Abstract
Cancer is one of the major health and social-economic problems despite considerable progress in its early diagnosis and treatment. Owing to the emergence and increase of multi drug resistance to various conventional drugs, and the continuing importance on health-care expenditure, many researchers have focused to develop novel and effective anticancer compounds. In the present study, a series of in-house synthesized quinazoline and quinazolino-benzothiadiazine derivatives were investigated for their anticancer efficacy against a panel of five cancer (DU145, MCF7, HepG2, SKOV3 and MDA-MB-231) and one normal (MRC5) cell lines. Among all the tested compounds, fifteen of them exhibited promising growth-inhibitory effect (0.15 - 5.0 μM) and induced cell cycle arrest in G2/M phase. In addition, the selected compounds inhibited the microtubule assembly; altered mitochondrial membrane potential and enhanced the levels of caspase-9 in MCF-7 cells. Furthermore, the active compound with combination of drugs showed synergistic effect at lower concentrations and the drug uptake was mediated through clathrin mediated endocytic pathway. Our results indicated that quinazoline and quinazolino-benzothiadiazine conjugates could serve as potential leads in the development of personalized cancer therapeutics.
Summary The present study describes the exploration of small molecules based on heterocyclic scaffolds for tubulin target based development of anticancer agents.
Introduction
Cancer, the condition resulting from unbridled progression of cell division, is a fatal affliction to human kind causing one death out of 7 total deaths worldwide. The gloomy prediction of high surge in new cancer cases to 21.7 million by 2030 further underlines the need of concerted endeavours to contain and cure this disease (Kamal et al., 2015). The seminal discovery of “Salvarsan - Magic bullet” laid the foundation for the present day targeted anticancer chemotherapy (http://www.hematology.org/Thehematologist/Profiles/4513.aspx). Tremendous advances achieved in cancer research in the past few decades have also been accompanied by major challenges such as statistical increase in the development of resistance to chemotherapy, limitations in selectivity and associated side effects of anticancer agents (Tiwary et al., 2015) (https://mlsmr.evotec.com/MLSMR_HomePage/index.html). In the development of novel therapeutic agent for cancer, both empirical and rational aspects of drug design are being pursued (Hoskin and Ramamoorthy., 2008). On global level for the last few decades, different organizations have started and are maintaining repositories of diverse synthetic and natural chemical compound to facilitate in the identification and development of potent and nontoxic drugs for pernicious diseases (Holbeck et al., 2010). Most of these repositories like Developmental Therapeutics Program (DTP) by NCI-USA, Molecular Libraries-Small Molecule Repository by NIH-South San Francisco, Open source drug discovery (OSDD) by CSIR-India are focusing on cancer and other diseases like tuberculosis, malaria etc.(http://www.osdd.net/research-development/open-access-repositories). Similarly, the Chemical Synthesis and Drug Supply Program (CSDSP) by NIMH-Bethesda was started with primary purpose to support the scientific research groups working on neurological diseases by synthesizing, purifying and distributing non-commercial and novel chemical entities to assist in psychopharmacology studies and mental health (https://nimh-repository.rti.org/assets/catalog.pdf) (Liu and Campillos., 2014; Srivastava and Srivastava., 2015).
