Thamizh Selvam N1, Acharya MV 2, Venkatakrishnan V3, Murugesan S4
1Assistant Director-Biochemistry, Central Ayurveda Research Institute- N&MSD, (CCRAS, Ministry of AYUSH, Government of India), Cheruthuruthy, Thrissur, Kerala.
2Director, CARI-N&MSD (CCRAS, Ministry of AYUSH, Govt. of India), Cheruthuruthy.
3Faculty, Department of Food Science and Technology, Pondicherry University, Pondicherry.
4Associate Professor, Department of Botany and Plant Biotechnology, Pachaiyappa’s College, Chennai.
Address for Corresponding Author:
Dr. N. Thamizh Selvam Ph.D.,
Central Ayurveda Research Institute- N&MSD, (CCRAS, Ministry of AYUSH, Government of India), Cheruthuruthy, Thrissur, Kerala-679 531.
Objective: Cancer is a complex disease representing uncontrolled cell proliferation form unwanted mutation or defects in many classes of genes. There are numerous methods of treatment and opted based on stage of the disease and physical condition of the patients. Indigenous medicine system comprising of plants and plant based formulations are also used for treatment and management of cancer. Material and methods: The present study was carried out on one of the Western ghat plants Syzygium jambos Linn Alston, for its effect on HepG2 liver cancer cell line and for understanding its molecular level action. The inter-nucleosomal DNA fragmentation, Nuclear localisation, Bcl-2 and Bax gene expression studies were carried out as per the standard protocols. Results: Study showed that S. jambos extract is working at intra cellular level and it influenced the apoptotic cell cycle pathway positively through Bcl-2 and Bax gene expression. The study vividly explained the mode of action and potency of the test extract for its cytotoxic property in cancer cell line.
Key words: Syzygium jambos, cytotoxic activity, Nuclear localization, DNA fragmentation
Carcinogenesis, transformation of normal cells into cancerous cells, is a multistep process wherein the genomes of new cancer cells acquire mutant alleles of the proto-oncogenes, tumour-suppressor genes, and other genes that control, cell proliferation (Lowe and Lin, 2000). Tumours which are confined to the site of origin are generally called benign, while those having metastatic ability are called malignant tumours. The causes of cancer are varied and include genetic, environmental factors, infectious agents, nutritional factors and radiation. Despite significant investments in capital, manpower, and intellectual innovations for the development of cancer therapies over the past several decades, cancer still remains a powerful threat with high mortality rates across the globe (Bartek and Lukas, 2001; Nebert, 2002). The magnitude of the problem of cancer in India is most alarming (Bobba and Khan, 2003).
Chemotherapy has become an essential component of treatment of most solid-organ cancers and is the mainstay of the treatment for patients with advanced or recurrent disease. Complementary and alternative medicine (CAM) is becoming increasingly popular, particularly among patients with advanced stages of cancer, and shares a sizeable percentage in the treatment methods employed in cancer cure which generally given alone or along with conventional therapies (Gerber et al., 2006; Pal, 2002). In India, use of Ayurvedic medicines like Valipani, Navjeevan and Kamdudha have shown efficacy in some leukemia patients.
Syzygium jambos (Linn) Alston, known as Rose-Apple in English (Champeyah-in Sanskrit) under the family Myrtaceae, is a large shrub or small tree with spreading branches, leaves simple, opposite, lanceolate, narrowed into short petioles. The leaves, bark and fruits are used for treatment of various illnesses including haemorrhages, syphilis, leprosy, dermatopathy, diarrhoea, colic helminthiasis, wounds and ulcers (Warrier et al., 1996). The present study has been taken up to evaluate the cellular and molecular level action of methanolic extract of S. jambos Leaves in the selective liver cancer cell line.
Materials and Methods
The plant material was collected from the western ghat region of Kerala. The extract was prepared by soxhlet method using methanol as solvent. After the extraction, the solvent was removed and solvent free extract was stored in refrigerator and used for the present study.
Chemicals and Reagents
The chemicals and reagents of Analytical grade of Hi Media, Spectrum and Merck India were used. Molecular works have been carried out with the technical support of ACTREC, Mumbai, Amala cancer centre, Thrissur and Anna University, Chennai.
DNA Fragmentation Assay
Internucleosomal cleavage of DNA was analyzed as described by Kim et al., 2001. Briefly, 1 x 106 HepG2 cells/ml seeded in a 6 well plate were treated with methanoilc extract (25 μg/ml) of S. jambos. The pure compound β-sitosterol (15 μg/ml) was also used for reference. Etoposide (15 μg/ml) treated HepG2 cells served as positive controls. Untreated HepG2cells served as control. The plate was incubated for 24 hours. For analysis of caspase-3 mediated DNA fragmentations, 100 μM of caspase 3 inhibitor (AC-DEVD-CHO), was added to the cells and incubated for 3 hours followed by treatment with methanol extracts and pure compound for 24 hours. DNA was run on a 2% agarose gel with ethidium bromide staining. After electrophoresis, DNA was visualized under UV-spectrophotometer and documented.
