Comparison of antiproliferative and apoptotic effects of a novel proteasome inhibitor MLN2238 with bortezomib on K562 chronic myeloid leukemia cells
Selin Engu¨r, Miri¸s Dikmen, and Yusuf O¨ ztu¨rk
Department of Pharmacology, Faculty of Pharmacy, Anadolu University, Eski¸sehir, Turkey
Abstract
Inhibition of the proteasome has emerged as a clinically effective anticancer therapeutic approach in recent years. Bortezomib (Velcadeti ) showed extremely high potency against a wide range of cancer cell lines. Ixazomib (MLN9708-MLN2238), the second-generation proteasome inhibitor, selectivity and potency were similar to that of bortezomib, is currently being investigated in phase I studies. It shows superior antitumor activity in hematologic malignancy, especially multiple myelomas. In this study, for the first time, we evaluated and compared the antiproliferative and apoptotic effects of the novel proteasome inhibitor MLN2238 (the active form of MLN9708) with bortezomib using in vitro chronic myeloid leukemia. Cytotoxic and apoptotic effects of MLN2238 and bortezomib were determined by trypan blue dye exclusion assays, WST-1 cell proliferation assay, increased AnnexinV-PI binding capacity, changes in caspase-3 activity and loss of mitochondrial membrane potential (JC-1). Associated with proteasome pathway NFkB1 and c-myc mRNA expression levels were examined by the qRT-PCR method. We observed that cytotoxic and apoptotic effects on K562 cells were started at 5 mm of MLN2238 and 1 mm of bortezomib after 24 and 48 h. Also, MLN2238 and bortezomib downregulated NFkB1 and c-myc mRNA expression at 24 h. Our result revealed that MLN22238 and bortezomib had significant cytotoxic and apoptotic effects on K562 cells. Here, we first demonstrate in vitro data that support the development of MLN2238, by direct comparison with bortezomib on K562 cells.
Keywords
Apoptosis, bortezomib, K562, MLN9708,
proteasome inhibitor
History
Received 12 August 2015 Revised 3 November 2015 Accepted 17 November 2015
Published online 10 December 2015
Introduction
Chronic myeloid leukemia (CML) is a clonal disorder of the pluripotent hematopoietic stem cell and the first type of leukemia with pathogenesis originates from a chromosomal translocation. The resultant reciprocal translocation structure is called a Philadelphia chromosome and occurs between the long arms of chromosomes 9 and 22 generates breakpoint cluster region/Abelson leukemia gene (BCR/ABL) oncogene, which is the main cause of CML. This translocation leads the transformation to malignancy. BCR/ABL inhibits apoptosis and induces cell growth and proliferation1–3. Activation of apoptosis by novel proteasome inhibitors is a new therapeutic strategy for the treatment of CML. The major pathways to apoptosis associated with caspase activation. For this reason, proteasome inhibition which results with caspase activation is an attractive target4. The ubiquitin–proteasome system is a major pathway for protein degradation. Targeting this path- way using proteasome inhibitors represents a novel approach for the treatment of cancer. Proteasome inhibitors lower cell
proliferation and induce apoptosis in solid and hemato- logic malignancies through multiple mechanisms. Therapeutically, proteasome inhibitors display a broad- spectrum of antiproliferative or proapoptotic activity in vitro against different types of hematological malignancies5, including multiple myelomas (MM)6, mantle cell lymphoma7, B-cell lymphoma8, T-cell leukemia9, Hodgkin lymophoma10, Burkitt’s lymphoma11, acute and CML12,13, chronic lympho- cytic leukemia14.
Bortezomib is a well-characterized antitumor drug and from first-generation proteasome inhibitor family which was approved by Food and Drug Administration (FDA) for clinical use for the treatment of refractory MM and mantle cell lymphoma15. It is a specific and reversible inhibitor of the chymotryptic activity of the 26S proteasome and has a wide range of molecular effects, including inhibition of nuclear factor kB (NFkB), abrogation of tumor growth and survival, induction of apoptosis16. Bortezomib, not only prolonged life span and was shown to be superior to high-dose dexametha- sone for relapsed MM patients17, but also induced cell apoptosis in CML and lymphoma cells18. Despite the clinical success of bortezomib, it has been associated with many
Address for correspondence: Assoc. Prof. Dr. Miri¸s Dikmen, Department of Pharmacology, Faculty of Pharmacy, Anadolu University, Eskisehir 26470, Turkey. Tel: +90 222 3350750. E-mail: [email protected]
deficiencies, including cytotoxic side effects and the devel- opment of drug resistance19.
