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Combination of phenylpropanoids with 5-fluorouracil as anti-cancer agents against human cervical cancer (HeLa) cell line.

ARTICLE INFO

Keywords: Phenylpropanoids Synergy Quantitative structure activity relationship 5-Fluorouracil Combination index

ABSTRACT

Combination therapy is the most effective treatment strategy in cancer to overcome drug toxicity and drug induced resistance. The effect of eight phenylpropanoids in combination with 5-fluorouracil against the cervical cancer cells (HeLa) is reported here. The cytotoxic activity of these phenylpropanoids against HeLa cells is in the order of eugenol > ferulic > cinnamic > caffeic > chlorogenic > p-coumaric > 3,4-dimethoxycinnamic > 2,4,5-trimethoxycinnamic acids. Eugenol, ferulic and caffeic acids interacted synergistically with the drug, in bringing about a reduction

in the amount of the latter. Flow cytometry results indicated that the combination of eugenol and 5-fluorouracil increased the number of cells in the S and G2/M phases when compared to treatment with the individual compounds alone. This indicates that they possess different cell cycle targets and induce apoptosis in the cancer cells. In vitro hemolytic activity of phenylpropanoids on human erythrocytes showed that the compounds possessed minimum amount of hemolytic activity, indicating that they can be used as drugs without causing adverse toxicity. 3D-quantitative structure activity relationship studies indicate the importance of electrostatic region near the substitutions present in the benzene ring and near the double bond of the compounds for anticancer and hemolytic activities, respectively. The models derived had good statistical predictive capability.

[c]2012 Elsevier GmbH. All rights reserved.

Introduction

Despite advances in therapy, the treatment of cancer remains poor due to resistance developed by the cancer cells to conventional chemotherapeutic drugs. So the search for new alternatives is needed. Conventional chemotherapy of cancer with 5-fluorouracil (5-FU) in combination with other agents improves the overall and disease-free survival of patients after surgery (Machover 1997). The response of 5-fluorouracil when given alone produces a survival rate between 11 and 17% with a median of approximately 1 year (The Advanced Colorectal Cancer Meta-Analysis Project 1992). In combination with newer medicines including irinotecan or oxaliplatin, it improves the response rates for advanced cancer to 40-50% and increases the median survival time to 15-20 months (Bouzid et al. 2003; Giacchetti et al. 2000). Combination of 5-fluorouracil with relatively nontoxic phytochemicals from fruits and vegetables enhances the efficacy of chemotherapy with a lower toxicity to normal cells (Akao et al. 2008). Use of phytochemicals or herbal extracts for the treatment of cancer appears to be very promising and is being researched (Hemalswarya and Doble 2006). Natural products acting in synergy with commercial drugs to bring about a positive or enhance effect is well documented (Hemalswarya and Doble 2006, 2008).

The phenylpropanoid pathway is responsible for the biogenesis of several structurally diverse groups of compounds. Many phenyl-propanoids have the potential to act as antioxidants, modulate the activity of cytochrome P450 and the enzymes in the arachidonic acid cascade, activate phase II reactions and affect cell signalling. In vitro, many have been shown to inhibit bacterial and viral replications (Ralph and Provan 2005). Phenylpropanoids including flavanoids, catechins, gingerol, coumarins, plant sterols and phytoestrogens have a variety of therapeutic properties ranging from antibacterial and antiviral, and ability to protect against heart disease and various cancers (Ralph and Provan 2005). For the current study, a group of simple phenylpropanoids namely cinnamic, p-coumaric, caffeic, chlorogenic, ferulic, 3,4-dimethoxycinnamic and 2,4,5-trimethoxycinnamic acids and eugenol were selected. Their interaction with the anticancer synthetic drug, 5-fluorouracil, was studied against the human cervical cancer HeLa cell line. These phenylpropanoids are rich in diet and reach high concentrations in the blood plasma where there is a chance of interaction with synthetic drug to bring about either positive or negative response. The cell cycle kinetics was studied in flow cytometer.

