Apoptotic Effects Of Cycloartane Triterpenoid Glycoside Biology Essay

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Plants provide us with many biologically active compounds that have potential therapeutic effect on a myriad of diseases. Leea indica (Burm. f.) Merrill (Leeaceace) is a traditional Chinese medicinal plant widely used for the treatment of various illnesses including inflammations, diabetes, skin diseases and cancer. Previous in vitro cytotoxicity results have verified its anti-cancer activity against Ca Ski cervical cancer cell lines.

To isolate the active constituents from the ethyl acetate fraction obtained from the leaf extract of Leea indica via cytotoxicity guided approach. The effect of the compounds on the regulation of cell proliferation, cell cycle and apoptosis in Ca Ski cells was evaluated.

The compounds were characterized by 1D-NMR (1H NMR, 13C NMR and DEPT-135), 2D-NMR (COSY, HMQC and HMBC) and LC-MS methods. The cytotoxic and anti-proliferative effects were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion (TBE) assays. The cell cycle distribution and apoptosis induction were analyzed by flow cytometry. Quantitative real-time PCR (Q-PCR) was used to measure the expression of proliferative cell nuclear antigen (PCNA).

Two cycloartane triterpenoid glycosides, mollic acid arabinoside (MAA) and mollic acid xyloside (MAX) were isolated for the first time from Leea indica. These compounds were showed to have anti-cancer activity for the first time. They demonstrated strong dose-dependent cytotoxic effects on Ca Ski cells with high selectivity over the normal MRC-5 cells. The more potent compound, MAA caused a decrease in the viability and proliferation of Ca Ski cells as assessed by MTT and TBE assays. The cytostatic (anti-proliferative) and cytocidal (apoptotic) effects of MAA were reflected by S-phase arrest, decreased of PCNA expression and induction of sub G1cells.

The MAA isolated from Leea indica showed potential anti-cancer activity, which is cytotoxic, anti-proliferative, and apoptotic on Ca Ski cervical cancer cells. Thus, this study provides the scientific evidence for the ethno-medicinal use of Leea indica as anticancer plant and may pave the way for future development as a cancer chemotherapeutic agent.

Leea indica (Burm. f.) Merrill is a leafy plant belonging to the Leeaceae family, which is grown in South East Asia, China and India. The leaves, roots and whole plant of L. indica are widely used for many medicinal values. The leaves are used to treat diabetes and the ointment prepared from roasted leaves is used to relief symptoms of vertigo (Chatterjee and Prakashi, 1994; Prajapati et al., 2003; Pullaiah and Naidu, 2003). The leaves paste is topically used to relieve itching, soreness, eczema, leprosy, sprain and bone fracture (Rahman et al., 2007; Yusuf et al., 2007). On the other hand, the root is claimed to have sudorific, anti-diarrhoeal, anti-dysenteric, anti-spasmodic effects and are often used to treat cardiac and skin diseases (Chatterjee and Prakashi, 1994). All part of the plant is used to treat headache, body pains and skin complaints (Burkill, 1966; Lattif et al., 1984).

In view of the traditional use of L. indica for various medicinal purposes, some phytochemical and biological studies have been conducted. The earliest phytochemical study on the leaves of L. indica reported the isolation of α-tocopherol, β-amyrin and β-sitosteryl-β-D-glucopyranoside (Saha et al., 2005). This was followed by the identification of eleven hydrocarbons, phthalic acid, palmitic acid, 1-eicosanol, solanesol, farnesol, phthalic acid esters, gallic acid, lupeol and ursolic acid (Srinivasan et al., 2008). Meanwhile, the essential oil extracted from the flower of L. indica was found to contain di-isobutylphthalate, di-n-butylphthalate, n-butylisobutylphthalate and butylisohexylphthalate and monobutyl carbonotrithioate ((Srinivasan et al., 2009). Anti-microbial activity has been reported in the leaf, root and essential oil from the flower of L. indica (Srinivasan et al., 2009; Srinivasan et al., 2010). Furthermore, the leaf was found to have hypo-glycemic and anti-strychnine activities (Dhar et al., 1968) while the root was found showing phosphodiesterase inhibitory activity (Temkitthawon et al., 2008). L. indica was also possessed remarkable anti-oxidant and anti-inflammatory activities, as shown by strong DPPH free radical scavenging and strong nitric oxide inhibitory effects (Saha et al., 2004). Recently, the high anti-oxidant activity of L. indica was also reported by other scholars (Li and Liu, 2009; Krishnaiah et al., 2010).

