Alternatives Therapies For Cancer As Conventional Therapies Decline Biology Essay

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Abstract

Conventional therapies for cancer are on the decline as a result of their escalating limitations and side effects. Conversely, natural products are of increasing importance as prospective anticancer agents. Phyllanthus spp., widely distributed throughout tropical and subtropical regions, had been studied extensively for its various medicinal usages. However, their anticancer potential has not been fully elucidated. Yet, a drug that destroys the healthy cells although it acts effectively on the cancerous cells has little benefits. In this study, both aqueous and methanolic extracts of four Phyllanthus spp., namely P. niruri, P. urinaria, P. watsonii, and P. amarus, are tested for their anticancer properties. Two commercialized anti-cancer drugs, namely Doxorubicin and Cisplatin were used as positive controls in this study to compare the efficacy of the extracts. Phyllanthus plants have the potential to inhibit the growth of MCF-7 and A549 cells with IC50 values ranging from 50g/ml to 180g/ml and 65g/ml to 470g/ml for both methanolic and aqueous extracts respectively. On top of that, they have lesser toxicity on normal cells with cell viability percentage remaining above 50% when treated up to 1000g/ml for both extracts. In contrast, Doxorubicin and Cisplatin have potent toxicity on both human cancer and normal cell lines at concentrations < 10g/ml. Results obtained through cell cycle analysis indicate that Phyllanthus plants caused cytotoxicity without a cell cycle arrest effect on the cancerous cells. Instead, Phyllanthus has shown to induce apoptosis in cancerous cells with several fold increase of caspases-3 and -7, presence of DNA-fragmentation and TUNEL-positive cells. Compiling these data, Phyllanthus might be a potential and novel chemotherapeutic agent, due to its cytotoxic potency as well as induction of apoptosis of abnormal cancer cells.

Introduction

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Worldwide, lung cancer remains as the leading cause of mortality due to cancer in both men and women with breast cancer a close second. Incidence of lung cancer mortality in women is still on a rise although death due to lung cancer has been declining in men (Didkowska et al., 2005). The incidence of cancer is expected to rise with an increase in aging population (Jemal et al., 2008). In Malaysia, the proportion of cancer patients aged more than 60 years was 4.6% in 1957, increased to 5.7% in 1990 and is projected to be 9.8% in 2020 (Lim, 2002). These might be due to people who have adopted unhealthy diets and lifestyle habits of developed and industrialized countries (Parkin et al., 2002).

Cancers when left untreated ultimately result in serious illness and most often, death. Unfortunately, there is still no absolute cure for cancer diseases yet (Jemal et al., 2008). However, most of the cancers can be controlled by adopting appropriate conventional treatments such as surgery, radiation therapy and chemotherapy. But, this treatment may cause a range of side effects such as pain, nausea, vomiting, immune system depression, alopecia, ulcers, and others to the patient with varying degrees of severity (Vasilis et al., 2007). For these reasons, the use of conventional therapies may decline with their escalating limitations and side effects. Hence this has resulted in scientists worldwide searching for alternative cures in which a back-to-nature approach might yield better possibilities to look for other alternatives. Natural products are carving a path as prospective anti-cancer agents (Pederson et al., 1997).

During the mid 20th century, the development of medical treatments for human disease was intimately connected with a variety of products derived largely from the plant kingdom. Despite recent developments in combinatorial chemistry that can rapidly generate thousands of new chemicals, the pharmaceutical industry still relies heavily on a staggering array of undiscovered possibilities from the natural environment (Cooper, 2004). The discovery of several well-known anti-cancer agents from plant sources such as vincristine and vinblastine in 1950s initiated the United States National Cancer Institute (NCI) to begin a plant collection program. These led to the discovery of numerous novel compounds with a range of cytotoxic effects. Yet, there is not a plant-derived anticancer agent that has reached the stage of general use (Cragg and Newman, 2006).

Plant-derived drugs which have an important role in herbal medicine, is a traditional form of remedy to cure various diseases especially in developing countries (Rauh et al., 2007; Ramzi et al., 2008). This sojourn into the world of drug discovery is an area pertinent to complementary and alternative medicine (CAM). This appears almost inevitable with the practice of western medicine either shifting, or bridging the divide between western and eastern medicine through the intervention of CAM. Traditional Chinese Medicine (TCM) approach for cancer treatment by using natural herbal sources will kill only the cancerous cells without harming the healthy cells as well as to strengthen the body's immune system to fight off the cancerous cells (Lin, 1996). However, ethnopharmacological knowledge is essential to lead the search for plants with potential cytotoxic activity (Galvez et al, 2003).

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The genus Phyllanthus, is one of the most widely distributed plants throughout the rainforests of The Amazon as well as other tropical and subtropical regions. Numerous researches on Phyllanthus spp. began in the late 1980's (Paranjape, 2001) when clinical efficacy of P. niruri against viral Hepatitis B was observed. P. niruri, locally known as dukung anak, was thought to originate from India. It can be found in almost every tropical country due to its wide medicinal usages without showing toxicity (Paranjape, 2001; Barros et al, 2003). Some of the studied medical benefits include hepatoprotective agent, to eliminate gallstone and kidney stones, to treat intestinal infections, diabetes, dermatosis, diarrhea, diuretic, itch, as well as against viral Hepatitis B (Paranjape, 2001; Bagalkotkar et al., 2006). In addition, P. amarus had been employed for treatment of nervous debility, epilepsy, medhya (intellect promoting), and anti-amnesic (Joshi and Parle, 2007).