Materials and methods

Cell lines

For anticancer studies Human cervical HeLa cell lines were purchased from NCCLS, Pune, India. The cells were grown in Dulbecco's modified Eagle's medium (DMEM, Himedia, India) supplemented with 10% foetal bovine serum (FBS, Pa nBiotech, Germany). They were incubated at 37 C in a humidified atmosphere consisting of 10% CO2 in air for 2-3 days before use. In order to subculture the cells, they were washed with phosphate buffered saline (PBS) and incubated with 0.25% of trypsin, 1 mM of EDTA for 3-5 min. The detached cells were resuspended in fresh serum-containing medium to inactivate the trypsin, and then they were transferred to new flasks as required.

Cytotoxicity of phenylpropanoids

The thiazolyl blue method was used for the assessment of cytotoxic effects of the compounds (Mosmann 1983). The tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is reduced by living cells, and this reaction is used as the end point. After trypsinisation, cells were resuspended in 10 ml of complete culture medium and repeatedly pipetted to ensure a homogeneous mixture during dispensing of 5000 cells into each well. HeLa cells were then seeded at a density of 5 x 103 cells/well in a 96-well tissue culture plate. They were then allowed to attach and grow for 24h. After this period, cells were incubated separately with increasing concentrations of 5-fluorouracil or phenylpropanoids (0.002, 0.004, 0.007, 0.015, 0.03, 0.06, 0.13, 0.25, 0.5, 1.0 and 2.0 mM) for 24 h.

MIT dye (5 mg, Sigma, Germany) was dissolved in 1 ml of PBS, passed through a 0.2 [micro],m filter to sterilize and remove undissolved particles, and used freshly before each test. 50 [mu]l of MIT solution was then added to each well and plates were incubated for 4 h at 37(degrees)C. During this period, living cells produce blue insoluble formazan from the yellow soluble MIT. The reaction was stopped by the addition of DMSO (200 [mu]l/well) and the contents of the wells were spontaneously dissolved in 2-3 min. The optical density of each well was measured in a spectrophotometer at 570 nm using an ELISA plate reader (BioRad Laboratories, Inc., California, USA). All the tests were performed in triplicates. The amount of formazan produced correlates with the number of live cells (Prabhakar et al. 2011), so this MIT assay was performed before the cells reached confluency. The absorbance of the compound treated wells was converted to percentage of living cells in comparison to the control. The absorbance at 570 nm (A570) was measured in each well, and the fractional survival was calculated from the ratio: [( mean A570 treated cells - mean A570 blank wells )/(mea n A570 control cells - mean A570 blank wells)]. The concentration ([IC.sub.50]) of the compounds required to inhibit half the maximum value was estimated by fitting the data to a four-parameter logistic function using SIGMAPLOT 10.0 (Systat Software, Germany).

Estimation of combination index to determine interaction of phenylpropanoids with 5-fluorouracil

All the combination studies were performed over a range of concentrations of each individual phenylpropanoid and also in combination with 5-fluorouracil at a fixed concentration ratio. The concentration chosen for combination studies is based on the [IC.sub.50] values of the compounds when used alone, as identified in preliminary experiments. Median effect analysis using the combination index (Cl) was employed to determine whether the compounds interacted synergistically, additively or antagonistically (Chou and Talalay 1984).

The Cl is estimated according to the following equation:

C1 = [(D).sub.1]/[(DX).sub.1] + [(D).sub.1]/[(DX).sub.2] + [a(D)1(D).sub.2]/[(DX)1(D).sub.2]

in which [(D).sub.1] is the dose necessary for a particular effect in the combination, [(Dx).sub.1].sub.1] is the dose of the same drug which will produce the identical level of effect by itself, [(D)).sub.12] is the dose of a second drug which will produce a particular effect in the combination and [(Dx)).sub.12] is the dose of the second drug which will produce the same level of effect by itself. When the drugs are mutually exclusive (i.e. with similar modes of action (alpha) = 0, or if they are mutually non-exclusive (i.e. with independent modes of action) (alpha)= 1.

In the current study, a value of (alpha)=0 was chosen. This is the most conservative for identifying synergy. A Cl of <1 would indicate synergy and a Cl of >1 would mean antagonism. CI is calculated for every fraction affected (Fa) value, where the fraction affected (Fa) is defined as 1 (OD of treated cells)/(OD of control cells). For example a Fa of 0.9 indicates that the cell growth is inhibited by 90s. The concept of combination index has been explored in the treatment of infectious diseases (Hemaiswarya and Doble 2010).

