ANTI-TUMOUR ACTIVITY OF BROMOPYRROLE ALKALOIDS AGAINST HUMAN BREAST TUMOUR (MCF-7) THROUGH APOPTOSIS INDUCTION

Objective: The present study was conducted to screen the cytotoxicity and initiation of apoptosis in MCF-7 human breast tumour cells using the Indian sponge Acanthostylotela cornuta of the Gulf of Mannar. Methods: The crude methanol extract of A. cornuta was fractionated using a reversed phase silica gel column chromatography. The purity of the active fraction of bromopyrrole alkaloids was confirmed with Gas Chromatography-Mass Spectrometry (GCMS). The bromopyrrole alkaloids induce apoptotic changes in MCF-7 cells were studied with electrophoresis, caspase assay, and different staining analysis. The MCF-7 cells were analyzed by Flow cytometry to determine their DNA content. Results: Bromopyrrole alkaloids of A. cornuta showed cytotoxicity against MCF-7 human breast tumour cells with the IC 50 value of 8.0μg/ml. The bromopyrrole compounds induced cells exhibited scatted red fluorescence, showing the presence of several residual bodies and condensation of chromatin. Caspase-3, pro-caspase-9, caspase-9 and Poly-ADP-Ribose-Polymerase (PARP) activity were occurred in bromopyrrole alkaloids treated tumour cell. The cell cycle arrest is conceivable that the compound prevented the progression of cell cycle through the G phase resulting from inhibition of survival, leading them to undergo apoptosis. Conclusion: Bromopyrrole alkaloids of A. cornuta possess antitumor activity which was arrested the G phase in cell cycle that clearly indicated its nature as that of antitumour drugs.


INTRODUCTION
Tumour is considered as one of the most serious threats to humankind. Annually, it causes more than 13% of all human deaths, among them, 70% of all tumour deaths occurred in low and middleincome countries. WHO estimates of death due to tumour worldwide are an estimated 12 million deaths in 2030. Breast tumour is the most common in women and one-third of the women with breast tumour develop metastases that ultimately results in death. In 1970, the MCF-7 cell line was noticed after that MCF-7 cell has become a prominent model system for the study of breast tumour. Most of the tumors initially respond to chemotherapy, similarly breast tumour cells can subsequently survive and gain resistance to the chemo treatment [1][2][3]. The discovery of new antitumor drug with low side effects on the human system has become an essential goal in many studies.
Marine organisms are the rich sources of structurally novel and biologically active metabolites. So far, many unique compounds of marine origin with different biological activities have been isolated and a number of them are under investigation [4][5][6][7]. The discovery of antitumor drugs derived from natural marine products has enjoyed in the past few years, especially after the approval of trabectedin [8]. As the first marine-derived antitumor drug to reach the market, trabectedin was first identified to be a tetrahydroisoquinoline alkaloid derived from a Caribbean tunicate in 1990 [9]. Alkaloids of marine origin are widely distributed in marine sponges with widespread biological activity [10].
Screening of organic solvent extracts from marine sponges and other marine organisms is a common approach to identify compounds of biomedical importance. Marine sponges have been considered as a goldmine during the past 50 y, with respect to the diversity of their secondary metabolites. The biological effects of new metabolites from sponges have been reported in many scientific papers. Sponges have the potential drugs of the future to act against important diseases, such as tumour, a range of viral diseases, malaria and inflammations. In recent years, there has been an increased need for new chemotherapeutic agents, as the emergence of drug resistance in tumour poses a significant clinical problem. The present study was conducted to screen the cytotoxicity and initiation of apoptosis in MCF-7 breast tumour cells using the Indian sponge Acanthostylotela cornuta of the Gulf of Mannar. from hexane to acetone to methanol. The different column fractions were collected and the purity of the active fraction was confirmed by Gas Chromatography-Mass Spectrometry (GCMS) library. Each fraction was used for cytotoxicity against MCF-7 breast tumour cells with 10µg/ml concentration.

DNA fragmentation
Bromopyrrole compounds treated cells as well the untreated cells of 106cells were pelleted and re-suspended in 1 ml of lysis buffer for cellular DNA extraction by proteinase K digestion [13]. The mixture was incubated overnight at 55 °C and then at 37 °C for 30 min with 0.25 mg/ml of RNase. DNA was extracted by phenol: chloroform: isoamyl alcohol (25:24:1) mixture, precipitated in ethanol and loaded on a 2% Agarose gel containing ethidium bromide (0.6 μg/ml), at 80 Volts. The gels were then photographed under UV light.