Although Phyllanthus spp. has been investigated for its various medicinal properties, little has been done to study their anticancer activities. In this study, the growth inhibitory potential of the Phyllanthus plants on various human normal (184B5: breast epithelium and NL20: lung epithelium) and cancer (MCF-7: breast carcinoma and A549: lung carcinoma) cell lines were assessed and subsequently the possible mode of cell death on treated cancer cells was investigated.

Materials and Methods

High performance liquid chromatography (HPLC) coupled with Electron Spray Ionization (ESI) and Mass Spectrometry (LC-MS- MS) analysis

Supernatant of the water extract sample was dried using a vacuum concentrator (concentrator 5301 eppendorf, Germany). For LC-MS-MS analysis, the lyophilised sample was redissolved into 20mg/ml with 30% methanol. Meanwhile, the total supernatant of methanolic extract samples were evaporated using rotary evaporator (rotavapor RII, BUCHI, Switzerland) and re-dissolved with 20% methanol. It were then subjected to solid phase extraction (SPE) column (LiChrolut RP-18 1000mg/6ml, Merck Germany) with mobile phase of 60% and 70% methanol. All elutes collected were first concentrated to 0.5ml, followed by 8 times dilution using 40% methanol before LC-MS-MS analysis was perfomed.

HPLC system used consist a HPLC binary pump, diode array detector (DAD), and an auto-sampler injector compartment (1200 series, Agilent Technologies, Germany). For separation, C-18, 150mm x 4.6mm i.d, 5µm particle size Thermo Hypersil GOLD column (Thermo Scientific, UK) were chosen for reverse phase while mobile phase of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) with a gradient setting of solvent B: 5% (5min), 5-90% (60 min), 5% (4min) at flow rate of 1ml/min was used. Detection wavelengths were both set at 280nm and 360nm with constant injection volume at 20µl. 3200 QTrap LC/MS/MS system (Appiled Bioscience - MDS Sciex) was used for mass spectrometry analysis, with the iron source and voltage maintained at 500 ËšC and -4.5 kV for negative ionization, respectively. Nitrogen generator was set at 60 psi curtain gas flow, 60 psi exhaust gas flow, and 90 psi source gas flow. Scanning modes chosen were Enhance Mass Spectrometer (EMS) and Enhance Ion Product (EIP) for full scan mass spectra that range from mass to charge ratio (m/z) 100-1200.

Plant extracts and Standard Drugs

The aqueous and methanolic extracts as well as fractions of the four Phyllanthus spp., namely P. niruri, P. urinaria, P. watsonii and P. amarus, were kindly extracted and provided by Dr Indu Bala Jaganath from MARDI, Serdang, Malaysia. The aqueous and methanolic extracts were prepared by dissolving 10mg and 40mg in 1ml of sterile PBS and DMSO to produce a stock concentration of 10mg/ml and 40mg/ml respectively. Meanwhile, the fractions were prepared by dissolving 10mg in 1ml of sterile PBS. The standard drugs used in this study are Cisplatin and Doxorubicin (MERCK). These standard drugs were prepared by dissolving 1mg in 1ml of sterile PBS to achieve a stock concentration of 1mg/ml. The extracts and drugs were wrapped with the aluminium foil and kept at -20°C freezer until further use.

Cell Culture

The cancer cell lines used in this study includes lung carcinoma (A549) and breast carcinoma (MCF-7) purchased from American type Culture Collection (Rockville, MD). They were grown in Roswell Park Memorial Institute 1640 (RPMI-1640) and Dulbecco's modified Eagles medium (DMEM) respectively, supplemented with 10% Fetal Calf Serum and incubated in a humidified atmosphere with 5% CO2 at 37°C. Two normal cell lines, bronchus epithelium (NL20) and breast epithelium (184B5) were also purchased from the American type Culture Collection (Rockville, MD) and cultured according to the manufacturer's instructions.

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Morphological Analysis

Cells were cultured in 6-wells plate in appropriate medium supplemented with 10% FCS overnight. After 24 hours, cultured cells were treated with Phyllanthus extracts, Cisplatin, and Doxorubicin at their respective Half Maximal Inhibitory Concentrations [IC50] (Budzinski et al., 2000; Salvatore et al., 2004) and further incubated for 72 hours. Effect of the extracts and drugs on the cells was then observed under a light microscope (Olympus) and photographs were taken at a magnification of 200X.

MTS Cytotoxicity Assay

Cells were seeded at their optimal cell density (1 x 104 cells/well) into a 96-wells microtiter plate followed by overnight incubation to allow cell attachment. They were then treated with Phyllanthus fractions as well as both the aqueous and methanolic Phyllanthus extracts at a 6-points serial dilution up to a final concentration of 1000µg/ml. Vehicle-control wells with cells only and compound-control wells with extracts only are included. Plates are incubated at 37°C, 5% CO2 and 100% humidity at three different time periods, which are 24, 48, and 72 hours. At the end of each incubation period, the cell viability was determined using Cell Titer96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, USA) according to the manufacturer's instructions. Absorbance was measured using GloMax Multi Detection System (Promega, USA) at a wavelength of 490nm with a reference wavelength of 750nm.

