A naturalproduct is a chemical compound or substance produced by a living organism-foundin nature that usually has a pharmacological or biological activity for use inpharmaceutical drug discovery and drug design. This product can be consideredas such even if it can be prepared by total synthesis. Its may be extractedfrom tissues of terrestrial plants, marine organisms or microorganismfermentation broths. A crude (untreated) extract from any one of these sourcestypically contains novel, structurally diverse chemical compounds (Lahlou.,2013) The main advantage which increase its use in drug synthesis is thatthose chemicals evolved to interact efficiently with their biological targets,that approximately half of the 20 best-selling nonprotein drugs are related tonatural products (Harvey.

, 2000). But the challenge of using naturalproducts in drug discovery is their complex structure and the limited supplybut this problem has been overcome with the synthesis of the analogues of thesenatural compounds           Furocoumarins are example of thecompounds which present naturally in many plant families including Apiaceae,Umbelliferae, and Rutaceae (Ojala, 2001) and synthesized chemically in order to avoid the difficulties that arisein case of using the natural products.  Thosecompounds were found to have many biological activities including: anticancer(Guitto et al.

, 1995), antifungal (Martin et al., 1966) and antibacterialactivities (Orchidee et al., 1982). Furocoumarins are selective coronary vasodilators (Manuel Campos- Toimil et al.

, 2002), they are also used incardiovascular diseases and acts as spasmolytic agents. (Manuel Campos- Toimilet al., 2002), utilized in leucodermic vitiligo therapy (Ortel et al, 1988,Laskin et al., 1989), Psoriasis treatment (Hashimoto et al.

, 1978).            From the results of this study wefound that compound 9 (4, 9-dimethoxy-5-methyl-7-oxo-7H-furo 3, 2-gchromene-6-carbonitrile) exerted the highest cytotoxic activity against HepG-2cells with IC50=11.9, while the other 9 compounds exhibited less activity.

  Other studies using  furocoumarins as DMFC(3,5-dimethyl-7H-furo3,2-gchromen-7-one) to treat HepG-2 revealed that this  compound induced significant cytotoxic activity and apoptosis  through bothextrinsic and intrinsic apoptotic pathways in a P53-dependent manner (Sun  et al., 2011) Furocoumarins exhibitphototoxic properties. Phototoxic reaction takes place because of theintercalation of furocoumarins between base pairs of DNA to form a non-covalentpsoralen-DNA complex. In addition, this property is useful in cancer therapy asit can prevent DNA replication (Ranganath et al., 2011).  Because of the high cytotoxic activity exertedby compound 19 we gave it a special attention to understand its mechanism ofaction using other tools like: molecular docking to recognize its inhibitoryeffect on Topoisomerase I which is a target of many anticancer drugs, chromosomalaberration assay: to assess their mutagenic and cytotoxic activity and RT-PCRto examine its effect on the expression level of cell cycle regulatory geneslike: cyclin b and cyclin D.          Topoisomerases are universal and present in eukaryotes, archaebacteriaand eubacteria.

They control DNA topology by cleaving and rejoining DNA strandsand play an important role in regulation of the physiological function ofgenome. Beyond their normal functions, topoisomerases are important targets,especially in the treatment of human cancer. They are essential enzymes thatrelax DNA supercoiling inside cells during several processes like replication,recombination, and transcription. The opening of duplex DNA and separation ofits two strands during transcription and replication generate supercoiling(torsional tension) on both sides of the open DNA segment. Excessive positivesupercoiling tightens the DNA and prevents further strand separation, therebystalling the polymerase. Negative supercoiling behind the polymerases, on theother hand, tends to extend DNA strand separation and facilitate the formationof abnormal nucleic acid structures. Topoisomerases prevent the formation ofsuch potentially deleterious structures by removing free supercoiling.

Thereare 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 Iinitiates the cleavage of a strand of DNA molecule while Topo II cleaves bothDNA strands, that the inhibitors of topo I interrupt the DNA replication, whichis a useful in cancer therapy.          The molecular dockingapproach can be used to model the interaction between a small molecule (likefurocoumarin compounds) and a protein (like topoisomerase I) at the atomiclevel, which allow us to characterize the behavior of small molecules in thebinding site of target proteins  as wellas to elucidate fundamental biochemical processes (McConkey et al.

