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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 1  |  Page : 71-77

Phyllanthus fraternus manifests potent anti-proliferative activity on cultured Daudi cells


Department of Zoology, Biomedical Technology and Human Genetics, School of Sciences, Gujarat University, Ahmedabad, Gujarat, India

Date of Submission20-May-2017
Date of Acceptance25-Feb-2018
Date of Web Publication12-Apr-2018

Correspondence Address:
Felisa P Parmar
Department of Zoology, Biomedical Technology and Human Genetics, School of Sciences, Gujarat University, Ahmedabad - 380 009, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_429_17

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 > Abstract 


Objective: The aim of this study is to observe the apoptosis of Phyllanthus fraternus Webster against Daudi cells and to study its primary mechanism.
Materials and Methods: Antiproliferative activity of cultured Daudi cells was evaluated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay in a dose- and time-dependent manner after treatment with the hydroalcoholic extract of P. fraternus . Trypan blue viability assay was also performed. Apoptosis induction in the cells posttreatment was determined by DNA fragmentation assay, Agarose gel electrophoresis, and Acridine orange/Ethidium bromide dual staining. Protein isolation and analysis was carried out using the standard polyacrylamide gel electrophoresis protocols.
Results: The extracts inhibited the growth and proliferation of Daudi cells through induced cell death, which was dose-dependent and time-dependent. The IC50 value was found to be 220 μg/ml after 72 h of treatment. The induction of DNA fragmentation and increase in a number of apoptotic cells posttreatment suggest the possibility of apoptosis induction. A significant decrease in protein level was also observed.
Conclusion: The results raise the possibility that the hydroalcoholic extract of P. fraternus could be a potent chemotherapeutic agent for the treatment of various cancers. Further evaluation of its potency as a chemotherapeutic agent is imperative.

Keywords: Antiproliferation, apoptosis, Daudi cell line, Phyllanthus fraternus


How to cite this article:
Parmar FP, George LB, Highland HN. Phyllanthus fraternus manifests potent anti-proliferative activity on cultured Daudi cells. J Can Res Ther 2020;16:71-7

How to cite this URL:
Parmar FP, George LB, Highland HN. Phyllanthus fraternus manifests potent anti-proliferative activity on cultured Daudi cells. J Can Res Ther [serial online] 2020 [cited 2020 Jun 6];16:71-7. Available from: http://www.cancerjournal.net/text.asp?2020/16/1/71/230013




 > Introduction Top


Cancer, a severe metabolic disease, is the leading cause of mortality and morbidity worldwide. The number of cancer cases is continuously increasing.[1],[2] Cancer is serious health problem showing significant impact on human health-care system, even with advancements in diagnosis, prevention, and therapy.[3] Cancer is a complex group of diseases with many possible causes, including both, external and internal factors and may act together or in a sequence to induce cancer. Treatment for cancer involves surgery, chemotherapy, radiation therapy, immunotherapy, targeted therapy, hormonal therapy or combinations thereof, depending on the type, location, and grade of cancer. Cancer chemoprevention which is the pharmacological interference with synthetic or naturally occurring compounds is now recognized as a better alternative since it may prevent, inhibit, or reverse carcinogenesis or prevent the development of invasive cancer. The side effects of synthetic chemotherapeutic agents are innumerable, and hence, the search for safer anti-cancer therapeutic agents proves challenging.

Plants are the rich source of medicine from ages. Over the last few decades, there has been increased interest by pharmaceutical industries to discover new drugs from the ethnobotanicals to provide new and alternative drugs to synthetic drugs for the treatment of dreadful diseases.[4]

In India, from ancient times a number of plant extracts are used against various diseases treatments in different systems of medicine such as Siddha, Ayurveda, and Unani. Only a few plants have been significantly explored regarding their medicinal uses. Our earlier studies have revealed the efficacy of Mimosa pudica , Xanthium strumarium, and Tinospora cordifolia plant extract as well as Kaempferol and L-mimosine compounds as potent anti-proliferative agents.[5],[6],[7],[8]

