Abstract: In the systematic screening programme for cytotoxic compound from marine actinomycetes, the compound furan-2-yl acetate (F2A) from Streptomyces VITSDK1 spp. The structure of the compound was unequivocally determined by spectral studies. It was previously found that F2A has potential antiviral activity against fish nodavirus. In the present study, the cytotoxicity of F2A was studied using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay which showed the IC50 values were less than 15 μg mL-1 against various tumor cell lines, whereas it was >25 μg mL-1 against non-tumor cell lines. F2A inhibited the cell proliferation in a dose-and time-dependent manner. Furthermore, the cytotoxic mechanism was determined in HeLa cells. The morphological analysis, Hoechst staining and DNA fragmentation studies revealed the apoptosis mediated cell death. The cytosolic protein analysis of F2A treated HeLa cells by immunoblotting showed the mitochondrial cytochrome c release, increased expression of caspase 3 and caspase 9 with PARP cleavage. There was no change in the caspase-8 levels. The Bcl-2 was found to be down regulated and Bax was up regulated in the F2A treated cells. Further, the apoptosis induction and cell death was found to be mediated by Reactive Oxygen Species (ROS) and lipid peroxidation. A molecular docking study of F2A with 28 selected cancer drug target enzymes provides some insight on mode of activity of the compound. The findings showed that the F2A exhibits selective cytotoxicity towards tumor cells at a lower concentration via apoptosis.
INTRODUCTION
In the continuing search for more effective therapeutical agents including HDAC inhibitors, marine microorganisms and marine invertebrates have emerged as a promising new resource yielding unusual chemical structures with potent biological activities (Williams, 2009). Marine extremophiles serve as valuable natural resource for the discovery of novel unique bioactive chemical scaffolds and marine natural products are very promising drug candidates for the treatment of cancer development (Amador et al., 2003).
The order actinomycetales, characteristic of its high G+C content in DNA plays an important role among the bacterial communities of marine origin because of its diversity and ability to produce novel chemical compounds of high commercial value. The marine actinomycetes are getting importance for their unique metabolites and enzymes (Mann, 2001). Hence, it is anticipated that the isolation, characterization and the pharmacological study on marine actinomycetes can be useful in the discovery of antibiotics and novel species of marine microorganisms. The actinomycetes, particularly Streptomyces have provided several bioactive compounds including, antibiotics (Berdy, 2005), novel thiol-antioxidants (Fahey, 2001), immune enhancers (Abe et al., 1989), immuno suppressive agents and enzymes and enzyme inhibitors (Imada, 2005). Several cytotoxic compounds have been reported from marine actinobacteria (Maskey et al., 2003; Feling et al., 2003; Song et al., 1994). However, in Indian peninsula, the reports of anticancer compounds from marine actinomycetes are very scanty and yet to be explored.
Furan compounds are very widespread in nature usually found to be in plant sources, especially food stuffs. Furan and its derivatives were reported from fungi (Song et al., 1994) and recently obtained from Streptomycete (Mukku et al., 2002; Fotso et al., 2008; Umezawa et al., 1971). Furan fatty acids have been isolated from other marine bacteria (Shirasaka et al., 1995). There are several reports on the biological activities of furan derivatives (Meotti et al., 2003) including antiviral (Kittakoop et al., 2001) insecticidal, nematocidal (Hayashi et al., 1981), inflammatory (Ciminiello et al., 1991) and cytotoxic activity (Mayer et al., 1996; Shen et al., 2001).
In the systematic screening programme for cytotoxic compound from marine actinomycetes, furan-2-yl acetate (F2A) was extracted from Streptomyces VITSDK1 spp. The structure of the compound was unequivocally determined by spectral studies which showed anti fish noda viral activity (Suthindhiran et al., 2011). The present manuscript deals with the cytotoxic activity and mechanism of novel furan-2-yl acetate on various cell lines and its possible mechanism of inducing cell death.
MATERIALS AND METHODS
Strain and compound: The strain Streptomyces VITSDK1 spp. was isolated from the Bay of Bengal coast, southern India. Isolation and characterization of the strain was previously reported (Suthindhiran and Kannabiran, 2009). The compound extracted from Streptomyces VITSDK1 spp. was elucidated as furan 2-yl acetate and reported earlier (Suthindhiran et al., 2011).