Demonstration of apoptotic morphology

For semi-quantitative assessment of apoptosis, cells were stained with 4',6-diamidino-2-phenylindole (DAPI), which detects the DNA in them. Treated and untreated cells, were washed with PBS and fixed in 4% of paraformaldehyde. Then, they were washed with PBS and centrifuged at 1500 rpm for three times, permeabil-ized with 0.1% of Triton X-100 and stained with 2 [micro]g/m1 of DAPI (Merck, Germany) at 37 C for 10 min. About 700 cells from each treatment were examined and counted under a fluorescent microscope (Nikon TE-Eclipse 300) with a peak-excitation wave length of 340 nm. Chromatin condensation and nuclear fragmentation were the criteria used to demonstrate apoptosis (Yang et al. 2005).

Effect of combination and individual treatment on cell cycle

The cells (1 x 106) were seeded onto 10 cm dishes, treated with or without eugenol (31611M), 5-fluorouracil (21 [J.,M) or combination (153 and 10.5 RM of eugenol and 5-fluorouracil, respectively) for 48 h and then washed twice with ice-cold PBS and collected by centrifugation at 200 x g for 5 min at 37 C. The cells were fixed in 70% (v/v) of ethanol at 37 C for 30 min. After fixation, cells were resuspended in 1 ml of propidium iodide staining buffer (0.1% of Triton X-100, 100 pg/ml of RNase A. 500 [micro]g/mlof propiclium iodide in PBS) at 37 C for 30 min (Yang et al. 2005). The DNA content was analysed using a Beckman-Coulter Quantirm SC MPL flow cytometer. Data presented here are representative of those obtained in at least three independent experiments done in triplicates.

Compatibility of phenylpropanoids with human erythrocytes

Human erythrocytes (blood type A) were freshly prepared from the blood taken from a healthy 24-year-old person prior to the experiments. The blood was centrifuged (800 x g for 10 min) and washed three times with PBS (pH 7.4) to remove the plasma and the buffy coat. Erythrocyte specimens were kept on ice throughout the experiments. Various concentrations of phenylpropanoids were incubated with the erythrocyte suspension [final erythrocyte concentration of 1% (v/v)] for 1 h at 37 C. The percentage of haemolysis was determined from the optical density measured at 540 nm of the supernatant after centrifugation (800 x g for 10 min) (Manabe et al. 1987). Hypotonically lysed erythrocytes were used as the standard which represented 100% haemolysis. Data was plotted as percent haemolysis as a function of concentration of the phenylpropanoid. The curve was fitted to a four-parameter logistic function using SIGMAPLOT 10.0 (Systat Software) and the concentration of the compound required to produce a haemolysis of 50% [IC.sub.50] was estimated.

3D QSAR

3D quantitative structure activity relationships (3D QSAR) were carried out on phenylpropanoids to understand the structural features which determine their anticancer and hemolytic activities. This understanding may help in designing new structures which may exhibit higher anticancer activity and lower hemolytic activity than the current set of compounds. The study was performed using vLifeMDS 3.5 software (vLife Sciences Technologies Pvt. Ltd., India, http://www.vlifesciences.com). It was used to calculate electrostatic, steric and hydrophobic descriptors for the given set of molecules and to perform regression to build a model by selecting a set of descriptors that describe the activity of the molecule. Thus the model can be used for predicting the activity of any new derivatives of molecules.

Molecular structures of phenylpropanoids were drawn Hyper-Chem Professional 7.1 (Hypercube, Inc., USA). Then they were optimized using molecular mechanics (MM+) and semiempirical (PM3) force fields. The optimized structures were then exported to vLifeMDS software. 3D QSAR was performed by comparative molecular field analysis (CoMFA) to obtain a quantitative model. The alignment was performed based on the common phenyl moiety that phenylpropanoids have. The aligned molecules were placed in a 3D cubic lattice grid. The descriptors were calculated by placing a probe [sp.sup.3] carbon atom, with a van der Waals radius of 1.52 A and charge of +1.0, at each grid point. The grid points were spaced at a distance of 2A from any neighbouring grid point (Kubinyi 1997). The electrostatic and steric energy values were truncated at a default value of 10 kCal/mol and 30 kCal/mol, respectively.