In the last several years, the utilization of the chemical compounds from these repositories to evaluate a specific or shared biological activities/selectivity of chemical entities is being explored in the quest of new drug development. Numerous reports highlight the different biological activities exhibited by a single class of compounds, suggesting that a compound reported for a specific biological activity may or may not be limited to that particular activity with a possibility of it to bind at more than one target at molecular level. Moreover, biological profiles of many compounds in repositories are available in public domain and some of the compounds could be obtained for research purpose. This provides a remarkable and unprecedented support to researchers working in the area of drug development.7
Chemical compounds with heterocyclic scaffolds form the pivot of pharmaceutical drug industry as well as new drug discovery and development programs for various diseases. Different heterocyclic moieties are well known for diverse range of pharmacological properties and one of them is nitrogen containing quinazoline pharmacophore. Alkaloids from natural sources contains basic quinazoline skeletal, which is a benzene ring fused to heteroatom containing pyrimidine ring. Peganine is a first quinazoline alkaloid isolated from plant and indolopyridoquinazolinone alkaloid isolated from the plant Evodia rutaecarpa in the early 19th century is extensively used as tradition medicine for various ailments related to gastro intestine, absence of menstruation, and postpartum haemorrhage in china (Son et al., 2015; Zayed et al., 2015; Kamal et al., 2011). Extensive reports are available in literature highlighting wide biological activities of quinazoline based derivatives, which includes antimicrobial, anti-inflammatory, anti-malarial, anti HIV, anti-depressant and anti-cancer properties (Kuroiwa et al., 2015). In recent years, the quinazoline based intermediates are reported to exhibit anti-cancer properties by targeting the mitotic spindle during cell division via inhibition of tubulin polymerization, protein kinase of epidermal growth factor receptor (EGFR) (Wang et al., 2014) Erlotinib and Afatinib, the drugs approved by FDA in 2013 are quinazoline derivatives that target EGFR kinases, thereby preventing the growth of cancer cells (Zahedifard et al., 2015). Benzothiadiazines, resulting from the addition of a sulphur atom on quinazoline skeleton, are also reported to exhibit wide range of pharmacological potency. 1,2,4-Benzothiadiazine-1,1-dioxide derivatives are used as diuretic medication and recently the moiety has been explored for different pharmacological properties such as antibacterial, antiviral, anticancer, inhibition of ATP-sensitive potassium channel inhibitors and allosteric modulation of AMPA receptors (Sak 2012; Manasa et al., 2011; Nerkar et al., 2009).
Exploring heterocylic scaffolds for pharmacological potency in our previous studies, we have synthesized a library of quinazoline and benzothiadiazine derivatives and studied their anti-inflammatory and anti-depressant activities in rats (Reddy et al., 1985, 1987). Of late in our laboratory, we explore and investigate novel chemical compounds with varied heterocyclic scaffolds for arresting unregulated growth in cancer cells and the mechanism of action of these new entities (Kamal et al., 2007). The recent advances in the library screening and exploitation of these core moieties have encouraged us to further investigate the quinazoline and benzothiadiazine derivatives stored in our in-house chemical repository for their anticancer activity. For this purpose, we have screened over sixty derivatives for antiproliferative activity against selected human cancer cells to identify promising derivatives. This is followed by mechanistic studies for tubulin polymerization at molecular level for a more focused target based screening for potent anticancer agents.
Microtubule is a dynamic protein made of monomeric α and β tubulin. Many cellular functions are dependent on this microtubule, which includes cell division, cell signalling, cilia and flagellar motions, intracellular transport and intracellular structure etc. Because of its major vital functions in cells it is also called as cytoskeleton and is considered as a desirable target for anticancer agents (Wang and Xie., 2012). In cancer cells, the dynamic stability of tubulin is altered and the tubulin targeting agents act as spindle poison by triggering a mitotic catastrophy resulting in apoptotic cell death. To date several antimitotic compounds are developed which binds to the tubulin at a specific binding site like colchicine, vinca alkaloid, taxol and peloruside binding sites, however most of the compounds bind at colchicine binding site (Wang et al., 2012, 2013).
Results and discussion
In vitro antiproliferation activity
From the past few years several research groups are focusing on the quinazoline and benzothiadiazine scaffold to exploit their vast biological activities, especially relating to cancer. This encouraged us to explore the anticancer acitivity of quinazoline (ATS 1-11) and benzothiadiazine-1,1-dioxide derivatives (ATS 12-53) stored in our in-house chemical repository. Moreover, some of the sulphonamides intermediates (ATS 54-62) required in the synthesis of benzothiadiazines were also evaluated. The compounds were carefully selected and were previously reported for their in vivo anti-inflammatory and anti-depression activities. The selected compounds were screened for their anticancer activity on a panel of human cancer cell lines MCF7 and MDA MB-231 (breast carcinoma), DU145 (prostate carcinoma), SKOV3 (ovarian carcinoma) and HepG2 (hepato carcinoma) (ST-1). Among over sixty compounds, four showed potent anti-proliferative activity with GI50 values ranging from 0.15 μM to 0.81 μM concentration whereas the other compounds showed moderate activity on the tested cell lines. Interestingly, selectivity towards particular cancer cell line was observed and compounds ATS 1, 2, & 24 exhibited GI50 values 0.15, 0.45, & 0.57, respectively against ovarian cancer SKOV3. Whereas the compounds ATS-4 and ATS-7 exhibited GI50 of 0.81 and 0.6 μM on DU145 and MCF7, respectively. The GI50 values of all the tested compounds are presented in the Table 1. Compound ATS-7 showed high potency against MCF7 compared to MDA MB-231, in previous literature it was reported that quinazolines exhibited similar activity and many of them are in clinical studies. This encouraged us to select ATS-7 for further biological studies to understand the mechanism of action.
