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Biomedicine & Pharmacotherapy 131 (2020) 110717
Available online 23 September 2020
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Original article
Rosemary (Rosmarinus ofcinalis L.) extract inhibits prostate cancer cell
proliferation and survival by targeting Akt and mTOR
Alina Jaglanian
a
, Deborah Termini
a
, Evangelia Tsiani
a
,
b
,
*
a
Department of Health Sciences, Brock University, St. Catharines, ON, L2S 3A1, Canada
b
Centre for Bone and Muscle Health, Brock University, St. Catharines, ON, L2S 3A1, Canada
ARTICLE INFO
Keywords:
Rosemary extract
Prostate cancer
Proliferation
Survival
Akt
mTOR
ABSTRACT
Prostate cancer is the most commonly diagnosed type of cancer in North American men and is typically classied
as either androgen receptor positive or negative depending on the expression of the androgen receptor (AR). AR
positive prostate cancer can be treated with hormone therapy while AR negative prostate cancer is aggressive
and does not respond to hormone therapy. It has been previously reported that rosemary extract (RE) has
antioxidant, anti-inammatory and anti-cancer properties. In the present study, we found that treatment of the
androgen-insensitive PC-3 prostate cancer cells with RE resulted in a signicant inhibition of proliferation,
survival, migration, Akt, and mTOR signaling. In addition, treatment of the androgen-sensitive 22RV1 prostate
cancer cells with RE resulted in a signicant inhibition of proliferation and survival while RE had no effect on
normal prostate epithelial PNT1A cells. These ndings suggest that RE has potent effects against prostate cancer
and warrants further investigation.
1. Introduction
Prostate cancer accounted for roughly 1.3 million cases and 359,000
deaths globally in 2018, and is the second leading cause of death in
North American men despite all available treatment strategies including
surgery, radiotherapy, and chemotherapy [1]. Finding novel approaches
to prevent and treat prostate cancer effectively is highly desirable. Major
disruptions of cellular homeostasis of the prostate gland leads to prostate
cancer [2]. The growth of prostate epithelial cells is inuenced by
growth factors, the expression and function of androgen receptors (AR)
and by the hypothalamic-pituitary axis [2,3]. Androgens provide
important growth stimuli for prostate cells, and ARs are typically
expressed in the stromal and epithelial compartments of the prostate
gland [2,4]. Production of luteinizing hormone (LH)-releasing hormone
(LHRH) by the hypothalamus induces the production of LH by the pi-
tuitary gland [5] leading to increased androgen production. Hypotha-
lamic LHRH and pituitary LH production are regulated by a negative
feedback mechanism [5]. Androgen binding to AR leads to transcrip-
tional activation of AR target genes which are involved in various bio-
logical processes such as proliferation and apoptosis [2–4]. Overall, AR
signaling is directly involved in maintaining normal prostate tissue ho-
meostasis [2].
Cancer cells are characterized by their ability to proliferate uncon-
trollably and evade apoptosis [6,7]. These characteristics are often ac-
quired as a result of mutations in key proteins involved in the signaling
pathways responsible for regulating cellular function and maintaining
homeostasis [8–13]. Molecular signaling pathways of growth factor
receptors; such as Epidermal Growth Factor (EGF) Receptor (EGFR)
initiate signal transduction pathways that lead to increased cell prolif-
eration and survival [8,14–16]. The phosphatidylinositol 3-kinase
(PI3K)/Akt pathway is activated by growth factor [17] and androgen
receptor [18] signaling and plays a prominent role in prostate cancer.
Mutations that result in the overactivation of this cascade, activation of
other oncogenes, and/or the inactivation of proteins that serve as tumor
suppressors such as p53, p27, and phosphatase and tensin homologue
(PTEN) [19,20], contribute to carcinogenesis and the development of
prostatic tumors [21–23].
Increased Akt activation is associated with carcinogenesis as well as
increased resistance to chemotherapeutic agents such as cisplatin,
methotrexate, and paclitaxel [24,25]. Akt expression/activation is often
elevated in human prostate cancers [26,27]. Aberrations in the
PI3K/Akt pathway have been reported in approximately 40 % of early
prostate cancer and 70–100 % of advanced cases [28,29]. Specically,
the loss of PTEN leads to the constitutive activation of the PI3K/Akt
* Corresponding author at: Department of Health Sciences, Brock University, St. Catharines, ON, L2S 3A1, Canada.
