A natural
product is a chemical compound or substance produced by a living organism-found
in nature that usually has a pharmacological or biological activity for use in
pharmaceutical drug discovery and drug design. This product can be considered
as such even if it can be prepared by total synthesis. Its may be extracted
from tissues of terrestrial plants, marine organisms or microorganism
fermentation broths. A crude (untreated) extract from any one of these sources
typically contains novel, structurally diverse chemical compounds (Lahlou.,
2013) The main advantage which increase its use in drug synthesis is that
those chemicals evolved to interact efficiently with their biological targets,
that approximately half of the 20 best-selling nonprotein drugs are related to
natural products (Harvey., 2000). But the challenge of using natural
products in drug discovery is their complex structure and the limited supply
but this problem has been overcome with the synthesis of the analogues of these
natural compounds

 

          Furocoumarins are example of the
compounds which present naturally in many plant families including Apiaceae,
Umbelliferae, and Rutaceae (Ojala, 2001) 
and synthesized chemically in order to avoid the difficulties that arise
in case of using the natural products.  Those
compounds were found to have many biological activities including: anticancer
(Guitto et al., 1995), antifungal (Martin et al., 1966) and antibacterial
activities (Orchidee et al., 1982). Furocoumarins are selective coronary vaso
dilators (Manuel Campos- Toimil et al., 2002), they are also used in
cardiovascular diseases and acts as spasmolytic agents. (Manuel Campos- Toimil
et 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 we
found that compound 9 (4, 9-dimethoxy-5-methyl-7-oxo-7H-furo 3, 2-g
chromene-6-carbonitrile) exerted the highest cytotoxic activity against HepG-2
cells 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 both
extrinsic and intrinsic apoptotic pathways in a P53-dependent manner (Sun  et al., 2011) Furocoumarins exhibit
phototoxic properties. Phototoxic reaction takes place because of the
intercalation of furocoumarins between base pairs of DNA to form a non-covalent
psoralen-DNA complex. In addition, this property is useful in cancer therapy as
it can prevent DNA replication (Ranganath et al., 2011).

  Because of the high cytotoxic activity exerted
by compound 19 we gave it a special attention to understand its mechanism of
action using other tools like: molecular docking to recognize its inhibitory
effect on Topoisomerase I which is a target of many anticancer drugs, chromosomal
aberration assay: to assess their mutagenic and cytotoxic activity and RT-PCR
to examine its effect on the expression level of cell cycle regulatory genes
like: cyclin b and cyclin D.

 

         
Topoisomerases are universal and present in eukaryotes, archaebacteria
and eubacteria. They control DNA topology by cleaving and rejoining DNA strands
and play an important role in regulation of the physiological function of
genome. Beyond their normal functions, topoisomerases are important targets,
especially in the treatment of human cancer. They are essential enzymes that
relax DNA supercoiling inside cells during several processes like replication,
recombination, and transcription. The opening of duplex DNA and separation of
its two strands during transcription and replication generate supercoiling
(torsional tension) on both sides of the open DNA segment. Excessive positive
supercoiling tightens the DNA and prevents further strand separation, thereby
stalling the polymerase. Negative supercoiling behind the polymerases, on the
other hand, tends to extend DNA strand separation and facilitate the formation
of abnormal nucleic acid structures. Topoisomerases prevent the formation of
such potentially deleterious structures by removing free supercoiling. There
are two major classes of topoisomerases, type I and type II, that are
distinguished by the number of DNA strands that they cleave and the mechanism by
which they alter the topological properties of the genetic material. Topo I
initiates the cleavage of a strand of DNA molecule while Topo II cleaves both
DNA strands, that the inhibitors of topo I interrupt the DNA replication, which
is a useful in cancer therapy.

 

         The molecular docking
approach can be used to model the interaction between a small molecule (like
furocoumarin compounds) and a protein (like topoisomerase I) at the atomic
level, which allow us to characterize the behavior of small molecules in the
binding site of target proteins  as well
as to elucidate fundamental biochemical processes (McConkey et al., 2002).

          From the results obtained using
molecular docking it was found that compound 19 have a good ability to inhibit
topoisomerase I which in turn will lead to cancer inhibition through the inhibition
of DNA replication. The inhibition of Topoisomerase I by linear furocoumarin
compounds (like compound  19) was
reported previously by  Diwan and  Malpathak.,
2009 who examined the
inhibitory effect of the crude extract of 
Ruta graveolens and the isolated furocoumarin compounds on the
Topisomerase I  enzyme and they concluded
that psoralen, bergapten and xanthotoxin (linear furocoumarins isolated from
Ruta graveolens) are potent topoisomerase I inhibitors which promote
Stabilization of DNA–topoisomerase covalent complex.

 

           On the other hand other studies
suggested the inhibitory effect of furocoumarin compound like
4-hydroxymethyl-4′,5′-benzopsoralen on topoisomerase ii). Topoisomerase II
inhibitors exert their biological activity via increasing the covalent
enzyme-cleaved DNA complexes that normally are intermediates in the catalytic
cycle of topoisomerase II. As a result of their action, these drugs generate
breaks in the genetic material of treated cells which induce cell death
pathways (Fortune et al., 2000).

