Induction of Apoptotic Effects of Antiproliferative Protein from the Seeds of Borreria hispida on Lung Cancer (A549) and Cervical Cancer (HeLa) Cell Lines

A 35 KDa protein referred to as F3 was purified from the seeds of Borreria hispida by precipitation with 80% ammonium sulphate and gel filtration on Sephadex G-100 column. RP-HPLC analysis of protein fraction (F3) on an analytical C-18 column produced a single peak, detected at 220 nm. F3 showed an apparent molecular weight of 35 KDa by SDS PAGE and MALDI-TOF-MS analyses. Peptide mass fingerprinting analysis of F3 showed the closest homology with the sequence of 1-aminocyclopropane-1-carboxylate deaminase of Pyrococcus horikoshii. The protein (F3) exhibited significant cytotoxic activity against lung (A549) and cervical (HeLa) cancer cells in a dose-dependent manner at concentrations ranging from 10 µg to 1000 µg/mL, as revealed by the MTT assay. Cell cycle analysis revealed the increased growth of sub-G0 population in both cell lines exposed to a concentration of 1000 µg/mL of protein fraction F3 as examined from flow cytometry. This is the first report of a protein from the seeds of Borreria hispida with antiproliferative and apoptotic activity in lung (A549) and cervical (HeLa) cancer cells.


Introduction
Cancer is the largest single cause of death in men and women, and chemoprevention has been a promising anticancer approach aimed at reducing the morbidity and mortality of cancer by delaying the process of carcinogenesis. Among many recent advances in cancer chemotherapy, plant natural products play an important role in having contributed considerably to approximately 60% of available cancer chemotherapeutic drugs [1]. A new strategy in cancer therapy is to induce the apoptosis in tumor cells which is characterized by early activation of endogenous proteases, cell shrinkage, membrane blebbing, and DNA fragmentation [2].
Plant-derived compounds and their semisynthetic and synthetic analogs serve as major source of pharmaceuticals for human diseases [3]. One of the most active areas of research in the field of cancer therapy is the search for plant proteins with potent cytotoxic activity and low toxicity with varied mechanisms of action on tumors. Plant seeds are an enormously rich source of proteins with the potential to be developed as anticancer agents, for example, Violaceae [4], Rubiaceae [5], and Cucurbitaceae [6] families and certain marine plants [7].
B. hispida (Rubiaceae) is a promising medicinal plant which is widely used in folk medicine to treat fever due to primary complex, ulcer, and glandular swellings [8]. Ethnobotanically, B. hispida has been used as therapeutic agent in the treatment of various pathological conditions. It is used as an antieczemic and antibacterial and also used to treat cardiovascular disorders [9,10]. The methanolic extract of seeds of B. hispida inhibited the growth of human lung carcinoma (A549) and breast carcinoma (MCF-7) cell lines [11]. The aim of this study is to isolate and purify the proteins exhibiting cytotoxic activity from the seeds of Borreria hispida. The significant effect of purified protein on cell proliferation and cell cycle arrest of lung (A549) and cervical (HeLa) cancer cell lines is also investigated.

Protein Extraction.
Seeds of B. hispida were washed with distilled water and shade dried. The dried seeds were ground to fine powder and the proteins were extracted with extraction buffer [12] consisting of 10 mM Na 2 HPO 4 , 15 mM NaH 2 PO 4 , 10 mM KCl, and 2 mM EDTA (pH 7.0) by constant stirring overnight at 4 ∘ C. The crude protein extract was filtered using the muslin cloth and centrifuged at 10,000 rpm for 10 minutes. Proteins were precipitated from the crude supernatant using ammonium sulphate up to 80% saturation, overnight at 4 ∘ C. The precipitated proteins were collected by centrifugation at 12,000 rpm for 20 min. The protein concentration was determined according to Lowry et al. (1951) [13], using bovine serum albumin as standard.

Purification of Proteins.
The precipitated protein fractions obtained by 80% ammonium sulphate saturation were applied onto Fast Protein Liquid chromatography (Akta purifier GE Healthcare) using Sephadex G-100 gel filtration column (1.5 × 50 cm) equilibrated with 50 mM Tris-HCl buffer (pH 7.5). The protein fractions were eluted using the same buffer at a flow rate of 0.1 mL/min and detected at 280 nm [14]. 2 mL fractions were collected and screened for cytotoxic activity against the selected cancer cell lines. The protein fraction exhibiting increased cytotoxic activity warranted further studies.