, 2002).           From the results obtained usingmolecular docking it was found that compound 19 have a good ability to inhibittopoisomerase I which in turn will lead to cancer inhibition through the inhibitionof DNA replication. The inhibition of Topoisomerase I by linear furocoumarincompounds (like compound  19) wasreported 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 fromRuta graveolens) are potent topoisomerase I inhibitors which promoteStabilization of DNA–topoisomerase covalent complex.             On the other hand other studiessuggested the inhibitory effect of 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 themechanisms which also can explain the ability of furocoumarin to inhibit cancerdevelopment is that furocoumarins able to activate the production of singletoxygen which react with proteins and inactivate from these proteins as provedby Lu et al., 2005 Topoisomerase enzyme which will in turn trigger theapoptosis, this may be the other way by which furocoumarin inhibittopoisomerase, That furocoumarins may inactivate topo enzyme directly by itsinteraction with the enzyme or in directly via activating the production ofreactive oxygen species.            Many studies suggested thatfurocoumarins in combination with UV induce DNA-protein crosslinking which canlead to cell death or stop cell cycle progression by impeding DNA and RNAsynthesis. But our study made in the dark.

(Bordin F et al., 1993)             Many of the assays have beendeveloped to investigate the mutagenic activity of the tested chemicals asRodent micronucleus assay, Bone marrow, peripheral blood (mouse) assay forclastogenicity with kinetochore and centromeric staining, Comet assay,Salmonella test for gene mutation, and Allium cepa assay (Bajpayee et al.,2005). Allium cepa test have been developed by (Levan., 1938), and then manyplants have been used for chromosomal aberration assay as Vicia faba, Zeamays, Tradescantia, Nicotiana tabacum, Crepis capillaries, Hordeum vulgare andPisum sativum. The International Program on Chemical Safety (IPCS) permitted assessmentthe availability of plant assays to determine the mutagenicity andclastogenicity, and their studies concluded that plant assays are efficient andreliable test systems for rapid screening of chemicals for mutagenicity andclastogenicity (Ma, 1999).The in vivo root-tip assay is known to givesimilar results to in vitro animal cytotoxicity tests (Chauhan et al.,1999; Vicentini et al.

, 2001, Teixeira et al., 2003, Eren andOzata, 2014, Khalifa et al., 2014).           Treatment of Pisumsativum root tips with different concentrations of the two furocoumarincompounds under investigation resulted in a significant decrease in mitoticindex at the high concentrations.

The decrease in mitotic index indicated thatthe experimental material exhibited mitodepressive effect which had beenassumed to result from the inhibition of cells access to mitosis (Badr andIbrahim 1987). Such an antimitotic effect is most likely attained by preventingDNA biosynthesis or/ and microtubule formation (Yüzba??o?lu et al. 2003). Thismight be attributed 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 ofdifferent types of chromosomal abnormalities was recorded, the types of theseabnormalities include C-metaphase, chromosomal breaks, and chromosomalstickiness.

These results are in agreement with those obtained from treatingonion root tips with coumarin and coumarin derivatives, including furocoumarincompounds, (Dolcher. 1960; Mongelli et al., 2000; Vera et al., 2001, Riveiro etal.

, 2004; Knoll et al., 2006). The appearance of apoptotic cells has beenrecorded in the root cells treated with compound 16 which agreed with theresults of Xia et al. 2014 who showed how the photoactivated PUVA activateapoptosis in breast cancer cells which is recognized with the over expressionof the ErbB2 receptor tyrosine kinase oncogene.

Kim  et al. 2014 also showed that Bergamottin(linear furocoumarin) induced apoptosis through the inhibition of STAT3signaling 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 many anticancerdrugs.            The chromosomal aberration assayindicated the presence of anaphase and telophase cells with lagging chromosomesand bridges. 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 resulting incell death or the formation of aneuploid cells which controlled with tumorsuppressor genes as P53 and Rb to prevent tumor cell formation (Castedo et al., 2004;Thompson and Compton 2010; Vitale et al.

, 2011). SACpathway disruption may be among the mechanisms by which furocoumarins 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 stoppingof mitosis in the mitotic phase and the inhibition of cytokinesis and theformation of polyploid cells (Kim et al., 2004). The binding ofthe drugs to tubulin preventing their polymerization to micrtubules is thetarget for many anticancer agents as it leads finally to cell death from theseanticancer agents Taxol and Vinca alkaloids (Cutler and ?Cutler.

, 1999).  The appearance of chromosomalstickiness was also recorded in the present work. Stickiness is characterisiticof DNA intercalators (like furocoumarins). This type of abnormalities isattributed to the failure in topoisomerase II and the peripheral proteins whosefunction is necessary for separation and segregation of chromatids duringanaphase. In addition, the changes being caused either by mutation instructural genes for the proteins (heritable stickiness) or by direct action ofmutagens on the proteins (induced stickiness) (Gaulden et al.

, 1987).Topoisomerase poisoning has biomedical effect in certain diseases as cancer(Liu, 1989; Fortune et al., 2000; Pommier et al., 2010 ; Ashour et al., 2015). Many of furocoumarincompounds proved to have inhibitory effect on Topoisomerase II enzyme (Marzano  et al., 1997, Pani  etal.