Cell Cycle Analysis

Cells were seeded at 105 cells/well, treated with extracts at their IC50 values, and incubated at various time periods from 0 to 72 hours. At the end of each incubation period, cells treated with or without Phyllanthus extracts were harvested and fixed with ice-cold 70% ethanol for at least 1 hour at -20°C. Cells were then pelleted, washed once with PBS, resuspended in the Propidium Iodide (PI) solution [10µg/ml PI (Sigma) and 1mg/ml RNase A in PBS], and incubated in a 37°C water bath for 30minutes. Data acquisition was performed using a Becton Dickinson FACSCalibur flow cytometer and CellQuest software and subsequently analysed using WinMDI 2.9 software. The distribution of cell percentages in each cell cycle phase is determined by setting gates based on their amount of DNA content.

DNA Fragmentation Analysis

DNA fragmentation was carried out as described by Lin J et al. (2003) with some modifications. Briefly, 5 x 105 treated cells in 500µl were lysed in 55µl DNA lysis buffer [1M Tris-HCI (pH8.0), 0.5M EDTA, and 100% Triton X-100] and incubated at 4°C for 30 minutes. Supernatant was then collected in a new tube and DNA was extracted with an equal volume of phenol/choloroform/isoamyl alcohol (25:24:1). Sample was spun and only the upper aqueous layer was transferred into a new tube with subsequent addition of equal volume of ice-cold 100% ethanol and 1/10 volume of 3M sodium acetate (pH5.2) for DNA precipitation at -20°C for overnight. Sample was spun, supernatant was decanted without affecting the pellet (DNA), and dried in air. After that, DNA was dissolved in deionized water-RNase solution [10mg/ml RNase I] and incubated at 37°C for 30 minutes. Equal amounts of DNA (10µg/well) were electrophoresed in 1.2% agarose gel impregnated with ethidium bromide at 5V for first 5 minutes and increased to 100V for 1 hour. DNA fragments were then visualized using a UV transilluminator.

Caspase assay

Cells were seeded, treated with extracts at their IC50 values, and incubated at 37°C, 5% CO2 and 100% humidity for 72 hours. Caspases activity was then determined using Caspase-Glo 3/7 Assay (Promega, USA) according to the manufacturer's instructions. Briefly, lyophilized Caspase-Glo 3/7 substrate was resuspended in its buffer and 100µl of this reagent was added into each well. Contents of wells were gently mixed and incubated at room temperature for 1 hour. Luminescence of each sample was measured using a plate-reading luminometer.

TUNEL assay

Terminal Deoxynucleotidyl-Transferase mediated dUTP Nick End Labelling (TUNEL) assay was performed using ApopTag® Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon® International). Briefly, 105 cells were harvested, fixed in 1% paraformaldehyde in PBS, pH 7.4 and subsequently dried on a silanized microscope slide. After that, it was post-fixed in pre-cooled ethanol/acetic acid (2:1) and quenched in 3% hydrogen peroxidase in PBS. Excess liquid was tapped off before 75µl/5cm2 of equilibration buffer was immediately applied on specimen. Next, 55µl/5cm2 of working strength TdT enzyme was added onto the specimen and incubated at 37°C for 1 hour. After incubation, specimen was placed in a coplin jar containing working strength stop/wash buffer followed by addition of 65µl/5cm2 of anti-digoxigenin peroxidase conjugate. Specimen was washed in four changes of PBS and stained with 75µl/5cm2 of peroxidase substrate. The specimen was counterstained in 0.5% (w/v) methyl green followed by several washes with distilled water, n-butanol, and xylene. Finally, the specimen was mounted under a glass coverslip in Permount and observed under a light microscope (Olympus BX51) at a magnification power of 200X. Photos are captured using Olympus U-CMAD3 at three fields per slide.

Lactate Dehydrogenase (LDH) assay

LDH assay was performed using CytoTox-ONE® Homogeneous Membrane Integrity Assay purchased from Promega, USA. Cells were seeded, treated, and incubated for 72 hours. No-cell control, untreated cells control, and maximum LDH release control wells were included in each plate. At the end of incubation, lysis solution was added to positive wells and further incubated for 30 minutes to generate maximum LDH release. After that, an equal volume of CytoTox-ONE® Reagent was added into each well and incubated at room temperature for 10 minutes with subsequent addition of stop solution. Fluorescence was recorded with an excitation wavelength of 560nm and an emission wavelength of 590nm within 1 hour to avoid increased background fluorescence.

Statistical Analysis

Results are expressed as mean standard deviation for at least three independent experiments. Differences between the means were deemed to be significant if p < 0.05 according to Students t-test.

Results

Polyphenols identification in Phyllanthus spp.

Tables 1 and 2 showed the polyphenol compounds present in both methanolic and water-soluble extracts obtained from various species of Phyllanthus after analysis by HPLC coupled with photodiode array (PDA) and MS-MS detection. There are twelve main compounds identified based on their retention times, UV spectra, parent mass spectra and secondary fragmentation patterns, namely gallic acid, galloylglucopyronside, digalloylglucopyronside, trigalloylglucopyronside, tetragalloylglucopyronside, corilagen, geraniin, rutin, quercetin glucoside, quercetin diglucoside, quercetin rhamnoside, and caffeolquinic acid.

