numerous (many) studies have been performed in the investigation of
anticancer drugs (conducted to investigate the anticancer
drugs and cancer therapy). Though, because of intolerance
or resistance, many treatments
have failed so far and progression of the disease is growing. Recently interests in the use of medicinal plants have increased significantly
due to their lesser side effects. The aim of this
study was to initially evaluate the anticancer characteristics of essential oil
of Oliveria decumbense (OEO), a plant from iranica flora, and thymol, a monoterpene found a lot in OEO, followed by understanding
of the mechanisms underlying the activities of these drugs in cancer cell
death. As we know this is the
first report of (We have demonstrated
for the first time— Here, we present the
first evidence regarding the inhibitory effects
of) the effect of Oliveria
decumbense on cancer cell lines. Although, recently it has been demonstrated that
this plant has some anti-microbial characteristics. Indeed, the GC-MS analysis of the OEO
identified thymol, carvacrol, p-cymen and ?-Terpinene as major
components while other separated components including sesquiterpenes, phenylpropens, and
non-phenolic portions weret1  accounted for
less than 15% of the oil. Of course these contents change with geographical variation, collection time, preparation process and other aspects. Some of these components such as thymol
has been known for their antioxidant, anti-inflammatory, and anti-microbial
activity. Thymol has also been found to possess some anti-cancer activity on 2D
cancer cell culture, but its anti-cancer mechanism has not yet been fully
elucidated. In this research,
after the identification of main components of essential oil obtained by hydrodistilation (OEO), thymol, as a main component of OEO with high percentage,
was selected to evaluate along with OEO.  For
investigation of the growth-inhibitory effect of OEO, two lines of breast
cancer, MDA-MB 231 (a triple negative breast cancer,
invasive, intermediate response to chemotherapy) and T47D (ER+
and PR+, non-invasive, responsive to
chemotherapy) were studied. Obtained results of Mtt assay showed
that OEO is capable to inhibit the proliferation of MDA-MB231 and T47D with
IC50 … and ? while thymol suppresses the growth in these cell lines with IC50 ?
and? sequentially in a dose dependent mannert2 . In addition, OEO did not
display any significant
toxicity in treated normal fibroblast cells (L929). This confirms that OEO possesses appropriate selectivity
between cancer and normal cells. Despite of valuable information accumulated from traditional
cell culture, increasing failure rate of designed drugs can be attributed to unreliable and defective results of 2D systems. Therefore, since 3D
cell culture is able to recapitulate native tumor microenvironments and provide
more accurate information, MDA-MB231 spheroids was formed and subsequently,
treatment of spheroids with OEO and thymol was performed. Our data from elicitation of
the cells responses in 3D and comparison with conventional monolayer cultures highlighted the profound differences
in responses from 3D and 2D models and showed the lower cytotoxicity of OEO and
thymol in formed spheroids rather than on 2D cell culture as expected. Regardless of the specific action site of pharmacologic agents,
cell death by most useful drugs is mediated by stimulation of the apoptotic
signaling pathways. To consider the inhibitory mode induced by OEO and thymol in the MDA-MB231 cells
cultured in 2D and separated cells from spheroids (in 3D system), flow cytometry was employed, by
incubation of the cells with AnnexinV-(FITC) to quantify phosphatidylserine exposure at the cells surface. Our results of this study confirmed the apoptotic
mode in the cells in compatible with the morphological changes of the cells
under confocal microscope.

DNA damaging is
an established strategy in cancer therapy. Cleavage of DNA into oligonucleosomal
size fragments is a vital part of apoptosis and is detectable by resolving isolated DNA by gel
electrophoresis, which results in a ladder pattern. However, this method is not useful to
quantitate the number of apoptotic cells. Terminal transferase mediated
DNA nick end labelling TUNEL assay
of fragmented DNA allows to recognize DNA fragmentation at the single
cells.  Therefore, in approving the apoptotic process, the
DNA ladder was demonstrated in the treated cells and the cells incubated with TUNEL reagent were counted
by flowcytometry.
