|Year : 2016 | Volume
| Issue : 2 | Page : 811-817
Flavopiridol's antiproliferative effects in glioblastoma multiforme
Gozde Cobanoglu1, Irem Dogan Turacli2, Ayla Cihan Ozkan1, Abdullah Ekmekci1
1 Department of Medical Biology, Faculty of Medicine, Gazi University, Ankara, Turkey
2 Department of Medical Biology, Faculty of Medicine, Ufuk University, Ankara, Turkey
|Date of Web Publication||25-Jul-2016|
Irem Dogan Turacli
Department of Medical Biology, Faculty of Medicine, Ufuk University, Ankara
Source of Support: None, Conflict of Interest: None
Aim of Study: Glioblastoma multiforme (GBM) is largely refractory to surgical operation, radiotherapy, and chemotherapy in use today. Remaining lifetime accounting for the GBM-affected patients varies between 12 and 16 months generally. The most frequently altered genes in GBM are p53, epidermal growth factor receptor, PTEN, and cyclin-dependent kinase inhibitor 2A. Our aim is to investigate the antiproliferative and apoptotic effects of flavopiridol, a cyclin-dependent kinases and specific phosphokinase inhibitor, on glioblastoma cell lines having different genetic profiles: U87MG, U118MG, and T98G.
Materials and Methods: Cell viability and IC50 values were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, protein expressions were determined by Western blot and caspase activities were analyzed by activity kit.
Results: Western blot analysis showed down-regulation of the cyclin D1, c-Myc, and p53 protein activities, and up-regulation of p27KIP1 activity after flavopiridol treatment. Additionally, flavopiridol diminished p-Akt protein levels generally which induces inhibition of proliferation.
Conclusion: The present study demonstrated that flavopiridol did not induce caspase-3/7 activation, BIM, and BAX pro-apoptotic proteins but it leads to the expression changes of various proteins that inhibit proliferation and eternity in glioblastoma cell lines which have different genetic alterations.
Keywords: Apoptosis, flavopiridol, glioblastoma multiforme, proliferation
|How to cite this article:|
Cobanoglu G, Turacli ID, Ozkan AC, Ekmekci A. Flavopiridol's antiproliferative effects in glioblastoma multiforme. J Can Res Ther 2016;12:811-7
| > Introduction|| |
Gliomas are the tumors arising from glia cells or their precursors in the central nervous system. Gliomas are divided into four subgroups as astrocytoma Grades I, II, III, and IV, oligodendrogliomas, ependymomas, and mixed gliomas. Grade IV astrocytoma is known as glioblastoma multiforme (GBM), which is the most aggressive and common form of glioma.,
There are several genetic alterations associated with GBM formation and progression such as mutations, amplifications, epigenetic, and gene expression changes. The common genetic alterations include epidermal growth factor receptor (EGFR) amplification, INK4a-ARF loss, RB loss, p53 mutations, MET amplification, PIK3CA mutations, ERBB2 mutation, PDGFR overexpression, cyclin-dependent kinase 4 (CDK4) and CDK6 amplifications.,,, One of the well-characterized mutations in the EGFR gene generates a truncated EGFR protein known as EGFRvIII, which lacks exons 2–7 of the EGFR gene. Also, amplification or overexpression of EGFR is a hallmark of GBM. EGFR amplification is associated with deletion of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene locus.
The PI3K/Akt pathway is usually activated in glioblastoma cells. Growth factor receptor mutations (EGFRvIII), PTEN, and PIK3CA mutations, Akt activation induce these pathway alterations. Phosphorylation of Akt at S473 and T308 is required for full activation of Akt., When Akt is activated, it induces activation of several proteins that play key roles in cell proliferation, apoptosis, and metabolism. PRAS40, FOXOs, MDM2, p21, p27, BAD, NF-κb, caspase-9, TSC2 are a few examples of Akt substrates.
Cell cycle control is another antitumorigenic approach in glioblastoma therapy. p53 tumor suppressor protein has pivotal functions in the signaling pathways required to mediate cell cycle and apoptosis. It interacts with the promoters of p21, BAX, and many regulatory genes that have roles on apoptosis and cell cycle., Among the cell cycle proteins, the cyclins, CDKs, and CDK inhibitors have roles in glial transformation. In response to stimulating signaling from upstream pathways, cyclin D/CDK4, cyclin D/CDK6, and cyclin E/CDK2 complexes sequentially coordinate to phosphorylate and inactivate RB. These phosphorylations disrupt RB-E2F cooperation leading to induction of E2F-regulated gene expression and cell proliferation. Due to RB repression, the cell progresses into S-phase. The activity of CDKs is suppressed by CDK inhibitors such as CIP/KIP (p21WAF1/CIP1, p27KIP1, and p57KIP2) and INK4 family (p15INK4B, p16INK4A, p18INK4C, and p19INK4D). So, CDK inhibitor pathway activation might be a promising strategy to improve efficacy of cancer treatments in GBM.
