EVALUATION OF PRO-APOPTOTIC ACTIVITY OF Chelidonium majus L . AND / OR GAMMA-RADIATION AGAINST EHRLICH AS CARCINOMA TRANSPLANTED IN MICE

This study was to evaluate, the pro-apoptotic activity of ethanolic Chelidonium majus L. (CM) leaf extract and/or gamma-radiation in Ehrlich ascites carcinoma (EAC) bearing mice by measuring tumor volume, apoptotic factors (caspase-3, Bcl-2 and Bax proteins, tumor necrosis factor-alpha (TNF-α), angiogenesis factors (matrix metalloproteinase (MMP-2 and MMP-9); tissue inhibitor of metalloproteinases (TIMP-1), DNA fragmentation, besides histopathological examination. After tumor inoculation, mice received CM (100 mg/body weight/day) on the 10 th day and were exposed to whole body γ-radiation (6Gy) on the 17 day. The data obtained reveales that combining CM with γradiation exposure induce significant regression in tumor growth, up-regulate Bax, caspase-3, TIMP-1 and inhibit Bcl-2, TNF-α, MMP-(2 and 9). Cytotoxicity is substantiated by increased DNA-fragmentation (comet assay and DNA content) and histological examination. It could be suggested that combining CM with gamma-radiation increased apoptosis of tumor cells which would help to increase the lifespan of mice.


INTRODUCTION
Cancer is one of the major causes of death, the general conventional treatment and standard care for clinical oncology remains surgery followed by radiation and/or systemic chemotherapy (ABD EL-WAHAB; FOUDA, 2009).Apoptosis is a mode of cell death used by multicellular organisms to remove cells in diverse physiological and pathological settings (BHATTACHARYYA et al., 2003).Modulation of signal transduction pathways, gene expression and arrest in cycle progression or apoptosis suppress tumorigenesis (CHATTERJEE et al., 2013).
Radiation therapy is considered to be one of the most popular and important tools to cure cancer.The killing action of ionizing radiation (X-rays, γrays) is through free radicals produced from the radiolysis of water in the cells.Free radicals by interacting with critical targets like DNA and membranes bring about irreversible damage leading to cell death (HANAFI; MANSOUR, 2010).The study of radio-sensitizers has emerged as a persistent hot spot in radiation oncology (SU et al., 2014).
The herbal drugs have gained attention and popularity because of their negligible toxicity and possibly with a ray of hope that they may replace some of the available toxic antineoplastic drugs.The Celandine (Chelidonium majus L., CM) is a plant of the Papaveraceae family, known for its richness in specific alkaloids such as sanguinarine, chelidonine, chelerythrine, coptisine or berberine.The plant contains also several secondary metabolites for example isoquinoline.Caffeic acid derivatives, several flavonoids and phenolic acids are present.The plant was shown to possess antitumor, antiinflammatory, anti-arthritic and antimicrobial properties which influence diverse cellular process (NADOVA et al., 2008).
The aim of this study was to evaluate the pro-apoptotic potential of ethanolic extract of CM leaves and/or gamma radiation in Ehrlich ascites carcinoma (EAC) bearing mice.This goal was achieved by measuring tumor volume, apoptotic factors such as caspase-3, Bcl-2 proteins, Bax proteins and tumor necrosis factor-alpha (TNF-α), angiogenesis factors including matrix metalloproteinase (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinases (TIMP-1), DNA fragmentation and histopathological examination.20 °C in a dark place.After drying, the material was fragmented and two gm of dried CM leaves were extracted with 50 ml of ethyl acetate using a Soxhlet apparatus.After 24 h, the infusions were filtered through Whatman No.1 filter paper and the residue re-extracted with 50 ml of ethyl acetate.After 48 h, the process was repeated.The combined supernatants were evaporated to dryness under vacuum at 40 °C using Rotatory evaporator and stored in sterile sample tubes in a refrigerator at 4 °C (JAKOVLJEVIĆ et al., 2013).

Cytotoxicity Assay:
The MTT assay to determine the growth and survival of Ehrlich Ascites Carcinoma cells (EAC) was performed in vitro according to Freimoser et al. (1999).The method is based on the reduction of )-2, 5-diphenyltetrazolium bromide) a water soluble salt to non-water-soluble violet formazan crystals.The amount of these crystals can be determined spectrophotometrically and serve as an estimate for the mitochondrial activity and hence the number of living cells in the sample.

