Discussion use in drug industry, about 50% of

Discussion      Natural products are a group of compoundsproduced by living organisms and having medicinal effects, so it can be used indrug discovery and design.

Its may be extracted from tissues of terrestrialplants, marine organisms or microorganism. The crude extracts of these sourcescontain biologically active compounds (Lahlou., 2013) whichinteract  with its target while causingless side effects, that increased its use in drug industry, about 50% ofbest-selling drugs are related to natural products (Harvey.

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, 2000). Butthe challenge of using natural products in drug discovery is their complexstructure and the limited supply but this problem has been overcome with thesynthesis of the analogues of these natural compounds as the compounds used inthis thesis.       Coumarins are type ofnatural compounds produced by many plants as defense mechanism, it was firstlyisolated from tonka bean, coumarin is a member of benzopyrone family which isdivided into benzo-?-pyrones and benzo-?-pyrones, that coumarin is belonging tobenzo-?-pyrones while flavonoids belong to benzo-?-pyrones. Coumarins showeddifferent medical activities including: anticancer activity (Rohini  and Srikumar, 2014).         The results of this studyshowed that furocoumarin compound number 9 (4,9-dimethoxy-5-methyl-7-oxo-7H-furo 3, 2-g chromene-6-carbonitrile) exertedthe highest cytotoxic activity against HepG-2 cells with Ic50=11.9, while theother synthetic coumarin derivatives exhibited less cytotoxic activity. Manystudies proved the cytotoxic activity of coumarin derivatives against differentcancer cell lines including HepG-2 like the study Sun et al (2011)made using coumarins derivative DMFC (3, 5-dimethyl-7H-furo3,2-gchromen-7-one)to treat HepG-2 showed similar results and proved that apoptosis was induced throughboth extrinsic and intrinsic apoptotic pathways in a P53-dependent manner.

         AlsoEl-Nakkady et al  (2015) demonstratedthat the synthesized furocoumarin compounds showed significant cytotoxicactivity against HepG-2 cell line and compound 4-bromo-9-methoxy-7H-furo 3,2-g chromen-7-one exerted the highest activity with IC50=10.5µg/ml. Manystudies proved the anticancer activity exerted by synthesized coumarin derivatives(Reddy et al., 2004; Goelet al., 2007; Liu et al., 2010; Ranganath et al.

,2011; Musaet al., 2012 and Morsy et al., 2017).        The results of SRB assay made using plantextracts containing coumarins revealed that Verbascum thapsus seeds and Achilleamillefolium leaves showed the highest cytotoxic activity against HepG-2cell line with IC50=27µg/ml and 32µg/ml, respectively.

While Ammimajus L. exerted the lowest cytotoxic activity with IC50=511.8.      Verbascumspp. contain high content ofcoumarin and the highest concentration present in plant seeds. Lin et al.,(2002) study had proved that the hot water crude extract of Verbascumthapsus prevent hepatoma in five human liver-cancer cell lines HepG2/C3A,SK-HEP-1, HA22T/VGH, Hep3B and PLC/PRF/5.

              Talib and Mahasneh (2010) studied thecytotoxic effect of the related species Verbascum sinaiticum andthey found that the methanol flower extracts showed significant cytotoxicactivity against MCF-7 and Vero cell lines (African monkey kidney cell line) withIC50= 25.15 and 17.68µg/ml, respectively. In addition Verbascumspp. showed anticancer activity against different cell lines SK-MEL, P-388, Ehrlichascites carcinomas in CF1 mice cell lines (Afifi et al., 1993 andTatl? and Akdemir 2006).

                Navaieet al (2016) showed that the methanol, extract of leaves of Achilleamillefolium exerted significant cytotoxic activity against MCF-7 (breast cancercell line) with IC50= 40.54µg/ml. In agreement with our results Ghavamiet al (2010) reported that methanol extract of Achilleamillefolium exerted significant cytotoxic activity against number of cancercell lines with IC50 ranged from 22.051 to 66.00µg/ml. Ahmed etal (2014) proved that the related species Achillea aleppicaexerted significant cytotoxic activity against HepG-2 cell line.      The polar extract of Achilleamillefolium contain number of coumarin derivatives as scopoletin I,umbelliferone II and aesculetin III, the anticancer activity of these coumarincompounds was reported in many studies. They exert their anticancer effect through inhibiting cyclins,upregulating apoptosis genes and cell cycle arrest (Elinos-Baez et al.

