Doxorubicin Changes Bax / Bcl-x L Ratio , Caspase-8 and 9 in Breast Cancer Cells

Samadi, Tel: +98 (41) 33355789, Fax: +98 (41) 33355789, Email: drnsamadi@yahoo.com © 2015 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2015, 5(3), 351-359 doi: 10.15171/apb.2015.049 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
Apoptosis is the process of programmed cell death that occurs in response to environmental stimuli.Regulation of apoptosis is important for normal growth and homeostasis, during development, embryogenesis, and modification of normal tissues, also cancer treatment.Disorders of abnormal induction of apoptosis can cause severe outcomes. 1Impaired regulation of apoptotic mechanisms provides an opportunity to growth cancerous cells and chemo-resistance.Apoptosis plays an important role in the treatment of cancer as it is the general goal of many treatment strategies. 2,3n mammalian cells, there are at least two main pathways that lead to apoptosis including extrinsic death-receptor dependent apoptosis and intrinsic mitochondrial dependent apoptosis. 4,5In both extrinsic and intrinsic apoptotic pathways induction of cell death is associated with the activation of caspases. 6,7Caspases are two types, initiator caspases (e.g.caspase-8 and -9) and effector caspases (e.g.caspase-3, -6, and -7).Caspase-8 is mainly activated in extrinsic apoptotic pathway, but caspase-9 activation is related to the mitochondrial or intrinsic pathway. 8In anticancer chemotherapy, activation of caspases can be started through stimulating of the extrinsic pathway or by activation of intrinsic pathway at the mitochondria. 9,10ne of the key regulators in the molecular mechanisms of apoptosis is the family of Bcl-2. 11These proteins are attached to cell membrane or free in cytosol.They are operating in intracellular membranes of mitochondria that organize intrinsic apoptosis. 12,135][16] The localization of certain Bcl-2 family proteins changes when stimulation of apoptosis occurs.For example, Bax moves from cytosol to the membrane of mitochondria followed induction of apoptotic signals by an apoptotic stimulant. 17he Bcl-2 gene is important in regulation of apoptosis that encodes variety of proteins that play key roles in regulation of cell apoptosis.Bax is another pro-apoptotic gene that meaningfully is homologous with Bcl-2. 14owever, Bax operates as an enhancer of apoptosis in contrast to Bcl-2 with antiapoptotic properties.Complex network of interactions between Bcl-2 family members both in the cytosol and on mitochondria determines the fate of the cell for death or survive. 13,18Enhanced expression of Bax in breast cancer cells increases sensitivity to apoptotic stimuli and decreases tumour enlargement. 19Bcl-x L is another anti-apoptotic protein that inhibits apoptosis. 20Progress of breast cancer has been associated with of Bcl-x L expression. 21,22Furthermore, it has been associated in advanced grade and development of metastatic cancer. 22Since Bcl-x L prevents apoptosis, this protein is considered as a key molecule to induce chemoresistance. 23 variety of stimuli and the molecular mechanisms of apoptosis are explained intensively. 24Several chemotrapoetic agents have been identified to persuade apoptosis in cancer cells. 25Doxorubicin is one of anthracycline drugs that used for treatment of variety of tumors especially solid tumors. 26The mechanism for cytotoxic effects of doxorubicin is free radical production, DNA intercalating and blocking of topoisomerase II followed by preventing DNA replication and eventually DNA breakage. 27Since doxorubicin is extensively utilized as a first line treatment of variety of different cancers including breast cancer, investigating the mechanisms of apoptosis induced by this chemotherapeutic agent is important. 27reast cancer is second prevalent and deadly cancer in women worldwide. 28Breast cancer treatment consists of radiotherapy, hormonal therapy and chemotherapy that stimulate apoptotic pathways followed by cancer cell death. 295][36] Although the extensive clinical application of doxorubicin for cancer patients has been studied, its anti-proliferative and death-inducing signalling cascades are yet unclear.In this study, we investigated the molecular basis of induction of apoptosis by doxorubicin on MCF-7 cells.Cytotoxic effects of doxorubicin have been evaluated with MTT assay.Alteration in the expression of pro-apoptotic Bax and anti-apoptotic Bcl-x L genes and Bax/Bcl-x L ratio were investigated via Real-time RT-PCR method.Furthermore, the levels of caspase-8 and caspase-9 proteins have been evaluated with ELISA method.