, 1994).             Chromosomal breaks were alsorecorded among chromosomal abnormalities. It is produced as a result of seriesof mechanisms beginning with liaison followed by DNA strand breakage by(formation of pyrimidine dimmer, alkylating or inter and intrastrand crosslinkage (Therman et al.

, 1993). This is usually formed by polyfunctionalalkylating agents as the compounds used in our study. Recent researches showedthat the anticancer activity of intercalating agents resulted from theirinterfering with the action of topoisomerase during their binding to nuclearDNA, thus causing chromosomal break (Ralph  et al., 1993). Topoisomerase II inhibitorsexert their biological activity via increasing the covalent enzyme-cleaved DNAcomplexes that normally are intermediates in the catalytic cycle oftopoisomerase II. As a result of their action, these drugs generate breaks inthe genetic material of treated cells which induce cell death pathways (Fortuneet al., 2000).           The induction of chromosomalaberration by furocoumarins using mammalian models was investigated by manyscientists as the induction of chromosomal breaks by isopimpinellin(furocoumarin isolated from Heracleum mantegazzianum, Fam.

Apiaceae)  plus UVA (Abel et al., 1985).  Chromosomal breaks were recorded as a resultof treatment of Chinese hamster ovary (CHO) cells with the methanolic andaqueous extract of Bryopteris gaudichaudii which contains highconcentration of furocoumarin. (Varanda et al., 2002).              Furocoumarins caninterfere with the action of cytochrome p450 which is a main target for manydrugs that.  Cytochrome P450 is a familyof isozymes responsible for the biotransformation of several drugs.

 In recent study it was found that fourfurocoumarins angelicin, bergamottin, isopimpinellin, and 8-methoxypsoralen actas potent inhibitors of CYP 1A1 (this gene encodes a member of the cytochromeP450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenaseswhich catalyze many reactions involved in drug metabolism and synthesis ofcholesterol, steroids and other lipids) in dark and light (Baumgart  et al., 2005) Drug metabolism via thecytochrome P450 system is an important determinant in the occurrence of severaldrug interactions that can result in drug toxicities reduced pharmacologicaleffect, and adverse drug reactions (Ogu et al., 2000).            Cyclinsare proteins which form complexes with cyclin dependent kinases to activate theprogression of cell cycle from stage to another in the proliferating cells.

Wherecyclin b is able to activate the progression of cell cycle from G2 to mitoticphase cyclin D stimulate progression from G1 to S phase.  cyclin D1 forms active complexes that promotecell cycle progression by phosphorylating and inactivating the retinoblastomaprotein (RB)(which is an important tumor suppressor protein) (Kato et 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 et al., 1996, Knudsen et al., 2006, Motokura and Arnold., 1993; Musgrove.

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

Cyclin D1 overexpressionis a common event in cancer. The importance of cyclin D1 in cancer makes it anattractive target for anti-cancer therapy (Yu et al., 2001,Dragnev et al., 2007, Huang et al., 2006).

Several anticancer agentshave been observed to induce cyclin D1 degradation in a many cancer cell lines,many of them are naturally derived compounds induce cyclin D1 degradation incancer cells (Alao., 2007).             One of the features that distinguishcancer cells from normal cells is uncontrolled cell division, likely resultingfrom the overexpression of cyclins and the abnormal control of cyclin-dependentkinases (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 Kawamoto et al., 1997). The deregulated expression of cyclin B1 seems to beclosely associated with early events in neoplastic transformation (Chang  and Schlegel., 1996). As cyclin b is an important protein in cancer development there are manydrugs had been developed in order to decrease its level in the cell to restorethe control over the rate of cell division, in addition there is a negativecorrelation between cyclin b and p53 (a tumor suppressor gene), it was observedthat the decrease in cyclin b increase the level of p53. P53 has many roles inthe cell from them repairing the DNA damage, induce apoptosis, and controllingthe cell division, when there is DNA damage P53 induce  the production of p21 and WAF1 proteins which prevents cyclinB/CDK1 complex activationand therefore stop the progression 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 onlycompound 19 caused the decrease in cyclin D. that Kim et al had reported thatBergamottin (furocoumarin compounds) inhibited STAT3 signaling pathway andreduced STAT3-regulated gene products such as COX-2, VEGF, cyclin D1, survivin,IAP-1, Bcl-2, and Bcl-xl in tumor cells. That affecting STAT3 signallingpathway may be from the mechanisms by which compound 19 exert its anticanceractivity.     Fromthese results it can be concluded that furocoumarins had antiproliferativeactivity against cancer cells and they exerting this effect through manymechanisms including their reducing effect on cyclins which lead to arrest ofthe cell cycle and controlling the cell division.

      

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