, 2005; Jeon et al., 2015; Li et al.,2015 and Lim et al., 2016).          Murraya paniculata leaf extract in thisstudy exerted high cytotoxic activity with IC50=35.

The study madeby Noolu et al. (2015). In this study M. paniculataexhibited high cytotoxic activity against different cell lines including HepG-2cell line and its IC50 was 17.55?g/mL on contrast to ourresults this difference in IC50 value may be due to the variance inthe quality of methanol used or season of plant collection. The different typesof Murraya paniculata extracts showed cytotoxic activity againstdifferent cancer cell lines.       Themain coumarin compounds isolated from M. paniculata were7-methoxy-8-(3-methyl-2-oxobutoxy)-2H-chromen-2-one, 6-hydroxycoumurrayin (Liet al.

, 2016), 2′-O-ethylmurrangatin (Choudharyet al., 2002),  murrayanoneand murraculatin (Wu, 1988). Murrmeranzin, murralonginal, minumicrolin,murrangatin, meranzin hydrate and hainanmurpanin (Saied et al.

, 2008).Omphamurrayone (Kinoshita et al., 1996), Paniculacin (Saeedet al., 2011).         Umbelliferone and scopoletin from the isolated compounds from Murrayapaniculata (Saeedet al., 2011).

They exhibit anticancer activity against different cancercell lines. Murata et al. (2008) demonstrated that furocoumarincompound murrayacoumarin B present in Murraya spp. as Murrayapaniculata showed significant anticancer activity against humanleukaemia HL-60 cells due to induction of apoptosis via caspase-9/caspase-3pathway.        Ruta graveolens is old medicinal plantwhich contains many active constituents including various furanocumarins,carotenoids, chlorophyll, furanoquionolones (Hale et al.

, 2004; Eickhorstet al., 2007; Malik et al., 2013).

      Ruta graveolens showed anticanceractivity against different cell lines including MCF7, Dalton’s lymphoma ascites(DLA), Ehrlich ascites carcinoma (EAC), L929 cells and brain cancer cells (Pathak etal., 2003; Preethi et al., 2006; Pushpa et al., 2015).   Ruta graveolens exertits anticancer activity through inducing DNA damage and blocking Akt activation,induction of P53 pathway and inhibiting topoisomerase I (Fadlalla et al.,2014; Manjulaand Mani,2016).

 The activation of p53 pathwaywas reported by many furocoumarins like bergapten which present in this plant,also bergapten in addition to xanthotoxin and psoralen present in this plantact as potent inhibitors of topoisomerase I (Manjulaand Mani,2016).         Theresults showed that Ferula asafoetida exerted low anticancer activity with IC50=412.2 µg/ml against HepG-2 cell line, In agreement withthese results (Unnikrishnanand Kuttan (1988) proved that Ferula asafoetida extract exerted its cytotoxicactivity against different cancer cell lines as human lymphocytes, Vero cells,Chinese hamster ovary (CHO) cells and Dalton’s lymphoma with IC50ranged from 150µg/ml to 600 µg/ml. Mahendra and Bisht (2012) mentionedthat the anticancer activity of F. asafetida extract is attributed to presenceof number of compounds with anticancer activity as isopimpnillin (furocoumarincompound), Umbelliferone (furocoumarin compound), ?-pinene,  Luteolin.      Thisplant also contain five sesquiterpene coumarins namely, conferone, badrakemin,feslol, isosamarcandin and samarcandin (Ashgari et al., 2016).

Theanticancer activity of conferone and samarcandin was recorded in studies madeby(Valiahdi et al., 2013 and Ghoran et al., 2016) using different cancer cell lines.

        Apium graveolens and Ammi majusshowed the lowest cytotoxic activity against HepG-2 cell line on contrast they showedhigh cytotoxic activity in other studies against different cancer cell lines,the varied responces of different cancer cells to the same drug is attributedto the Heterogeneity in cancer cells (Arul etal., 2017) that tumor heterogeneity mean the observation that differenttumour cellscan show distinct morphological and phenotypicprofiles, including cellular morphology, gene expression, metabolism, motility,proliferation, and metastatic potential (Marusykand  Polyak , 2010).           Because of the high cytotoxic activityexerted by compound 9 it was given a special attention to understand itsmechanism of action using other tools like: molecular docking to recognize itsinhibitory effect on Topoisomerase I which is a target of many anticancerdrugs, chromosomal aberration assay: to assess their mutagenic and cytotoxicactivity, RT-PCR to examine its effect on the expression level of cell cycleregulatory genes like: cyclin b and cyclin D and ISSR to understand the effectof them on genome stability.