Cell culture
The human MCF-7 breast cancer cells were obtained from National Cell Bank of Iran (Pasteur Institute, Iran).The MCF-7 cells were cultivated in RPMI 1640 medium (Sigma, St Louis, MO, USA) enriched with 10% fetal bovine serum (FBS; Invitrogen), 100 mg/ml streptomycin and 100 units/ml penicillin G (Sigma, St Louis, MO, USA).Cells were incubated in a humidified atmosphere with 5% CO2 at 37°C.

Cell viability assessment using MTT assay
Cell proliferation and viability of doxorubicin treated MCF-7 cells determined using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) which evaluates the percentage of viable cells.The MCF-7 cells with 70% confluency were detached from the dish with 0.05% trypsin/EDTA solution.Cells were seeded into 96well plate with concentration of 4×10 4 cells/cm 2 .Cells with about 50% confluency were exposed with increasing concentrations of the doxorubicin (Ebewe, Unterach, Austria) 0.1-10 μM to induce cytotoxicity.Four wells were remained untreated as control.MTT assay was carried out 24, 48 and 72 hours after treatments.To prepare MTT (Sigma-Aldrich, St. Louis, MO) reagent, 2mg of MTT powder was dissolved in 1 ml PBS.The culture medium was exchanged with 150 μl fresh media plus 50 μl MTT reagent (2mg/ml in PBS); the cell-free wells were considered as blank controls.Cells were incubated in 37°C with 5% CO2 and humidified atmosphere for 4 hours.Then the MTT solution was removed and 200μl of DMSO were added to each well.The plate was maintained for 15 min at37 °C and then the optical density (OD) of the wells was determined at 570 nm through a spectrophotometric microplate reader (Biotek, EL x 800.USA).For PCR thermal cycling program, the samples were included 10 min at 95 °C, 35 cycles of 30 s at 95 °C for denaturation step, 30 s at annealing temperature, and 30 s at 72°C for the extension respectively.Final 10 min incubation at 72°C was carried out to completion of amplicons.mRNA expressions were calculated in compare with 18S rRNA (housekeeping gene) expression.Melting-curve analysis was carried out after amplification to verification the validity of the amplicons.For reporting of gene expression level Pfaffl method was applied.

Analysis of Caspase-8 and Caspase-9 production
To measure Caspase-8 and Caspase-9 protein levels, MCF-7 cells were treated with various concentrations of doxorubicin (0.1, 0.5, 1 μM) and incubated for 24, 48 and 72 hours.Then Caspase-8 and Caspase-9 levels were measured using enzyme-linked immunosorbent assay (Platinum ELISA; eBioscience©) according to the instructions of the manufacturer.

Morphology of doxorubicin treated cells
To study the alteration in morphology of the cells, doxorubicin were applied with concentrations of 0.1, 0.5, 1 μM and the cells were incubated for 24, 48 and 72 h.Light Microscopy was applied to study the morphology of alive and dead cells.

Statistical analysis
All statistical analyses were performed using SPSS 16.0 software.Data obtained from three or more individual experiments were expressed as mean ± SD.Data were analyzed by One-Way ANOVA and Tukey post Hoc tests.p-values less than 0.05 were considered as statistically significant.

Cell viability and IC50 values in doxorubicin treated MCF-7 cells
MCF-7 cells were treated with various concentrations of the doxorubicin ranging from 0.1-10 μM for 24, 48 and 72 hours, and cell viability was measured by MTT assay.MTT assay showed that increased concentrations of the doxorubicin decreased the viability of cells in a time and concentration dependent manner.IC 50 values were 0.75 μM for the cells incubated for 24 h and 0.25 μM for the cells incubated for both 48 and 72 h (Figure 1).Highest decrease in Bcl-x L mRNA level was shown after 48 h incubation.Change in Bcl-x L gene expression was not dose dependent.Our results also revealed a significant increase in Bax /Bcl-x L ratio in all doxorubicin concentrations (p<0.05).Highest increase in Bax/Bcl-x L ratio (more than 10 fold) was revealed after 48 h incubation (Table 2).

