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mTOR signaling pathway in human breast cancer cells
Bioorganic Chemistry 66 (2016) 124–131
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Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg
Benzo[b]furan derivatives induces apoptosis by targeting the PI3K/Akt/ mTOR signaling pathway in human breast cancer cells Ahmed Kamal a,b,⇑, V. Lakshma Nayak a, Narayana Nagesh c, M.V.P.S. Vishnuvardhan a, N.V. Subba Reddy a a
Medicinal Chemistry and Pharmacology, CSIR – Indian Institute of Chemical Technology, Hyderabad 500 007, India Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Hyderabad 500 037, India c CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India b
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Article history: Received 3 February 2016 Revised 13 April 2016 Accepted 25 April 2016 Available online 26 April 2016 Keywords: Benzo[b]furan derivatives Breast cancer Cell cycle PI3K/Akt/mTOR pathway Apoptosis
a b s t r a c t The PI3K/Akt/mTOR signaling pathway plays a key regulatory function in cell survival, proliferation, migration, metabolism and apoptosis. Aberrant activation of the PI3K/Akt/mTOR pathway is found in many types of cancer and thus plays a major role in breast cancer cell proliferation. In our previous studies, benzo[b]furan derivatives were evaluated for their anticancer activity and the lead compounds identified were 26 and 36. These observations prompted us to investigate the molecular mechanism and apoptotic pathway of these lead molecules against breast cancer cells. Benzo[b]furan derivatives (26 and 36) were evaluated for their antiproliferative activity against human breast cancer cell lines MCF-7 and MDA MB-231. These compounds (26 and 36) have shown potent efficiency against breast cancer cells (MCF-7) with IC50 values 0.057 and 0.051 lM respectively. Cell cycle analysis revealed that these compounds induced cell cycle arrest at G2/M phase in MCF-7 cells. Western blot analysis revealed that these compounds inhibit the PI3K/Akt/mTOR signaling pathway and induced mitochondrial mediated apoptosis in human breast cancer cells (MCF-7). Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Breast cancer is the most common cause of cancer death among women worldwide, and especially young women in the developing world. If current rates of increase remain constant, a woman born today has a 1 in 10 chance of developing breast cancer [1,2]. The discovery and development of new, more active, more selective compounds for the treatment of breast cancer is one of the most important goals in medicinal chemistry. Therefore this needs extensive research that will lead to more effective strategies for breast cancer treatment and prevention. The phosphatidylinositol 3-kinase (PI3K/AKT)/mammalian target of rapamycin (mTOR) pathway is known to be aberrantly activated in numerous human cancers including breast cancer [3]. This pathway plays a central role in diverse cellular functions including proliferation, growth, survival and metabolism. Accumulating data from genetic and cancer biology studies have indicated that the PI3K/AKT/mTOR pathway has a prominent role in cancer cell growth and survival, and there is now extensive evidence validating various components of this pathway as potential molecular ⇑ Corresponding author at: Medicinal Chemistry and Pharmacology, CSIR – Indian Institute of Chemical Technology, Hyderabad 500 007, India. E-mail address: [email protected] (A. Kamal). http://dx.doi.org/10.1016/j.bioorg.2016.04.004 0045-2068/Ó 2016 Elsevier Inc. All rights reserved.
targets for treating cancer [4–6]. Therefore, PI3K/AKT/mTOR signaling pathway is considered as an attractive target for the development of newer anticancer agents that could be used alone or in combination with other targeted therapies for the treatment of breast cancer patients. The mechanisms underlying the development of breast cancer is complex [7]. Breast epithelial cell homeostasis requires the balance of cell proliferation with a type of cell death called apoptosis and defects in apoptosis (programmed cell death) can contribute to tumor formation and progression [8,9]. Benzofurans and their derivatives possess a broad range of important biological activities including anticancer, antibacterial, antifungal and antiviral properties. They have attracted considerable attention among organic and medicinal chemists in the last few years [10,11]. In our earlier publication [12], we have synthesized a series of benzo[b]furan derivatives and evaluated for their antiproliferative activity and this investigation provided two interesting lead compounds 26 and 36. Therefore it was considered to further worthwhile study of this class of compounds and to understand their mechanism of action. The encouraging results obtained in our earlier studies [12], prompted us to evaluate these potent molecules for their antiproliferative activity against human breast cancer cell lines, MCF-7 and MDA MB-231. These conjugates showed significant antiproliferative activity against human breast cancer cell line, MCF-7. The prominent anticancer activity of these
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conjugates encouraged us to explore the molecular mechanism against breast cancer cell line (Fig. 1).
chemiluminescence reagent (Thermo Scientific Inc.). Images were captured by using the chemiluminescence (vilber lourmat) [15].
2. Materials and methods
2.4. Hoechst staining for apoptosis
2.1. MTT assay
MCF-7 cells were seeded at a density of 10,000 cells over 18-mm cover slips and incubated for 24 h. After incubation, cells were treated with compounds 26 and 36 at 50 nM concentration for 48 h. After treatment, cells were stained with Hoechst 33,258 (Sigma-Aldrich) and incubated for 30 min at 37 °C. Later, cells were washed with phosphate buffered saline (PBS). Cells from each cover slip were captured from randomly selected fields under a fluorescence microscope to qualitatively determine the proportion of viable and apoptotic cells based on their relative fluorescence and nuclear fragmentation [16].