Flavopiridol is a semisynthetic flavonoid obtained from dysoxylum binectariferum. Flavopiridol inhibits in vitro cell growth through CDKs (CDK2, CDK4, and CDK6) in G1/S or G2/M of cell cycle., Flavopiridol has been shown to delay tumor growth and cell migration and it depletes cyclin D1, CDK4, and Bcl-2 expression in GL261 murine glioma cells. Also, flavopiridol treatment induces caspase-dependent and independent apoptosis pathways. While flavopiridol induces caspase and cytochrome c independent pathway in human glioma cells, it is mitochondrial mediated in murine glioma cells., Also, flavopiridol plays an important role in the progression to treatment resistance by reducing JUNB gene expression in multiple breast cancer cell lines.
The genetic alterations on constitutively activated/inactivated signal transduction pathways such as p16, p53, PTEN, EGFR, and their aberrant protein expressions can be rational targets for GBM. Despite several agents that are developed for targeted therapies, tumors got resistance to current therapeutics. Today, alkylating agent temozolomide is used for the treatment of GBM after tumor resection and radiotherapy. However, median survival is still limited to 15 months. Flavopiridol, inhibitor of broad-spectrum CDKs, causes apoptosis and cell cycle arrest at the G1/S and G2/M boundaries. In this study, we investigated the antiproliferative and apoptotic effects of flavopiridol on GBM cell lines based on their genetic background [Table 1].
| > Materials and Methods|| |
Cells and reagents
All the cell lines used in this study (U87MG, T98G, and U118MG) were grown in DMEM (Lonza) supplemented with 5% heat-inactivated fetal bovine serum (Lonza), 100 U/ml penicillin and 100 μg/ml streptomycin (Lonza). Cells were grown in a humidified incubator containing 5% CO2 and 95% air at 37°C.
Flavopiridol was obtained from Enzo. A 10 mM stock solution was prepared in dimethyl sulfoxide (DMSO). Flavopiridol (150 nM-10 µM) was diluted and added to cells that were growing on cell culture dishes. All assays were performed at 24, 48, and 72 h of incubation.
Cell viability was determined by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in DMEM at 7 × 103 cells/200 µl per well in 96-well plates for 24 h at 37°C. When cells were attached after 24 h, cells were treated with DMSO (control group) and flavopiridol (156 nM; 312 nM; 625 nM; 1.25 µM, 2.5 µM, 5 µM, and 10 µM) for 24, 48, and 72 h periods. After incubation time, 10 µl MTT solution (5 mg/ml) was added to each well. After 4 h of MTT incubation at 37°C, 100 µl crystal dissolving buffer was added and the plates were gently shaked on an orbital shaker for 5 min. The absorbance at 570 nm was measured with a microplate reader. Each treatment was repeated 4 times. The mean absorbance of four wells was used as an indicator of relative cell growth.
Cells were plated at a density of 5 × 105 per well in six-well plates. The following day, cells were treated with drug or equal volume of DMSO for 6, 24, and 48 h. Cell extracts were prepared by adding 2x Laemmli sample buffer supplemented with a phosphatase and protease inhibitor cocktail. Lysates were sonicated and the protein concentration was quantified using bicinchoninic acid (BCA) kit (Thermo, MA, USA). Equivalent protein was loaded and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis then transferred to 0.45 µm nitrocellulose membranes. Equivalent loading was confirmed by staining membranes with Ponceau S dye. Membranes were blocked for 1 h in blocking buffer (5% milk, 1× TBS, 0.1% Tween 20) and placed in primary antibody (5% bovine serum albumin, 1× TBS, 0.1% Tween 20) overnight at 4°C. The following day, membranes were washed thrice in wash buffer (1× TBS, 0.1% Tween 20). Primary antibodies against Akt, p-Akt, p53, p-p53, EGFR, p-EGFR, p27, BIM, BAX, cyclin D1, c-Myc, GAPDH, secondary anti-rabbit IgG, and anti-mouse IgG were purchased from Cell Signaling Technologies (Danvers, MA, USA). Proteins were detected using horseradish peroxidase-linked secondary antibodies and visualized with the enhanced chemiluminescent detection system (GE Healthcare Biosciences, Pittsburgh, PA, USA). Immunoblot experiments were performed at least twice.
