Discussion use in drug industry, about 50% of

Discussion

      Natural products are a group of compounds
produced by living organisms and having medicinal effects, so it can be used in
drug discovery and design. Its may be extracted from tissues of terrestrial
plants, marine organisms or microorganism. The crude extracts of these sources
contain biologically active compounds (Lahlou., 2013) which
interact  with its target while causing
less side effects, that increased its use in drug industry, about 50% of
best-selling 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 as the compounds used in
this thesis.

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      Coumarins are type of
natural compounds produced by many plants as defense mechanism, it was firstly
isolated from tonka bean, coumarin is a member of benzopyrone family which is
divided into benzo-?-pyrones and benzo-?-pyrones, that coumarin is belonging to
benzo-?-pyrones while flavonoids belong to benzo-?-pyrones. Coumarins showed
different medical activities including: anticancer activity (Rohini  and Srikumar, 2014).

         The results of this study
showed that furocoumarin compound number 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 synthetic coumarin derivatives exhibited less cytotoxic activity. Many
studies proved the cytotoxic activity of coumarin derivatives against different
cancer 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 through
both extrinsic and intrinsic apoptotic pathways in a P53-dependent manner.

 

        Also
El-Nakkady et al  (2015) demonstrated
that the synthesized furocoumarin compounds showed significant cytotoxic
activity 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. Many
studies proved the anticancer activity exerted by synthesized coumarin derivatives
(Reddy et al., 2004; Goel
et al., 2007; Liu
 et al., 2010; Ranganath et al.,
2011; Musa
et al., 2012 and Morsy et al., 2017).

 

      The results of SRB assay made using plant
extracts containing coumarins revealed that Verbascum thapsus seeds and Achillea
millefolium leaves showed the highest cytotoxic activity against HepG-2
cell line with IC50=27µg/ml and 32µg/ml, respectively. While Ammi
majus L. exerted the lowest cytotoxic activity with IC50=511.8.

 

     Verbascum
spp. contain high content of
coumarin and the highest concentration present in plant seeds. Lin et al.,
(2002) study had proved that the hot water crude extract of Verbascum
thapsus 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 the
cytotoxic effect of the related species Verbascum sinaiticum and
they found that the methanol flower extracts showed significant cytotoxic
activity against MCF-7 and Vero cell lines (African monkey kidney cell line) with
IC50= 25.15 and 17.68µg/ml, respectively. In addition Verbascum
spp. showed anticancer activity against different cell lines SK-MEL, P-388, Ehrlich
ascites carcinomas in CF1 mice cell lines (Afifi et al., 1993 and
Tatl? and Akdemir 2006).

        

        Navaie
et al (2016) showed that the methanol, extract of leaves of Achillea
millefolium exerted significant cytotoxic activity against MCF-7 (breast cancer
cell line) with IC50= 40.54µg/ml. In agreement with our results Ghavami
et al (2010) reported that methanol extract of Achillea
millefolium exerted significant cytotoxic activity against number of cancer
cell lines with IC50 ranged from 22.051 to 66.00µg/ml. Ahmed et
al (2014) proved that the related species Achillea aleppica
exerted significant cytotoxic activity against HepG-2 cell line.

 

     The polar extract of Achillea
millefolium contain number of coumarin derivatives as scopoletin I,
umbelliferone II and aesculetin III, the anticancer activity of these coumarin
compounds 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 this
study exerted high cytotoxic activity with IC50=35. The study made
by Noolu et al. (2015). In this study M. paniculata
exhibited high cytotoxic activity against different cell lines including HepG-2
cell line and its IC50 was 17.55?g/mL on contrast to our
results this difference in IC50 value may be due to the variance in
the quality of methanol used or season of plant collection. The different types
of Murraya paniculata extracts showed cytotoxic activity against
different cancer cell lines.

 

      The
main coumarin compounds isolated from M. paniculata were
7-methoxy-8-(3-methyl-2-oxobutoxy)-2H-chromen-2-one, 6-hydroxycoumurrayin (Li
et al., 2016), 2′-O-ethylmurrangatin (Choudhary
et al., 2002),  murrayanone
and murraculatin (Wu, 1988). Murrmeranzin, murralonginal, minumicrolin,
murrangatin, meranzin hydrate and hainanmurpanin (Saied et al., 2008).
Omphamurrayone (Kinoshita

et al., 1996), Paniculacin (Saeed
et al., 2011).

 

       Umbelliferone and scopoletin from the isolated compounds from Murraya
paniculata (Saeed
et al., 2011). They exhibit anticancer activity against different cancer
cell lines. Murata et al. (2008) demonstrated that furocoumarin
compound murrayacoumarin B present in Murraya spp. as Murraya
paniculata showed significant anticancer activity against human
leukaemia HL-60 cells due to induction of apoptosis via caspase-9/caspase-3
pathway.