In Leeaceae family, L. guineense and L. macrophylla were ethno-medicinally used to treat cancer disease (Graham et al., 2000; Choudhary et al., 2008). The leaf decoction of L. indica is consumed by woman during pregnancy and delivery, or for birth control (Bourdy and Walter, 1992). The leaves are also used to treat obstetric diseases and body pain (Srithi et al., 2009). It is also an ingredient in the preparation to treat leucorrhea, intestinal cancer and uterus cancer (Saralamp, 1997). Moreover, the dried leaves are used as a tea beverage to prevent cancer and relieve cancer-related symptoms. In our previous cytotoxicity screening, the crude ethanol extract and fractions (ethyl acetate, hexane and water) were found to inhibit the growth of Ca Ski cervical cancer cell line (Wong and Kadir, 2011). This further provides the evidence for the use of L. indica as folkloric treatment of cancer.

To the best of our knowledge, the anti-cancer potential of L. indica has not yet been studied extensively. In the present study, we report the further progress in an ongoing research on L. indica whereby the active fraction (ethyl acetate) was subjected to bioassay-guided approach in order to isolate the active chemical constituents and further evaluate its cytotoxic, anti-proliferative and apoptotic effect on Ca Ski cells

From the previous report (Wong and Kadir, 2011), the fresh leaves of L. indica were collected, authenticated, extracted and fractionated. A voucher specimen (47365) was deposited at the herbarium of the Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.

The active ethyl acetate fraction (50g) was dissolved in MeOH and loaded onto Diaion HP-20 SS (Supelco, Bellefonte) column, eluted using a gradient solvent system of 40% MeOH and 60% H2O with 10% MeOH increment. Thin layer chromatography (TLC) analysis were performed on pre-coated silica gel 60 F254 plates (0.2mm thick, Merck) and spots were detected by UV illumination after spraying with 10% H2SO4 followed by heating. Based on the TLC profiles, a total of nine combined fractions (designated F1-F9) were pooled together. MTT assay was performed on each fraction. The active F8 was further subjected to silica gel (200-400 mesh, Merck) column chromatography. The mobile phase was consisted of CHCl3: MeOH: H2O (C: M: H, v/v). The initial solvent composition was 100% C, and then it was changed to C: M (9.5: 0.5), followed by C: M: H (9: 1: 0.1), C: M: H (8.5: 1.5: 0.1), C: M: H (8: 2: 0.2), C: M: H (7: 3: 0.5), C: M: H (6.5: 3.5: 0.5), C: M: H (6: 4: 1) and finally to 100% M. A total of six fractions (F81-F86) were obtained. The active F83 was further fractionated again on silica gel 60 column using C: M: H system. The initial solvent was 100% C, and then it was changed to C: M: H (9: 1: 0.1), followed by C: M: H (8.5: 1.5: 0.1), C: M: H (8: 2: 0.2), C: M: H (7: 3: 0.5), and finally to 100% M. Another six fractions (F831-F836) were obtained. The active F835 was further purified by prep-TLC (silica gel 60 F254 glass plates, size 20cm x 20cm, Merck) using C: M: H (7: 3: 0.5) as solvent system and yielded compounds 1 (55.9mg) and 2 (26.6mg).

Structure elucidation of the compounds was determined by spectral techniques. The compounds were dissolved in pyridine-d5 solution. The 1H, 13C and DEPT-135 NMR data were recorded on a Bruker DRX 300 spectrometer, while the 2D-NMR spectra (COSY, HMQC and HMBC) were obtained by JEOL ECA 400. LC-MS analysis was performed using LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific) fitted with an electrospray interface.

