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Novel angiotensin receptor blocker, azilsartan induces oxidative stress and NFkB-mediated apoptosis in hepatocellular carcinoma cell line HepG2
Biomedicine & Pharmacotherapy 99 (2018) 939–946
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Novel angiotensin receptor blocker, azilsartan induces oxidative stress and NFkB-mediated apoptosis in hepatocellular carcinoma cell line HepG2
T
Elham Ahmadiana,b,c,e,1, Ahmad Yari Khosroushahid,1, Aziz Eftekharib,e,f, Safar Farajniaa, ⁎ Hossein Babaeia,g, Mohammad Ali Eghbala,g,h, a
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Pharmacology and Toxicology Department, Maragheh University of Medical Sciences, Maragheh, Iran c Students’ Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran d Department of Pharmacognosy, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran e Department of Basic Sciences, Maragheh University of Medical Sciences, Maragheh, Iran f Toxicology Research Center, Maragheh University of Medical Sciences, Maragheh, Iran g Department of Pharmaclogy and Toxicology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran h Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran b
A R T I C L E I N F O
A B S T R A C T
Keywords: Liver cancer Angiotensin Apoptosis Azilsartan NF-kB pathway
Overexpression of renin angiotensin system (RAS) components and nuclear factor-kappa B (NF-kB) has a key role in various cancers. Blockade of RAS and NF-kB pathway has been suggested to reduce cancer cell proliferation. This study aimed to investigate the role of angiotensin II and NF-kB pathway in liver hepatocellular carcinoma cell line (HepG2) proliferation by using azilsartan (as a novel Ag II antagonist) and Bay 11-7082 (as NF-kB inhibitor). HepG2 cells were treated with different concentrations of azilsartan and Bay 11-7082. Cytotoxicity was determined after 24, 48, and 72 h by MTT assay. Reactive oxygen spices (ROS) generation and cytochrome c release were measured following azilsartan and Bay11- 7082 treatment. Apoptosis was analyzed qualitatively by DAPI staining and quantitatively through flow cytometry methodologies and Bax and Bcl-2 mRNA and protein levels were assessed by real time PCR and ELISA methods, respectively. The cytotoxic effects of different concentration of azilsartan and Bay11- 7082 on HepG2 cells were observed as a reduction in cell viability, increased ROS formation, cytochrome c release and apoptosis induction. These effects were found to correlate with a shift in Bax level and a downward trend in the expression of Bcl-2. These findings suggest that azilsartan and Bay117082 in combination or alone have strong potential as an agent for prevention or treatment of liver cancer after further studies.
1. Introduction Cancer, as a major public health problem, is the second leading cause of death in the world [1]. According to world health organization (WHO) report, there is an estimation of more than 21 million cancer cases and 13 million deaths worldwide [2]. Hepatocellular carcinoma (HCC) has been previously reported to be the third most common cause of death from cancer [3]. Although surgical resection is considered as the standard curative treatment of HCC, the majority of patients are not candidates for this type of treatment mainly due to the advanced tumor extension at the first diagnosis and/or insufficient liver functionality reservoirs [4]. In addition, chemotherapy regimes in suitable candidates have often been restricted because of major organ damages,
inefficient drugs and poor prognosis [5]. Thus, search for novel anticancer agents or regimes with higher efficacy and minimal side effects is continued. Angiotensin II (Ang II), is a multifunctional octapeptide component of the renin angiotensin system (RAS) with modulatory effects on angiotensin receptors type 1 (AT1) and type 2 (AT2) [6]. Ang II is a potent vasoconstrictive agent which regulates blood pressure and body fluid homeostasis [7]. Apart from the well-known systemic role of the RAS in cardiovascular and renal systems, Ang II has been shown to have various pleiotropic functions one of which is to meet the hallmarks of the cancer [8]. Recent researches have shown the overexpression of RAS in different cancer cells and tissues such as lung, breast, pancreas and liver [9]. Ang II has also been linked to various signaling pathways to play a
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Correspondence author at: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Postal Code 51664-14766, Tabriz, Iran. E-mail addresses: [email protected] (E. Ahmadian), [email protected] (A.Y. Khosroushahi), [email protected] (A. Eftekhari), [email protected] (S. Farajnia), [email protected] (H. Babaei), [email protected] (M.A. Eghbal). 1 These authors contributed equally to this manuscript. https://doi.org/10.1016/j.biopha.2018.01.117 Received 23 November 2017; Received in revised form 13 January 2018; Accepted 24 January 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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microplate reader (Biotek, ELx800, USA). Combination index (CI) analysis based on Chou and Talalay method [18] was performed using CompuSyn software for Azilsartan and Bay 11-7082 combinations, determining synergistic, additive, or antagonistic cytotoxic effects against HepG2 cancer cells. Values of CI < 1 demonstrate synergism while CI = 1 and CI > 1 values represent additive and antagonistic effects of drug combination, respectively.
key role in tumor cell growth and migration [10]. AT1 downstream signaling is documented to lead to the activation of nuclear factorkappa B (NF-kB) [11]. NF-kB is a transcription factor involved in the cellular survival pathway that activated in response to several stimuli such as hypoxia, bacterial endotoxins and inflammatory cytokines. Also, NF-kB is a key regulator of gene expression in inflammatory-related malignant tumors [12] and inhibition of this pathway can be a potential option for treatment of cancer. Angiotensin receptor blockers (ARBs) are considered with very low side effects compared to chemotherapeutic agents and many ARBs have been addressed to possess potential for suppressing tumor growth in vitro and in vivo [13]. Azilsartan, as a potent and highly selective ARB, is a novel long lasting drug approved for the treatment of hypertension [14] and at its maximal dose has superior efficacy to other ARBs such as valsartan and olmesartan at their maximal approved doses without increasing adverse events [15]. Azilsartan potently inhibits aortic endothelial and vascular cell proliferation in the absence of exogenous Ang II supplementation [16]. However, apoptotic and anti-proliferative impacts of azilsartan in cancer cell lines have not been examined yet. This study aimed to assess the possible cytotoxicity mechanism of azilsartan in hepatocellular carcinoma cell line (HepG2) in association with NF-kB signaling pathway. Therefore, the cytotoxic and apoptotic roles of Ang II and NF-kB pathways in HepG2 cell line were studied using azilsartan and Bay 11-7082.
2.4. Determination of ROS formation
2. Materials and methods
The non-fluorescent dye, 2′,7′dichlorofluorescin diacetate (DCFHDA), (which is oxidized to fluorescent dichlorofluorescein (DCF) by hydroperoxides) was utilized to determine relative levels of cellular ROS generation [19,20]. Cells (3 × 104 cells/well) were treated with azilsartan (0–200 μM) for 24 h at 37 °C. Also ROS formation in azilsartan–treated (200 μM) cells were measured in the presence of Nacetyl cysteine (NAC) as a potent well-known antioxidant. All treated/ untreated control cells were detached by trypsin-EDTA (0.25% trypsin and 0.02% EDTA) and were washed by PBS (0.1 M, pH 7.2) and then were incubated in FBS free culture medium containing 50 μM dye for 30 min. The washing process was performed once more and then the cell suspensions were centrifuged at 412g for 10 min. the supernatant was removed and cell plates were dissolved with 1% Triton X100. Fluorescence changes were measured using a Jasco R_FP-750 spectrofluorometer (Jasco Corporation, Tokyo, Japan) with excitation and emission wavelengths of 485 and 530 nm, respectively [21].
