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ORIGINAL ARTICLE
Year : 2017  |  Volume : 13  |  Issue : 2  |  Page : 346-350

Effects of EF-24, RAD001, and paclitaxel on the expression profiles of apoptotic and anti-apoptotic genes


1 Department of Medical Biology, Faculty of Medicine, Giresun University, Giresun, Turkey
2 Department of Medical Biology, Faculty of Medicine, Hitit University, Corum, Turkey
3 Department of Medical Biology and Genetics, Faculty of Medicine, Gazi University, Besevler, Ankara, Turkey

Date of Web Publication23-Jun-2017

Correspondence Address:
Ebru Alp
Department of Medical Biology, Faculty of Medicine, Giresun University, 28200 Giresun
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.176172

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 > Abstract 

Context: Cancer cells exert differential responses to chemotherapeutics and inhibitors. To the best of our knowledge, a few or no research has been performed until now to determine the effect of EF-24 and RAD001 on MDA-MB-231 breast cancer cells with regard to mRNA expression of apoptotic and anti-apoptotic genes.
Aims: In this study, we aimed to investigate the mRNA expression levels of apoptotic (caspase 2 [CASP2], CASP8, and CASP9) and anti-apoptotic (B-cell lymphoma 2 [BCL2] and BCL2-like protein 1 [BCL2L1]) genes after exposure to paclitaxel, EF-24, and RAD001 in MDA-MB-231 cells.
Materials and Methods: After treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to measure cell viability. mRNA expressions were analyzed using quantitative real-time polymerase chain reaction.
Results: Decrease in cell viability ratios was seen in a dose-dependent manner for all chemicals. MDA-MB-231 cells responded slightly different to paclitaxel, EF-24, and RAD001 at the transcriptional level of apoptotic and anti-apoptotic genes.
Conclusions: Our results showed that response of these cells to paclitaxel, EF-24, and RAD001 was found different at the transcriptional level of apoptotic and antiapoptotic genes. Therefore, understanding transcriptional changes after these drug exposure may give us a change to figure out more realistic results of the apoptotic pathway inhibition.

Keywords: Apoptosis, breast cancer, EF-24, paclitaxel, RAD001


How to cite this article:
Alp E, Yilmaz A, Onen HI, Menevse ES. Effects of EF-24, RAD001, and paclitaxel on the expression profiles of apoptotic and anti-apoptotic genes. J Can Res Ther 2017;13:346-50

How to cite this URL:
Alp E, Yilmaz A, Onen HI, Menevse ES. Effects of EF-24, RAD001, and paclitaxel on the expression profiles of apoptotic and anti-apoptotic genes. J Can Res Ther [serial online] 2017 [cited 2019 Nov 22];13:346-50. Available from: http://www.cancerjournal.net/text.asp?2017/13/2/346/176172


 > Introduction Top


Breast cancer is one of the most common cancers with poor prognosis and the second leading cause of cancer-related deaths in women.[1] Advances in the diagnosis and treatment notwithstanding, breast cancer treatment remains a major challenge for physicians due to considerable side effect of current therapies.[2] Hence, new drugs are developing to diminish the adverse effect of classical chemotherapeutic agents.[3]

While numerous factors contribute to the growth of cancer, one hallmark is the loss of balance between cell proliferation and cell death.[4] Apoptosis or programed cell death is regulated by complex interactions between anti-apoptotic and pro-apoptotic proteins.[5] Overexpression of anti-apoptotic B-cell lymphoma 2 (BCL2) or BCL2-like protein 1 (BCL2L1) (known as BCL-xL) likely appears in more than half of all cancers.[6] Caspases (CASPs) are another group of proteins which is responsible for the regulation of apoptosis. Dysregulation of CASPs has been induced a number of diseases including cancers.[7]

Profiling transcriptional changes after drug exposure is a powerful approach for investigating cellular responses to drugs. This approach has paved the way for greater understanding of pathway inhibition and off-target drug effects.[8] In addition, functional dysregulation in apoptotic signal transduction may translate into drug resistance of cancer as well.[6] It is known that transcript levels are known to exhibit dose-responsive behavior against pharmacological agents.[9],[10],[11]