The human cervical epidermoid carcinoma cell line (Ca Ski, ATCC number CRL-1550) and human fibroblast cell line (MRC-5, ATCC number CCL-171) were purchased from the American Type Culture Collection (ATCC, USA). Ca Ski cells were maintained in RPMI 1640 Medium (Sigma) and MRC-5 cells in EMEM (Eagle Minimum Essential Medium) (Sigma). The media were supplemented with 10% (v/v) heat-inactivated fetal bovine serum (PAA Lab, Austria), 100µg/ml streptomycin and 100unit/ml penicillin (PAA Lab, Austria) and 50µg/ml amphotericin B (PAA Lab, Austria). The media were filter sterilized using a 0.22µm filter membrane (Minisart, Sartorius stedim). The cells were cultured in 5% CO2 incubator at 37°C in a humidified atmosphere.

MTT assay was modified from Mossmann and used to evaluate the cytotoxic effects of each fraction and compounds. MTT assay is widely used to assess the viability and/or the metabolic state of the cells based on mitochondrial respiratory activity (Mossmann, 1983). A total of 5x103 Ca Ski or MRC-5 cells were seeded into 96-well plates and allowed to adhere for 24h. After 24h, the cells were treated with the fractions or compounds in the final concentrations ranging from 3.125 to 100μg/ml. Control cells were treated with vehicle DMSO to get the final concentration of 0.5% v/v. The cells were then incubated for 72h. After 72h exposure period, MTT (5mg/ml) was added and further incubated for 4h at 37°C. The medium was then aspirated and the formazan crystals generated from the viable cells were dissolved in 150μl DMSO. The absorbance was measured at 570nm against the reference wavelength of 650 nm. The percentage of viability was calculated based on the formula: Viability (%) = (absorbance of treated cells/ absorbance of control cells) x 100%. The IC50 (concentration that reduces cell viability to 50%) was derived from the dose-response curves. The selectivity index (SI) was calculated by divided the IC50 value of the normal MRC-5 cell line with the IC50 value of the cervical cancer Ca Ski cell line. Sample which shows SI > 3 will be considered to have high selectivity (Bézivin et al., 2003; Aljewari et al., 2010).

In order to determine whether the cytotoxicity is cytostatic or cytocidal, a recovery assay was conducted whereby after the 72h incubation (exposure period), the medium containing the compound was removed, washed with medium and replaced with medium alone for a recovery period of 72h followed by addition of MTT and measurement as described earlier. A sample is showing cytostatic effect when the IC50 in the recovery assay was higher than that of the exposure assay. Whereas for cytocidal effect, the IC50 obtained in the recovery assay is about similar to that shown by the exposure assay (Lee and Houghton, 2005; Ashidi et al., 2010).

Ca Ski cells were cultured at a density of 1-106cells/ml in 60mm culture dishes. After 24h of attachment, the cells were treated with 0.5% DMSO (control) or 60μM of MAA for different time periods. After 6-72h, the cells were harvested, washed with medium and the cell pellets were resuspended in medium. After incubation in 0.4% trypan blue for 5min, viable cells were counted using a hemocytometer. At the indicated time point straight before the cells were harvested, the cells were visualized under an inverted phase-contrast microscope (Motic) to observe the cell morphological changes upon treatment and photographs were taken.

The cell cycle distribution and apoptosis were assessed using propidium iodide (PI) staining (Nicoletti et al., 1991). Ca Ski cells were seeded in 60mm culture dishes (1x106cells) and left 24h for attachment. The cells were then treated with 0.5% DMSO (control), 60, 80 and 100µM of MAA for 24h. The cells were also treated with 60µM MAA for 6-72h. After the designated treatment period, both adherent and floating cells were harvested and washed twice with PBS. Cell pellets were resuspended in 100ul of PBS and fixed with absolute ethanol and stored at -20°C for 24h. Fixed cells were washed twice with PBS and the cell pellets were incubated in a buffer containing 50µg/ml PI, 0.1% sodium citrate, 0.1% Triton-X-100 and 100µg/ml RNase A for 45min in the dark at room temperature. The percentage of cells in the Sub-G1, G1, S, and G2/M-phases of the cell cycle was then analyzed using a FACSCalibur flow cytometer (Beckton Dickinson). Data were acquired and analyzed using CellQuest software (Becton Dickinson).