Phyllanthus fraternus Webster is an important medicinal plant, commonly known as bhoiamli, belongs to family Euphorbiaceae. It has been used in traditional system of medicines for over 2000 years.[9] It is widely distributed in tropical and subtropical countries of the world, for example, India, Pakistan, China, Cuba, the Caribbean, Central and South America, Brazil, Nigeria, East and West Africa, West Indies and introduced to Saudi Arabia. Bhuiamla or P. fraternus is one of the valuable medicinal plants. Conventionally, the plant P. fraternus is widely used in folk medicine for treatment of various liver diseases.[10] The plant extract shows antiviral activity, especially against hepatitis virus.[11] The plant was used for different purposes such as a diuretic, antidiabetic, hypotensive, hypoglycemic, antihyperlipidemic, antihepatotoxic, and antioxidant activities.[12],[13] It is used in the treatment of kidney and bladder complications and to control intestinal parasites.[14] Antimicrobial activity of P. fraternus has also been reported.[15] Further studies have also shown that P. fraternus to be effective in the treatment of Diabetes.[16]

Therefore, this study is aimed at investigating the antiproliferative potential of P . fraternus against Daudi lymphoma cell line.


 > Materials and Methods Top


Preparation of plant extracts

P. fraternus was collected from University campus, and the herbarium specimens were authenticated at the Botany Department. Whole plant, namely, stems, leaves, roots, and seeds of P . fraternus were collected, washed, and shade-dried under ambient temperature. After complete drying plants were powdered and defatted with petroleum ether (40°C–60°C) for 24 h at room temperature with constant shaking. A total of 50 g of the defatted powdered material was capsulated in filter paper and kept in the thimble, 500 ml solvent (water:ethanol 70:30) was added into the flask, and continuous extraction was carried out in the Soxhlet apparatus for 72–74 h at 60°C (till the color in the siphon became colourless). The crude solvent collected in the flask was dried at reduced pressure and kept at 4°C until further use.[17]

Cancer cell culture

For cancer cell culture, Daudi cell line was obtained from the National Centre for Cell Science (NCCS), Pune. Cells were cultured in RPMI 1640 media with 10% fetal bovine serum (FBS) and antibiotic-antimycotic solution (Hi-Media, Mumbai, Maharashtra, India). Cell cultures were maintained in a CO2 incubator at 5% CO2 and 37°C.

Cell viability assay by trypan blue dye exclusion technique

Any compound, which is cytotoxic to cells, inhibits the cell growth proliferation and kills the cells. Trypan blue is a supravital dye, used to estimate the number of cells present in the population.[18] It can penetrate dead cells and give it a blue color. This method gives a score of dead and viable cells.[19]

The cellular viability of the cells was measured using trypan blue exclusion assay. Sterility was maintained throughout the procedure. In brief, 2 × 106 cells were seeded into 24-well plates and treated with or without (as control) hydroalcoholic extract of P . fraternus (12.5–400 μg/ml) for 24, 48 and 72 h. After the incubation period, the cultures were harvested and washed twice with phosphate buffered saline (PBS). The cell pellet was then resuspended with 0.5 ml PBS. Then, 20 μl of cells were mixed with equal volume of 0.4% trypan blue and was counted using a Neubauer hemocytometer by clear field microscopy. Viable and nonviable cells were counted. The percentage of cell viability was calculated using the equation shown below:



3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay

The ability of cells to survive a toxic insult is the basis of most cytotoxic assays. This assay is based on the assumption that dead cells or their products do not reduce tetrazolium. It is described by the modified method of Mosmann and Wilson.[20],[21]

The assay detects the reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) by mitochondrial dehydrogenase to blue formazan product, which reflects the normal function of mitochondria. 2 × 106 viable cells/ml were plated into the 96-well cell culture plate. The hydroalcoholic extract was added with the concentrations (12.5–400 μg/ml), respectively, for 24, 48, and 72 h and incubated at 37°C. After incubation, the supernatants were removed and incubated with MTT (0.5 v/v) in RPMI 1640 without FBS and without phenol red for 4 h in a humidified atmosphere at 37°C and 5% CO2 incubator. The absorbance (A) of the colored solution was quantified at 540 nm wavelengths by an enzyme-linked immunoabsorbent assay reader (ELISA READER, MERCK MIOS mini). Each extract and control were assayed in triplicate in three independent experiments. Percent growth inhibition of cells exposed to treatments was calculated as follows:



Agarose gel electrophoresis

DNA fragmentation was assessed using Agarose Gel Electrophoresis of extracted genomic DNA from Daudi cells.[22] 2 × 106 cells/ml were taken and treated with the extract at its IC50 concentration. After 72 h, the culture was harvested and washed twice with PBS. The cell pellet was then resuspended with DNA-Xpress reagent (Hi-Media, Mumbai, Maharashtra, India), homogenized and centrifuged for 10 min at 13,000 rpm. The upper aqueous phase was transferred to another tube and 1 ml ethanol (100%) was added. The sample was mixed by inverting the tube several times, allowed to stand for 5 min (25°C) and centrifuged at 8500 rpm for 4 min. The supernatant was discarded; 1 ml of ethanol was added and mixed by inverting the tube several times. Allowed to stand for 3–4 min and DNA became visible. DNA was washed twice with 1 ml 95% ethanol and dissolved in 8 mM NAOH. Electrophoresis was carried out on a 2% agarose gel. The gel was stained with 1 μg/ml ethidium bromide (EB) and photographed using gel documentation system.

Acridine orange/ethidium bromide fluorescence staining

Analysis of changes in cell morphology was evaluated using acridine orange/ethidium bromide (AO/EB) fluorescence staining according to the method described by Jimenez et al ., with slight modification.[23] In brief, 2 × 106 cells were plated into 24 well cell culture plates and incubated for 72 h with or without (as control) hydroalcoholic extract of P . fraternus . The cells were removed from the plate in 1 ml microfuge in PBS pH 7.4. To each sample, 1 μl of AO/EB was added only before microscope examination. A volume of 10 μl of cell suspension was placed on a microscope slide and observed under a fluorescence microscope under ×40 objective.

Protein estimation

For protein estimation, cells after dosing with an IC50 concentration of the extract for 72 h, were then removed and washed with PBS twice. PBS was discarded, and cells were suspended in 500 μl RIPA Buffer (G-Biosciences, St. Louis, USA) and kept on ice for 20 min. Cells centrifuged for 15 min at 14,000 rpm at 4°C. The supernatant was taken into another tube for further analysis.

The concentration of protein was determined using Lowry's method using the Folin–Ciocalteu reagent, with crystalline bovine serum albumin as the standard.[24]

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis (PAGE) was performed for protein separation. Cells were treated with the extract at IC50 concentration for 72 h and protein was extracted from the cells by the above-mentioned protocol. The protein sample was prepared by mixing sample obtained, with sample buffer containing glycerol, 20% sodium dodecyl sulfate (SDS), Bromophenol blue dye, 1 M Tris-Cl, and β-Mercaptoethanol. A volume of 20 μl of samples were loaded into the wells after the polymerization of gel and were run on the electrophoresis unit. The gels were fixed and then stained 0.25% Coomassie Brilliant Blue. After overnight staining, the gels were destined and then photographed and analyzed.

Statistical analysis

Each parameter was performed in triplicate, and the results were expressed as a mean ± standard error. The data were statistically analyzed using Student's t -test and the values of P < 0.05 were considered statistically significant.


 > Results Top


Cell viability assay

We evaluated the cell viability assay by trypan blue dye exclusion technique [Figure 1]. This assay showed that there was a highly significant ( P < 0.001) decrease in viability with an increase in time and concentration in extract treated Daudi cells as compared to untreated controlled cells [Figure 1].
Figure 1: The effect of Phyllanthus fraternus extract on the percentage viability of Daudi cells. Values are mean ± standard error for the three individual experiments. ** P < 0.005, *** P < 0.001

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3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay

Using MTT assay, the effect of the hydroalcoholic extract of P. fraternus on the proliferation of Daudi cell line was studied after 24, 48, and 72 h of incubation. As shown in [Figure 2], the treatment of Daudi cells with the extract lead to inhibition in the cell proliferation as concluded by the IC50 value 220 μg/ml. This revealed a moderate antiproliferative activity of the extract against Daudi cells. Increasing the concentrations of the extract in the culture medium of Daudi cells resulted an increase in the percentage of inhibition of proliferation with increased time duration as compared to untreated control cells ( P < 0.001).
Figure 2: The effect of Phyllanthus fraternus extract on the percentage decrease of proliferation in Daudi cells. Values are mean ± standard error for the three individual experiments. *** P < 0.001

