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PMC3124362

Anticancer Res. Author manuscript; available in PMC Jan 1, 2012.

Published in final edited form as:
Anticancer Res. Jan 2011; 31(1): 233241.
PMCID: PMC3124362
NIHMSID: NIHMS300578

Ruta Graveolens Extract Induces DNA

Damage Pathways and Blocks Akt Activation

to Inhibit Cancer Cell Proliferation and

Survival
KHALDA FADLALLA,1 ANGELA WATSON,1 TESHOME YEHUALAESHET,1 TIMOTHY
TURNER,2 and TEMESGEN SAMUEL1
Author information Copyright and License information
The publisher's final edited version of this article is available at Anticancer Res
See other articles in PMC that cite the published article.

Abstract
Background
Ruta graveolens is a medicinal herb that has been used for centuries against various ailments.
This study examined the anticancer properties of the herb using cancer cell lines.

Materials and Methods

Methanolic extract of R. graveolens was tested on colon, breast and prostate cancer cells.
Viability, cell cycle profiles, clonogenicity and capase activation were measured. Induction and
subcellular localizations of p53, 53BP1 and -H2AX proteins were examined.

Results
the extract dose-dependently decreased the viability and the clonogenicity of treated cells and
induced G2/M arrest, aberrant mitoses, and caspase-3 activation. It also induced the p53 pathway
and focal concentration of the DNA damage response proteins 53BP1 and -H2AX. Moreover,
the levels of phospho-Akt and cyclin B1 were reduced by treatment, whereas only cyclin B1 was
reduced in normal dermal fibroblasts.

Conclusion
R. graveolens extract contains bioactive compounds which, independently of known
photoactivatable mechanisms, potently inhibit cancer cell proliferation and survival through
multiple targets.
Keywords: Medicinal herb, bioactivity, p53 pathway, apoptosis
Plants are important sources of medicinal compounds worldwide. A few examples are: aspirin
from willow tree, digitalis from foxglove, artemisinin from wormwood, taxol from the pacific

yew tree, vinblastine and vincristine from periwinkle, etoposide from mayapple, etc. More than
60% of cancer therapeutics on the market or in pre-clinical trials are based on natural products
(1, 2). Despite the decline of interest by the pharmaceutical industry in research and development
of natural compounds, these unrefined compounds from terrestrial and aquatic sources continue
to serve as the chemical foundations from which modified or synthetic versions can be derived.
Ruta graveolens is a medicinal and culinary plant that is native to the Mediterranean region of
southern Europe and northern Africa. Widely grown in different parts of the world, this herb has
historically been in use since the ancient times (3). Its documented therapeutic uses include the
treatment of inflammatory conditions, eczema, ulcers, arthritis, fibromyalgia, antidote for
venoms, insect repellent, and as an abortifacient (46). The plant has also been commonly used
to season some food items such as soup, cheese, butter, coffee, and tea, and in medicinal
preparations such as rue oil and infusions that are used as antispasmodics and emmenagogues
(7).
Chemical compounds so far known to be present in R. graveolens include furanocumarins,
carotenoids, chlorophyll, and furanoquinolones (4, 8). Psoralens, a family of furanocumarins in
R. graveolens, have been widely studied for their DNA inter-strand cross-linking activity when
exposed to ultra violet (UV) light. This photoactivation property of psoralens has been utilized in
the treatment of various skin malignancies including psoriasis, vitiligo and cutaneous lymphoma
as a psoralen plus UV-A or UV-B (PUVA or PUVB) regimen (911).
Although some bioactive components in the herbal extracts and infusions made from the herb R.
graveolens plant have been suggested to be responsible for the plants beneficial or phototoxic
effects, molecular studies that extensively decipher the activities of most of its bioactive
ingredients are scarce. While the phytophototoxicity caused by R. graveolens has been known for
long time, the bioactivities of R. graveolens extracts or its various preparations against tumor
cells or pathogenic microbes have attracted attention only recently (1217).
This study examined the potency of an extract from R. graveolens on cancer cell lines. This
study shows that this extract has potent anti-cancer activity, exhibited through strong antiproliferative and ant-survival effects on cancer cells.