Recently, several new agents have been introduced into the field, including Ixazomib (MLN9708-MLN2238), and trials investigating these second-generation proteasome inhibitors have demonstrated promising results20. MLN9708 is an investigational small-molecule proteasome inhibitor currently being developed for a broad range of human malignancies. In several studies, MLN9708 showed extremely high potency against a wide range of cancer cell lines and it has selectivity and potency similar to that of bortezomib21–24. MLN9708 is an improved second-generation new proteasome inhibitor and is currently being evaluated in multiple phase I clinical studies for both solid tumor and hematologic malignancy. Preclinical studies show that MLN9708, which is a dipeptidyl boronic acid, immediately hydrolyzed to MLN2238, the biologically active form, when it is exposed to aqueous
21,22,24
solutions or plasma . It potently, reversibly and select- ively inhibits the proteasome and has developed as an orally bioavailable drug to improve the toxicity profile23.
Our aim in this study was to investigate the in vitro effects of a new second-generation proteasome inhibitor MLN2238 on K562 CML cell line. Here, we first demonstrate in vitro data that support the development of MLN2238, by direct comparison with bortezomib on K562 cell line.
Materials and methods
Cell culture and treatment
K562 cells were obtained from American Type Culture Collection (ATCCti , Manassas, VA, CCL-243ti). The cells
were grown in RPMI 1640 medium supplemented with 2 mm L-glutamine and 10% fetal bovine serum, 1% penicillin/
streptomycin at a temperature of 37 ti C in a humidified incubator with a 5% CO2 atmosphere. MLN2238 and bortezomib were dissolved in dimethyl sulfoxide (DMSO) at a stock solution and it was diluted to the required concentra- tions. A total of 70–80% confluent cells were treated with concentrations (0.01, 0.1, 1, 5, 10, 20 and 30 mm) of MLN2238 and bortezomib for 24 and 48 h in the growth medium.
Cell viability/cell cytotoxicity assay
The effect of MLN22238 and bortezomib on the viability of K562 cells was determined by trypan blue dye exclusion assays. The cells were plated (at a density of 5 ti 103 cells/
100 ml medium/well) in 96-well plates and after 24 h in 100 complete medium containing different concentrations (0.01, 0.1, 1, 5, 10, 20 and 30 mm) of MLN2238 and bortezomib. After incubation for 24 and 48 h, cells were collected and 10 ml of cell suspension was mixed with 10 ml of trypan blue and cells were counted with Cedex XS Image-based cell analysis system according to Cedex XS Measurement Data Software (Roche Innovatis, Bielefeld, Germany)25.
The cytotoxicity of cells was measured using a 4-[3-(4- Iodo-phenyl)-2-(4-nitrophenyl)-2H-5 tetrazolio]-1,3-benzene disulfonate (WST-1) assay (Roche, Germany). The test is based on the cleavage of the tetrazolium salt WST-1 in formazan by mitochondrial dehydrogenases in viable cells. The formazan dye was quantified by a scanning multi-well spectrophotometer by measuring the absorbance of the dye at 420 nm. K562 cells were inoculated into 96-well culture
plates at densities of 5 ti 103 cells/100 ml medium/well. After 24 h, they were treated within 100 ml complete medium containing different concentrations of MLN2238 and borte- zomib for 24 and 48 h. After the incubation period, cell proliferation reagent WST-1 (10 ml per well) was added and the absorbance was measured with an ELISA reader (at a wavelength of 420 nm) after 3 h. The measured absorbance directly correlates to the number of viable cells. Cell viability rates were expressed as the percentage of the controls26,27.
Graphics (trypan blue dye exclusion and WST-1 assay results) were drawn with GraphPad Prism version 5.0 software (San Diego, CA) and were statistically analyzed using one-way ANOVA and Tukey’s post hoc test. Results are expressed as mean ± standard deviation and the means of
three independent experiments (n ¼ 8), n.s.; p40.05, *p50.05, **p50.01 and ***p50.001 were considered to be significant compared to control group.