Two-thousand two-hundred and sixty-nine descriptors were calculated for these eight molecules, which include electrostatic, steric and hydrophobic descriptors. These were used as the independent variables. For QSAR studies, the cytotoxic and hemolytic activities were converted into respective negative logarithmic forms, i.e.--log(IC50) or--log(H50%). The observed biological activity is assumed to be a function of the molecular properties or descriptors (Verma et al. 2010), and so it is considered as a dependent variable.

The data was divided into training and test sets using random selection method. The partial least squares regression genetic algorithm method for anticancer and multiple regression stepwise forward method for hemolytic activity were used. A cross correlation limit of 1.0, [r.sup.2] as selection criteria, variance cut off value as zero and autoscaling method was used for building the regression models. Statistical parameters such as [r.sup.2] (square of correlation coefficient), [q.sup.2] (validation based on leave one out method), [r.sub.se.sup.2],[q.sub.2.sup.se] (square of correlation coefficient after adjusting for number of data points and number of parameters in the model), qe, 4 (standard errors corresponding to [r.sup.2] and [q.sup.2]) and F (ratio of the variances corresponding to residual and error) were estimated for determining the quality of the model fit and its predictive ability. r2, [ra.sup.2dj], and [q.sup.2] above 0.70, large F value and small standard errors indicate good model. A parity plot with 95% confidence levels was plotted using Sigma plot 11.0 (Systat Software, Inc.). More details about these statistical parameters are described elsewhere (Sivakumar et al. 2009).

Results and discussion

Antiproliferative effect of phenylpropanoids

MTT assay was used to assess the antiproliferative effect of phenylpropanoids against this cervical cancer cell line. Phenylpropanoicis inhibit the cell growth in a dose dependent manner (see figures in supporting information). The [IC.sub.50] (percentage at which 50% of HeLa cells are dead) values for the phenyl-propanoids are listed in Table 1. In contrast to phenylpropanoid treated cells, those treated with DMSO showed little or no cytotoxicity. Moreover, it was found that HeLa cells were more sensitive to eugenol when compared to other phenylpropanoids. The subsequent interaction studies between the synthetic drug and the phenylpropanoids were carried out at these [IC.sub.50 ]concentrations.

Table 1 Cytotoxicity ([IC.sub.50]) of phenylpropanoids
and 5-fluorouracil

Compounds                      [IC.sub.50](in [micro]M) [+ or -] SE

Cinnamic acid                                  484.98 [+ or -] 4.3

p-Coumaric acid                               728.52 [+ or -] 13.2

CafTeic acid                                    546.5 [+ or -] 2.3

Chlorogenic acid                                677.7 [+ or -] 4.3

Fugenol                                        315.79 [+ or -] 9.1

Ferulic acid                                   323.49 [+ or -] 3.5

3,4-Dimethoxycinnamic acid                      759.4 [+ or -] 7.0

2,4,5-Trimethoxycinnamic acid                   877.8 [+ or -] 1.8

5-Fluorouracil                                   20.8 [+ or -] 3.9


The cytotoxic activity of phenylpropanoids against the cervical cancer HeLa cells was in the order of eugenol > ferulic > cinnamic > caffeic > chlorogenic >p-coumaric > 3,4-dimethoxycinnamic > 2,4,5-trimethoxycinnamic acids. Similar reports are available for the cytotoxic activities of phenylpropanoids against different cell lines. The cytotoxicity of the components of Cinnamomum bark namely, cinnamic acid and eugenol, were tested against model tumour cells (SK-OV-3, XF-498, HeLa, SK-MEL-2 and HCT-15 cells). The results showed that eugenol exhibited moderate cytotoxic activity with IC50 of <200 [micro]g/m1 (<1 mM) towards HeLa cells (Lee et al. 2004). Cinnamic acid showed cytotoxicity at concentrations more than 200 [mu]g/ml. The IC50 values calculated from the current study were high and were in close agreement with the reported literature (Ng and Wu 2011). The IC50 of cinnamic acid against HepG2 (human hepatocarcinoma) cells was 34.2 [mu]M when treated for 24h (Ng and Wu 2011), although there was no cytotoxicity observed till 8 mM against Caco-2 (human colon adenocarcinoma) cells (Ekmekcioglu et al. 1999). Cinnamic acid induces cytostasis and a reversal of malignant properties in vitro in the human tumour cells. The [IC.sub.50] concentration against glioblastoma, melanoma, prostate and lung carcinoma cells ranged from 1 to 4.5 mM (Liu et al. 1995). The antiproliferative activity of cinnamic, ferulic, 3,4-dimethoxycinnamic and caffeic acids which constitute the majority of propolis (a resinous hive product) was determined against different cell lines. The cytotoxic activity of these compounds was found to be more than 200[mu]m (Banskota et al. 2002).