E-mail addresses: aj11fo@brocku.ca (A. Jaglanian), dt14@brocku.ca (D. Termini), etsiani@brocku.ca (E. Tsiani).
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
https://doi.org/10.1016/j.biopha.2020.110717
Received 28 July 2020; Received in revised form 28 August 2020; Accepted 30 August 2020
Biomedicine & Pharmacotherapy 131 (2020) 110717
2
pathway [4]. Due to the importance of Akt in prostate cancer, several
small molecules that target/inhibit Akt are currently in clinical devel-
opment [30,31].
Mechanistic target of rapamycin (mTOR), a 289 kDa serine/threo-
nine kinase, is a downstream effector of the PI3K/Akt pathway, and is
involved in the control of cell growth [32,33]. Twice the levels of total
and phosphorylated mTOR have been reported in prostate cancer tissue
when compared to normal prostate epithelium [34]. As a result mTOR is
an appealing therapeutic target and mTOR inhibitors such as sirolimus,
deforolimus, everolimus, and temsirolimus are used as a monotherapy
or combined therapy for various types of cancers [24,33,35].
The process of programmed cell death, known as apoptosis, is an
essential process in the maintenance of cell homeostasis. The most
common signaling cascades involved in regulating cellular apoptosis
promote the downstream activation of caspases and Poly (ADP-ribose)
polymerase-1 (PARP-1) cleavage to form 89 and 24 kDa fragments [36].
PARP-1 is an enzyme responsible for DNA repair and therefore, plays a
role in genomic maintenance [37,38]. Cleaved PARP is an established
indicator of apoptosis [36,38].
Over 50 % of modern chemotherapeutic agents that are used for
cancer treatment are derived from natural products [39,40]; for
example, the chemotherapeutic drug paclitaxel was originally isolated
from the bark of the Pacic yew tree (Taxus brevifolia) and the chemo-
therapeutic drug docetaxel was originally isolated from the needles of
the European yew tree (Taxus baccata) [41].
Scientic interest in nding chemicals in plants with anti-cancer
potential continues today. The rosemary (Rosmarinus ofcinalis L.)
plant, native to Mediterranean countries, contains the polyphenols
carnosic acid (CA), rosmarinic acid (RA), and carnosol (COH) in high
concentrations [42,43]. In recent years, rosemary extract (RE) and RE
polyphenols have been reported to have antioxidant, antimicrobial, and
anti-cancer properties [44–48]. Limited data exists regarding the effects
of rosemary extract in prostate cancer [49–52], and little is known about
the underlying signaling mechanisms involved in mediating its
pro-apoptotic and anti-proliferative effects.
In the present study, we investigated the effects of rosemary extract
in PC-3 and 22RV1 prostate cancer cells, as well as in PNT1A normal
prostate epithelial cells.
2. Materials and methods
2.1. Materials
The PC-3 human epithelial prostate cancer cells were obtained from
American Type Culture Collection (ATCC) (Manassas, VA, USA). The
Roswell Park Memorial Institute (RPMI) 1640 Medium, fetal bovine
serum (FBS), 0.25 % trypsin and the antibiotic–antimycotic solution
were purchased from GIBCO Life Technologies (Burlington, ON, Can-
ada). Akt (#9272) (1:1000 dilution), p-Akt (Ser473) (#9271) (1:1000
dilution), mTOR (#2972) (1:1000 dilution), p-mTOR (Ser2448)
(#2971) (1:1000 dilution), PARP (#9542) (1:1000 dilution), β-actin
(#8457) (1:1000 dilution), as well as secondary anti-rabbit IgG HRP-
linked antibodies (#7074) (1:2000 dilution) were from Cell Signaling
Technology via New England Biolabs (Mississauga, ON, Canada).
22RV1 human epithelial prostate cancer cells, PNT1A normal prostate
epithelial cells, bovine serum albumin (BSA), dimethyl sulfoxide
(DMSO), docetaxel and paclitaxel were from Sigma (Oakville, ON,
Canada). Clarity western enhanced chemiluminescence (ECL) reagent,
30 % acrylamide/bis solution 37 (5:1), ammonium persulfate (APS),
polyvinylidene diuoride (PVDF) membranes and reagents for electro-
phoresis were purchased from Bio-Rad (Hercules, CA, USA).