Experimental animals
Adult female Swiss Albino mice (22-25g) were obtained from the breeding unit of the Egyptian Organization for Biological Products and Vaccines (Cairo) and used in this study.The animals received a commercial standard pellet diet and water ad libitum.Animal care and handling were performed according to the guidelines set by the world health organization, Geneva, Switzerland and approved from the committee for animals care at National Centre for Radiation Research and Technology (NCRRT), Atomic Energy Authority.

Tumor transplantation:
A cell line of EAC was used in this study.The parent line was supplied from the Egyptian National Cancer Institute (NCI), Cairo University.The EAC cells originated from breast cancer which were adjusted to grow in female Swiss albino mice and maintained by intraperitoneal injection (IP) of 2.5x10 6 EAC cells in the mice.The EAC cells were counted before their IP administration to mice using the bright line haemocytometer and diluted with physiological sterile saline solution.To induce Ehrlich Solid Tumor (EST), 0.2 ml (2.5x 10 6 EAC cells) was inoculated intramuscularly in the right thigh of the lower limb of female mouse (GUPTA et al., 2004).The day of EAC inoculation was referred to as day zero of experiment.

Radiation process:
Whole body γ-radiation was performing using a Canadian gamma cell-40 ( 137 CS) at the NCRRT.Cairo, Egypt.Mice were exposed to 6 Gy applied in one shot dose at a dose rate of 0.49 Gy/ min, on the 17 th day after EAC inoculation.

In vivo study:
70 Female Swiss albino mice were divided randomly into 5 groups.Normal group: 10 mice considered as normal control, E group: 15 mice bearing EAC, E+R group: 15 mice bearing EAC and exposed to γ-radiation, E+CM group: 15 bearing EAC mice treated with CM extract at a dose of 100 mg/kg b.wt (ZAREI et al., 2014) on the 10 th day after EAC inoculation and E+CM+R group: 15 bearing EAC mice treated with CM extract and exposed to γ-radiation.On the 24 th day, animals were fasted for 16 hrs prior to sampling.Then the animals were sacrificed and blood samples and tumor tissues collected for further biochemical and histopathological examinations.

Tumor volume monitoring
Tumor growth analysis was performed by measuring tumor volume, using vernier calliper on the 8 th , 16 th and 23 rd day.The volume of solid tumor was calculated using the formula [A X B 2 X 0:52], where A and B are the longest and the shortest diameter of tumor, respectively (O'REILLY et al., 1997).

Experimental parameters: Detection of caspase-3 and TIMP-1 gene expression by quantitative real time PCR (qRT-PCR):
RNA isolation and reverse transcription: RNA was extracted from the tumor tissue homogenate using the RNeasy plus mini kit (Qiagen, Venlo, The Netherlands), according to the manufacturer's instructions.Genomic DNA was removed by DNase-on-column treatment supplied with the kit.The RNA concentration was determined spectrophotometrically at 260 nm using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher scientific, Waltham, USA) and RNA purity was checked by absorbance ratio at 260/280 nm.RNA integrity was assessed by electrophoresis on 2% agarose gels.One µg of RNA was used in the subsequent cDNA synthesis reaction, using the Reverse Transcription System (Promega, Leiden, The Netherlands).Total RNA was incubated at 70 °C for 10 min to prevent secondary structures.The RNA was supplemented with MgCl 2 (25mM), RTase buffer (10X), dNTP mixture (10mM), oligod(t) primers, RNase inhibitor (20 U) and AMV reverse transcriptase (20 U/µl).This mixture was incubated at 42°C for 1 h .
Quantitative real time PCR: qRT-PCR was performed in an optical 96-well plate with an ABI PRISM 7500 fast sequence detection system (Applied Biosystems, Carlsbad, California) and universal cycling conditions of 40 cycles of 15 s at 95°C and 60 s at 60°C after an initial denaturation step at 95°C for 10 min.Each 10 µl reaction mixture contained 5 µl SYBR Green Master Mix (Applied Biosystems), 0.3 µl gene-specific forward and reverses primers (10 µM), 2.5 µl cDNA and 1.9 µl nuclease-free water.The sequences of PCR primer pairs used for each gene are shown in Table 1.Data were analyzed with the ABI Prism sequence detection system software and quantified using the v1•7 Sequence Detection Software from PE Biosystems (Foster City, CA).Relative expression of studied genes was calculated using the comparative threshold cycle method.All values were normalizing to the endogenous control GAPDH (LIVAK;SCHMITTGEN, 2001).