Cell cycle analysis
Bromopyrrole compounds treated and untreated MCF-7 cells were harvested and centrifuged at 3000 rpm for 3 min at 4 °C. The cells were washed once with cold PBS (phosphate buffer saline) and harvested again by centrifugation under the same condition. Then, 150 µl of propidium iodide in water solution (propidium iodide 50µg/ml, sodium citrate 0.1% and nonidet P-40 0.2%) was added into each tube and the cells were stained at 4 °C for 30 min. Then the cells were analyzed by Flow cytometry to determine their DNA content (using the computing system, XLII (Coulter)).

Hoechst staining
Bromopyrrole compounds treated and untreated cells were harvested by centrifugation; the obtained pellet was resuspended in 1 ml PBS. Each sample was stained with 10μl Hoechst solution (dissolved in double distilled water at a stock concentration of 0.1 mg/ml) for 5 min at 37 °C in the dark. The dye was removed by centrifugation (110xg; 10 min; 4 °C) and washing with PBS. Condensed chromatin was visualized by confocal laser scanning microscopy.

Propidium iodide staining
Propidium iodide is used as a marker of cell death and as well as for analysis of nuclear morphology of dead cells. Bromopyrrole compounds treated cells were stained with propidium iodide (20µg/ml, 5 min) on glass coverslips. The cells were visualized by confocal laser scanning microscopy and apoptotic nuclei were observed.

Determination of caspase activity
Caspase activation was measured using a caspase colorimetric assay kit as described by the manufacturer (Geno Technology). Cultured monolayer of cells in the dishes was exposed to 1 ml of MEM medium containing defined different concentrations of the bromopyrrole compounds then protein was estimated using bicinchoninic acid assay (BCA). 100µg protein (cellular extract) was diluted in 50 µl cell lysis buffer and then incubated in 96-well microtiter plates with 5 µl of the 4 mmol p-nitroanilide (pNA) substrates (DEVD-pNA for caspase-3 activity and Ac-LEHD-pNA for caspase-9 activity) for 2h at 37 °C. Caspase activity was measured by cleavage of the substrates to free pNA. Free pNA (cleaved substrates) was measured by absorbance at 405 nm in a microtiter plate reader. Relative caspase-3 and caspase-9 activities were calculated.

Western blotting analysis
20μg aliquots of protein extracts from bromopyrrole compounds treated and untreated MCF-7 cells were separated on 10% SDS polyacrylamide gels. After electrophoresis, the proteins were blotted onto polyvinylidene difluoride membranes (PVDF). The membranes were blocked with 5% non-fat milk in PBS plus 0.1% Tween-20 and then incubated with primary antibodies (monoclonal rabbit anti-PARP) to the Poly-ADPribose-polymerase (PARP). The secondary antibody (anti-mouse coupled to horse-radish peroxidase) was added (1:2000 dilution) and kept for 1h at room temperature. Finally, the protein was visualized using the ECL detection kit (Amersham) and exposing the membranes to X-ray film (HYPERFILMTM, Amersham).

Cell viability assay
This assay is a quantitative colorimetric method for the determination of cell survival and cytotoxicity. The crude methanol extract of the marine sponge A. cornuta in different concentrations (7,14,21,28,35 and 42 µg/ml), and its nine column fractions with 10 µg/ml and different concentrations of the 9 th column fraction (Bromopyrrole compounds) (1, 3, 9, 12, 15, 18 and 21 µg/ml) were subjected to MTT assay. 1 ml of MCF-7 cell suspension (10 5 cells/ml) were seeded in each well and incubated at 37 °C for 48 h in 5% CO2 for the formation of a confluent monolayer. 200 µl of MTT (5 mg/ml) solution and 1 ml of DMSO with a particular concentration of the extract were added to each well and left for 45 sec. Controls were maintained with only DMSO throughout the experiment. Absorbance was measured at 570 nm with an ELISA plate reader. The bromopyrrole alkaloid concentrations required to inhibit growth by 50% (IC50) were calculated.

