CC BY-NC-ND
0J Acupunct Meridian Stud 2013;6(3):149-158
ELSEVIER
Available online at www.sciencedirect.com
Journal of Acupuncture and Meridian Studies
journal homepage: www.jams-kpi.com
I RESEARCH ARTICLE I
Anticancer Activity of Zea mays Leaf Extracts on Oxidative Stress-induced Hep2 Cells
Kiruthika Balasubramanian, Palghat Raghunathan Padma*
Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Deemed University, Tamil Nadu, India
Available online Feb 11, 2013
Received: Aug 9, 2012 Revised: Nov 23, 2012 Accepted: Nov 28, 2012
KEYWORDS
apoptosis; fluorescent staining
techniques; Hep2 cells; viability assays; Zea mays leaves
Abstract
Cancer is one of the leading causes of death in humans. It is believed that plants can provide potential bioactive compounds for the development of "new leads" to combat cancer and other diseases. The present study focuses on the ability of the different extracts (aqueous, methanol, and chloroform) of the leaves of Zea mays in influencing the process of apoptosis induced by hydrogen peroxide (H2O2) in Hep2 (laryngeal carcinoma) cells. Various apoptosis-related parameters, such as cell viability, morphological changes, nuclear changes, and apoptotic index were characterized. sulforhodamine B and MTT assays were used to quantify the extent of cell death in the group exposed to H2O2, plant extracts, and their combination. Treatment with H2O2 caused cytotoxicity in cancer cells. The administration of leaf extract also caused an increase in the death of cancer cells. Oxidatively stressed cancer cells co-treated with all the Z. mays leaf extracts (except the chloroform extract) demonstrated cytotoxicity on a par with the H2O2-treated groups. This indicated that the aqueous and methanol leaf extracts did not influence the cytotoxic action of H2O2 in the cancer cells. Thus, various apoptosis-related events in Hep2 cells exposed to leaf extract throw light on the potential anticancer activity of the Z. mays leaves. The maximum activity was exerted by the methanolic extract followed by the aqueous and chloroform extracts.
* Corresponding author. Department of Biochemistry, Biotechnology and Bioinformatics, Faculty of Science, Avinashilingam Deemed University, Coimbatore, Tamil Nadu 641 043, India. E-mail: prpadma@yahoo.co.in. Copyright © 2013, International Pharmacopuncture Institute pISSN 2005-2901 elSSN 2093-8152 http://dx.doi.org/10.1016/j.jams.2013.01.015
1. Introduction
Chemoprevention, a relatively new and promising strategy to prevent cancer, is defined as the use of natural dietary compounds and/or synthetic substances to block, inhibit, reverse, or retard the process of carcinogenesis. Various cancer chemopreventative agents can encourage apoptosis in premalignant and malignant cells in vivo and/or in vitro, which is conceivably another anticancer mechanism [1].
Thus, the search for natural products represents an area of great interest in which the plant kingdom has been documented as the most important source to provide many antioxidant and cancer chemopreventative agents with novel structures and unique mechanisms of action. With this backdrop, the present study was formulated to analyze the anticancer potential of Zea mays leaves. Z. mays is commonly known as makkacholam or maize. It belongs to the family Gramineae. Our preliminary studies with three different extracts (aqueous, chloroform, and methanol) revealed that it possesses strong antioxidant properties.
2. Materials and methods
2.1. Preparation of plant extracts
Z. mays seeds were procured from Tamil Nadu Agriculture University (TNAU) in Coimbatore district, Tamil Nadu. They were grown within the university campus in pots. Plant growth was observed at 5, 10, 15, 20, 25 and 30 days after sowing. The leaves on the 10th day of growth were found to have maximum antioxidant activity [2]. Hence, 10-day-old plants were selected for the present study. The plantlets were uprooted and washed thoroughly with running tap water. Then the leaves were blotted dry between folds of filter paper to remove excess water.
2.2. Aqueous extract
The leaves were homogenized in water (1 g/mL) using a micropestle in a microfuge tube. They were centrifuged at low speed to clarify the extract. The supernatant corresponding to the concentration of 20 mg/20 mL was used for the assay.