Paclitaxel and its derivatives are broadly used in the treatment of breast cancer [12] and several other solid tumors, such as nonsmall cell lung cancer [13] and ovarian cancer.[14] In addition to stabilization of microtubules, it induces apoptosis by not only CASP-dependent but also CASP-independent pathways and inhibits the anti-apoptotic protein BCL2. Despite its successful use as an anti-cancer drug, raising the resistance of cancer cells to paclitaxel has created a drawback. On the other hand, the major limitation is extreme toxicity of paclitaxel. To overcome the problem, studies have been focused on identifying natural compounds that could increase the therapeutic index.[15] Curcumin, obtained from the rhizome of the perennial herb Curcuma longa, has been shown significant therapeutic potential against carcinogenesis in several cancer cell types in vitro because it suppresses cancer cell proliferation, induces cell cycle arrest and apoptosis by means of the CASP cascade.[16],[17] Unfortunately, bioavailability of curcumin is low due to very low absorptive capacity, therefore its therapeutic benefit is limited.[18] In light of these findings, design and synthesis of novel structural analogs have been investigated for cancer therapy.[19] One novel analog is EF-24 which exhibited approximately 10- and 20-fold enhanced cytotoxic activity against various cancer cell lines relative to curcumin.[17]

RAD001 (everolimus), one of the mechanistic targets of rapamycin (mTOR) inhibitor, is recently accepted for the treatment of renal cell carcinoma and breast cancer.[20] RAD001 has shown broad anti-tumor activities in preclinical models.[21],[22] Dysregulation of the mTOR signaling pathway is closely associated with tumorigenesis because the mTOR plays a central role in cell growth and many biological activities including gene transcription, protein translation initiation, ribosome biosynthesis, and cellular apoptosis. As a result, mTOR represents a valuable target for anti-tumor therapy.[23]

In this study, we aimed to investigate the mRNA expression levels of apoptotic (CASP2, CASP8, and CASP9) and anti-apoptotic (BCL2 and BCL2L1) genes after exposure to paclitaxel, EF-24, and RAD001 in breast cancer cell line (MDA-MB-231).


 > Materials and Methods Top


Cell line and cell culture

MDA-MB-231 breast cancer cell line was purchased from the Foot-and-Mouth Disease Institute of Ministry of Agriculture and Rural Affairs of Turkey. Cells were routinely cultured in Dulbecco's modified Eagle's medium containing 10% heat inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin G, and 100 mg/ml streptomycin (all obtained from HyClone, Thermo, USA) at 37°C in a humidified atmosphere of 5% CO2.

Cell viability assay

The growth inhibitory effect of paclitaxel (Sigma-Aldrich, St. Louis, Missouri, USA), EF-24 (Sigma-Aldrich, St. Louis, Missouri, USA), and RAD001 (Fluka, Hamburg, Germany) were evaluated on MDA-MB-231 cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, Missouri, USA). Cells were suspended in 96-well tissue culture plates (containing 104 cells, per well) and seeded in culture medium at 37°C for overnight. After overnight culture, the cells were incubated with paclitaxel (ranges 25–500 nM), EF-24 (ranges 0.125–8 μM), and RAD001 (ranges between 25 and 500 nM) for 24 h and then MTT assay was used to measure cell viability. After the incubation period, 10 μl MTT solution (5 mg/ml) was added to the medium and kept at 37°C for 4 h. To dissolve formazan crystals, dimethyl sulfoxide (Sigma-Aldrich, St. Louis, Missouri, USA) was used and the absorbance values were measured with a SpectraMax M3 microplate reader (Molecular Devices, California, USA) at 570 nM. The experiments were performed in quadruplicate. As a result of MTT assay data, 100 nM paclitaxel, 2 μM EF-24, and 50 nM RAD001 concentrations (IC20) were selected for mRNA analysis.

