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Journal of Cell Communication and Signaling logoLink to Journal of Cell Communication and Signaling
. 2017 Aug 10;12(2):467–478. doi: 10.1007/s12079-017-0404-8

Novel combination of 2-methoxyestradiol and cyclophosphamide enhances the antineoplastic and pro-apoptotic effects on S-180 ascitic tumour cells

Srabantika Mallick 1, Atish Barua 2, Goutam Paul 3, Samarendra Nath Banerjee 1,✉
PMCID: PMC5910319  PMID: 28795302

Abstract

Sarcoma 180 (S-180) tumour cell line is a stable murine tumour cell line with 98–99% stumour takes capacity in Swiss albino mouse - Mus musculus. 2 Methoxyestradiol (2ME) - a promising anti-neoplastic and anti-angiogenic agent, showed toxicity to host body in higher concentration. Cyclophosphamide (CP), the anti-neoplastic agent has long been used as a chemotherapeutic drug for treatment of different cancers. Our studies have shown that the combination effect of 2ME and CP on S-180 tumour cell line is anti-proliferative and less toxic. The treatment with lower concentrations of 2ME and CP (6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight) antagonistically increased the life span of tumour bearing mice and synergistically inhibited the viable cell population. 2ME or CP treatment individually induces G2/M arrest. The combination treatment of 2ME + CP (6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight) produced a significant increase of cells in the G0 which is the indication of cell arrest or apoptosis. Reduction of cell viability by 2ME + CP treatments is due to apoptotic cell death. This combination therapy produced a significant inhibitory effect of cell proliferation and augmentation of cell accumulation in the G0 phase (i.e. apoptosis). Apoptosis is validated by Fluorescence staining of control and treated S-180 tumour cells with Acridine Orange and EtBr dye. Moreover, a steady increase in the frequency of complex chromosomal aberrations (i.e. tri-, qudri-radial translocations) in tumour cells was noted in that particular concentration of combination therapy treated series along with the increase in dead cell frequency and tumour regression pattern. It is assumed that, these chromosomal abnormalities or damages recorded in higher frequency prevent the affected metaphases to enter into the next cell cycle through apoptosis or necrosis. This study introduces a novel combination, where this particular concentration of 2ME + CP (i.e. 6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight) not only enhanced the life span of tumour bearing mouse and decreased the tumour volume antagonistically but also inhibited the viable cell population synergistically, which could serve as a potential effective regimen for cancer treatment.

Keywords: Apoptosis, Chromosome, Chou-Talalay method, Combination effect, Combination index, Cyclophosphamide, 2 Methoxyestradiol, S-180 tumour cell line

Introduction

The concept of use of combination therapy is a rapidly growing area in cancer treatment rather than surgery, radiation therapy, chemotherapy etc. which establishes new therapeutic approaches to reduce cancer growth. Since cancer is a disorder of uncontrolled, uncoordinated cell proliferation and cell survival, preventing cell proliferation and increasing apoptotic cell death in tumour are effective strategies for treatment of cancer. There is evidence that several common antineoplastic agents including cyclophosphamide (CP), paclitaxel (Taxol), mitomycin-C, cisplatin have antitumour effects in different animal tumour models (Pal et al. 1984; Chakrabarti et al. 1985; Chakrabarti and Chakrabarti 1987; Klauber et al. 1997; Colleoni et al. 2002; Mallick et al. 2015a, b). In addition, different chemotherapeutic drugs destroy tumour cells or inhibit tumour cell proliferation primarily by inducing tumour cell apoptosis (Yamamoto et al. 1999). CP has been used as a single as well as combination chemotherapeutic drug against different human cancers (Friedman et al. 1979; Adler 1982; Das and Chakrabarti 1989; Huang et al. 2000; Duncan et al. 2012) since last four decades. The antineoplastic effect of cyclophosphamide has also been reported in different animal tumour models (Curtis et al. 1992; Kimura et al. 1998). However, its use causes severe cytotoxicity to normal cells in human (Culo et al. 1977; Anton 1987). 2 Methoxyestradiol (2ME) is a potent and novel agent that has shown a promising antineoplastic and antiangiogenic activity in a number of tumour cell lines in vivo and in vitro (Zoubine et al. 1999; Sattler and Salgia 2003; LaVallee et al. 2003; Banerjee et al. 2003; Banerjee and Banerjee 2005, 2008; Ray et al. 2005; El Naga et al. 2009; Banerjee and Mallick 2013; Mallick et al. 2015b; Banerjee 2017). It also induces G2/M arrest and apoptosis in many actively dividing cell types (Pribluda et al. 2000). These findings on the use of cyclophosphamide as a single as well as combination chemotherapeutic drug on one side and antiproliferative effects of 2 Methoxyestradiol on the other generated interest to investigate the effect of 2 Methoxyestradiol in combination with CP against the S-180 tumour cell line. Therefore, the aim of this study was to evaluate the potential cytotoxicity of the combination effect of 2ME and CP in different concentrations on S-180 tumour cell line as well as the possible underlying mechanisms, particularly the antiproliferative and apoptotic effects. We have also explored the mechanism of antagonism and synergism for these two components 2ME and CP quantitatively through the use of the multiple drug effect analysis of Chou-Talalay (Chou and Talalay 1984; Chou 1991). Moreover, the study has also been oriented to observe whether the combination therapy is less toxic or safe in comparison to other therapies.

