Chalepin: isolated from Ruta angustifolia L. Pers induces mitochondrial mediated apoptosis in lung carcinoma cells

The whole plant of Ruta angustifolia L. Pers was obtained from a plant nursery near Sungai Buloh, Selangor, Malaysia. The plant was identified by Slamet Wahyono from the Research Station of Medicinal Plant and Traditional Medicine Research and Development Centre, Tawangmangu, Central Java, Indonesia. A voucher specimen numbered KLU48128 was deposited at the Herbarium of the Institute of Biological Sciences, Faculty of Science, University of Malaya on 26th April 2014.

The human cell lines A549 (lung cancer) and Ca Ski (cervical cancer) were cultured as monolayer in RPMI 1640 growth media whereas HCT-116 (colon cancer) and MRC5 (human normal lung fibroblasts) was cultured in McCoy’s 5A and EMEM medium, respectively. All cells were purchased from the American Tissue Culture Collection (ATCC, USA). All the media were supplemented with 10 % v/v foetal bovine serum (FBS), 100 μg/mL penicillin/streptomycin, and 50 μg/mL amphotericin B. The cells were cultured in a 5 % CO₂ incubator at 37 °C.

The monolayer cell culture was detached using accutase and the cell count was adjusted to 2.5 x10⁵ -3.0 x10⁵ cells/mL using medium containing feotal bovine serum. The cells were then plated into 96 wells with a cell density of 5000-7000 cells/well and then incubated for 24 h to allow adherence of cells to the base of well of the plates. The media was then removed and 200 μl of fresh media containing different concentration of various extracts or isolated compounds were added. Cisplatin was used as the positive control. The plates were incubated for 24, 48 and 72 h at 37 °C and 5 % CO₂. The cytotoxicity of the extracts and isolated compounds were screened using the Sulphoramide B (SRB) assay. This assay was first described by Houghton in 2007 [8]. The incubation was halted by gentle addition of 50 μl ice cold 4 % trichloroacetic acid (TCA) to each well and the plates were incubated at 4 °C for 1 h. Then, the supernatant was discarded and the plates were washed with distilled water for 5 times and then air dried. SRB dye, 50 μl, (0.4 % w/v) was added into each well and the treated cells and control were incubated at room temperature for 30 min. The unbound dye was removed by rapid washing four times with 1 % acetic acid. The plates were then air dried and 100 μl of tris base (10 mM unbuffered, pH 10.5) was added and the plates were shaken at 500 rpm for 5 min to solublize the bound SRB stain. The plates were then read with an ELISA reader (Synergy H1 Hybrid) at an absorbance of 492 nm.

From the HPLC analysis, twenty peaks were observed, and the eluents of the peaks were collected and then pooled to give twenty fractions (1-20) based on similarity of spots on TLC. Fractions that showed single spot on TLC were subjected to analytical HPLC analysis for determination of purity.

All the compounds were identified through their mass spectral and NMR data. Some of the compounds were further confirmed by their Rf values from the TLC analysis by comparison with authenticated compounds. TLC precoated plates (silica gel 60F254) of 20-25 mm thickness were used and a solvent system consisting of chloroform with a few drops of methanol was used. Identification of compounds using gas chromatography-mass spectrometry (GCMS) was done using the Agilent Technologies 6980N gas chromatography equippped with a 5979 mass selective detector (70 eV direct inlet) was used. The column used was HP-5MS (5 % phenylmethyl siloxane) capillary column (30.0 mm × 25 mm × 25 μm) with helium as carrier gas at flow rate of 1mL min^-1. The column temperature was programmed initially at 100 °C, then increased to 300 °C, at 3 °C per minute afterwhich the temperature was kept isothermally for 10 min. The compounds were identified by comparison of their mass spectral data with an accompanying mass spectral library NIST08 Spectral Library.

This study was carried out to observe changes in cell nucleus. 5000-7000 cells were seeded into each 6 cm culture dish and allowed to adhere overnight. The cells were subsequently treated with various concentrations of chalepin for 48 and 72 h. At the end of the incubation period, the media in the culture dishes were discarded and the cells were washed twice with PBS, after which 1mL of PBS was added to each culture dish. To stain the cells with fluorescent dyes, 100 μ L of Hoechst solution (100μ g/mL) and 25 μL of PI solution (100 μ g/mL) were added to each culture dish and incubated for 15min. Finally, the cells were photographed using Leica DM-16000B fluorescent microscope.