The results showed that treatment with OEO and thymol significantly increased
TUNEL-positive dead cells. A large proportion of
anticancer drugs are agents that interact with DNA directly however the
long-term usage of these drugs is restricted by their toxicity. Thus, the discovery and applications of
appropriate DNA-targeting drugs is of crucial importance.  Activation of
caspases, through selective cleavage of vital cellular substrates, results in
apoptotic morphologic changes and DNA fragmentation. Accordingly, caspase 3
activity, responsible for triggering of DNase enzymes and consequently DNA
damage, was considered in untreated and treated monolayer cells and spheroids.
Our findings showed that treatment with both OEO and thymol leads to caspase 3
activation significantly that eventually result in apoptosis. Cancer cells typically exhibit a high levels of ROS. This
may contribute to the progression of cancer, but may make cancer cells more
vulnerable to extra (excessive) ROS. Recently, many drugs, mostly natural
products, have been known to promote ROS overload specifically
in cancer cells. Enhancement
of oxidative stress induced by current anticancer drugs is associated with blocking cancer development
and apoptosis. However, the biochemical mechanisms linking ROS level to apoptosis is various
and not exactly clear. ROS-induced oxidative stress directly or through DNA
damage and cell cycle arrest, increasing the TNF-? level, hyperpolarization of
mitochondrial membrane potential and cytochrome C discharge can provoke apoptosis.
In this research, we investigated whether OEO and thymol-induced apoptosis was
mediated by ROS generation in MDA-MB-231 cells. Collectively, our results indicated that treatment of cells with
OEO and thymol markedly increased ROS
level after ? h in these cells. An important target of ROS is the guanine nucleotide pool as
well as guanine nucleobase in DNA and RNA. The oxidative damage generates damaged guanine nucleotides
such as 8-oxo- dGTP and
8-oxo-GTP. The existence of these molecules
in genomic DNA are severely mutagenic and provoke cell death. We showed that
the OEO and thymol-induced ROS in MDA-MB231 cells leads
to accumulation of oxidized guanine (8-oxo-dG),
confirming that treatment with OEO and thymol results in cell death through enhancement
of ROS and DNA strand breaks. Although mitochondria
membrane depolarization precedes the ROS production, ROS accumulation results
in the loss of mitochondrial membrane potential (??m). These procedures are
associated with activation of mitochondrial
apoptotic pathway. A common feature of early apoptosis is the active mitochondrial
dysfunction, including changes in the redox potential of mitochondria. Our findings
demonstrated that various doses of OEO and thymol were able to decrease MMP of
cells in a concentration-dependent manner, indicating that the ROS accumulation
induced by OEO and thymol reproducibly triggered abrupt mitochondrial
depolarization. Therefore, there was both an
accumulation of ROS and a loss of ??m in response to OEO and thymol in treated
cells leading to mitochondrial apoptotic pathways, resulting in apoptosis of
MDA-MB231 cell lines. Induction of cell cycle arrest to prevent
the cell proliferation is one of the effective ways used by cytotoxic drugs. These
drugs play an anticancer role via arresting G0/G1, S or G2/M phases, thereby
inhibiting cell growth, and eventually leading to apoptosis. Inducing the cell cycle arrest achieves by
establishment of DNA damage triggered by ROS generation. Following
treatment of MDA-MB231 with various concentrations of OEO and thymol, our results, obtained from flowcytometery, clearly verified that OEO
and thymol have the potential to arrest MDA-MB231 cell lines at S-phase significantly.