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Agarose gel electrophoresis

Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. The conventional agarose gel electrophoresis was performed on the Daudi cells treated at IC50 dose concentration of hydroalcoholic extract for 72 h. The results showed that DNA cleavage produced no ladder pattern for the hydroalcoholic extract-treated cells [Figure 3]. The DNA may be intact, or no DNA fragmentation was detected.
Figure 3: DNA pattern of treated and untreated Daudi cells at 72 h after dosing, along with 100 blood pressure DNA ladder on Agarose gel electrophoresis. Lane 1 - Showing 100 blood pressure DNA ladder, Lane 2 - effect of Phyllanthus fraternus at IC50concentration on Daudi cell DNA, Lane 3 - Untreated control Daudi cell DNA

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Acridine orange/ethidium bromide double staining

The control and treated Daudi cells were stained with AO/EB dual stain where the live cells took up green color, and the apoptotic cells/nonproliferative cells were seen as yellowish orange. The result obtained depicted that there was a significant increase ( P < 0.001) in a number of apoptotic cells/nonproliferative cells with increased dose and time duration as compared to untreated control cells [Figure 4].
Figure 4: Apoptotic changes of Daudi cells detected with acridine orange/ethidium bromide staining and visualized under fluorescence microscope (×40) L-Live cells and A-apoptotic cells/nonproliferative cells

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Protein estimation

To further confirm the inhibitory effect of hydroalcoholic extract at the cellular level, Daudi cells were treated with an IC50 dose concentration of extract followed by protein estimation. Cells treated with the extracts showed a significant decrease in the protein levels as compared to untreated control Daudi cells [Figure 5].
Figure 5: Alterations in levels of total proteins after treatment with Phyllanthus fraternus extract compared with untreated control Daudi cells. Values are mean ± standard error for the three individual experiments. *** P < 0.001

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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The results of SDS PAGE had shown clear dark bands of proteins having an approximate molecular weight of 200, 180, 75, 63, 50, 45, 35, 15, and 11 kDa in control Daudi cells. Two prominent bands between 63 and 35 kDa as well as one prominent band of 11 kDa were observed in the gel pattern after cells were treated with IC50 concentration for 72 h with the extract of Phyllanthus [Figure 6].
Figure 6: Separated protein pattern of treated and untreated Daudi cells at 72 h after treatment, along with marker on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Lane 1 - Protein marker, Lane 2 - Untreated control Daudi cell proteins, Lane 3 - Showing Phyllanthus fraternus treated at IC50concentration on Daudi cell for 72 h

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 > Discussion Top


Plants are important and supplementary sources of drugs. It can provide biologically active molecules which are used for treatments of varieties of disease. P. fraternus Webster is a well-known herbal medicine globally. Many studies have reported the pharmacological efficacies and benefits of P . fraternus .

Trypan Blue staining for cell viability was used to verify the influence of the plant extracts in mediating cell death. As observed in the present study cell survival was lowered in a time and dose-dependent manner. It was reported that hairy root extract of Phyllanthus amarus induced apoptosis of MCF-7 cells establishing the potential anticancer activity.[25] Other in vitro study results showed the antimetastatic potentials of Phyllanthus emblica extract on fibrosarcoma cells through the modification of matrix metalloproteinase (MMP2) and MMP9 expression.[26] As observed in another study, phyllanthin and hypophyllanthin, plant compounds, exhibited a strong inhibitory effect on the growth of MCF-7 and MDA-MB-231 human breast cancer cells in vitro and inhibited the growth of mammary carcinoma in vivo .[27] Hence, it could be hypothesized that decrease in cell viability could be attributed to P. fraternus extract/compounds induced cell death as confirmed by the cell viability trypan blue assay.