2.1. Chemicals
2.5. Determination of cytochrome c amount
Glycine, NaCl, chloroform, and diethylpyrocarbonate (DEPC) were obtained from Merck (Merck, Darmstadt, Germany). 3-(4,5-Dimethyl-2thiazolyl)-2,5-diphenyltetra-zoplium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640, penicillin and streptomycin and trypsin-EDTA solutions, L- glutamine, phosphate buffered saline (PBS), and fetal bovine serum (FBS) were purchased from Gibco Life Technologies Ltd. (Tulsa, OK, USA). Azilsartan, 2′,7′dichlorofluorescin diacetate (DCFH-DA), Bay 11-7082 were purchased from Sigma-Aldrich (St Louis, MO, USA). Azilsartan and Bay 117082 were pharmaceutical grade and all other materials were analytical/cell culture tested grade. All materials were used without further purification.
Enzyme linked immunosorbent assay (ELISA) kit (Cytochrome C ELISA Kit, Human - Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify cytosolic cytochrome c. The cells in the density of 5 × 105 were lysed in lysis buffer according to the manufacturer’s user instructions and samples were centrifuged at 500g, room temperature for 10 min. The supernatants and cytochrome c conjugate were added to the 96 well microplates coated with human cytochrome c monoclonal antibody. The absorbance of samples was measured at 450 nm in a microplate reader (Biotek, ELx800, USA). A standard curve was prepared by plotting the absorbance values of diluted solutions of a human cytochrome c standard and the cytochrome c concentration was expressed as ng/ml [22].
2.2. Cell culture
2.6. DAPI staining method
HepG2 (liver hepatocellular adenocarcinoma) and normal epithelial KDR cell lines were obtained from Pasture Institute, Tehran, Iran. Cell lines were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 μg/ml of streptomycin and were incubated in humidified atmosphere containing 5% CO2 at 37 °C according to the guidebook of the cell line bank.
All treated and normal control cells groups were stained using DAPI staining method to determine qualitatively the presence of apoptotic cells. After placing sterile cover slips into each well of a 6-well culture plate, wells were seeded by HepG2 cells with seeding density of 120 × 104 cell/well. After overnight of post-seeding, the cells were treated with azilsartan and were incubated for 24 h. For apoptosis detection, all treated/untreated control cells were fixed with paraformaldehyde (4%) for 5 min then the fixed cells were permeabilized with 0.1% Triton-X100 at 37 °C for 5 min and finally the permeabilized cells were stained with 50 μl DAPI dye (1:2000 dilution) per well at room temperature for 3 min. The stained cells on slips were washed with PBS (0.1 M, pH 7.2) thrice and were utilized for apoptosis assessments by fluorescent microscopy (BX63, Olympus, Japan) equipped with U-MWU2 fluorescence filter set (excitation filter BP 330-385, dichromatic mirror DM 400, and emission filter LP 420) [23].
2.3. Determination of cell proliferation The effects of azilsartan on cell proliferation of HepG2 and KDR cells were determined by MTT assay [17]. Cells, with seeding density 1.2 × 104 cells/well, were seeded in 96-well microplate containing 200 μl RPMI growth medium. The cells were then treated with various concentrations of azilsartan (5, 25, 50, 100 and 200 μM) and/or Bay 117082 (5, 10 and 25 μM) over different incubation time points (24, 48, and 72 h). After treatment time point, cells (treated/untreated control cells) were incubated with 150 μl fresh medium plus 50 μl MTT solutions (2 mg/ml in PBS) for 4 h at 37 °C. After incubation time, the MTT contained medium was removed and the mixture of 200 μl DMSO and 25 μl ml Sorenson’s glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5) was added to each well and the cells were again incubated for 30 min at 37 °C. The absorbance of plates was measured at 570 nm using a
2.7. Apoptosis detection by flow cytometry assay Apoptosis was quantitatively determined using annexin V apoptosis detection kit (BD Biosciences Pharmingen, San Diego, CA, USA) according to the manufacturer’s user instructions. Cells (1.2 × 105 cells/ well) were seeded into a six-well culture plate. The cells were treated 940
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with azilsartan (100 μM) or Bay 11-7082 (25 μM) and/or the combination of these two agents at reaching 50% confluence. After 24 h, the treated/untreated control cells were detached by trypsin and then washed with cold PBS (pH 7.2) subsequently centrifuged at 1100 rpm for 7 min at 28 °C. The cell pellet of HepG2 was re-suspended in 500 μl of 1X binding buffer, the tubes were centrifuged again at previous conditions and the supernatants were replaced with100 μl binding buffer (1X). This tubes were added with 5 μl of FITC-conjugated Annexin V, and then incubated for 15 min at room temperature under dark conditions. Finally, 5 μl of propidium iodide solution was added to the cells, and quadrant settings were fixed with untreated, singlestained controls and copied to dot plots of the treated cells. Quadrant statistic calculations were performed using CELLQuest Pro software (BD Bioscience, San Diego, CA, USA) on data obtained from two times repeated experiments with triplicate samples for each experiment. Analyses were accomplished using 100,000 cells at a rate of 500 cells/s. FL-1 and FL-3 were represented in dot plots illustrating the viable, apoptotic, and necrotic cells. 2.8. Real time All untreated/treated cells were washed with phosphate buffer saline (pH 7.2) then 1 ml ice cold RNX-plus solution (SinaClone, Iran) was added into cells according the kit user instruments. Complementary DNA (cDNA) was synthesized using PrimeScript RT Reagent kit (Takara Bio Inc., Tokyo, Japan). The cDNA synthesis was confirmed by usual agarose gel electrophoresis method, then cDNAs were stored at −20 °C for Real-Time PCR experiments. Each Real-Time PCR experiment, triplicate for each sample, was subjected to ABI‑step I plus (Applied Biosystems, Forster City, CA, USA) instrument. Three gene‑specific sets of primers for Bax (F 5′-CCCGAG AGGTCTTTTTCCGAG-3′and R 5´- CCAGACCATAGCACACTCGG-3´), BCL-2 (F 5′- GGTGGGGTCATGTGTGTGG-3′and R 5′-CGGTTCAGGTAC TCAGTCATCC-3′) and housekipping gene GAPDH (F5´-AAGCTCATTT CCTGGTATGACAACG-3´ and R5´-TCTTCCTCTTGTGCTCTTGCTGG-3´) were used. The relative expression is commonly used where the expression of target genes are standardized by GAPDH as reference gene and was expressed as fold change from the GAPDH level and quantified using the ΔCt method [24].
Fig. 1. Effect of azilsartan and Bay 11-7082 (Bay) on cytotoxicity against HepG2 and KDR cell lines. (A) and (B) represent HepG2 and KDR cells treated with azilsartan, respectively. (C) Represents HepG2 cells treated with Bay 11-7082. Results are in mean ± SD (n = 6). X axis shows the concentration and Y axis shows the viability percentage. * Statistically significant compared to 24 h control. (p < .05). + Statistically significant compared to 24 h control. (p < .05). # Statistically significant compared to 24 h control. (p < .05).
