Background
In the last era, many diseases have overcome through the significant improvement of biomedical science. However, cancer remains elusive, especially from a therapeutic viewpoint. Currently, cancer is treated using radiotherapy, chemotherapy and surgery that are related with severe side effects [
1]. Even a large number of tumours are insufficiently responsive to cancer therapeutic drugs and radiotherapy due to multi-drug resistance. Epidemiologic studies suggests that in dietary supplement, increasing consumption of fruits, vegetables, whole grains and related lifestyles is a practical approach for significantly reducing the incidence of cancer. Numerous individual phytochemicals (>5000) are isolated from the plant-based foods [
2]. Before we can fully understand the health benefits in humans, their identification, characterization and mechanism of action need to be determined. In mammalian cells, biological targets of phytochemicals were found to be involved in antiproliferative, anti-inflammatory, antiangiogenic, antimetastatic and pro-apoptotic effects, or the ability to reduce oxidative stress [
3,
4]. In vitro studies on different cancer cell lines proved the role of polyphenols as growth inhibitors, either by induction of G1-cell cycle arrest [
5], G2/M arrest [
6], or cell death [
7]. Similarly, different carotenoids demonstrated G-1 arrest [
8] and apoptosis [
9] in various cancer cells. So, identification and development of natural resources used for cancer treatment have attracted a lot of attention globally.
Reactive oxygen species (ROS), a regular by-product of the normal breakdown of oxygen, play a significant role in normal biochemical functions and abnormal pathological processes [
10]. There is a subtle balance between intracellular ROS and antioxidant capacity in normal cells, which determines their destiny [
11]. Yet cancer cells are characterised by elevated intracellular ROS stress, resulting from carcinogenesis stimulation, abnormal metabolic activity and mitochondrial malfunction. The limited capacity of tumor cells to deal with the elevated ROS levels makes them vulnerable to oxidative stress [
12]. This embarks a novel strategy for cancer therapy to promote apoptosis in cancer cells by eliminating the ability of antioxidant defence systems through inducing ROS production [
13].
Euglena tuba (Carter) (Family, Euglenaceae) is one of the most widespread microalga in the Rarh region of West Bengal, India. Cells of
E.
tuba are normally green, having periplast without spiral rows of granules and more than one chloroplast. Paramylon bodies are present but margins not convolute, with cylindrical and highly metabolic cells that constantly change shapes in movements as they have stiff blue pellicle outside the cell membrane that are flexible in nature [
14]. Euglena mainly grows in aquatic bodies with algal bloom all over the year mostly in winter. Various
Euglena sp. have a broad range of medicinal properties, such as antimicrobial, anti-mutagenic, anti-HIV, immunopotentiating and antitumor activity [
15‐
20] with numerous isolated bioactive compounds such as, vitamin C, vitamin E and β-carotene that can be harnessed for commercial use [
21]. In our previous study, we reported that
E. tuba possess potent in vitro antioxidant and in vivo iron chelation activity [
22,
23] and numerous phytochemicals such as phenolics, flavonoids, alkaloids, tannins, terpenoids, triterpenoids, saponin, glycoside and carbohydrates are present in adequate amount in the extract which was confirmed by phytochemical analysis [
22]. In this study, we first demonstrated the antiproliferative effect of 70 % methanolic extract of
E. tuba (ETME) on lung and breast cancer cells and normal fibroblast cells in vitro. Moreover, we have studied that ROS accumulation caused by ETME leads to the activation of apoptosis and inhibition of metastasis through regulation of MAPK pathways.
Discussion
Algae are an important source of different bioactive compounds such as antioxidants, antivirals, antimicrobials etc. [
25]. Many algae have shown cytotoxic and antitumor activities against different types of cancers [
26]. These antitumor activities can play a significant role in development of new pharmaceutical compounds as antitumor drugs [
27]. In our study, ETME had been considered for its probable anticancer activity against two kinds of human tumor cell lines: A549, MCF-7. Results from this study demonstrated that ETME inhibited the growth of A549 and MCF-7 cells through the induction of apoptosis, while showed negligible cytotoxicity towards non-malignant cells (WI-38). ETME promoted apoptosis in both type of cells with an effective dose 200 μg/ml for A549 and 150 μg/ml for MCF-7. Further, morphological study from DAPI staining also supported that the above-mentioned doses of ETME are effective in inducing apoptosis in both types of cells.