Discussion
Cytotoxic agents induce apoptosis by initiating death signalling pathways in susceptible target cells. 37ctivating of apoptosis by chemotherapeutic agents is involved in simultaneous or consequent activation of death receptor systems, disturbance in mitochondrial function and proteolytic processing of caspases. 38Thus, the cell death pathway may occur in several locations; however the accurate molecular mechanisms for each particular drug and special target cell have not been determined completely.In this study, the alteration in Bcl-x L (anti-apoptotic), Bax (pro-apoptotic) genes' expression, Bax /Bcl-x L ratio and caspase doxorubicin on MCF-7 was about 0.5 μM which is consistent with our findings. 39Moreover, the reported IC50 of doxorubicin on MCF-7 were approximately 0.  The Bcl-2 family proteins play a significant role in apoptosis, either as apoptotic activators (Bax) or as apoptotic inhibitors (Bcl-x L ). 42,43 Bax and Bak are the effectors of the Bcl-2 family as upon activation, they change conformation, insert into the outer mitochondrial membrane, oligomerize, and induce mitochondrial outer membrane permeabilization (MOMP). 11,18On the other hand, Bcl-x L is a strong inhibitor of apoptosis that heterodimerize with Bax and neutralizes the effects of the latter.When Bcl-x L is present in excess, cells are protected against apoptosis.In contrast, when Bax is in excess and the homodimers of Bax dominate, cells are susceptible to programmed death.
High expression of Bax gene in the cells incubated with doxorubicin demonstrated that doxorubicin has Bax enhancer effects followed by induction in intrinsic apoptosis pathway on MCF-7 breast cancer cells.April measured Bax in rats and declared that Bax declined markedly in the cells after treatment with doxorubicin. 44ur data showed that doxorubicin was reduced expression of Bcl-x L protein, suggesting the importance of Bcl-2 family proteins for breast cancer cell survival.Clinical studies have showed that increased level of Bclx L is related to a poor response to chemotherapy in breast cancer. 22,45n this study, expression of Bcl-x L gene in doxorubicin treated MCF-7 cells was not time and dose dependent.The most decrease in expression of this gene was in the treated cells with 0.1, 0.5, 1 μM doxorubicin for 48 h which showed more than 5 fold decrease compared with non-treated MCF-7 cells (p<0.05).Several apoptosis regulators have been associated with various human malignancies.For example, studies on human tumors have demonstrated an overall positive correlation between increased expression of Bcl-x L , decreased expression of Bax and uncontrolled tumour cell growth (due to suppressed apoptosis).A wide range of experimental and clinical reports have demonstrated biological effects for doxorubicin.Similar results were shown with doxorubicin, which causes a decrease in Bcl-2 expression and increase in Bax expression. 46Other chemotherapeutic agents such as paclitaxel and thiotepa up-regulate several proapoptotic Bcl-2 proteins and down-regulate antiapoptotic Bcl-2 proteins. 47dditionally, the present study evaluated the effects of doxorubicin on Bcl-x L and Bax expression ratio in induction of apoptosis in vitro.The Bax/Bcl-x L ratios determine the fate of a cell rather than the absolute concentration of either. 48The treatment of MCF-7 cells with doxorubicin originate a significant percentage in the Bax/Bcl-x L ratios (p<0.05).Thus, the imbalance between Bcl-x L and Bax expressions during doxorubicin treatment is believed to play important role in doxorubicin-induced apoptosis as observed in this study and in other investigation. 49ariana and Gonzalez suggest that Bax/Bcl-x L expression ratio may be a sensitive monitor of cancer progression and an early predictor for cancer patients. 50n this study, we investigated at least three distinct parameters: absolute expression levels of Bax and Bcl-x L genes and the ratio of these pro and antiapoptotic proteins.Our results support the existence of a specific apoptotic mechanism in MCF-7 cells that increasing in Bax/Bcl-x L ratio causes the loss of cell viability and enhanced apoptosis.Doxorubicin-induced apoptosis is regulated by the Bcl-2 family of proteins upstream of caspase activation.2][53] Caspases are members of a family of cysteine proteases that are divided into initiator caspases, such as caspase-8 and -9 and executioner caspases such as caspase-3 or -7. 54itiator caspase-8 is known to be activated through extrinsic pathway, whereas caspase-9 is activated in the event of mitochondrial cytochrome c leakage. 54,55n our study, treatment of the cells with doxorubicin caused a time dependent increase in caspase-9 levels, while caspase-8 level didn't follow time and dose dependency pattern.Liang et al. 56 showed that MCF-7 cells can undergo apoptosis by the sequential activation of caspases-9, -7, and -6.An increased level of caspase-9 is similar to Bax gene expression, therefore, both of these proteins increased with increase in doxorubicin dose and incubation time.Up regulation of caspase-9 takes place in doxorubicin treated MCF-7 cells.Bcl-x L interacts with Apaf1 to prevent apoptosis by inhibiting Apaf1 dependent activation of caspase-9. 57These results suggest that doxorubicin induced apoptosis via mitochondrial dependent intrinsic pathway.In this study, decreased level of Bcl-x L caused increase in caspase-9 levels consequently in apoptotic cells.These results are compatible with our other findings for the Bax /Bcl-x L ratio, since BCL-2 family controls the intrinsic apoptotic pathway in doxorubicin treated MCF-7 breast cancer cells.MCF7 cells are deficient in capase-3 expression because of a deletion mutation in their caspase-3 gene, suggesting the existence of caspase-3-independent apoptotic pathways. 58,59Participation of activated caspase-3 is crucial for activation of caspase-8 which explains our results on caspase 8 protein level in the cells with lack of caspase-3 protein level. 60Therefore, increased level of caspase-8 couldn't trigger extrinsic apoptosis pathway in MCF-7 cells.This is compatible with other studies on a disabled Fas pathway in this cell line. 61he presence of caspase-3 can increase cell sensitivity to apoptosis-inducing agents.MCF-7 cells, which are relatively insensitive to many chemotherapeutic agents, acquire greater sensitivity to doxorubicin and etoposideinduced apoptosis when caspase-3 is reconstituted. 62uture research may show whether maximizing crosstalk between the intrinsic and extrinsic pathways of apoptosis and up-regulation of caspase-3 are potential targets for breast cancer therapy.In conclusion, our data suggest that increase in the Bax/Bcl-x L ratio and caspase-9 level can play a key role in doxorubicin-induced apoptosis in MCF-7 cells, however further studies are warranted to validate these findings.