Apoptosis detection by staining with Annexin V-FITC and propidium iodide (PI)
The Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen, Franklin Lakes, NJ, cat. no. 556547) was used to detect apoptosis as described by the manufacturer. K562 cells (1 ti 105 cells/well) were seeded in six-well plates and treated with different concentrations (1, 5 and 10 mm) of MLN2238 and bortezomib for 24 and 48 h. After incubations, cells were washed twice with cold phosphate-buffered saline (PBS). The cells were resuspended in 100 ml of PBS and were stained with 5 ml of Annexin V-FITC solution and 5 ml propidium iodide (PI) solution for 20 min at room temperature in the dark. Then, the samples were diluted with 250 ml of the 1 ti binding buffer, processed for data acquisition and analyzed on a Becton-Dickinson FACS Aria flow cytometer using FACSDiva version 6.1.1 software. At least 10 000 cells were analyzed per sample27.
Apoptosis detection by caspase-3 activation
Changes in the caspase-3 activity of the cells were examined by PE Active Caspase-3 Apoptosis Kit (BD Pharmingen, cat. no. 550914). Caspase-3 is a key protease that is activated during the early stages of apoptosis and, like other members of the caspase family, is synthesized as an inactive proenzyme that is processed in cells undergoing apoptosis by self- proteolysis and/or cleavage by another protease. In short, the cells (1 ti 105 cells/well) were seeded in six-well plates and treated with different concentrations (1, 5 and 10 mm) of MLN2238 and bortezomib for 24 and 48 h. After the incubations, the analysis was performed according to the kit procedure and processed for data acquisition, and analyzed on a Becton-Dickinson FACS Aria flow cytometer using FACSDiva version 6.1.1 software. At least 10 000 cells were analyzed per sample27.
Mitochondrial membrane potential determined by JC-1 dye on flow cytometer
Mitochondria play a critical role in apoptosis so we examined the loss of mitochondrial membrane potential in response to MLN2238 and bortezomib in cells by Flow Cytometry
Mitochondrial Membrane Potential Detection Kit (BD Pharmingen, cat. no. 551302). K562 cells (1 ti 105 cells/
well) were seeded in six-well plates and treated with different concentrations (1, 5 and 10 mm) of MLN2238 and bortezomib for 24 and 48 h. At the end of the treatment period, 1 ml of each cell suspension was transferred into a sterile 15 ml polystyrene centrifuge tube and centrifuged at 1500 rpm for 5 min. Each pellet were resuspended with JC-1 Working Solution and incubated for 15 min at 37 ti C in CO2 incubator. After the incubation period, cell pellets were washed with 1000 ml assay buffer and centrifuged again. Finally, the samples were resuspended with 250 ml assay buffer and processed for data acquisition and analyzed on a Becton- Dickinson FACS Aria flow cytometer using FACSDiva version 6.1.1 software. At least 10 000 cells were analyzed per sample28,29.
RNA isolation and reverse transcription of the isolated RNA from K562 cells
K562 cells (1 ti 106 cells/flask) were seeded in 25 cm2 flask and treated with different concentrations (1, 5 and 10 mm) of MLN2238 and bortezomib for RNA isolation. After 24 h, cells were prepared and transferred into a tube of MagNA Lyser Green Beads and the cell homogenization process started by using a MagNA Lyser Instrument. Then MagNA Pure Compact RNA Isolation Kit (Roche, Lot: 13243700) procedure was performed by using the MagNA Pure LC 2.0 system. The high quality of the RNA samples was confirmed by using the NanoDrop Instrument. From each RNA popu- lation, 500 ng total RNA was used for cDNA synthesis with the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Lot: 14856520).
qRT-PCR analysis
The resulting total cDNA was then used in PCR to measure the mRNA levels of NFkB1 (Roche, Lot: 90007129, Accession ID: ENST00000394820, Amplicon Length: 71 bps), c-myc (Roche, Lot: 90007128, Accession ID:
ENST00000377970, Amplicon Length: 92 bps) and
GAPDH (Roche, Lot: 90006719, Accession ID: ENST00000229239, Amplicon Length: 112 bps) genes. The mRNA levels of GAPDH were used as an internal positive control.