RP-HPLC Analysis.
The purity of protein fraction F3 which showed potent cytotoxic activity was tested using analytical reverse phase-HPLC-C 18 column (250 × 4.6 mm, 5 m, 300Å), with solution A being 0.1% TFA and solution B being acetonitrile (90%) as the mobile phase at a flow rate of 1 mL/min, and detected at 220 nm [15].

SDS-PAGE Analysis.
12% SDS-PAGE analysis of active protein fraction F3 was performed according to Laemmli et al. [16]. About 20 g/mL of F3 fraction was loaded in Lane 1 ( Figure 1) and 20 g/mL of protein marker with the molecular weight in the range of 14.3 KDa-66 KDa was loaded in Lane 2.
Electrophoresis was run at 100 V for 2 hrs. The protein bands were visualized using Coomassie Brilliant Blue (CBB) R-250 [17].

Identification of Protein Fraction F3 by MALDI-TOF Mass
Spectrometry. The gel band of F3 was excised from SDS-PAGE and the tryptic digests were analysed using MALDI-TOF mass spectrometry which acts as a platform to generate the molecular mass of purified F3 protein [18]. In proteomics research, trypsin is commonly used for protein digestion to produce peptides with molecular masses in the optimal range for MS analysis. Prior to tryptic digestion, purified F3 protein was reduced and alkylated with tributylphosphine (TBP) and 15 mM iodoacetamide, respectively. About 2 g trypsin predissolved in 1 mM HCl was added to F3 and digested for 12-18 hr at 37 ∘ C. An equal volume of cyano-4-hydrocinnamic acid solution (10 mg/mL CHCA in 70% ACN, 0.03% TFA) was added to the trypsinized protein fraction F3. MALDI-TOF spectrum depicts the fragmentation of F3 in to various peptides. Peptide mass fingerprinting (PMF) was carried out to examine the sequence of the peptide fragments of F3 using SWISS-PROT sequence database.

Evaluation of Cytotoxic Activity of Protein Fractions.
The isolated protein fractions were tested for cytotoxic activity against lung (A549) and cervical (HeLa) cancer cell lines. A549 and HeLa cells were cultured in Dulbecco's modified eagle medium (GIBCO), supplemented with 10% fetal bovine serum (GIBCO) and 1% antibiotic antimycotic solution at 37 ∘ C in a humidified atmosphere of 5% CO 2 . A549 and HeLa cells were separately seeded at densities of 3500 and 3000 cells/well, respectively, in 96-well microtitre plates in a total volume of 200 L. The protein fractions were added at concentrations ranging from 10 g to 1000 g and cells were incubated. After 24 hours, the cellular morphology was observed using a phase contrast microscope (Leica, Wetzlar, Germany). MTT assay [19] was performed to evaluate the cytotoxic activity of the protein fractions. To each of the wells, 50 L (5 mg/mL) of 0.5% MTT was added and incubated for 4 h. The formazan crystals formed were dissolved in dimethyl sulfoxide (DMSO) and the absorbance was read at 570 nm using a microplate reader (Bio-Rad, CA). Wells without protein fractions served as blank. Doxorubicin (Sigma) was used as positive control. Percentage inhibition of cell viability was determined by using the formula control − treatment / control × 100.