2.3. Methanolic/chloroform extract
Z. mays leaves (1.0 g) were homogenized in approximately 1 mL of the solvent (methanol/chloroform). The supernatant was collected and dried at 60°C, well protected from light. The residue obtained after drying the chloroform and methanol extracts was weighed and dissolved in a known amount of dimethylsulfoxide (DMSO) to yield a concentration of 20 mg/5 mL. DMSO was maintained at a minimum level to avoid DMSO-induced events, if any. The extract preparation took around 2 hours.
2.4. Hep2 cell line culture
The cell line was procured from the National Centre for Cell Science, Pune, India. The cells were maintained in a CO2
incubator with 5% CO2 and 95% humidity, and supplemented with Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum (FBS). Penicillin and streptomycin (PAA) were also added to the medium to 1 x final concentration from a 100 x stock.
Once the cells had attained confluent growth, the cells were trypsinized using Trypsin-EDTA (PAA) and the number of cells needed for carrying out various assays was seeded into sterile six-well and 96-well plates. In each well of the six-well plates, a clean, dry, sterile coverslip was placed before the cells were seeded. Then the plates were incubated in a CO2 incubator with 5% CO2 and 95% humidity.
Hydrogen peroxide (H2O2) at a concentration of 200mM was used as an oxidant. The concentration of plant extract used was 20 mg. The cells were treated with the oxidant, both in the presence and absence of the leaf extracts. The cells were exposed to H2O2 for 1 hour at 37°C. The time points were arrived at by conducting a time-related response analysis of each cell type.
After treatment, the coverslips from the six-well plates were removed and placed on a glass slide and sealed with Vaseline. These slides were used for various staining techniques; however in 96-well plates, the medium was removed and replaced with fresh medium. These were used for checking the viability status.
2.5. Parameter analysis
The apoptotic events that occurred after the oxidant treatment were analyzed using various parameters [viability assays and Giemsa, propidium iodide (PI), acridine orange/ethidium bromide (AO/EtBr), 4,6-diamino-2-phenylindol dihydro chloride (DAPI), and EtBr staining], as described below.
2.6. MTT assay
A total of 106/107 cells per Eppendorf were seeded into 96-well plates and exposed to H2O2/plant extracts for 1 hour. The cytotoxicity of the drugs was assessed by the MTT assay according to the procedure used by Igarashi and Miyazawa
[3]. MTT (50 mL) was added to the treated wells. The plate was incubated at 37°C for 3 hours with mild shaking. The cells were re-suspended in 200 mL of 2-propanol containing 0.04N HCl overnight in the dark. The absorbance was read at 650 nm, in a microtiter plate reader (Anthos, Germany).
2.7. Sulforhodamine B assay
Sulforhodamine B (SRB) binds to basic amino acid residues in TCA-fixed cells to provide a sensitive index of cellular protein content that is linear over a range of cell densities
[4]. The cell survival was measured at 492 nm.
2.8. Giemsa staining
The incubated cells were spread on the microscopic slides with a drop of diluted Giemsa stain. The slides were mounted with cover slips and observed under a phase contrast microscope (Nikon, Japan) for morphological changes as described by Chih et al [5]. The number of cells showing apoptotic morphological changes was counted in
each experimental group per 100 cells in 10 different fields and the experiment was repeated five times.
2.9. PI staining
PI staining was employed to discriminate apoptotic cells from normal cells, which reflects the nuclear changes during apoptosis using the protocol developed by Sarker et al [6]. After incubation with H2O2 and/or plant extracts, the cells were permeabilized with a mixture of acetone and methanol (1:1) at —20oC for 10 minutes. Then, 10 mLof 5 mg/mL PI was added to each slide, spread with a cover slip and incubated at 37oC for 30 minutes in the dark. The apoptotic cells were detected using the green filter of a fluorescence microscope (Nikon, Japan).