The primer sequences were NFkB1 forward: 50 -CTGG CAGCTCTTCTCAAAGC-30 , reverse: 50 -TCCAGGTCATAG AGAGGCTCA-30 ; c-myc forward: 50 -GCTGCTTAGACGCT GGATT T-30 , reverse: 50 -TAACGTTGAGGGGCATCG-30 and GAPDH forward: 50 -CTCTGCTCCTC CTGTTCGAC- 30 , reverse: 50 -ACGACCAAATCCGTTGACTC-30 .
A qPCR using the Universal ProbeLibrary detection format was performed on the LightCyclerti 480 Instrument to quantify gene expression. The PCR protocol is as follows:
The real-time PCR mixture contained 10 ml 2 ti LightCyclerti 480 Probes Master, 1 ml of each primer (Real-Time Ready Assay, Roche), 4 ml PCR grade water and 5 ml of cDNA. The cycling conditions included initial incubation step at 95 ti C for 10 min, followed by a 45 cycles of amplification with 10 s at 95 ti C, 30 s at 60 ti C and 1 s at 72 ti C. The final cooling step was 40 ti C for 30 s.
Results
MLN2238 and bortezomib-reduced viability and cytotoxic effects on K562 cells
In order to detect the potential viability and cytotoxic effects of MLN2238 and bortezomib on human K562 cells, we used both trypan blue dye exclusion and WST-1 cell proliferation assays. The results of the trypan blue dye exclusion assay showed that there was a dose and time-dependent reduction in cell viability as compared to untreated control (Figure 1A).
Cytotoxic effects of MLN2238 and bortezomib on K562 cells were determined by WST-1 cell proliferation assay. MLN2238 and bortezomib inhibited growth depending on concentration and time. The cell viability significantly decreased with 1, 5, 10, 20 and 30 mm MLN2238 concentra- tions as 89.56, 47.60, 46.16, 38.27 and 37.17%, respectively for 48 h in K562 cells (p50.001). Similar to the cytotoxic effect of MLN2238, the cell viability decreased with 1, 5, 10, 20 and 30 mm bortezomib concentrations as 49.83, 34.53, 32.64, 29.31 and 28.29%, respectively for 48 h in K562 cells (p50.001) (Figure 1B).
MLN2238 and bortezomib-induced apoptosis
Apoptotic effects of MLN2238 and bortezomib on K562 cells were determined by Annexin-V/PI binding capacity method. The percentage of the apoptotic cell population of cells which were treated with increasing concentrations of MLN2238 and bortezomib was determined by flow cytometry. Ten microm- eters of MLN2238 caused 13.76-fold increase in apoptotic K562 cell population for 48 h as compared to untreated controls. In addition, 5 mm bortezomib caused 10.21-fold increase in apoptotic K562 cell population for 24 h as compared to untreated controls (Figure 2). The contour plots with quadrant gates also indicated that cells in the lower left quadrant Annexin-negative/PI negative, viable cells. The cells in the upper left quadrant indicate Annexin-negative/PI positive, necrotic cells. The cells in the lower right quadrant indicate Annexin-positive/PI negative, early apoptotic cells. The cells in the upper right quadrant indicate Annexin- positive/PI positive, late apoptotic cells (Figure 3 and Table 1).
MLN2238 and bortezomib increased caspase-3 activity in K562 cells
Caspase-3 enzyme has an important role in the mitochondrial- mediated cell death. In order to determine apoptotic effects of MLN2238 and bortezomib on K562 cells, these cells were incubated with increasing concentrations of MLN2238 and bortezomib for 24 and 48 h changes in caspase-3 activities were analyzed by flow cytometer. Caspase-3 activity analysis result and findings related to percent values are shown in Figure 4 and the quadrant gates also shown in Figure 5. So there were 7.25, 14.52 and 17.61-fold increases in caspase-3 activity in response to 48-h incubation time, respectively, as compared to untreated cells. The same concentrations of bortezomib increased caspase-3 activity 15.30, 19.69 and 18.69-fold after 48 h incubation, respectively (Figures 4 and 5).