RESULTS
The crude methanol extract of the marine sponge Acanthostylotela cornuta showed dose-dependent cytotoxic activity against MCF-7 cells ( fig. 1). Out of nine column fractions, 9th column fraction showed the highest activity of 52% inhibition of cells at 10µg/ml concentration ( fig.  2). The activity of 9th column fraction was dose-dependent and the IC50 value of the9th column fraction (bromopyrrole compounds) concentration inhibiting the mean growth value by 50% was calculated as 8.0μg/ml ( fig. 3). GCMS spectrum of the bioactive 9th column fraction showed molecular ions at m/z 225, 190 and 268 suggesting the presence of 2-Bromo-6-chloro-4-fluoroaniline, 5-bromopyrrole-2-carboxylic acid and 3-Acetyl, 6-bromocoumarin respectively, with reference to previous literature of GC-MS library ( fig. 4).    . 9). The percentages of caspase-9 activity of treated MCF-7 breast tumour cells were 115.3, 176 and 238.66 in 5, 8 and 11µg/ml respectively. Statistical analysis between treatments with IC50 (8µg/ml) concentration and without inhibitor (control cells) using paired two-tailed Student's t-test with P-values<0.05 were considered as significant. The significant caspase activities of the tumour cells were comparable to the standard antibiotic camptothecin (10µg/ml).
Apoptosis characteristics were further verified by Fluorescence Activated cell sorter (FACS) analysis, which showed the presence of apoptotic peak in sub-Go/G1 and Go/G1 phases ( fig. 11). MCF-7 cells treated with 6, 8, and 10 µg/ml of the bromopyrrole compounds displayed a dose-dependent. Accumulation of apoptotic cells and the apoptosis percentage of cells at the sub GO/G1 population increased by 1.82%, 7.2% and 8.35% respectively as compared to the untreated control (2.3%). Similarly, the apoptosis percentage of cells at the GO/G1 population increased by 61.72%, 65.9% and 67.29% respectively as compared to the untreated control (55.12%).