A total of 1x106 Ca Ski cells were seeded into 60mm culture dishes and incubated for 24h for attachment. Cells were then treated with DMSO (0.5%) or 20-100µM of MAA for 24h. For time-course study, cells were treated with 60µM MAA for 0, 3, 6, 12 and 24h. After specific treatment period, cells were harvested and total RNA was isolated using the RNAqueous-4PCR kit (Applied Biosystem) according to the manufacturer's directions. The RNA concentration was determined using spectrophotometry. The gene expression of PCNA was assessed by one-step SYBR Green relative real-time PCR (RotorGene System, Qiagen) and normalized to GAPDH reference control amplifications. The primer sequences for PCNA and GAPDH were forward 5'-GCCTGCTGGGATATTAGCTC-3'; reverse 5'-CATACTGGTGAGGTTCACGC-3' and forward 5'-CCAGGGCTGCTTTTAACTCTG-3'; reverse 5'-CGTTCTCAGCCTTGACGGTG-3' respectively. The PCR amplification reactions was carried out in a total volume of 25μl for 30 cycles of 45 seconds at 95°C, 45 seconds at 56°C and 120 seconds at 72°C. The mean fluorescence threshold value (CT) of each sample was obtained according to the manufacturer's guidelines, and used to determine ΔCT values whereby ΔCT = CT target gene (PCNA) - CT reference gene (GAPDH). The relative fold change in PCNA expression in the treated sample over the untreated control was calculated with the comparative ∆∆CT method where ∆∆CT = ∆CT sample - ∆CT control and calculated using formula 2-ΔΔCT (Livak and Schmittgen, 2001).

All the results were presented as mean ± standard error (S.E.) of three experiments. Significant difference was analyzed by Student t-test. A p value < 0.05 was regarded as a significant difference from the corresponding control group.

Based on our previous study, the ethyl acetate fraction of L. indica demonstrated the strongest cytotoxic effect on Ca Ski cells (Wong and Kadir, 2011). Hence, it was subjected to MTT assay-guided isolation. The results were summarized in Fig. 1. MTT test on the first 9 fractions showed that Ca Ski cells were most susceptible to F8. Further separation of F8 yielded another 6 fractions (F81 to F86). Among the fractions, F83 was found to be the most effective. Subsequent fractionation of F83 yielded another 6 fractions (F831 to F836). The active F835 was subjected to prep-TLC and this led to the isolation of two compounds. They were identified by spectroscopic methods (1H NMR, 13C NMR, DEPT-135) and by comparison with the published data in literature (Pegel and Rogers, 1985; Rogers and Thevan, 1986; Rogers, 1989), to be (1) mollic acid arabinoside (MAA) and (2) mollic acid xyloside (MAX). The chemical structures were shown in Fig. 2.

Compound 1 was identified as 1α,3β-dihydroxy-cycloart-24-ene-28-oic acid 3-O-[α-L-arabinopyranoside], C35H56O8. Positive ESI-MS m/z: 627.3851 [M+Na]+. 1H NMR (125MHz, C5D5N): δ 0.47(1H, d, J=4.0Hz, H-19A), 0.75 (1H, d, J=4.0Hz, H-19B), 0.85-1.66 (6 x CH3), 3.39-4.42 (arabinose protons; H-1of aglycone), 5.01 (1H, d, J=6.6Hz, H-1' of arabinose), 5.21-5.5(2H, m, H-3α, H-24). Compound 2 was identified as 1α,3β-dihydroxy-cycloart-24-ene-28-oic acid 3-O-[β-D-xylopyranoside], C35H56O8. Positive ESI-MS m/z: 627.3851 [M+Na]+. 1H NMR (125MHz, C5D5N): δ 0.44(1H, d, J=4.0Hz, H-19A), 0.72 (1H, d, J=4.0Hz, H-19B), 0.91-1.68 (6 x CH3), 3.39-4.42 (xylose protons; H-1of aglycone), 5.08 (1H, d, J=6.6Hz, H-1' of xylose), 5.20-5.48(2H, m, H-3α, H-24). The 13C NMR data of compounds 1 and 2 were shown in Table 1. The position of the sugar moieties were confirmed by HMBC correlation of the anomeric protons to C3 of the aglycones and vice versa. Their LC-MS spectra showed the same molecular ion peak at m/z 627.3851, which corresponds to a molecular formula of C35H56O8.