Materials and Methods

Cell culture and treatments
The human colorectal cancer cell line HCT116 was a generous donation from Dr. Bert Vogelstein
(Johns Hopkins University, MD, USA). The cell line was maintained in McCoys medium
(Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS) and 10,000
U/ml penicillin/10mg/ml streptomycin. The MCF7 cell line was a gift from Dr. Leslie Wilson
(University of California at Santa Barbara, CA, USA). RKO (purchased from ATCC, Manassas,
VA, USA) and MCF7 cells were grown in Dulbeccos Modified Eagles Medium (DMEM,
Invitrogen Corp, Carlsbad, CA, USA) supplemented with FBS and penicillin/streptomycin.
Prostate cancer cell lines PC3 and DU-145 cells, purchased from American Type Culture
Collection (ATCC, Manassas, VA, USA), were cultured in T-medium (Invitrogen Corp, Carlsbad,

CA, USA), supplemented with 10% FBS and penicillin/streptomycin. For experimental
treatments, cells were seeded in dishes with 96-well (for viability assays), 12-well (for colony
formation assays), 6-well (for flow cytometry and clonogenicity assays), 6 cm culture plates (for
cell lysate preparations), or 4-well chamber slides (for immunofluorescence staining). All cell
cultures were incubated at 37C and 5% CO2 in a humidified incubator.

Extract preparation
Fresh leaves of Ruta graveolens were minced finely in a food processor and extracted in 80%
methanol for 24 hours. Particulate matters were removed by two rounds of centrifugations at
1000 g and 10,000 g for five and ten minutes, respectively. The soluble fraction was
desiccated in a rotary evaporator, and the evaporated solids were resuspended in DMSO at a final
concentration of 60 mg/ml.

Antibodies
Anti-p53, -actin, phospho--H2AX ser139, 53BP1, Akt, phospho-Akt, cyclin B1, CDK1
antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-p21WAF1
was purchased from Invitrogen Corporation (Carlsbad, CA, USA). Monoclonal antibody to tubulin (clone DM1A) was purchased from Sigma (St Louis, MO, USA).

Clonogenicity assays
Clonogenicity assay was performed as described elsewhere (18). The clonogenic potential of
untreated and treated cells was determined by seeding approximately 150 cells per well of a 6well dish. Cells were allowed to adhere for approximately 24 hours and then treated with varying
concentrations (0300 g/ml) of the R. graveolens extract in culture medium. Colony formation
was examined daily by light microscopy. The assay was terminated by fixing the cells when the
control treated cells formed visible discrete colonies. Formed colonies were stained with 10%
crystal violet in methanol for 1520 minutes, washed to remove excess dye, and air-dried.
Colonies were counted using AlphaImager (AlphaInnotech, Santa Clara, CA, USA) in colony
counting mode. The relative clonogenicity of the treated cells was computed as percentage of the
number of colonies that formed in the control DMSO treated wells.

Flow cytometry
Cells were harvested and prepared for flow cytometry as described elsewhere with some
modifications (19). Cells were harvested by trypsinization using 0.25% trypsin-EDTA
(Invitrogen Corporation, Carlsbad, CA, USA) and centrifuged. Pellets were resuspended in 300
l phosphate buffered saline (PBS, Invitrogen Corp, Carlsbad, CA, USA), fixed by addition of
700 l 100% ethanol while vortexing, and stored at 20C for a minimum of 12 hours. Fixed
cells were centrifuged, and stained in FACS staining solution (320 mg/ml RNase A, 0.4 mg/ml
propidium iodide) in PBS without calcium and magnesium for 15 minutes at 37C. Stained cells
were filtered through a 70 m pore size filter and analyzed by flow cytometry on C6 Accuri
flow cytometer (Accuri Cytometers, Ann Arbor, MI, USA). Data was analyzed and histograms
were prepared using CFlow software (Accuri Cytometers, Ann Arbor, MI, USA).