DISCUSSION
Marine sponges contain a number of biodynamic compounds of therapeutic value. These compounds are providing valuable ideas for the development of new drugs against tumour. Many compounds from marine sponges have been reported to have antitumour activity in vitro [14], but there are no reports about Indian sponge Acanthostylotela cornuta against tumour cells. The results of this study indicate that there may be bromopyrrole compounds in A. cornuta which induce cytotoxic action against MCF-7 tumour cells and initiate cell death. The American national tumour institute guidelines have guideline for extracts at 50% inhibition (IC50 value) of proliferation as less than 30μg/ml [15]. A. cornuta methanol extracts exhibited antitumour activity against MCF-7 tumour cells, with an IC50 value of less than 30μg/ml on the cell lines used. IC50 value of A. cornuta column fraction of bromopyrrole compounds as 8µg/ml for MCF-7 tumour cell, though considered potent, was high when compared to IC50 of IC50 values 0.28µg/ml of marine sponge Dactylia sp., which was interestingly cytotoxic against murine leukemia cell line P388 by Sato et al., [16]. This may be because, the column fraction may be only partially pure and the observation of an IC50 of 8µg/ml indicated that it might yield potent and more active pure compound, as evidenced from the increase in activity from crude extract to column fraction. However, it was low IC50 when compared to the Mediterranean sponge Axinella damicornis (IC50 of>40µg/ml) against human cervix carcinoma HeLa S3 cells reported by Aiello et al., [17] and the Mediterranean sponge Axinella verrucosa having the IC50 of 10μg/ml against PC12, HeLa, and L5178y cells reported by Aiello et al., [18].
The overall investigation of cytotoxic activities of the bromopyrrole compounds of A. cornuta revealed that, the active constituents obtained by the more polar solvent in the extraction process were responsible for the antitumor properties in this sponge. The biologically selective activity of any compound might depend on the type of chemical composition and the concentration of active constituents as well as the type and developmental stages of the tumour [19]. One of the biochemical characteristics of apoptosis is the cleavage of DNA into fragments consisting of 180-200 base pairs and the generation of typical DNA ladder on gel electrophoresis [20]. These cleavages in DNA of MCF-7 tumour cells may be induced by the bromopyrrole compounds from A. cornuta.
The present study reports, the evidence that a bromopyrrole compounds of A. cornuta induce apoptosis in tumor cells by caspase activation that has cytotoxic effect in vitro against tumour cells at least in part through induction of apoptosis via caspase-9 (mitochondrial) pathway. Induction of apoptosis in vitro was associated with caspase-3 activation [21]. According to Reiner et al., [22], caspase-3 was responsible for many of the morphological and biochemical features of apoptosis, where the process occurs in a systematic and deliberate manner which involves a series of activation of enzymes such as cysteine proteases (caspases) and endonucleases. The present study reveals that the bromopyrrole compounds induce apoptosis with affecting mitochondrial regulation.
According to Nicholson et al., [23] fragmentation of 116 kDa PARP proteins into 85 kDa fragments was the evidence of apoptosis.
Results of the present study also showed that active fractions of A. cornuta cleaved 116 kDa PARP protein into 85 kDa fragments. Thus the bioactive bromopyrrole compounds in A. cornuta are capable of inducing apoptosis in MCF-7 cells via the mitochondria-mediated pathway, which results in the activation of caspase-9, caspase-3 and degradation of PARP. This study also defines those events, most of which are used as biomarkers of apoptosis, that are associated with bromopyrrole compounds of A. cornuta induced apoptotic cell death. Activated caspase-3 was responsible for the proteolytic degradation of poly ADP-ribose polymerase, which occurs at the onset of apoptosis [24]. It has been reported that the central mechanism of apoptosis is evolutionarily conserved, and that caspase activation is an essential step in the complex apoptotic pathway [25]. The presented data, therefore, provides important evidence that compounds of A. cornuta induced MCF-7 cell death in apoptosis.
The results of annexin-V staining analysis revealed that the bromopyrrole compounds of A. cornuta induced the early stage of apoptosis due to membrane phospholipids redistributing from the inner to the outer leaflet of the membrane bilayer. One of the hallmarks of the early stage of apoptosis is that membrane phospholipids such as phosphatidyl-serine and phosphatidylethanolamine redistribute from the inner to the outer leaflet of the membrane bilayer where they are exposed on the cell surface [26]. Externalization of phosphatidylserine residues to the outer plasma membrane leaflet al. lows their detection via their high affinity for annexin-V, a phospholipid binding protein.
The results of Hoechst staining revealed that the bromopyrrole compounds of A. cornuta induced the nuclear condensation. In the same manner, the apoptosis induced by the bromopyrrole compounds of A. cornuta was also confirmed by staining the cells using Propidium iodide (PI) fluorescence dye. Additionally, the results of the PI staining revealed the nuclear condensation and cell fragmentation into apoptotic bodies. These distinct characteristics of apoptotic process in all the treated tumour cells suggest that the mode of cell death triggered by the bromopyrrole compounds of A. cornuta might be the process of apoptosis. These processes in apoptosis are recognized as a novel strategy for identification of antitumor drugs [27].
Regulation of the tumour cell cycle is one of the strategies in the development of antitumor drugs [28]. In the present study, the result of cell cycle analysis determined by flow cytometry analysis showed that the compound could arrest the cell cycle in a dosedependent manner in MCF-7 cells. The apoptotic rate contrarily increased after treatment with high concentration. The observations with cell cycle progression revealed that pre-exposure to the compound induced GO-G1-phase cell cycle arrest. This showed that the compound inhibited the proliferation of MCF-7 cells through the inducement of cell apoptosis. According to Ormerod et al., [29] the visibility of "sub-GO" peak (hypodiploid DNA peak) by flow cytometry with other supporting information can be taken as definite evidence for the apoptotic cell population. Results of this method of measuring apoptosis would provide stronger evidence that apoptosis indeed was induced in the cell lines after treatment with the compound. The cell cycle arrest is conceivable that the compound prevented the progression of cell cycle through the G phase resulting from inhibition of survival, leading them to undergo apoptosis. The arrest of G phase by the bromopyrrole compounds of A. cornuta clearly indicated its nature as that of antitumor drugs.

CONCLUSION
In conclusion, the data reported here indicate that the bromopyrrole compounds of A. cornuta inhibits growth and proliferation by arresting the cell cycle at the G phase, and induces apoptosis in MCF-7 cells. This apoptosis was mediated by the activation of intrinsic caspase cascade, in MCF-7 cells. Thus the results of the present study provide supportive data for the antitumor potential of the bromopyrrole compounds of A. cornuta.