2.9. Measurement of Bax and Bcl‐2 protein levels Bax and Bcl‐2 protein levels were measured using ELISA kits (Gibco, invitrogen) according to the manufacturer user guide. Briefly, treated and untreated control cells were lysed according to the ELISA kit instruction then100 μL of samples and standards were added into the appropriate wells and the plate was shacked (500 rpm) at room temperature for 1 h. After that, each content of wells were carefully emptied and were washed with 400 μL washing buffer (1X). After removing any remaining wash buffer, 100 μL of the Bax and/or Bcl-2 antibody were added into each appropriate well subsequently the plate was shacked (500 rpm) at room temperature for 1 h then the plate was washed (washing buffer 1X) and subjected for adding 100 μL human Bax and Bcl-2 Horseradish peroxidase (HRP)‐conjugated secondary antibody and was incubated at similar condition. After incubation time Bax and/or Bcl‐2 protein levels were determined by measuring the absorbance at 450 nm using a microplate reader (Biotek, ELx800, USA).
prepared using Microsoft Office Excel 2013 software. 3. Results 3.1. Cytotoxicity The MTT assay was performed using different concentrations of azilsartan (5, 25, 500, 100 and 200 μM) for 24, 48 and 72 hrs in cancerous/normal treated/untreated human cell lines to identify the capability of the drug to inhibit the cell growth. As illustrated in Fig. 1 part A, azilsartan gradually decreased the viability of HepG2 cells by increasing the incubation time and dose. Based on these findings, the inhibitory concentration of azilsartan (IC 50%) against HepG2 cells was 100 μM for 24 h treatment time point (Fig. 1, part A) while in KDR epithelial normal cells no significant cytotoxic effect was observed during the similar treatment conditions (Fig. 1, part B). As shown in Fig. 1 part C, Bay 11-7082 decreased the cell proliferation in a time and dose dependent manner. To elucidate the potential role of NF-KB in the cytotoxic activity of azilsartan, the combination of different concentrations of azilsartan (5–200 μM) with Bay117082 (10–25 μM) were added to HepG2 cells and the viability was
2.10. Statistical analysis The normal distribution of data was tested by Kolmogorov-Smirnov test and ANOVA and Tukey's post hoc test were used for analyzing data and multiple mean comparisons, respectively. Statistical significance was considered as a value of P ≤ .05 and quantitative data were reported as mean ± SD. The statistical analysis was performed by SPSS software version 16.0 (SPSS Inc, Chicago, IL, USA) and all graphs were 941
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Fig. 4. Effect of azilsartan and Bay 11-7082 (Bay) on cytochrome c release in HepG2 cells. HepG2 cells were treated with azilsartan (100 μM) and Bay 11-7082 (10, 25 μM) and the combination of azilsartan and Bay 11-7082 for 24 h and cytochrome c release was measured with an ELISA kit. Data represent the mean ± SD (n = 6). *p < .05 compared with control. +p < .05 compared with azilsartan (100 μM) and Bay 11-7082 (25 μM) alone.
Fig. 2. The effect of combination of azilsartan with Bay 11-7082 (Bay) on cell viability of HepG2. Cells were treated with different concentrations of combination of azilsartan with Bay 11-7082 for 24 h. Cell toxicity was assessed by MTT assay. Results are in mean ± SEM (n = 6). X axis shows the concentration and Y axis shows the viability percentage. * Statistically significant versus azilsartan (0). (p < .05). + Statistically significant versus Bay 11-7082 (10 μM). (p < .05). # Statistically significant versus Bay 11-7082 (25 μM). (p < .05).
the amount of ROS in comparison with both of them alone (Fig. 3).
determined 24 h after treatment. As shown in Fig. 2, treatment with the combination of azilsartan (50–200 μM) and Bay 11-7082 (10 μM) caused substantially more decrease in cell proliferation compared to Bay 11-7082 alone (10 μM). In addition, treatment with combination of azilsartan (5–200 μM) with Bay 11-7082 (25 μM) caused significantly more decrease in the viability of HepG2 cells compared to Bay 11-7082 (25 μM) alone. Furthermore, the CI value at IC50 of azilsartan along with Bay 11-7082 (25 μM) calculated to be 0.54 indicating synergism.
3.3. The effect of azilsartan and Bay 11-7082 on cytochrome c release Induction of mitochondrial permeability transition (MPT) is supposed to be ensued from opening of the related pores in mitochondrial membrane and further release of cytochrome c [25]. The incubation of HepG2 cells for 24 h with azilsartan (100 μM) and/or Bay 11-7082 (25 μM) induced the release of cytochrome c from mitochondria into the cytosol (p ≤ 005) (Fig. 4). Additionally, the combination of azilsartan (100 μM) and Bay 11-7082 (10 or 25 μM) caused a more potentiated cytochrome c release compared to azilsartan and/or Bay 117082 alone (Fig. 4).
3.2. Azilsartan and Bay 11-7082 induced ROS formation To determine whether the azilsartan-associated cytotoxicity is related to oxidative stress, azilsartan effect on the formation of ROS in HepG2 cells was examined. As shown in Fig. 3, treatment of HepG2 cells with azilsartan (100 μM) resulted in an upward trend in the formation of ROS after 24 h. Bay 11-7082 caused a significant increase in production of ROS at the concentration of 25 μM whereas at the concentration of 10 μM it did not have any significant impact on the amount of intracellular ROS production (Fig. 3). Although Bay 11-7082 did not increase intracellular ROS significantly at the concentration of 10 μM, the combination of azilsartan (100 μM) with Bay 11-7082 (10 μM) resulted in potentiated ROS formation for 24 h. Thus, the combination of azilsartan with Bay 11-7082, led to further increment in
3.4. DAPI staining The apoptosis incidence was qualitatively determined in HepG2 carcinoma cells treated with azilsartan (100 μM) and/or Bay 11-7082 (25 μM) and the combination of these two agents for 24 h. The presence of intact nucleus shows the healthy intact cells, whereas shrinking cells with condensed nucleus illustrates early apoptosis and fragmented nucleus indicates late apoptosis (Fig. 5, part A). The outcomes of this experiment revealed that the apoptotic cells with condensed nuclei, membrane blebbing, generation of micronuclei, and cell shrinkage were markedly higher in HepG2 cells treated with (100 μM) and Bay 11-7082 (25 μM) compared to the control (Fig. 5, parts B&C). In the groups treated with combination of (100 μM) and Bay 11-7082 (25 μM), the initiation of late apoptosis was observed which shows the synergistic effects of azilsartan and Bay 11-7082 in induction of apoptosis as a possible main mechanism of cytotoxicity pattern (Fig. 5, part D). 3.5. Apoptosis detection by flow cytometry The HepG2 cells treated with azilsartan (100 μM), Bay 11-7082 (25 μM) and combination of them for 24 h were analyzed by using Annexin V-FITC/PI kit. The dual parameter fluorescent dot blots show the viable cells in the lower left quadrant (Annexin V−/PI−), the cells at the early apoptosis in the lower right quadrant (Annexin V+/PI−), the cells at the late apoptosis in the upper right quadrant (Annexin V+/ PI+), and the necrotic cells in the upper left quadrant (Annexin V−/ PI+) (Fig. 5). Based on the findings, azilsartan at the concentration of 100 μM induced 57.2% early and 0.52% late apoptosis respectively after 24 h (Fig. 5 Part F). Also, Bay 11-7082 at the concentration of 25 μM could induce 40.72% early and 4.62% late apoptosis (Fig. 5 Part G). However, the initiation of late apoptosis stage was observed when
Fig. 3. The effect of azilsartan (100 μM), Bay 11-7082 (Bay) (10, 25 μM) and the combination of azilsartan and Bay 11-7082 on the amount of intracellular ROS. HepG2 cells were treated with azilsartan and Bay 11-7082 on for 24 h and changes in 2′, 7′dichlorofluorescin (DCF) fluorescence were measured. Data represent the mean ± SD (n = 6). *p < .05 compared with control. +p < .05 compared with azilsartan (100 μM) alone.
