell membrane in both early and late apoptotic cells. Apoptotic cells were detected using Annexin V‐FITC Kit, and necrotic cells were detected by a different dye, propidium iodide.
2.6 | Cell cycle assay
After 72 hours of BIBR1532 treatment, cell cycle analysis was performed for K‐562 and U87MG cell lines. Cycletest Plus DNA Reagent Kit (BD Biosciences, UK) was used to determine the cell cycle arrest, and the results were measured by flow cytometry.
2.7 | Gene expression analyses
Total RNA isolation was performed using RNeasy Plus Mini Kit (Qiagen, Germany). The amount and purity of RNA samples were checked by a spectrophotometer (NanoDrop 1000, USA). Complementary DNA synthesis was performed using the RT2 First Strand Kit (Qiagen).
Real‐time polymerase chain reaction array was used for gene expression analysis (Qiagen). Gene expression changes occurring through the treatment with BIBR1532 on K‐562 and U87MG cells were determined with Light-Cycler 480 Instrument II (Roche). Data analyses were achieved by 2−ΔΔCt method (LightCycler 480 Quantifica- tion Software, USA).
TeloTAGGG hTERT Quantification Kit (Roche) was used for hTERT activity on cell lines of the IC50 dose of the BIBR1532 agent. hTERT gene expression changes were studied via real‐time polymerase chain reaction using LightCycler 480 Instrument II (Roche). Data analyses were achieved by relative quantification (LightCycler 480 Quantification Software).
3 | RESULTS
3.1 | Optimum cell concentrations
K‐562 and U87MG cell numbers and absorbance values are shown in Figure 1. Optimum cell concentrations were determined as 1.25 × 105 cells/mL for both K‐562 and U87MG cell lines with the WST‐1 assay for 72‐hour incubation period.
3.2 | Cytotoxicity results
The IC50 of the BIBR1532 inhibitor in the K‐562 and U87MG cell lines was calculated to be 8.56 and 32 μM at 72 hours, respectively (Figure 2). BIBR1532 has the cytotoxic effect on K‐562 and U87MG.
3.3 | Apoptosis results
Apoptosis results were evaluated using annexin V method by flow cytometry (Figure 3). We observed that BIBR1532 treatment increased apoptosis by 2.41‐fold in U87MG cells compared with the control. The survival rate of the cells was determined to be 79.9% in the control cells and 55.8% in the BIBR1532‐treated cells. Apoptosis rates of control and dose were determined as 17.8% and 42.9%, respectively. We observed that apoptosis was not induced too much in K‐562 cells with BIBR1532 treatment compared with control cells. The survival rates of the cells were determined to be 98.4% in the control cells and 97.5% in the BIBR1532‐treated cells. Apoptosis rates of control and dose groups were determined as 1.1% and 1.9%, respectively.
FIGURE 1 Optimal cell concentration in 72‐hour incubation periods for K‐562 (A) and U87MG (B) cell lines.
FIGURE 2 IC50 values of K‐562 (A) and U87MG (B) cell lines.
FIGURE 3 Apoptotic effect of BIBR1532 agent in U87MG (A) and K‐562 (B) cell lines. BIBR1532, (2‐[(E)‐3‐naphtalen‐2‐yl‐ but‐2‐enoylamino]‐benzoic acid).
3.4 | Cell cycle analysis results
Cycletest Plus DNA Reagent Kit was used for cell cycle analysis (Figure 4). We observed that the IC50 dose of BIBR1532 blocked the cell cycle in the G0/G1 phase in both cell lines. According to cell cycle results of K‐562 cell lines, control cells accrued 73.3% in the G0/G1 phase, 10.6% in the S phase, and 15.4% in the G2/M phase. BIBR1532‐treated cells accumulated 80.0% in the G0/G1 phase, 7.6% in the S phase, and 8.3% in the G2/M phase.
According to cell cycle results of U87MG cell lines, control cells accrued 62.2% in the G0/G1 phase, 22.7% in the S phase, and 13.2% in the G2/M phase. BIBR1532‐ treated cells accumulated 71.6% in the G0/G1 phase, 13.4% in the S phase, and 12.4% in the G2/M phase.