Effect of Phyllanthus extracts on cell morphology

Upon treatment with Phyllanthus extracts and standard drugs for 72 hours, both A549 and MCF-7 cells had displayed significant morphological changes characteristic of apoptosis. Some of them had been detached from the monolayer and were rounded up (Figure 1a). Besides that, some of the cells had also become granulated (Figure 1b), possessed condensed chromatin (Figure 1c), as well as membrane blebbing or with the presence of apoptotic bodies (Figure 1d).

Effect of Phyllanthus extracts, fractions and Standard Drugs on the growth of different cell lines

The MTS assay was used in order to investigate the potential cytotoxic effects of Phyllanthus extracts and fractions on the different cell lines, where the cells were treated at increasing concentrations up to 1000µg/ml for 24, 48, and 72 hours. Two standard drugs, namely Cisplatin and Doxorubicin were used as positive controls in this study, where the cells were treated at increasing concentrations up to 10µg/ml for 24, 48, and 72 hours. The cytotoxicity was recorded as IC50 (µg/ml) values, which resembles the concentration of extracts, fractions or drugs that kills or inhibits the growth of 50% of the population (Table 3). Data obtained showed that Phyllanthus extracts have the potential to inhibit growth of A549 and MCF-7 with minimal effect on the NL20 and 184B5. As a comparison, growth inhibition of Phyllanthus extracts on MCF-7 cells is shown to be more effective than on A549 cells. From the data, methanolic extracts of Phyllanthus exhibited greater cytotoxicity compared to the aqueous extracts for both cancer cell lines. Among the four Phyllanthus species, P. watsonii showed the strongest cytotoxicity with lowest IC50 values for both aqueous and methanolic extracts on A549 and MCF-7 respectively, followed by P. urinaria, P. amarus, and P. niruri. In contrary, fractions of Phyllanthus are not as effective as Phyllanthus extracts. Fractions 1 showed very low toxicity to both cancer cell lines. Fractions 2 were more toxic to cancer cells compared to fractions 1, but not as toxic as Phyllanthus extracts. At the same time, fractions 2 were toxic to the normal cells also. Both standard drugs also showed strong cytotoxicity on A549 and MCF-7 cells with IC50 values < 10g/ml. However, they are also very toxic to the normal cell lines with IC50 values comparable to their IC50 values for cancer cells.

Figure 2 illustrates the growth inhibitory curves of P. watsonii on both A549 and MCF-7 cells. Results for the other species are similar and hence not shown here (results are shown in supplementary data). Cells treated at low concentrations (lesser than 50g/ml for aqueous extracts and lesser than 25g/ml for methanolic extracts) of Phyllanthus extracts did not show obvious reduction in growth. But, the growth of the cancer cells is significantly decreased as the extracts concentrations increases. In addition, it was also found that cell growth inhibition effect increased with time for 24, 48, and 72 hours.

Effect of Phyllanthus extracts on cell cycle distribution

In repeated experiments, our data did not demonstrate any cell cycle phase arrest in both the A549 and MCF-7 cells treated with Phyllanthus extracts (Figure 3 and 4). However, at increasing incubation time points (24, 48, and 72 hours), the percentage of gated cells for each cell cycle phases (G0/G1, S, and G2/M) decreased for both A549 and MCF-7 cell lines with an increase in the number of dead cells (increase in Sub G1 phase). On the other hand, there is an accumulation of cells at G2/M phase for both A549 and MCF-7 cells treated with Cisplatin and Doxorubicin. Similarly, an increase in the number of cells arrested at Sub G1 phase was noted, as this is one of the indicators for the presence of apoptotic cells.

Effect of Phyllanthus extracts on Caspase-3 and -7 activities

Caspase-3 and -7 play crucial roles as early apoptosis biochemical markers in mammalian cells. Caspase-Glo® 3/7 Assay uses a luminogenic substrate containing the DEVD sequence which is selective for caspase-3 and -7. The caspases activity in both non-treated and Phyllanthus-treated cancer cells were measured after 72 hours and are shown in Figure 5. Caspases activity level detected in non-treated control cells correspond to the portion of apoptotic cells present in the naturally growing population due to natural aging. In treated cells, activities of Caspase-3 and -7 increased from 3-fold to 5-fold over basal levels signifying an activation of these caspases in both A549 and MCF-7 cells treated with Phyllanthus extracts.

Effect of Phyllanthus extracts on nuclear fragmentation

DNA fragmentation in condensed chromatin and formation of apoptotic bodies are some of the events of late apoptosis (Qiu et al., 1998). In order to further demonstrate the ability of Phyllanthus extracts to induce apoptosis, DNA fragmentation assay with Doxorubicin and Cisplatin as the positive controls was used to verify the presence of apoptotic cells in the treated cultured cells. Figure 6 showed a typical ladder-like pattern of DNA fragments with multiples of approximately 180-200 bps after A549 and MCF-7 cells were treated with the extracts and standard drugs, one of the hallmarks of apoptosis.

In addition to that, the cell apoptosis was determined in situ based on the enzymatic labelling of free 3'-OH terminus of non-random DNA single-stranded and double-stranded breaks with modified nucleotides, resulting in the apoptotic cells staining brown. After 72 hours treatment of the A549 and MCF-7 cells with Phyllanthus extracts and standard drugs, the percentage of apoptotic cells increased tremendously as compared to the untreated control cells. Since the cells were treated with the IC50 concentrations of extracts and drugs, the mean percentage of apoptotic cells observed from 3 views per slide varied from 28% up to 55%. Figure 7 and 8 showed the TUNEL-positive cells after treatment with aqueous and methanolic extracts of Phyllanthus, Cisplatin, and Doxorubicin.