Determination of Serum MMP-2 and -9
Determination of MMP-2 and MMP-9 was carried out using polyacrylamide gel electrophoresis according to Birkedal-Hansen and Taylor (1982).MMP-2 and MMP-9 protein levels are quantitatively assessed by vertical slap SDS-PAGE containing 1 mg/ml type B gelatine as a substrate for the enzymes.MMP-2 and MMP-9 detected on the SDS gel as transparent bands.The serum samples were used without boiling, MMP-2 (72 kDa) and MMP-9 (92 kDa) proteins can be defined on slap gel by their gelatinolytic enzyme activity, which appeared as bands on background and compared to standard human purified collagenase IV (Sigma Co., USA) and a protein marker (Sigma Co., USA) the gel system adapted as described by Davis (1964).

Single cell gel electrophoresis (SCGE) Comet assay
The alkaline comet assay was performed on tumor tissue.Tissues were dissected and homogenized in ice-cold buffer (0.075 M NaCl and 0.024 M Na 2 EDTA; 1 g tissue ⁄ 1 ml buffer).The comet assay was carried out under alkaline conditions, as described by Singh et al. (1988).The slides were stained with ethidium bromide (20 lg ⁄ ml) and examined using a 250 X magnification fluorescence microscope (Zeiss, Jena, Germany) equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm.A computer-based image analysis system (Comet Assay II; Perceptive Instruments Ltd., Suffolk, UK) was used.A total of 50 comets per sample per animal were scored, and data were pooled (250 comets per group).Tail intensity (% DNA in the comet tail) and tail length (expressed in micrometres) were evaluated.The values of tail length were used for the determination of the long-tailed nuclei count as described previously (BENKOVIC´et al., 2009).

Histopathological Estimation of Tumor
Portions of the formalin-fixed tumor were setting in paraffin, sectioned and stained with hematoxylin and eosin stain (H&E) for histopathological examination under a light microscope (BANCROFT et al., 1996).

DNA content
Total protein was detected by mercurybromophenol blue stain (MAZIA et al., 1953) and DNA content was examined by Feulgen reaction (PEARSE, 1985).

Immunohistochemistry
Immunostaining technique of TNF-α, Bcl-2 and Bax proteins were tested by the method of Teerds and Dorrington (1995).

Statistical Analysis
The data were statistically analyzed using SPSS computer Program (v.17).The results were presented as Mean ± SE.The differences between mean values were analysed by one way analysis of variance (ANOVA test) followed by LSD as a post Evaluation of pro-apoptotic activity… THABET, N. M.; MOUSTAFA, E. M.; EL-KHASHAB, I. H. Biosci.J., Uberlândia, v. 32, n. 6, p. 1657-1668, Nov./Dec.2016 hoc test for multiple comparisons.All statements of significance were based on probability of P ≤ 0.05.

Cytotoxicity Assay:
To show the effect of CM on cell viability, EAC cells were incubated with CM at different concentration (50, 75,100 and 150 µg/mL) for 48 hours.The results revealed that, CM at a dose of 100 µg/mL was the most efficient in decreasing the viability of EAC cells (Table 2).Tumor Volume: The results revealed that, treatment of EAC bearing mice with CM and/or gamma-radiation caused a marked decrease in tumor volume compared to E-group (Figure 1).

Detection of Caspase-3 and TIMP-1 Gene Expression:
In the present study, the results showed that in the control group the gene expression of caspase-3 and TIMP-1 was 1±0.13 fold and 5.03±0.59fold, respectively.In E-group, the data revealed nonsignificant change in gene expression of caspase-3 mRNA compared with control.But in EAC mice treated with CM and/or exposed to γ-radiation, it was significantly higher compared to E-group (P< 0.05) (Table 3).On the other hand, the results displayed a significant decrease (P<0.05) in TIMP-1 mRNA in E-group compared with control group.Meanwhile, a significant increase in TIMP-1 mRNA was recorded in E+CM and E+CM+R groups compared to E-group (P < 0.05) (Table 3).

Detection of serum MMP-(2 and 9) by gel electrophoresis:
The experimental data for MMP-(2 and 9) in different mice groups is presented in Table 4 and Figure 2. The concentrations of MMP-2 and MMP-9 in active form were 117.12±1.26 and 86.35±0.431U/mg proteins in control group, respectively.A significant increase in MMP-(2 and 9) concentration was observed in samples isolated from the serum of E-groups as compared to control group.The treatment with CM and/or γ-radiation induced a remarkable modulation in MMP-(2and9) concentration as compared to E-group.The qualitative analysis of the zymographs showed that the majority of E-groups serum showed gelatinolytic bands for both the active enzymatic forms of MMP-2 and MMP-9 when compared with normal serum (Figure .2).Legend as Table 3 Comet Assay: The results displayed the DNA strand breaks by CM or ionizing radiation, in isolated lymphocyte cells, whereas damaged nuclei consist of a head with DNA migrating into the tail region because of extensive single-strand breaks.The results showed normal nuclei in the control group (Figure 3-C), different levels of DNA damage in Egroup (Figure 3-1), and apoptotic nucleus in tumor cells in E+R, E+CM and E+CM+R groups (Figure 3-2, 3-3 and 3-4), respectively.Average background DNA damage in lymphocytes was significantly higher in the E+CM+R group than those of E-group (Table 5).Legend as Table 3;