2.10. EtBr staining
The treated cells were incubated for 5 minutes with 10 mL of EtBr (50 mg/mL) and spread by placing a cover slip over it. The apoptotic cells were scored by counting the cells with condensed chromatin and fragmented nuclei under a fluorescence microscope (Nikon, Japan) using UV 2A filter at 400x magnification. The ratios of apoptotic cells to normal cells were calculated in each staining method [7].
2.11. DAPI staining
The apoptotic ratios of the oxidant-treated cells with or without the leaf extract to the untreated cells were calculated by DAPI staining [8]. The cells were then transferred to slides and were immediately fixed with 3% paraformaldehyde and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 10 minutes at room temperature. They were then incubated with 5 mg/mL of DAPI. The apoptotic nuclei were observed under a fluorescence microscope (Motic, Germany) using a DAPI filter.
2.12. AO/EtBr staining
AO (0.1 mg/mL) and EtBr (0.1 mg/mL) were used to label nuclear DNA in Hep2 cells. Both solutions were prepared in PBS buffer (pH 7.4). PBS buffer was used to preserve normal physiological activity for unicellular cells. For cell staining, Hep2 cell samples (100 mL) were stained with AO/EtBr (5 mL) and observed under a fluorescence microscope (Nikon, Japan) with a B-2A filter [9].
2.13. Statistical analysis
Statistical significance was determined by a two-way analysis of variance (p < 0.01) using Agress statistical software (version 3.01).
3. Results and discussion
3.1. Cell viability assays
Cell viability was assessed by MTT assay, which is a colori-metric assay for measuring the activity of cellular enzymes
100 90 80 70 60 50 40 30 20 10 0
□ Control
□ H2O2 treated
No extract Aqueous Methanol
extract extract
Chloroform extract
Figure 1 Effect of Zea mays leaf extracts on the viability of Hep2 cells subjected to oxidative stress as determined by the MTT assay. The values of the negative (untreated) control group were fixed as 100% viability and the percentage viabilities in the other groups were calculated relative to this.
that reduce the tetrazolium dye, MTT, to its insoluble for-mazan, giving a purple colour. Fig. 1 shows the results of evaluation of cytotoxicity of all three extracts of Z. mays at a concentration of 20 mg/mL and an exposure time of 1 hour. These data showed that the treatment with H2O2 exhibited cytotoxicity in the cancer cells. The administration of leaf extracts also caused increased death of cancer cells. Oxidatively stressed cancer cells co-treated with the Z. mays leaf extracts (apart from the chloroform extract) showed cytotoxicity on a par with H2O2-treated groups. This indicates that the aqueous and methanol leaf extracts did not influence the cytotoxic action of H2O2 in the cancer cells. Using the MTT assay, many researchers found time- and dose-dependent inhibition in different cells with different agents.
Inhibition of cell proliferation by sesquiterpene lactones (tomentosin and inuviscolide) from Inula viscosa (Compo-sitae) leaves against human melanoma cell lines was reported by Rozenblat et al [10].
An SRB assay was also performed to assess cell survival. The results of this assay showed a similar trend to that
90 ■■ 80 ■■ 70 ■■ 60 ■■ 50 ■■ 40 ■■ 30 ■■ 20 -10 ■■ 0 J"
□ Control
□ H2O2 treated
No extract Aqueous Methanol Chloroform
extract extract extract
Figure 2 Effect of Zea mays leaf extracts on the viability of Hep2 cells subjected to oxidative stress as determined by the SRB assay. The values of the negative (untreated) control group were fixed as 100% viability and the percentage viabilities in the other groups were calculated relative to this.
Table 1 Effect of Zea mays determined by Giemsa staining. leaf extracts on the morphological changes in Hep2 cells subjected to oxidative stress as
Sample No. of apoptotic cells/100 cells Apoptotic ratio
Control H2O2 treated Control H2O2 treated
No extract 9 ± 1 52 ± 2* 0.10 1.10
Aqueous extract 45 ± 2* 60 ± 1*'14 0.81 1.50
Methanol extract 63 ± 3* 68 ± 2*'14 1.70 2.13
Chloroform extract 40 ± 2* 54 ± 2*'z 0.67 1.17
The values are means ± SD of triplicates. CD value = 4.7. * Statistically significant (p < 0.01) compared with untreated control group. y Statistically significant (p < 0.01) compared with oxidant treated group. z Statistically significant (p < 0.01) compared with respective plant extract treated group.