Immunofluorescent staining and microscopy

Phase contrast images of cells were taken at 20X and 40X magnification objectives (and 10X
eyepiece) using an Olympus IX71 inverted microscope (Olympus America Inc, Melville, NY,
USA) fitted with digital image capture cameras (Digital Microscopy Lab, College of Veterinary
Medicine, Nursing and Allied Health, Tuskegee University). HCT116 cells for
immunofluorescent staining were grown in 4-well chamber slides. Staining was performed as
described previously (19). Confocal images were taken using an Olympus DSU spinning disk
confocal microscope (Olympus America Inc, Melville, NY, USA) at the Research Center at
Minority Institutions (RCMI) core-facility using a 40X dry objective. Images were captured
using Metamorph Premium software (Strategic Triangle Inc, Toronto, Canada), stored in TIFF
format and further processed in Adobe Photoshop.

Immunoblotting
Cell lysates were prepared in NP-40 lysis buffer (20 mM Tris-Cl pH 7.5, 150 mM NaCl, 10%
Glycerol and 0.2% NP-40 plus protease inhibitor cocktail) and protein concentrations were
determined using DC detergent compatible protein assay (BioRad Laboratories Inc, Hercules,
CA, USA). Samples containing equivalent protein concentrations were mixed with Laemmli
buffer, and boiled for five minutes. Proteins were resolved by SDS-PAGE, transferred to PVDF
membranes (GE Healthcare Life Sciences, Piscataway, NJ, USA) and blocked in 5% non-fat dry
milk. Primary antibodies (p53, p21, and cyclin B1) were used at 1:1000 dilutions. Peroxidase
conjugated anti-rabbit and anti-mouse IgG secondary antibodies were purchased from GE
Healthcare Life Sciences (Piscataway, NJ, USA) and used at a 1:5000 dilution.
Chemiluminescent detections were performed using Chemiluminescent HRP Substrate (GE Life
Sciences, Piscataway, NJ, USA)

Caspase 3 activation assay

The assay was performed using a colorimetric Caspase 3 activation assay kit (Caspase-3-C,
Sigma, St. Louis, MO, USA). Briefly, treated and control cells were lysed in 1X lysis buffer.
Thirty micrograms of total protein were used to perform the assay according to the
manufacturers recommendations. Recombinant Caspase 3 was added to untreated (control) cell
lysates and used as a positive control for the assay. A separate set of cells were incubated with 1
M staurosporine for three hours to activate endogenous Caspase 3 as another control.
Absorbance was read in a kinetic mode every 1.5 minutes at 405 nm using PowerWave XS plate
reader and Gen5 software (Biotek Instruments Inc, Winooski, VT, USA).

Results
Dose dependent inhibition of cell viability by R. graveolens extract
To determine the bioactivity of a methanolic extract of R. graveolens against cancer cells,
HCT116 (colorectal cancer), MCF7 (breast cancer), DU-145 and PC3 (both prostate cancer)

cancer cells were treated with varying concentrations of the extract and examined the viability in
treated versus control cells by MTS assay after 24 hours.
As shown in Figure 1A, R. graveolens extract reduced the viability of treated cancer cells in a
dose dependent manner. Based on the cell viability measured after only 24 hours, the
approximate 50% inhibitory concentration (IC50) of R. graveolens extract was approximately 75
g/ml (PC3 cells), 150 g/ml (MCF7), 200 g/ml (HCT116), 300 g/ml (DU-145 cells), with
PC3 cells being the most sensitive. However, as described below, colony formation by the cell
lines was completely inhibited by even smaller doses of the extract, suggesting even lower
effective growth inhibitory concentration of the extract.

Figure 1
A. PC3 (prostate), DU-145 (prostate), HCT116 (colorectal) or MCF7 (breast) cancer cells were
exposed to varying concentrations of R. graveolens extract. Viability of control (untreated or
DMSO) or experimental (R. graveolens extract 37 g/ml to ...