           Topoisomerases are enzymeswhich help in completing the process of DNA replication through overcoming theproblem of DNA supercoiling which arise during DNA replication by cleaving andrejoining DNA strands. They are present in eukaryotes, archaebacteria andeubacteria. Because of its importance not only in the process of DNAreplication but also in recombination, and transcription, they became target ofmany anticancer drugs. The opening of double stranded DNA generate supercoilingin the replication fork which prvent further separation of two strandsresulting in stopping of replication process (Kellner et al., 2002).

There are two major classes of topoisomerases, type I and type II, that aredistinguished by the number of DNA strands that they cleave and the mechanism bywhich they alter the topological properties of the genetic material. Topo I cleaveone strand of DNA molecule while Topo II cleaves both DNA strands, that theinhibitors of topo I interrupt the DNA replication, which is a useful in cancertherapy   (Ewesuedoaand Ratain,1997).         The molecular docking approach can beused to model the interaction between a small molecule (like coumarincompounds) and a protein (like topoisomerase I) at the atomic level, whichallow us to characterize the behavior of small molecules in the binding site oftarget proteins  as well as to elucidatefundamental biochemical processes (McConkey et al., 2002).           From the resultsobtained using molecular docking it was found that compound 9 have a goodability to inhibit topoisomerase I which in turn leads to cancer inhibitionthrough the inhibition of DNA replication.

The inhibition of Topoisomerase I bylinear furocoumarin compounds (like compound 9) was reported previously by  Diwan and  Malpathak(2009) who examined theinhibitory effect of the crude extract of Ruta graveolens and the isolated furocoumarin compounds on theTopisomerase I  enzyme and they concludedthat psoralen, bergapten and xanthotoxin (linear furocoumarins isolated from Rutagraveolens) are potent topoisomerase I inhibitors which promoteStabilization of DNA–topoisomerase covalent complex.            On the other hand otherstudies suggested the inhibitory effect of a furocoumarin compound like4-hydroxymethyl-4′,5′-benzopsoralen on topoisomerase ii). Topoisomerase IIinhibitors exert their biological activity via increasing the covalentenzyme-cleaved DNA complexes that normally are intermediates in the catalyticcycle of topoisomerase II.

As a result of their action, these drugs generatebreaks in the genetic material of treated cells which induce cell deathpathways (Fortune et al., 2000).   From the mechanisms which also can explainthe ability of furocoumarin to inhibit cancer development is that furocoumarinsable to activate the production of singlet oxygen which react with proteins andinactivate  these proteins as topoisomeraseenzyme proved by Lu et al (2005) thus inactivation oftopoisomerase enzyme in turn triggers the apoptosis, this may be the other wayby which furocoumarin inhibit topoisomerase, That furocoumarins may inactivatetopo enzyme directly by interaction with the enzyme or indirectly viaactivating the production of reactive oxygen species.            Many studies suggestedthat furocoumarins in combination with UV induce DNA-protein crosslinking whichcan lead to cell death or stop cell cycle progression by impeding DNA and RNAsynthesis. But our study made in the dark (Bordin  et al., 1993).             Many of the assayshave been developed to investigate the mutagenic activity of the testedchemicals as Rodent micronucleus assay, Bone marrow, peripheral blood (mouse)assay for clastogenicity with kinetochore and centromeric staining, Cometassay, Salmonella test for gene mutation, and Allium cepa assay (Bajpayeeet al., 2005).

Allium cepa test have been developed by (Levan,1938), and then many plants have been used for chromosomal aberration assayas Vicia faba, Zea mays, Tradescantia, Nicotiana tabacum,Crepis capillaries, Hordeum vulgare and Pisum sativum. TheInternational Program on Chemical Safety (IPCS) permitted assessment theavailability of plant assays to determine the mutagenicity and clastogenicity,and their studies concluded that plant assays are efficient and reliable testsystems for rapid screening of chemicals for mutagenicity and clastogenicity (Ma,1999).The in vivo root-tip assay is known to give similar results to invitro animal cytotoxicity tests (Chauhan et al., 1999; Vicentini etal., 2001, Teixeira et al.