Caspase 3/7 protein activity
About 1× 106 cells were seeded in 6-well plates and incubated with DMSO (control group) and flavopiridol for 6, 24, and 48 h. Cell extracts were prepared by adding 2× Laemmli sample buffer supplemented with a phosphatase and protease inhibitor cocktail. Lysates were sonicated and protein concentration of each sample was determined by BCA kit (Thermo, MA, USA). Caspase 3/7 protein activity was measured by “AnaSpec Sensolyte Homogeneous AFC Caspase 3/7 Assay Kit” (AnaSpec, CA, USA) according to the assay protocol.
Differences in cytotoxicity and protein activity levels were analyzed using SigmaStat software (version 12.3) by Systat Software, Inc. by using Student's t-test. P < 0.05 was considered statistically significant.
| > Results|| |
Antiproliferative effects of flavopiridol (10 µM, 5 µM, 2.5 µM, 1.25 µM, 625 nM, 312 nM, 156 nM) was tested on T98G (mt-p53), U118MG (mt-p53), and U87MG (wt-p53) cells at 24, 48 and 72 h periods, respectively. For U118MG cells, although flavopiridol decreased cell viability even at 0.156 µM at 72 h, its IC50 value was seen at 0.6 µM at 48 h. Also, there was no significant death rate at the highest concentration (10 µM) at 24 h [Figure 1]a. Although it was not significant at 0.156, 0.312, 0.625, and 2.5 µM, the proliferation rate was decreased according to increased flavopiridol doses at T98G cells at 24 h. The IC50 value was determined around 300 nM at 48 h for T98G cells and all applied doses of flavopiridol decreased cell viability significantly at both 48 and 72 h periods [Figure 1]b. For U87MG cells, flavopiridol decreased proliferation significantly at 300 nM and higher doses at all-time points studied. The IC50 value was determined around 300 nM at 48 h for U87MG cells [Figure 1]c.
|Figure 1: The proliferation inhibitory effects of flavopiridol on glioblastoma multiforme cells followed by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay. Viability rates of flavopiridol were shown in a time- and dose-dependent manner. Glioblastoma multiforme cells were treated with 0.156, 0.312, 0.625, 1.25, 2.5, 5, and 10 μM of flavopiridol for 24, 48, and 72 h. (a:U118MG, b:T98G, c:U87MG cells) *P < 0.05|
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To investigate the protein expression changes between control and flavopiridol treated groups, Western blot analysis was performed for several proliferative and apoptotic proteins at 6, 24, and 48 h. Western blot analysis showed that while 600 nM flavopiridol decreased p-Akt, c-Myc, and cyclin D1 expression in a time-dependent manner, it increased p27KIP1 expression at all-time points at U118MG cells. However, p-p53 expression was not orderly changed with flavopiridol treatment. Also, the pro-apoptotic marker expressions were not coordinated with each other, whereas BAX expression was increased by flavopiridol application at 24 and 48 h, BIM expression was decreased at all time points at U118MG cells [Figure 2].
|Figure 2: Immunoblot analysis and expression changes of several proliferative and apoptotic proteins at U118MG, U87MG, and T98G cells treated with varying doses of flavopiridol for 6, 24, and 48 h. C = Vehicle control, F = Flavopiridol|
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For T98G cells, although p-Akt signal was decreased at 6 h, it was increased by 300 nM flavopiridol application at 24 and 48 h. Also, cyclin D1 expression was only decreased by flavopiridol at 48 h, where c-Myc expression was decreased at all time points. While p-p53 signal was increased at all time points, p27 protein expressions were increased with flavopiridol treatment at 24 and 48 h. Interestingly, BAX pro-apoptotic protein expression was not changed by flavopiridol, where BIM expression was decreased at 24 and 48 h [Figure 2].
Treatment with 300 nM flavopiridol reduced p-Akt signal only at 48 h at U87MG cells. Also, important cell cycle protein cyclin D1 and c-Myc expression was decreased by flavopiridol treatment at all time points. Parallel to these results, p-p53 signal was induced with flavopiridol application. On the other hand, p27 protein expression was decreased as the opposite of p-p53 signal. Also, BIM and BAX pro-apoptotic protein expressions were reduced by flavopiridol treatment at U87MG cells [Figure 2].