Cell culture, chemicals and antibodies: HeLa (Human cervical carcinoma), HT-29 (Human colon carcinoma), A549 (Human lung adenocarcinoma epithelial), NFFs (neonatal foreskin fibroblasts), HEK (Human embryonic kidney), Vero (green monkey kidney), cell lines obtained from ATCC and MDA-MB 435S, MCF7 (Human breast adenocarcinoma), HepG2 (hepatocellular carcinoma) obtained from NCCS (India) were maintained in RPMI 1640/DMEM/L-15 (Himedia/Gibco, Mumbai, India) medium supplemented with 10% FBS (v/v) and 100 mg L-1 streptomycin and 100 IU mL-1 penicillin (Himedia, India), at 37°C in 5% carbon dioxide. The (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed using CellQuanti-MTT cell viability assay kit (Bioassay Systems). Antibodies against cytochrome c, caspase 3, caspase 8, caspase 9, β-actin, PARP, Bax, Bcl-2 and the dye Dichlorodihydrofluorescein diacetate (DCHF-DA) were purchased from Sigma. All other chemicals and reagents used in this study were of analytical grade. The western blot kit and nitrocellulose membrane was purchased from Medox, India.
MTT cell proliferation assay: The cytotoxic activity of the F2A (0 to 25 μg mL-1) on various celllines (1x105 cells well-1) were determined as per user manual. The optical density was measured at 570 nm for each well on a multiwell plate reader (Biorad). The assay was carried out in triplicates. The wells with only culture medium or treated with 0.1% of Dimethyl sulfoxide (DMSO) served as control. The average of the blank controls were determined and subtracted from the absorbance values. The same protocol was followed for 5-fluorouracil (5-FU) (positive control). The graph was plotted with cell viability against the time period with various concentrations of the compound.
Morphological alterations: Cells treated with different concentration of (0-7.5 μg mL-1) F2A were observed for morphological alterations. The cell morphology was observed and photographed under an inverted microscope (Hund Wetzlar, Germany).
Detection of apoptosis: Apoptosis in F2A treated HeLa cells were assessed by Hoechst dye method. Cells (2x105) were seeded in 35 mm culture dishes and F2A was added to the cells at 6 μg mL-1; appropriate controls were also maintained. At various time points (3, 6, 12, 24 and 48 h) cells were harvested and washed free of medium. The cells were resuspended in 1 mL of PBS. The contents were centrifuged at 1600 rpm for 4 min at 4°C and the supernatant was discarded leaving 100 μL in the tube to resuspend the pellet. Hoechst dye (1 mg mL-1) 2 μL was added to 100 μL cell suspension and incubated in the dark at 37°C for five minutes. Approximately 50 μL of the Hoechst stained cell suspension was observed under inverted fluorescence microscope (Olympus IX70); the morphology and DNA fragmentation was visualized and the same was followed for control cells.
DNA extraction and electrophoresis: After 24 h of incubation of cells (5x105 cells mL-1) with F2A, cells were subjected to DNA isolation as described earlier (Allen et al., 1997) and DNA fragmentation was analyzed by agarose gel (1.5%) electrophoresis in both control and treated cells. After electrophoresis, the gels were stained with ethidium bromide and visualized with UV light.
Mitochondrial cytochrome c release assay: To assess mitochondrial cytochrome c release, HeLa cells were treated with F2A and the cytosolic protein extracts were collected as described earlier (Jun et al., 2003) Briefly, 5x106 HeLa cells were treated with F2A, washed twice with cold PBS and then suspended in 0.25 mL lysis buffer (250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 1 mM PMSF, 2.5 mg mL-1 E-64 and 20 mM HEPES, pH 7.2). The cells were allowed to swell on ice for 30 min and homogenized with 20 strokes. The homogenates were centrifuged at 3500 rpm for 10 min at 4°C. The supernatants were collected and centrifuged again at 13,700 rpm for 15 min at 4°C. The supernatants were harvested as mitochondrial free cytosolic extracts. The cytosolic fraction of the samples was analyzed for mitochondrial cytochrome c release by western blot using an antibody against cytochrome c.
Determination of apoptosis related proteins: The apoptotic mechanisms were elucidated by Western blot using HeLa cells. The cells were incubated with various concentrations (0, 1.5, 3, 4.5, 6 μg mL-1) and harvested after 12 h. The cells were washed with ice-cold PBS and suspended in a lysis buffer {20 mM Tris, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 1 mM glycerophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, leupeptin (10 mg mL-1), aprotinin (20 mg mL-1)} to obtain the whole cell extract. The contents were vortexed for 30 min and centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was collected and the protein content was measured (Bradford, 1976). The lysates were resolved on 10% SDS-PAGE gels and then transferred on to nitrocellulose membranes. The membranes were then incubated with primary antibodies (Cyt-c, Bcl-2, Bax, caspase 3, caspase 8, caspase 9 and PARP) in 10 mL of antibody-dilution buffer (Tris-buffered saline and 0.05%, Tween-20 with 5% milk) with gentle shaking at 4°C for 8-12 h. After incubation it was washed and then incubated with respective conjugated secondary antibodies. Monoclonal β-actin was used as an internal control. Bands were visualized by using Western blot detection reagents.