Effect of compounds on tubulin polymerization
Assay was performed in 384 well black plate contacting 3mg/ml tubulin in the presence of 1 mM GTP and with or without test compound. Fluorescence was measured at 360 nm excitation and 420 nm emission wavelengths. ATS-7 showed 59% tubulin polymerization inhibition at 3 μM concentration whereas the positive control, nocodazole, showed 67% inhibition at the same concentration. Tubulin IC50 was also determined for this molecule (Figure 1).
Cell cycle analysis
Most of the anticancer agents arrest the cancer cells at a particular cell cycle phase leading to either apoptosis or necrosis, however some drugs directly induce apoptosis.9, 10 Anti proliferative activity and tubulin inhibition studies revealed that compound ATS-7 showed significant anticancer as well as tubulin inhibition properties. In order to determine whether the effect of ATS-7 on tubulin polymerization inhibition is translated to similar effect in the cancer cells, cell cycle analysis was performed at two different concentrations in time dependent manner. MCF7 cells were treated with the ATS-7 and nocodazole at 0.6 and 1.2 μM concentrations, after 12 and 48 h of incubation cells were stained with propidium iodide and analysed for their effect on cell cycle. Clear evidence was observed with the data obtained by cell cycle analysis that ATS-7 compound arrest the MCF7 cells at G2/M phase. After 12 h of incubation ATS-7 blocked 16.03%, and 21.24% of MCF7 cells at G2/M phase at 0.6 and 1.2 μM concentration, respectively whereas nocodazole blocked 18.5% and 30.3% cells at G2/M phase under conditions. At 48 h of incubation ATS-7 and nocodazole showed two-fold enhanced effect by blocking 42.39 %, 48.86%, 22.87%, and 39.73% cells at G2/M phase at 0.6 and 1.2 μM concentrations, respectively (Figure 2). These results demonstrated that ATS-7 showed comparable and superior activity than nocodazole with the increase in exposure time on cancer cells. It also demonstrated that ATS-7 anticancer activity of ATS-7 is due to inhibition of tubulin polymerization and arresting of the cells at G2/M phase as a time and dose dependent manner.
Immunohistochemistry
Compound ATS-7 with in vitro tubulin inhibition and cell cycle arrest properties, was expected to disrupt microtubules in cells. To demonstrate cellular microtubule disruption, MCF7 cells were grown on coverslips and treated with ATS-7 and nocodazole at 0.6 and 1.2 μM concentration. After 48 h of incubation, cells were fixed and treated with primary anti tubulin and rhodamine conjugated secondary antibodies and DAPI for DNA staining as described in experimental section. These cover slips were mounted on slides and analysed under confocal microscope to photograph the tubulin network in cells. Cells treated with ATS-7 showed altered microtubule network compare to control cells, where well organized microtubule was observed. In both the concentrations ATS-7 and nocodazole both showed disorganized microtubule network at centre of the cells with dense peripheral microtubules (Figure 3). This demonstrated that ATS-7 has antimitotic activity comparable with the positive control nocodazole.
Soluble versus polymerized tubulin analysis by westernblotting
Inhibition of tubulin polymerization and arrest of cells at G2/M phase in cell cycle analysis was encouraged to analyse the levels of soluble versus polymerized forms of tubulin in MCF7. After incubation of MCF7 cells with compound ATS-7 (0.6 μM and 1.2 μM) for 48 h, cells were washed with PBS and soluble (free tubulin) and insoluble (polymerized form of tubulin as microtubule) fractions were isolated as described in materials and methods section. Nocodazole and taxol (1 μM) was used as tubulin destabilising and stabilising agents (positive and negative controls respectively). Immunoblot was performed with the isolated fractions and results indicate that cells treated with ATS-7 and nocodazole showed increased levels of tubulin in soluble fraction compared to the DMSO control, where as in cells treated with taxol showed increased levels of tubulin polymerised fraction (insoluble fraction) (Figure 4). Results suggest that the compound ATS-7 was a microtubule destabilizing agent.