Materials and methods

Experimental animal

Male Swiss Albino adult mice (Mus musculus) 20 g approximately of 9 weeks of age were grouped and housed in polyacrylic cages. Mice were maintained under standard laboratory conditions (temperature 24 °C – 25 °C). The mice were provided with standard mice food (dry pellet) and water ad libitum. Before experiment the mice were kept for 10 days under standard laboratory conditions for acclimatization.

Selection of animal tumour model

Sarcoma-180 cell line was maintained in the inbred Swiss albino mice (inoculums 106 cells per animal) by serial intraperitoneal transplantation. S-180 tumour cells (106 cells were suspended in 0.5 ml sterile 0.9% NaCl solution) were injected intraperitoneally for development of ascitic form of tumour (Chakrabarti and Chakrabarti 1987). Tumour cells showing 85% - 90% viability as assessed by Trypan blue exclusion test were used for transplantation. Full grown ascitic form of tumour was developed within 7 days after transplantation. All experiments were done in accordance with the guidelines framed by the IAEC (Institutional Animal Ethics Committee) of Rammohan College, Kolkata (Animal House Registration No.- 1795/PO/ERe/S/14 CPCSEA) for the care and use of Laboratory animals.

S-180 tumour transplantation

Ascitic form of Sarcoma 180 tumour cell line is maintained in the Zoology Department laboratory, Rammohan College, Kolkata-700009, India and propagated into transplantable tumours in the peritoneal cavity of male Swiss albino mice. Freshly aspirated S-180 tumour cells from ascitic fluid of the mouse peritoneum were washed with 0.9% NaCl under sterile condition. Then tumour cells (inoculum 106 cells per animal) were injected intraperitoneally to normal healthy mice for induction of ascitic tumour (Chakrabarti and Chakrabarti 1987; Banerjee and Banerjee 2005).

Selection of drug

2 Methoxyestradiol (2ME) is an endogenous estrogen metabolite produced by sequential hydroxylation of parent compounds followed by methylation in the liver (Brueggemeier and Singh 1989; Zhu and Conney 1998a, b; Zoubine et al. 1999). It is a potent inhibitor of tumour angiogenesis as well as tumorigenesis (D’Amato et al. 1994; Banerjee et al. 2003). A stock solution of 2ME was prepared by following the method as practised in the laboratory (Mallick et al. 2015b). Cyclophosphamide, the alkylating, antineoplastic DNA cross-linking agent has been found to be one of the most effective standard chemotherapeutic drugs in the treatment of variety of human tumours (Friedman et al. 1979; Shand 1979).

However, its use causes severe, undesirable side effects due to the formation of reactive oxygen species (Patel 1987; Shokrzadeh et al. 2014) which can damage to normal cells. But this drug can be used at lower doses, reducing toxicity.

Preparation of drug solution

200 mg CP was dissolved in 13.4 ml. of 0.9% NaCl solution to make the CP stock solution. 5 mg 2ME was dissolved in 5 ml absolute alcohol and then the solution was diluted in 5 ml normal saline to make the 2ME stock solution (Mallick et al. 2015b). A parallel vehicle (positive control) was prepared by absolute alcohol and normal saline in 1:1(v/v) ratio. In combination therapy 2ME and CP drugs were given in different concentrations as mentioned in the Table 1. Tumor-transplanted mice (68 male mice) were randomly divided into seventeen (17) groups (with 04 mice in each group) depending on their allocated treatment (Table 1).

Table 1.