Interaction of phenylpropanoids with 5-fluorouracil against HeLa cells

The phenylpropanoids were cytotoxic only at higher concentrations (350-880 p,M) when compared to 5-fluorouracil (20.8 [micro]M). The phenylpropanoids are available in high concentrations in human diet. The specific mechanism of action of most of the phenyl-propanoids in cancer prevention is not yet clear but they appear to be varied from that of 5-fluorouracil.

Combination of eugenol and 5-fluorouracil at 0.5, 0.6, 0.8, 0.9 and 1.5 times their respective [IC.sub.50] values were tested against the HeLa cells. Eugenol at 1 [m.sub.M] and 50 [mu]m of 5-fluorouracil when used alone brought about nearly 75% growth inhibition. The same amount of inhibition was observed with combination of eugenol and 5-fluorouracil at 315.8 and 21 [mu]M, respectively. Thus, eugenol diminished the dose of 5-fluorouracil by half. The Cl for this combination is 0.74. Similarly other combinations of eugenol and 5-fluorouracil were tested keeping the molar ratio constant (1:0.07) and the correspondinggrowth inhibition were experimentally measured. Ferulic, caffeic and cinnamic acids were also tested in combination with 5-fluorouracil in the same manner.

The combination index (Cl) values for different fractional inhibition rates of growth of HeLa cell were calculated and shown as the median-effect plot (Fig. 1). Cl decreases as a function of increasing fraction affected and above 0.7, Cl drops below a value of 1, indicating synergy. The Cl values for the combination of phenylpropanoids and 5-fluorouracil for 0.25, 0.5 and 0.75 fractional inhibition values are presented in Table 2. AC! of 1 is additive, less than 1 is synergistic and greater than 1 is antagonistic. At 0.75 fractional inhibition of growth of HeLa cells there is a strong synergy between eugenol, ferulic and caffeic acids with 5-fluorouracil. The molar ratios of the two compounds used in these experiments are also given at which synergy is observed is given in the same table.

The current results show that the combination of eugenol with 5-fluorouracil in vitro is highly synergistic against HeLa. Eugenol is found to enhance the anticancer activity of 2-methoxyestradiol against hormone refractory prostate cancer (Ghosh et al. 2009) cells and a recent study shows that a combination of sulforaphane (SFN) and eugenol is synergistic on HeLa cells (Hussain et al. 2011). Combined effect of ferulic acid with cisplatin or carboplatin showed effectiveness on human erythroleukemic cell line, 1(562, demonstrating that the phytochemical could potentiate the activity of platinum complexes (Indap et al. 2006). Apart from the above reports, studies on interaction of the phenylpropanoids with anticancer agents are minimal.

Analysis of apoptotic cells by DAPI staining

The apoptosis of HeLa was induced by eugenol (316 [mu]m), 5-fluorouracil (21 [mu],M) or their combination (158 +10.5 [mu]m of eugenol and 5-fluorouracil, respectively). The morphological features including cell and nuclear shrinkage, fluorescence intensity strength, nucleoli disappearance with crescent-like changes, and apoptosis-formed bodies were observed under a fluorescence microscope (Fig. 2). The number of necrotic cells was not significant as identified by propidium iodide staining. The mean percentage of apoptotic cells for these treatments and control are given in Fig. 3. There was significant increase in the number of apoptotic cells in combination when compared to the treatments with individual compounds when used alone.