In the modulation of apoptotic signalling pathways, caspases play important roles in cells and hence their survival and death. Moreover, the fate of cells towards survival or death relies on critical balance between Bcl-2 (antiapoptotic) and Bax (pro-apoptotic) proteins. Time-dependant increase of Bax/Bcl-2 ratio after the treatment of ETME resulted in apoptosis through intrinsic pathway. ETME treatment further led to the downregulation of pro-caspase 8 and upregulation of its active form along with formation of t-Bid on ETME treatment. Caspase 8 has been known to connect intrinsic and extrinsic apoptotic pathways via Bid cleavage to t-Bid, thus suggesting its importance in activation of both the pathways [
28]. These results suggest that ETME regulates various proteins involved in intrinsic and extrinsic pathways thereby inducing apoptosis. Ineffectiveness of presently existing treatments is generally due to the invasive and metastatic abilities of malignant cancer cells. Numerous studies have established that suppression of these stages results in the prevention of metastasis and they are the targets of anticancer drug development. We found that ETME-treated cells exhibited downregulated MMP-9 expression in both carcinomas, compared to the control cells. Additionally, a remarkable decrease of cell motility was also seen at a lesser toxic concentration of ETME, signifying its capability to inhibit cell’s motility. This capability of ETME to inhibit cell’s motility can be interconnected with its ability to inhibit the invasiveness of cells since suppression of MMP-9 activities had also been found to be able to reduce migration of cells. Evidences proposed that various natural compounds, for example curcumin and resveratrol, behave either as antioxidant or pro-oxidant depending on the concentration used and the aimed cells [
29,
30]. Neferine could trigger mitochondrial-mediated ROS formation in inducing apoptosis in HepG2 cells [
31]. Here, we observed that while ETME had an effective cytotoxicity on lung and breast cancer cells, it showed a negligible effect on normal human fibroblast cells. At the same time, our result suggested that ETME significantly increased enormous ROS generation in both cancer cells, but failed to generate in WI-38 cells. A549 cells treated with ETME displayed increase in levels of GST and GSH as well as production of TBARS (cell damaging index) while SOD and CAT activities decreased over 48 h of treatment. In case of MCF-7 cells exposed with ETME, SOD, CAT, GST activity and TBARS production is increased, while GSH level remained unaffected. There is a threshold of ROS above which cells cannot tolerate. A minimum increase of ROS levels in cancer cells can be toxic, making these cells more susceptible to ROS-induced cell death due to the failure of antioxidant defence mechanism [
12]. But in case of catalase in A549 cells, probably H
2O
2 suppress the transcription factor FoxO1 by PI 3 kinase/Akt-dependent phosphorylation, where PI 3 kinase/Akt signaling is essential for this negative regulation of catalase [
32]. We speculated that suppression of SOD gene transcription in A549 cells results possibly in lower activity of SOD leading to increase intracellular
\( {\text{O}}_{{2^{ - } }} \) levels and furthermore the OH production through Fenton reaction known to initiate lipid peroxidation. Additionally, superoxide ions can prevent GPx activity through oxidative alterations [
11]. GSH is an antioxidant and its reduction in intracellular levels in MCF-7 cells are connected with enriched susceptibility to ROS-induced apoptosis [
33]. Surprisingly, in normal cells, the balance of antioxidants is maintained.
The MAPK family have important roles in cell survival and death. It is familiar that JNK, p38 and p53 trigger apoptosis while ERK helps cell survival [
34]. Previous studies have shown that ERK is specially stimulated by mitogen through the involvement of Ras/Raf/MEK in MAPK pathways, thus leading to cell growth and survival [
35]. Furthermore, JNK and p38 MAPK signalling molecules are predominantly activated depending upon the inflammatory cytokines and environmental stress, which ultimately helps in cell differentiation and apoptosis [
36]. In this study, we observed a decreased p-ERK1/2, JNK and p38 in response to ETME treatment. In addition, JNK may have a role as an antiapoptotic protein kinase in few tumors. Antisense oligonucleotides, specific to JNK can lead to the reduction of its mRNA level, thereby restricting the growth of A549 cells, probably by promoting apoptosis [
37]. Our report showed that phosphorylation of p38 was inhibited dose-dependently by ETME. This discrepancy may be due, at least in part, to the variation of cell type’s. In case of MCF-7 cells p53 was upregulated up to 12 h followed by downregulation after treatment with ETME might be due to the loss of cells indicating that ETME induces apoptosis in MCF-7 cells by activating p53. However, in case of A549 cells time dependent downregulation of p53 expression was observed suggesting ETME induce p53 independent apoptosis in this type of cells. This regulation in MAPK pathways could be responsible for the activation of apoptotic pathways and inhibition of MMPs expression since inhibition of Ras in vitro has been shown to stop MMPs formation [
38].