Figure 1. Effect of MLN2238 and bortezomib on K562 cells. (A) Trypan blue exclusion assay. The cells were treated with MLN2238 and bortezomib concentration for 24 and 48 h and the viability of cells was determined by trypan blue exclusion assay. (B) WST-1 assay. The cells were treated with MLN2238 and bortezomib concentration for 24 and 48 h and cell cytotoxicity was determined by WST-1 assay. Results are expressed as mean ± standard deviation and the means of three independent experiments (n ¼ 8), n.s. p40.05, *p50.05, **p50.01 and ***p50.001 was considered to be significant compared to control group.
MLN2238 and bortezomib-induced the loss of mito- chondrial membrane potential in K562 cells
In order to determine the loss of mitochondrial membrane potential effects of MLN2238 and bortezomib on K562 cells, these cells were incubated with increasing concentrations of MLN2238 and bortezomib for 24 and 48 h changes in JC-1 mitochondrial membrane potential were analyzed by flow cytometer. JC-1 analysis result and findings related to percentage values are shown in Figures 6 and 7. So there were 1.06, 5.5 and 7.32-fold increase in the loss of mitochondrial membrane in response to 48-h incubation respectively, as compared to untreated cells. The same concentrations of bortezomib increased the loss of mitochon- drial membrane 15.30, 19.69 and 18.69-fold after 48 h incubation, respectively (Figures 6 and 7).
MLN2238 and bortezomib-induced to mRNA expression levels of NFkB1
K562 cells were treated with concentrations (1, 5, 10 and 20 mm) of MLN2238 and bortezomib and expression levels of NFkB1 and c-myc genes were determined by the qRT-PCR method. According to the qRT-PCR results, the expression
levels of NFkB1 and c-myc genes were changed significantly. The expression levels of NFkB1 gene decreased by approxi- mately 1.6-fold in response to 10 mm MLN2238 and 1.5-fold in 10 mm bortezomib concentration. Similarly, the expression levels of c-myc gene decreased by approximately 2.42-fold in response to 10 mm MLN2238 and 2.93-fold in 10 mm bortezomib concentration (Figure 8).
Discussion
The ubiquitin-proteasome system is one of the major processes during protein homeostasis and forms by targeting specific proteins for destruction via ubiquitin attachment. Highly regulated members of the critical signaling cascades, including proteins involved in cell cycle regulation, growth control and apoptosis and misfolded proteins are substrates of these proteasomes. The stabilization and accumulation of these substrates end up with the activation of antiproliferative signals and apoptotic pathways and ultimately cell death by proteasome inhibition22. MLN2238 is a selective, potent and reversible inhibitor of proteasome which is derived from bortezomib. In this study, we demonstrate the antiproliferative and apoptotic effects of MLN2238 on CML K562 cells for the
Figure 2. Percentages of the apoptotic cell population in K562 cell line according to the
control group. The result are the means of three independent experiments and the error bars shows the standard error, ***p50.001 was considered to be significant.
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first time. In addition, this is the first study that compares the antiproliferative effect of MLN2238 and bortezomib on this cell line.
Cell viability and cytotoxicity of these drugs on K562 cell line were conducted by trypan blue dye exclusion and WST-1 assays, respectively. MLN2238 showed significantly antipro-
Annexin V-PI, measuring the activation of caspase-3 and loss of mitochondrial membrane potential (JC-1) on flow cytometry.
MLN2238 is a second-generation small-molecule prote- asome inhibitor orally bioavailable being developed for the treatment of a broad range of human hematologic malig-
liferative and cytotoxic effects in both 24 and 48 h depends on
nancies
21,22
. It specifically binds to and inhibits the chymo-
increasing concentration and time. Especially, after 48 h of incubation, the cell viability decreased under 50%. There is no present study about cytotoxic effects of MLN2238 on K562 cell line, but there is another study in which these effects were investigated on human MM cell lines, including MM.1S, MM.1R, RPMI-8226, OPM1, OPM2, H929 and INA-6, by using MTT method which resulted with a significant concen- tration-dependent decrease in viability of all cell lines24.