The antiproliferative activity of the compounds (26 and 36) was determined by employing MTT assay [13]. Cells were seeded in 200 ll of DMEM (Dulbecco’s Modified Eagle Medium) or MEM (Minimum Essential Medium), supplemented with 10% FBS in each well of 96-well microculture plates and incubated for 24 h at CO2 incubator. After 24 h of incubation, cells were treated with the test compounds for 48 h. After 48 h of incubation, 20 ll MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (5 mg/mL) was added to each well and the plates were further incubated for 4 h. Then the supernatant from each well was carefully removed, formazon crystals were dissolved in 200 ll of DMSO and absorbance at 570 nm wavelength was recorded. 2.2. Cell cycle analysis Flow cytometric analysis (FACS) was performed to evaluate the distribution of the cells through the cell cycle phases. MCF-7, human breast cancer cells were incubated with compounds 26 and 36 at 25 and 50 nM concentrations for 48 h. Untreated and treated cells were harvested, washed with PBS, fixed in ice-cold 70% ethanol and stained with propidium iodide (Sigma Aldrich). Cell cycle was performed by flow cytometry (Becton Dickinson FACS Caliber) as earlier described [14]. 2.3. Protein extraction and Western blot analysis MCF-7 cells were treated with compounds 26 and 36 at 50 nM concentration for 48 h. The cell lysates were obtained by lysing the cells in ice-cold radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific Inc.). After centrifugation at 12,000 rpm for 20 min, the protein in the supernatant was quantified by the Bradford assay method by using a multimode plate reader, Varioskan instrument (Thermo Fischer Scientifics Ltd.). Proteins were separated using Sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinyl difluoride (PVDF) membrane. The membrane was blocked at room temperature for 2 h in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST/Phosphate buffered saline with tween 20 (PBST)) containing 5% bovine serum albumin (BSA). The membrane was washed with TBST for 5 min, then primary antibody was added and the membrane was incubated at 4 °C overnight. All the antibodies were purchased from Cell Signaling Technology (CST). The membrane was incubated with the corresponding horseradish peroxidase labeled secondary antibody and incubated for another 1 h. Membranes were washed with TBST/PBST three times for 15 min and the protein blots were visualized with
MeO
MeO Me
2.5. Measurement of mitochondrial membrane potential (DWm) MCF-7 cells were cultured in six-well plates. After 24 h of incubation cells were treatment with compounds 26 and 36 at 25 and 50 nM concentrations for 48 h. After 48 h of treatment, cells were collected by trypsinization and washed with PBS (Phosphate buffered saline) followed by resuspending in JC-1 (5,5, 6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolocarbocyanine iodide5 lg/mL) and incubated at 37 °C for 20 min. The cells were then subjected to flow cytometric analysis on a flow cytometer (Becton Dickinson) in the FL1H, FL2H channel to detect mitochondrial potential [17]. 2.6. Annexin V-FITC staining assay for apoptosis MCF-7 cells were seeded in six-well plates and allowed to grow overnight. The medium was then replaced with complete medium containing compounds 26 and 36 at 25 and 50 nM concentrations. After 48 h of drug treatment, cells were harvested by trypsinization, washed with PBS at 3000 rpm. Then the cells were stained with Annexin V-FITC (Annexin V-Fluorescein isothiocyanate) and PI (Propidium iodide) using the Annexin-V-FITC apoptosis detection kit (Sigma-Aldrich). Flow cytometry was performed for this study as described earlier [18,19]. The percentages of viable (Annexin V /PI ), early apoptotic (Annexin V+/PI ), late apoptotic (Annexin V+/PI+) and necrotic (Annexin V /PI+) cells were analyzed. 3. Results 3.1. Antiproliferative activity Test compounds 26 and 36 were evaluated for their antiproliferative activity against MCF-7 and MDA MB-231 cell lines by employing MTT assay [13]. These compounds (26 and 36) showed significant activity against MCF-7 cell line with IC50 values 0.057 and 0.051 lM respectively (Table 1).
MeO
OMe
Me
OMe HO
MeO
O
O
MeO
26 Fig. 1. Chemical structure of compounds 26 and 36.
O 36
OMe OMe
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3.2. Cell cycle analysis
Table 1 IC50a values (expressed in lM) of compounds 26 and 36.
a b c
Compound
MCF-7b
MDA MB-231c
26 36
0.057 ± 0.008 0.051 ± 0.002
0.168 ± 0.008 0.093 ± 0.006
50% inhibitory concentration after 48 h of drug treatment. ER positive breast cancer cell line. ER negative breast cancer cell line.
Many anticancer compounds exert their growth inhibitory effect either by arresting the cell cycle at a particular checkpoint of cell cycle or by induction of apoptosis or a combined effect of both cycle block and apoptosis [14]. The in vitro screening results revealed that, compounds 26 and 36 showed significant antiproliferative activity against human breast cancer cell line MCF-7. Therefore, it was considered of interest to understand whether this
Fig. 2a. Effect of compounds 26 and 36 on the cell cycle phase distribution of MCF-7 cells. Cells were treated with these compounds at 25 and 50 nM concentrations for 48 h. Then the cells were fixed and stained with Propidium iodide (PI) to analyze DNA content by flow cytometry. A: Untreated control cells (MCF-7), B: 26 (25 nM), C: 26 (50 nM), D: 36 (25 nM) and E: 36 (50 nM).