Reactive oxygen species (ROS) formation assay: The intracellular ROS production was assayed using 2, 7-dichlorofluorescein diacetate (DCFH-DA) method as previously described (Sohn et al., 2005) . Briefly, HeLa cells (5x105) were incubated with different concentrations (0, 1.5, 3, 4.5, 6, 7.5, 9 μg mL-1) of F2A at 37°C for 12 h. After incubation the cells were washed twice with cold PBS, suspended in PBS at 5X105 cells mL-1 and added with DCFH-DA (5 μM). The mixture was incubated at 37°C in the dark for 40 min. H2O2 (20 μM) was used as a positive control. The fluorescent intensity of the cell suspensions was detected using a fluorescence spectrophotometer (Fluostar optime, BMG Biotech, Germany) at wavelengths of 485 nm (excitation) and 550 nm (emission). The results were expressed as fluorescent intensity per 1x106 cells.
Nitric oxide (NO) releasing assay: The nitric oxide (NO) release was assayed by measuring the production of nitrite/nitrate as described previously (Cavalcanti et al., 2008). The cells were exposed to F2A (0, 1.5, 3, 4.5, 6, 7.5, 9 μg mL-1) and 100 μL of the cell supernatant were added to 100 μL of the Griess reagent (1% sulfanilamide in 1% H3PO4/0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride/1% H3PO4/distilled water, 1:1:1:1). The mixture was incubated at room temperature for 10 min. Blank was prepared with 100 μL of the Griess reagent added with 100 μL of the culture medium. The absorbance was measured at 560 nm in a multiwall plate reader (Biorad, India). A standard curve was prepared with various concentrations (0.5 to 10 μg mL-1) of NaNO2 under the same conditions. Experiments were performed in triplicate and results were expressed as Mean±SD.
Lipid peroxidation assay: Lipid peroxide was measured as described by Draper and Hadely (1990). Briefly, the HeLa cells were treated with F2A at various concentrations (0, 1.5, 3, 4.5, 6, 7.5 and 9 μg mL-1). After incubation the cells were trypsinised and lysed with Triton X-100. The lysed cells were then added with 400 μL of 35% perchloric acid and incubated in a water bath at 37°C for 1 h to stop lipid peroxidation. The mixture was centrifuged at 3500 rpm for 10 min and to the supernatant (1800 μL), 600 μL of 1.2% sodium 2-thiobarbiturate solution was added. The glass tubes were then placed in a water bath and heated at 95°C for 30 min. The tubes were then cooled and the absorbance was measured at 535 nm in a multiwall plate reader (Biorad, India). The mean and standard deviation was calculated from three individual experiments.
In vitro hemolytic assay: The hemolytic activity of the compound was evaluated as described earlier (Suthindhiran and Kannabiran, 2009).
Molecular docking
Target enzymes and ligand: Cancer drug target enzymes (28 Nos.) were selected
for docking studies (Imming et al., 2006) and
the 3D structures were downloaded from Protein Data Bank (PDB). The water molecules
were removed during modeling. The modeling methods follow standard protocols
and include minimization of the protein structure prior to docking to accommodate
hydrogen atoms not included in the crystal structure coordinates. The ligand
(F2A) was built by Chem3D Ultra (Version 8.0) and has been MM2 optimized.
Functional site identification: Predictions of functional sites in target proteins were performed based on Conserved Functional Group (CFG) analysis using siteFiNDER|3D server. siteFiNDER|3D is a fully integrated, web-based implementation of the CFG analysis method for functional site prediction (Innis, 2007).
Molecular docking: In order to carry out the docking simulation, AutoDock 4.0 suite molecular-docking tool was used as per the user manual (Morris et al., 1998). The F2A was manually docked into functional sites of all the enzymes individually and the docking energy was monitored to achieve a minimum value. AutoDock 4.0 is widely distributed public domain molecular docking software which perform the flexible docking of the ligands into a known protein structure. The default parameters of the automatic settings were used. Each docking experiment consisted of 10 docking runs with 150 individuals and 500,000 energy evaluations. The search was conducted in a grid of 40 points per dimension and a step size of 0.375 centered on the binding site of enzyme. The result of Auto Dock gives the binding position and bound conformation of the peptide, as well as a rough estimate of its activity. The docked conformation which had the minimum binding energy was selected to analyze the mode of binding.