 

  From the
mechanisms which also can explain the ability of furocoumarin to inhibit cancer
development is that furocoumarins able to activate the production of singlet
oxygen which react with proteins and inactivate from these proteins as proved
by Lu et al., 2005 Topoisomerase enzyme which will in turn trigger the
apoptosis, this may be the other way by which furocoumarin inhibit
topoisomerase, That furocoumarins may inactivate topo enzyme directly by its
interaction with the enzyme or in directly via activating the production of
reactive oxygen species.

 

           Many studies suggested that
furocoumarins in combination with UV induce DNA-protein crosslinking which can
lead to cell death or stop cell cycle progression by impeding DNA and RNA
synthesis. But our study made in the dark. (Bordin F et al., 1993)

 

            Many of the assays have been
developed to investigate the mutagenic activity of the tested chemicals as
Rodent micronucleus assay, Bone marrow, peripheral blood (mouse) assay for
clastogenicity 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 many
plants have been used for chromosomal aberration assay as Vicia faba, Zea
mays, Tradescantia, Nicotiana tabacum, Crepis capillaries, Hordeum vulgare and
Pisum sativum. The International Program on Chemical Safety (IPCS) permitted assessment
the availability of plant assays to determine the mutagenicity and
clastogenicity, and their studies concluded that plant assays are efficient and
reliable test systems for rapid screening of chemicals for mutagenicity and
clastogenicity (Ma, 1999).The in vivo root-tip assay is known to give
similar results to in vitro animal cytotoxicity tests (Chauhan et al.,
1999; Vicentini et al., 2001, Teixeira et al., 2003, Eren and
Ozata, 2014, Khalifa et al., 2014).

 

          Treatment of Pisum
sativum root tips with different concentrations of the two furocoumarin
compounds under investigation resulted in a significant decrease in mitotic
index at the high concentrations. The decrease in mitotic index indicated that
the experimental material exhibited mitodepressive effect which had been
assumed to result from the inhibition of cells access to mitosis (Badr and
Ibrahim 1987). Such an antimitotic effect is most likely attained by preventing
DNA biosynthesis or/ and microtubule formation (Yüzba??o?lu et al. 2003). This
might 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 of
different types of chromosomal abnormalities was recorded, the types of these
abnormalities include C-metaphase, chromosomal breaks, and chromosomal
stickiness. These results are in agreement with those obtained from treating
onion root tips with coumarin and coumarin derivatives, including furocoumarin
compounds, (Dolcher. 1960; Mongelli et al., 2000; Vera et al., 2001, Riveiro et
al., 2004; Knoll et al., 2006). The appearance of apoptotic cells has been
recorded in the root cells treated with compound 16 which agreed with the
results of Xia et al. 2014 who showed how the photoactivated PUVA activate
apoptosis in breast cancer cells which is recognized with the over expression
of the ErbB2 receptor tyrosine kinase oncogene. Kim  et al. 2014 also showed that Bergamottin
(linear furocoumarin) induced apoptosis through the inhibition of STAT3
signaling pathway in tumor cells, which is related to growth, survival,
proliferation, metastasis, and angiogenesis of various cancer cells in addition
to 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 anticancer
drugs.

        

  The chromosomal aberration assay
indicated the presence of anaphase and telophase cells with lagging chromosomes
and bridges. This type of mitotic abnormality results from the SAC pathway
disruption (which is responsible for stopping the premature separation of
sister chromatids and formation of premature anaphase cells, thus resulting in
cell death or the formation of aneuploid cells which controlled with tumor
suppressor genes as P53 and Rb to prevent tumor cell formation (Castedo et al., 2004;
Thompson and Compton 2010; Vitale et al., 2011). SAC
pathway disruption may be among the mechanisms by which furocoumarins exert
their action. On the other hand, the C-metaphase cells are known to appear
because of the failure of the separated chromatids to reach the two poles, as a
result of complete inhibition of spindle formation accounting for the stopping
of mitosis in the mitotic phase and the inhibition of cytokinesis and the
formation of polyploid cells (Kim et al., 2004). The binding of
the drugs to tubulin preventing their polymerization to micrtubules is the
target for many anticancer agents as it leads finally to cell death from these
anticancer agents Taxol and Vinca alkaloids (Cutler and ?
Cutler., 1999).

 

 The appearance of chromosomal
stickiness was also recorded in the present work. Stickiness is characterisitic
of DNA intercalators (like furocoumarins). This type of abnormalities is
attributed to the failure in topoisomerase II and the peripheral proteins whose
function is necessary for separation and segregation of chromatids during
anaphase. In addition, the changes being caused either by mutation in
structural genes for the proteins (heritable stickiness) or by direct action of
mutagens 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 furocoumarin
compounds proved to have inhibitory effect on Topoisomerase II enzyme (Marzano  et al., 1997, Pani  et
al., 1994).