, 2003, Eren and Ozata, 2014, Khalifa etal., 2014).           Treatment of Pisum sativumroot tips with different concentrations of the synthetic coumarin compounds andtwo plant extracts containing coumarin derivatives under investigation resultedin a significant decrease in mitotic index at the high concentrations. Thedecrease in mitotic index indicated that the experimental materials exhibitedmitodepressive effect which had been assumed to result from the inhibition ofcells access to mitosis (Badr and Ibrahim 1987). Such an antimitoticeffect is most likely attained by preventing DNA biosynthesis or/ andmicrotubule formation (Yüzba??o?lu et al., 2003).

This might beattributed to a slower progression of cells from S (DNA synthesis) phase to M(mitosis) phase of the cell cycle (Blakemore et al., 2013).          In addition the appearnance of different types of chromosomalabnormalities was recorded, the types of these abnormalities include C-metaphase,chromosomal breaks and chromosomal stickiness. These results are in agreementwith those obtained from treating onion root tips with coumarin and coumarinderivatives (Dolcher. 1960; Mongelli et al.

, 2000; Vera et al.,2001, Riveiro et al., 2004; Knoll et al., 2006). Theappearance of apoptotic cells has been recorded in the root cells treated withcompound 8.

This may be due to the ability of furocoumarins to induce reactiveoxygen species which able to induce apoptosis in plant cells (Breusegem and Dat ?2006).  These results agreed with the results of Xiaet al. (2014) who showed how the photoactivated PUVA (psoralen andUVA) activate apoptosis in breast cancer cells which is recognized with theover expression of the ErbB2 receptor tyrosine kinase oncogene. Kim  et al. (2014) also showed thatBergamottin (linear furocoumarin) induced apoptosis through the inhibition ofSTAT3 signaling pathway in tumor cells, which is related to growth, survival,proliferation, metastasis, and angiogenesis of various cancer cells in additionto down regulation of STAT3-regulated genes COX-2, VEGF, cyclin D1, survivin,IAP-1, Bcl-2, and Bcl-xl. Thus, STAT3 is an important target for manyanticancer drugs.

           The chromosomal aberration assay indicatedthe presence of anaphase and telophase cells with lagging chromosomes andbridges. This type of mitotic abnormality results from the SAC pathwaydisruption (which is responsible for stopping the premature separation ofsister chromatids and formation of premature anaphase cells) thus disturbingthis pathway resulting in cell death or the formation of aneuploid cells (Castedoet al., 2004; Thompson and Compton 2010; Vitale et al., 2011).SAC pathway disruption may be among the mechanisms by which coumarins exerttheir action. On the other hand, the C-metaphase cells are known to appearbecause of the failure of the separated chromatids to reach the two poles, as aresult of complete inhibition of spindle formation accounting for the stopping ofmitosis in the mitotic phase, the inhibition of cytokinesis,  formation of polyploid cells and apoptosis(Kim et al.

, 2004). So that Spindle fibers are target for anticancerdrugs blocking its formation as Taxol and Vinca alkaloids (Cutler and ? Cutler., 1999).  The appearance of chromosomal stickiness wasalso recorded in the present work. This type of abnormalities is attributed tothe failure in topoisomerase II and the peripheral proteins whose function isnecessary for separation and segregation of chromatids during anaphase. Inaddition, the changes being caused either by mutation in structural genes forthe proteins (heritable stickiness) or by direct action of mutagens on theproteins (induced stickiness) (Gaulden et al.

, 1987). Topoisomerasepoisoning has biomedical effect in certain diseases as cancer (Liu, 1989;Fortune et al., 2000; Pommieret al., 2010 ; Ashour et al., 2015). Many of naturaland synthetic coumarin derivatives proved to have inhibitory effect onTopoisomerase II enzyme (Marzano  et al.

, 1997, Lewis et al., 1996; Pani  et al., 1994, Hueso-Falcónet al., 2017).          Chromosomal breaks werealso recorded among chromosomal abnormalities. It is produced as a result ofseries of mechanisms beginning with liaison followed by DNA strand breakage by(formation of pyrimidine dimmer, alkylating or inter and intrastrand crosslinkage (Therman et al., 1993). Recent researches showed that theanticancer activity of intercalating agents resulted from their interferingwith the action of topoisomerase during their binding to nuclear DNA, thuscausing chromosomal break (Ralph  et al.