FITC annexin V apoptosis detection kit (BD) was used to detect apoptosis. Cells were seeded in tissue culture dishes (5000-7000 cells/dish) and allowed to adhere overnight. Next, the cells were treated with various concentrations of chalepin for 48 and 72 h. The cells were harvested, washed, and resuspended in 1x binding buffer according to the manual provided in the kit. In the next step, the cells were stained in the ratio of per 100,000 cells; staining of cells with 5 μL of FITC-annexin V and 5 μL of propidium iodide was done for 15 min. Then, 200 μL of 1× binding buffer was added to each sample. Unstained and single-stained untreated cells were also included as controls. A minimum number of 10,000 events was acquired for each replicate using Accuri C6 flow cytometer. A quadrant statistic was used to measure the population of viable, early apoptotic, late apoptotic and secondary necrotic cells. The determination betweent these different populations were obtained by estimating quantitatively the cells that were AnnexinV/PI stained.

DNA strand breaks in apoptotic cells caused by the activation of endonucleases, were detected by APO-BrdU TUNEL assay kit. After incubation with various concentrations of chalepin at 48 and 72 h, cells were harvested and washed with PBS. Next, the cells were fixed with 1 % (w/v) paraformaldehyde for 15 mins, then washed with PBS, and fixed with 70 % (v/v) ethanol overnight. Ethanol was removed by centrifugation and DNA labeling steps were performed according to the manual provided in the kit. Samples were analyzed by flow cytometer and a minimum of 10,000 events were acquired for each replicate.

The assay was conducted as described by Ling et al. [9] with some minor modifications. The ROS measurement was done using the fluorescent probe 2’,7’-dichlorofluorescein diacetate (DCFH-DA) that enabled the monitoring of intracellular accumulation of ROS. The A549 cells were seeded into a 96 well plate with a density of 2×10⁴ cells per well and allowed to attach overnight. Treatment with chalepin at various concentrations were done the next day and incubated for different time periods. At the end of the incubation period, the media was removed and 5 μM of DCFH-DA which is diluted in media was added to the cells and incubated for 40 min at 37 °C. The cells were then washed three times with clear media and the fluorescence intensity (excitation = 485 nm and emission = 530 nm) was measured using a microplate reader. The morphology of the cells were observed using a fluorescence microscope.

Caspase activity assays were performed according to the instructions in the manual (CaspILLUME, Genetex). Cells were seeded at a density of 1×10⁶ cells per culture dish. After being subjected to treatment for 48 and 72 h, the cells were detached with accutase, washed, and resuspended in PBS. Next, 300 μL of each sample was aliquoted into centrifuge tubes, after which 1 μL of fluorescent substrate (FITC-DEVD-FMK for caspase 3 and FITC-LEHD-FMK for caspase 9 activity) was added to each tube and incubated for 1 h at 37°C incubator. At the end of the incubation period, the cells were centrifuged at 3000 rpm for 5 min and the supernatant was removed. After that, the cells were resuspended in 0.5 mL wash buffer (provided in the kit) and centrifuged at 3000 rpm for 5 min. This step was repeated before performing the analysis with flow cytometer (Accuri C6) and a minimum of 10,000 events were acquired for each replicate.

For this analysis, 1× 10⁵ of A549 cells were plated per tissue culture dish. Chalepin treated whole-cell extracts were lysed in lysis buffer (20 mM Tris (pH 7.4), 250 mM NaCl, 2 mM EDTA (pH 8.0), 0.1 % Triton X-100, 0.01 mg/ml aprotinin, 0.005 mg/ml leupeptin, 0.4 mM PMSF, and 4 mM NaVO4). Lysates were then spun at 15,000 rpm for 10 min to remove insoluble material and resolved on a 10.0 % SDS-PAGE gel. After electrophoresis, the proteins were electrotransferred to a nitrocellulose membrane, blocked with Blocking One (Nacalai Tesque, Inc.), and probed with various antibodies (1:1000) overnight at 4 °C. The blot was washed, exposed to HRP-conjugated secondary antibodies for 1 h, and finally examined by chemiluminescence (ECL, Advansta).