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Fig. 2b. Effect of compounds 26 and 36 on cell cycle progression of MCF-7 cells. Data are expressed as % of cell count in each phase of cell cycle induced by each compound. Statistical analysis was performed using GraphPad Prism software version 5.01. (⁄ p < 0.05 vs control).
inhibition of cell growth was on account of cell cycle arrest. In this study, MCF-7 cells were treated with test compounds at 25 and 50 nM concentrations for 48 h. The data obtained clearly indicated that these compounds show G2/M cell cycle arrest in comparison to the untreated control cells. The test compounds (26 and 36) showed 36.4 and 37.1% of cell accumulation in G2/M phase at 25 nM concentration, whereas they exhibited 47.6 and 50.5% of cell accumulation in G2/M phase at 50 nM concentration respectively (Figs. 2a and 2b). 3.3. Compounds 26 and 36 inhibits PI3K/Akt/mTOR signaling pathway in MCF-7 cells The PI3K/Akt/mTOR signaling pathway has been well documented for playing a major role in carcinogenesis in the breast cancer cells [20]. It has been shown that the breast tumor cell growth is closely related to the activation of PI3K/Akt/mTOR pathway [21,22]. Among molecules of PI3K/Akt/mTOR cascade, it has been reported that 4E-BP1, a direct downstream of mTOR plays an important role for cell proliferation, while p70S6K is critical for cell size regulation [23]. The in vitro results revealed that these compounds (26 and 36) possessed significant activity in human breast cancer cell line MCF7. Therefore, we next investigated the effects of these compounds on the PI3K/AKT/mTOR pathway in breast cancer cells, MCF-7. When these cells were treated with test compounds (26 and 36) at 50 nM concentration for 48 h, the expression level of p-Akt, p-mTOR, p-p70S6K, and p-4E-BP1 was effectively suppressed (Fig. 3). These results indicated that both the compounds were found to be potent inhibitors of PI3K/Akt/mTOR pathway. 3.4. Hoechst staining for apoptosis Apoptosis is one of the major pathways that lead to the process of cell death. Chromatin condensation and fragmented nuclei are known as the classic characteristics of apoptosis. It was considered of interest to investigate the apoptotic inducing effect of these compounds (26 and 36) by Hoechst staining (H33258) method in breast cancer cells. In this study, MCF-7 cells were treated with these compounds at 50 nM concentration for 48 h. Results indicated that these compounds showed potent effect on nuclear condensation when compared with untreated control cells and the results demonstrate that these compounds were effective in inducing cellular apoptosis (Fig. 4). 3.5. Measurement of mitochondrial membrane potential (DWm) The maintenance of mitochondrial membrane potential (DWm) is significant for mitochondrial integrity and bioenergetic
Fig. 3. Effect of compounds 26 and 36 on the PI3K/Akt/mTOR pathway. MCF-7 cells were treated with these compounds for 48 h at 50 nM concentration before being harvested. Expression of Akt, mTOR, p70S6K, and 4E-BP1 along with their phosphorylated forms were then measured by Western blot analysis. b-Actin was used as an equal loading control.
function [24]. Mitochondrial changes, including loss of DWm, are key events that take place during drug induced apoptosis. In order to further investigate the apoptosis inducing effect these conjugates 26 and 36, mitochondrial membrane potential (DWm) changes were designed and detected, using the fluorescent probe JC-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimida zolocarbocyanine iodide-5 lg/mL). It is commonly used dye to detect mitochondrial depolarization that occurs in the apoptosis [25]. In healthy cells, JC-1 accumulates in the mitochondria as JC-1 aggregates (whose fluoresce is red) and also in the cytoplasm as JC-1 monomers (whose fluoresce is green). During apoptosis, the Dwm collapses. Consequently, JC-1 aggregates cannot accumulate within the mitochondria and dissipate into JC-1 monomers leading to loss of red fluorescence. Therefore, collapse of the Dwm is signified by decrease in the ratio of red to green fluorescence [25]. The red (JC-1 aggregate) and green (JC-1 monomer) fluorescence were detected at FL-2H and FL-1H channel, respectively. In this study we investigated the involvement of mitochondria in the induction of apoptosis by these compounds and the results indicated that these conjugates significantly reduce the mitochondrial membrane potential (DWm) of MCF-7 cells (Fig. 5). 3.6. Annexin V-FITC assay for apoptosis It is well known that phosphatidylserine (PS) externalization is an early feature of apoptosis and can be detected by the binding of Annexin V to PS on the cell surface [26–28] and the apoptosis assessment method by Annexin V-FITC/PI assay is well recognized
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Fig. 4. Detection of apoptotic morphological changes in MCF-7 cells treated with compounds 26 and 36 at 50 nM concentration for 48 h. Nuclei were stained with Hoechst 33258 and examined by fluorescence microscopy. A: Untreated control cells (MCF-7), B: 26 (50 nM) and C: 36 (50 nM).