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Fig. 5. DAPI staining and flow cytometric analysis of HepG2 cells after incubation without any treating (A and E), with azilsartan 100 μM (B and F) with Bay 11-7082 (25 μM) (C and G), with azilsartan 100 μM + Bay 11-7082 (25 μM) (D and H) for 24 h. Dots with Annexin V−/PI− (lower left), Annexin V+/PI− (lower right), and Annexin V+/PI+ (upper right) Annexin V−/PI+ (upper left) features represent viable intact, early apoptotic, late apoptotic cells, and necrotic cells, respectively.
the mixture of these two agents was used to treatment of HepG2 cells. As shown in Fig. 5 Part H, a total of 66.39% of apoptosis occurred in cells treated with combined azilsartan 100 μM with Bay 11-7082 (25 μM).
through real time PCR assay. As illustrated in Fig. 6(B), the relative expression of Bax mRNA was increased in the presence of azilsartan and/or Bay 11-7082 and the addition of azilsartan and Bay 11-7082 together augmented the expression rate (p ≤ .05). Bcl-2 mRNA expression decreased markedly in azilsartan (100 μM) and/or Bay 117082 (25 μM)-treated cells. The combination of azilsartan and Bay 117082 had a more significant effect (p ≤ .05) in attenuation of Bcl-2 expression (Fig. 6, A). The levels of Bax and Bcl-2 proteins followed the similar expression pattern which were measured with ELISA technique (Fig. 7, parts A&B). Also, Bax/Bl2 ratio was evaluated acording to the
3.6. The effect of azilsartan and Bay 11-7082 on the expression of Bax and Bcl-2 Bax and Bcl-2 mRNA expression was measured following 24 h of treatment with azilsartan (100 μM) and/or Bay 11-7082 (25 μM) 943
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Fig. 6. Effect of azilsartan and Bay 11-7082 (Bay) on Bax and Bcl-2 mRNA expression in HepG2 cells. HepG2 cells were treated with azilsartan (100 μM) and Bay 11-7082 (10, 25 μM) and the combination of azilsartan and Bay 11-7082 for 24 h. Data represent the mean ± SD (n = 6). *p < .05 compared with control. +p < .05 compared with azilsartan (100 μM) and Bay 11-7082 (25 μM) alone.
candidate in pancreas cancer treatment [30]. Telmisartan as a peroxisome proliferator activated receptor (PPAR) agonist class of ARBs has been shown to inhibit prostate cancer cell growth in a dose and time dependent manner, since PPAR receptors are highly expressed in prostate cancer [28]. Losartan has been reported to plummet the pancreatic tumor progress in vivo and also has extended survival rate of SPARC-null mice during the experiment [13]. Kosugi et al. demonstrated anti-tumoral function of candesartan via the blockade of angiogenesis in xenograft model of bladder cancer [31]. In addition to ARBs, long term treatment with angiotensin converting enzyme inhibitors has also gained prominence against some human cancers [32]. Captopril has been shown to exert toxic effect on lung cancer cell lines and also in xenograft model via the proliferation and metastasis inhibiting impact. Therefore, captopril could be a promising treatment option for lung cancers [33]. Downstream signaling pathway of RAS has been linked to the activation of NF-kB [11] which is an important mediating factor in various cancers [34]. Bay 11-7082 as a potent irreversible inhibitor of NFkB has been shown to possess anti-inflammatory and neuroprotective effects [35]. This study’s outcomes are in line with other researches in which the cytotoxicity and apoptotic effects of Bay 11-7082 has been studied in some cancerous cells while no other study has been reported regarding the mentioned effects in HepG2 cells yet. Bay 11-7082 has been shown to increase the cytotoxic activity of imatinib in human leukemic cells. Furthermore, Bay 11-7082 has the ability to sensitize multi-drug resistant cells to apoptosis [36]. Chen et al. demonstrated the apoptotic effect of Bay 11-7082 in gastric cancer cells [37].
observed data. As shown in Fig. 7 part C, Bax/Bcl2 ratio is increased markedly in azilsartan- and/or Bay 11-7082 - treated cells. 4. Discussion HCC as a major health problem is the third most common cause of death from cancer worldwide [3]. Based on previous findings, RAS is overexpressed in different cancer cells such as cervical and hepatocellular carcinomas [26,27]. Hence, inhibition of RAS components may be considered as a favorable approach in cancer treatment thus ARBs as blocking agents of RAS are considered as novel anti-cancer drugs [28]. This current study for the first time represents the cytotoxic and apoptotic properties of azilsartan against HepG2 cell line with no significant cytotoxic effect on non-cancerous KDR cells. Azilsartan, a newly approved ARB, has been reported for lowering blood pressure more efficiently than maximally recommended doses of prior ARBs. Although azilsartan is considered as a remarkably potent ARB, little is known about the potential pleiotropic effects of this drug. Azilsartan has been reported to potently inhibit vascular cell proliferation. Besides, azilsartan can stimulate the expression of genes encoding PPAR receptors [16]. However, cytotoxic and apoptotic effects of azilsartan in cancer cell line have not been investigated so far. Antitumoral activity of ARBs and other RAS inhibitors have been reported in several in vivo and in vitro studies. For instance, Kurikawa et al. reported that olmesartan suppresses cell proliferation in activated hepatic satellite cells [29]. Olmesartan prevents pancreatic cancer cell growth through blockade of satellite cell activity in mice and is deemed to be a good
Fig. 7. Effect of azilsartan and Bay 11-7082 (Bay) on Bax and Bcl-2 protein expression in HepG2 cells. HepG2 cells were treated with azilsartan (100 μM) and Bay 11-7082 (10, 25 μM) and the combination of azilsartan and Bay 11-7082 for 24 h. Data represent the mean ± SD (n = 6). *p < .05 compared with control. +p < .05 compared with azilsartan (100 μM) and Bay 11-7082 (25 μM) alone.
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ROS formation has been implicated to the initiation of apoptosis as an early event [38,39]. Also, oxidative stress has shown to be prominent mechanism of drug-induced cytotoxicity [40,41]. Cancer cell death is regulated by elevated expression of mitochondrial apoptosis related proteins and loss of the mitochondrial membrane potential leading to activation of caspases [42]. Furthermore, apoptosis induced by many chemotherapeutic agents requires activation of oxidative signaling pathway [43]. ROS generation leads to the opening of mitochondrial transition pores and release of cytochrome c into the cytosol which in turn activates caspase cascade and apoptosis process [44,45]. Cytochrome c is normally located in the intermembrane space of the mitochondrion, loosely bound to the inner membrane. This study proved a significant generation of ROS and also liberation of cytochrome c into the cytosol in the azilsartan and Bay 11-7082 treated cells. Apoptosis stands for an organized and energy-dependent process leading to cell death which is of central significance to tissue survival and homeostasis [46]. Membrane blebbing, nuclear condensation, and the formation of apoptotic bodies are morphological signs of apoptosis. Also, a biochemical hallmark of apoptosis is the cleavage of chromatin into small fragments [47]. DAPI staining of azilsartan and Bay 11-7082 treated HepG2 cells was also associated with signs of apoptosis occurrence. Dual staining with FITC-conjugated Annexin-V allows discrimination of healthy viable, early apoptotic cells, and late apoptotic/ necrotic cells. Flow cytometry findings as well as DAPI staining support the conclusion that azilsartan and Bay 11-7082 induce cytotoxicity mainly through the apoptotic pathway. Members of the Bcl-2 family of proteins are critical mediators of the apoptotic process. Bcl-2 as a potent suppressor of apoptosis is an upstream effector molecule in the apoptotic pathway [48]. Bcl-2 has been shown to form a heterodimer with the pro-apoptotic member Bax and may neutralize its pro-apoptotic effects. Thus, changes in the levels of Bax and Bcl-2 has a decisive role in determining whether cells will undergo apoptosis or not [49]. In this study, a decrease in Bcl-2 mRNA/ protein expression and an increase in the mRNA/protein expression of Bax was observed in azilsartan- and/or Bay 11-7082 -treated HepG2 cells for 24 h. By considering these two genes expression, up-regulation of Bax and down-regulation of Bcl-2 may be involved in the molecular mechanism of azilsartan- and/or Bay 11-7082 -induced cell apoptosis. As a first report, this research’s data showed that Bay 11-7082 increased azilsartan-associated toxicity against HepG2 cell line. In conclusion, based on our knowledge, it is the first time to demonstrate that azilsartan and Bay 11-7082 have cytotoxic and apoptotic properties against HepG2 cancer cells and combination treatment with azilsartan and Bay 11-7082 led to higher levels of cytotoxicity in HepG2 cell line. Therefore, we could propose that combination use of azilsartan and Bay 11-7082 might be a novel aid in liver cancer treatment.