Therefore one of the toxicological mechanisms of OEO and thymol for inhibition
of MDA-MB231 is S-phase arrest, resulting in blockage
of DNA replication and ultimately apoptosis. Chemotherapy and other
therapies of cancer cells has been linked to stimulation of apoptosis signaling
pathways such as the extrinsic (death
receptor-mediated) and/or intrinsic (mitochondria-initiated
apoptotic) pathway. Although, some anticancer
drugs initiate cell death through death receptors on cell membrane, most studies
suggest a caspase-dependent pathway in mitochondria-based
apoptosis by drugs. However, the relationship between these paths has
been observed at different levels. In addition, it is obvious that the simultaneous stimulation of the
extrinsic and intrinsic pathway by drugs cause to an increase in apoptosis. In
the intrinsic pathway of apoptosis, the mitochondrial membrane permeability is
a basic event resulting in caspase
activation. This permeability can be linked to ROS generation, DNA damage
mitochondrial lipids, proteins, and also proapoptotic members of the Bcl
family. Bcl-2 family including pro-apoptotic proteins (Bax, Bak, Bad, and
Bcl-Xs) and antiapoptotic proteins (Bcl-2 and Bcl-XL) plays an important role
in regulating apoptosis induced by caspases. Main antiapoptotic protein, Bcl-2, located in cytoplasm, nucleus and
mitochondria, stabilizes the mitochondrial membrane integrity and inhibits the
cell death. While by activation of apoptosis signaling, proapoptotic
Bax, located in cytoplasm, is transmitted to mitochondria which ultimately
enhances the mitochondrial membrane permeability. Therefore upregulation
of Bax and downregulation of Bcl-2 are associated with the enhanced levels of activated
caspase and ultimately cell death. Indeed,
with upregulation of Bax and mitochondrial membrane damage, caspase-9, an upstream initiator protease,
is activated and is followed by activation of caspase-3.  In extrinsic apoptotic pathway, stimulation of
death receptors results in activation of the initiator caspase-8. This activated
caspase-8 can amplify the apoptosis induced by cytotoxic drugs through cleavage
of effector caspases such as caspase-3. Caspase-3 is a frequently downstream effector caspase
catalyzing the cleavage of numerous vital cellular
proteins. Based
on this, in the following of
our research, to determine whether OEO and thymol induce apoptosis in MDA-MB231 cell lines through the
intrinsic and/or extrinsic pathway, western blot analysis was performed. The
results showed that the protein levels of Bcl-2 were decreased,
while those of Bax were increased, resulting in an elevation of the Bax/Bcl-2
ratio in MDA-MB231 cell lines. These results suggested that the cell apoptosis
induced by treatment with OEO and thymol is dependent on alterations in the
expression of Bcl-2 family proteins. Furthermore, consideration of caspase-9, 3
and 8 clarified that the expression levels of cleaved caspase-9, 3 and 8 were
significantly decreased with treatment. Collectively, these findings suggested
that OEO and thymol not only stimulate the mitochondrial pathway in OEO and
thymol-induced apoptosis in MDA-MB231 cell lines, but also may
amplify apoptosis through extrinsic pathway.  

Ability of a
drug to interact with DNA has been proposed as an important feature in the discovery
of new anti-cancer agents. Thus, the finding the DNA-binding drugs that improve
the cytotoxicity is valuable. To elucidate whether OEO and
thymol have a cytotoxic effect based on interaction with DNA, OEO/thymol
and rat hepatocyte genomic DNA
interaction was studied via UV–visible and fluorescence
spectroscopic techniques, CD spectral analysis and in silico molecular
docking. Based
on the results, by adding increasing amounts of OEO and thymol, alterations in
UV-Vis spectra at 260 nm such as Hyperchromicity and red shift
were observed and confirmed conformational changes in DNA by forming
DNA-OEO/thymol complexes. In addition, competitive displacement assays with EB showed
that OEO and thymol interacts with DNA through groove binding and does not intercalate into
the DNA base pairs (or can intercalate in DNA). These data (Or intercalation in DNA) was completed by
CD spectral analysis, with observing the hyperchromicity at 275 nm positive
band and 245 nm negative band. In
silico molecular docking, the four main compounds in the OEO,
thymol, carvacrol, p-cymen and ?-terpinene was considered. Obtained data confirmed
that thymol and carvacrol can bind to the minor groove of the DNA with relative
binding energy of ? and ? kcal/mol sequentially while p-cymen and ?-terpinene bind to the minor groove with lower
binding affinity. Collectively, these findings showed
that OEO through interaction with DNA is able to behave as an anti-cancer
agent. And in this interaction two components of thymol and carvacrol are

Collectively, despite
some differences, based on the relatively similar obtained results from thymol
and OEO, it can be explained that the some parts of effectiveness of OEO in
cancer cells probably belongs to thymol. However better results obtained from
OEO can suggest that the involvement of other components such as carvacrol is probably
vital and researchable in anti-cancer effects of OEO.


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