Histological Studies: Solid tumor in muscle tissue
Histopathological examination of the solid tumor in muscle section of E-group, showed tumor cells characterized by their large, round and polygonal with polymorphic shape, basophilic cytoplasm, hyper-chromatic nuclei and mitosis (Figure 4A) and many islets of tumor cells surrounding muscular fibres (Figure 4B).It was observed also that, islets of tumor cells were enclosed by necrotic rupture muscle fibres replacing the normal fibres (Figure 4C) and such islets were invaginated by newly formed blood capillaries "angiogenesis" surrounded by a thick zone of tumor cells (Figure 4D).Nevertheless, solid tumor of E+R and E+CM groups showed small regression in volume of islets of tumor cells that were surrounded by muscular tissue capsule (Figure 5A and 5B, respectively).While, the solid tumor of E+CM+R group, showed more regression in tumor volume than the previous groups (Figure 5C).

DNA content in solid tumor:
Solid tumor sections of E-group showed DNA chromatin forms well-defined irregular clumps with variable sizes, shapes and sharp pointed projections as well as tumor nuclei with multiple nucleoli and irregular nuclear membrane (Figure 6A).On the other hand, E+CM+R group revealed a few neoplastic cells and some apoptotic cells identified by DNA fragmentation (Figure 6B).

Immunohistochemistry TNF-α
Immunostaining of TNF-α was identified by the brown coloration in the cytoplasm of tumor tissue.Its overexpression elevation is clearly indicated in E-group (Figure 7A and 7B).Whereas, in E+R (Figure 7C and 7D) and E+CM (Figure7E and 7F) groups, TNF-α expression was slightly reduced in the cytoplasm of solid tumor cells compared to the previous group.While, in E+CM+R group (Figure7G and 7H) TNF-α expression was highly reduced compared to the previous groups.

Bcl2
The accumulation of Bcl-2 protein is indicated by a brown coloration in the cytoplasm of tumor tissue.Strong immune reactivity for Bcl-2 was detected in the E-group (Figure 9A and 9B).While, the E+R (Figure 9C and 9D) and E+CM groups (Figure 9E and 9F) revealed weak expression in Bcl-2 protein compared with the previous group.On the other hand, strong down expression of Bcl-2 protein was observed in the E+CM+R group (Figure 9G and 9H) compared with the previous groups.