Figure 1 .
Figure 1.Column charts of MTT assay results of MCF-7 cell treated with different concentrations of doxorubicin.Expression of Bax, Bcl-x L genes and Bax /Bcl-x L ratio in doxorubicin treated MCF-7 cellsExpression of Bax Gene significantly increased by increasing concentrations of doxorubicin (0.1, 0.5, 1 μM) after 24, 48 and 72 hours incubations (p<0.05)(Figur 2).Highest increase (4.5 fold) in expression of Bax revealed with 1μM doxorubicin after 72 h incubation.Bcl-x L gene expression was decreased in 48 and 72 h (Figur 2. b).Highest decrease in Bcl-x L mRNA level was shown after 48 h incubation.Change in Bcl-x L gene expression was not dose dependent.Our results also revealed a significant increase in Bax /Bcl-x L ratio in all doxorubicin concentrations (p<0.05).Highest increase in Bax/Bcl-x L ratio (more than 10 fold) was revealed after 48 h incubation (Table2).

Figure 2 .
Figure 2. Expression of Bcl-xL and Bax genes in apoptosis pathway were investigated to demonstrate the induction of apoptosis by doxorubicin.Cultivated MCF-7 cells were treated with three concentrations of doxorubicin (0.1, 0.5, 1 μM) and incubated for 24, 48 and 72 hours.
Doxorubicin displayed morphological alterations in MCF-7 cells.Adherent cells after treated with doxorubicin became rounded, condensed and detached from the dish.Number of detached and rounded cells was increased by increase in the dose of doxorubicin and incubation time.Doxorubicin (0.1 μM) after 24 h incubation showed least and 1 μM doxorubicin with 72 h incubation showed the most rounded and detached cells in comparison with other doxorubicin concentrations and incubation times (Figure.4 a-j).
8 and 9 proteins' level following the stimulation of apoptosis by doxorubicin in MCF-7 cells were reported.For evaluation of cytotoxicity induced by doxorubicin MTT assay was applied.The results demonstrated that the viability of MCF-7 cells was clearly decreased in a time and dose-dependent manner.IC50 values were 0.75, 0.25 and 0.25 μM for 24, 48 h and 72 h incubations respectively.Based on Lukyanova study, the IC50 of Advanced Pharmaceutical Bulletin, 2015, 5(3), 351-359

Figure 4 .
Figure 4. Morphology and cell death of doxorubicin treated MCF-7 cells was investigated via light Microscopic image.a: untreated MCF-7 cell line, b: MCF-7 cells treated with 0.1 μM for 24 h, c: cells treated with 0.5 μM for 24 h, d: cells treated with 1 μM for 24 h, e: treated with 0.1 μM for 48 h, f: cells treated with 0.5 μM for 48 h, g: cells treated with 1 μM for 48 h, h:cells treated with 0.1 μM for 72 h, i: cells treated with 0.5 μM for 72 h, j: cells treated with 1 μM for 72 h.

Table 1 .
Applied Primer sequences for Quantitative PCR of Bax and Bcl-xL genes.

Table 2
40,41and 1.19 μM using MTT assay based on Fornari et al. and Taherian et al. studies, respectively.The mentioned differences can be explained regarding different brands of doxorubicin or technical strategies.40,41