Mitochondrial membrane potential
Mitochondria is considered as “power house of cells” but studies on mitochondrial function in cells revealed that they are involved in many cellular activities such as lipid modification, calcium level maintenance and cellular death signalling etc. Change in mitochondrial membrane potential (ΔψM) because of any type of cellular stress leads to the activation of mitochondrial mediated apoptosis. This can be measured by using JC-1 dye (cytofluorimetric, lipophilic cationic dye, 5, 5’, 6, 6’-tetraethylbenzimi-diazolylcarbocyanine iodide) which aggregates when cells are healthy where as in cells with altered MMP it is in monomeric form. After incubating the MCF7 cells with ATS-7, nocodazole and doxorubicin (0.6 and 1.2 μM concentration) for 48 h, cells were stained with JC-1 dye. The cells were analysed in tecan multimode reader as mentioned in experimental section. Cells treated with ATS-7 showed 40% decreased signal compared to the healthy control cells (Figure 5). It was evident that ATS-7 alters the MMP in the MCF7 cells.
Effect of compound on Caspase 9 activation
Caspases are cysteine-aspartic proteases, activated during abnormal cell functions due to several factors such as radiation, cellular stress, sudden lethal mutations in genomic DNA and chemotherapy leading to apoptosis (Irene et al, 2009). Executioner caspases (caspase 3, 6 and 7) are present in the cells and are activated by the initiator caspases like caspase 2, 8, 9, & 10. Executioner caspases are activated by both caspase 9 and caspase 8 which mediates intrinsic and extrinsic apoptotic pathways, respectively. MCF7 has a defective caspase-3, and caspase 9 plays a crucial role in drug induced apoptosis by activating the other executioner caspases. Therefore, MCF7 cells were treated with ATS-7 at 0.6 and 1.2 μM concentrations where as positive control nocodazole and doxorubicin were used at 0.6 μM for 48 h. The cells were harvested and analysed for the release of fluorophore from the AFC conjugated LEHD substrate by the enzyme present in the cell lysate. ATS-7 showed significant caspase 9 activation compared to the positive controls as shown in the Figure 6, which indicated that this compound ATS-7 induces the mitochondrial mediated apoptosis in MCF7 cells (Figure 6).
Cooperative index of combination drug treatment
In this investigation combination treatment was studied and MCF7 cells were treated either in combination or alone with ATS-7, nocodazole and doxorubicin at GI50 concentration of test compound and it double concentration to get final concentrations of 0.6 and 1.2 μM. MTT assay was performed after incubating the cells at four different time intervals like 3, 8, 12 and 24 h. Cooperative index is calculated according the formula given in the experimental section and the death percentage of drug combination is represented in figure 7 whereas CI index is presented in table 2. The data demonstrated that at the lower concentration of ATS-7/nocodazole and ATS-7/doxorubicin combinations up to 12 h incubation showed synergistic effect whereas at 24 h of incubation showed antagonistic effect and at higher concentration showed antagonistic effect.
Endocytic pathway of ATS-7
Bioavailability of drug by cellular uptake and internalization is crucial for its efficacy. Macromolecules cellular uptake is achieved by various mechanisms and internalized drugs/molecules fate is dependent on its uptake mechanism. Drug transport in to the cells are determined by using endocytic pathway inhibitors.30 Generally, liquids are transported in to the cells by pinocytosis and it is further classified as macro pinocytosis, clathrine mediated pinocytosis and caveolae mediated pinocytosis. MCF7 cells were incubated with the inhibitors of three endocytic pathways as described in the experimental section for 1 h and then coincubated with ATS-7, nocodazole and doxorubicin individually at two different concentrations i.e. 0.6 and 2.4 μM for 2 h. At the end of the experiment MTT assay was performed and internal percentage drug uptake was determined by using multimode plate reader. It was observed that the ATS-7 uptake was restricted when clathrin mediated endocytic pathway was inhibited, the positive control doxorubicin may also use the clathrin mediated endocytic pathway as shown in the Figure 7. However, nocodazole uptake was restricted in all the endocytic pathway inhibitors treated wells.