2.2. Rosemary extract preparation
Whole dried rosemary (Rosmarinus ofcinalis L.) leaves (purchased
from Compliments/Sobey’s, Mississauga, ON, Canada) were used, and
the rosemary extract was prepared as previously reported [53]. Briey,
dried rosemary leaves were ground and steeped overnight (16 h) in
dichloromethane: methanol (1:1) followed by ltration the next day.
After ltering, the solvent was set aside while the leaves were boiled in
methanol for 30 min. The solvent obtained after boiling was combined
with the ltered solvent. The combined solvent was removed from the
nal extract by rotary evaporation and the green powder was collected
and stored at −20 ◦C, protected from light. Aliquots were prepared in
dimethyl sulfoxide (DMSO) to yield a stock concentration of
100 mg/mL, stored at −20 ◦C and protected from light.
2.3. Cell culture and treatment
The cells were cultured in RPMI 1640 media supplemented with 1%
(v/v) antibiotic–antimycotic solution (containing 100 units/mL of
penicillin, 100
μ
g/mL of streptomycin, and 0.25
μ
g/mL of Amphotericin
B) and 10 % (v/v) FBS in an incubator at 37 ◦C.
Cells were treated with a working stock of RE (400
μ
g/mL in cell
culture media) and the nal concentration of DMSO in the RE-treated
cells was less than 0.1 %. Exposure of the cells to DMSO to match the
concentration of DMSO seen by cells exposed to RE (vehicle control) did
not have any effect on any assays/measurements used in the current
study.
2.4. Cell proliferation assay
Cells were seeded (1000 cells/well) in a 96-well plate supplemented
with DMEM and treated as indicated in the gures for 72 h. The cells
were xed with 10 % formalin and stained using 0.5 % crystal violet
stain. The next day solubilizer solution containing 0.05 M NaH
2
PO
4
was
added into each well and the absorbance was read at 570 nm using the
KC4 microplate reader.
2.5. Clonogenic survival assay
Cells were seeded (1000 cells/well) in six-well plates and allowed to
adhere for 24 h followed by treatment as indicated in the gures for
seven days. At the end of the treatment, the cells were washed twice with
sterile phosphate-buffered saline (PBS) and stained with 0.05 % w/v
methylene blue. The next day, colonies greater than 50 cells were
counted under the microscope.
2.6. Wound healing assay
The wound healing assay was used to assess cell migration [54,55].
Cells were seeded at a density of 2.5 ×10
5
cells/mL into a 6-well plate
and the media was replaced every 48 h until the cells reached 90–100 %
conuency. When conuency was reached the cells were incubated with
mitomycin-C (MMC) (1
μ
g/mL) for 1 h to prevent cell proliferation.
After the incubation period, a vertical line was drawn in the centre of
each well using a 100
μ
L pipette tip. The wells were drained of media
and washed twice with PBS to get rid of oating cells, followed by
treatment as indicated in the gures. Before taking photographs, hori-
zontal lines were drawn underneath the well plates to be used as a
reference for future time points. Photos were taken at 0 and 40 -h time
points. Wound closure percentage was calculated using the equation
0hr Area—40 hr Area
0 Hr Area x 100. The area of each wound was measured using the
ImageJ software.
2.7. Immunoblotting
Cell lysate samples containing 20
μ
g of protein, determined using the
Bradford assay [56], were loaded onto 10 % polyacrylamide gel and
separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE). The separated proteins were then transferred onto a
A. Jaglanian et al.
Biomedicine & Pharmacotherapy 131 (2020) 110717
3
polyvinylidene diuoride (PVDF), membrane which was exposed to
blocking buffer (5% (w/v) dry milk in Tris-buffered saline) for 1 h and
incubated with the primary antibody overnight at 4 ◦C. The following
day the membrane was incubated with horseradish peroxidase
(HRP)-linked IgG anti-rabbit secondary antibody for 1 h at room tem-
perature. Enhanced chemiluminescence (ECL), the Bio-Rad clarity
western solution, was used to detect the bands corresponding to the
proteins of interest. Densitometric analysis was performed using ImageJ
software. The data (arbitrary densitometric units) were corrected to
β-actin levels and expressed as a percentage of untreated control cells.