Statistical analysis: All the experiments were repeated in three independent trials and the data were expressed as Mean±standard error and analyzed with ANOVA followed by student’s t test. p-values below 0.05 were considered statistically significant. The IC50 values and their respective 95% confidence intervals were calculated using Bliss and Finney methods.
RESULTS
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay: Furan-2-yl acetate (Scheme 1) was found to be cytotoxic on all the tested celllines. The cytotoxicity of the compound was evaluated at various concentrations at different time points. The effect of various concentrations of the compound on the tested cells revealed concentration and time dependent inhibition. The IC50 values of F2A and 5-FU against the tumor and normal cell lines were tabulated (Table 1). The HeLa cells were found to be highly sensitive to this F2A compound. The strongest cytotoxicity was observed in HeLa, MCF7, MDA-MB 435S and HT-29 with the IC50 values of 5.3, 6.5, 7.2 and 7.9 μg mL-1, respectively. The compound also showed significant cytotoxicity towards HepG2 and A549 with IC50 more than 10 μg mL-1. When compared with cancer cell lines the compounds showed less cytotoxicity towards the tested normal cells. The compound exhibited cytotoxicity on cancer cell lines with IC50 values less than 15 μg mL-1 whereas it was more than 25 μg mL-1 for normal cells.
| Scheme 1: | Structure of Furan-2-yl acetate |
| Table 1: | Cytotoxicity of F2A against different tumor and normal cell lines in vitro. 5-fluorouracil (5-FU) was used as control drug |
| IC50: Concentration, at which half of the cells were inhibited in their growth. The IC50 values and their respective 95% confidence intervals were calculated using Bliss and Finney methods, HeLa: Human cervical carcinoma, HT-29: Human colon carcinoma, A549: Human lung adenocarcinoma epithelial, NFFs: Neonatal foreskin fibroblasts, HEK: Human embryonic kidney, Vero: Green monkey kidney, MDA-MB 435S: Melanoma, MCF7: Human breast adenocarcinoma, HepG2: Hepatocellular carcinoma | |
| Fig. 1: | Dose response relationship of F2A on HeLa and NFF cells determined by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Normal (NFF) and tumor cells (HeLa) treated with F2A at various concentrations |
The cell viability was further assessed at different time points (0, 6, 12, 24, 48) and concentrations (0, 1.5, 3, 4.5, 6, 7.5, 9) which showed dose-and time-dependent inhibition of cells. It was observed that 30% cell death was seen after 24 h when treated with 3 μg mL-1 of compound at 24 h. The percentage of cell death was doubled when incubated for 48 h at same concentration. Further, the dose dependent toxicity was observed in both NFF and HeLa cells (Fig. 1).
Morphological observations: The observation of F2A (6 μg mL-1) treated HeLa cells under light and fluorescent microscopy for 12 h revealed considerable swelling and nuclear condensation. The cells were detached from the culture dish and appeared with rounded morphology (Fig. 2). The treated cells showed the characteristic morphological features of apoptosis such as chromatin condensation, membrane blebbing, fragmentation of nuclear chromatin and membrane-bound apoptotic bodies. DNA fragmentation was seen in 80% of treated cells as compared to 6% of control cells. After 24 h of post treatment of the compound (6 μg mL-1) on HeLa cells showed clear DNA fragmentation visualized with the staining of Hoechst staining under UV filter (Fig. 3). An apoptotic response upon the compound treatment was initially observed at 6-8 h.
| Fig. 2(a-b): | Morphological alterations in HeLa cells treated with compound, (a) Control HeLa cells and (b) HeLa cells treated with compound for 24 h with 4.5 μg mL-1 concentration. Datas determined by three individual experiments and one of the pictures shown. (Images on 40X) |
| Fig. 3(a-b): | Single cell imaging of HeLa control and treated cells. Images taken after 24 h incubation with 4.5 μg mL-1 of compound. DNA fragmentation was clearly seen on the treated cells, (a) Control cells and (b) Treated cell (Images on 100X) |
For the better understanding of induction of apoptosis in the compound treated cells, the DNA fragmentation in compound treated and control cells were determined by gel electrophoresis. The effect of compound on DNA fragmentation was given in Fig. 4 which shows the treated HeLa cells with extensive DNA fragmentation and appeared as typical DNA ladder.