Effect of Phyllanthus extracts on Cellular Membrane Integrity

Lactate Dehydrogenase (LDH) is a cytosolic enzyme released into cell culture supernatant due to compromised membrane integrity, which is associated with necrotic cell death. Its extent of activity of converting tetrazolium salt into red formazan product is proportional to the number of necrotic cells (Yang et al., 2004). Figure 9 showed the percentage of LDH released from the A549 and MCF-7 cells after 72hours treatment with each aqueous and methanolic extracts of Phyllanthus. From the data, the LDH amount released by Phyllanthus-treated cells remained at low level (< 20%) comparable to the untreated cells. Similarly, treatment with Cisplatin and Doxorubicin which are well-known apoptosis-inducing drugs (Abdolmohammadi et al., 2008) caused a low LDH level release. Therefore, it suggests that Phyllanthus induces minimal cytotoxicity by disrupting membrane integrity which leads to necrosis.

Discussion

The plants with genus Phyllanthus which comes from the family Euphorbiaceae have a long history in folks medicine. It has gained world-wide attention ever since the first report on the effectiveness of P. niruri against Hepatitis B was published (Venkateswaran et al., 1987). Subsequently, its various therapeutic properties had been reported including anti-hepatotoxic, anti-lithic, anti-hypertensive, and most recently anti-HIV (Bagalkotkar et al., 2006; Sabir and Rocha, 2008). Besides that, there are also some reports on the antitumor activity of various Phyllanthus plants. P. emblica demonstrated growth inhibitory activity on A549 and HepG2 (Pinmai et al., 2008) while toxicity of P. polyphyllus on MCF-7, HT-29, and HepG2 cells were reported with IC50 values of 27, 42, and 38μg/mL respectively (Rajkapoor et al., 2007). However, the anticancer potential of Phyllanthus plants was not completely elucidated yet. In our study, we evaluated the toxicity of both aqueous and methanolic extracts of four different species of Phyllanthus plants, namely P. niruri, P. urinaria, P. watsonii, and P. amarus on two human cancer cell lines (A549 and MCF-7) and two normal human cell lines (184B5 and NL20). Our data showed that Phyllanthus selectively exhibits cytotoxicity to the MCF-7 and A549 human cancer cells with IC50 values ranging from 50g/ml to 180g/ml and 65g/ml to 470g/ml respectively for both methanolic and aqueous extracts while having lower toxicity to the normal cell lines.

Most anticancer activities exhibited by natural products are attributed to the presence of phytochemicals within the plant extracts (Gopalakrishnan and Tony Kong, 2008; Russo, 2007; Issa et al., 2006). High Performance Liquid Chromatography studies showed presence of several major phytochemicals in various parts of P. niruri, including phyllanthin, gallic acid, geraniin, ascorbic acid, lignans, quercetin, and estradiol (Murugaiyah and Chan, 2007; Bagalkotkar et al., 2006; De Souza et al., 2002; Jerrold J. Simon, 2002). Complete recovery of these bioactive compounds from a multi-components plant sample is not possible as a single solvent may not be sufficient to select every single component (Markom et al., 2007). Most of these phytochemicals dissolve better in organic solvents such as methanol and ethanol while they are only partially soluble in polar solvents such as water (Ojala et al., 2000). Therefore, most studies reported the higher effectiveness of ethanolic or methanolic extracts in inhibiting the growth or being toxic to the various cancerous cells in vitro (Saetung et al., 2005; Wu et al., 2009). Their results are consistent with the findings of this study in which methanolic extracts of Phyllanthus spp. demonstrated greater toxicity to the A549 and MCF-7 cells in vitro compared to aqueous extracts.

In addition, Sun and Liu (2006) reported that not any individual class of components in their extract could be entirely accountable for the activity produced by the whole extract itself. We previously tested the toxicity of two Phyllanthus fractions on both cancer (A549 and MCF-7) and normal (NL20 and 184B5) cell lines. The first fraction of Phyllanthus plants were either not toxic or showed very little toxicity to cancer cells. Although the second fraction of Phyllanthus showed toxicity to the cancer cells, but they are not as strong as the Phyllanthus extracts tested and are also being toxic to the normal cells. Therefore, it is more meaningful to assess the activity of Phyllanthus as a complete mixture of phytochemicals rather than evaluating them as single components. Some of the components discovered within the Phyllanthus species are geraniin, corilagen, galloylglucopyronside, digalloylglucopyronside, trigalloylglucopyronside, tetragalloylglucopyronside, corilagen, rutin, caffeolquinic acid, quercetin glucoside, quercetin diglucoside, and quercetin rhamnoside. Different species of Phyllanthus plants will have variation in the percentage of composition of each phytochemical component, therefore giving rise to the different extent of cytotoxicity to cancer cells. Among the four Phyllanthus species being studied, P. watsonii showed the highest cytotoxicity to both A549 and MCF-7 cells in vitro. The percentage of compounds present in this particular Phyllanthus species that contributes to its cytotoxicity cannot be disclosed as application of patency is in progress.