Propidium Iodide Staining
Cell death was assessed using the uptake of the fluorescent dye propidium iodide by the fragmented DNA in dying cells that lack plasma membrane integrity and intact DNA in control untreated cells. 1x106 HepG2 cells/ml seeded in a 6 well plate were treated with methanolic extract (25 μg/ml) of S. jambos and pure compound β-sitosterol (15 μg/ml) respectively. Etoposide treated HepG2 cells served as positive control. Untreated cells served as control. The treatment was continued for 24 hours and then cells were pelleted by centrifugation at 10,000 rpm for 10 minutes. Cell pellets were re-suspended in 50 ml of PBS. 5 ml of RNase (1 mg/ml) and 5 ml of propidium iodide (25 mg/ml in PBS) was added and incubated at 37oC for one hour. Fluorescence was excited with an Argon ion laser at 488 nm and visualized under Nikon Fluorescence microscope.
Flow Cytometry Analysis
Apoptosis induced by methanoilc extract (25 μg/ml) of S. jambos and pure compound β-sitosterol (15 μg/ml) respectively in HepG2 cells was determined by DNA content analysis (sub-G1) by flow cytometry using propidium iodide. Briefly 1 x 106 cells/ml seeded in a 6 well plate was treated with methanolic extract (25 μg/ml) and pure compound (15 μg/ml). Untreated HepG2 cells served as control. The plate was incubated for 24 hours and then cells were pelleted by centrifugation at 10,000 rpm for 10 minutes. The cells were washed once with PBS. Cells were then fixed with 70% ethanol for 2 hours at -20ºC and again washed with PBS. Cells were re-suspended in 300 μl of PBS containing 0.5 mg/ml RNase A and 0.1 mg/ml PI and incubated at dark, 37ºC for 30 min. The cells were analyzed for DNA content using fluorescence activated cell sorter (FACS Calibur System, BD Biosciences) equipped with Cell Quest software (Becton Dickinson, USA). PI fluorescence was collected through an FL-2 filter (585 nm).
Reverse Transcription and Polymerase Chain Reaction (RT-PCR)
RT-PCR was carried out as described by Hall et al., (1998). Briefly, 1 x 106 HepG2 cells/ml were grown in a 6 well plate and treated with methanolic extract of S. jambos and pure compound β-sitosterol (15 μg/ml). The HepG2 cells were incubated for varying time points (3, 6, and 12 hours) to study the mRNA expression of various cellular targets such as Bcl-2 and Bax using semi quantitative RT-PCR. Etoposide treated HepG2 cells served as positive control and untreated cells served as control. RNA extraction was carried out using TRIZOL. Reverse transcription was carried out as follows: 200 ng of random hexamer or 200 ng/ml oligo d[T] was added to 4.5 ml of total RNA and was made up to 10 ml using deionised water. The cDNA was further used for PCR with different primers for the Bax and Bcl-2 gene. PCR products were checked by agarose gel electrophoresis.
Western Blot Analysis
The plates treated with extract and standard compound were incubated for varying time points of 18 and 36 h in HepG2 cells to study the protein expression of various cellular targets such as Bcl-2, Bax, and cytochrome-c. The expression levels were normalized using b-actin as internal. The bands were detected using chromogenic substrate NBT-BCIP in alkaline phosphate buffer.
Results and Discussion
The in vitro study of methanolic extracts of Syzygium jambos Linn leaves in Hep G2 cell showed the cytotoxic activity.
Fragmentation of chromatin to units of single or multiple nucleosomes that form the nucleosomal DNA ladder in agarose gel is an established hallmark of programmed cell death or apoptosis. The gels showed unambiguously that the genomic DNA of the extract treated cells was severely fragmented, at the optimized concentration. Untreated control cells which served as control, showed no fragmentation.
To characterize the cell death induced by test extract the nuclear morphology of dying cells with a fluorescent DNA-binding agent, propidium iodide (PI) was studied. Loss of plasma membrane integrity allowed uptake of propidium iodide, which subsequently entered the cells, bound stoichiometrically to DNA and specifically did stain of DNA. Propidium iodide staining was carried out to check the localization of DNA in the nucleus of HepG2 cells after 40 hours treatment with methanol extract of S. jambos and β-sitosterol. Untreated cells did not show any nuclear fragmentation (Figure 1).