UV spectroscopy is widely adapted as a useful technique for studying drug–DNA and drug–protein interactions. The UV–visible spectra of ds and ss DNA show a prominent absorption peak at 205 nm and slight red shift also observed after addition of increasing concentrations of ETME. Moreover, another peak i.e. 260 nm also increase in its intensity after ETME addition suggesting hyperchromicity [
39]. A continuous hyperchromicity of the soret band upon addition of ETME to ds DNA solution may be an indication of electrostatic binding or partial unwinding or intercalative mode of binding involving a strong stacking interaction between an aromatic chromophore and the base pairs of DNA [
40‐
42]. In order to understand the interaction of the ETME with denatured DNA, the DNA-compound mixture was heated at 95 °C for 15 min. The resulting spectra (Fig.
6b) revealed a moderately lower hyperchromic shift than the native conditions. Under denatured conditions the DNA double helix uncoils, exposing more number of nitrogen bases to the medium which loss interface electrostatic interactions with the complexes present in ETME, indicating the presence of DNA intercalating agents in ETME.
In case of protein, UV–vis absorption measurement is a simple and relevant method that is used to investigate structural changes and to discover complex formation [
43,
44]. The elevated levels of absorbance after the addition of ETME indicate the formation of a ground state complex. As dynamic quenching does not affect the absorption spectrum of a molecule and it only affects the excited states of a molecule, the observed changes in BSA absorbance in the presence of different concentrations of ETME could be an indicative of occurrence of static quenching interaction between the compounds present in ETME and BSA [
45]. Also, fluorescence spectroscopy has been regarded as the most comprehensive method for studying protein–ligand interactions especially in dynamic states [
46]. Generally, BSA fluorescence absorption originates from Trp, Tyr and Phe residues, whereas its intrinsic fluorescence can be mainly attributed to the Trp residue alone [
47]. The fluorescence intensity of BSA was decreased dose-dependently with increase in ETME concentrations (Fig.
6d) and no emission spectral shifting was observed, indicating that ETME could interact with BSA, and that the fluorescence chromophores of BSA were not exposed to an obvious polarity change with ETME titration.
It was also described that no single class of compounds in an extract could be completely held accountable for the activity produced by the total extract itself [
48]. Our result suggested that probable bioactive compounds present in ETME, individually are not specific to kill cancer cells, they also affected the growth of normal cells. Therefore, it is more significant as well as prudent to assess the activity of ETME as a complete mixture of interacting bioactive compounds rather than evaluating them as a breakup of their individual components.
Methods
Collection, extract and sample preparation of microalga
Microalgal sample were gather from various ponds in the Bankura district in the state of West Bengal, India in April 2014. This algal sample was authenticated by Dr. R. K. Gupta, Botanical Survey of India, Kolkata, India and preserved in formalin (4 %) for identification and noticed under the light microscope using standard methods [
49]. The procedure of extract preparation of
E. tuba followed exactly is same like previous [
23]. The microalgal extract was taken and dissolved in sterile distilled water followed by filter sterilization using 0.22 μm syringe filter and a stock of 2 mg/ml was prepared for conducting all experiments.
Cell lines and culture
Human lung adenocarcinoma (A549), human breast adenocarcinoma (MCF-7) and human lung fibroblast (WI-38) cell line were obtained from the National Centre for Cell Science (NCCS), India and kept in the ideal laboratory condition. A549 cells were cultured as monolayers in Ham’s F-12 medium whereas MCF-7 and WI-38 cells were cultured as monolayers in DMEM. Both the media were supplemented with 10 % (v/v) fetal bovine serum (FBS), 100 U/ml Penicillin G, 100 µg/ml Streptomycin, 50 µg/ml Gentamycin sulphate and 2.5 µg/ml Amphotericin B. All the cell lines were kept in a humidified atmosphere containing 5 % CO2 in incubator at 37 °C and passaged tri-weekly.
Determination of cytotoxicity using WST-1 Assay
Cell viability were analysed using the WST-1 cell proliferation reagent according to the earlier described technique [
50]. All cells (1 × 10
4 cells/well) were treated for 48 h with concentrations ranging from 0–200 µg/ml of ETME, ascorbic acid, catechin, tannic acid, reserpine, methyl gallate and rutin. Cell proliferation and viability were measured by measuring absorbance of the coloured product spectrophotometrically at 460 nm using a microplate ELISA reader MULTISKAN EX (Thermo Electron Corporation, USA).
Cell cycle analysis
According to a previously described method, cell cycle analysis was studied by flow cytometry [
50]. Cell cycle phase distribution of treated and untreated cells was examined by flow cytometry [FACS Verse (Becton–Dickinson) equipped with 405 nm (Violet), 488 nm (Blue) and 640 nm (Red) solid state laser light using FACSuite software Version 1.0.3.2942] by acquiring at least 10,000 cells per sample. The percentage of populations in the G0/G1, S and G2/M phases were determined using similar software.