\ ,, -% ^ < y
I V r ■
J NT* 2 ^ Ï : 'S ' '
Aqueous extract
Aqueous extract + H202
y . ' ■ 'IT ' . V m ^
5»—I
Methanol extract
Methanol extract + H20,
Chloroform extract
Chloroform extract + H,0,
Figure 3 Apoptotic Hep2 cells showing membrane blebbing when stained with Giemsa exposed to 200 mM of H2O2 at 100x magnification. The number of shrunken cells was found to increase in the groups co-treated with the leaf extracts. The apoptotic cells with blebbing, cell shrinkage, nuclear fragmentation, and margination of the nucleus are indicated by arrows.
Table 2 Effect of Zea mays leaf extracts on nuclear changes in Hep2 cells subjected to oxidative stress as determined by PI
staining.
Sample No. of apoptotic cells/100 cells Apoptotic ratio
Control H2O2 treated Control H2O2 treated
No extract 8 ± 3 51 ± 2* 0.09 1.04
Aqueous extract 48 ± 2* 59 ± 2*'t4 0.92 1.44
Methanol extract 61 ± 1* 73 ± 1*'t4 1.56 2.70
Chloroform extract 34 ± 2* 49 ± 1*'} 0.52 0.96
The values are means ± SD of triplicates. CD value = 4.46.
* Statistically significant (p < 0.01) compared with the untreated control group.
y Statistically significant (p < 0.01) compared with the oxidant-treated group.
z Statistically significant (p < 0.01) compared with the respective plant extract-treated group.
Aqueous extract
Aqueous extract + H202
Methanol extract
Methanol extract + H,0,
Chloroform extract
Chloroform extract + H,0,
Figure 4 Photographic record of the apoptosing cells in each treatment group stained by PI. A greater number of apoptotic cells were observed in the leaf extract-treated groups exposed to oxidative stress. The apoptotic cells with blebbing, cell shrinkage, nuclear fragmentation, and margination of the nucleus are indicated by arrows.
Table 3 Effect of Zea mays leaf extracts on nuclear changes in Hep2 cells subjected to oxidative stress as determined by EtBr staining.
Sample No. of apoptotic cells/100 cells Apoptotic ratio
Control H2O2 treated Control H2O2 treated
No extract 5 ± 3 48 ± 2* 0.05 0.92
Aqueous extract 47 ± 2* 55 ± 2*'14 0.89 1.22
Methanol extract 61 ± 2* 70 ± I*'1"4 1.56 2.33
Chloroform extract 35 ± 2* 50 ± 2*'z 0.54 1.00
The values are means ± SD of triplicates. CD value = 4.9. * Statistically significant (p < 0.01) compared with the untreated control group. y Statistically significant (p < 0.01) compared with the oxidant-treated group. z Statistically significant (p < 0.01) compared with the respective plant extract-treated group.
Figure 5 Apoptotic Hep2 cells showing membrane and nuclear changes when stained with ethidium bromide exposed to 200 mM of H2O2 at 100x magnification. These pictures indicate that the Zea mays leaves were highly efficient at inducing apoptosis in cancer cells, both in the presence and absence of H2O2. The apoptotic cells with blebbing, cell shrinkage, nuclear fragmentation, and margination of the nucleus are indicated by arrows.
Table 4 Effect of Zea mays leaf extracts on nuclear changes in Hep2 cells subjected to oxidative stress as determined by DAPI
staining.