Bortezomib is the most studied and best-characterized proteasome inhibitor from the first generation and the first approved drug that use for the treatment of refractory MM and mantle cell lymphoma15. Antiproliferative effects of bortezomib on K562 cell line was studied and 64 nm concentration caused an 80% decrease in cell viability29.
In addition to this data, bortezomib’s antiproliferative effects were investigated on various cell lines and it is found
15,30–36
effective as a potent inhibitor against cell viability . Therefore, in this study, bortezomib was chosen to compare the antiproliferative and apoptotic effects of MLN2238 and it is determined that these drugs have similar antiproliferative effects on K562 leukemia cell line. Both trypan blue dye exclusion and WST-1 assays data supported to antiprolifera- tive effects in our study.
Cancer involves uncontrolled growth and spread of abnor- mal cells and apoptosis is the process to promote the killing of these abnormal cancer cells. However, cancer cells often suppress apoptosis because of their dysregulated apoptotic signaling pathways. The key factors, that control apoptosis, are regulated by the 26S proteasome complex37. The inhib- ition of the proteolytic functions of the 26S proteasome has been shown to induce apoptotic factors, such as regulation of caspases and inhibition of NFkB, therefore, the proteasome
trypsin-like proteolytic (b5) site of the 20S proteasome22. Our results showed that the apoptotic effects started with 5 mm of MLN2238 and 1 mm of bortezomib. According to the Annexin-V PI experiment results, after 24 h incubation period with the treatment of 5 mm concentration, bortezomib was found more effective than MLN2238, but after 48 h of incubation MLN2238 was found almost twofold more effect- ive than bortezomib. At 10 mm concentration, after 24 h, the apoptotic effect of MLN2238 increased with concentration, on the other hand, bortezomib decreased and at 48 h two drugs had shown almost the same apoptotic effect. In summary, in increasing concentrations MLN2238 continues to be effective, however, bortezomib loses its apoptotic effect dependent on time. At high concentrations, bortezomib has caused necrosis more than MLN2238. This difference may depend on the higher toxicity of bortezomib. MLN2238 was derived from bortezomib, these compounds have a selectivity and potency similar to each other, but the physicochemical profile of them is different. According to the results of Kupperman’s and friends’ proteasome-glo assay, MLN2238 dissociated more rapidly from the proteasome than bortezo- mib22. Both MLN2238 and bortezomib showed time-depend- ent reversible proteasome inhibition; however, the proteasome dissociation half-life for MLN2238 was determined to be sixfold faster than that of bortezomib22,23. Our studies about the activation of caspase-3 and loss of mitochondrial mem- brane potential (JC-1) results also support that these two drugs have apoptotic effects on K562 leukemia cell line. In summary, MLN2238 has caused an increase in the caspase-3 amount and also the loss of mitochondrial membrane potential similar to bortezomib. There are several other studies that have similar results were obtained in various cell
inhibitors have shown a broad spectrum of antiproliferative
lines
24,30,31,33,38
. However, this is the first study that demon-
and proapoptotic activities against hematological malignan- cies36. In this study, we compared the apoptotic effects of MLN2238 and bortezomib in various concentration, defined with WST-1 results, by determining the binding capacity of
strates the apoptotic effects of MLN2238 on K562 CML cell line.
NFkB is a transcription factor and is involved in the activation of the genes encoding for cytokines, growth factors,
Figure 3. Evaluation of apoptosis in K562 cells induced by MLN2238. The percentage of cells undergoing apoptosis in a dose-dependent manner as compared to control and FACS analysis via Annexin V-FITC/propidium iodide (PI) staining (Control, 1, 5, 10 mm MLN2238 at 24 h in line A and 48 h in line B. Control, 1, 5, 10 mm bortezomib at 24 h in line C and 48 h. in line D). Cells in the lower left quadrant indicate Annexin-negative/PI negative, viable cells. The cells in the upper left quadrant indicate Annexin-negative/PI positive, necrotic cells. The cells in the lower right quadrant indicate Annexin-positive/PI negative, early apoptotic cells. The cells in the upper right quadrant indicate Annexin-positive/PI positive, late apoptotic cells.