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Novel angiotensin receptor blocker, azilsartan induces oxidative stress and NFkB-mediated apoptosis in hepatocellular carcinoma cell line HepG2
T
Elham Ahmadiana,b,c,e,1, Ahmad Yari Khosroushahid,1, Aziz Eftekharib,e,f, Safar Farajniaa, ⁎ Hossein Babaeia,g, Mohammad Ali Eghbala,g,h, a
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Pharmacology and Toxicology Department, Maragheh University of Medical Sciences, Maragheh, Iran c Students’ Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran d Department of Pharmacognosy, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran e Department of Basic Sciences, Maragheh University of Medical Sciences, Maragheh, Iran f Toxicology Research Center, Maragheh University of Medical Sciences, Maragheh, Iran g Department of Pharmaclogy and Toxicology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran h Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran b
A R T I C L E I N F O
A B S T R A C T
Keywords: Liver cancer Angiotensin Apoptosis Azilsartan NF-kB pathway
Overexpression of renin angiotensin system (RAS) components and nuclear factor-kappa B (NF-kB) has a key role in various cancers. Blockade of RAS and NF-kB pathway has been suggested to reduce cancer cell proliferation. This study aimed to investigate the role of angiotensin II and NF-kB pathway in liver hepatocellular carcinoma cell line (HepG2) proliferation by using azilsartan (as a novel Ag II antagonist) and Bay 11-7082 (as NF-kB inhibitor). HepG2 cells were treated with different concentrations of azilsartan and Bay 11-7082. Cytotoxicity was determined after 24, 48, and 72 h by MTT assay. Reactive oxygen spices (ROS) generation and cytochrome c release were measured following azilsartan and Bay11- 7082 treatment. Apoptosis was analyzed qualitatively by DAPI staining and quantitatively through flow cytometry methodologies and Bax and Bcl-2 mRNA and protein levels were assessed by real time PCR and ELISA methods, respectively. The cytotoxic effects of different concentration of azilsartan and Bay11- 7082 on HepG2 cells were observed as a reduction in cell viability, increased ROS formation, cytochrome c release and apoptosis induction. These effects were found to correlate with a shift in Bax level and a downward trend in the expression of Bcl-2. These findings suggest that azilsartan and Bay117082 in combination or alone have strong potential as an agent for prevention or treatment of liver cancer after further studies.
1. Introduction Cancer, as a major public health problem, is the second leading cause of death in the world [1]. According to world health organization (WHO) report, there is an estimation of more than 21 million cancer cases and 13 million deaths worldwide [2]. Hepatocellular carcinoma (HCC) has been previously reported to be the third most common cause of death from cancer [3]. Although surgical resection is considered as the standard curative treatment of HCC, the majority of patients are not candidates for this type of treatment mainly due to the advanced tumor extension at the first diagnosis and/or insufficient liver functionality reservoirs [4]. In addition, chemotherapy regimes in suitable candidates have often been restricted because of major organ damages,
inefficient drugs and poor prognosis [5]. Thus, search for novel anticancer agents or regimes with higher efficacy and minimal side effects is continued. Angiotensin II (Ang II), is a multifunctional octapeptide component of the renin angiotensin system (RAS) with modulatory effects on angiotensin receptors type 1 (AT1) and type 2 (AT2) [6]. Ang II is a potent vasoconstrictive agent which regulates blood pressure and body fluid homeostasis [7]. Apart from the well-known systemic role of the RAS in cardiovascular and renal systems, Ang II has been shown to have various pleiotropic functions one of which is to meet the hallmarks of the cancer [8]. Recent researches have shown the overexpression of RAS in different cancer cells and tissues such as lung, breast, pancreas and liver [9]. Ang II has also been linked to various signaling pathways to play a
⁎
Correspondence author at: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Postal Code 51664-14766, Tabriz, Iran. E-mail addresses: [email protected] (E. Ahmadian), [email protected] (A.Y. Khosroushahi), [email protected] (A. Eftekhari), [email protected] (S. Farajnia), [email protected] (H. Babaei), [email protected] (M.A. Eghbal). 1 These authors contributed equally to this manuscript. https://doi.org/10.1016/j.biopha.2018.01.117 Received 23 November 2017; Received in revised form 13 January 2018; Accepted 24 January 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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microplate reader (Biotek, ELx800, USA). Combination index (CI) analysis based on Chou and Talalay method [18] was performed using CompuSyn software for Azilsartan and Bay 11-7082 combinations, determining synergistic, additive, or antagonistic cytotoxic effects against HepG2 cancer cells. Values of CI < 1 demonstrate synergism while CI = 1 and CI > 1 values represent additive and antagonistic effects of drug combination, respectively.
key role in tumor cell growth and migration [10]. AT1 downstream signaling is documented to lead to the activation of nuclear factorkappa B (NF-kB) [11]. NF-kB is a transcription factor involved in the cellular survival pathway that activated in response to several stimuli such as hypoxia, bacterial endotoxins and inflammatory cytokines. Also, NF-kB is a key regulator of gene expression in inflammatory-related malignant tumors [12] and inhibition of this pathway can be a potential option for treatment of cancer. Angiotensin receptor blockers (ARBs) are considered with very low side effects compared to chemotherapeutic agents and many ARBs have been addressed to possess potential for suppressing tumor growth in vitro and in vivo [13]. Azilsartan, as a potent and highly selective ARB, is a novel long lasting drug approved for the treatment of hypertension [14] and at its maximal dose has superior efficacy to other ARBs such as valsartan and olmesartan at their maximal approved doses without increasing adverse events [15]. Azilsartan potently inhibits aortic endothelial and vascular cell proliferation in the absence of exogenous Ang II supplementation [16]. However, apoptotic and anti-proliferative impacts of azilsartan in cancer cell lines have not been examined yet. This study aimed to assess the possible cytotoxicity mechanism of azilsartan in hepatocellular carcinoma cell line (HepG2) in association with NF-kB signaling pathway. Therefore, the cytotoxic and apoptotic roles of Ang II and NF-kB pathways in HepG2 cell line were studied using azilsartan and Bay 11-7082.
2.4. Determination of ROS formation
2. Materials and methods
The non-fluorescent dye, 2′,7′dichlorofluorescin diacetate (DCFHDA), (which is oxidized to fluorescent dichlorofluorescein (DCF) by hydroperoxides) was utilized to determine relative levels of cellular ROS generation [19,20]. Cells (3 × 104 cells/well) were treated with azilsartan (0–200 μM) for 24 h at 37 °C. Also ROS formation in azilsartan–treated (200 μM) cells were measured in the presence of Nacetyl cysteine (NAC) as a potent well-known antioxidant. All treated/ untreated control cells were detached by trypsin-EDTA (0.25% trypsin and 0.02% EDTA) and were washed by PBS (0.1 M, pH 7.2) and then were incubated in FBS free culture medium containing 50 μM dye for 30 min. The washing process was performed once more and then the cell suspensions were centrifuged at 412g for 10 min. the supernatant was removed and cell plates were dissolved with 1% Triton X100. Fluorescence changes were measured using a Jasco R_FP-750 spectrofluorometer (Jasco Corporation, Tokyo, Japan) with excitation and emission wavelengths of 485 and 530 nm, respectively [21].
2.1. Chemicals
2.5. Determination of cytochrome c amount
Glycine, NaCl, chloroform, and diethylpyrocarbonate (DEPC) were obtained from Merck (Merck, Darmstadt, Germany). 3-(4,5-Dimethyl-2thiazolyl)-2,5-diphenyltetra-zoplium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640, penicillin and streptomycin and trypsin-EDTA solutions, L- glutamine, phosphate buffered saline (PBS), and fetal bovine serum (FBS) were purchased from Gibco Life Technologies Ltd. (Tulsa, OK, USA). Azilsartan, 2′,7′dichlorofluorescin diacetate (DCFH-DA), Bay 11-7082 were purchased from Sigma-Aldrich (St Louis, MO, USA). Azilsartan and Bay 117082 were pharmaceutical grade and all other materials were analytical/cell culture tested grade. All materials were used without further purification.