, 1993). Topoisomerase IIinhibitors exert their biological activity via increasing the covalentenzyme-cleaved DNA complexes that normally are intermediates in the catalyticcycle of topoisomerase II. As a result of their action, these drugs generatebreaks in the genetic material of treated cells which induce cell deathpathways (Fortune et al., 2000).       Cyclinsare group of proteins which form complexes with cyclin dependent kinases toactivate the progression of cell cycle from stage to another in theproliferating cells. Cyclin b is able to activate the progression of cell cyclefrom G2 to mitotic phase while, cyclin D stimulate progression from G1 to Sphase.

 Cyclin D1 forms active complexesthat promote cell cycle progression by phosphorylating and inactivating theretinoblastoma protein (RB) (which is an important tumor suppressor protein) (Katoet al., 1993; Lundberg et al., 1998; Weinberg.,  1995)        Theoverexpression of cyclin D can lead to the uncontrolled cell division whichmake it act as an oncogene, that the over expression of cyclin D can occur byone of three ways which are: gene amplification, impaired protein degradation,or chromosomal translocation. Cyclin D1 is important for the development andprogression of several cancers including those of the breast, oesophagus,bladder and lung (Hall and Peters.

, 1996; Vermeulen et al., 2003, Gillett etal., 1996, Knudsen et al., 2006, Motokura and Arnold.

, 1993; Musgrove., 2006; Sicinskiet al., 1995; Sutherland and Musgrove, 2002; Weinstat-Saslow et al., 1995; Yamamotoet al., 2006).

Overexpression of cyclin D1 has also been linked to thedevelopment of endocrine resistance in breast cancer cells and hepatocellularcarcinoma (Hodges et al., 2003, Hui et al., 2002, Kenny et al.

, 1999; Zhang etal., 1993). Cyclin D1 overexpression is a common event in cancer.

Theimportance of cyclin D1 in cancer makes it an attractive target for anti-cancertherapy (Yu et al., 2001, Dragnev et al., 2007, Huang et al., 2006). Several anticanceragents have been observed to induce cyclin D1 degradation in a many cancer celllines, many of them are naturally derived compounds induce cyclin D1degradation in cancer cells (Alao., 2007).             One of the features thatdistinguish cancer cells from normal cells is uncontrolled cell division,likely resulting from the overexpression of cyclins and the abnormal control ofcyclin-dependent kinases (CDK) (Singhal et al., 2005).

Cyclins are a family ofproteins whose levels vary during the cell cycle to activate specific CDKsrequired for the proper progression through the cell cycle. Cyclin B1, which isessential for cell cycle progression through mitosis, is overexpressed in avariety of cancers compared with normal cells and tissues (Pines., 2005 Kawamotoet al., 1997).

The deregulated expression of cyclin B1 seems to be closelyassociated with early events in neoplastic transformation (Chang  and Schlegel., 1996). As cyclin b is an important protein in cancerdevelopment there are many drugs had been developed in order to decrease itslevel in the cell to restore the control over the rate of cell division, inaddition there is a negative correlation between cyclin b and p53 (a tumorsuppressor gene), it was observed that the decrease in cyclin b increase thelevel of p53. P53 has many roles in the cell from them repairing the DNAdamage, induce apoptosis, and controlling the cell division, when there is DNAdamage P53 induce  the production of p21and WAF1proteins which prevents cyclinB/CDK1 complex activation and therefore stop theprogression through the cell cycle (Nigam et al.

, 2009).    Our results indicated that the two usedfurocoumarin compounds caused the decrease in the level of cyclin b theseresults were in agreement with the results obtained from many studies madeusing furocoumarin compounds (Kang et al., 2009; Fadlalla,et al., 2011), on the other hand only compound 19 caused the decrease in cyclinD. that Kim et al had reported that Bergamottin (furocoumarin compounds)inhibited STAT3 signaling pathway and reduced STAT3-regulated gene productssuch as COX-2, VEGF, cyclin D1, survivin, IAP-1, Bcl-2, and Bcl-xl in tumorcells. That affecting STAT3 signalling pathway may be from the mechanisms bywhich compound 19 exert its anticancer activity.

     Fromthese results it can be concluded that furocoumarins had antiproliferativeactivity against cancer cells and they exerting this effect through many mechanismsincluding their reducing effect on cyclins which lead to arrest of the cellcycle and controlling the cell division.