Cell cycle disorder plays a critical role in cancer progression. Thus, modulation of cell cycle by phytochemicals from natural product sources is gaining worldwide attention to control carcinogenesis (Abdolmohammadi et al., 2008). In the present study, insignificant shift in the cell cycle phases for the cells treated with Phyllanthus extracts suggests that there is no direct correlation between extracts' cytotoxicity and specific cell cycle arrest. In contrast, Cisplatin and Doxorubicin caused a G2/M phase arrest for both A549 and MCF-7 cells (Fornari et al., 1999; Otto et al., 1996; Sorenson et al., 1990; Dan and Yamori, 2002). Cisplatin works via formation of DNA adducts resulting in the blockage of replication, transcription and repair mechanisms due to inhibitory action of the DNA adduct on the DNA polymerase (Zamble et al., 1998). Meanwhile, Doxorubicin (Adriamycin) acts as a topoisomerase II poison, intercalating between the DNA bases, and sequestering irons followed by the generation of free radical leading to disruption of DNA (Swift et al., 2006). However, an increase in the cell percentage at Sub G1 phase for MCF-7 cells treated with Phyllanthus extracts may signify an increase in the number of apoptotic cells. This therefore suggests apoptosis as the mode of cell death in the extracts-treated cells. However, this data is not conclusive enough and need to be further confirmed with other apoptotic assays such as Caspases assay, TUNEL assay, and DNA fragmentation assay which are discussed below. Although Phyllanthus extracts also caused apoptosis in the treated A549 cells, but the cell distribution in Sub G1 phase is not very high and this explained the vague DNA ladder bands on the agarose gel.

Cytotoxicity on A549 and MCF-7 cells treated with Phyllanthus extracts may occur via two possible modes of cell death, either through apoptosis or necrosis. The former typically involves a multistage of DNA fragmentation (Qiu et al, 1998) beginning with the release of cytochrome c from mitochondria, activation of a cascade of caspases, degradation of PARP, and fragmentation of chromosomal DNA (Pojarova et al., 2007; Yang et al., 2006). Meanwhile, the latter is most oftenly associated with external damage leading to accidental cell death, resulting in mitochondrial and cytoplasmic swelling, followed by compromised membrane integrity that eventually burst releasing cytoplasmic contents (Woo et al., 2008; Yang et al., 2004). An effective and successful anticancer drug should has the ability to kill or hinder the growth of cancer cells without causing severe harm to the healthy cells which could only be achieved by triggering apoptosis in cancer cells (Abdolmohammadi et al., 2008).

Apoptosis can be diverged into two pathways; extrinsic and intrinsic pathways which involve caspases that are constitutively expressed during apoptosis. Extrinsic pathway begins with the formation of a death-inducing complex to activate caspase-8 while intrinsic pathway involved release of cytochrome c from mitochondria membrane into cytosol, formation of apoptosome, and activation of caspase-9 (Samali et al., 1998; Kiechle and Zhang, 2002, Jin et al., 2006). Irrespective of which pathways, both will eventually converge and activate the Caspases-3 and -7 which are the execution caspases (Wu et al., 2006). Based on our data, apoptosis occurs in the cells treated with Phyllanthus plants since the level of these execution caspases increased several folds over the basal level of untreated cells. Presence of these caspases indicates that the cells are proceeding towards a terminal pathway of cell death and is therefore an early biochemical marker for apoptosis.

Activation of caspase-3 will subsequently trigger the proteolytic cleavage of poly (ADP-ribose) polymerase resulting in DNA fragmentation that usually occurs during the late apoptosis (Yang et al., 2006; Wu et al., 2009). The DNA fragments will appear as a DNA ladder on an agarose gel as shown in Figure 5 instead of a randomized DNA breakdown which is observed as a smear for necrosis. However, internucleosomal DNA fragmentation is not universal as it may not always occur during apoptosis (Vinatier et al., 1996). Thus, an in situ staining of the DNA breaks further confirmed induction of apoptosis by Phyllanthus plants with the presence of TUNEL-positive cells shown in Figure 6.

Although these studies strongly suggest apoptosis as the mode of cell death induced by Phyllanthus extracts, necrosis as the competing action cannot be ruled out. Some of the cytotoxic agents have the ability to activate both apoptotic and necrotic cell death pathways (Woo et al., 2008). Hence, release of LDH by control and Phyllanthus-treated cells which is an indicator of necrosis was assessed. Our data revealed that LDH level released from both the control and Phyllanthus-treated cells remained at low level. Therefore, the possibility of necrosis as the major mode of cell death used by Phyllanthus can be excluded.

A schematic diagram depicting possible mechanism of action of Phyllanthus extracts on cancer cells is presented in Figure 10. The anticancer activities of Phyllanthus plants were skewed towards its ability to cause cytotoxicity on the cancer cell lines without significant correlation with the induction of cell cycle arrest. Its cytotoxicity potential is attributed to its capability to induce apoptosis which is associated with the activation of caspases-3 and -7 as well as DNA fragmentation with low LDH level released. This finding further provides scientific evidences to prove the traditional claims of Phyllanthus plants. Nevertheless, apoptosis is the end result of sequential events which begin with the effect of natural plant extracts on the cancer cells. However, the upstream signaling that stimulates apoptosis is yet unclear and further investigations are needed. There are several possibilities; (1) induction of tumour suppressor genes such as p53, resulting in the overexpression of p21 that triggers growth arrest or apoptosis in cancer cells (2) downregulation of proto-oncogenes such as c-myc that causes growth arrest (3) activation of binding of Fas ligand to its receptor, therefore inducing apoptosis of Fas-bearing cells (Vinatier et al., 1996). Knowledge of the mode of actions of these extracts would definitely help to identify their possible applications in cancer prevention. The main concern before application of the anti-proliferative agents as anticancer drugs is their in vivo effect. Thus, further testing of the extracts activity in vivo is a necessity to exploit it is a chemotherapeutic agent.