chemokines, cell adhesion molecules and surface recep- cancer36. The transcription factor NFkB has received consid-
39–42
tors
. IkB binds to NFkB and inhibits the translocation of
erable attention for its role in cancer cell survival. Bortezomib
NFkB to the nucleus for gene activation in the cytoplasm. External stimuli, including pathogens, stress, free radicals and cytokines, initialize phosphorylation of IkB and this phos- phorylation induces polyubiquitylation of IkB for degradation by the 26S proteasome complex. IkB promotes the transloca- tion of NFkB to the nucleus to switch on the transcription of its target genes by proteasomal degradation. NFkB controls various immune and inflammatory responses, through tran- scriptional regulation of a number of genes and it suppresses apoptosis and induces cell proliferation. Therefore, misregu- lation of NFkB function would lead to various types of cancers, including myeloma, leukemias, breast and prostate
is a powerful inhibitor of NFkB activation via stabilization of NFkB’s inhibitor, IkBa and its effects as an NFkB inhibitor have been used to sensitize cancer cells to other death stimuli43. Besides NFkB c-myc mRNA expression also increases during cancer formation. The proto-oncogene c- myc encodes a transcription factor c-myc. The c-myc oncogene is a ‘‘master regulator’’ which controls many aspects of cellular growth regulation and vitality44. c-Myc plays a pro-oncogenic role in tumors such as Burkitt’s lymphoma in which it is translocated under the promoter regions of the heavy- or light-chain immunoglobulin genes45. Drug treatments that cause apoptotic death of several cell
Table 1. Percent of typical quadrant analysis of Annexin V-FITC/propidium iodide flow cytometry of K562 cells treated with MLN2238 and bortezomib.
K562 cells
Viable cells % (Q3)
Necrotic cells % (Q1)
Early apoptotic cells % (Q4)
Late apoptotic
cells % (Q2)
24 h Control (%0.1 DMSO) 93.2 ± 2.8 0.8 ± 0.05 2.9 ± 1.15 3.1 ± 1.7
MLN2238 1 mm 87.1 ± 4.21 1.4 ± 0.5 7.4 ± 2.3 4.1 ± 2.1
MLN2238 5 mm 57.3 ± 1.15 3.0 ± 1.7 28.6 ± 2.30 11.1 ± 3.46
MLN2238 10 mm 53.90 ± 2.3 2.6 ± 1.15 29.5 ± 5.19 14.0 ± 2.3
Bortezomib 1 mm 54.6 ± 2.10 1.2 ± 0.5 32.4 ± 2.8 11.8 ± 2.5
Bortezomib 5 mm 36.6 ± 3.4 2.1 ± 1.1 37.4 ± 4 23.9 ± 1.7
Bortezomib 10 mm 42.9 ± 1.1 3.9 ± 1.15 29.7 ± 5.1 23.6 ± 2.8
48 h Control (0.1% DMSO) 92.1 ± 2.8 3.6 ± 0.5 1.8 ± 2.3 2.5 ± 0.5
MLN2238 1 mm 91.20 ± 2.8 1.9 ± 0.5 4.4 ± 2.3 2.5 ± 0.5
MLN2238 5 mm 29.8 ± 5.19 11 ± 1.7 33.2 ± 1.7 26 ± 3.4
MLN2238 10 mm 39.10 ± 5.2 27.1 ± 4.04 20 ± 5.7 13.8 ± 1.8
Bortezomib 1 mm 43.10 ± 1.7 23.4 ± 1.5 19.3 ± 5.1 14.3 ± 2.3
Bortezomib 5 mm 37.7 ± 4.0 33.20 ± 1.7 17.7 ± 4.1 11.3 ± 0.5
Bortezomib 10 mm 37.4 ± 1.15 25.9 ± 2.8 22.7 ± 1.7 14 ± 2.2
The result are the means of three independent experiments and the ± shows the standard error.
Figure 4. Typical quadrant analysis of caspase-3 flow cytometry of K562 cells treated with MLN2238 and bortezomib. K562 cells were cultured for 24 and 48 h in medium with MLN2238 and bortezomib at concentration of 1, 5 and 10 mm (Control, 1, 5, 10 mm MLN2238 at 24 h in line A and 48 h in line B. Control, 1, 5, 10 mm bortezomib at 24 h in line C and 48 h in line D). At least 10 000 cells were analyzed per sample and quadrant analysis was performed. The proportion of cell number is shown in each quadrant, Q3, viable cells and Q4, caspase-3 positive cells.