R. graveolens extract inhibits the proliferation of cancer cells

Unlike normal cells, cancer cells have the capacity to proliferate and form cell colonies even
when they are seeded as individual cells after being separated from other cancer or stromal cells.
It was assessed by colony formation assay whether R. graveolens extract would inhibit the ability
of HCT116, RKO or DU-145 cancer cells to establish cell colonies from individual cells. As
shown in Figure 1B, the colony forming ability of all tested cancer cells was inhibited by R.
graveolens extract. Colony formation was nearly 100% inhibited in all cell lines at the 60 g/ml
dose. The inability of the cells to form colonies indicates the presence of very potent antiproliferative compounds in the extract.
Cell cycle arrest is one of the mechanisms by which cell proliferation is blocked. Stressed cells,
which possess functional cell cycle checkpoints, activate cell cycle arrest, thus allowing the cells
to recover from the stress before the cell division. To examine the effect of R. graveolens extract
on the cell cycle, MCF7 breast cancer cells were treated with vehicle (control), 37 g/ml, 75
g/ml or 150 g/ml of extract for 24 hours and the cell cycle profile of the cells was analyzed by
flow cytometry. As shown in Figure 2A-B, cells treated with R. graveolens extract arrested
mostly in the late S to G2/M phases of the cell cycle. HCT116 and DU-145 cells also showed
similar cell cycle arrest (data not shown). In addition to the cell cycle arrest, it was also observed
that cells treated with R. graveolens extract also displayed increased number of aberrant mitoses
and post-mitotic nuclear integrity, particularly when exposed to higher doses of the extract. The
proportion of observed aberrant mitoses for HCT116 cells is shown in Figure 2C. Similarly,
MCF7 and other treated cells also showed a higher rate of aberrant mitosis at similar doses.
These aberrant phenotypes included tripolar or multipolar spindles, short or disarrayed spindle

fibers, lagging chromosomes at metaphase-anaphase transition and micronuclei formation. These

data suggests cell cycle arrest and disruption of mitosis are two of the mechanisms by which
compounds in R. graveolens extract inhibit cell proliferation.

Figure 2
Cell cycle arrest and aberrant cell division phenotypes induced by R. graveolens extract. A. cell
cycle arrest in MCF7 cells. B. Bar graph showing the proportion of aberrant mitoses in control
and treated HCT116 cells.

R. graveolens extract activates the p53 pathway

Various cellular stressors induce the stabilization and activation of the p53 tumor suppressor
protein. The p53 protein regulates critical cellular mechanisms that modulate the cell cycle,
apoptosis, senescence, and various signaling pathways. Therefore, it was examined whether the
p53 protein is activated after the treatment of cells with R. graveolens extract. HCT116
colorectal cancer cells, which have an intact p53 pathway, were treated with the extract and the
induction of p53 was examined by immunofluorescence staining. As shown in Figures 3A and B,
exposure of HCT116 cells to R. graveolens extract, but not the vehicle DMSO, led to the
accumulation of nuclear p53. The induction of p53 was also accompanied by the induction of the
cell cycle regulator CDK inhibitor protein p21 (Figure 3B), indicating the activation of p53mediated transcription. This transcriptional activity suggests that compounds in R. graveolens
extract activate p53-regulated pathways, which include cell cycle arrest that can be mediated by
the p21 protein.

Figure 3
A. Induction of the p53 tumor suppressor protein by R. graveolens extract. HCT116 cells were
treated with carrier control (DMSO) or the indicated concentrations of the R. graveolens extract
in culture medium for 24 hours. Nuclear p53 was visualized by ...

R. graveolens extract induces the DNA-damage response pathway

The tumor suppressor protein p53 can be activated by signals originating from activated DNA
damage response (DDR) (20, 21). ATM, Chk1, Chk2, p53, 53BP1, MRN complex and other
proteins are components of the DDR signaling network. 53BP1 is a large (450kDa) protein that
was initially identified by its ability to bind to p53. Subsequent studies have shown that 53BP1
protein is part of the DNA-damage response (DDR) protein complex, and co-localizes with the