 

       Ruta graveolens is old medicinal plant
which contains many active constituents including various furanocumarins,
carotenoids, chlorophyll, furanoquionolones (Hale et al., 2004; Eickhorst
et al., 2007; Malik et al., 2013).      Ruta graveolens showed anticancer
activity against different cell lines including MCF7, Dalton’s lymphoma ascites
(DLA), Ehrlich ascites carcinoma (EAC), L929 cells and brain cancer cells (Pathak et
al., 2003; Preethi et al., 2006; Pushpa et al., 2015).   Ruta graveolens exert
its anticancer activity through inducing DNA damage and blocking Akt activation,
induction of P53 pathway and inhibiting topoisomerase I (Fadlalla et al.,
2014; Manjula
and Mani,
2016).  The activation of p53 pathway
was reported by many furocoumarins like bergapten which present in this plant,
also bergapten in addition to xanthotoxin and psoralen present in this plant
act as potent inhibitors of topoisomerase I (Manjula
and Mani,
2016).

 

       The
results showed that Ferula asafoetida 
exerted low anticancer activity 
with IC50=412.2 µg/ml against HepG-2 cell line, In agreement with
these results (Unnikrishnan
and Kuttan (1988) proved that Ferula asafoetida extract exerted its cytotoxic
activity against different cancer cell lines as human lymphocytes, Vero cells,
Chinese hamster ovary (CHO) cells and Dalton’s lymphoma with IC50
ranged from 150µg/ml to 600 µg/ml. Mahendra and Bisht (2012) mentioned
that the anticancer activity of F. asafetida extract is attributed to presence
of number of compounds with anticancer activity as isopimpnillin (furocoumarin
compound), Umbelliferone (furocoumarin compound), ?-pinene,  Luteolin.

 

     This
plant also contain five sesquiterpene coumarins namely, conferone, badrakemin,
feslol, isosamarcandin and samarcandin (Ashgari et al., 2016). The
anticancer activity of conferone and samarcandin was recorded in studies made
by
(Valiahdi et al., 2013 and Ghoran et al., 2016) using different cancer cell lines.

 

       Apium graveolens and Ammi majus
showed the lowest cytotoxic activity against HepG-2 cell line on contrast they showed
high cytotoxic activity in other studies against different cancer cell lines,
the varied responces of different cancer cells to the same drug is attributed
to the Heterogeneity in cancer cells (Arul et
al., 2017) that tumor heterogeneity mean the observation that different
tumour cells
can show distinct morphological and phenotypic
profiles, including cellular morphology, gene expression, metabolism, motility,
proliferation, and metastatic potential (Marusyk
and  Polyak , 2010).

 

         Because of the high cytotoxic activity
exerted by compound 9 it was given 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, RT-PCR to examine its effect on the expression level of cell cycle
regulatory genes like: cyclin b and cyclin D and ISSR to understand the effect
of them on genome stability.

 

          Topoisomerases are enzymes
which help in completing the process of DNA replication through overcoming the
problem of DNA supercoiling which arise during DNA replication by cleaving and
rejoining DNA strands. They are present in eukaryotes, archaebacteria and
eubacteria. Because of its importance not only in the process of DNA
replication but also in recombination, and transcription, they became target of
many anticancer drugs. The opening of double stranded DNA generate supercoiling
in the replication fork which prvent further separation of two strands
resulting in stopping of replication process (Kellner et al., 2002).
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 cleave
one 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   (Ewesuedoa
and Ratain,
1997).

         The molecular docking approach can be
used to model the interaction between a small molecule (like coumarin
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 9 have a good
ability to inhibit topoisomerase I which in turn leads to cancer inhibition
through the inhibition of DNA replication. The inhibition of Topoisomerase I by
linear furocoumarin compounds (like compound 
9) 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 a 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  these proteins as topoisomerase
enzyme proved by Lu et al (2005) thus inactivation of
topoisomerase enzyme in turn triggers the apoptosis, this may be the other way
by which furocoumarin inhibit topoisomerase, That furocoumarins may inactivate
topo enzyme directly by interaction with the enzyme or indirectly 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  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 synthetic coumarin compounds and
two plant extracts containing coumarin derivatives under investigation resulted
in a significant decrease in mitotic index at the high concentrations. The
decrease in mitotic index indicated that the experimental materials 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 (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 8. This may be due to the ability of furocoumarins to induce reactive
oxygen species which able to induce apoptosis in plant cells (Breusegem and Dat ?2006).  These results agreed with the results of Xia
et al. (2014) who showed how the photoactivated PUVA (psoralen and
UVA) 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 disturbing
this pathway resulting in cell death or the formation of aneuploid cells (Castedo
et al., 2004; Thompson and Compton 2010; Vitale et al., 2011).
SAC pathway disruption may be among the mechanisms by which coumarins 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, the inhibition of cytokinesis,  formation of polyploid cells and apoptosis
(Kim et al., 2004). So that Spindle fibers are target for anticancer
drugs blocking its formation as Taxol and Vinca alkaloids (Cutler and ? Cutler., 1999).

 

 The appearance of chromosomal stickiness was
also recorded in the present work. 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 natural
and synthetic coumarin derivatives proved to have inhibitory effect on
Topoisomerase II enzyme (Marzano  et al., 1997, Lewis et al., 1996; Pani  et al., 1994, Hueso-Falcón
et al., 2017).

 

         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). 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).

 

      Cyclins
are group of proteins which form complexes with cyclin dependent kinases to
activate the progression of cell cycle from stage to another in the
proliferating cells. Cyclin b is able to activate the progression of cell cycle
from G2 to mitotic phase while, 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.