Annexin V/propidium iodide staining
Apoptotic cells were quantified by Propidium iodide (PI) and Annexin V-FITC double staining, using an Annexin-V-FLUOS Staining Kit, Roche Diagnostics according to manufacturer’s protocols. Treated and untreated cells were stained with PI and FITC according to the manufacturer protocol. The distribution of apoptotic cells was quantified by flow cytometer. A total 10,000 events were acquired.
DAPI (4′,6′-diamidino-2 phenylindole) staining
According to an earlier described method, cell morphology was observed by DAPI staining [
51]. The undergoing apoptosis cells, represented by the morphological alteration of apoptotic nuclei, were observed and taken image from ten eye views at 630× magnifications under a laser scanning confocal microscope Leica TCS SP8 (Leica, Illinois, United States) and the data was analyzed using Leica Application Suite X software.
Immunoblot analysis
Both A549 and MCF-7 cells were treated with 200 and 150 µg/ml ETME respectively for different time intervals (6–36 h) for testing apoptotic and MAPK related protein study. Protein was quantified by the Folin-Lowry method. Equal quantities of proteins (40 mg for apoptotic related and 30 mg for MAPK) were taken for gel electrophoresis. The blot was developed using the previous protocol [
51]. The photographs were taken using imaging system EC3 Chemi HR (UVP, USA). The developed blots were then analysed for densities of bands by ImageJ 1.45 s software.
Scratch motility assay
Both malignant cells were seeded and grown for overnight to make confluent. The monolayer was then scratched vertically using pipette tip followed by washing with PBS two times to eliminate detached cells and incubated with media containing extracts at their respective less-toxic dose for 48 h. After incubation, cells migration on these scratched zones was pictured at five randomly selected fields.
Gelatin zymography assay
MMP-9 activity was checked by gelatin zymography as described previously [
52]. After 48 h treatment with several concentrations of ETME (0–200 µg/ml) both A549 and MCF-7 cells were harvested, total protein was isolated and gelatin gymography was performed. Appearance of unstained bands on a blue background, indicates gelatinolytic activity.
Measurement of intracellular ROS using DCFH-DA staining
For DCFH-DA staining treated and untreated cells were washed with PBS and loaded with 20 µM DCFH-DA diluted in clear serum free media for 30 min at 37 °C. After that cells were washed twice with PBS and intensity of the intracellular ROS was measured by FACS in all three cell lines and also morphology of cells (A549 and MCF-7 cells) was photographed (400× magnifications) using a previously mentioned laser scanning confocal microscope.
Measurement of antioxidants by different biochemical assays
After ETME treatment, all the cells were harvested and washed with PBS. The cell pellet was suspended in cold PBS and were lysed on ice using a sonicator and centrifuged at 13800
g for 20 min at 4 °C. The supernatant was taken for testing antioxidant enzyme assays, such as SOD, CAT, GST and also measuring the production of GSH and TBARS. SOD as checked by measuring the inhibition of the formation of blue colour formazan at 560 nm with slight modification from a previous study [
53]. CAT activity was measured with modified protocol continuous time course decaying of H
2O
2 at 240 nm [
54]. Earlier described method was followed to examine glutathione-
S-transferase GST based on the formation of GSH-CDNB conjugate [
55]. The reduced glutathione (GSH) level was determined spectrophotometrically at 412 nm according to the standard protocol [
56]. The level of lipid peroxide in cell homogenates were determined in conditions of thiobarbituric acid reactive substances (TBARS) as an index of accumulation of malondialdehyde [
57]. All biochemical tests were assayed in hexaplate.
DNA and protein binding studies
DNA and protein binding characteristics of the compounds present in ETME were investigated by UV–Visible spectroscopy using a Shimadzu UV-2401 PC UV–VIS recording spectrometer (Shimadzu Corporation, Kyoto, Japan) according to previously described method [
58]. In addition, for protein binding study the fluorescence emission spectra was also measured at 25 °C. Spectra were recorded in the wavelength range of 200–500 nm setting the excitation at 288 nm, and emission at 340 nm according to the previously described method [
59].
Statistical analysis
All spectrophotometric data were presented as the mean six times ±SD and cell cycle and ROS analysis data by FACS was introduced as the mean three times ±SD. The statistical analysis was determined by KyPlot version 2.0 beta 15 (32 bit). When A1 = IC50, Y = response (Y = 100 % when X = 0), X = inhibitory concentration, then IC50 values were calculated by the following formula, \( {\text{Y}} = {{100 * {\text{A}}1} \mathord{\left/ {\vphantom {{100 * {\text{A}}1} {\left( {{\text{X}}\text{ + }{\text{A1}}} \right)}}} \right. \kern-0pt} {\left( {{\text{X}}\text{ + }{\text{A1}}} \right)}} \). The IC50 values were compared by paired t test. P < 0.05 was considered significant.