When the GBM cells used in this study were treated with flavopiridol (T98G (300 nM), U118MG (600 nM) and U87MG (300 nM)) at 6, 24 and 48 h, the caspase 3/7 activity was not changed significantly [Figure 3]a, [Figure 3]b, [Figure 3]c. Our results indicate that flavopiridol induced down-regulation of important regulators in cell cycle and proliferation in all cell lines independent of genetic status.
|Figure 3: In vitro caspase 3/7 protein activity at U118MG (a), U87MG (b) and T98G (c) cells in a time- and dose-dependent manner after flavopiridol administration. *P < 0.05|
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| > Discussion|| |
Understanding the mutations, cell cycle regulation and cell death will be beneficial for identifying the pathogenesis and the appropriate treatment of GBM. PTEN, EGFR, p53 mutations, and loss of chromosome 9p, which codes for CDKN2A and CDKN2B genes, are common genetic alterations in GBM. CDKN2A locus has two overlapping genes p14ARF and p16INK4a, which are entirely different tumor suppressor proteins., While p16INK4a regulates RB activation, p14ARF regulates p53 pathway. P16INK4a binds and inhibits cyclin D/CDK4/6 complex thereby preventing RB phosphorylation and causes G1/S cell cycle arrest. Thus, the loss of p16INK4a and/or p14ARF which is important for GBM formation and treatment directly affects cell cycle control. Flavopiridol is a CDK inhibitor that can be used to inhibit cell cycle progression. It has been used in clinical trials either as a single agent or in combination with other agents., Although flavopiridol is known to inhibit CDKs, it still has to be clarified how flavopiridol causes apoptosis.
In our study, we treated the GBM cell lines with various doses of flavopiridol for 24, 48, and 72 h and got IC50 values at 300 nM for U87MG and T98G, and at 600 nM for U118MG. However, Brüsselbach et al. reported IC50 concentrations were 144 nM for A549 nonsmall cell lung cancer (NSCLC) cells, 16 nM for LNCaP, 94 nM for HuVEC cells and 154 nM for LoVo cells. On the other hand, for A549 cells, 200–500 nM continuous flavopiridol resulted in reduced cell growth at 72 h. IC50 value of flavopiridol was 500 nM for CD133+/CD44+ human prostate cancer stem cells. Even the IC50 values of flavopiridol change according to cell type, in parallel with our study, Alonso et al. found flavopiridol inhibited cell proliferation at 300 nM concentration at glioma cell lines independent of p53 status.
There are distinctive studies demonstrating how flavopiridol leads to apoptosis in various cell lines. Although the tumor suppressor gene p53 is required for the efficient activation of apoptosis and p-p53 levels were upregulated by flavopiridol in T98G and U87MG cells in our study. Shapiro et al. suggested flavopiridol-mediated apoptosis was independent of p53 by using p53 wild type A549 lung carcinoma cells. Alonso et al. showed flavopiridol decreased XIAP, Bcl-XL, p21, MDM2, IAP2, and survivin mRNA levels and induces apoptosis independent of p53 and caspase-related mechanism in glioma cells. Also, overexpression of MDM2 resulted in resistance of p53 wild type U87MG cells to cisplatin-induced apoptosis but did not change p53 expression. Demidenko and Blagosklonny showed low doses of flavopiridol induces p53 by inhibiting MDM2 and p21 at HCT116 cells and also promotes apoptosis in p53 null cells. So, it still remains unclear how p53 joined flavopiridol's antitumorigenic activities in our and other studies.
In parallel with Alonso et al., the caspase 3/7 levels were not changed significantly by flavopiridol treatment in GBM cells in our study. However, Takada et al. showed flavopiridol suppressed tumor necrosis factor (TNF)-induced activation of Akt, c-Myc, Bcl-2, Bcl-XL, and several anti-apoptotic proteins. Moreover, they showed TNF-related apoptosis depends on caspase activation in human myeloid cells. Flavopiridol-induced growth inhibition and apoptosis at 500 nM results in a significant increase in immunofluorescence staining of caspase-3, caspase-8, and p53 in human prostate cancer stem cells. This discrepancy on dependence on caspase activation may be due to cancer stem cell dynamics or different cancer cell types. Also, treatment of multiple myeloma cell lines with 200 nM flavopiridol induced apoptotic cell death but decreased anti-apoptotic Mcl-1 protein and mRNA expression. Also, 200 nM flavopiridol treatment resulted in evaluation of E2F1 protein which represses Mcl-1 and induce apoptosis in H1299 lung carcinoma cell line.