Cell Cycle Analysis by Flow
Cytometry. A549 and HeLa cells were separately seeded in 6-well culture plates at a density of 0.3 × 10 6 cells/well and allowed to attach to the plates by incubating at 37 ∘ C, 5% CO 2 in an incubator for 24 h. The cells were treated with F3 at the concentration of 1000 g/mL using fresh media and incubated for 24, and 48 h for A549 and HeLa cells, respectively. The cells were trypsinized, washed with media, and centrifuged at 1000 rpm for 5 min at 4 ∘ C. The supernatant was discarded and cells were suspended in 300 L of PBS and 700 L of ethanol. The contents were kept at 4 ∘ C and then processed for cell cycle analysis by flow cytometry [20]. The cells were washed and centrifuged at 1500 rpm for 10 min at 4 ∘ C. The supernatant obtained was discarded and cells were resuspended in 600 L of PBS containing 0.5% Triton X-100 and 20 g RNase and incubated at room temperature for 1 h. To the cells, 24 L (40 g/mL) of propidium iodide (1 mg/mL stock) was added and again incubated at room temperature for 45 min in dark. The cells were detected in a flow cytometer (FACSCalibur, Beckton Dickinson) equipped with an air cooled argon laser providing 15 mW at 488 nm (blue laser) with standard filter setup. Ten thousand events were acquired and the percentage of cells in each phase of the cell cycle was analyzed using CellQuest Pro software (Becton Dickinson, USA).

Protein Extraction and Purification.
Purification of the precipitated proteins [21] with Sephadex G-100 gel filtration chromatography (FPLC, Akta, and GE) using Tris HCl (pH 7.5) resulted in the elution of protein fractions designated as F1 eluted at 15 min, F2 at 25 min, and F3 at 45 min detected at 280 nm ( Figure 1). The protein concentration in each fraction was determined according to Lowry et al. (1951). The elution volume of the protein fractions was compared with that of standard molecular weight markers of cytochrome c, -lactalbumin, trypsinogen, ovalbumin and bovine serum albumin. The results obtained in this study suggested that plant proteins with molecular masses of 4 KDa-150 KDa can be separated using Sephadex G-100 chromatography [22]. Earlier Etlzer (1985) reported the purification of plant lectins consisting of subunits ranging in molecular mass from 25 KDa to 35 KDa using Sephadex G-100 chromatography [23]. The purified peak fraction eluted at 45 min was found to possess maximum cytotoxic activity and was named as "protein fraction F3" which was then carried on for further analyses.

Reverse
Phase-HPLC Analysis. RP-HPLC of cytotoxic protein fraction F3 showed a single peak (Figure 2)

Identification of Protein Fraction (F3) by SDS PAGE.
The solubilizing denaturing agents such as sodium dodecyl sulphate (SDS) are widely used in the separation of proteins by gel electrophoresis. Identification of approximate mass of the cytotoxic protein fraction F3 was analysed by SDS-PAGE. A single band corresponding to the molecular weight of F3 approximately 35 KDa was observed when compared with the standard molecular weight markers (Figure 3).

Peptide Mass Fingerprinting of Protein Fraction (F3).
Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) has become an important tool of choice for large molecular analyses, especially for proteins. The proteome profiling technique by MALDI-TOF-MS provided a broad-base and effective approach for the identification of proteins with biological activity [24,25]. In the present investigation the gel band of F3 was excised from Coomassie Blue-stained gel and further processed for MALDI-TOF-MS analysis. Each peak in the MALDI-TOF-MS spectrum as given in Figure 4 represents a peptide present in the tryptic digest of protein fraction F3. The size of the peptide ion varies from m/z 507. 21 Figure 4. The nominal mass of the active protein F3 was found to be 35166 Da as obtained from peptide mass fingerprinting analysis. Identification of F3 using SWISS-PROT database which contains theoretical tryptic digests of all known proteins revealed the sequence similarity with 1-325 amino acids of 1-aminocyclopropane-1-carboxylatedeaminase (ACCD) of hyperthermophilic archearon, Pyrococcus horikoshii OT3 (Box 1). The molecular mass of mapped peptides of ACCD with F3 is given in Table 1. ACCD is a pyridoxal 5 -phosphate dependent enzyme that shows deaminase activity toward ACC, a precursor of plant hormone ethylene. ACCD has been reported to break the cyclopropane ring of ACC to yield -ketobutyrate and ammonia [26].