Extraction and fractionation was done on the leaves of R. angustifolia. The yield of the methanol extract was 55.0 g which is 31.4 % of the total dried ground leaves. The methanol extract was further partitioned with solvents of different polarity. Water extract gave the highest yield followed by chloroform extract. Ethyl acetate extract had the lowest yield from methanol extract, as compared to the other fractions. The percentage of the fractionated extracts (hexane, chloroform, ethyl acetate and water) were calculated based on the methanol extract (Table 1).

Selected cancer cell lines (A549, Ca Ski and HCT-116) were used in the screening of the crude and fractionated extracts of R. angustifolia for their cytotoxic potency. Toxicity towards normal cells were tested against MRC5 cell line. All the extracts showed no toxicity towards MRC5 with an IC₅₀ value of more than 100.00 μg/ml. The chloroform extract (without chlorophyll), showed slight toxicity with an IC₅₀ value of 65.7 ± 0.5 μg/ml. It was found that the activity of all the extracts were time and concentration dependant in which the lowest IC₅₀ value was observed when treatment was done over 72 h. The methanol extract exhibited good cytotoxic activity in all the cancer cell lines tested and without toxicity to normal cells. The chloroform extract showed the highest activity and the cytotoxicity was even better when chlorophyll was removed; however it exhibited slight toxicity to MRC5 cell. The chloroform extract without chlorophyll exhibited the best cytotoxicity in A549 cell line with an IC₅₀ of 8.8 μg/ml; hence it was chosen as a candidate for further isolation of the chemical components (Table 2) (Fig. 1).

The chloroform extract was subjected to isolation and purification using HPLC. Fractions were repeatedly collected using preparative HPLC method. The HPLC profile of the chloroform extract was shown in Fig. 2. Twenty fractions were collected and twelve compounds were successfully identified using GCMS analyses. The mass spectral data of the isolated compounds obtained were found to be consistent with previous reports [6, 7, 10‐12]. The compounds which has been successfully identified are graveoline (1), psoralen (3), kokusaginine (4), methoxsalen (5), bergapten (7), arborinine (8), moskachan B (9), chalepin (10), moskachan D (12), chalepensin (13), rutamarin (14) and neophytadiene (16). The structures of these compounds are illustrated in Fig. 3.