Treatment schedule (treatment was started on and from 7th day of tumour cell transplantation intraperitoneally (i.p) and continued for 5 consecutive days, Average weight of each mice = 20 g)

Groups Doses
(mg/kg body weight) (μM/ml)
Control (Negative) - -
Vehicle (Positive) 0.2 ml. -
2ME (Monotherapy) 2.5 0.83
6.5 2.15
25 8.30
CP (Monotherapy) 50 19
75 30
100 38
2ME + CP (Combination Therapy) 2.5 + 50 0.83 + 19
2.5 + 75 0.83 + 30
2.5 + 100 0.83 + 38
6.5 + 50 2.15 + 19
6.5 + 75 2.15 + 30
6.5 + 100 2.15 + 38
25 + 50 8.3 + 19
25 + 75 8.3 + 30
25 + 100 8.3 + 38
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2ME was injected 4 h before CP to minimize potential for drug interactions in combination therapy.

The mice of all experimental groups were monitored regularly for signs of toxicity and were fed with normal food.

Pharmacologic analysis of antagonism and synergism

The interaction between two compounds 2ME and CP was analyzed quantitatively through the use of the method – the multiple drug effect analysis of Chou- Talalay (Chou and Talalay 1984; Chou 1991). This method is a most generalized method that determines the expected effect of a given drug combination and provides a continuous assessment of whether the interaction is additive, synergistic or antagonistic. In this experiment, agents, 2ME and CP are then combined at varying doses as depicted in the Table 1. In all cases, CI < 1 indicates synergism, CI = 1 indicates additivity and CI > 1 indicates antagonism.

Tumour volume determination

The control and treated mice were dissected and ascitic fluid was collected from peritoneal cavity. The i.p fluid volume was measured by taking in graduated tube and expressed in milliliter (Prasad and Giri 1994; Dolai et al. 2012). For analysis of possible enhanced (i.e. antagonistic) effects of combination treatment in the S-180 tumour model, the mean tumour volumes were calculated and Chou-Talalay analysis was performed.

Determination of survival time of control and treated tumour bearing mice

Both control and treated mice were monitored for their survival regularly until their death and life span of tumour bearing mouse in both control and treated groups was calculated according to the specific protocol (Mallick et al. 2015b) and quantitatively analyzed by Chou-Talalay method (Chou and Talalay 1984; Chou 1991).

Cell viability test by trypan blue exclusion method

The numbers of viable and dead cells in control, vehicle and different treated series were measured by Trypan Blue Exclusion test (Das and Chakrabarti 1989; Mallick et al. 2015a). The ascitic fluid was taken in WBC pipette and diluted 100 times with 0.9% NaCl. One drop of the Trypan blue stained (0.4%) cell suspension was kept on the Neubauer counting chamber. The viable and dead cells were counted to study the regression pattern of the tumour (Chakrabarti and Chakrabarti 1987; Das and Chakrabarti 1989) and quantitatively analyzed by Chou-Talalay method (Chou and Talalay 1984; Chou 1991).

Bone marrow toxicity assessment by chromosomal aberration analysis

Bone marrow toxicity was assessed by chromosomal aberration study. Chromosome preparation from bone marrow cells of tumour bearing mice in both control and treated series was performed by following the mitotic division inhibition technique in practice in this laboratory (Chakrabarti et al. 1985). The technique is described in brief: i) Both control and treated specimens received injection of 0.04% colchicine solution intraperitoneally at a rate of 1 ml/100 g body weight for 2 h. ii) Cells from bone marrow were collected in hypotonic solution (0.075 M KCl) and aspirated gently. Then cell suspension was incubated for 30 min at 37 °C and centrifuged for 15 min at 1500 rpm to collect the cell sediment. The sediment was fixed in aceto-alcohol fixative (3:1, methanol: glacial acetic acid, v/v) and centrifuged for 8 min. The sediment was fixed again and kept for chromosomal slide preparation. The flame dried chromosome prepared slide was stained with 5% Giemsa diluted in phosphate (pH 6.8) buffer. Only well spread metaphases were analyzed under the Binocular Research Microscope (10x100magnifications).

Tumour chromosomal aberration analysis for mitotic catastrophe

Chromosome preparation from S-180 ascitic cells in control and treated series was done after slight modifications of the original technique as described by Pal et al. 1984 and Chakrabarti et al. 1998. The technique in brief: i) after completion of treatment, on 6th day all specimens received i.p injection of 0.04% colchicine (Sigma, St Louis, U.S.A) solution at a rate of 1 ml/100 g body weight 2 h prior to sacrifice, ii) Cells in the ascitic fluid were collected in hypotonic solution (0.075 M KCl) and aspirated gently to form a homogenous cell suspension. Then cell suspension was incubated for 30 min at 37 °C and followed by centrifugation at 1500 rpm for 15 min, iii) The pellet was fixed in fresh methanol: acetic acid fixative (3:1 v/v). The whole process was repeated thrice. Three drops of cell suspension were dropped on clean grease-free slide (soaked previously in chilled 50% ethanol) and allowed to dry in flame, iv) For conventional staining, slides were dipped into 5% phosphate buffered Giemsa stain (pH 6.8) for 40 min and washed in tap water for observation under the Binocular Research Microscope (10 × 100 magnifications).