A novel strategy for the control of tumor growth in cancer cells is control of cell proliferation.[28],[29] As the molecular analysis of human cancers have revealed that the cell cycle regulators are frequently mutated in most common malignancies.[30],[31] In a related study, Lee et al . reported cytotoxicity effect on human lung (A549) and breast (MCF-7) cancer cell lines that were determined using MTS reduction assay.[32] Our in vitro data indicated that treatment of Daudi cells with Phyllanthus extract resulted in inhibition of proliferation of Daudi cells. Any decline in the number of metabolically active proliferating cells might mean that the proliferation pathway itself was halted (mitotic arrest), or that fraction of the cells went through a death pathway. Hence, it could be postulated that the reduction in cell proliferation, as registered by the MTT assay could be attributed to P. fraternus extract induced cell death.

A typical ladder pattern was not evident in Daudi cells treated with P . fraternus extract. This may be indicative that the cells die by apoptosis, where DNA damage is not only a unique feature of apoptosis but can also occur in necrosis.[33] DNA extracted from apoptotic cells often show a ladder pattern and the presence of smear pattern may indicate that apoptotic cells enter into late apoptosis (secondary necrosis) because of the absence of phagocytosis to remove cell remnants[34] or some cell lines, such as MCF-7, can undergo apoptosis without showing DNA fragmentation due to the lack of caspase-3 which is responsible for this feature.[35],[36] Another explanation may be that the long incubation time of DNA led to lyses of a substantial part of the cell population. The DNA isolated from the control cells exhibited one clear band that pointed to the presence of living cells with intact DNA strand.

The changes that occurred in apoptotic cells/nonproliferative cells were perceived through AO/EB staining, and this helped in deducing that the cell death observed was not due to necrosis, but may be due to apoptosis. Once the cell has begun to die, the earliest observable ultra-structural event is the complication and segregation of the nuclear chromatin, with the formation of sharply delineated and finely uniform granular masses that become marginated against the nuclear envelope with simultaneous condensation of the cytoplasm. Progression of the condensation is accompanied by convolution of the nuclear and cell outlines, and this is followed by breaking up of the nucleus into discrete fragments that are surrounded by a double-layered envelope and by budding of the cell as a whole to produce membrane-bound apoptotic bodies.[37] In our study, when cells stained with AO/EB, it was observed that there was a higher percentage of cell with orange–red nuclei with condensed chromatin which possibly indicate apoptotic cells.

To further determine the effect of P . fraternus extract at the cellular level, Daudi cells were treated with IC50 dose concentration of extract followed by SDS PAGE analysis of its cell lysates. Administration of the P. fraternus extract brought about a significant decrease in the total protein content indicated loss of protein as depicted on the scanned gels by the loss of protein bands.

A protein, whose apparent molecular weight is between 30 and 50 kDa was identified, showed that medium conditioned from the male cell line Daudi causes ovarian dysgenesis closely linked to the death of the Drosophila melanogaster female germ cells.[38] Another H-Y antigenic plasma membrane protein was excreted into the culture medium was identified as a series of polymers of 18 kDa. It has testis organizing function.[39] In addition, however, it is not known whether those proteins have any activities.

Tang et al . have shown a positive result with various extract of Phyllanthus against MeWo cells. The possible mechanism behind the anti-cancer activity of Phyllanthus extract inspection of their regulation in multiple signaling pathways that involves NFkB, Myc/Max, Hypoxia, MAPK/ERK and MAPK/JNK and protein-protein interaction in melanoma MeWo cells. The identified differentially expressed proteins could become potential target development.[40] In another study, Tang et al ., have also shown a positive result with various extract of Phyllanthus against PC-3 cell line. Phyllanthus exert its antiproliferative and apoptotic effects through suspension of MAPKs, PI3K/Akt, Wnt, Myc/Max, Hypoxia, and NFkB signaling cascade in vitro .[41] Our in vitro data indicate that treatment of Daudi cells with Phyllanthus extract resulted into significant inhibition of proliferation could be due to the suspension of multiple signaling pathways as well as protein-protein interaction or with phytoconstituent of the extract.


 > Conclusion Top


The in vitro cytostatic activity of P . fraternus extract against Daudi cells was positive and translated into potent antiproliferative action. The results reveal that P . fraternus has a strong antiproliferative effect by inducing apoptotic cell death. The mode of action of the phytoconstituents present in this plant is still unclear and understanding the detailed mechanism will help in improving useful information for their possible application in cancer prevention and perhaps also in cancer therapy.

Acknowledgment

This study was supported by university grant commission, in the form of Maulana Azad National Fellowship.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]



 

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