The compound graveoline, psoralen, kokusaginine, methoxsalen, bergapten, arborinine, moskachan B, moskachan D, chalepensin, rutamarin and neophytadiene were identified through their mass spectral data which were found to be consistent with published data [6, 7, 10‐12]. However chalepin was identified through its’ mass spectral which were consistent with published data [12] and NMR data. Some collected peaks of HPLC were not identified due to insufficient amount. The following compounds analysed were identified from their EI mass spectra by their m/z values: Graveoline, colorless powder, EIMS m/z (%), 279 ([M]⁺, 100), 251 ([M-CO]⁺, 92), 192 (22), 125 (25). The mass spectral data obtained was in agreement with published data [6]. Psoralen, yellowish powder, EIMS m/z (%), 186 ([M]⁺, 100), 158 ([M-CO]⁺, 65), 130 ([M-CO-CO]⁺, 19) and 102 ([M-CO-CO-CO]^+, 32). The mass spectral data was in agreement with published data of psoralen [6, 11]. Kokusaginine, white crystals, EIMS m/z (%), 259 ([M]⁺, 100), 244 ([M-Me]⁺, 43), 216 ([M-Me-CO]⁺, 15), 201 ([M-Me-CO-Me]⁺, 19), 186 ([201-Me]⁺, 25) and 173 ([186-CH]⁺, 16). The obtained mass spectral data is in agreement with published data [6]. Methoxsalen, yellowish oil, EIMS m/z (%), 216 ([M]⁺,100), 201 ([M-Me]⁺, 35), 188 ([M-CO]⁺,14), 173 ([188-CO]⁺, 54) and 145 ([173-CO]⁺, 21). The data obtained was consistent with the mass spectral data of methoxsalen from NIST webbook. Bergapten, white powder, EIMS m/z (%), 216 ([M]⁺, 100), 201 ([M-Me]⁺, 35), 188 ([M-CO]⁺, 15), 173 ([M-Me-CO]⁺, 64) and 145 ([M-Me-CO-CO)]⁺, 21). The data obtained was consistent with published data [12]. Arborinine, yellow crystal, EIMS m/z (%), 285 ([M]⁺, 66), 270 ([M-Me]⁺, 100), 242 ([M-Me-CO]⁺, 49). The mass spectra was in agreement with published data [11]. Moskachan B, brownish oil, EIMS m/z (%), 220 ([M]⁺, 21), 135 ([M-CH₂CH₂CH₂COCH₃]⁺, 100). The mass spectra was in agreement with published data [7]. Chalepin, white crystals, EIMS m/z (%), 314 ([M]⁺, 88), 299 ([M-Me]⁺, 100), 281 ([M-Me-H₂O]⁺, 34) and 255 ([M-(CH₃)₂COH]⁺. The mass spectral data in the present study corresponds to the data reported in published data [12]. The GCMS analysis of chalepin is shown in Fig. 4. ¹H NMR data (600 MHz, CDCl₃): δ 7.48 (1H, s, H-5), δ 7.20 (1H, s, H-4), δ 6.71 (1H, s, H-9), δ 6.17 (1H, dd, J = 18.00, 12.00 Hz, H-2’), δ 5.09 (2H, overlapping dd, H-3’), δ 4.72 (1H, dd, J = 12.00, 6.00 Hz, H-2), δ 3.21 (2H, overlapping dd, J = 18.00, 12.00, 6.00 Hz, H-3), δ 1.47 (6H, s, 4’,5’-CH₃), δ 1.37 (3H, s, 3”-CH₃), δ 1.23 (3H, s, 2”-CH₃). The ¹³C NMR data (600 MHz, CDCl3): δ 162.25 (C-9a), δ 160.21 (C-7), δ 154.64 (C-8a), δ 145.61 (C-2’), δ 138.09 (C-5), δ 130.87 (C-6), δ 124.58 (C-3a), δ 123.26 (C-4), δ 113.14 (C-4a), δ 112.09 (C-3’), δ 97.14 (C-9), δ 90.91 (C-2), δ 71.70 (C-1”), δ 40.30 (C-1’), δ 29.61 (C-3), δ 26.11 (C-3”,4’,5’), δ 24.21 (C-2”). The NMR spectra of isolated chalepin is shown in Fig. 5. Moskachan D, yellowish oil, EIMS m/z (%), 248 ([M]⁺, 30), 148 ([M-CH₂-CH₂-CH₂-CH₂-COCH₃]⁺, 15), 135 ([M- CH₂-CH₂-CH₂-CH₂-COCH₃-CH]⁺,100),91([M-CH₂-CH₂-CH₂-CH₂-COCH₃-CH-COO]⁺,3), 77([C₆H₅]⁺, 11). The obtained data is consistent with published data [7]. Chalepensin, yellowish solid, EIMS m/z (%), 254 ([M]⁺, 100), 239 ([M-CH₃]⁺, 95), 211 ([M-CH₃-CH-CH₃]⁺, 70), 199 ([M-CH₃-CH-CH3-C]⁺, 85). The mass spectra is in agreement with published data [6]. Rutamarin, colorless crystals, EIMS m/z (%), 356 ([M]⁺, 8), 341 ([M-CH₃]⁺, 4), 296 ([M-CH₃-COO]⁺, 19), 281 ([M-CH₃-COO-CH₃]⁺, 100), 253 ([M-CH₃-COO-CH₃-CO]⁺, 14). The mass spectral data is in agreement with published data [6]. Neophytadiene, light yellow oil, EIMS m/z (%), 278 ([M]+, 15), 137 (14), 123 (68), 109 (42), 95 (71), 82 (70) and 71 (100). The mass spectral from GCMS analysis is identified through comparison with NIST MS Library data (Table 3).