and accurate. In this study, MCF-7 cells were treated with test compounds (26 and 36) at 25 and 50 nM concentrations for 48 h to examine the apoptotic effect. It was observed that both the compounds showed significant apoptosis against MCF-7 cells as shown in Fig. 6). As shown in Fig. 6, the population of apoptotic cells (Lower right-early apoptotic and Upper right-late apoptotic) significantly increased after treatment with these conjugates. Results indicated that the test compounds (26 and 36) showed 60.20 and 70.14% and 78.60 and 89.96% of apoptosis at 25 and 50 nM concentrations respectively, whereas 0.13% of apoptosis was observed in control cells (untreated cells). From this experiment it was revealed that these compounds significantly induce apoptosis in human breast cancer cell line, MCF-7. 3.7. Regulation of apoptosis by compounds 26 and 36 Apoptosis (programmed cell death) is a normal physiological phenomenon that can be observed in various tissues. Cells undergoing apoptosis are characterized by distinct biochemical and morphological changes. Failure to undergo apoptosis has been implicated in tumor development. The activation of caspases represents a crucial step in the induction of drug induced apoptosis and cleavage of poly ADP-ribose polymerase (PARP) by caspase-3 is considered to be one of the hallmarks of apoptosis [29–31]. In our earlier studies [12], we observed that, benzofuran derivatives activate the caspase 3 activity in human lung cancer cells A549, but MCF-7 cell line lacks endogenous caspase-3 [32], whereas caspase-9 plays an important role in mediating drug induced apoptosis [33]. In this context, we investigated the apoptosis inducing capability of these potent molecules by performing western blot analysis of pro apoptotic protein Bax and antiapoptotic protein Bcl-2, Cytochrome c, procaspase-9 activity, cleavage of Poly (ADP-ribose) polymerase (PARP) and tumor suppressor gene p53 in human breast cancer cell line MCF-7 for 48 h. The results demonstrated that the typical 89 kDa cleaved fragment of PARP
and cytochrome c levels increased in treated cells. Further, after treatment with these conjugates the expression level of p53 was increased followed by decrease in the levels of procaspase-9. Finally, up regulation of pro apoptotic protein Bax and down regulation of antiapoptotic protein Bcl-2 (Fig. 7), suggesting that these compounds induced mitochondrial mediated apoptosis in human breast cancer cells. 4. Discussion Benzofuran scaffolds have drawn considerable attention over the last few years due to their profound physiological and chemotherapeutic properties as well as their widespread occurrence in nature [34]. Identification of novel and selective anticancer agents remains an important and challenging goal in pharmacological research. As a result, there is a need towards the development of newer molecules that are not only effective but safer. In our previous study [12], we have synthesized a series of benzo[b]furan derivatives and evaluated for their antiproliferative activity and this investigation provided two interesting lead compounds 26 and 36. These findings prompted us to further study of this class of compounds for their anticancer efficiency in breast cancer cell lines, MCF-7 and MDA MB-231. Anticancer agents generally alter the regulation of the cell cycle resulting in the arrest of cell division in various phases, thereby decreasing the growth and proliferation of cancerous cells. The results of the cytotoxicity assay suggest that these compounds (26 and 36) possess significant antiproliferative activity against the human breast cancer cell line MCF-7. The promising activity of these lead molecules prompted us to examine its influence on cell-cycle progression. Cell cycle analysis results suggest that these compounds arrested the cell cycle at G2/M phase (Figs. 2a and 2b). Studies like, Hoechst staining, mitochondrial membrane potential (DWm) and Annexin V-FITC assay revealed that these compounds induced mitochondrial mediated apoptosis in MCF-7 cells.
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Fig. 5. Compounds 26 and 36 triggers mitochondrial injury. Drops in mitochondrial membrane potential (DWm) was assessed by JC-1 staining of MCF-7 cells treated with test compounds and samples were then subjected to flow cytometry analysis on a FACScan (Becton Dickinson) in the FL1H and FL2H channel to detect mitochondrial membrane potential (DWm). A: Untreated control (MCF-7); B: 26 (25 nM); C: 26 (50 nM); D: 36 (25 nM) and E: 36 (50 nM).
The signaling pathway involving phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt/PKB) and mammalian target of rapamycin (mTOR) regulates several cellular functions that are critical to tumorigenesis such as cellular proliferation, growth, survival and mobility [35]. The recent development and clinical testing of PI3K/Akt/mTOR inhibitors has led to the conclusion that targeting the PI3K/Akt/mTOR pathway is a promising approach for
the treatment of breast cancer. After treatment with these compounds (26 and 36), the expression levels of p-Akt and p-mTOR were effectively suppressed (Fig. 3). Further, we have studied the effect of these compounds on the expression levels of p-p70S6K and p-4E-BP1, which are important in cell proliferation [23]. As expected, after treatment with these compounds (26 and 36), the expression levels of p-p70S6K and p-4E-BP1 were effectively
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Fig. 6. Effect of compounds 26 and 36 on the induction of apoptosis in MCF-7 cells. MCF-7 cells were treated with these compounds at 25 and 50 nM concentrations for 48 h, subsequently stained with Annexin V-FITC and propidium iodide (PI). The population of apoptotic cells was determined using flow cytometry. Quadrants: UL (upper left)necrotic cells; LL (lower left)-live cells; LR (lower right)-early apoptotic cells and UR (upper right)-late apoptotic cells. A: Untreated control (MCF-7); B: 26 (25 nM); C: 26 (50 nM); D: 36 (25 nM) and E: 36 (50 nM).
suppressed (Fig. 3). Thus, revealing a potent inhibitory effect of these compounds on PI3K/Akt/mTOR signaling pathway. Franke and coworkers reported that PI3K/Akt/mTOR pathway is thought to transduce anti-apoptotic signals in tumor cells [36,37]. In light of the key role of PI3K/Akt/mTOR signaling pathway in governing apoptosis, our study showed that inhibition of PI3K/Akt/mTOR pathway by these compounds increase the expression of apoptotic markers such as release of cytochrome c, up regulation of p53, down regulation of procaspase-9, cleavage of Poly (ADP-ribose)
polymerase (PARP), up regulation of Bax and down regulation of Bcl-2 (Fig. 7), revealed that the inhibition of PI3K/Akt/mTOR pathway might be the main mechanism underlying the apoptosis of breast cancer cells induced by these test compounds (26 and 36). In conclusion, this investigation showed that compounds 26 and 36 inhibit the PI3K/Akt/mTOR signaling pathway in the breast cancer cell line (MCF-7) by inducing mitochondrial mediated apoptosis. Hence, benzo[b]furan derivatives can be considered as potential anticancer drugs for the effective treatment of breast cancer.
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Fig. 7. Effect of compounds 26 and 36 on the expression levels of cytochrome c, Bax, Bcl-2, p53, procaspase-9 and PARP. The expression levels of the proteins were analyzed through western blot analysis. b-Actin was used as an equal loading control.