Cell cycle analysis by flow cytometry (FACS)

Sarcoma 180 cells from control and treated mice were collected and washed twice with cold PBS according to the protocol as described by (Banerjee et al. 2011; Imreh et al. 2011). Subsequently, cells were resuspended in 10 μg/ml RNase A to destroy RNA in the cells and incubated for 30 min at 37 °C. Then cells were stained with 50 μg/ml Propidium iodide and data were acquired immediately after addition of dye by Fluorescence-activated cell sorting (FACS) flow cytometer (BD FACSCalibur) for anti-neoplastic and pro-apoptotic effect of combination therapy.

Assays for apoptosis by acridine orange (AO) and ethidium bromide (EtBr) double staining

The morphology of viable, apoptotic and necrotic cells was detected using DNA binding dye acridine orange (AO) and ethidium bromide (EtBr) staining method (Kasibhatla et al. 2006; Kuan et al. 2015; Krishnamoorthy and Mirunalini 2016). S – 180 cells from control and treated mice were collected, washed by PBS and stained with acridine orange (100 μg/ml) and ethidium bromide (100 μg/ml) at 25 °C for 10 min. The stained slides were mounted in glycerol and observed with a fluorescence microscope (Leica DM4000 B) at 40X magnifications. The cells were divided into four categories as follows: living or viable cells (bright green nuclei with intact cells), early apoptotic (dense green nucleus with condensed or fragmented chromatin or dense green patches), late apoptotic (orange-stained nuclei with highly fragmented chromatin) and necrotic cells (uniformly red or orange-stained cell nuclei with intact structures). In each experiment, more than 300cells/sample were counted.

Statistical analysis

Statistical analysis was performed using Student’s t-test. All these results were expressed as mean ± standard error (SE) of three samples in each group. P < 0.05 and P < 0.001 were considered as statistically significant (Panse and Sukhatme 1985).

Results

Tumour volume

The tumour volume in some groups of monotherapy and combination therapy treated series was found to have decreased as compared to that of control group. But it is interesting to note that the tumour volume had significantly (p < 0.001) reduced in a particular concentration of combination therapy (i.e. 6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight concentration) series as shown in the Table 2. These studies demonstrate that moderate antagonism occurred when tumour volume is reduced with the treatment of that particular concentration of 2ME + CP as shown in the Table 3 and Fig. 1.

Table 2.

Determinations of Tumour Volume, Survivability Rate, Total Cells (Viable + Non-Viable) and Percentage of Viable and Non-Viable Cells in Control, Vehicle and Treated Groups. 0.2 ml solutions of 2ME/CP injected in each mouse (Average weight of each mice = 20 g)

Groups Dose (mg/ml) Ascitic Cell or Tumour Vol. (ml.) Survivability Rate Tumour Cells (×106/ml.) % Viable Cells (×106/ml.) % Non-Viable Cells (×106/ml.)
Control - 7.5 ± 0.24 74.25 ± 0.48 65.23 ± 16.4 67.44 ± 4.24 32.56 ± 4.24
Vehicle - 7.8 ± 0.23 73.0 ± 0.41 54.4 ± 14.24 68.47 ± 1.93 31.53 ± 1.93
2ME 0.25 6.97 ± 0.03 73.75 ± 0.48 28.13 ± 1.16 57.67 ± 0.90 42.33 ± 0.90
0.65 5.47 ± 0.18 * 78.25 ± 0.48 * 35.4 ± 7.61 52.85 ± 2.56 * 47.15 ± 2.56 *
2.50 # 70.25 ± 0.48 * # # #
CP 5.0 6.13 ± 0.03 * 73.25 ± 0.48 30.9 ± 2.06 60.70 ± 1.45 39.30 ± 1.45
7.5 5.80 ± 0.12 * 76.25 ± 0.48 * 35.6 ± 8.85 56.35 ± 2.72 43.65 ± 2.72
10 3.80 ± 0.15 ** 75.25 ± 0.48 26.7 ± 4.40 74.64 ± 1.04 25.36 ± 1.04
2ME + CP 0.25 + 5.0 8.27 ± 0.07 * 80.0 ± 0.41 ** 34.67 ± 3.39 79.77 ± 0.28 * 20.23 ± 0.28 *
0.25 + 7.5 7.93 ± 0.09 75.25 ± 0.48 38.5 ± 1.61 74.48 ± 1.50 25.52 ± 1.50
0.25 + 10 7.90 ± 0.06 77.0 ± 0.91 50.83 ± 10.99 77.58 ± 1.41 22.42 ± 1.41
0.65 + 5.0 6.37 ± 0.03 * 84.5 ± 0.65 ** 28.5 ± 1.90 77.90 ± 4.98 22.10 ± 4.98
0.65 + 7.5 3.53 ± 0.03 ** 87.0 ± 0.71 ** 26.07 ± 3.16 28.61 ± 1.94 * 71.39 ± 1.94 *
0.65 + 10 4.63 ± 0.09 ** 76.5 ± 0.65 * 36.03 ± 3.79 76.77 ± 1.14 23.23 ± 1. 41
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Values are expressed as mean ± SE (n = 3). **p < 0.001,*p < 0.05 compared with control group