Cytotoxic Effect of Protein Fractions on Lung (A549)
and Cervical (HeLa) Cancer Cells. Cultured cancer cells are valuable reagents not only for rapid screening of potential anticancer agents but also for elucidation of mechanism of their activity. In this study, cytotoxic activity of purified protein fractions was determined using MTT assay on  human lung (A549) and cervical (HeLa) cells. The results of the study clearly marked that only F3 fraction exerted more significant cytotoxic activity than F1 and F2 fractions against tested cell lines at concentrations ranging from 10 g −1000 g/mL as shown in Tables 2 and 3. Doxorubicin at concentrations ranging from 0.1 g/mL to 10 g/mL inhibited the cell proliferation of A549 and HeLa cells and served as a positive control. Untreated A549 and HeLa cells served as cell control exhibiting 100% cell viability (Tables 2 and 3).
Morphological changes consistent with apoptotic cell death were seen in A549 and HeLa cells treated with F3 isolated from seeds of B. hispida as evidenced by microscopic observations shown in Supplementary Material available online at http://dx.doi.org/10.1155/2014/179836. Cell shrinkage and rounding, were observed in A549 cells indicating 59% cell inhibition, when treated with F3 at the concentration of 10 g/mL as given in Table 2. Increased inhibition of cell proliferation was observed in A549 cells treated with F3 at concentrations of 30 g/mL showing 78% inhibition and 100-1000 g/mL of F3 showing >90% inhibition of cell proliferation in A549 cells as evidenced in the Supplementary Material. Further the results of MTT assay also showed that protein fraction F3 effectively suppressed the proliferation of HeLa cells with 61.38% inhibition (Table 3) at the concentration of 10 g/mL depicting chromatin condensation (pyknosis) in the nucleus of cells undergoing apoptosis as shown in the Supplementary Material. Similar morphological alterations associated with pyknosis and karyorrhexis (nuclear fragmentation) were observed in cancer cells treated with cytotoxic proteins isolated from the various plant sources [27,28]. Apoptosis is an important homeostatic mechanism that balances cell division and cell death for maintaining  the appropriate cell number in the body. Induction of apoptosis in cancer cells is one of the strategies for anticancer drug development [29,30].

Effect of F3 on Cell Cycle of A549 and HeLa Cells.
Disturbance of the cancer cell cycle is one of therapeutic targets for development of new anticancer drugs [31]. The distribution of cell population in each phase of cell cycle was examined by flow cytometry. Our results revealed that apoptosis induced by F3 fraction was not triggered at a specific phase of the cell cycle. However, F3 fraction increased the number of apoptotic cells of sub-G0 population in a time-dependent manner. Cell cycle analysis performed on A549 cells treated with F3 at a concentration of 1000 g/mL for different time periods revealed interesting observations. Marked changes in the sub-G0 population (hypodiploid cells) of A549 cells were observed recording 2.11% and 2.71% increases (Table 4) at 24 and 48 h as given in the Supplementary Material. A successful anticancer compound should kill or incapacitate cancer cells without causing excessive damage to normal cells. It is evident that there is no growth in sub-G0 phase of cell cycle in control cells of both the A549 and HeLa cells which indicated the nonapoptotic population.
The most pronounced cytotoxic effect was obtained with HeLa cells treated with F3 at concentration of 1000 g/mL, leading to increased cell population in sub-G0 phase as 3.07% and 3.42% (Table 5) at 24 and 48 h, indicating increased apoptotic-associated chromatin degradation in HeLa cells as shown in the Supplementary Material. Similar observations were noted with nebrodeolysin, isolated from Pleurotus nebrodensis which induced apoptosis in HeLa cancer cells [32]. The protein fraction isolated from the leaves of Mirabilis  jalapa L. was cytotoxic to HeLa cells causing DNA fragmentation and apoptosis [33]. Reports have demonstrated that the toxin abrin, a type of ribosome inactivating protein, induced cell cycle arrest and apoptotic cell death in HeLa cells [34]. Other proteins isolated from different plant sources also have been found to exhibit antiproliferation action involving cell cycle arrest in HeLa cells [35,36].

Conclusion
This study is the first report of identification of the cytotoxic protein with molecular mass of 35 KDa from the seeds of Borreria hispida showing sequence similarity with 1-325 amino acids of 1-aminocyclopropane-1-carboxylate deaminase (ACCD) of hyperthermophilic archearon, Pyrococcus horikoshii OT3. The cytotoxic protein F3 is found to exhibit growth inhibition (in vitro cytotoxicity assay) and cell cycle arrest in sub-G0 population of human lung (A549) and cervical (HeLa) cancer cells as examined by flow cytometer.