The isolated compounds were tested for their cytotoxicity effect against A549 human lung carcinoma cells and the normal human lung fibroblasts MRC5 cells. Results showed that the cytotoxicity effect against A549 cell was dose and time dependent. Chalepin exhibited the highest cytotoxicity against A549 cell line after 72 h incubation with a IC₅₀ value of 8.69 ± 2.43 μg/ml. It was also mildly toxic to the normal cell line with a IC₅₀ value of 23.4 ± 0.6 μg/ml. Arborinine, chalepensin and also moskachan D also exhibited promising cytotoxic property against A549 cell line with IC₅₀ values of 13.1 ± 0.06, 14.0 ± 0.15 and 18.5 ± 0.65 μg/ml respectively. However, arborinine and chalepensin showed moderate toxicity towards MRC5 normal human lung fibroblast cell line with IC₅₀ values of 20.8 ± 0.15 and 23.3 ± 0.55 μg/ml respectively. Moskachan D was non-toxic towards normal cell with an IC₅₀ value of 90.8 ± 0.8 μg/ml. Other isolated compounds showed no toxicity towards the normal cells with IC₅₀ values ranging more than 50.0 μg/ml. The results are represented in Table 4.

The nuclear morphological changes after treatment with chalepin at various concentrations were observed using Hoesct 33342 and propidium iodide (PI) dye, and was observed under a fluorescent microscope. The control showed only low blue color. After treatment of chalepin for 72 h at various concentrations, observation of cells fluorescing bright blue colored nucleus was observed. The higher the concentration of chalepin, the higher the number of cells that emits a bright blue signal. In 40x magnification, chromatin condensation and nuclear cleavage were observed (Fig. 7).

The phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane is a hallmark of apoptosis. Annexin V is a protein which has high affinity for PS. Thus the binding of Annexin V to PS provides a very sentitive method for detecting cellular apoptosis. A population of cells undergoing apoptosis may contain necrotic cells due to their damaged plasma membrane. To distinguish between Annexin V positive apoptotic and necrotic cells, the fluorescent dye propidium iodide was used. Membranes of damaged cells are permeable to propidium iodide. Thus, using Annexin V conjugated to FITC (a fluorescent dye) enables apoptotic cells to be identified and quantified on single cell basis by flow cytometry. In this study, it can be observed that as the treatment concentration of chalepin increased, the density plot showed increase in the cell population at the lower right quadrant (Q1-LR) which represents the early apoptotic cells and also subsequently towards the upper right quadrant (Q1-UR) which represents population of late apoptotic/secondary necrotic cells. Comparison between 48 h and 72 h showed that at longer incubation, the cell population in early, late apoptosis/secondary necrosis (upper right quadrant – Q1-UL) increased with dose. But the highest increase was observed in the late apoptosis quadrant represented by upper right quadrant in the density plot. The untreated, control cells which stayed at lower left quadrant (Q1-LL) showed low staining with both annexin V and PI showing viable cells. The total AnnexinV positive cells represents a total of early and late apoptotic cells in A549 cells, shows an increase in dose and time dependant manner. The Annexin V positive cells showed an increase from 7.27 ± 0.16 % at 9 μg/ml to 20.6 ± 0.53 % at 45 μg/ml of chalepin. The population of AnnexinV positive cells observed was significantly higher after 72 h incubation. These results are shown in Fig. 8.

DNA fragmentation is one of the main distinctive feature of apoptosis. DNA fragmentation is activated by nucleases which would degrade the nuclear DNA into fragments of about 200 base pairs in length. This feature is measured by using APO-BrDU TUNEL assay kit. In this study, chalepin was treated at various concentrations and incubated for 48 and 72 h to measure the ability to induce DNA fragmentation. It was found that chalepin induces DNA fragmentation in A549 cell in a dose and time dependent manner (Fig. 9). DNA fragmentation is represented in the density plot above with R3 representing percentage of cells that has nuclear DNA fragmentation for treatment at 48 h and 72 h. The cell population would move to the upper region in the density plot when DNA fragmentation is labelled. Upper region in the density plot represents cells with fragmented DNA. Based on the results obtained, after 48 h incubation, the DNA fragmentation increased from 1.0 ± 0.08 % for untreated control cells to 38.5 ± 0.70 % for cells treated with chalepin at 45 μg/ml. At 72 h, the DNA fragmentation increased from 1.50 ± 0.35 % for untreated control cells to 73.50 ± 0.20 % at 36 μg/ml however a drop was observed at 45 μg/ml to 66.50 ± 0.29 %. This result indicates that upon treatment of chalepin, DNA fragmentation is initiated in the cell by endonucleases to enable cells to undergo apoptosis. With increasing concentration of treated chalepin, the percentage of DNA fragmentation in the treated cells increases. This trend is also true for higher incubation time. The DNA fragmentation that occurs in the cells is dependant on the concentration of chalepin and the incubation hours.