Sample No. of apoptotic cells/100 cells Apoptotic ratio
Control H2O2 treated Control H2O2 treated
No extract 6 ± 2 68 ± 2* 0.06 2.13
Aqueous extract 47 ± 2* 56 ± 1*'t4 0.89 1.27
Methanol extract 50 ± 3* 58 ± 3*'t4 1.00 1.38
Chloroform extract 43 ± 3* 53 ± 1*'y'} 0.75 1.13
The values are means ± SD of triplicates. CD value = 5.4.
* Statistically significant (p < 0.01) compared with the untreated control group.
y Statistically significant (p < 0.01) compared with the oxidant-treated group.
z Statistically significant (p < 0.01) compared with the respective plant extract-treated group.
Figure 6 Apoptotic shrunken Hep2 cells with nuclear changes when stained with DAPI exposed to H2O2. The most apoptotic cells were found in the leaf extract-treated groups. The apoptotic cells with blebbing, cell shrinkage, nuclear fragmentation, and margination of the nucleus are indicated by arrows.
Figure 7 Hep2 cells showing red fluorescence indicated that they are undergoing apoptosis with acridine orange/ethidium bromide stain and normal cells showed green fluorescence. This staining method clearly indicates the anticancer effect of Zea mays leaf extracts. The apoptotic cells with blebbing, cell shrinkage, nuclear fragmentation, and margination of the nucleus are indicated by arrows.
observed in the MTT assay, and are presented in Fig. 2. An extract of the rhizomes of Iris tectorum Maxim exhibited a very high cytotoxicity against MCF-7 (breast cancer) and C32 (melanoma) cell lines [11].
3.2. Membrane morphological characteristics
Morphological changes, including plasma membrane bleb-bing, changes to the cell membrane, such as loss of membrane asymmetry and attachment and cell shrinkage, are the early stages of apoptosis that can be analyzed by Giemsa staining. The ratios of cells that undergo apoptosis to normal cells, when exposed to H2O2 in both the presence and absence of Z. mays leaf extracts, are presented in Table 1.
The results revealed that the occurrence of morphologically altered cells was greatest in the H2O2-treated group. Treatment with the leaf extracts also exerted an
apoptosis-inducing effect in the cancer cells (Fig. 3). The number of apoptotic cells increased further upon co-exposure of the leaf extract with H2O2. The methanolic extract of Z. mays leaves was found to be very active against Hep2 cells. A crude ethanolic extract of the plant Lycopodium clavatum considerably inhibited the growth of HeLa cells through the induction of apoptosis via caspase-3 activation, as determined by morphological changes [12].
4. Nuclear changes
Apoptosis is a form of cell death that allows for the elimination of damaged or unwanted cells without damaging the organism. The most obvious characteristics of this form of cell death are cytoplasmic and nuclear condensation, followed by internucleosomal DNA cleavage, membrane blebbing, and finally cell fragmentation [13].
Table 5 Effect of Zea mays leaf extracts on nuclear changes in Hep2 cells subjected to oxidative stress as determined by AO/
EtBr staining.
Sample No. of apoptotic cells/ 100 cells Apoptotic ratio
Control H2O2 treated Control H2O2 treated
No extract 8 ± 2 54 ± 3* 0.09 1.17
Aqueous extract 47 ± 3* 65 ± 1*'t4 0.89 1.85
Methanol extract 64 ± 1* 72 ± 1*'t4 1.78 2.58
Chloroform extract 39 ± 1* 54 ± 3*'} 0.64 1.17
The values are means ± SD of triplicates. CD value = 4.98.
* Statistically significant (p < 0.01) compared with the untreated control group.
y Statistically significant (p < 0.01) compared with the oxidant-treated group.
z Statistically significant (p < 0.01) compared with the respective plant extract-treated group.
Nuclear changes were observed in Hep2 cells exposed to H2O2 in both the presence and absence of the Z. mays leaf extracts using various staining techniques (PI, EtBr, DAPI, and AO/EtBr).