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Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg
Benzo[b]furan derivatives induces apoptosis by targeting the PI3K/Akt/ mTOR signaling pathway in human breast cancer cells Ahmed Kamal a,b,⇑, V. Lakshma Nayak a, Narayana Nagesh c, M.V.P.S. Vishnuvardhan a, N.V. Subba Reddy a a
Medicinal Chemistry and Pharmacology, CSIR – Indian Institute of Chemical Technology, Hyderabad 500 007, India Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Hyderabad 500 037, India c CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India b
a r t i c l e
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Article history: Received 3 February 2016 Revised 13 April 2016 Accepted 25 April 2016 Available online 26 April 2016 Keywords: Benzo[b]furan derivatives Breast cancer Cell cycle PI3K/Akt/mTOR pathway Apoptosis
a b s t r a c t The PI3K/Akt/mTOR signaling pathway plays a key regulatory function in cell survival, proliferation, migration, metabolism and apoptosis. Aberrant activation of the PI3K/Akt/mTOR pathway is found in many types of cancer and thus plays a major role in breast cancer cell proliferation. In our previous studies, benzo[b]furan derivatives were evaluated for their anticancer activity and the lead compounds identified were 26 and 36. These observations prompted us to investigate the molecular mechanism and apoptotic pathway of these lead molecules against breast cancer cells. Benzo[b]furan derivatives (26 and 36) were evaluated for their antiproliferative activity against human breast cancer cell lines MCF-7 and MDA MB-231. These compounds (26 and 36) have shown potent efficiency against breast cancer cells (MCF-7) with IC50 values 0.057 and 0.051 lM respectively. Cell cycle analysis revealed that these compounds induced cell cycle arrest at G2/M phase in MCF-7 cells. Western blot analysis revealed that these compounds inhibit the PI3K/Akt/mTOR signaling pathway and induced mitochondrial mediated apoptosis in human breast cancer cells (MCF-7). Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Breast cancer is the most common cause of cancer death among women worldwide, and especially young women in the developing world. If current rates of increase remain constant, a woman born today has a 1 in 10 chance of developing breast cancer [1,2]. The discovery and development of new, more active, more selective compounds for the treatment of breast cancer is one of the most important goals in medicinal chemistry. Therefore this needs extensive research that will lead to more effective strategies for breast cancer treatment and prevention. The phosphatidylinositol 3-kinase (PI3K/AKT)/mammalian target of rapamycin (mTOR) pathway is known to be aberrantly activated in numerous human cancers including breast cancer [3]. This pathway plays a central role in diverse cellular functions including proliferation, growth, survival and metabolism. Accumulating data from genetic and cancer biology studies have indicated that the PI3K/AKT/mTOR pathway has a prominent role in cancer cell growth and survival, and there is now extensive evidence validating various components of this pathway as potential molecular ⇑ Corresponding author at: Medicinal Chemistry and Pharmacology, CSIR – Indian Institute of Chemical Technology, Hyderabad 500 007, India. E-mail address: [email protected] (A. Kamal). http://dx.doi.org/10.1016/j.bioorg.2016.04.004 0045-2068/Ó 2016 Elsevier Inc. All rights reserved.
targets for treating cancer [4–6]. Therefore, PI3K/AKT/mTOR signaling pathway is considered as an attractive target for the development of newer anticancer agents that could be used alone or in combination with other targeted therapies for the treatment of breast cancer patients. The mechanisms underlying the development of breast cancer is complex [7]. Breast epithelial cell homeostasis requires the balance of cell proliferation with a type of cell death called apoptosis and defects in apoptosis (programmed cell death) can contribute to tumor formation and progression [8,9]. Benzofurans and their derivatives possess a broad range of important biological activities including anticancer, antibacterial, antifungal and antiviral properties. They have attracted considerable attention among organic and medicinal chemists in the last few years [10,11]. In our earlier publication [12], we have synthesized a series of benzo[b]furan derivatives and evaluated for their antiproliferative activity and this investigation provided two interesting lead compounds 26 and 36. Therefore it was considered to further worthwhile study of this class of compounds and to understand their mechanism of action. The encouraging results obtained in our earlier studies [12], prompted us to evaluate these potent molecules for their antiproliferative activity against human breast cancer cell lines, MCF-7 and MDA MB-231. These conjugates showed significant antiproliferative activity against human breast cancer cell line, MCF-7. The prominent anticancer activity of these
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conjugates encouraged us to explore the molecular mechanism against breast cancer cell line (Fig. 1).
chemiluminescence reagent (Thermo Scientific Inc.). Images were captured by using the chemiluminescence (vilber lourmat) [15].
2. Materials and methods
2.4. Hoechst staining for apoptosis
2.1. MTT assay
MCF-7 cells were seeded at a density of 10,000 cells over 18-mm cover slips and incubated for 24 h. After incubation, cells were treated with compounds 26 and 36 at 50 nM concentration for 48 h. After treatment, cells were stained with Hoechst 33,258 (Sigma-Aldrich) and incubated for 30 min at 37 °C. Later, cells were washed with phosphate buffered saline (PBS). Cells from each cover slip were captured from randomly selected fields under a fluorescence microscope to qualitatively determine the proportion of viable and apoptotic cells based on their relative fluorescence and nuclear fragmentation [16].