Discussion
Several literature precedence highlights the anticancer properties of quinazoline derivatives and their advantages over the present clinical agents (Sak 2012; Manasa et al., 2011; Nerkar et al., 2009). In one of the studies, Zahedifard et al reported that the modified quinazolines exhibited significant cytotoxicity against MCF7 compared to MDA MB 231 breast cancer cell lines (Zahedifard et al., 2015). In agreement with this, in the present investigation the quinazoline derivatives also exhibited better activity in MCF7 compared to MDA MB-231. Compound ATS-7 showed high potency against MCF7 with little activity against MDA MB-231 cell line.
Several reports have been found that quinazoline based anti-inflammatory, anti-depressant and VDA’s showed potent anti-tubulin activity. Recently Kenta Kuroiwa et al, Xiao-Feng Wang et al and Virginia Spanò et al. reported anti mitotic activity of quinazoline scaffold molecules (Wang et al., 2012, 2012, 2013, 2013) which encouraged us to investigate the active compounds for their in vitro tubulin polymerization inhibition property (Kuroiwa et al., 2015; Wang et al., 2014, 2013, 2013). As previous reports these quinazolin based derivatives are also showed anti mitotic activity. Inhibition of cell cycle progression and apoptosis are the most common causes of cell growth inhibition. Cell cycle progression is induced by various cell cycle proteins such as CDKs and cyclins as they are the key regulators of cell cycle (Park and Lee 2013). Previous studies reported that treatment of quinazoline derivatives arrests cancer cells in G2/M phase (Cubedo et al., 2006). In the present study, the exposure of MCF7 cells to ATS-7 compound induced cell cycle arrest in G2/M phase and also induces mitotic arrest. Furthermore, the western blot analysis and immuno cycochemistry studies clearly indicated that ATS-7 alters the spatial arrangement of tubulin and increased the amount of soluble tubulin in MCF7 cells. Some of the quinazoline derivatives induce cellular apoptosis by altering the MMP, which activates the release of cytochrome C from mitochondria to cytosol. The cytosolic cyt C subsequently activates the levels caspase-9 which leads to apoptosis (Nerkar et al., 2009). In addition, several studies (Iyer et al., 1998; Weir et al., 2007) reported that cell cycle arrest at G2/M phase takes place by the induction of cellular apoptosis. Considering this fact, we further investigated the effect of ATS-7 compound on the levels of MMP and caspase-9 in MCF7 cells. In general, caspases (cysteine aspartase enzyme), a group of intracellular proteases, are responsible for the induction of apoptosis. In the present investigation, the compound ATS-7 reduced the levels of MMP and increased the levels of caspase-9 in cancer cells which indicated that these derivatives have the capacity to induce apoptosis in MCF7 cells.
Presently, most of the chemotherapeutic treatments are administered in two or three drug combinations for effective chemotherapy. Several in vitro and clinical investigations on the combination drug treatment suggested that various molecular targets are involved in cancer treatment. Riva and co-workers employed a combination treatment of anti-mitotic compound paclitaxel and VPA (valproic acid) on GSC (Glio stem cells) and observed the synergistic effect to develop a useful combination treatment (Riva et al., 2014). In a similar manner, Konecny and workers also performed the combination drug treatment of paclitaxel with other agents such as epirubicin, carboplatin, gemcitabine and vinorelbine separately on MCF7 cells at lower drug concentrations and observed various effects like synergistic, additive and antagonistic of different combinations (Konecny et al., 2001). It was observed that at higher concentrations the effect of combinations was changed. Effect of ATS-7 compound in combination with nocodazole and doxorubicin on MCF7 cells was performed. Results demonstrated that ATS-7-N, and ATS-7-D both showed synergistic effect at lower concentration and incubation time whereas, at lower concentration and higher incubation time (24 h) as well as at higher concentration at all tested incubation time antagonistic effect was observed on MCF7 cells.
Cell membrane is the important barrier which allow entry to various intra and extracellular molecules, further it controls the cell signalling and fate of the internalized molecules (Khalil et al., 2006; Haucke 2015). Previously researchers encountered various challenges during high throughput screening of compound libraries in the early steps of drug discovery. Many molecules fail in clinical phase trials because of unavailability of molecule internalization after cellular uptake and accumulation as well as on how the cells respond to its entry (McShane et al., 2015). Many studies revealed that failed or reduced cellular uptake and accumulation of drug is one of the reasons for drug resistance. Romero and co-workers studied the cisplatin and chloride complex cellular uptake mechanism to understand the cellular influx and efflux equilibrium of these anticancer agents in a time dependent manner. Further, they have also discussed the caveolae mediated endocytosis of these anticancer compounds (Romero et al., 2012).33 In this connection in the present investigation quinazoline derivative ATS-7 cellular uptake is observed by clathrin mediated endocytosis whereas standard nocodazole cellular uptake is mediated by all three endocytic pathways.