# The life span of the particular treated group of tumour bearing mice (i.e. 25 mg/kg body weight 2ME) is very low in comparison to control group, so data of tumour volume determination and viability test are not displayed in the table

The mortality rate is very high in case of 2.5 mg/ml 2ME + 5.0 mg/ml CP, 2.5 mg/ml 2ME + 7.5 mg/ml CP and 2.5 mg/ml 2ME + 10 mg/ml CP treated groups, so these are excluded

Table 3.

Combination Index (CI) value of the combination therapy (2ME + CP) at different combinations

2ME + CP (mg/ml) Combination Index (CI)
Tumour Volume Survivability Rate Viable Cells
0.25 + 5.0 2.63067 1.40681 0.45762
0.25 + 7.5 3.57474 2.42531 4.32109
0.25 + 10 4.26134 2.09979 0.52920
0.65 + 5.0 2.31154 1.88209 4.69372
0.65 + 7.5 1.27255 2.59988 0.28479
0.65 + 10 1.75046 3.16201 1.14316
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CI > 1.3 indicates antagonism, CI = 1.1 to 1.3 moderate antagonism, CI = 0.9 to 1.1 additive effect, CI = 0.8 to 0.9 slight synergism, CI = 0.6 to 0.8 moderate synergism, CI = 0.4 to 0.6 synergism, and CI = 0.2 to 0.4 strong synergism

The mortality rate is very high in case of 2.5 mg/ml 2ME + 5.0 mg/ml CP, 2.5 mg/ml 2ME + 7.5 mg/ml CP and 2.5 mg/ml 2ME + 10 mg/ml CP treated groups so, these are excluded from our combination index

Fig. 1.

Fig. 1

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Effect of different combinations of 2ME + CP on S-180 tumour bearing mice displayed by the Combination Index (CI) of tumour volume. The Combination Index value was determined by Chou-Talalay Method. a. Graph shows log fa –CI plot of tumour volume of different combinations of 2ME + CP doses where all values are greater than 1 indicating antagonism, fa = Fraction affected. b. Graph indicate CI plot of tumour volume. In this plot the values of tumour volume indicate antagonistic effect of all combinations of 2ME + CP as mentioned in the Table 1 of ‘Materials and Method’ section

Determination of survival time

Treatment with monotherapy as well as combination therapy showed considerable increase of life span of tumour bearing mouse when compared with control. But the survival time of 6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight treated combination therapy series was significantly longer (p < 0.001) than control and other treated groups. The survival time was 87.0 ± 0.71 (days, mean ± standard error) as shown in the Table 2. The treatment with that particular concentration of combination therapy (i.e. 6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight) antagonistically increased the life span of tumour bearing mice as shown in the Table 3 and Fig. 2.

Fig. 2.

Fig. 2

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Effect of different combinations of 2ME + CP on S-180 tumour bearing mice displayed by the Combination Index (CI) of survivability rate. The Combination Index value was determined by Chou-Talalay Method. a. Graph shows log fa –CI plot of survivability rate of different combinations of 2ME + CP doses where all values are greater than 1 indicating antagonism, fa = Fraction affected. b. Graph indicate CI plot of survivability rate. In this plot the values of survivability rate indicate antagonistic effect of all combinations of 2ME + CP as described in the Table 1 of ‘Materials and Method’ section

Viable and non- viable cells assay by trypan blue exclusion test

Scoring of viable and non viable or dead cells of control and different treated series was done. We counted unstained cells as an index of viable or proliferating cells using Cell Counter. As depicted in the Table 2 and Fig. 3 combination therapy particularly the concentration of 6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight significantly (p < 0.05) inhibits or reduces cell proliferation as compared to other therapies or control series. Maximum tumour regression (potential cure) in relation to cell proliferation was noted at 5th day of 2ME and CP treatment demarcated by an apparent absence of tumour cells in the peritoneal cavity of the host subjected to therapy. A maximum frequency of dead cells was noted at late hours of combination therapy. This particular dose and concentration of combination therapy proved to be strongly synergistic (CI values 0.28) as shown in the Table 3, Fig. 3. These studies demonstrate that strong synergism occurred when tumour cells are treated with combinations of the concentration of 6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight as defined by the Chou-Talalay analysis.