RNA isolation and cDNA synthesis

Total cellular RNA was isolated after drug treatment of cells using High Pure RNA Isolation Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's protocol. Then, quantitation of RNA purity and concentration of each sample was measured using NanoDrop spectrophotometer (NanoDrop ND-1000, Montchanin, DE, USA). The isolated RNA samples were stored at −80°C until required for further experiments. cDNA was synthesized from 1 μg of total RNA via random hegzamer primers using Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. Eppendorf Mastercycler ep gradient S thermal cycler (Eppendorf, Hamburg, Germany) was used for cDNA synthesis reaction. The samples were kept at −20°C after reaction.

Quantitative real-time polymerase chain reaction analysis

CASP2, CASP8, CASP9, BCL2, and BCL2L1 mRNA expression levels were quantified using Light Cycler ® 480 (Roche Diagnostics GmbH, Mannheim, Germany). Suitable probes and gene-specific primers spanning exon–exon boundaries were designed at the Universal Probe Library (UPL) Assay Design Center (https://www.universalprobelibrary.com). Primer pairs and UPL probe numbers were indicated in [Table 1]. Each target gene expression level was normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase. Polymerase chain reaction (PCR) efficiency of each gene was determined by serial dilutions. The real-time PCR conditions were as follows: 10 min preincubation at 95°C, followed by 50 cycles of 10 s at 95°C, 20 s at 60°C, and finally a cooling step to 40°C. Each sample was measured in triplicate and mean value was used for further calculation.
Table 1: mRNA specific primer sequences of studied genes and Universal Probe Library probe numbers

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Statistical analysis

Cell viability experiments were repeated 4 times, and data were expressed as the mean ± standard deviation from a representative experiment. Differences of viability were assessed by one-way ANOVA followed by post hoc Tukey's test using SigmaStat version 3.5 software (Systat Software, Inc, Point Richmond, CA, USA). Statistical significance levels of differences in mRNA expressions were analyzed by REST 2009 version 2.0.13 (Qiagen, Hilden, Germany).[24] P< 0.05 was considered as significant.


 > Results Top


Decrease in cell viability ratios was seen in a dose-dependent manner for all chemicals as determined by MTT assay [Figure 1]. Nearly, similar cell viability responses were found at the highest concentration of EF-24 (8 μM) and paclitaxel (500 nM) and about 40% cells death were found at these concentrations after 24 h. On the other hand, RAD001 caused less cell death at the highest concentration as compared with other chemicals (EF-24 and paclitaxel) and 30% cells death were obtained at 500 nM concentrations for 24 h. At 100 nM paclitaxel, 2 μM EF-24, and 50 nM RAD001 concentrations, agents caused 20% cell death (IC20) after 24 h incubation.
Figure 1: Effects on cell viability of paclitaxel (a) EF-24, (b) and RAD001, (c) on MDA-MB-231 cells measured by 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay. The results are expressed as a percent of control. Data shown are the means of four independent experiments. *P < 0.05

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In [Figure 1]a, MDA-MB-231 cell viability significantly reduced after 24 h paclitaxel incubation at doses higher than 100 nM (P < 0.05) and it was found 63.8% at highest concentration (500 nM). Another agent EF-24 at doses of 2, 4, and 8 μM significantly decreased the cell viability after 24 h incubation (P < 0.05) [Figure 1]b. After 24 h RAD001 incubation at doses higher than 50 nM, cell viability was significantly reduced (P < 0.05); it was found that 79.6% and 70.2% at 50 nM and highest concentration (500 nM), respectively [Figure 1]c. To continue for further analysis, IC20 values were selected.

We treated the cells with paclitaxel, EF-24, and RAD001 at selected doses for the mRNA expression analysis of the apoptotic (CASP2, CASP8, and CASP9) and anti-apoptotic (BCL2 and BCL2L1) genes in MDA-MB-231 cell line. The results of quantitative PCR analysis of the CASP2, CASP8, CASP9, BCL2, and BCL2L1 genes were given in [Figure 2] for paclitaxel, EF-24, and RAD001.
Figure 2: mRNA expression of caspase 8, caspase 9, B-cell lymphoma 2, B-cell lymphoma 2-like protein 1, and caspase 2 genes in MDA-MB-231 cells treated with paclitaxel, EF-24, and everolimus (RAD001) for 24 h. Glyceraldehyde-3-phosphate dehydrogenase used as a housekeeping gene to normalize data