2.8. Statistical analysis
The data are the mean ±standard error mean (SEM) of the indicated
number of independent experiments. Analysis of variance (ANOVA)
followed by Bonferroni’s post-hoc test was used to determine the sig-
nicance of the differences between groups. Signicance was assumed
at p <0.05. Statistical tests were performed using GraphPad Prism 8
software.
3. Results
3.1. Inhibition of PC-3 prostate cancer cell proliferation by rosemary
extract
The antiproliferative effects of RE were evaluated in the androgen
receptor negative PC-3 prostate cancer cells. PC-3 cells were exposed to
5, 10, 25, 50, 75, 100, or 150
μ
g/mL RE for 72 h and cell proliferation
was assessed using the crystal violet assay. The RE powder was dissolved
in dimethyl sulfoxide (DMSO) to create a stock solution (100 mg/mL).
This solution was diluted using cell culture media to create a working
stock (400
μ
g/mL) that was used to treat the cells. Treatment with RE
resulted in a dose–dependent inhibition of cell proliferation (Fig. 1A). A
signicant inhibition (80.25 ±4.44 % of control, p <0.01) was seen
with 10
μ
g/mL RE while maximum inhibition (26.51 ±2.95 % of con-
trol, p <0.0001) was seen with 50
μ
g/mL RE (Fig. 1A-C). Higher RE
concentrations (75, 100 and 150
μ
g/mL) did not result in a statistically
greater inhibition of cell proliferation compared to 50
μ
g/mL (Fig. 1A).
The data from Fig. 1A were plotted on a log scale (Fig. 1B) and the
calculated RE concentration for the half maximal inhibition (IC
50
) of cell
proliferation was 19.72
μ
g/mL. Docetaxel (DTX), derived from Taxus
baccata and paclitaxel (PTX), derived from Taxus brevifolia, are estab-
lished medications used clinically in the treatment of prostate cancer
[57] and we used them in the present study to compare the effects of RE
to their effects. We used two different concentrations (5 and 10 nM) of
PTX and DTX based on other in vitro studies [58,59]. Treatment of the
cells with 5 nM PTX did not result in any signicant inhibition of cell
proliferation (91.27 ±2.79 % of control, p >0.05) (Fig. 1C), while
treatment with 10 nM of paclitaxel showed signicant inhibition of cell
proliferation (67.63 ±4.24 % of control, p <0.01) (Fig. 1C). Treatment
of the cells with 5 and 10 nM DTX resulted in signicant inhibition of
cell proliferation (38.11 ±1.16, 32.08 ±0.84 % of control respectively,
both p <0.0001) (Fig. 1C). The inhibition of cell proliferation seen with
50
μ
g/mL of rosemary extract (26.51 ±2.95 % of control, p <0.0001)
was greater than that seen with 10 nM PTX treatment and at the same
level achieved with DTX treatment.
Fig. 1. Inhibition of PC-3 prostate cancer cell proliferation by RE. PC-3 cells were treated with 5, 10, 25, 50, 75, 100, or 150
μ
g/mL of rosemary extract (RE) (A, B,
C), 5, 10 nM paclitaxel (PTX) (C), or 5, 10 nM docetaxel (DTX) (C) for 72 h, followed by xing and staining with 0.5 % crystal violet. The stain was solubilized, and
absorbance was read at 570 nm. Data are expressed as percent of control, untreated cells. Data are the mean ±SEM of 6 independent experiments.
**p <0.01, ****p <0.0001.
A. Jaglanian et al.
Biomedicine & Pharmacotherapy 131 (2020) 110717
4
3.2. Inhibition of PC-3 prostate cancer cell survival by rosemary extract
The ability of cancer cells to survive and form colonies was also
assessed through a clonogenic survival assay. Exposure of PC-3 cells to
0.5, 1, 2.5, 5, or 10
μ
g/mL of RE resulted in a concentration-dependent
inhibition of survival (Fig. 2A) with a signicant inhibition
(80.20 ±4.60 % of control, p <0.01) seen with 0.5
μ
g/mL RE. The
greatest inhibition (15.27 ±3.80 % of control, p <0.0001) of cell sur-
vival was seen at 10
μ
g/mL RE (Fig. 2A and 2C). The data from Fig. 2A
were plotted on a log scale and the calculated RE concentration for the
half maximal inhibition (IC
50
) of cell survival was 2.43
μ
g/mL (Fig. 2B).