Fig. 3.

Fig. 3

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Effect of different combinations of 2ME + CP on S-180 tumour bearing mice displayed by the Combination Index (CI) of viable cells. The Combination Index value was determined by Chou-Talalay Method. a. Graph shows log fa –CI plot of viable cells of different combinations of 2ME + CP doses, fa = Fraction affected. b. Graph indicates CI plot of viable cells. In this plot the value of viable cells indicates synergistic effect particularly in the concentration of 6.5 mg 2ME /kg body weight + 75 mg CP/kg body weight as described in the Table 1 of ‘Materials and Method’ section

On the basis of Chou-Talalay analysis (Chou and Talalay 1984; Chou 1991) in the tumour volume determination and survival test groups, it is established that the effects of combination therapy (particularly the concentration of 6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight) appeared to be antagonistic where as in case of viable and non- viable cells assay the effects of the two components appeared to be synergistic. So other experiments i.e. Bone marrow toxicity assessment by Chromosomal Aberration Analysis, Tumour Chromosomal Aberration Analysis for Mitotic Catastrophe, Cell Cycle Analysis by Flow cytometry (FACS) and Assays for Apoptosis by Acridine Orange (AO) and Ethidium Bromide (EtBr) double staining were performed using only with that particular concentration of drug (i.e. 6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight) treated tumour bearing mouse and compared with the 2ME, CP monotherapy and control groups in this paper.

Bone marrow chromosomal aberration analysis

Bone marrow toxicity was analyzed through the scoring of different types of aberrations. Most aberrations recorded in the form of simple chromosomal aberrations (i.e. chromosome break, gap, lesion etc.) and complex chromosomal aberrations (i.e. centric fusion or submetacentric and metacentric chromosome etc.). It is interesting to note that the frequencies of chromosomal aberrations in 2ME and 2ME + CP treated series in bone marrow cells of S-180 tumour bearing mice are not high in comparison to control series but in CP treated series the aberrations are comparatively higher (Fig. 4).

Fig. 4.

Fig. 4

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a. Metaphase chromosomes (Giemsa stained) prepared from bone marrow cells of control group (all chromosomes are normal), b. Metaphase chromosomes (Giemsa stained) prepared from bone marrow cells of 2ME + CP (6.5 mg 2ME /kg body weight + 75 mg CP/kg body weight) treated series showing small metacentric chromosome (arrowed), c. Distribution of normal metaphase cells (NC) and affected metaphase cells (AC) in control, vehicle, and in 2ME, CP monotherapy treated and 2ME + CP (6.5 mg 2ME /kg body weight + 75 mg CP/kg body weight) treated series (300 metaphases studied from three specimens in each case), d. Histogram showing frequency distribution of different chromosomal aberrations recorded in control and treated series. Each graph represents the mean ± SE. P value was determined by Student’s t-test (*p < 0.05).NC = Normal Cells, AC = Affected Cells, SCA = Simple Chromosome Aberrations, CCA = Complex Chromosome Aberrations

Tumour chromosome for mitotic catastrophe analysis

S-180 tumour cell-line is a hypo-tetraploid chromosomal constitution with a modal number variable in between 73 and 75 (Chakrabarti et al. 1998; Pal et al. 1984) with three submetacentric or metacentric markers and a few minute chromosomes. The most frequent chromosome number observed in a tumour is chromosome modal number. The analysis of data revealed that single and combination therapy or 2ME, CP and 2ME + CP induced different types of chromosomal abnormality in form of simple and complex chromosomal aberrations. Most of the chromosomal aberrations were recorded in form of breaks, deletions, centric fusions, fragments in control and treated series whereas bi-radial, quadri-radial translocations or configurations were maximum in 2ME + CP combination therapy (Fig. 5a,b,c,d).

Fig. 5.