Effect of F2A on mitochondrial cytochrome c release: To determine the involvement of cytochrome c in apoptosis induction, the HeLa cells were incubated with various concentrations of the compound (0, 1.5, 3, 4.5, 6 μg mL-1) for 12 h and the cytochrome c release was analyzed by immunoblotting with β-actin as internal control. The accumulation of cytochrome c was found in the dose-dependent manner (Fig. 5).
| Fig. 4: | Analysis of DNA fragmentation in F2A treated cell. Electrophoresis was carried out in 1.5% agarose gel. M: DNA marker (100 bp), Lane 1: Control DNA, Lane 2: 4.5 μg mL-1 for 12 h, Lane 3: 6 μg mL-1 for 12 h |
| Fig. 5: | Effect of F2A on the expression of Cytochrome c dependent caspase cascade in HeLa cells. Western blot analysis of Bcl-2, Bax, cytochrome c, caspase-3, caspase-8, caspase-9 and PARP protein levels exposed to various concentrations of F2A for 12 h. β-actin served as the loading control. Representative results from three independent experiments are shown |
The results indicate that the compound induced the release of cytochrome c from mitochondria into the cytosol.
Effect of F2A on caspase cascade: To investigate the effect of F2A on the caspase cascade, we analyzed the expression of caspase 3, 8 and 9 in treated HeLa cells. The cells were treated with the compound for 12 h at various concentrations (0, 1.5, 3, 4.5 and 6 μg mL-1) and immunoblotting was performed with respective antibodies. The results showed the concentration dependent increase in the caspase proteins.
| Fig. 6: | Effect of F2A on ROS formation in HeLa cells. The cells were treated with F2A or carrier vehicle (DMSO) for 12 h and the fluorescence intensity was measured. Datas are presented as Mean±SD calculated from three independent experiments. *p<0.05 vs. control, **p<0.01 vs. control |
As shown in Fig. 5 there was a considerable increase in the levels of caspase 3 and caspase 9, notably the caspase 3 has shown a 1.5 fold increase at 7.5 μg mL-1 concentration. But there was no changes observed in the expression levels of caspase 8. Since the caspase 3 is an enzyme which cleaves the DNA repair protein PARP, we also examined the cleavage of PARP with specific antibody. The western blot results showed the gradual increase in the cleaved PARP fragments when treated with F2A. Figure 4 shows the cleaved Poly (ADP-ribose) polymerase (PARP) fragments from the treated HeLa cells.
Effect of F2A on Bax and Bcl-2 expression: To determine the involvements of Bcl-2 and Bax proteins on the F2A induced apoptosis in HeLa cells, the expression levels were studied by Western blot. Exposure of HeLa cells with 6 μg mL-1 of F2A for 12 h caused a significant decrease in Bcl-2 and increase in Bax protein levels (Fig. 5). The expression of Bcl-2 was markedly decreased with 6 μg mL-1 of F2A. These results indicate that the F2A can dysregulate the expression levels of Bax and Bcl-2. The F2A induced changes in the expression levels of Bax and Bcl-2 are dose-dependent.
Effect of F2A on ROS, TBARS and NO: In the further studies on ROS formation, DCFH-DA assay was performed with or without compound in HeLa cells. The results were given in mean fluorescence intensity. There was no significant increase in ROS level when the cells were treated with 1.5 μg mL-1 of F2A when compared with untreated control. However, there was a slight increase in the intensity when the cells were treated with 3 μg mL-1 of F2A (Fig. 6). A gradual increase in higher ROS generation was seen when the concentrations were raised more than 3 μg mL-1. There was three fold increases in the ROS level between 3 and 9 μg mL-1. The F2A has a notable activity on the ROS formation in HeLa cells in a dose-dependent manner. Similarly there was also a significant (p<0.01) increase in the intracellular lipid peroxidation products when compared with the control. There was a 2 fold increase in the TBARS level in the increasing concentration of the F2A. As Fig. 7 shows, the F2A has a strong effect in the production of TBARS.
Nitric Oxide (NO) production was examined in the F2A treated HeLa cells to examine the role of NO in apoptosis induction. The NO levels were measured indirectly by means of nitrate/nitrite presence in both control and treated cells. As shown in Fig. 8, the NO levels significantly raised with increasing concentration. Maximum increase in the NO was determined with 9 μg mL-1 of F2A incubated for 12 h.