Mitochondrial membrane potential (MMP) may be disrupted during early apoptosis. The loss of MMP in A549 cells which were treated with various concentrations of chalepin was observed using the JC-1 dye. During the early stage of apoptosis, mitochondrial depolarisation occurs and this enabled fluorescence of JC-1 to turn from red aggregates to green monomers. Figure 10a showed untreated control cell did not show any increase in green monomer formation. Upon treatment with chalepin at different concentrations, the percentage of green monomer increases in cytoplasm (Fig. 10b). This shows that, chalepin significantly induced reduction of mitochondrial membrane potential in A549 cells in a concentration dependant manner. Figure 10c shows that at 48 h incubation time, the untreated cells gives red fluorescence which indicates that there is high membrane potential in the mitochondrial membrane which enables the JC-1 dye to pass through the mitochondrial membrane and form aggregates which could give the red fluorescence. This shows that the cells are healthy. As the cells are treated with chalepin, there is increase in the presence of the green fluorescence as the concentration of chalepin treated is increased. When a cell undergoes apoptosis, the mitochondrial membrane potential would drop and this would inhibit the influx of JC-1 dye into mitochondria. It would remain in its initial monomeric form at cytoplasm which would give the green fluorescence that is observed in cells that were treated with high concentration of chalepin i.e., 36 μg/ml and 45 μg/ml. This observation is similar at 72 h incubation time with a difference of more fluorescence intensity at higher treatment time. We observed that the cells at 45 μg/ml treatment dosage at 72 h, almost all gives a green fluorescence. This indicates that almost all the cells that were observed is undergoing apoptosis.

The effect of chalepin in inititating the generation of intracellular ROS in A549 cells was detected using the DCFH-DA dye. Observation through fluorescent microscope was done on lung carcinoma cells after treatment of chalepin for 24 and 48 h showed an increase in intensity of green fluorescence of DCF in a dose and time dependant manner in (Fig. 11a). It was observed that, as the concentration of chalepin that was treated to the cells was increased, there was an increase in the intensity of green fluorescence by the DCFH-DA dye. This trend was also true for an increase in the incubation time. The DCFH-DA is a non-polar dye which would be converted into DCFH by cellular esterases. DCFH is non-fluorescent but is switched to fluorescent DCF when it is oxidized by ROS in the cell. Hence, the qualitative imagining showed that the increase in the intracellular ROS content is dependant upon the concentration of chalepin and also the incubation time of the treatment. Besides the qualitative imaging, the quantification of the intracellular content of ROS by measurement of the fluorescence of the DCF was also conducted. The fluorescence were then calculated in the ratio of fold increase of the fluorescence intensity in the treated cells as compared to the untreated negative control cells. The fold increase of intracellular ROS was calculated based on fluorescence value obtained from microplate reader. It was observed that in comparison to the control, the treated cells showed a gradual increase in the fold increase of intracellular ROS content starting at 2 h up till 6 h. This increase was time and dose dependant. However at 8 h, there was a drop in the fluorescent measurement of the treated cells (Fig. 11b). This observation could be due to the reason where cell death has commenced and thus there is a drop.

Caspase 9 is associated with the activation of the intrinsic mitochondrial mediated pathway. CaspILLUME fluorescein active caspase-9 staining kit was used to determine the caspase 9 activity in regards to the treatment of chalepin in A549 cells. Cells with activated caspase 9 would move to the right side (V1-R) of the density plot. The results shows that there is a remarkable increase in the caspase 9 activity of cells treated with chalepin as compared to the control (Fig. 12). It was observed that after 48 h of incubation period there is a five-fold increase in activation of caspase 9 from 4.5 ± 0.35 % in untreated control to 22.57 ± 0.21 % in cells treated with 45 μg/ml of chalepin (Fig. 12a). As for the 72 h incubation period, there was an increase from 3.97 ± 0.15 % for untreated control cells to 33.5 ± 0.35 % for treated cells with 45 μg/ml of chalepin and this is a 8.44 fold increase (Fig. 12b). When cells receive apoptotic stimuli, the mitochondria releases cytochrome c which then binds to Apaf-1, together with dATP. The resultant complex recruits caspase-9 leading to its activation. Activated caspase-9 cleaves downstream caspases such as caspase-3, -6 and -7 initiating the caspase cascade [13]. This results shows that chalepin treated cells undergo apoptosis through the mitochondrial pathway.