Enzyme linked immunosorbent assay (ELISA) kit (Cytochrome C ELISA Kit, Human - Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify cytosolic cytochrome c. The cells in the density of 5 × 105 were lysed in lysis buffer according to the manufacturer’s user instructions and samples were centrifuged at 500g, room temperature for 10 min. The supernatants and cytochrome c conjugate were added to the 96 well microplates coated with human cytochrome c monoclonal antibody. The absorbance of samples was measured at 450 nm in a microplate reader (Biotek, ELx800, USA). A standard curve was prepared by plotting the absorbance values of diluted solutions of a human cytochrome c standard and the cytochrome c concentration was expressed as ng/ml [22].
2.2. Cell culture
2.6. DAPI staining method
HepG2 (liver hepatocellular adenocarcinoma) and normal epithelial KDR cell lines were obtained from Pasture Institute, Tehran, Iran. Cell lines were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 μg/ml of streptomycin and were incubated in humidified atmosphere containing 5% CO2 at 37 °C according to the guidebook of the cell line bank.
All treated and normal control cells groups were stained using DAPI staining method to determine qualitatively the presence of apoptotic cells. After placing sterile cover slips into each well of a 6-well culture plate, wells were seeded by HepG2 cells with seeding density of 120 × 104 cell/well. After overnight of post-seeding, the cells were treated with azilsartan and were incubated for 24 h. For apoptosis detection, all treated/untreated control cells were fixed with paraformaldehyde (4%) for 5 min then the fixed cells were permeabilized with 0.1% Triton-X100 at 37 °C for 5 min and finally the permeabilized cells were stained with 50 μl DAPI dye (1:2000 dilution) per well at room temperature for 3 min. The stained cells on slips were washed with PBS (0.1 M, pH 7.2) thrice and were utilized for apoptosis assessments by fluorescent microscopy (BX63, Olympus, Japan) equipped with U-MWU2 fluorescence filter set (excitation filter BP 330-385, dichromatic mirror DM 400, and emission filter LP 420) [23].
2.3. Determination of cell proliferation The effects of azilsartan on cell proliferation of HepG2 and KDR cells were determined by MTT assay [17]. Cells, with seeding density 1.2 × 104 cells/well, were seeded in 96-well microplate containing 200 μl RPMI growth medium. The cells were then treated with various concentrations of azilsartan (5, 25, 50, 100 and 200 μM) and/or Bay 117082 (5, 10 and 25 μM) over different incubation time points (24, 48, and 72 h). After treatment time point, cells (treated/untreated control cells) were incubated with 150 μl fresh medium plus 50 μl MTT solutions (2 mg/ml in PBS) for 4 h at 37 °C. After incubation time, the MTT contained medium was removed and the mixture of 200 μl DMSO and 25 μl ml Sorenson’s glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5) was added to each well and the cells were again incubated for 30 min at 37 °C. The absorbance of plates was measured at 570 nm using a
2.7. Apoptosis detection by flow cytometry assay Apoptosis was quantitatively determined using annexin V apoptosis detection kit (BD Biosciences Pharmingen, San Diego, CA, USA) according to the manufacturer’s user instructions. Cells (1.2 × 105 cells/ well) were seeded into a six-well culture plate. The cells were treated 940
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with azilsartan (100 μM) or Bay 11-7082 (25 μM) and/or the combination of these two agents at reaching 50% confluence. After 24 h, the treated/untreated control cells were detached by trypsin and then washed with cold PBS (pH 7.2) subsequently centrifuged at 1100 rpm for 7 min at 28 °C. The cell pellet of HepG2 was re-suspended in 500 μl of 1X binding buffer, the tubes were centrifuged again at previous conditions and the supernatants were replaced with100 μl binding buffer (1X). This tubes were added with 5 μl of FITC-conjugated Annexin V, and then incubated for 15 min at room temperature under dark conditions. Finally, 5 μl of propidium iodide solution was added to the cells, and quadrant settings were fixed with untreated, singlestained controls and copied to dot plots of the treated cells. Quadrant statistic calculations were performed using CELLQuest Pro software (BD Bioscience, San Diego, CA, USA) on data obtained from two times repeated experiments with triplicate samples for each experiment. Analyses were accomplished using 100,000 cells at a rate of 500 cells/s. FL-1 and FL-3 were represented in dot plots illustrating the viable, apoptotic, and necrotic cells. 2.8. Real time All untreated/treated cells were washed with phosphate buffer saline (pH 7.2) then 1 ml ice cold RNX-plus solution (SinaClone, Iran) was added into cells according the kit user instruments. Complementary DNA (cDNA) was synthesized using PrimeScript RT Reagent kit (Takara Bio Inc., Tokyo, Japan). The cDNA synthesis was confirmed by usual agarose gel electrophoresis method, then cDNAs were stored at −20 °C for Real-Time PCR experiments. Each Real-Time PCR experiment, triplicate for each sample, was subjected to ABI‑step I plus (Applied Biosystems, Forster City, CA, USA) instrument. Three gene‑specific sets of primers for Bax (F 5′-CCCGAG AGGTCTTTTTCCGAG-3′and R 5´- CCAGACCATAGCACACTCGG-3´), BCL-2 (F 5′- GGTGGGGTCATGTGTGTGG-3′and R 5′-CGGTTCAGGTAC TCAGTCATCC-3′) and housekipping gene GAPDH (F5´-AAGCTCATTT CCTGGTATGACAACG-3´ and R5´-TCTTCCTCTTGTGCTCTTGCTGG-3´) were used. The relative expression is commonly used where the expression of target genes are standardized by GAPDH as reference gene and was expressed as fold change from the GAPDH level and quantified using the ΔCt method [24].
Fig. 1. Effect of azilsartan and Bay 11-7082 (Bay) on cytotoxicity against HepG2 and KDR cell lines. (A) and (B) represent HepG2 and KDR cells treated with azilsartan, respectively. (C) Represents HepG2 cells treated with Bay 11-7082. Results are in mean ± SD (n = 6). X axis shows the concentration and Y axis shows the viability percentage. * Statistically significant compared to 24 h control. (p < .05). + Statistically significant compared to 24 h control. (p < .05). # Statistically significant compared to 24 h control. (p < .05).
2.9. Measurement of Bax and Bcl‐2 protein levels Bax and Bcl‐2 protein levels were measured using ELISA kits (Gibco, invitrogen) according to the manufacturer user guide. Briefly, treated and untreated control cells were lysed according to the ELISA kit instruction then100 μL of samples and standards were added into the appropriate wells and the plate was shacked (500 rpm) at room temperature for 1 h. After that, each content of wells were carefully emptied and were washed with 400 μL washing buffer (1X). After removing any remaining wash buffer, 100 μL of the Bax and/or Bcl-2 antibody were added into each appropriate well subsequently the plate was shacked (500 rpm) at room temperature for 1 h then the plate was washed (washing buffer 1X) and subjected for adding 100 μL human Bax and Bcl-2 Horseradish peroxidase (HRP)‐conjugated secondary antibody and was incubated at similar condition. After incubation time Bax and/or Bcl‐2 protein levels were determined by measuring the absorbance at 450 nm using a microplate reader (Biotek, ELx800, USA).