Hemolytic activity: Hemolytic assay was performed to verify the ability of F2A on membrane disruption. The F2A has weak activity on the lysis of erythrocytes.
| Fig. 7: | TBARS formation in HeLa cells treated with F2A or carrier vehicle (DMSO) for 12 h. Datas are presented as Mean±SD calculated from three independent experiments, *p<0.05 vs. control, **p<0.01 vs. control |
| Fig. 8: | Effect of F2A on NO release. The production of NO was measured indirectly by measuring the nitrate/nitrite levels determined by a calorimetric method based on the Griess reaction as described in methods section. Datas are presented as Mean±SD calculated from three independent experiments, *p<0.05 vs. control, **p<0.01 vs. control |
The EC50 value was found to be 288 μg mL-1 with F2A exposure and the total hemolysis (positive control) was obtained with 20 μL of Triton X-100 (0.1%) and 1 h incubation. The EC50 and 95% confidence interval (CI 95%) was obtained by non-linear regression analyses.
Molecular docking: The docking results are ranked according to the ascent of the binding energies for each of the enzymes investigated (Table 2). Docking of F2A into the active site of target enzymes resulted in 10 docked confirmations. Among these the lowest energy confirmation were chosen for further analysis. Among the 28 enzymes screened, cathepsin K showed the least binding energy of-5.18. The F2A interacted with LYS181, VAL31, SER183, LEU165, THR14, LYS17, PHE28, GLY32 of the cathepsin K protein and the hydrogen bond formation occurred in LYS17 and LYS181 residues. Also, topoisomerase II, Raf kinase, HDAC, HAT, CHK1-serine/threonine kinases, COX1, COX 2, Cytochrome P450 and Aromatase p450 showed good binding efficiency. Histone methyl transferase (2.84) and ubiquitin ligase (5.84) were found to have the lesser efficiency among the screened enzymes. It was also observed that minimum one hydrogen bond was formed between the F2A and the receptors.
DISCUSSION
It was already been reported that marine actinomycetes produce many novel pharmacologically potential compounds with antibiotic and antitumor properties such as salinosporamide (Feling et al., 2003), sporolides (Buchanan et al., 2005), the terpenoid chloro-dihydroquinones (Soria-Mercado et al., 2005) and marinomycins (Kwon et al., 2006).
| Table 2: | Summary of molecular docking results of F2A with various drug target |
However, there are no extensive research being carried out on marine derived actinobacteria from the Indian sub-continent and the reports on anticancer compounds are very scanty. The marine actinomycete, Streptomyces VITSDK1 spp. was isolated from marine sediments collected at the Bay of Bengal coast, India which produced a novel compound, furan-2-yl acetate. The structure of the compound was unambiguously established by spectroscopy.
Furans and compounds with furan moiety are commonly isolated from plant sources (Hannemann et al., 1989) but also isolated from soft corals (Ishii et al., 1988) and other marine bacteria (Shirasaka et al., 1995). A cytotoxic furan derivative has been isolated from New Zealand sponge Hymeniacidon huraki1 (P-388; IC50 13.4 μg mL-1) (Prinsep et al., 1994) and recently a novel furan type cytotoxic metabolite was isolated from soil-derived Streptomyces sp. HS-HY-071, with an IC50 of 18.2 μg mL-1 in HCT-116 cells (Wang et al., 2008). A novel proximicin A, B and C, aminofuran anticancer compounds were isolated from marine derived actinomycete Verrucosispora, which shows a strong cytostatic effect to various human tumor cell lines (Fiedler et al., 2008). Similarly F2A exhibits cytotoxic potential against tested cancer cell lines under in vitro conditions. Also, F2A is more toxic and selectivity inhibits the proliferation of human cancer cells leaving normal cells less toxic. The focus of the present investigation was to evaluate the cytotoxic potential of F2A on various cancer and normal celllines. With this, molecular mechanism of F2A in inducing cell death was also determined. This is the first report on the characterization and pharmacology of F2A.
Initially, the cytotoxic potential of F2A was determined by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in a 96 well plate with various concentrations of F2A and at different time points. For the determination of IC50, five cancerous cell lines were used and in order to find out the tumor cell specificity of F2A, three normal cells were assessed for the inhibition of cell proliferation. For all the tested cancer cells the IC50 values was found to be less than 15 μg mL-1. The HeLa cells were most sensitive, whereas the IC50 for the HepG2 cells was 13.2 μg mL-1, which was higher among the all tested cancer cells. When the F2A checked against the normal cells the IC50 values were more than 25 μg mL-1 for all the 3 cells. The HEK-293 showed the least IC50 of 26.3 μg mL-1. Based on these findings it is concluded that the cancer cells are more sensitive to F2A than normal cells and needs to be investigated in depth.