The antiproliferative activity of the compounds (26 and 36) was determined by employing MTT assay [13]. Cells were seeded in 200 ll of DMEM (Dulbecco’s Modified Eagle Medium) or MEM (Minimum Essential Medium), supplemented with 10% FBS in each well of 96-well microculture plates and incubated for 24 h at CO2 incubator. After 24 h of incubation, cells were treated with the test compounds for 48 h. After 48 h of incubation, 20 ll MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (5 mg/mL) was added to each well and the plates were further incubated for 4 h. Then the supernatant from each well was carefully removed, formazon crystals were dissolved in 200 ll of DMSO and absorbance at 570 nm wavelength was recorded. 2.2. Cell cycle analysis Flow cytometric analysis (FACS) was performed to evaluate the distribution of the cells through the cell cycle phases. MCF-7, human breast cancer cells were incubated with compounds 26 and 36 at 25 and 50 nM concentrations for 48 h. Untreated and treated cells were harvested, washed with PBS, fixed in ice-cold 70% ethanol and stained with propidium iodide (Sigma Aldrich). Cell cycle was performed by flow cytometry (Becton Dickinson FACS Caliber) as earlier described [14]. 2.3. Protein extraction and Western blot analysis MCF-7 cells were treated with compounds 26 and 36 at 50 nM concentration for 48 h. The cell lysates were obtained by lysing the cells in ice-cold radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific Inc.). After centrifugation at 12,000 rpm for 20 min, the protein in the supernatant was quantified by the Bradford assay method by using a multimode plate reader, Varioskan instrument (Thermo Fischer Scientifics Ltd.). Proteins were separated using Sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinyl difluoride (PVDF) membrane. The membrane was blocked at room temperature for 2 h in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST/Phosphate buffered saline with tween 20 (PBST)) containing 5% bovine serum albumin (BSA). The membrane was washed with TBST for 5 min, then primary antibody was added and the membrane was incubated at 4 °C overnight. All the antibodies were purchased from Cell Signaling Technology (CST). The membrane was incubated with the corresponding horseradish peroxidase labeled secondary antibody and incubated for another 1 h. Membranes were washed with TBST/PBST three times for 15 min and the protein blots were visualized with
MeO
MeO Me
2.5. Measurement of mitochondrial membrane potential (DWm) MCF-7 cells were cultured in six-well plates. After 24 h of incubation cells were treatment with compounds 26 and 36 at 25 and 50 nM concentrations for 48 h. After 48 h of treatment, cells were collected by trypsinization and washed with PBS (Phosphate buffered saline) followed by resuspending in JC-1 (5,5, 6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolocarbocyanine iodide5 lg/mL) and incubated at 37 °C for 20 min. The cells were then subjected to flow cytometric analysis on a flow cytometer (Becton Dickinson) in the FL1H, FL2H channel to detect mitochondrial potential [17]. 2.6. Annexin V-FITC staining assay for apoptosis MCF-7 cells were seeded in six-well plates and allowed to grow overnight. The medium was then replaced with complete medium containing compounds 26 and 36 at 25 and 50 nM concentrations. After 48 h of drug treatment, cells were harvested by trypsinization, washed with PBS at 3000 rpm. Then the cells were stained with Annexin V-FITC (Annexin V-Fluorescein isothiocyanate) and PI (Propidium iodide) using the Annexin-V-FITC apoptosis detection kit (Sigma-Aldrich). Flow cytometry was performed for this study as described earlier [18,19]. The percentages of viable (Annexin V /PI ), early apoptotic (Annexin V+/PI ), late apoptotic (Annexin V+/PI+) and necrotic (Annexin V /PI+) cells were analyzed. 3. Results 3.1. Antiproliferative activity Test compounds 26 and 36 were evaluated for their antiproliferative activity against MCF-7 and MDA MB-231 cell lines by employing MTT assay [13]. These compounds (26 and 36) showed significant activity against MCF-7 cell line with IC50 values 0.057 and 0.051 lM respectively (Table 1).
MeO
OMe
Me
OMe HO
MeO
O
O
MeO
26 Fig. 1. Chemical structure of compounds 26 and 36.
O 36
OMe OMe
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3.2. Cell cycle analysis
Table 1 IC50a values (expressed in lM) of compounds 26 and 36.
a b c
Compound
MCF-7b
MDA MB-231c
26 36
0.057 ± 0.008 0.051 ± 0.002
0.168 ± 0.008 0.093 ± 0.006
50% inhibitory concentration after 48 h of drug treatment. ER positive breast cancer cell line. ER negative breast cancer cell line.
Many anticancer compounds exert their growth inhibitory effect either by arresting the cell cycle at a particular checkpoint of cell cycle or by induction of apoptosis or a combined effect of both cycle block and apoptosis [14]. The in vitro screening results revealed that, compounds 26 and 36 showed significant antiproliferative activity against human breast cancer cell line MCF-7. Therefore, it was considered of interest to understand whether this
Fig. 2a. Effect of compounds 26 and 36 on the cell cycle phase distribution of MCF-7 cells. Cells were treated with these compounds at 25 and 50 nM concentrations for 48 h. Then the cells were fixed and stained with Propidium iodide (PI) to analyze DNA content by flow cytometry. A: Untreated control cells (MCF-7), B: 26 (25 nM), C: 26 (50 nM), D: 36 (25 nM) and E: 36 (50 nM).
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Fig. 2b. Effect of compounds 26 and 36 on cell cycle progression of MCF-7 cells. Data are expressed as % of cell count in each phase of cell cycle induced by each compound. Statistical analysis was performed using GraphPad Prism software version 5.01. (⁄ p < 0.05 vs control).