Fig. 5

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Giemsa stained tumour chromosome from ascitic fluid of S-180 tumour bearing mice in control, vehicle, 2ME, CP monotherapy and 2ME + CP (6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight) treated series with histographic representation. a. Normal chromosome complement of ascitic fluid in control series, b. Abnormal metaphase chromosome complement of ascitic fluid in 2ME + CP treated series showing chromosome break, exchanges (i.e. bi-, tri-, quadri-radial translocations). Red arrow showed chromatid break, black arrow showed chromatid exchange, c. Histogram showing frequency distribution of normal and affected metaphase cells in control and different treated series, d. Histogram showing frequency distribution of different chromosomal aberrations recorded in control and treated series. P value was determined by Student’s t-test (*p < 0.05and **p < 0.001). NC = Normal Cells, AC = Affected Cells, SCA = Simple Chromosome Aberrations, CCA = Complex Chromosome Aberrations

Analysis of cell cycle

The relative percentages of control and treated cells were analyzed in each phase of cell cycle. The increase of cell population at G2/M phase in 2ME, CP and 2ME + CP treatment was accompanied by a decrease of cell population in the G1 phase of the cell cycle. After 2ME treatment cells in G2/M population increased from 17.80% to 70.11% compared to control series whereas, in case of CP and 2ME + CP treatment cell in the G2/M population increased to 36.08% and 16.47% respectively. The increase of cell number or percentage in the G2/M was resulted or accompanied by the reduction of cell population in the G1 phase of cell cycle. The apoptotic cell population in 2ME, CP and 2ME + CP treated series increased to 2.28%, 1.55% and 5.37% respectively compared to 0.94% control group (Fig. 6).

Fig. 6.

Fig. 6

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FACS analysis of the cell cycle distribution of control and different treated series in S-180 tumour cell line. The increase of cell population at G2/M phase in 2ME, CP, 2ME + CP (6.5 mg 2ME /kg body weight + 75 mg CP/kg body weight) treatment was accompanied by a decrease of cell population in the G1 phase of cell cycle. Figure showing dotted plot (a = Con, b = Veh, c = 2ME, d = CP, e = 2ME + CP) and bar-diagram (f) showing distribution of cells in different stages of cell cycle of control and different treated series. Con = Control, Veh = Vehicle, 2ME = 2 methoxyestradiol, CP = Cyclophosphamide, 2ME + CP = 2 methoxyestradiol + Cyclophosphamide

Assay of apoptosis by AO-EtBr staining

The morphological changes associated with apoptosis and necrosis was investigated in control and treated series of S-180 tumour cells by AO-EtBr staining method. The viable or proliferating cells, apoptotic (early and late) and necrotic cells of control and treated series were studied on the basis of their staining nature (Fig. 7) and were statistically evaluated using Student’s t-test. It was interesting to note that 2ME + CP combination treatment revealed a large number of apoptotic cells (89.61 ± 2.35) and less number of proliferating or viable cells (5.49 ± 2.16) in comparison with control (AC = 27.51 ± 3.71, VC = 68.52 ± 3.99), vehicle (AC = 25.75 ± 10.55, VC = 68.84 ± 12.92), 2ME (AC = 48.75 ± 2.59, VC = 27.71 ± 6.79) and CP (AC = 67.66 ± 2.83, VC = 27.73 ± 3.00) treated series (Fig. 7a,b,c,d,e,f). So, combination therapy potently induces apoptosis and cell proliferation inhibition in Sarcoma 180 tumour cell lines. But 2ME or CP alone also exhibit inhibitory effect of cell proliferation through apoptosis but these effects were significantly less than combination treatment. But in Positive and Negative Control apoptotic cells are significantly less or minimum and proliferating or viable cells are maximum indicating that they do not undergo apoptosis.

Fig. 7.

Fig. 7

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AO-EtBr stained cells of control and different treated series in S-180 tumour cell line showing viable (yellow arrow) apoptotic (blue arrow) and necrotic cells (white arrow). a = Control, b = Vehicle, c = 2ME, d = CP, e = 2ME + CP (6.5 mg 2ME /kg body weight + 75 mg CP/kg body weight), f. Histogram showing percentages of proliferating, apoptotic and necrotic cells of control and different treated series.VC = Viable Cells, AC = Apoptotic Cells, NC = Necrotic Cells, Con = Control, Veh = Vehicle, 2ME = 2 methoxyestradiol, CP = Cyclophosphamide, 2ME + CP = 2 methoxyestradiol + Cyclophosphamide

Discussion

Combination therapy or the use of two or more anticancer drugs together is often a more effective in cancer treatment than monotherapy. Moreover, the use of anticancer drug at significantly lower dose than maximum tolerable dose may reduce serious toxic side effects. Browder et al. (2000) demonstrated that low dose chemotherapy has significant anti-angiogenic effect. According to Bello et al. (2001), low dose chemotherapy combined with an anti-angiogenic drug reduces tumour growth. Therefore, administration of lower dose of anti-cancer drugs in combination may be a better therapeutic strategy for cancer treatment due to significant tumour growth retarding property.