Exposure of the cells to 0.5 nM (64.12 ±6.94 % of control, p <0.05) and
5 nM paclitaxel (23.81 ±11.92 % of control, p <0.0001) both resulted
in a signicant inhibition of cell survival (Fig. 2C). In addition, treat-
ment with both 0.5 nM (59.18 ±9.45 % of control p <0.01) and 5 nM
docetaxel (8.50 ±8.50 % of control p <0.0001) resulted in a signicant
inhibition of cell survival.
3.3. Inhibition of Akt signaling in PC-3 prostate cancer cells by rosemary
extract
We also examined the effects of RE treatment on Akt and measured
the levels of total Akt and Akt phosphorylation on the serine 473 res-
idue, an established indicator of Akt activity [60]. Treatment of PC-3
prostate cancer cells with 50
μ
g/mL RE for 24 and 48 h both
signicantly reduced Akt phosphorylation/activation (60.65 ±11.84 %
of control, p <0.001) and (36.46 ±4.79 % of control, p <0.0001),
respectively (Fig. 3A and B). The total Akt levels were signicantly
reduced by 48 h (69.93 ±4.66 % of control, p <0.01) but not 24 h
(87.52 ±9.50 % of control, p >0.05) RE treatment.
3.4. Inhibition of mTOR signaling in PC-3 prostate cancer cells by
rosemary extract
Next, we examined the effects of RE on mTOR activation by utilizing
an antibody that recognizes phosphorylation of the serine 2448 residue,
an established marker of mTOR activation [61]. Treatment of PC-3 cells
with RE for 24 (49.41 ±6.60 % of control, p <0.0001) or 48 h
(38.82 ±6.69 % of control, p <0.0001) resulted in a signicant
decrease in mTOR phosphorylation (Fig. 4A and B). Treatment with RE
for 24 and 48 h also showed a signicant decrease of total mTOR levels
(71.74 ±10.38 % of control, p <0.01) and (60.55 ±9.02 % of control,
p <0.001), respectively.
3.5. Increased apoptosis of PC-3 prostate cancer cells by rosemary extarct
The effect of RE on cell apoptosis was examined by measuring the
levels of cleaved PARP, an established indicator of apoptosis [37].
Exposing PC-3 prostate cancer cells to rosemary extract (50
μ
g/mL) for
24 h resulted in a signicant increase in cleaved PARP (177.9 ±14.50 %
Fig. 2. Inhibition of PC-3 prostate cancer cell survival by rosemary extract. PC-3 cells were seeded (1000 cells/well) in six-well plates and exposed to 0.5, 1, 2.5, 5, or
10
μ
g/mL of rosemary extract (RE) (A, B, C), 0.5, 5 nM paclitaxel (PTX) (C), or 0.5, 5 nM docetaxel (DTX) (C) for 7 days followed by xing and staining with 0.05 %
methylene blue. Colonies of more than 50 cells were counted. Data are expressed as percent of control, untreated cells. Data are the mean ±SEM of 6 independent
experiments. *p <0.05, **p <0.01, ****p <0.0001.
A. Jaglanian et al.
Biomedicine & Pharmacotherapy 131 (2020) 110717
5
of control, p <0.01) relative to the control, indicating enhanced
apoptosis (Fig. 5A and B).
We routinely examined microscopically the morphology of cells
before and after treatments. Fig. 6 shows a representative image of RE-
and DTX-treated PC-3 cells compared to the control untreated cells. No
changes in cell morphology were observed with any of the treatments. It
is important to note that the same number of cells were seeded in all
wells (six-well plates were used). As it can be seen from Fig. 6, treatment
with 50
μ
g/mL of RE for 24 or 48 h resulted in a substantially reduced
cell density relative to the control untreated group. Treatment with DTX
for 24 or 48 h showed a reduction in cell density that was not as great as
the reduction seen with RE.