Figure 1: Morphological changes of cells after treated with Phyllanthus extracts and standard drugs for 72 hours. Red arrows are pointing to each of these morphologies; (a) detached and rounded cells, (b) granulated and vacuolated cells, (c) cells with condensed chromatin, and (d) membrane blebbing or apoptotic bodies. Colour differences are due to changes in light source of microscope. Typical result from three independent experiments was shown. (Magnification power= 200x)

Table 1: Polyphenol compounds detected in water extracts of Phyllanthus species

Compound

Retention time

[M-H]

m/z

MS-MS

fragmentation

Phyllanthus species

Gallic acid

3.8

169

125,169

P. amarus, P. niruri,

P. urinaria, P. watsonii

Galloylglucopyronside

2.8

331

125, 169, 211, 271

P. amarus, P. niruri,

P. urinaria, P. watsonii

Digalloylglucopyronside

15.0

483

125, 169, 211, 271, 313

P. amarus, P. niruri,

P. watsonii

Trigalloylglucopyronside

23.0

635

125, 169, 211, 271, 313, 465

P. urinaria

Corilagen

18.0

633

301, 125, 169

P. amarus, P. niruri,

P. urinaria, P. watsonii

Geraniin

22.0

951

301, 125, 169, 463

P. amarus, P. niruri,

P. urinaria, P. watsonii

Rutin

26.0

609

301, 179, 151

P. amarus, P. niruri,

P. urinaria, P. watsonii

Quercetin glucoside

27.0

463

301, 179, 151

P. amarus, P. niruri,

P. urinaria, P. watsonii

Quercetin rhamnoside

30.0

447

301, 151

P. urinaria, P. watsonii

Caffeolquinic acid

23.0

353

191

P. amarus, P. niruri,

P. urinaria, P. watsonii

Table 2: Polyphenol compounds detected in methanolic extracts of Phyllanthus species

Compound

Retention time

[M-H]

m/z

MS-MS

fragmentation

Phyllanthus species

Trigalloylglucopyronside

13.0

635

125, 169, 211, 271, 313, 465

P. urinaria

Tetragalloylglucopyronside

15.0

787

169, 211, 313, 465

P. urinaria

Geraniin

12.0

951

301, 125, 169, 463

P. amarus, P. niruri,

P. urinaria, P. watsonii

Quercetin diglucoside

9.0

625

463, 301

P. niruri

Table 3: Cytotoxicity effect [IC50 (g/ml)] of Phyllanthus extracts against two cancer cell lines (A549, MCF-7) and two normal cell lines (NL20, 184B5) after 72 hours incubation time. Data is expressed as a mean of three independent experiments  Standard Error Mean (SEM).

Solvents

IC50 (g/ml)  SEM

Cancer Cell Lines

Normal Cell Lines

A549

MCF-7

NL20

184B5

Plant Extracts

P. niruri (P.n)

Aqueous

466.7  41.63

179.7  0.58

> 500

> 500

Methanol

128.3  17.56

62.3  9.07

> 500

> 500

P. urinaria (P.u)

Aqueous

215.0  21.79

139.3  1.16

> 500

> 500

Methanol

69.0  11.53

48.7  10.02

> 500

> 500

P. watsonii (P.w)

Aqueous

198.3  10.41

104.0  10.39

> 500

> 500

Methanol

61.3  16.17

49.0  8.19

> 500

> 500

P. amarus (P.a)

Aqueous

240.0  26.46

156.7  5.77

> 500

> 500

Methanol

126.7  7.64

56.3  6.66

> 500

> 500

Standard Drugs

Cisplatin

7.6  1.10

1.4  0.54

0.9  0.05

3.0  0.03

Doxorubicin

0.6  0.08

0.4  0.05

0.3  0.02

0.6  0.03

Fraction 1

P. niruri

Aqueous

380.0  18.03

438.3  11.55

266.7  41.93

283.3  25.17

P. urinaria

Aqueous

> 500

> 500

> 500

231.7  18.93

P. watsonii

Aqueous

395.0  8.66

376.7  2.89

241.7  20.21

230.0  26.46

P. amarus

Aqueous

> 500

> 500

> 500

> 500

Fraction 2

P. niruri

Aqueous

228.3  5.77

81.7  16.07

108.3  5.77

230.0  50.74

P. urinaria

Aqueous

225.0  13.23

61.7  12.58

95.0  5.00

230.0  13.23

P. watsonii

Aqueous

225.0  43.30

46.7  10.41

105.0  5.00

201.7  20.21

P. amarus

Aqueous

264.3  45.24

70.0  17.32

106.7  7.64

213.3  54.85

(a) (b)

(c) (d)

Figure 2: Growth inhibition effect of (a) aqueous extracts of P. watsonii on A549, (b) methanolic extracts of P. watsonii on A549, (c) aqueous extracts of P. watsonii on MCF-7, and (d) methanolic extracts of P. watsonii on MCF7. Each cell lines were treated at varying concentrations for 24, 48, and 72 hours. Error bar indicates the standard error of the mean of three independent experiments.