In order to determine the cytotoxicity of F2A was due to membrane disruption, the hemolytic assay was performed using erythrocytes. The F2A was found to be inactive and the EC50 was 288 μg mL-1 for human erythrocytes. Evaluation of membrane stability during exposure to newer drugs is important and the erythrocytes represent a good model for the study of membrane stability since their lysis releases the protein hemoglobin which can be readily measured spectrophotometrically. The mechanical stability of the erythrocytic membrane is a good indicator of the effect of various bioactive compounds and our results showed that the F2A is inactive in inducing hemolysis at low concentrations. It should also be noted that the crude extract isolated from the strain Streptomyces spp. showed EC50 value of 127 μg mL-1 on human erythrocytes and 168 μg mL-1 on mouse erythrocytes (Suthindhiran and Kannabiran, 2009). But the purified F2A doesn’t exhibit hemolytic activity on human erythrocytes. Based on the findings we can conclude that the cytotoxicity of F2A was not related to the lytic properties or membrane instability.
Since the F2A showed the strong inhibitory activity on HeLa cells, the further studies on the mechanism of F2A on cell death was investigated by taking HeLa as a model system. Apoptosis is a genetically controlled response of cells to commit suicide, especially unwanted and damaged cells and characterized by a distinct set of morphological events involving plasma membrane blebbing, loss of cell volume, nuclear condensation, fragmentation of DNA and ultimate fragmentation of the cell into membrane enclosed apoptotic bodies (Hacker, 2000). In F2A treated HeLa cells, the cytoplasmic changes including cytoplasmic shrinkage was clearly seen. The nuclear condensation was also seen in the treated cells which are due to the break down of chromatin in the nucleus. The success of an anticancer drug is greatly depends on the apoptosis induction. Apoptosis was observed in HeLa cells after F2A (6 μg mL-1) treatment for 12 h and the DNA ladder was observed in gel electrophoresis with the same concentration after 24 h treatment.
There are various mechanisms through which apoptosis can be induced in cells. Many cytotoxic and DNA targeting drugs induce apoptosis through the mitochondrial pathway (Green and Reed, 1998). Pro (such as Bad, Bax or Bid) and antiapoptotic (such as Bcl-2 and Bcl-XL) members of the Bcl-2 protein family may regulate mitochondrial participation in cell death. So during induction of apoptosis there is excess of Bax and down regulation of Bcl-2 proteins (Blatt and Glick, 2001). It was observed that there was down regulation of anti-apoptotic Bcl-2 proteins and up regulation of pro-apoptotic Bax proteins in the F2A treated cells. Results of our observations revealed that an elevated expression of Bax in cells pretreated with F2A for 24 and 48 h. Further, decreased in the expression level of antiapoptotic-Bcl-2 was observed. The down regulation of Bcl-2 proteins can lead to the formation of pores in the mitochondria and facilitates the release of cytochrome c and other pro-apoptotic molecules from the inter membrane space. This in turn leads to the formation of the apoptosome and the activation of the caspase cascade (Adams and Cory, 2001).
To determine whether the Bcl2 down regulation can cause the cytochrome c release, the presence of cytochrome c in the cytosol was examined. In the present study, cytochrome c in the cell lysate from F2A treated cells showed a notable increase as compared with the control. The cytochrome c release was dose-dependent and it showed a 3 fold increase. The release of cytochrome c is important in initiating the caspase cascade as it interacts with Apaf-1 and this leads to the recruitment of pro-caspase 9 and finally forms the apoptosome. Formation of the apoptosome leads to the activation of caspase 9 and subsequently caspase 3 (Thornberry, 1998). In addition, activated caspase-3 can activate caspase-6 which in turn activates caspase-8. These caspases cleave the key cellular proteins, such as cytoskeletal proteins, causing typical morphological changes observed in cells undergoing apoptosis (Chen and Wang, 2002).