DNA repair complex proteins such as Mre11-Rad50-NBS1 (MRN) at distinct nuclear foci (22
26).
To examine if the activation of p53 pathway was the result of DDR signaling, the nuclear
distributions of 53BP1 and -H2AX proteins were analyzed by immunofluorescenCE staining of
control or R. graveolens treated HCT116 cells 24 hours after exposure to the extract. As shown
in Figure 3C and D, treatment with R. graveolens extract induced the activation and the
recruitment of 53BP1 and -H2AX proteins to distinct nuclear foci, typically evident in cellular
response to DNA-damage. The relocalization of both 53BP1 and -H2AX proteins into subnuclear foci indicates that compounds in R. graveolens have the capacity to induce DNA damage
foci, to which damage-repair proteins are recruited.
The Akt/PKB protein is a pro-survival signaling molecule constitutively activated in a number of
solid tumors, and it cross-talks with the p53 pathway (27). To examine if R. graveolens extract
modulates this critical signaling pathway, the phosphorylation of the Akt protein as a marker of
Akt activation in DU145 prostate cancer cells was examined. As shown in Figure 4A, treatment
of the cells with R. graveolens extract reduced the levels of phospho-Akt, which suggests that the
activity of the Akt signaling pathway may be inhibited by the extract. In contrast to the
phosphorylated Akt, the total cellular levels of the Akt protein were not affected by the treatment.
Additionally, the level of the mitotic cyclin B1 was significantly reduced by R. graveolens
extract, further indicating the inhibitory effect of the extract on cell cycle transition. To examine
the effect of R. graveolens extract on untransformed cells, normal human dermal fibroblasts were
treated with vehicle or the extract. Interestingly, in contrast to cancer cell lines, no major changes
were observed in the levels of phospho-Akt and p53 in fibroblasts exposed to up to 150g/ml of
the extract (Figure 4B). However, the levels of cyclin B1 were markedly reduced in treated
fibroblasts, similarly to the cancer cells. Higher than 150g/ml doses of the extract were nonspecifically cytotoxic also to the fibroblasts.

Figure 4
A. R. graveolens extract suppresses Akt activation and cyclin-B1 levels. DU145 prostate cancer
cells were treated with carrier only or 150 g/ml R. graveolens extract for 48 hours. The
expression of the indicated proteins was analyzed by immunoblotting. ...

R. graveolens extract induces caspase activation

During the experiments, it was observed that reduction in cell viability or induction of cell death
was associated with increased number of cells with membrane blebbing, which is a characteristic
microscopic phenotype of cells undergoing apoptosis. This was especially evident in cells
exposed to R. graveolens extract at doses between 150 g/ml to 300 g/ml for 24 hours. Figure
5A shows the microscopic appearance of HCT116 cells treated with DMSO (control) or 300
g/ml R. graveolens extract. To examine if the membrane blebbing phenotype was also
associated with the activation of intracellular caspases, the activity of caspase 3 in the lysates of

HCT116 cells treated with DMSO, or 75 g/ml, 150 g/ml, or 300 g/ml R. graveolens extract
was assayed. In parallel, HCT116 cells were treated with 1 M staurosporine for three hours as a
positive control to induce endogenous caspase 3. Recombinant caspase 3 was used as an assay
control. As shown in Figure 5B, the activity of caspase 3 in the lysates of cells treated with 150
300 g/ml R. graveolens extract was markedly enhanced compared to control cells. This result
confirmed that the membrane blebbing phenotype observed in cells treated with R. graveolens
extract was coincidental to the execution of apoptosis, and the induction of apoptotic phenotype
correlated with enhanced intracellular caspase activity.

Figure 5
Activation of caspase 3 and induction of apoptosis by R. graveolens extract. A. HCT116 cells
were treated for 24 hours with the carrier DMSO or 300 g/ml R. graveolens extract in culture
medium. Phase contrast pictures of treated cells were taken ...