 

            Chromosomal breaks were also
recorded among chromosomal abnormalities. It is produced as a result of series
of mechanisms beginning with liaison followed by DNA strand breakage by
(formation of pyrimidine dimmer, alkylating or inter and intrastrand cross
linkage (Therman et al., 1993). This is usually formed by polyfunctional
alkylating agents as the compounds used in our study. Recent researches showed
that the anticancer activity of intercalating agents resulted from their
interfering with the action of topoisomerase during their binding to nuclear
DNA, thus causing chromosomal break (Ralph  et al., 1993). Topoisomerase II inhibitors
exert their biological activity via increasing the covalent enzyme-cleaved DNA
complexes that normally are intermediates in the catalytic cycle of
topoisomerase II. As a result of their action, these drugs generate breaks in
the genetic material of treated cells which induce cell death pathways (Fortune
et al., 2000).

 

          The induction of chromosomal
aberration by furocoumarins using mammalian models was investigated by many
scientists 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 result
of treatment of Chinese hamster ovary (CHO) cells with the methanolic and
aqueous extract of Bryopteris gaudichaudii which contains high
concentration of furocoumarin. (Varanda et al., 2002).

 

 

           Furocoumarins can
interfere with the action of cytochrome p450 which is a main target for many
drugs that.  Cytochrome P450 is a family
of isozymes responsible for the biotransformation of several drugs.  In recent study it was found that four
furocoumarins angelicin, bergamottin, isopimpinellin, and 8-methoxypsoralen act
as potent inhibitors of CYP 1A1 (this gene encodes a member of the cytochrome
P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases
which catalyze many reactions involved in drug metabolism and synthesis of
cholesterol, steroids and other lipids) in dark and light (Baumgart  et al., 2005) Drug metabolism via the
cytochrome P450 system is an important determinant in the occurrence of several
drug interactions that can result in drug toxicities reduced pharmacological
effect, and adverse drug reactions (Ogu et al., 2000).

 

          Cyclins
are proteins which form complexes with cyclin dependent kinases to activate the
progression of cell cycle from stage to another in the proliferating cells. Where
cyclin b is able to activate the progression of cell cycle from G2 to mitotic
phase cyclin D stimulate progression from G1 to S phase.  cyclin D1 forms active complexes that promote
cell cycle progression by phosphorylating and inactivating the retinoblastoma
protein (RB)(which is an important tumor suppressor protein) (Kato et al.,
1993; Lundberg et al., 1998; Weinberg.,  1995)

 

       The
overexpression of cyclin D can lead to the uncontrolled cell division which
make it act as an oncogene, that the over expression of cyclin D can occur by
one of three ways which are: gene amplification, impaired protein degradation,
or chromosomal translocation. Cyclin D1 is important for the development and
progression 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 the
development of endocrine resistance in breast cancer cells and hepatocellular
carcinoma (Hodges et al., 2003, Hui et al., 2002, Kenny et al., 1999;
Zhang et al., 1993). Cyclin D1 overexpression
is a common event in cancer. The importance of cyclin D1 in cancer makes it an
attractive target for anti-cancer therapy (Yu et al., 2001,
Dragnev et al., 2007, Huang et al., 2006). Several anticancer agents
have been observed to induce cyclin D1 degradation in a many cancer cell lines,
many of them are naturally derived compounds induce cyclin D1 degradation in
cancer cells (Alao., 2007).

    

       One of the features that distinguish
cancer cells from normal cells is uncontrolled cell division, likely resulting
from the overexpression of cyclins and the abnormal control of cyclin-dependent
kinases (CDK) (Singhal et al., 2005). Cyclins are a family of
proteins whose levels vary during the cell cycle to activate specific CDKs
required for the proper progression through the cell cycle. Cyclin B1, which is
essential for cell cycle progression through mitosis, is overexpressed in a
variety of cancers compared with normal cells and tissues (Pines., 2005 Kawamoto et al., 1997). The deregulated expression of cyclin B1 seems to be
closely associated with early events in neoplastic transformation (Chang  and Schlegel., 1996).

 

As cyclin b is an important protein in cancer development there are many
drugs had been developed in order to decrease its level in the cell to restore
the control over the rate of cell division, in addition there is a negative
correlation between cyclin b and p53 (a tumor suppressor gene), it was observed
that the decrease in cyclin b increase the level of p53. P53 has many roles in
the cell from them repairing the DNA damage, induce apoptosis, and controlling
the cell division, when there is DNA damage P53 induce  the production of p21 and WAF1 proteins which prevents cyclinB/CDK1 complex activation
and therefore stop the progression through the cell cycle (Nigam et al.,
2009).

    Our results indicated that the two used
furocoumarin compounds caused the decrease in the level of cyclin b these
results were in agreement with the results obtained from many studies made
using furocoumarin compounds (Kang et al., 2009; Fadlalla, et al., 2011), on the other hand only
compound 19 caused the decrease in cyclin D. that Kim et al had reported that
Bergamottin (furocoumarin compounds) inhibited STAT3 signaling pathway and
reduced 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 signalling
pathway may be from the mechanisms by which compound 19 exert its anticancer
activity.

     From
these results it can be concluded that furocoumarins had antiproliferative
activity against cancer cells and they exerting this effect through many
mechanisms including their reducing effect on cyclins which lead to arrest of
the cell cycle and controlling the cell division.

 

 

 

 

 

 

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