Experimental methods
In vitro anti-proliferative activity
Anticancer properties of the selected compounds from in house library were evaluated by a standard sulpha rhodamine B assay (SRB) on a panel of selected human epithelial cancer cell lines such as MCF7 (Breast carcinoma), DU145 (Prostate carcinoma), SKOV3 (Ovarian carcinoma), HepG2 (Hepato carcinoma), and MDA MB-231 (Breast carcinoma). The assay was performed in 96 well plate and the cells were seeded with a density of 3.5 × 104 in 100 μL in each well in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal bovine serum, 1% antibiotic solution (PSK) in cell culture incubator at 37 °C temperature supplied with 5% CO2 and 100% relative humidity. After 24 h of incubation, the cells were treated with the experimental compounds and positive controls nocodazole and doxorubicin of 2 μl diluted in DMSO at different concentrations (5 × 10-8, 1 × 10-7, 1 × 10-6, 5 × 10-6, & 1 × 10-5 M). Drug exposure was continued for 48 h in CO2 incubator, at the end of incubation cells were fixed with 50 μl of 10% TCA (w/v) at 4 °C for 1 h and followed by washing the cells with water for three times to remove the TCA and then 100 μl of 0.4% SRB (w/v) prepared in 1% acetic acid was added in each well and further incubated for 30 min at RT. The plates were washed with 1% acetic acid for three to five times to remove excess and unbound dye from the wells and then plates were air dried. SRB dye bound to the protein was dissolved by 10 mM Trizma base, optical density of the dissolved dye was measured at 565 nm in a multimode plate reader (Tecan multimode reader). The experiment was carried out in triplicates. Increased absorbance of the SRB dye is relative to the percentage growth of the cells and 50% growth inhibition concentration of the compounds were calculated by the formula described in NCI protocol, GI50= (T-T0/C-T0)*100 where T is the absorbance of treated wells with test compounds at 48 h, T0 is the absorbance of wells at time zero and C is the absorbance of the control wells at 48 h (Ashraf et al., 2016; Kamal et al., 2010).
Tubulin polymerization assay
BK011P kit from Cytoskeleton, Inc. was used to perform fluorescent based tubulin polymerization inhibition assay. According to the manufacturer’s protocol inhibitor detection reaction mixture was prepared for a total volume of 10 μl containing 3 mg/ml porcine brain tubulin, 1 mM GTP and a fluorescence reported in PEM buffer at pH −6.9 (80 mM PIPES, 0.5 mM EGTA, 2 mM MgCl2). In the presence and absence of test compounds at a concentration of 3 μM the increase in fluorescence of reaction mixture was measured at 37 °C at 420 nm emission wavelength by exciting at 360 nm wavelength for every 1 min till 60 min in multimode plate reader. Nocodazole was used as a positive control. Increase in fluorescence was due to the incorporation fluorophore in to the microtubules polymerization.
Tubulin IC50 was also determined in the same experimental condition as mentioned above. The concentration of compound where 50% of tubulin polymerization was inhibited compared to the control is known as tubulin IC50. Various concentrations like 0.5, 1, 3, 6, and 9 μM of compound ATS-7 was added in the reaction mixture. Increase in fluorescence as the polymerization progresses was monitored by tecan multimode plate reader at 37 °C for 1 h at 1 min time interval at 420 nm emission wavelength when excited at 360 nm wave length. Nocodazole is used as a positive control (Tecan multimode reader) (Kamal et al., 2012; Kamal et al., 2011).
Cell cycle analysis
Human breast cancer cells MCF7 were seeded in 6 well plates, after 24 h the cells were treated with the test compound ATS-7 and a standard drug nocodazole at 0.6 & 1.2 μM concentration for 12 h and 48 h. DMSO was used for appropriate controls. At the end of incubation, cells were trypsinized and harvested by centrifugation for 1 to 2 min at 2000 rpm. The pellets were washed with PBS for 3 to 4 times and fixed in ice cold 70% ethanol for 30 min at 4 °C. Ethanol was removed by centrifugation and cells were stained with 1 ml of DNA staining solution containing 10 mg propidium iodide, 0.1 mg trisodium citrate, 0.03 ml triton X 100 in 100 ml of sterile water. After 30 min of incubation in the staining solution the cells were analysed for the DNA content by flow cytometry (Becton Dickinson FACS Caliber instrument). Histograms were analysed with the software provided by the instrument (Ashraf et al., 2016; Kamal et al., 2011).