Discussion
This study showed that the medicinal and culinary herb Ruta graveolens contains bioactive
compounds that inhibit cell proliferation, reduce cell viability, and induce DNA damage response
and apoptosis. This is particularly interesting since this herb has been used for centuries as a
spicing ingredient for food and to treat various illnesses. R. graveolens preparations can be
readily commercially obtained as infusions and oils for medicinal use.
These data suggest that R. graveolens extract contains candidate compounds that deserve not
only to be further evaluated for their mechanisms of action, but for their chemotherapeutic or
adjuvant therapy potentials as well. Importantly, treatment with the extract induced nuclear foci
formation that is characteristic of the typical DNA-damaging agents used in cancer therapy, such
as irradiation and platinum compounds. Moreover, even the smaller doses that were tested still
inhibited cell proliferation in colony formation assay, suggesting the presence of long-acting
components that inhibit cancer cell proliferation at low doses of the extract. At such low doses,
aberrant mitoses were not significantly evident, suggesting a potentially different mechanism of
activity at low doses compared to high doses, where DNA damage and aberrant mitoses were
evident. Therefore, investigating the therapeutic (high dose) or chemopreventive (low dose)
potentials of the extract and its components is warranted. It is currently unknown, however, if the
same pathways involved in DDR are also responsible for the anti-proliferative activity of the
extract observed at lower doses. It is possible that other survival pathways may be inhibited at
lower doses without inducing a strong DDR. The ingredients that inhibit the long-term survival
of treated cancer cells need to be examined in detail, since low doses of R. graveolens
compounds may be beneficial as chemopreventive agents in high-risk cancer subjects.
The induction of DDR by R. graveolens extract raises a question about the role of DDR as a
checkpoint in the current context, i.e. if compounds in R. graveolens would be suitable for

chemoprevention, or as sensitizers to chemotherapy. Activated DDR serves as a checkpoint to

allow damaged cells to repair their DNA, or to induce apoptosis or senescence, as a primary line
of defense against neoplastic transformation of cells (28). Although initiation of DDR is
suggested to be a checkpoint to prevent carcinogenesis, persistent DDR may have undesirable
outcomes through chronic inflammatory signaling (29) that may in turn promote carcinogenic
transformation. To address these issues, it is necessary to identify the bioactive ingredients in R.
graveolens extract in order to delineate those with DDR-inducing activities, and those with
potential effects on other survival signaling pathways. On the other hand, the DDR-inducing
property of the R. graveolens extract might be expected to sensitize cancer cells to
chemotherapeutic drugs that target survival-signaling pathways. Recently, the inhibition of
CYP3A by compounds in grapefruits was used to reduce the therapeutic dose of the anti-cancer
drug rapamycin (30, 31), illustrating the potential use of natural compounds as adjuvants or
supplements to conventional therapeutic drugs. On the other hand, the DDR-inducing property of
R. graveolens extract should also raise caution about the potential risks of prolonged use of high
doses of medicinal preparations from the herb.
Ruta species and many other plants such as grapefruit, parsley, celery, and parsnip contain a
group of phytochemicals called furanocoumarins (furocoumarins) that have been associated with
phytophotodermatitis (4, 8, 32), or the grapefruit juice effect (33, 34). The phototoxic effects of
furanocoumarin compounds have been associated with topical application of products containing
the compounds followed by exposure to sources of ultraviolet light (4, 5, 35). However,
photoactivation-independent bioactivities of these compounds, including p53 induction and in
vivo CYP450 inhibition, have been reported (14, 36). Therefore, further studies are needed to
identify the individual molecules, the molecular targets for each of the bioactive compounds in
R. graveolens, and to suggest the rational conditions under which the plant products or the
isolated compounds might be used beneficially. Since treated cells were not exposed to known
sources of UV light in this study, it is proposed that the DDR and anti-proliferative effects that
are reported here are independent of the UV-inducible type of photophytotoxicity. A similar
result has been reported for bergapten, one of the photoactivatable furanocumarines, where the
compound induced the p53 pathway and apoptosis of breast cancer cells independent of
photoactivation (14). In conclusion, the current data suggest that extracts from the medicinal and
culinary herb R. graveolens contain bioactive compounds that activate specific molecular
signaling pathways and significantly interfere with the survival and proliferation of cancer cells,
warranting further investigations.

Acknowledgments
We thank Bert Vogelstein and Leslie Wilson for kind provision of cell lines, Cesar Fermin for
help with microscopy and digital imaging, Clayton Yates for help with confocal microscopy,
Sibyl Bowie for suggestions and editorial help during preparation of the manuscript. Research in
T.S. lab is supported by grant SC2CA138178 from the NIH/NCI/NIGMS. Support to T.T. was
through grant numbers 3P20MD000195 (NIH/NCMHD) and U54CA118623 (NIH/NCI). We
acknowledge the CVMNAH Center of Excellence (grant # 5D34HP00001-20-00) summer
research support (to AW, TY, and TS).

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