prepared using Microsoft Office Excel 2013 software. 3. Results 3.1. Cytotoxicity The MTT assay was performed using different concentrations of azilsartan (5, 25, 500, 100 and 200 μM) for 24, 48 and 72 hrs in cancerous/normal treated/untreated human cell lines to identify the capability of the drug to inhibit the cell growth. As illustrated in Fig. 1 part A, azilsartan gradually decreased the viability of HepG2 cells by increasing the incubation time and dose. Based on these findings, the inhibitory concentration of azilsartan (IC 50%) against HepG2 cells was 100 μM for 24 h treatment time point (Fig. 1, part A) while in KDR epithelial normal cells no significant cytotoxic effect was observed during the similar treatment conditions (Fig. 1, part B). As shown in Fig. 1 part C, Bay 11-7082 decreased the cell proliferation in a time and dose dependent manner. To elucidate the potential role of NF-KB in the cytotoxic activity of azilsartan, the combination of different concentrations of azilsartan (5–200 μM) with Bay117082 (10–25 μM) were added to HepG2 cells and the viability was
2.10. Statistical analysis The normal distribution of data was tested by Kolmogorov-Smirnov test and ANOVA and Tukey's post hoc test were used for analyzing data and multiple mean comparisons, respectively. Statistical significance was considered as a value of P ≤ .05 and quantitative data were reported as mean ± SD. The statistical analysis was performed by SPSS software version 16.0 (SPSS Inc, Chicago, IL, USA) and all graphs were 941
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Fig. 4. Effect of azilsartan and Bay 11-7082 (Bay) on cytochrome c release in HepG2 cells. HepG2 cells were treated with azilsartan (100 μM) and Bay 11-7082 (10, 25 μM) and the combination of azilsartan and Bay 11-7082 for 24 h and cytochrome c release was measured with an ELISA kit. Data represent the mean ± SD (n = 6). *p < .05 compared with control. +p < .05 compared with azilsartan (100 μM) and Bay 11-7082 (25 μM) alone.
Fig. 2. The effect of combination of azilsartan with Bay 11-7082 (Bay) on cell viability of HepG2. Cells were treated with different concentrations of combination of azilsartan with Bay 11-7082 for 24 h. Cell toxicity was assessed by MTT assay. Results are in mean ± SEM (n = 6). X axis shows the concentration and Y axis shows the viability percentage. * Statistically significant versus azilsartan (0). (p < .05). + Statistically significant versus Bay 11-7082 (10 μM). (p < .05). # Statistically significant versus Bay 11-7082 (25 μM). (p < .05).
the amount of ROS in comparison with both of them alone (Fig. 3).
determined 24 h after treatment. As shown in Fig. 2, treatment with the combination of azilsartan (50–200 μM) and Bay 11-7082 (10 μM) caused substantially more decrease in cell proliferation compared to Bay 11-7082 alone (10 μM). In addition, treatment with combination of azilsartan (5–200 μM) with Bay 11-7082 (25 μM) caused significantly more decrease in the viability of HepG2 cells compared to Bay 11-7082 (25 μM) alone. Furthermore, the CI value at IC50 of azilsartan along with Bay 11-7082 (25 μM) calculated to be 0.54 indicating synergism.
3.3. The effect of azilsartan and Bay 11-7082 on cytochrome c release Induction of mitochondrial permeability transition (MPT) is supposed to be ensued from opening of the related pores in mitochondrial membrane and further release of cytochrome c [25]. The incubation of HepG2 cells for 24 h with azilsartan (100 μM) and/or Bay 11-7082 (25 μM) induced the release of cytochrome c from mitochondria into the cytosol (p ≤ 005) (Fig. 4). Additionally, the combination of azilsartan (100 μM) and Bay 11-7082 (10 or 25 μM) caused a more potentiated cytochrome c release compared to azilsartan and/or Bay 117082 alone (Fig. 4).
3.2. Azilsartan and Bay 11-7082 induced ROS formation To determine whether the azilsartan-associated cytotoxicity is related to oxidative stress, azilsartan effect on the formation of ROS in HepG2 cells was examined. As shown in Fig. 3, treatment of HepG2 cells with azilsartan (100 μM) resulted in an upward trend in the formation of ROS after 24 h. Bay 11-7082 caused a significant increase in production of ROS at the concentration of 25 μM whereas at the concentration of 10 μM it did not have any significant impact on the amount of intracellular ROS production (Fig. 3). Although Bay 11-7082 did not increase intracellular ROS significantly at the concentration of 10 μM, the combination of azilsartan (100 μM) with Bay 11-7082 (10 μM) resulted in potentiated ROS formation for 24 h. Thus, the combination of azilsartan with Bay 11-7082, led to further increment in
3.4. DAPI staining The apoptosis incidence was qualitatively determined in HepG2 carcinoma cells treated with azilsartan (100 μM) and/or Bay 11-7082 (25 μM) and the combination of these two agents for 24 h. The presence of intact nucleus shows the healthy intact cells, whereas shrinking cells with condensed nucleus illustrates early apoptosis and fragmented nucleus indicates late apoptosis (Fig. 5, part A). The outcomes of this experiment revealed that the apoptotic cells with condensed nuclei, membrane blebbing, generation of micronuclei, and cell shrinkage were markedly higher in HepG2 cells treated with (100 μM) and Bay 11-7082 (25 μM) compared to the control (Fig. 5, parts B&C). In the groups treated with combination of (100 μM) and Bay 11-7082 (25 μM), the initiation of late apoptosis was observed which shows the synergistic effects of azilsartan and Bay 11-7082 in induction of apoptosis as a possible main mechanism of cytotoxicity pattern (Fig. 5, part D). 3.5. Apoptosis detection by flow cytometry The HepG2 cells treated with azilsartan (100 μM), Bay 11-7082 (25 μM) and combination of them for 24 h were analyzed by using Annexin V-FITC/PI kit. The dual parameter fluorescent dot blots show the viable cells in the lower left quadrant (Annexin V−/PI−), the cells at the early apoptosis in the lower right quadrant (Annexin V+/PI−), the cells at the late apoptosis in the upper right quadrant (Annexin V+/ PI+), and the necrotic cells in the upper left quadrant (Annexin V−/ PI+) (Fig. 5). Based on the findings, azilsartan at the concentration of 100 μM induced 57.2% early and 0.52% late apoptosis respectively after 24 h (Fig. 5 Part F). Also, Bay 11-7082 at the concentration of 25 μM could induce 40.72% early and 4.62% late apoptosis (Fig. 5 Part G). However, the initiation of late apoptosis stage was observed when
Fig. 3. The effect of azilsartan (100 μM), Bay 11-7082 (Bay) (10, 25 μM) and the combination of azilsartan and Bay 11-7082 on the amount of intracellular ROS. HepG2 cells were treated with azilsartan and Bay 11-7082 on for 24 h and changes in 2′, 7′dichlorofluorescin (DCF) fluorescence were measured. Data represent the mean ± SD (n = 6). *p < .05 compared with control. +p < .05 compared with azilsartan (100 μM) alone.