Fig. 1. Flow-chart of bioassay-guided isolation of mollic acid arabinoside (MAA) and mollic acid xyloside (MAX) from the ethyl acetate fraction of L. indica. Each of the fractions was evaluated its cytotoxic effect on Ca Ski cells using MTT assay. The IC50 values were means ± S.E. calculated from three experiments performed in triplicate.

Table 1. 13C-NMR spectra data (δ C in p.p.m,; 75 MHz ) of compounds 1 and 2(C5D5N).

Fig. 2. The chemical structures of the compounds isolated from L. indica via bioassay-guided approach.

MAA and MAX were evaluated for their cytotoxic effect on Ca Ski cervical cancer cells and MRC 5 normal cells using MTT assay. A 72h exposure to Ca Ski cells with MAA or MAX led to a significant dose-dependent reduction in cell viability. According to Fig. 3, the decrease in cell viability ranged from 20-95% and 6-90% when the cells were treated with 3.125-100µg/ml of MAA and MAX respectively. The IC50 values for MAA, MAX and camptothecin (CPT) were summarized in Table 2. MAA and MAX were much less cytotoxic to the normal cells, as revealed by the relatively higher IC50 values on MRC 5, whereas CPT displayed comparable IC50 value on Ca Ski and MRC 5 cells. The small different in IC50 value led to our deduction that CPT cannot differentiate between normal and cancer cells and killed both cells at almost the same efficiency. This lack of tumor cell-specificity was also reported by other scholar (Iwasaki, 2006).

The primary goal of cancer chemotherapy is to target specific to cancer cells and innocuous to normal cells. However, many anti-cancer drugs fail to meet this criterion, as they cannot discriminate between cancer and normal cells, which make them cytotoxic not only to cancer cells, but also to normal cells. Therefore, development of novel cancer chemotherapeutic agent with a higher potency and specificity against cancer cells are urgently needed. It is interesting to note that MAA and MAX exhibited approximately 20-fold and 17-fold higher IC50 values against MRC-5 when compared to CPT (Table 2). Moreover, we also determined the degree of their selectivity against Ca Ski cells over MRC-5 cells, by comparing the SI values of the compounds. As shown in Table 2, MAA exhibited the greatest selectivity (highest index), indicating that the cytotoxicity to Ca Ski cells was about 8 times higher in selectivity over the normal cells. Good SI (> 3) was also demonstrated by MAX, where Ca Ski cells was about 4 times more sensitive than normal cells to the cytotoxic effect of MAX.

Fig. 3. Cytotoxic effect of mollic acid arabinoside (MAA) and mollic acid xyloside (MAX) on Ca Ski cells. Cells were treated with 0.5% DMSO (control) or increasing doses (3.125-100µg/ml) of MAA or MAX for 72h. The cytotoxicity was measured by MTT assay as described in method. Camptothecin (CPT) was used as positive control. The data were mean values ± S.E. of three different experiments. The asterisks represented significantly different value from control (* p < 0.05).

Table 2. IC50 [in µg/ml (µM)] and SI values of MAA, MAX and camptothecin (CPT) determined by MTT assay as described in methods. Data were means ± S.E. calculated from three experiments.

Since MAA exhibited higher SI and lower IC50 compared with MAX, it was selected for further investigations. In order to explore its anti-cancer activity, the cytotoxicity, anti-proliferation and apoptosis were analyzed. Previously, we have shown that treatment of MAA resulted in a conspicuous dose-dependent reduction of formazan formation in Ca Ski cells. This indicated that the cytotoxic action was mediated via disruption of mitochondrial dehydrogenase system inside the cells. The IC50 value obtained in the MTT recovery assay is about the same to that shown by the exposure assay (data not shown). Hence, it can be assumed that cytotoxic effect appeared to be mainly cytocidal.