Immuno cytochemistry
MCF7 cells at a density of 1 × 104 cells were seeded on the 18 mm sterile coverslips placed in 6 well plates and incubated for 24 h in a CO2 incubator. In the presence and absence of the test compound ATS-7 at 0.6 and 1.2 μM concentrations, cells were incubated for 48 h in a CO2 incubator. Cells were incubated with DAPI for 30 min to stain the DNA. At the termination of experiment, cells were fixed with the mixture of paraformaldehyde, glutaraldehyde solution in 1 ml PBS at a concentration of 3% and 0.02%, respectively for 15 min and permeabilized by dipping them in ice cold 100% methanol for 10 sec. The coverslips were blocked by incubated for 1 h with 1% BSA in PBS and later coverslips were incubated with mouse monoclonal primary anti α tubulin antibody followed by rhodamine conjugated secondary anti-mouse IgG antibody. Coverslips were mounted on a glass slide using DPX fmount and analysed using Nikon A1R confocal microscopy, equipped with rhodamine and DAPI settings. Images were taken at a same scale to analyse the microtubule integrity in test compounds compared to control and positive control nocodazole at 0.6 μM.
Cellular levels of tubulin
MCF7 cells were seeded in 6-well plates at a density of 2 × 105 in DMEM and treated with or without test compfound ATS-7 at 0.6 & 1.2 μM concentration. The cells were harvested after 48 h of incubation followed by washing with 1X PBS at 24 °C. Further, the cells were lysed with tubulin lysis buffer (0.1 M Pipes, 1 mM EGTA, 1 mM MgSO4, 30% glycerol, 5% DMSO, 5 mM GTP, 0.125% NP-40, and protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany), including aprotinin [200 units/ml], pH 6.9) for 30 min at 24 °C temperature. Lysates were centrifuged at 15000g at 24 °C for 30 min, supernatant containing soluble tubulin was collected in separate tube and pellet was suspended with equal volume of lysis buffer containing polymerized tubulin. Pellet and soluble fractions were boiled in lamelli sample buffer for 10 min at 95 °C. Samples were resolved by 10% SDS-PAGE, further immunoblotting was performed after transferring the protein bands on to the nitrocellulose membrane with primary anti α tubulin antibody followed by HRP conjugated secondary anti-mouse IgG antibody.anti-body. Finally, the blot was analysed using ECL prime western blotting mixture supplied by GE health care on photographic film. Quantitative analysis of the soluble and polymer fractions was done by densitometry. Nocodazole and taxol were used as a positive and negative control, respectively at 0.6 μM. Amount of total protein in the soluble and pellet fractions were estimated by Broadford reagent from sigma to normalize the protein before loading the samples in SDS/PAGE gel (Ashraf et al., 2016; Kamal et al., 2012).
Mitochondrial membrane potential
One of the key functions that altered when the cancer cells are treated with anticancer agents is mitochondrial membrane potential. Most of the anticancer compounds that induce apoptosis will affect the integrity and functioning of mitochondrial membrane. MCF7 cells were analysed for the loss of mitochondrial membrane potential after treatment with the test compound ATS-7 at 0.6 and 1.2 μM concentrations. Assay was performed using JC-1 dye from Cyman chemicals (CAYM15003-1) and cells were incubated with 0.5 ml JC-1 dye at 37°C in a 5% CO2 incubator for 15-30 min. Cells were washed with PBS for 3 times and cells were harvested in 200 μl of assay buffer from each well, cell suspension was centrifuged for 5 min at RT at 1200 rpm and resulted pellet was suspended in 200 μl of 1x assay buffer. 100 μl of cell suspension was transferred in to the 96 well black plate and the cells analysed by fluorescence multimode plate reader. Healthy cells form JC-1 aggregates and strong red fluorescence intensity was measured at 595 nm emission wavelength when excited at 535 nm wavelength, unhealthy cells or altered mitochondrial membrane cells JC-1 monomers exists and strong fluorescence was measured at 535 nm emission wavelength when excited at 485 nm wavelength. The ratio J-aggregates to J-monomers directly implies to the mitochondrial membrane potential of the cells (Budihardjo et al., 1999).