Cell cycle kinetics

Cancer is precisely the imperfections at the cell cycle checkpoints, which prevents the cycle from stopping in the face of adverse conditions, making them vulnerable. The kinetics of the cell cycle was examined after 48 h by labelling the DNA with pro-pidium iodide and analysing with flow cytometer. Eugenol at a concentration of 316 [micro]M induced a minor but significant decrease (when compared to untreated control) in the number of cells in the G2/M phase after 48 h, and a significant decrease in the GO/G1 and S phases. Similarly 5-fluorouracil (21 [micro]M) reduced significantly the GO/G1 phase, whereas it significantly increased the S phase and had no effect on the G2/M phase (Fig. 4).

Table 2
Combination index of phenylpropanoids with 5-fluorouracil for
various fractional inhibition of Hela cells.C1<1 is synergy;
C1=1 is additive; C1>1 is antagonistic.

Phenylpropanoids          Molar ratios of           CI
                          phenylpropanoid   calculated
                                      and  for various
                           5-fluorouracil   fractional
                                            inhibition
                                              [+ or -]
                                                    SD

                                                  0.25     0.50   0.75

Cinnamic acid                      1:0.04   9.17 [+ or  1.58 [+      -
                                                -] 6.5    or -]
                                                              0

p-Coumaric acid                    1:0.03   7.23 [+ or  1.75 [+   1.04
                                                  -] 0    or -]     [+
                                                            0.5  or -]
                                                                   0.1

Caffeic acid                       1:0.04            -  2.04 [+   0.75
                                                          or -]     [+
                                                            0.7  or -]
                                                                  0.09
                                                                     *

Chlorogenic acid                   1:0.03    2.5 [+ or   1.9 [+      -
                                                -] 0.9    or -]
                                                           0.21

Eugenol                            1:0.07    2.4 [+ or   2.0 [+   0.64
                                               -] 0.07    or -]     [+
                                                            1.1  or -]
                                                                   0.7
                                                                     *

Ferulic acid                       1:0.06            -  1.57 [+   0.67
                                                          or -]     [+
                                                            0.2  or -]
                                                                  0.09
                                                                     *

3,4-Dimethoxycinnamic              1:0.03    2.7 [+ or   1.8 [+      -
acid                                           -] 0.04    or -]
                                                           0.78

2.4.5-Trimethoxycinnamic           1:0.02    2.6 [+ or   1,1 [+      -
acid                                            -] 1.8    or -]
                                                           0.15

p<0.05-synergy.


Combining eugenol with 5-fluorouracil resulted in a cell cycle distribution significantly different from that seen with the individual compounds. At 48th hour, the combination of eugenol and 5-fluorouracil (at half their respective [IC.sub.50] concentration, namely 158 and 10.5 [micro]M, respectively) significantly reduced the cells in the GO/G1 phase. The combination increased the number of cells in the S phase when compared to 5-fluorouracil treatment alone. Also an increase in the cells in the G2/M phase was also observed (Table 3).

In previous reports, eugenol was shown to cause cell cycle arrest in the GO/G1 phase (KaImes et at. 2006) or in the S-phase (Ghosh et at. 2009) and 5-fluorouracil was reported to arrest the cycle at the G1/S-phase (Liu et at. 2006). The combination of eugenol and 5-fluorouracil reduced cell proliferation when compared to the individual treatments and control. So the combination lead to fewer proliferating cells as well as enhanced the accumulation of cells in the S and G2/M phase which may be unable to divide.

Haemolysis

The choice of the erythrocyte as a model is due to the fact that it is a complete cellular system with high content of polyunsaturated fatty acids. Moreover, the membrane contains cytoskeleton proteins, interchelating with lipids and phospholipids, creating the typical flexible structure of the erythrocyte (AnseImi et al. 2004). The phenylpropanoids including cinnamic, caffeic and ferulic acids have been shown to possess antioxidant activities and inhibit metal induced haemolysis of erythrocytes (Cao et al. 1997). Though there are reports on the free radical induced haemolysis by adding catalysts, osmotic induced haemolysis has not been explored.