3.6. Inhibition of PC-3 prostate cancer cell migration by rosemary extract
The wound healing assay was used to assess the ability of prostate
cancer cells to migrate. The cells were seeded at a density of 2.5 ×10
5
cells/mL into a 6-well plate and grown until the cells reached 90–100 %
conuency. PC-3 cells were exposed to 1
μ
g/mL of mitomycin-C (MMC)
for 1 h to inhibit cell proliferation. After the MMC was removed a wound
was established by drawing a vertical line in the centre of each well
using a 100
μ
L pipette tip. The cells were then treated without (control)
or with either 50
μ
g/mL RE or 10 nM docetaxel for 40 h. Treatment with
rosemary extract was shown to signicantly inhibit wound closure
(56.14 ±3.48 % of control, p <0.0001) indicating properties against
Fig. 3. Inhibition of Akt signaling in PC-3 prostate cancer cells by rosemary extract. Cell lysates were prepared from PC-3 prostate cancer cells treated with 50
μ
g/mL
rosemary extract (RE) for either 24 or 48 h. Cell lysates (20
μ
g) were immunoblotted using specic antibodies against phosphorylated/activated Akt, total Akt, or
β-actin. A representative immunoblot is shown (A). The densitometry of the bands were corrected to β-actin levels and expressed in arbitrary densitometry units as
percent of control (B). The data are the mean ±SEM of 4 independent experiments. **p <0.01, ***p <0.001, ****p <0.0001.
Fig. 4. Inhibition of mTOR signaling in PC-3 prostate cancer cells by rosemary extract. Cell lysates were prepared from PC-3 cells treated with 50
μ
g/mL rosemary
extract (RE) for either 24 or 48 h. Cell lysates (20
μ
g) were immunoblotted using specic antibodies against phosphorylated/activated mTOR, total mTOR, or β-actin.
A representative immunoblot is shown (A). The densitometry of the bands were corrected to β-actin levels and expressed in arbitrary densitometry units as percent of
control (B). The data are the mean ±SEM of 5 independent experiments. **p <0.01, ***p <0.001 ****p <0.0001.
Fig. 5. Effect of rosemary extract on
PARP signaling in PC-3 prostate cancer
cells. Cell lysates were prepared from
PC-3 cells treated with 50
μ
g/mL rose-
mary extract (RE) for 24 h. Cell lysates
(20
μ
g) were immunoblotted using spe-
cic antibodies against cleaved PARP or
β-actin. A representative immunoblot is
shown (A). The densitometry of the
bands were corrected to β-actin levels
and expressed in arbitrary densitometry
units as percent of control (B). The data
are the mean ±SEM of 3 independent
experiments. *p <0.05.
A. Jaglanian et al.
Biomedicine & Pharmacotherapy 131 (2020) 110717
6
cell migration (Fig. 7B). A signicant inhibition of cell migration was
also seen when treating the cells with 10 nM docetaxel (70.92 ±2.35 %
of control, p <0.001), (Fig. 7A and B).
3.7. Inhibition of 22RV1 prostate cancer cell proliferation and survival by
rosemary extract
We also examined the effects of RE on the androgen receptor positive
22RV1 prostate cancer cells. A signicant inhibition of cell proliferation
was seen with 25
μ
g/mL RE (86.20 ±4.53 % of control, p <0.01) while
the highest level of inhibition was seen with 150
μ
g/mL RE
(49.80 ±2.289 % of control, p <0.0001), (Fig. 8A). The IC
50
value of RE
for cell proliferation, calculated by graphing the data from Fig. 8A on a
log scale, was 43.41
μ
g/mL (Fig. 8B). The effects of RE on 22RV1
prostate cancer cell survival was investigated by treating the cells with
2.5, 5, 10, 15, and 20
μ
g/mL RE for 7 days (Fig. 8C). A dose-dependent
inhibition of cell survival was seen. The calculated RE concentration for
the half maximal inhibition (IC
50
) of cell survival calculated using the
data from Fig. 8C and graphing it on a log scale was 4.17
μ
g/mL
(Fig. 8D).
3.8. Effect of rosemary extract on PNT1A normal prostate epithelial cell
proliferation
PNT1A prostate epithelial cells represent normal healthy prostate
epithelium. Treatment of PNT1A cells with 5, 10, 25, 50, 75, 100, or
150
μ
g/mL RE for 72 h did not result in any signicant changes in cell
proliferation (p >0.05) (Fig. 9).