DISCUSSION
In the present study, the results of E-group revealed increased angiogenesis and depressed apoptosis.the resistance of cancer cells to TNF-α induced death based on the fact that TNF-α induced survival signals such as nuclear factor-κB (NF-κB) that blunt the apoptotic pathway and activated the survival pathway ( WAJANT et al., 2003).In a concurrent study, Abdel-Rahman and Kabel (2012) noted that, immune-staining of TNF-α and showed over expression in the group of mice bearing tumour while down expression of Bax was observed in the same group.Meanwhile, Sun et al. (2010) reported that cancer cells down-regulate TIMP and produce extracellular MMP-inducer, a cell surface glycoprotein, which directly stimulates fibroblasts to produce MMP-1, 2, 3, 9 and 14.Changes occurring in the biochemical parameters were confirmed by the histopathological observation.The histopathological examination showed islets of tumor cells were invaginated by newly formed blood capillaries "angiogenesis".Active angiogenesis are identified by the presence of irregular blood capillaries to supply the tumours (GROSSMAN; MCNEIL, 2012).This leads to activation of NF-κB in cancer cells, which regulate transcription of genes involved in cell proliferation, anti-apoptosis and invasion.Activation of NF-κB induces tumour growth and reduces cytokinesinduced apoptosis (AGGARWAL et al., 2006) which is supported by the previous findings of Salah (2011) andHanafi (2012).the disappearance of dna fragments in e-group, postulates loss of apoptosis ended by tumour development.Ghobrial et al. (2005) reported that types of cancers characterized by defects in apoptosis leads to immortal clones of cells.
CM and/or γ-radiation treatment, in the present study, revealed regression in tumour volume.It is well known that exposure of mammalian cells to ionizing radiation induces several types of damage to DNA, including double and single-strand breaks, base and sugar damage, as well as DNA-DNA and DNA-protein cross-links.During radiotherapy, ionizing radiation interacts with biological system and induces excessive oxygen free radicals or reactive oxygen species (ROS) which attack cellular DNA, proteins and membrane lipids.This leads to significant cellular damage by increasing apoptotic cells % and decreasing the mitochondrial membrane potential (BEGUM et al., 2012).Further, Kitada et al. (1996) showed that exposure of mice to γ-radiation results in a rapid elevation in the levels of the Bax protein in lymphoid cells.However, ionizing radiation induces mitotic cell death in dividing cells and activates pathways that lead to death by apoptosis in interphase cells and differentiated cells.In addition, Lawrence et al. (2008) reported that, γ-radiation therapy shrink tumour and kill cancer cells through damaging their DNA directly or creates free radicals within the cells to damage the DNA.Susskind et al. (2003) found that, radiation provokes a sharp decrease in MMP-9 within 2 weeks, without change in TIMP-1, and that decrease reflects reduced neutrophil and macrophage involvement in the neoplastic process.
The modulation of tumor by CM treatment could be referred to its anti-inflammatory, antimicrobial, antiviral and antitumor activities.In addition, CM showed an anti-angiogenesis effect that was dependent on decreased activity of migration, inhibition of VEGF and MMP-2,-9 and increase of TIMP release (KIM et al., 2011).Beside and Park et al. (2015) found that, CM extract induce cell cycle arrest, activation of the caspase-3 dependent pathway, blocking of NF-κB activation as well as significantly decrease the mRNA levels of Bcl-2, survivin and increase Bax expression.Moreover, sanguinarine isolated from CM, suppresses NF-κB and sensitizes cancer cells to apoptosis by inducing ROS generation and activate TNF-related apoptosis-inducing ligand ( Gupta et al., 2010).In addition, Choi et al. (2009) found that, sanguinarine down regulates MMP-2 and MMP-9.
It is concluded that, CM as well as γradiation decrease tumor growth by initiating apoptosis, distribution of nuclear DNA fragmentation and histopathological investigations besides up-regulating Bax while down-regulating Bcl-2, by activation of caspase-3 and inhibition of TNF-α, by releasing TIMP-1 and preventing MMP-2,-9.The combination of both CM and γ-radiation has a better anti-tumor effect against tumour growth than CM or γ-radiation alone.

Figure 1 .
Figure 1.Effect of administration of CM (100 mg/kg b.wt) and/or exposed to γ-radiation on tumor volume in E treated mice.Values were represented as mean ± SE (n=10).a: Significant compared to the control group (P < 0.05).b: Significant compared to E group (P < 0.05).

Figure 3 .
Figure 3. Appearances of comet in different types of mice tumour tissue.All photomicrographs are acquired by image analysis system Comet Assay II (Perceptive Instruments Ltd., Suffolk, UK) under magnification 250x.Stained with ethidium bromide.

Figure 5 .
Figure 5. Photomicrograph of solid tumour section treated with γ-radiation (A) or CM (B) showed small regression in volume of islets of tumour cells (black star) that were surrounded by muscular tissue capsule (H&E× 400).(C): group treated with γ-radiation and CM showed more regression in tumour volume (black star) (H&E× 400).

Figure 6 :
Figure 6: Photomicrograph for the determination of DNA content in solid tumour tissue (A) showed DNA chromatin forms well-defined irregular clumps with variable sizes, shapes and sharp pointed projections (arrow) and tumour nuclei with multiple nucleoli and irregular nuclear membrane (arrow) in (E) group.(B) Showed some neoplastic cells (round arrow), some apoptotic cells illustrated by DNA fragmentation and some dead cells (white star) in E+CM+R.

Table 2 .
Effect of different concentrations of CM extract on the cell viability of EAC cells Values were represented as mean ± SE. a: Significant compared to EAC group (P < 0.05)

Table 3 .
Effect of CM and/or γ-radiation on gene expression of caspase-3 and TIMP-1 d Values are means ± SE (n = 10); Values with different superscripts significantly different, at P≤0.05;Values with same superscripts notsignificantly different, at P≤0.05.

Table 4 .
Effect of CM and/or γ-radiation on serum MMP-(2 and-9) (U/mg protein) in active forms analyzed by Gelatin zymography

Table 5 .
Comet assay expressed as tail length