PI3K-Akt signaling pathway inhibits apoptosis by regulating several cell survival molecules. In cerebellar granule neurons, flavopiridol has shown to be a neuroprotective agent in such a manner that it inhibited GSK3b and Akt and prevent apoptosis which is induced by a PI3K inhibitor. This finding has important implications on chemoresistance mechanisms in GBM. Also, Akt phosphorylation was decreased in a time-dependent manner with flavopiridol treatment in U118MG and U87MG cells in our study. It could be interpreted as flavopiridol has antiproliferative and kinase inhibiting effects on PI3K and PDK1 kinases.
BAX and BIM proteins are the markers which can induce apoptosis in cancer cells. However, we could not observe increased BAX protein expression with flavopiridol treatment. Similar to our study, in B-cell chronic lymphoblastic leukemia cells, flavopiridol administration inhibited Bcl-2 and Mcl-1 expression, but it did not induce BAX expression. On the other hand, Pei et al. showed flavopiridol and its combination with Bcl-2 inhibitor HA14-1 triggered mitochondrial damage and lead to mitochondrial translocation of BAX in human myeloma cells. Also, BAX was upregulated and cyclin D1 was downregulated by flavopiridol in Eca109 esophageal cancer cells. In line with previous work in wild type and mutant p53 glioma cell lines, Alonso et al. observed Bcl-2 protein expression was downregulated whereas BAX protein levels were upregulated at 24 and 48 h, resulting in a low Bcl-2/BAX ratio, which is an indicator of apoptosis. We could not observe BIM protein upregulation with flavopiridol administration in our study. However, combination of flavopiridol and obatoclax, a BH3-mimetic, inhibited Mcl-1 and triggered BIM transcription and induce apoptosis in human myeloma cells.
Although we could not see apoptotic stimulus in our study, proliferation markers were downregulated by flavopiridol application in glioblastoma cell lines. In parallel with our study, Alonso et al. showed cyclin D1 levels decreased after flavopiridol treatment. By decreasing cyclin D1 protein levels, CDK4 kinase activity is decreased, which results in the Rb hypophosphorylation that results in growth arrest at G1/S-phase. Also, it has been reported flavopiridol decreased cyclin D1 expression in MCF-7 cells. At the same time, c-Myc expression was inhibited by flavopiridol in all cell lines in our study. The decrease in c-Myc expression may be due to inhibition of cyclins and CDKs by flavopiridol in GBM cells. Similar to our findings, flavopiridol suppressed TNF-induced c-Myc expression in human myeloid cells.
p27KIP1, a CDK inhibitor and a negative regulator of cell cycle, was upregulated by flavopiridol in p53 mutant U118MG and T98G cells in our study. However, we observed it was upregulated by flavopiridol in p53 wild-type U87MG cells. Also, Shapiro et al. showed flavopiridol-induced p27KIP1 cleavage in p53 wild type A549 NSCLC cells, which may be a consequence of apoptosis. In B cell chronic lymphocytic leukemia cells, flavopiridol downregulated p27KIP1 expresssion. Lechpammer et al. showed N-methyl-N-benzylnitrosamine and gastroduodenal-esophageal reflux induces Barrett's esophagus and malignant transformation of the esophageal mucosa in p27 knockout mice. Flavopiridol treatment reduced cyclin D1 expression and Barrett's esophagus formation in those mice. In uterine leiomyoma cells, flavopiridol has been used as chemopreventive and therapeutic agent that induced G1 cell cycle arrest and increased p27KIP1 and p21 expressions.
In summary, the present study focused on the antiproliferative and apoptotic effects of flavopiridol on GBM cell lines based on their genetic background. The expression profiles of our apoptotic markers showed no sign of flavopiridol-induced apoptosis. Although we could not observe apoptotic stimulus, the proliferation rates were decreased with flavopiridol treatment in all cell lines used in this study regardless of their genetic makeup. It is important to emphasize that these results probably indicates other pathways or apoptotic markers that we did not search are changed by flavopiridol application. Further in vivo studies are necessary to clarify the possible mechanisms and other signals to solve flavopiridol's effects on cell cycle and apoptosis.
This study was supported by the Gazi University Research Fund as a research project with code number 01/2010-13.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]