The data obtained with PI staining and the apoptotic indexes calculated are presented in Table 2. As shown in Fig. 4, control cells exhibited intact nuclei, but cells treated with 200 mM of H2O2 showed significant nuclear fragmentation. The apoptotic ratio of cells treated with oxidant was 1.04, whereas the treated cells co-exposed to leaf extracts (water, methanol, and chloroform) were 1.44, 2.70, and 0.96, which indicates that the Z. mays leaf extracts did not influence the cytotoxic action of H2O2 in the cancer cells. The effect of an ethanolic extract of Tremella mesenterica on the induction of apoptosis in human lung carcinoma A549 epithelial cells was also assessed by PI staining [14].
In addition to the observation of nuclear fragmentation, the anticancer potential of leaf extracts was confirmed by EtBr staining in each treatment group and the results are given in Table 3. The nuclear changes observed are shown in Fig. 5.
H2O2-treated cells co-treated with leaf extract exhibited morphological changes typical of apoptosis, including cell shrinkage, plasma membrane blebbing, chromatin condensation, and nuclear fragmentation, compared with the control cells with prominent rounded nuclei and defined plasma membrane contours. The leaf extracts, by themselves, also caused an induction of apoptosis in Hep2 cells. The aqueous and chloroform extracts also exhibited considerable apoptotic effects in the cancer cells.
Two phytochemicals, resveratrol and citroflavon-3-ol, and four plant extracts (grape seed polyphenols, olive oil extract, bearberry, and Echinacea) examined for their effect on cell viability by the EtBr assay provided evidence for strong protection against oxidative stress in H2O2 stress-induced U937 cells [15].
DAPI staining is used to observe the nuclear contents in shrunken cells. DAPI has been used to study pamidronate, anti-proliferative, apoptotic, and anti-migratory effects in hepatocellular carcinoma cells [16]. The extent of nuclear changes observed during H2O2-induced apoptosis after DAPI staining in the different treatment groups of Hep2 cells are presented in Table 4.
As is deducible from the values listed in Table 4, a trend similar to that of EtBr was noted with DAPI staining. The cells with altered nuclear changes were found to be greatest in the H2O2-treated group, which shows the apoptotic ratio of 0.92. The extracts of Z. mays leaves were also highly efficient in inducing apoptosis in cancer cells, both in the presence and in the absence of H2O2 (Fig. 6). Human breast cancer cells treated with the extracts of Astrodaucus persicus also showed potential decrease in the cell proliferation by staining with DAPI. [17] The results with Giemsa and the nuclear stains were further affirmed by AO/EtBr staining (Fig. 7), as is evident from the data obtained (Table 5).
AO stain is membrane permeable and marks the nuclei green, and EtBr, which binds to DNA, is mainly taken up by cells when membrane integrity is lost and stains the nuclei red. Since AO intercalates in the DNA but only interacts with the RNA, viable cells do not uptake EtBr and these cells exhibit green nuclei. However, EtBr is taken up by dying cells, which turn red [18].
Treatment with a combination of AO/EtBr has been used as a reliable index of cellular degeneration [19]. Sangui-narine, a benzophenanthridine alkaloid derived from the root of Sanguinaria canadensis, induced apoptosis in human cancer cells, which was assessed by AO/EtBr staining [20].
Phytochemical screening of the leaves identified phe-nolics and flavonoids as the major antioxidant components in the leaves of Z. mays [21]. The cancer-protective effects of flavonoids and phenolics have been attributed to a wide variety of mechanisms, including free radical scavenging, modifying enzymes that activate or detoxify carcinogens [22].
5. Conclusion
The aim of anticancer agents is to trigger the apoptosis signaling system in these cancer cells while disturbing their proliferation. Plants have many phytochemicals with various bioactivities, including antioxidant, anti-inflammatory, and anticancer activities. Therefore, many plants have been examined to identify new and effective antioxidants and anticancer compounds, as well as to elucidate the mechanisms of cancer prevention and apoptosis. Our
findings suggest that the Z. mays leaf extracts most likely have anticancer properties. The qualitative analysis revealed the presence of phenolics and flavonoids in Z. mays leaves, which may contribute to their medicinal properties.
Acknowledgments
The authors thank Indian Council of Medical Research, New Delhi for financial assistance to BK in the form of an SRF.
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