So the expression levels of caspase proteins were investigated and the caspases 3, 8 and 9 in F2A treated HeLa cells showed over expression of caspase 3 and caspase 9 levels as compared with control. The increased level of Caspase-3 was 1.6 folds and caspase 9 was up to 2 folds in comparison with untreated control cells. However there was no change in the expression levels was observed for caspase 8. The enzyme PARP (poly (ADP-ribose) polymerase) is an important DNA repair enzyme and was identified as a substrate for caspases (Budihardjo et al., 1999) . The caspase-3 cleaves the PARP into two fragments and prevents the DNA repair (Thornberry and Lazebnik, 1998). In this study the cleaved fragments of PARP (115 kDa and 85 kDa) was seen in the HeLa cell lysates upon F2A treatment. The study demonstrates that increase in caspase 3 expression levels leads to the fragmentation of PARP and prevent DNA repair. This is an important event for tumor cells that have been treated with DNA-damaging compounds and undergoing apoptosis.
In addition the role of ROS and lipid peroxidation in apoptosis induction and cell death was also examined. Based on the earlier reports it is shown that Reactive Oxygen Species (ROS) can reduce tetrazolium compounds, such as MTT (Andrews et al., 1997). Since (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) based cytotoxicity assay was performed, we studied the ROS levels, nitrite/nitrate production and lipid peroxidation in F2A treated HeLa cells to find out the role of ROS mediated cytotoxicity. It is observed in our study that the F2A elevates the ROS, nitrite/nitrate and TBARS in a dose-dependent manner in HeLa cells. The findings suggest that ROS plays a major role in cytotoxicity and apoptosis induction in HeLa cells upon F2A exposure. The Reactive Oxygen Species (ROS) generation in mitochondria seems to play a role in cell death by initiating apoptosis signaling and cause DNA damage (Brand et al., 2004). Nitric oxide is a free radical generated in cells which might display cytotoxicity but the exact mechanisms of cell death by NO is not well documented. However it is thought that the Mitochondria comprise a target for NO and it triggers the caspase cascade and cause apoptosis (Brune, 2003).
Since many drugs are enzyme inhibitors which bind to the enzymes and arrest the activity, a molecular docking simulation study was undertaken to investigate the binding efficiency of F2A with the cancer drug target proteins. This study enables us to determine the possible role of F2A in inhibiting the key enzymes which in turn induce the apoptosis. Molecular docking using AutoDock 4.0 was carried out to perform binding scoring and mechanism. The results of AutoDock 4.0 results were verified by considering some top clusters of conformations in addition to the best scored one. The docking accuracy was evaluated in terms of the Root Mean Square Deviation (RMSD) and the prediction was considered successful if the RMSD value is less then 2.0 Å for the best scored conformation (Cole et al., 2005). The redox inhibitory conformations were energetically and statistically validated. The results indicate that F2A can able to bind to various enzymes in silico; especially it has strong binding property towards HDAC and protein kinases. Calculated binding energies from AutoDock for the selected enzymes ranged between -5.18 and +5.84 kcal mol-1. The docking studies confirm that the active sites of enzymes can accommodate F2A of different size and structure, adopting a variety of binding modes and interactions. This is demonstrated by the number of structurally diverse scaffolds. The F2A made at least two hydrogen bond contacts with active site residues of enzymes. The Lys and Arg residues are predominant in the receptor-ligand contacts. Furthermore, many of the compounds made additional hydrogen bonds with other residues such as Serine, Threonine and Glycine. Compounds with furan moiety have been showed to inhibit the cancer target enzymes efficiently (Engman et al., 2003; Girennavar et al., 2007; Qiu et al., 2008). It is suggested that the furan part of the compound is involved in the bio-activation and covalent interaction with the enzymes. The calculated binding energies of F2A with the receptors, however may not correlate well with experimental inhibitory concentrations and need to be investigate further.
CONCLUSION
Taken together, the furan 2-yl acetate extracted from marine derived Streptomyces spp. is found to be a potential cytotoxic and antiproliferative compound on the tested celllines. Furan 2-yl acetate induces the apoptosis by altering the Bax and a Bcl-2 protein expression which causes the mitochondrial cytochrome c release which in turn activates the series of caspase cascade with PARP cleavage (Fig. 9). Furthermore it is determined that the F2A causes the generation of ROS and mediates the mitochondrial mediated cell death. The docking study revealed the binding orientation of F2A with the functional sites of target enzymes could be responsible for inhibition of enzyme activity. However, the effect of F2A needs to be investigated in large set of tumor and normal celllines. The results of the studies also indicate that the marine actinomycetes from the unexplored Indian coast could provide lead compounds of biotechnological and biomedical value.
| Fig. 9: | The possible mechanism of cell death induced by furan-2-yl acetate |
ACKNOWLEDGMENT
The study was supported by the Institutional grant and the authors wish to thank the management of VIT University for providing necessary facilities for the completion of this study. The SAIF, IITM was gratefully acknowledged for providing analytical facilities.