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Fig. 5. DAPI staining and flow cytometric analysis of HepG2 cells after incubation without any treating (A and E), with azilsartan 100 μM (B and F) with Bay 11-7082 (25 μM) (C and G), with azilsartan 100 μM + Bay 11-7082 (25 μM) (D and H) for 24 h. Dots with Annexin V−/PI− (lower left), Annexin V+/PI− (lower right), and Annexin V+/PI+ (upper right) Annexin V−/PI+ (upper left) features represent viable intact, early apoptotic, late apoptotic cells, and necrotic cells, respectively.
the mixture of these two agents was used to treatment of HepG2 cells. As shown in Fig. 5 Part H, a total of 66.39% of apoptosis occurred in cells treated with combined azilsartan 100 μM with Bay 11-7082 (25 μM).
through real time PCR assay. As illustrated in Fig. 6(B), the relative expression of Bax mRNA was increased in the presence of azilsartan and/or Bay 11-7082 and the addition of azilsartan and Bay 11-7082 together augmented the expression rate (p ≤ .05). Bcl-2 mRNA expression decreased markedly in azilsartan (100 μM) and/or Bay 117082 (25 μM)-treated cells. The combination of azilsartan and Bay 117082 had a more significant effect (p ≤ .05) in attenuation of Bcl-2 expression (Fig. 6, A). The levels of Bax and Bcl-2 proteins followed the similar expression pattern which were measured with ELISA technique (Fig. 7, parts A&B). Also, Bax/Bl2 ratio was evaluated acording to the
3.6. The effect of azilsartan and Bay 11-7082 on the expression of Bax and Bcl-2 Bax and Bcl-2 mRNA expression was measured following 24 h of treatment with azilsartan (100 μM) and/or Bay 11-7082 (25 μM) 943
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Fig. 6. Effect of azilsartan and Bay 11-7082 (Bay) on Bax and Bcl-2 mRNA expression in HepG2 cells. HepG2 cells were treated with azilsartan (100 μM) and Bay 11-7082 (10, 25 μM) and the combination of azilsartan and Bay 11-7082 for 24 h. Data represent the mean ± SD (n = 6). *p < .05 compared with control. +p < .05 compared with azilsartan (100 μM) and Bay 11-7082 (25 μM) alone.
candidate in pancreas cancer treatment [30]. Telmisartan as a peroxisome proliferator activated receptor (PPAR) agonist class of ARBs has been shown to inhibit prostate cancer cell growth in a dose and time dependent manner, since PPAR receptors are highly expressed in prostate cancer [28]. Losartan has been reported to plummet the pancreatic tumor progress in vivo and also has extended survival rate of SPARC-null mice during the experiment [13]. Kosugi et al. demonstrated anti-tumoral function of candesartan via the blockade of angiogenesis in xenograft model of bladder cancer [31]. In addition to ARBs, long term treatment with angiotensin converting enzyme inhibitors has also gained prominence against some human cancers [32]. Captopril has been shown to exert toxic effect on lung cancer cell lines and also in xenograft model via the proliferation and metastasis inhibiting impact. Therefore, captopril could be a promising treatment option for lung cancers [33]. Downstream signaling pathway of RAS has been linked to the activation of NF-kB [11] which is an important mediating factor in various cancers [34]. Bay 11-7082 as a potent irreversible inhibitor of NFkB has been shown to possess anti-inflammatory and neuroprotective effects [35]. This study’s outcomes are in line with other researches in which the cytotoxicity and apoptotic effects of Bay 11-7082 has been studied in some cancerous cells while no other study has been reported regarding the mentioned effects in HepG2 cells yet. Bay 11-7082 has been shown to increase the cytotoxic activity of imatinib in human leukemic cells. Furthermore, Bay 11-7082 has the ability to sensitize multi-drug resistant cells to apoptosis [36]. Chen et al. demonstrated the apoptotic effect of Bay 11-7082 in gastric cancer cells [37].
observed data. As shown in Fig. 7 part C, Bax/Bcl2 ratio is increased markedly in azilsartan- and/or Bay 11-7082 - treated cells. 4. Discussion HCC as a major health problem is the third most common cause of death from cancer worldwide [3]. Based on previous findings, RAS is overexpressed in different cancer cells such as cervical and hepatocellular carcinomas [26,27]. Hence, inhibition of RAS components may be considered as a favorable approach in cancer treatment thus ARBs as blocking agents of RAS are considered as novel anti-cancer drugs [28]. This current study for the first time represents the cytotoxic and apoptotic properties of azilsartan against HepG2 cell line with no significant cytotoxic effect on non-cancerous KDR cells. Azilsartan, a newly approved ARB, has been reported for lowering blood pressure more efficiently than maximally recommended doses of prior ARBs. Although azilsartan is considered as a remarkably potent ARB, little is known about the potential pleiotropic effects of this drug. Azilsartan has been reported to potently inhibit vascular cell proliferation. Besides, azilsartan can stimulate the expression of genes encoding PPAR receptors [16]. However, cytotoxic and apoptotic effects of azilsartan in cancer cell line have not been investigated so far. Antitumoral activity of ARBs and other RAS inhibitors have been reported in several in vivo and in vitro studies. For instance, Kurikawa et al. reported that olmesartan suppresses cell proliferation in activated hepatic satellite cells [29]. Olmesartan prevents pancreatic cancer cell growth through blockade of satellite cell activity in mice and is deemed to be a good
Fig. 7. Effect of azilsartan and Bay 11-7082 (Bay) on Bax and Bcl-2 protein expression in HepG2 cells. HepG2 cells were treated with azilsartan (100 μM) and Bay 11-7082 (10, 25 μM) and the combination of azilsartan and Bay 11-7082 for 24 h. Data represent the mean ± SD (n = 6). *p < .05 compared with control. +p < .05 compared with azilsartan (100 μM) and Bay 11-7082 (25 μM) alone.
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ROS formation has been implicated to the initiation of apoptosis as an early event [38,39]. Also, oxidative stress has shown to be prominent mechanism of drug-induced cytotoxicity [40,41]. Cancer cell death is regulated by elevated expression of mitochondrial apoptosis related proteins and loss of the mitochondrial membrane potential leading to activation of caspases [42]. Furthermore, apoptosis induced by many chemotherapeutic agents requires activation of oxidative signaling pathway [43]. ROS generation leads to the opening of mitochondrial transition pores and release of cytochrome c into the cytosol which in turn activates caspase cascade and apoptosis process [44,45]. Cytochrome c is normally located in the intermembrane space of the mitochondrion, loosely bound to the inner membrane. This study proved a significant generation of ROS and also liberation of cytochrome c into the cytosol in the azilsartan and Bay 11-7082 treated cells. Apoptosis stands for an organized and energy-dependent process leading to cell death which is of central significance to tissue survival and homeostasis [46]. Membrane blebbing, nuclear condensation, and the formation of apoptotic bodies are morphological signs of apoptosis. Also, a biochemical hallmark of apoptosis is the cleavage of chromatin into small fragments [47]. DAPI staining of azilsartan and Bay 11-7082 treated HepG2 cells was also associated with signs of apoptosis occurrence. Dual staining with FITC-conjugated Annexin-V allows discrimination of healthy viable, early apoptotic cells, and late apoptotic/ necrotic cells. Flow cytometry findings as well as DAPI staining support the conclusion that azilsartan and Bay 11-7082 induce cytotoxicity mainly through the apoptotic pathway. Members of the Bcl-2 family of proteins are critical mediators of the apoptotic process. Bcl-2 as a potent suppressor of apoptosis is an upstream effector molecule in the apoptotic pathway [48]. Bcl-2 has been shown to form a heterodimer with the pro-apoptotic member Bax and may neutralize its pro-apoptotic effects. Thus, changes in the levels of Bax and Bcl-2 has a decisive role in determining whether cells will undergo apoptosis or not [49]. In this study, a decrease in Bcl-2 mRNA/ protein expression and an increase in the mRNA/protein expression of Bax was observed in azilsartan- and/or Bay 11-7082 -treated HepG2 cells for 24 h. By considering these two genes expression, up-regulation of Bax and down-regulation of Bcl-2 may be involved in the molecular mechanism of azilsartan- and/or Bay 11-7082 -induced cell apoptosis. As a first report, this research’s data showed that Bay 11-7082 increased azilsartan-associated toxicity against HepG2 cell line. In conclusion, based on our knowledge, it is the first time to demonstrate that azilsartan and Bay 11-7082 have cytotoxic and apoptotic properties against HepG2 cancer cells and combination treatment with azilsartan and Bay 11-7082 led to higher levels of cytotoxicity in HepG2 cell line. Therefore, we could propose that combination use of azilsartan and Bay 11-7082 might be a novel aid in liver cancer treatment.
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