Cyclophosphamide (CP) has been used in both single as well as combination therapy for treatment of cancer (Friedman et al. 1979; Ahmed and Hombal 1984; Huang et al. 2000; Khan et al. 2004). 2 Methoxyestradiol (2ME), the novel antiangiogenic, antineoplastic component, now a days, has been used for treatment of different types of cancer in preclinical and clinical level (Zoubine et al. 1999; Banerjee et al. 2003; Folkman 2008). The cytotoxic effect of anticancer drug on tumour cells has been studied earlier (Nowell et al. 1964; Pal et al. 1984; Chakrabarti and Chakrabarti 1987) but so far as we are aware, chronological analysis of complex chromosomal aberrations i.e. triradial, quadriradial configurations induced by combinational therapy (2ME + CP) and its relationship with tumour regression and apoptotic cell death using an in vivo murine S-180 model has not been studied earlier. An examination of the data showed in the histogram (Fig. 5) indicated that although good number of metaphases recorded in the 2ME and CP monotherapy with simple and complex chromosomal aberrations, yet the frequency of affected metaphases is significantly high (p < 0.001) in 2ME + CP combination therapy. Similar trend has not been noticed in the control system. The maximum number of chromosomal abnormality (mainly, quadriradial and triradial chromosomal aberration) was noted in the 2ME + CP treated S-180 tumour cells which is the indication of cell arrest or apoptosis. In the present paper a clear correlation between the rate of tumour regression with an increase in dead cell frequency and induction of chromosomal aberrations in tumour cells with apoptosis was noted. It is assumed that, these abnormalities in higher frequency prevent the affected metaphases to enter into the next cell cycle resulting into apoptotic cell death. In the present experiment, combination therapy with low dose of 2ME and CP (i.e. 6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight concentration) significantly increased the therapeutic potentiality as evident from increased tumour cell proliferation inhibition and increased survival days or life span of the tumour bearing host. It is interesting to mention that in control series of S-180 tumour bearing mouse a regular rapid increase of ascitic tumour volume was observed. Ascitic fluid is the source of nutrition for the rapid proliferation of tumour cells (Prasad and Giri 1994). In the present experiment, treatment with 2ME, CP and 2ME + CP, the tumour volume was decreased but it is interesting to mention that the tumour volume had significantly (p < 0.001) decreased in the 6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight concentration treated combination therapy series. This particular concentration of 2ME + CP (i.e. 6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight) not only enhanced the life span of tumour bearing mouse and decreased the tumour volume antagonistically but also inhibited the viable cell population synergistically. It may be assumed that, combination therapy increases the survival time by decreasing the ascitic fluid volume and arresting the tumour growth.

In addition, combination therapy does not produce any toxic effect to host body as determined through the analysis of chromosomal abnormalities of bone marrow cells. Importantly no major toxic side effects were observed during treatment of mice as evident from chromosomal abnormalities analysis (Fig. 4). As a result, it is known that combination therapy of 2ME + CP is less toxic and safe to the host body. Combination therapy of 2ME + CP treatment induces G2/M arrest and produced a significant increase of cells in the G0 which is the indication of apoptosis. These observations are validated by Fluorescence staining of treated S-180 tumour cells with Acridine Orange and Ethidium bromide dye. Results indicate that 2 Methoxyestradiol in combination with Cyclophosphamide not only gave protection to bone marrow chromosomes of mouse but also induced tumour regression through apoptosis and increased the life span of tumour bearing mouse.

So, these studies suggest that use of 2ME and CP (i.e. 6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight) in combination therapy may represent an ideal and powerful approach to the treatment of cancer.

Acknowledgements

Acknowledgement is due to UGC, New Delhi (Ref.F.42-602/2013 (SR) dated 22.03.2013 MRP) for financial support. The paper is dedicated to late Professor Samar Chakrabarti, Cancer Cytogenetic Unit, Department of Zoology, Burdwan University. Authors are grateful to Dr. R.N. Boral and Dr. C.K. Panda, CNCI, for help. Authors are thankful to Dr. Snigdha Banerjee and Prof. Sushanta K. Banerjee, Kansas University Medical Center, Kansas, U.S.A. for encouragement. Authors are also grateful to Dr. Samiran Mondal and Dr. Ashesh Garai, Department of Chemistry, Rammohan College, Kolkata for help. Authors are thankful to the Principal Dr. Saswati Sanyal of Rammohan College, Kolkata for support and encouragement.

Abbreviations

AO

Acridine orange

CP

Cyclophosphamide

EtBr

Ethidium bromide

2ME

2Methoxyestradiol

S-180

Sarcoma 180

CI

Combination Index

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