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Although CASP8 and CASP2 mRNA expression were not induced, CASP9 mRNA expression was induced after paclitaxel treatment, but not statistically significant (P > 0.05). In addition, paclitaxel was slightly decreased in BCL2 and BCL2L1 mRNA expression, but this decline was not statistically significant (P > 0.05). Even though EF-24 treatment increased CASP8, BCL2, BCL2L1, and CASP2 mRNA expression level, this treatment lightly decreased CASP9 mRNA expression. However, these changes not reached statistically significant value (P > 0.05). After RAD001 treatment, mRNA expression levels of CASP8, CASP9, and BCL2 were slightly increased, but CASP2 and BCL2L1 mRNA expressions were slightly decreased in MDA-MB-231 cell line. These changes were also not statistically significant (P > 0.05).


 > Discussion Top


In a recent study by performing microarray analysis, it has been shown that breast cancer cell lines showed quite distinct transcriptional responses to chemotherapeutics.[25] Despite the anti-tumor activity of paclitaxel toward cancer including breast cancer,[15] rising drug resistance to paclitaxel poses a challenge for future cancer treatment.[26] Liu et al. showed that very high concentrations of paclitaxel did not further increase growth inhibition and apoptosis.[27] Paclitaxel resistance was also found in breast cancer cell lines including MDA-MB-231.[28] In our study, cell viability was significantly reduced at doses higher than 100 nM paclitaxel.

Imbalance between pro- and anti-apoptotic members of BCL2 family may contribute to the progression of human cancers and rise drug resistance.[29] Ferlini et al. showed that BCL2 mRNA expression is down-regulated in resistant cell lines with respect to parental paclitaxel sensitive cells in human ovarian A2780 cell line.[30] In our study, despite the down-regulation tendency of BCL2 and BCL2L1 mRNA expression, statistically significant value was not found. CASP8 and CASP9 mRNA expression did not also significantly change after paclitaxel treatment. As for CASP2, to the best of our knowledge, there was no study with respect to CASP2 expression in MDA-MB-231 cell line after paclitaxel treatment. Jelínek et al. indicated that inhibition of CASP2 expression using siRNA resulted in increased number of surviving MCF-7 and SK-BR-3 cells and decreased cleavage of initiator CASP8, CASP9 in both cells after paclitaxel treatment.[31] In our study, CASP2 mRNA expression was not significantly altered after paclitaxel treatment. We recommended that further studies are needed to clarify the potential role of paclitaxel on mRNA expression of CASP2 in MDA-MB-231 cells.

Our previous report has shown that EF-24 significantly decreased MSTO-211H cell viability.[22] In addition, Tan et al. indicated that EF-24 significantly suppressed proliferation of ovarian cell lines at ≤2 μM concentration.[32] According to our results, it also significantly inhibited the proliferation of MDA-MB-231 cells at ≥2 μM concentration. With regard to CASP2, CASP8, CASP9, BCL2L1, and BCL2 mRNA expressions, we did not find significant changes after EF-24 treatment in MDA-MB-231. However, we previously showed [22] that BCL2L1 mRNA was significantly decreased in MSTO-211H cells after 2 μM EF-24 treatment.

The antiproliferative effects of mTOR inhibition have been shown in various cell lines.[22],[33],[34],[35] We showed that cell viability was significantly reduced after RAD001 incubation at doses higher than 50 nM. However, we did not find significant changes in the expression of CASP2, CASP8, CASP9, BCL2L1, and BCL2 mRNAs after RAD001 treatment and we are unable to compare our results due to lack of similar studies in the literature.

In general, mRNA analysis showed that MDA-MB-231 cells responded slightly different to paclitaxel, EF-24, and RAD001 at the transcriptional level of apoptotic (CASP2, 8, 9) and anti-apoptotic (BCL2 and BCL2L1) genes. This molecular study might uncover the need for further wide investigation on this pathway and approaches to the development of effective chemotherapy by targeting appropriate signal transducers.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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