inhibition of cell growth was on account of cell cycle arrest. In this study, MCF-7 cells were treated with test compounds at 25 and 50 nM concentrations for 48 h. The data obtained clearly indicated that these compounds show G2/M cell cycle arrest in comparison to the untreated control cells. The test compounds (26 and 36) showed 36.4 and 37.1% of cell accumulation in G2/M phase at 25 nM concentration, whereas they exhibited 47.6 and 50.5% of cell accumulation in G2/M phase at 50 nM concentration respectively (Figs. 2a and 2b). 3.3. Compounds 26 and 36 inhibits PI3K/Akt/mTOR signaling pathway in MCF-7 cells The PI3K/Akt/mTOR signaling pathway has been well documented for playing a major role in carcinogenesis in the breast cancer cells [20]. It has been shown that the breast tumor cell growth is closely related to the activation of PI3K/Akt/mTOR pathway [21,22]. Among molecules of PI3K/Akt/mTOR cascade, it has been reported that 4E-BP1, a direct downstream of mTOR plays an important role for cell proliferation, while p70S6K is critical for cell size regulation [23]. The in vitro results revealed that these compounds (26 and 36) possessed significant activity in human breast cancer cell line MCF7. Therefore, we next investigated the effects of these compounds on the PI3K/AKT/mTOR pathway in breast cancer cells, MCF-7. When these cells were treated with test compounds (26 and 36) at 50 nM concentration for 48 h, the expression level of p-Akt, p-mTOR, p-p70S6K, and p-4E-BP1 was effectively suppressed (Fig. 3). These results indicated that both the compounds were found to be potent inhibitors of PI3K/Akt/mTOR pathway. 3.4. Hoechst staining for apoptosis Apoptosis is one of the major pathways that lead to the process of cell death. Chromatin condensation and fragmented nuclei are known as the classic characteristics of apoptosis. It was considered of interest to investigate the apoptotic inducing effect of these compounds (26 and 36) by Hoechst staining (H33258) method in breast cancer cells. In this study, MCF-7 cells were treated with these compounds at 50 nM concentration for 48 h. Results indicated that these compounds showed potent effect on nuclear condensation when compared with untreated control cells and the results demonstrate that these compounds were effective in inducing cellular apoptosis (Fig. 4). 3.5. Measurement of mitochondrial membrane potential (DWm) The maintenance of mitochondrial membrane potential (DWm) is significant for mitochondrial integrity and bioenergetic
Fig. 3. Effect of compounds 26 and 36 on the PI3K/Akt/mTOR pathway. MCF-7 cells were treated with these compounds for 48 h at 50 nM concentration before being harvested. Expression of Akt, mTOR, p70S6K, and 4E-BP1 along with their phosphorylated forms were then measured by Western blot analysis. b-Actin was used as an equal loading control.
function [24]. Mitochondrial changes, including loss of DWm, are key events that take place during drug induced apoptosis. In order to further investigate the apoptosis inducing effect these conjugates 26 and 36, mitochondrial membrane potential (DWm) changes were designed and detected, using the fluorescent probe JC-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimida zolocarbocyanine iodide-5 lg/mL). It is commonly used dye to detect mitochondrial depolarization that occurs in the apoptosis [25]. In healthy cells, JC-1 accumulates in the mitochondria as JC-1 aggregates (whose fluoresce is red) and also in the cytoplasm as JC-1 monomers (whose fluoresce is green). During apoptosis, the Dwm collapses. Consequently, JC-1 aggregates cannot accumulate within the mitochondria and dissipate into JC-1 monomers leading to loss of red fluorescence. Therefore, collapse of the Dwm is signified by decrease in the ratio of red to green fluorescence [25]. The red (JC-1 aggregate) and green (JC-1 monomer) fluorescence were detected at FL-2H and FL-1H channel, respectively. In this study we investigated the involvement of mitochondria in the induction of apoptosis by these compounds and the results indicated that these conjugates significantly reduce the mitochondrial membrane potential (DWm) of MCF-7 cells (Fig. 5). 3.6. Annexin V-FITC assay for apoptosis It is well known that phosphatidylserine (PS) externalization is an early feature of apoptosis and can be detected by the binding of Annexin V to PS on the cell surface [26–28] and the apoptosis assessment method by Annexin V-FITC/PI assay is well recognized
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Fig. 4. Detection of apoptotic morphological changes in MCF-7 cells treated with compounds 26 and 36 at 50 nM concentration for 48 h. Nuclei were stained with Hoechst 33258 and examined by fluorescence microscopy. A: Untreated control cells (MCF-7), B: 26 (50 nM) and C: 36 (50 nM).
and accurate. In this study, MCF-7 cells were treated with test compounds (26 and 36) at 25 and 50 nM concentrations for 48 h to examine the apoptotic effect. It was observed that both the compounds showed significant apoptosis against MCF-7 cells as shown in Fig. 6). As shown in Fig. 6, the population of apoptotic cells (Lower right-early apoptotic and Upper right-late apoptotic) significantly increased after treatment with these conjugates. Results indicated that the test compounds (26 and 36) showed 60.20 and 70.14% and 78.60 and 89.96% of apoptosis at 25 and 50 nM concentrations respectively, whereas 0.13% of apoptosis was observed in control cells (untreated cells). From this experiment it was revealed that these compounds significantly induce apoptosis in human breast cancer cell line, MCF-7. 3.7. Regulation of apoptosis by compounds 26 and 36 Apoptosis (programmed cell death) is a normal physiological phenomenon that can be observed in various tissues. Cells undergoing apoptosis are characterized by distinct biochemical and morphological changes. Failure to undergo apoptosis has been implicated in tumor development. The activation of caspases represents a crucial step in the induction of drug induced apoptosis and cleavage of poly ADP-ribose polymerase (PARP) by caspase-3 is considered to be one of the hallmarks of apoptosis [29–31]. In our earlier studies [12], we observed that, benzofuran derivatives activate the caspase 3 activity in human lung cancer cells A549, but MCF-7 cell line lacks endogenous caspase-3 [32], whereas caspase-9 plays an important role in mediating drug induced apoptosis [33]. In this context, we investigated the apoptosis inducing capability of these potent molecules by performing western blot analysis of pro apoptotic protein Bax and antiapoptotic protein Bcl-2, Cytochrome c, procaspase-9 activity, cleavage of Poly (ADP-ribose) polymerase (PARP) and tumor suppressor gene p53 in human breast cancer cell line MCF-7 for 48 h. The results demonstrated that the typical 89 kDa cleaved fragment of PARP
and cytochrome c levels increased in treated cells. Further, after treatment with these conjugates the expression level of p53 was increased followed by decrease in the levels of procaspase-9. Finally, up regulation of pro apoptotic protein Bax and down regulation of antiapoptotic protein Bcl-2 (Fig. 7), suggesting that these compounds induced mitochondrial mediated apoptosis in human breast cancer cells. 4. Discussion Benzofuran scaffolds have drawn considerable attention over the last few years due to their profound physiological and chemotherapeutic properties as well as their widespread occurrence in nature [34]. Identification of novel and selective anticancer agents remains an important and challenging goal in pharmacological research. As a result, there is a need towards the development of newer molecules that are not only effective but safer. In our previous study [12], we have synthesized a series of benzo[b]furan derivatives and evaluated for their antiproliferative activity and this investigation provided two interesting lead compounds 26 and 36. These findings prompted us to further study of this class of compounds for their anticancer efficiency in breast cancer cell lines, MCF-7 and MDA MB-231. Anticancer agents generally alter the regulation of the cell cycle resulting in the arrest of cell division in various phases, thereby decreasing the growth and proliferation of cancerous cells. The results of the cytotoxicity assay suggest that these compounds (26 and 36) possess significant antiproliferative activity against the human breast cancer cell line MCF-7. The promising activity of these lead molecules prompted us to examine its influence on cell-cycle progression. Cell cycle analysis results suggest that these compounds arrested the cell cycle at G2/M phase (Figs. 2a and 2b). Studies like, Hoechst staining, mitochondrial membrane potential (DWm) and Annexin V-FITC assay revealed that these compounds induced mitochondrial mediated apoptosis in MCF-7 cells.