The time-dependent effect of MAA on cell growth was assessed by TBE assay. As shown in Fig. 4, the control cells proliferated much faster compared to the MAA-treated cells, as demonstrated by the rapid exponential growth of the cells in the presence of 0.5% DMSO. In contrast, when exposed to MAA, the cell growth was hindered. Treatment for 6h modestly inhibited the cell growth. Prominent cell growth retardation was observed at 12h and 24h. After 24h, the cell number started to decrease from the initial cell seeding density, indicating a more pronounced disruption of cell-membrane integrity. Several cellular morphological changes were observed during MAA treatment (Fig. 5). The cells remained elongated in shape and attached at 6 and 12h, while at 24-72h, the cells shrunk to smaller rounded shape and started to detach. This time-course TBE analysis and cell morphology observations suggested that MAA exerted both cytostatic and cytocidal effects on Ca Ski cells. Cytostatic effect was evident during early hours (6-24h) of treatment with MAA. After 24h onwards, the cells died as a result of cytocidal effect. This was in agreement with the MTT recovery assay which showed cytocidal effect at 72h.

Fig. 4. Anti-proliferative effect of MAA on Ca Ski cells. Cells were seeded into culture dishes and exposed to 0.5% DMSO or 60µM MAA for 6-72h. At each time point, the viable cell numbers were counted using a hemocytometer as described in TBE assay in the methods. Each point represents means ± S E from three experiments.

Fig. 5. Effect of MAA on the morphological changes of Ca Ski cells. Cells were seeded into culture dishes and after 24h attachment, the cells were incubated with 60µM MAA for 6-72h. The control represents cells without treatment at 0h. At each time point, the cells were visualized under an inverted phase-contrast microscope and photographed. Magnification: 100x.

Next, we investigated the effect of MAA on cell cycle distribution by flow cytometry to check whether the anti-proliferative effect was associated with any cell cycle phase-specific arrest. As depicted in Fig. 6, incubation with MAA at increasing exposure periods induced a sustained accumulation of cells in the S-phase, which was significantly higher than the control. This accumulation of cells in S-phase reached its maximum (2.5-fold from the control) at 12 and 24h, in which MAA was cytostatic and did not cause reduction of cell viability (Fig. 4). Meanwhile, the S-phase population was about 2-fold significantly higher compared to control when treated with 60-100µM MAA for 24h. These results indicated that the observed cytostatic (anti-proliferative) effect may be due to the S-phase arrest which caused the perturbation of cell cycle progression.

Furthermore, DNA cell cycle analysis was also performed to check whether the cell growth-inhibitory effect of MAA could be related to the induction of apoptosis. Our results indicated that MAA caused a significant increase of hypo-diploid cells in a time and dose-dependent manner with a concomitant decrease of the cells in the G1 phase (Fig. 6). These hypo-diploid cells, which revealed by the appearance of sub G1 peak, are an indicator of apoptotic cells (Darzynkiewicz et al., 2001). Notably, the presence of apoptotic cells starting from 12h and increased to 20-fold at 72h compared to the control. Moreover, a nearly 30-fold increase was achieved during 24h incubation with 100µM MAA. Taken together, the induction of apoptosis may be responsible for the observed cytocidal effect showed by MAA.

Fig. 6. Effect of MAA on cell cycle phase distribution and apoptosis in Ca Ski cells. (A) Cells were treated with 60µM MAA for 6-72h. Control corresponds to untreated cells for 6h and similar results were obtained at other incubation times. (B) Cells were treated with 60, 80 and 100µM or without (control) MAA for 24h. After treatment, cells were harvested, fixed, stained with PI and analyzed by flow cytometry as described in methods. Cell cycle distribution was shown in histograms from one representative experiment. (C) Percentage of cells in sub G1, G1, S, and G2/M phases of the cell cycle are showed in bar charts. Results are mean values ± S E of three experiments. Statistical significance in comparison with the corresponding control is indicated by * p < 0.05.