In vitro studies on the effect of phenylpropanoids on human erythrocytes of phenylpropanoids showed that these compounds possessed minimum amount of hemolytic activity (Fig. 5). The total haemolysis was obtained using a hypotonic NaC1 solution (0.1%) which showed 100% lysis. The hemolytic activity of the phenylpropanoids increased with increasing concentrations showing a dose dependent hemolytic behaviour. The hemolytic activities of the phenylpropanoids were in the following order: eugenol > 2,4,5-tri methoxycinnamic > 3, 4-d imethoxycinnamic > cinnamic > coumaric > ferulic > chlorogenic> caffeic acid. An [IC.sub.50] value lower than 200 [micro]g/m1 (~1 mM) is considered to be active. None of the phenylpropanoids possessed hemolytic activity at this concentration suggesting that they are not toxic hence, they may be suitable for the preparation of drugs for treating cancer.

Table 3
Effect of combined treatment of eugenol and 5-fluorouracil
on cell cycle progression (percentage distribution of cells
at various check points).

Treatments                  G0/G1          S     C2/M      Sub-Gl

Control                60.9 [+ or    12.7 [+  21.7 [+  5 [+ or -]
                           -] 1.2      or -]    or -]        0.05
                                         1.0      0.5

Eugenol (316           30.5 [+ or    15.1 [+  23.9 [+    31 [+ or
[micro]M)                 -] 2.05      or -]    or -]      -] 1.3
                                         3.1      1.1

5-Fluorouracil(21      66.3 [+ or  8.6 [+ or  16.3 [+  9 [+ or -]
[micro]M]                  -] 4.0     -] 4.7    or -]         0.2
                                                 3.1'

Eugenol +              52.4 [+ or    13.5 [+   14.0 t  20.1 [+ or
5-fluoratir.icil (158      -]4.1'   or -]3.7     2.4'    -] 0.3 *
+ 10.5 [micro]M)

p <0.05, for combination when compared to individual treatments.


3D-QSAR

In 3D-QSAR, the structures are aligned on a common template and the electrostatic, hydrophobic and steric features around these molecules are calculated. The best one descriptor regression model obtained for the anticancer activities of these phenylpropanoids and the corresponding statistics are given below

Activity =--2.7906 + 0.0289 * E_613 [r.sup.2] = 0.9079. [r.sup.2.sub.adj]. = 0.8772, [q.sup.2] = 0.7864. [F.sub.test] = 29.5706, [r.sup.2.sub.se] = 0.0508, [q.sup.2.sub.se] = 0.0774

The superposition of all the molecules on a common template and the location of this electrostatic descriptor are shown in Fig. 6. The parity plot comparing the experimental and predicted anticancer activities is shown in Fig. 7. All the data fall within the 95% confidence band, indicating the good predictive capability of the model. The statistical parameters are acceptable indicating that the model has a good predictive capability.

The best one descriptor regression model obtained for the hemolytic activity of these phenyl propanoids and the corresponding statistics are given below

Activity =-0.8165 + 0.1030 *E_370 [r.sup.2] = 0.9701, [r.sup.2.sub.adj]= 0.9601, [q.sup.2] = 0.9345. [F.sub.test] = 97.26, [r.sup.2.sub.se] = 0.0197, [q.sup.2.sub.se]= 0.0292

Superposition of all the molecules on a common template and the location of this electrostatic descriptor are shown in Fig. 8. The parity plot comparing the experimental and predicted anticancer activities is shown in Fig. 9. All the data fall within the 95% confidence band, indicating the good predictive capability of the model.

The statistical parameters are acceptable indicating that the model has a good predictive capability.

The anticancer and the hemolytic activities of the phenyl-propanoids are not correlated with each other. If one compares Figs. 6 and 8, it can be noticed that the hemolytic activity of phenylpropanoids against red blood cells is determined by the electrostatic energy near the double bond of the compounds, whereas the anticancer activity of the phenylpropanoids against HeLa cells is determined by the electrostatic energy near the substitutions present in the benzene ring of the compounds. This study indicates that new molecules could be designed so that their anticancer activities are enhanced but at the same time maintain low hemolytic activities.