Figure 5. Effects of MLN2238 and bortezo- mib on caspase-3 enzyme activity. The data are indicated as the mean of three independ- ent experiments and the error bars shows the standard error. ***p50.001 was considered to be significant.
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Figure 6. Typical quadrant analysis of JC-1 flow cytometry of K562 cells treated with MLN2238 and bortezomib. K562 cells were cultured for 24 and 48 h in medium with MLN2238 and bortezomib at concentration of 1, 5 and 10 mm (Control, 1, 5, 10 mm MLN2238 at 24 h in line A and 48 h in line B. Control, 1, 5, 10 mm bortezomib at 24 h in line C and 48 h in line D). At least 10 000 cells were analyzed per sample and quadrant analysis was performed. The proportion of cell number is shown in each quadrant, Q3, viable cells and Q4, JC-1 positive cells.
Figure 7. Effects of MLN2238 and bortezo-
mib on the loss of mitochondrial membrane potential. The data are indicated as the mean of three independent experiments and the error bars shows the standard error. *p50.05, **p50.01 and ***p50.001 were considered to be significant.
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Figure 8. Changes in mRNA levels of NFkB1
and c-myc genes. ***p50.001 was consid- ered to be significant.
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μ
MLN2238 Concentration (24h.) Bortezomib Concentration (24 h.)
types have been shown to first greatly lower c-myc expres- sion46. In our study, we compared the effects of MLN2238 and bortezomib on NFkB1 and c-myc mRNA expression levels in various concentrations by RT-PCR method. According to our results, the treatment with MLN2238 at 5 and 10 mm and bortezomib at 1, 5 and 10 mm concentrations downregulated the expression levels of these genes on K562 cell line dependent on increasing concentration after 24 h. The downregulation of NFkB1 and c-myc expression indicates the decrease in cell viability and the activation of apoptosis. There is no data about MLN2238 and bortezomib’s down- regulatory effect on NFkB1 and c-myc mRNA expression levels on K562 cell line which was investigated by RT-PCR method in the literature. However, there are other in vitro studies that have shown bortezomib induces apoptosis by
myelogenous leukemia and acute lymphoblastic leukemia activated NFkB, and that tumorigenesis is driven by BCR- ABL-expressing cells could be blocked by expression of the super-repressor IkBa50.
In summary, taking together all these data showed that MLN2238 has antiproliferative and apoptotic effects as potent as bortezomib, the first-in-class proteasome inhibitor that was approved by FDA. In addition, MLN2238 has lower cytotoxic effects, especially in higher concentrations, than bortezomib. MLN2238 showed more apoptotic effects while bortezomib caused necrosis. Almost all cases of CML are characterized by the expression of the BCR-ABL oncogene and exhibit a multiple drug resistance phenotype, making this tumor hard to treat, but it is possible to induce the apoptosis by proteasome inhibition50.
inhibiting NFkB on different cell lines and methods
43,47–49
.
In conclusion, we believe that our results may open the
The transcription factor NFkB is activated in certain cancers. The transcriptional activation of genes associated with cell proliferation, angiogenesis, metastasis and sup- pression of apoptosis appears to lie at the heart of the ability of NFkB to promote oncogenesis and cancer therapy resistance. Supporting these findings are recent experi- ments, performed in vitro models of cancer, which impli- cate NF-kB inhibition as an important new approach for the treatment of certain hematological malignancies and as an adjuvant approach in combination with chemotherapy or radiation for a variety of cancers. Clinical trials with drugs that block NF-kB are currently in progress with promising results. As important, it was shown that the oncogenic fusion protein BCR-ABL associated with chronic
way of the treatment for CML with MLN2238 and other novel proteasome inhibitors. Therefore, further molecular works to evaluate MLN2238 treatment on CML is needed.
Acknowledgements
The flow cytometry analysis of this research was studied in the Anadolu University Medicinal Plants, Drugs, and Scientific Research Center.
Declaration of interest
The authors declare no conflicts of interest. This study was carried out as a part and support of Anadolu University Scientific Research Projects numbered 1207S120.
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