3.5 | Gene expression analysis results
The expression profiles of genes belonging to epigenetic chromatin modification enzymes were determined as 8.56 and 32 μM doses of BIBR1532 treatment at the 72nd hour on K‐562 and U87MG cells according to the control group, respectively (Table 1). The hTERT activity on the cell lines treated with the BIBR1532 agent was assessed at 72nd hour. TeloTAGGG hTERT Quantification Kit (Roche) was used (Figure 5). hTERT activity in the K‐562 control cells was determined to be 9.36E−5, and BIBR1532 dose treated K‐562 cells was determined to be 1.25E−6. hTERT activity in the U87MG control cells were determined to be 6.19E−3, and BIBR1532 dose treated U87MG cells were determined to be 4.39E−3. The treatment of BIBR1532 significantly reduced hTERT gene expression in the K‐562 cell line. hTERT gene expression decreased in U87MG cells treated with BIBR1532.
FIGURE 4 Cell cycle analysis results of K‐562 (A) and U87MG (B) cell lines.
4 | DISCUSSION
In this study, we aimed to investigate the possible effects of telomerase inhibitor BIBR1532 on cell growth, apoptosis, cell cycle, viability, and gene expressions on K‐562 and U87MG cell lines. We observed the cytotoxic effect on our cell lines.BIBR1532 is a selective inhibitor of cancer cells that does not affect normal human cells.14 In a study carried out by Damm et al15 telomere restriction fragment was shown to be shortened from 4 to 1.5 kb after application of BIBR1532, while in untreated cells it was in stable length.Although there are studies on the IC50 dose of BIBR1532, the IC50 value has not been discovered yet for K‐562 and U87MG cell lines in the literature. In this study, the IC50 values of BIBR1532 were found to be 8.56 and 32 μM at the 72nd hour, respectively. A concentra- tion of 32 μM stopped cell growth and induced apoptosis in U87MG cell lines. BIBR1532 inhibited cell prolifera- tion in both of cells. The BIBR1532 agent was ineffective in the apoptosis of K‐562 cells.
FIGURE 5 hTERT gene expression changes in K‐562 (A) and U87MG (B) cell lines after BIBR1532 treatment.
Different studies on cancer cells exposed to BIBR1532 support the cytotoxic effect of this drug. We observed that the IC50 dose of BIBR1532 blocked the cell cycle in the G0/G1 phase in both cell lines. According to Ladetto et al16 BIBR1532 affects at G1 phase in most cancer types. The study of Shi et al17 shows that BIBR1532 inhibited cell proliferation and induced a G1‐phase cell cycle arrest in breast cancer cell lines.
It has been reported that the BIBR1532 telomerase inhibitor suppresses c‐Myc and hTERT expression in pre‐ B acute lymphoblastic leukemia cells.18 We observed that the treatment of BIBR1532 significantly reduced hTERT gene expression in the K‐562 cell line. Recent studies have shown that epigenetic modifica- tions have significant roles in the regulation of human telomeres. Telomeric and subtelomeric chromatin re- gions are enriched in chromatin modifications that are characteristic of constitutive heterochromatin domains in mammals.19 We found that telomere inhibition is associated with different epigenetic elements in both types of cells, such as HDAC10, DNMT1, KAT6B, PRMT3, PRMT5, USP21, and SUV420H1 genes.
Histone deacetylases (HDACs) provide for removing the acetyl groups on histones that is added by the histone acetyltransferases. HDACs are essential for epigenetic regulation of gene expression, chromosome structure, and control of cellular stability, and are associated with the loss of genomic integrity in cancer cells. In humans, 18 HDAC enzymes are divided into four classes. The class I proteins include HDAC1, HDAC2, HDAC3, and HDAC8. The class II proteins include HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10. The class III proteins include SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7. The class IV protein includes HDAC11.