Caspase 9 Assay
As previously reported that breast cancer cells MCF7 does not express caspase 3 but they undergo apoptosis via caspase 9. MCF7 cells were seeded in 6 well plates, after treatment with or without test compounds and positive control nocodazole for 48 h followed by harvesting of cells in PBS by centrifugation. The cells were suspended in 200 μl of ice cold 1X lysis buffer on ice for 10- 20 min. Supernatant was collected by centrifugation at 13,200 rpm for 20 min at 4°C. Caspase 9 expression in all the samples were measured in 96 well black polystyrene plate in 2X assay buffer containing 50μl cell lysate in the presence of 40 μM of caspase substrate. Increase in fluorescence was measured when caspase 9 cleaved the AFC from Ac-LEHD-AFC at emission wavelength of 505 nm when excited at 400 nm for a period of 1 h at an interval of 5 min. Amount of total protein in all the samples were estimated by Broadford reagent from sigma to normalize the protein (Ye et al., 2001; Mc Gee et al., 2002).
Combination drug treatment
Cooperative index (CI) is defined as the in vitro cytotoxicity determination of combination drug treatment on a human cancer cell lines. This assay was performed to determine the synergy, antagonism or additive effect of the test compound in combination with other drug at a given exposure time to cells. MCF7 cells were seeded in 96 well plate in a complete medium and treated with test compound and other positive controls nocodazole and doxorubicin individually at 0.6 μM (IC50 concentration of ATS-7) and 1.2 μM (fixed multiple 2 times of the IC50 concentration). Combination treatment also performed at the same concentrations mentioned above in 1:1 ratio. MTT assay was performed at various drug exposure times as previously described. Cooperative Index (CI) was calculated as mentioned in the previous literature, sum of cell death percentages obtained for individual drug treatment divided to the percentage of cell death of combination drug treatment (CI = ATS-7 cell death% + positive control cell death%/Combined treatment cell death%). If CI value is greater than 1 then it has an antagonism effect similarly for additive and synergy effect CI value should be less than 1 and equals to 1 respectively (Riva et al., 2014; Hahm et al., 2001).
Endocytic pathway determination
Basically two major endocytic pathways are known to exist, namely phagocytosis and pinocytosis. Phagocytosis is one in which larger and solid macromolecules/particles are engulfed by phagosome (referred as cell eating) where as in pinocytosis liquid/solvent material is uptaken by cells (referred as cell drinking). Further pinocytosis is classified to two major groups called micropinocytosis (clathrin-mediated and caveolae-mediated endocytosis) and macropinocytosis. The MCF7 cells were seeded in two replica plates of 24 well plates at a density of 1 × 105, the cells were co-incubate with 154 mg/mL of sucrose (inhibitor of clathrin-mediated endocytosis), 54 μg/mL of nystatin (inhibitor of caveolae-mediated endocytosis) and 133 μg/mL of amiloride (inhibitor of macropinocytosis) for 1 h at 37°C followed by test compounds and positive controls doxorubicin alone and in combination with test compound at 0.6 & 1.2 μM concentration for 2 hours. One replica plate was used for MTT assay and the cells in the other replica plate were then rinsed with PBS at 4°C (3 times). The amount of ATS-7 and doxorubicin inside the cells were analysed by multimode plate reader at 340 nm and 480 nm wave lengths respectively (Khalil et al., 2006; Ding et al., 2015).
Competing interests
The authors declare no competing or financial interests.
Author contributions
T.B.S. designed and performed the experiments. M.S.M and Z.S.S interpreted the experiments and also helped in the manuscript preparation. S.R.R. assisted in SRB and FACS experiments. A.K conceived, designed and supervised the study and all authors reviewed the manuscript.
Funding
This work was supported by a UKIERI council
Graphical Abstract
Acknowledgements
We thank Prof Andrew D. Miller, Dr. Maya Thanaou, Centelles Miguel, Michael Wright and Isma Ali from King’s College, London for their support and technical assistance in confocal microscopy & other experiments. T.B.S thank UKIERI council and CSIR-IICT.