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Fig. 5. Compounds 26 and 36 triggers mitochondrial injury. Drops in mitochondrial membrane potential (DWm) was assessed by JC-1 staining of MCF-7 cells treated with test compounds and samples were then subjected to flow cytometry analysis on a FACScan (Becton Dickinson) in the FL1H and FL2H channel to detect mitochondrial membrane potential (DWm). A: Untreated control (MCF-7); B: 26 (25 nM); C: 26 (50 nM); D: 36 (25 nM) and E: 36 (50 nM).
The signaling pathway involving phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt/PKB) and mammalian target of rapamycin (mTOR) regulates several cellular functions that are critical to tumorigenesis such as cellular proliferation, growth, survival and mobility [35]. The recent development and clinical testing of PI3K/Akt/mTOR inhibitors has led to the conclusion that targeting the PI3K/Akt/mTOR pathway is a promising approach for
the treatment of breast cancer. After treatment with these compounds (26 and 36), the expression levels of p-Akt and p-mTOR were effectively suppressed (Fig. 3). Further, we have studied the effect of these compounds on the expression levels of p-p70S6K and p-4E-BP1, which are important in cell proliferation [23]. As expected, after treatment with these compounds (26 and 36), the expression levels of p-p70S6K and p-4E-BP1 were effectively
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Fig. 6. Effect of compounds 26 and 36 on the induction of apoptosis in MCF-7 cells. MCF-7 cells were treated with these compounds at 25 and 50 nM concentrations for 48 h, subsequently stained with Annexin V-FITC and propidium iodide (PI). The population of apoptotic cells was determined using flow cytometry. Quadrants: UL (upper left)necrotic cells; LL (lower left)-live cells; LR (lower right)-early apoptotic cells and UR (upper right)-late apoptotic cells. A: Untreated control (MCF-7); B: 26 (25 nM); C: 26 (50 nM); D: 36 (25 nM) and E: 36 (50 nM).
suppressed (Fig. 3). Thus, revealing a potent inhibitory effect of these compounds on PI3K/Akt/mTOR signaling pathway. Franke and coworkers reported that PI3K/Akt/mTOR pathway is thought to transduce anti-apoptotic signals in tumor cells [36,37]. In light of the key role of PI3K/Akt/mTOR signaling pathway in governing apoptosis, our study showed that inhibition of PI3K/Akt/mTOR pathway by these compounds increase the expression of apoptotic markers such as release of cytochrome c, up regulation of p53, down regulation of procaspase-9, cleavage of Poly (ADP-ribose)
polymerase (PARP), up regulation of Bax and down regulation of Bcl-2 (Fig. 7), revealed that the inhibition of PI3K/Akt/mTOR pathway might be the main mechanism underlying the apoptosis of breast cancer cells induced by these test compounds (26 and 36). In conclusion, this investigation showed that compounds 26 and 36 inhibit the PI3K/Akt/mTOR signaling pathway in the breast cancer cell line (MCF-7) by inducing mitochondrial mediated apoptosis. Hence, benzo[b]furan derivatives can be considered as potential anticancer drugs for the effective treatment of breast cancer.
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Fig. 7. Effect of compounds 26 and 36 on the expression levels of cytochrome c, Bax, Bcl-2, p53, procaspase-9 and PARP. The expression levels of the proteins were analyzed through western blot analysis. b-Actin was used as an equal loading control.
Conflict of interest All the authors of this paper declare that they have no conflict of interest. Acknowledgments The authors V.L.N, M.V.P.S.V and N.V.S.R thank the Council of Scientific and Industrial Research (CSIR), New Delhi (India) for financial support under the 12th Five Year Plan project ‘‘Affordable Cancer Therapeutics (ACT)” (CSC0301). References [1] B.O. Anderson, C.H. Yip, R.A. Smith, R. Shyyan, S.F. Sener, A. Eniu, R.W. Carlson, E. Azavedo, J. Harford, Cancer 113 (2008) 2221–2243. [2] P. Porter, N. Engl, J. Med. 358 (2008) 213–216. [3] D.A. Altomare, J.R. Testa, Oncogene 24 (2005) 7455–7464. [4] T.A. Yap, M.D. Garrett, M.I. Walton, F. Raynaud, J.S. de Bono, P. Workman, Curr. Opin. Pharmacol. 8 (2008) 393–412.
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