Caspase 3 is a downstream caspase signal also known as the effector caspase. Upon activation of caspase 9, a signal cascade results in an activation of caspase 3 which in turn results in the hydrolysis of more than 100 target protein that subsequently results in apoptosis. Defect in caspase 3 activity results in cancer. In this study, it was observed that A549 cells treated with chalepin induces caspase 3 activation. CaspILLUME fluorescein active caspase-3 staining kit was used and the cell population was analysed using a flow cytometer. In the event of caspase 3 activation, the cell population would move to the right hand side (V1-R) of the density plot (Fig 13). It is observed that after 48 h of incubation period there is an increase of 2.83 ± 0.06 % for untreated control to 26.40 ± 0.79 % for cells treated with 45 μg/ml of chalepin and this is a 9.32 fold increase (Fig. 13a). As for the 72 h incubation period, there was an increase from 5.63 ± 0.21 % for untreated control cells to 49.07 ± 0.63 % for treated cells with 45 μg/ml of chalepin and this is a 8.71 fold increase (Fig. 13b). Caspase-3 is required for some typical characteristics of apoptosis, and is crucial for apoptotic chromatin condensation and DNA fragmentation in all cell types. Caspase-3 is essential for certain processes associated with the dismantling of the cell and the formation of apoptotic bodies, but it may also function before or at the stage when commitment to loss of cell viability is made [14]. Activation of caspase 3 in chalepin treated A549 cells therefore is an evidence that apoptosis has commenced.

Changes in the expression of proteins involved in regulation of survival and cell death following chalepin treatment were examined by Western Blot analysis. Activation of caspase cascade would trigger proteolytic degradation of PARP and DNA fragmentation by endonucleases, leading to apoptosis. The expression of PARP was studied in A549 cells treated with chalepin at different incubation time. It was observed that the quantity of PARP expressed decreased with time. This shows that the level of PARP decreased with time as shown by Fig. 14a.

The mitochondrial mediated pathway is also known as BCL-2 regulated pathway. The balance of function between proapoptotic and antiapoptotic BCL-2 protein family determines the fate of cells towards apoptosis [15]. Studies have shown Bax, Bad, Bak promote the release of cytochrome c, while bcl-2 and Bcl-X_L delays this response and promote cell survival. Bcl-2 family members regulate the apoptotic response by controlling the mitochondrial membrane permeabilization (MMP). Depolarisation of MMP and the formation of MPT (mitochondrial permeability transition) pore is a result of the tranlocation of Bax from mitochondria to cytosol [16].

Chalepin was found to downregulate anti-apoptotic gene products such as Bcl-2, survivin, XIAP, Bcl-xl and cFLIP as shown by Fig. 14b This clearly shows that it downregulates proteins that play a vital role in the cell survival and thus, induces apoptosis. An upregulation in the expression of Bax was observed (Fig. 14c). There was an upregulation in the expression of cytochrome c, Fig. 14c, which shows that the cytochrome c moved out of mitochondria to cytosol leading to an upregulation of cytochrome c. All these factors promotes cell cytolysis or cytostasis.

The cytochrome c release and binding with dATP and Apaf-1 results in formation of apoptosome which recruits and activates caspase-9. Activated caspase 9, results in activation of effector caspases such as caspase-3/7. These effector caspases then initiates apoptosis events. Results indicated that chalepin induces cleavage of procaspases 9 and 3 to the active cleaved form. Procaspase 9 showed a downregulation which indicated cleavage of the protein to an active form. Procaspase 3, also showed downregulation which indicated an activation. This result is further stregthened by detection of the cleaved caspase 3 which showed an upregulation. All these results are illustrated in Fig. 14d. This clearly indicates that the intrinsic pathway which is mediated by the mitochondria is activated. These results are consistent with earlier caspase 3 and caspase 9 activity determination using flow cytometer.