According to Lucio‐Eterovic et al21 HDAC II and IV were shown to be downregulated in high‐grade gliomas rather than astrocytomas in both protein and messenger RNA levels. This study supports the positive effect of our drug BIBR1532 that increases the expression levels of HDAC10 on U87MG glioma cells. Similar to this study, we observed that HDAC10 gene expression increased in both cells.
We found that BIBR1532 treatment increased HDAC4 and HDAC11 gene expression levels 3.00 and 2.50 folds, respectively. HDAC4 and HDAC11 gene expressions are downregulated in gliomas and might play pivotal roles in glioma malignancy. Recent studies have demonstrated a negative correlation between HDAC gene expression and glioma grade, suggesting that HDAC4 and HDAC11 might play important roles in glioma malignancy.21
According to Rajendran et al22 DNA methyltransferase 1 (DNMT1) is shown to be overexpressed in gliomas. DNMT1 is a tumor suppressor gene and essential for the sustain- ability of methylation. DNMT1 inhibits tumor growth, increases activation of p53, thus inducing apoptosis in glioma. Hypermethylation by DNMTs, especially DNMT1, is associated with epigenetic silencing of oncogenes, and upregulation of DNMT1 inhibits tumor growth.23 We found 2.50‐fold overexpression of DNMT1 gene expression in BIBR1532‐treated K‐562 and U87MG cells compared with
unexposed control cells. On the other hand, the expression level of DNMT1 significantly upregulated in patients with blast phase CML and K‐562 cells.24 DNMT1 expression increased in BIBR1532‐treated K‐562 cells.
DNMT3a and DNMT3b prevent malignant mouse lymphopoiesis by their tumor‐suppressor functions.25 Previous studies have shown that the inactivation of DNMT3a in hematopoietic cells related to the occurrence of chronic lymphocytic leukemia in mice.26 We observed that DNMT3a gene expression increased in BIBR1532‐ treated K‐562 cell lines.
Lysine acetyltransferases (KATs) family member KAT6B has tumor‐suppressor properties with histone H3 Lys23 acetyltransferase activity.27 We observed that BIBR1532 inhibitor had increased KAT6B gene expression levels by 3.31‐fold. Consequently, we thought that the inhibition of telomerase enzyme by BIBR1532 regulates epigenetic pathways and is crucial to target it for glioma treatment.
A study on small‐cell‐lung cancer, in vivo and in vitro, determined that a decreased expression of KAT6B enhances cancer growth, while restoration of it showed tumor‐suppressor effect.27 We observed high messenger RNA expression levels of KAT6B after treatment of BIBR1532 in both hematological malignancy (K‐562) and solid tumor (U87MG).
Histone methylation of hTERT promoter is vital in human cancer. Also, tumor suppressor gene SET domain containing protein 2 (SETD2) is downregulated in cancer cells. TERT is related with the SETD2, which is a histone methyltransferase and suppresses telomerase.28 RPS6KA3 (histone and TFs kinase) phosphorylates, activates p53 and induced apoptosis.29 We observed that BIBR1532 upregulated SETD2 gene expression
by 5.35‐fold (2−ΔΔCt log 2 value) and RPS6KA3 gene expression by 3.81‐fold (2−ΔΔCt log 2 value) in U87MG cells.
Protein arginine‐methyltransferase 1 (PRMT1) was upregulated in gliomas compared with normal brain cells and the inhibition of PRMT1 induces apoptosis.According to our data PRMT1 expression is decreased by 5.14‐fold ( 2−ΔΔCt log 2 value). PRMT5 is very important and necessary for p53 activity and the inhibition of PRMT5 prevent p53 protein synthesis.32 PRMT5 dysfunction is related to kidney disease, heart disease, neurological disorders, and many cancers such as lymphomas, lung cancer, and breast cancer. According to Peng et al33 ubiquitin‐specific protease 21 (USP21) is overexpressed in triple‐negative breast cancer lines. In contrast, BIBR1532‐treated cells dis- played higher levels of USP21 compared with both untreated U87MG and K‐562 cells. Consequently, we thought that BIBR1532 is effective on epigenetic mechanisms. Moreover, it is important to identify treatment targeting for hematological malignan- cies and solid tumors.
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