Previous reports have shown that PCNA is greatly expressed in most of the proliferating cancer cells including cervical cancer (Chan et al., 1983; Benbrook et al., 1995). PCNA is a cell proliferation biomarker which plays a pivotal role in the decision of the life or death of the mammalian cells (Hall et al., 1990; Kelma, 1997; Paunesku et al., 2001). Hence, the effect of MAA on the expression levels of PCNA was investigated. The cells were treated with 60µM MAA at different exposure time periods and the relative expression of PCNA was measured. Results showed that MAA significantly suppressed the expression of PCNA in a time-dependent manner (Fig. 7). Time course Q-PCR analysis showed PCNA suppression reached its peak at 24h treatment of MAA. Cells were also treated with increasing doses of MAA (20-100µM) for 24h and results showed significant suppression of PCNA in a dose-dependent manner. These data suggested that the S-phase arrest induced by MAA could be attributed to the down regulation of PCNA expression.

Fig. 7. Effect of MAA on the PCNA expression in Ca Ski cells. (A) Cells were treated with 60µM MAA for 0 (control), 3,6,12 and 24h. (B) Cells were incubated in the absence (control, 0.5% DMSO) or presence of 20-100µM MAA for 24h. After indicated time, cells were collected, total RNA was extracted and real-time PCR was performed as described in methods. The data were from three independent experiments, which were expressed as mean ± S.E. fold decrease in PCNA expression compared to control, normalized with GAPDH. Statistical significance in comparison with the corresponding control is indicated by * p < 0.05.

To the best of our knowledge, mollic acid glycosides have only been isolated from Combretum species (Pegel and Rogers, 1976; Pegel and Rogers, 1985; Rogers and Thevan, 1986; Rogers, 1989; Rogers and Coombes, 2001; Ahmed et al., 2004; Facundo et al., 2008). This is the first time that mollic acid glycosides were isolated from L. indica. It is noteworthy that diverse anticancer studies such as anti-proliferative, cytotoxic, anti-tumor, DNA-damaging and anti-mitotic activities have been reported in plants belonging to the family Combretaceae (Pettit et al., 1989; Dorr et al., 1996; McGaw et al., 2001; Fyhrquist et al., 2006). In one of the studies, the authors suggested the possibility that the isolated mollic acid glycoside was responsible for the observed strong cytotoxic effect of Crombetum molle on HeLa, T 24 and MCF 7 cancer cells (Fyhrquist et al., 2006). However, no further study was conducted to verify the compound(s) responsible for the cytotoxic action. Furthermore, the in vitro cytotoxic effects of mollic acid glycosides have not been studied. Here, we are the first to show the cytotoxic effect of mollic acid glycosides on cancer cells. Previously, these types of compounds are also known to exert anti-molluscicide, hypo-glycaemic, analgesic, anti-inflammatory, anti-hypertensive and cardiovascular effects (Pegel and Rogers, 1985; Ojewole, 2008a, b; Ojewole and Adewole, 2009).

MAA and MAX are belonging to the group of cycloartane triterpenoid glycoside. Recently, cycloartane-type triterpenes have received considerable attention for their cytotoxic potential (Wang and Ma, 2009; Nuanyai et al., 2009; Nian et al.,, 2010; Yokosuka et al., 2010; Fang et al., 2011). Therefore, our findings here warrant the need for further investigation on the anti-cancer potential of MAA, especially for cervical cancer. Elaborate studies to identify the mechanisms of action are in progress.

Mollic acid glycoside (MAA), a cycloartane triterpenoid glycoside was isolated for the first time from L. indica via bioassay-guided approach. It was found to possess anti-cancer effect for the first time, which demonstrated strong and selective cytotoxic effect on Ca Ski cervical cancer cells while posing lower toxicity to MRC-5 normal cells. The cytostatic (anti-proliferative) and cytocidal (apoptotic) effects of MAA with the simultaneous arrest of the S-phase and suppression of PCNA expression may explain that MAA blocks the cell cycle progression to prevent further cell proliferation accompanied by trigger of apoptotic response. These results showed that it is a strong candidates for further development as potential cancer chemotherapeutic drugs.

The authors would like to thank Miss Tan Hooi Poay from Forest Research Institute of Malaysia (FRIM) for her technical assistance in instrument operation and NMR spectral analyses. The authors would also like to express their gratitude to Mr. Yap Fon Kwei for supplying the plant material. This research was financially supported by a research fund from the University of Malaya (PS282/2009C).