Figure 1. Nuclear localization of HepG2 cells on treatment with crude test extracts by propidium iodide staining.(A-Control; B-Treated with Extract)
The measurement of cellular DNA content by flow cytometry after staining with fluorescent dye propidium iodide was carried out to further confirm and quantify the percentage of apoptotic cells upon incubation with crude extracts of S. jambos and pure compound β-sitosterol. HepG2 cells treated with crude extract and pure compound for various time points and the cells were subjected to FACS analysis. Cells in the G0/G1 phases of the cell cycle (falling under M2) are diploid (2n). Cells within the G2/M phases (M4) have a DNA content of 4n, while S-phase cells (M3) have a DNA content greater that 2n and less than 4n. Cells that are sub-diploid (<2n) are apoptotic (M1) and quantified.
In agreement with DNA fragmentation results, a typical sub-diploid apoptotic peaks were observed in HepG2 cells treated with plant extracts for 40 hrs. The FACS analysis of control cells, on the other showed prominent G1, followed by G2/M phase with only 2% of the cells falling in the sub G0/G1 phase. The value of 2.40% hypodiploid DNA in control cells increased to 44.0% and 63% in the treatment groups of S. jambos and β-sitosterol respectively. These studies thus reveal increase of hypodiploid apoptotic cells in response to test extracts treatment in a time dependent manner and the decrease of the cells at S-phase and G2 phase of cell cycle. Evasion of apoptosis is considered as one of the key factors that determines the transformation of normal cells to malignant cells. Hence, any agent or plant-derived molecule, that has the ability to induce apoptosis in cancer cells, can be considered as a viable drug. The above observation from propidium iodide staining, cell cycle analysis and DNA fragmentation observed by gel electrophoresis provides a potential proof that, the antiproliferative property induced by methanol extract of study plants in HepG2 cells could be due to induction of apoptosis.
Semi quantitative Reverse Transcriptase-PCR Analysis
The pro-apoptotic family member-Bax, under the influence of external stimuli, plays a pivotal role in inducing apoptosis aiding in the release of cytochrome-c. The ratio between anti-apoptotic Bcl-2 and pro-apoptotic Bax has been reported to be the crucial determinant of cellular sensitivity for the induction of apoptosis. A gradual increase in the expression of Bax upon treatment with test extracts was observed in a time dependent manner in HepG2 cells right from 6 hrs. A positive Bax: Bcl-2 ratio facilitates the release of cytochrome-c from mitochondria to cytosol. In the mitochondrial pathway, cytochrome-c is deemed as a potent mediator of apoptosis and appears to directly stimulate the activity of caspases, which then rapidly degrade cellular organelles and chromatin so as to induce cell destruction without inflammatory response.
For elucidating the role of cytochrome-c in the test extract induced apoptosis in HepG2 cells, the levels of cytochrome-c was determined. In HepG2 cells, the test extracts and pure compound were found to have marked increase the cytochrome-c levels compared to untreated control cells. Sub cellular fractionation studies also confirmed the release of cytochrome-c from mitochondria to cytosol (Figure 2). Aforesaid experimental evidences from this study clearly demonstrated the involvement of test extracts in eliciting apoptosis through the release of mitochondrial cytochrome-c in HepG2 cells.
Figure 2. Western blot analysis of apoptotic proteins in HepG2 cells treated with S. jambos and other plants and pure compounds. (Western blot analysis of apoptotic proteins A) Bcl-2, B) Cytochrome c, and C) Caspase -3 in HepG2 cells. B-actin was used as internal cell control. M-Marker 1- Control 2-S. jambos 3,4,5- known plants for comparison)
Mechanism based assays look for activity using isolated systems such as enzymes, receptors, etc. or simply the molecular markers that play a significant role in the signalling cascade of tumour induction and progression. Due to their selectivity and sensitivity combined with good reproducibility and high sample through put, these assays are preferred over other assays and animal models. Utilization of these assay systems and monitoring the after math effects should help in the identification of unique natural products that can be compared with known standard clinical drugs currently available for treating tumours. Since most of the clinically effective antitumour drugs belong to a small number of mechanistic classes, this approach seems reliable, reproducible, time saving, safe, and appropriate. Evidence has emerged from various studies that cell death can either be the consequence of a passive, degenerative process, or the consequence of an active process. The former type of cell death is termed necrosis; the latter is apoptosis (Lotem and Sachs, 1999; Robertson and StenOrrenius, 2000).
The studies such as DNA fragmentation assay, Fluorescent microscope assay, Flow cytometer assay, and gene expression of Bcl-2 and Bax showed that the methanolic extract of Syzygium jambos has potential anti-tumour activity and the mode of action was found to be induced apoptotic pathway.
First Author is thankful to the Director General, CCRAS (Ministry of AYUSH, Government of India, New Delhi) for his support and encouragement. Authors are also extending their regards to staff members of their concerned organizations for their kind support.
Conflict of Interest: Nil
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