(a)

(b)

(c)

Figure 3: Percentage of cell cycle phase distribution of A549 cells treated with both aqueous and methanolic Phyllanthus extracts and standard drugs at their IC50 (g/ml) concentrations for (a) 24 hours, (b) 48 hours, and (c) 72 hours. Error bar indicates the standard error of the mean of three independent experiments.

(a)

(b)

(c)

Figure 4: Percentage of cell cycle phase distribution of MCF-7 cells treated with both aqueous and methanolic Phyllanthus extracts and standard drugs at their IC50 (g/ml) concentrations for (a) 24 hours, (b) 48 hours, and (c) 72 hours. Error bar indicates the standard error of the mean of three independent experiments.

Figure 5: Activity level of Caspase-3 and -7 released from A549 and MCF-7 cells treated with both aqueous and methanolic Phyllanthus extracts and standard drugs at their IC50 (g/ml) concentrations after 72 hours. Error bar indicates the standard error of the mean of three independent experiments. P.N - P. niruri, P.U - P. urinaria, P.W - P. watsonii, P.A - P. amarus, CIS - Cisplatin, DOX - Doxorubicin, CTRL - Non-treated cells.

800 bp

600 bp

200 bp

1500 bp

a

M 1 2 3 4 5 6 7 8 9 10 11 11

800 bp

600 bp

200 bp

1500 bp

b

M 1 2 3 4 5 6 7 8 9 10 11 11

Figure 6: DNA fragmentation of (a) A549 and (b) MCF-7 cells treated with P.watsonii and standard drugs at their IC50 (g/ml) concentrations for 72 hours. Red arrows at the right are pointing to the bands of DNA fragments. Typical result from three independent experiments was shown. M: Molecular-weight marker, Lane 1 - 4: aqueous extracts of P. niruri, P. urinaria, P. watsonii, and P. amarus, Lane 5 - 8: methanolic extracts of P. niruri, P. urinaria, P. watsonii, and P. amarus, Lane 9: Cisplatin, Lane 10: Doxorubicin, and Lane 11: Untreated control.

%Apoptotic cells: 6.8 ± 0.01

%Apoptotic cells: 44.8 ± 0.05

%Apoptotic cells: 39.4 ± 0.04

%Apoptotic cells: 46.7 ± 0.11

%Apoptotic cells: 42.3 ± 0.15

%Apoptotic cells: 53.7 ± 0.05

%Apoptotic cells: 54.1 ± 0.11

%Apoptotic cells: 29.4 ± 0.08

%Apoptotic cells: 50.4 ± 0.08

%Apoptotic cells: 49.0 ± 0.08

%Apoptotic cells: 55.4 ± 0.03

Figure 7: Red arrows showing TUNEL-positive A549 cells; (a) non-treated and after treated with (b) aqueous P. niruri, (c) aqueous P. urinaria, (d) aqueous P. watsonii, (e) aqueous P. amarus, (f) methanolic P. niruri, (g) methanolic P. urinaria, (h) methanolic P. watsonii, (i) methanolic P. amarus, (j) Cisplatin, and (k) Doxorubicin at their IC50 (g/ml) concentrations for 72 hours. Typical result from three independent experiments was shown. (Magnification power: 200X)

%Apoptotic cells: 41.2 ± 0.10

%Apoptotic cells: 43.8 ± 0.09

%Apoptotic cells: 44.7 ± 0.05

%Apoptotic cells: 50.3 ± 0.05

%Apoptotic cells: 33.7 ± 0.11

%Apoptotic cells: 28.4 ± 0.04

%Apoptotic cells: 53.3 ± 0.12

%Apoptotic cells: 52.6 ± 0.03

%Apoptotic cells: 42.8 ± 0.01

%Apoptotic cells: 7.1 ± 0.04

%Apoptotic cells: 44.6 ± 0.10

Figure 8: Red arrows showing TUNEL-positive MCF-7 cells (a) non-treated and after treated with (b) aqueous P. niruri, (c) aqueous P. urinaria, (d) aqueous P. watsonii, (e) aqueous P. amarus, (f) methanolic P. niruri, (g) methanolic P. urinaria, (h) methanolic P. watsonii, (i) methanolic P. amarus, (j) Cisplatin, and (k) Doxorubicin at their IC50 (g/ml) concentrations for 72 hours. Typical result from three independent experiments was shown. (Magnification power: 200X)

Figure 9: Percentage of Lactate Dehydrogenase (LDH) released from A549 and MCF-7 cells treated with both aqueous and methanolic Phyllanthus extracts and standard drugs at their IC50 (g/ml) concentrations after 72 hours. Error bar indicates the standard error of the mean of three independent experiments. P.N - P. niruri, P.U - P. urinaria, P.W - P. watsonii, P.A - P. amarus, CIS - Cisplatin, DOX - Doxorubicin, CTRL - untreated cells, and MAX - Maximum Lysed Cells.

Apoptosis

PARP Cleavage

?

DNA fragmentation

Necrosis

LDH release

Phyllanthus extracts

Cytostatic

Cytotoxic

Growth Arrest

Activation of execution caspases

Figure 10: A Schematic Diagram depicting possible mechanism of action of Phyllanthus extracts on cancer cells. Based on this study, apoptosis is the end result due to toxicity of Phyllanthus. However, detailed mechanisms that lead to apoptotic events remained to be clarified.