Conclusions

Combination therapy is the most effective treatment strategy in cancer. The rationale for combination chemotherapy is to use drugs that work by different mechanisms of action, thereby decreasing the likelihood of development of resistant cancer cells. 5-Fluorouracil is associated with side effects such as myelosu-pression, mucositis, resistance, dermatitis, cardiac toxicity and is ineffective in subjects with dihydropyrimidine dehydrogenase deficiency. As an alternative strategy, 5-fluorouracil can be combined with relatively nontoxic phenylpropanoids which may enhance the efficacy of chemotherapy with reduced toxicity to normal cells. Therefore a combination of phenylpropanoids with 5-fluorouracil against cancer cell line under in vitro conditions was explored.

The cytotoxic activity was in the order of eugenol > ferulic > cinnamic > caffeic > chlorogenic >p-coumaric > 3,4- dimethoxycinnamic > 2,4,5-trimethoxycinnamic acids. Eugenol and ferulic, cinnamic and caffeic acids exhibited synergy with 5-fluorouracil. Synergy between compounds arises because of their different modes of action. There was significant increase in the number of apoptotic cells in the combination when compared to the individual treatments. Treatment with a combination of 5-fluorouracil and eugenol increased the number of cells in the GO/G1 and G2/1\4 phase when compared to control. An increase in cells in the sub-G1 phase was also observed.

The current study proposes that eugenol interacts synergistically with 5-fluorouracil against human cervical cancer, HeLa cell lines, by blocking the cell cycle and inducing apoptosis. Eugenol is shown to induce apoptosis by reducing the intracellular nonprotein thiols and increasing the earlier lipid layer break. Further events like dissipation of MMP and generation of reactive oxygen species (ROS) were accompanied in the eugenol-induced apoptosis (Jaganathan et al. 2011). Augmented ROS generation resulted in the DNA fragmentation of treated cells as shown by DNA fragmentation and TUNEL assay (Jaganathan et al. 2011). Further activation of poly-adenosine diphosphate-ribose polymerase (PARP), p53 and caspase-3 was observed in the Western blot analyses by others (Jaganathan et al. 2011). Experimental data suggested the involvement of p53, the Bc1-2 family and BAG-1 in of 5-fl uorouracil chemotherapy-induced apoptosis ( Hengstermann etal. 2001; Ding etal. 2000a,b). Resistance to apoptosis is correlated with the reduced caspase-3 activation and enhanced expression of antiapoptotic proteins in human cervical multidrug-resistant cells (Ding et al. 2000a,b; Violette et al. 2002). 5-Fluorouracil is shown to possess different targets when compared to that of eugenol, so multi-targeting by two different drugs brings about synergy.

Only with exact knowledge of the mechanism underlying the synergistic effects, it will be possible to develop a new generation of safe and standardized combination of drugs with higher efficacy than the current ones (Wagner and Ulrich-Merzenich 2009). Multi-targeting using combination of drugs is effective in the treatment of infectious diseases and cancer. Eugenol and 5-fluorouracil are shown to possess different cell cycle targets and induce apoptosis in the cancer cell. The anticancer and the hemolytic activities of the phenylpropanoids are not correlated with each other. 3D QSAR studies indicate that the hemolytic activity of phenylpropanoids against red blood cells is determined by the electrostatic energy near the double bond of the compounds. On the contrary the anticancer activity of the phenylpropanoids against HeLa cells is determined by the electrostatic energy near the substitutions present in the benzene ring of the compounds.

These findings can lead to new treatment strategies and could also pave the way in the reduction of the amount of anticancer agents required as therapeutic dose. A reduction in the amount of the synthetic drug can eventually lead to reduction in the toxicity and side effects caused to the patients. The next focus should be on the relative pharmacokinetic and pharmacodynamic behaviour of these natural compounds with reference to these antibiotics when they are used in combination.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phymed. 2012.10.009.

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* Corresponding author. Tel.: +91 44 2257 4107; fax: +91 44 2257 4102.

E-mail addresses: mukeshd@iitm.ac.in, iswarya_herna@yahoo.ac.in (M. Doble).

Shanmugam Hemaiswarya, Mukesh Doble *

Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India
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Author:Hemaiswarya, Shanmugam; Doble, Mukesh
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Jan 15, 2013
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