4. Discussion
The current treatment strategies for prostate cancer include surgery,
radiotherapy, and chemotherapy [62]. Patients with localized prostate
cancer are most often treated with radical prostatectomy or radical
radiotherapy, however advanced and metastatic prostate cancer is
treated with hormonal therapy [62]. Common hormonal therapies often
use androgen-receptor inhibitors or LHRH agonists (such as leuprolide,
goserelin, buserelin, or nafarelin) that initially increase testosterone
production, but with prolonged exposure downregulate the LHRH re-
ceptor and inhibit testosterone production [5]. LHRH antagonists (such
as cetrorelix, abarelix, or orgalutran) directly inhibit LHRH, which de-
creases testosterone production [5]. Surgical castration can also
decrease testosterone levels by removing the source of production. For
patients that do not respond to androgen therapies, cytotoxic chemo-
therapeutic agents, such as etoposide, doxorubicin, paclitaxel, and
docetaxel are used [5] but resistance often develops, indicating a need
for novel therapeutics to be used alone or in combination with existing
drugs to treat prostate cancer and improve patient outcome.
Plant extracts have been used traditionally for medicinal purposes
and more than half of all available chemotherapy agents used in cancer
treatment are derived from plants with paclitaxel and docetaxel repre-
senting two such chemotherapeutics [39–41]. Finding chemicals in
plants with anti-cancer potential is the focus of many research labs,
including ours. In recent years a few studies provided evidence of
anticancer properties of rosemary extract [42–48]. In the present study
we found a dose-dependent inhibition of PC-3 androgen-independent
and 22RV1 androgen-dependent prostate cancer cell proliferation with
rosemary extract treatment (Figs. 1,8, and 10). Similar to our ndings, in
other studies, treatment with RE dose-dependently inhibited the
viability of 22RV1 and LNCaP prostate cancer cells [51]. In addition, a
Fig. 6. Effect of rosemary extract on PC-3 prostate cancer cell morphology. Cells were seeded (200,000 cells/well) and after 24 h were treated without (control) or
with RE (50
μ
g/mL) or DTX (10 nM) for 24 or 48 h. Photographs were taken immediately after treatment using an EVOS XL Core Cell Imaging System by Life
Technologies (10×magnication).
A. Jaglanian et al.
Biomedicine & Pharmacotherapy 131 (2020) 110717
7
dose-dependent decrease in proliferation and viability of PC-3, DU145,
and LNCaP prostate cancer cells was seen by RE treatment [49,52].
Based on our data, we calculated the RE concentration for half maximal
inhibition (IC
50
) of cell proliferation and comparing these IC
50
values it
appears that the androgen-independent PC-3 cells (19.72
μ
g/mL) are
more sensitive to RE treatment than the androgen-dependent 22RV1
cells (43.41
μ
g/mL). Importantly, treatment of the PNT1A normal
prostate epithelial cells with RE did not signicantly affect their rate of
proliferation (Fig. 9). Similarly to our ndings, Petiwala et al. [51], as
mentioned above, found a signicant inhibition of 22RV1 and LNCaP
prostate cancer cell viability but not an effect on normal prostate
epithelial cells derived from two different patients undergoing radical
prostatectomy [51]. These data indicate that RE is able to discriminate
and preferentially target prostate cancer cells while sparing normal
healthy prostate epithelial cells.
Apart from cell proliferation, treatment with RE resulted in a dose-
dependent inhibition of cell survival with IC
50
values of 2.43
μ
g/mL
and 4.17
μ
g/mL for PC-3 and 22RV1 cells, respectively (Figs. 2,8, and
10). These data indicate a higher sensitivity of the androgen-
independent PC-3 cells than the androgen-dependent 22RV1 cells to
RE treatment and are in agreement with our proliferation data.
The inhibition of prostate cancer cell (PC-3) proliferation and sur-
vival seen with RE treatment was robust and comparable to the inhibi-
tion seen with docetaxel (DTX) and paclitaxel (PTX) both routinely used
in the treatment of prostate cancer [57].
Cleaved PARP is an established indicator of apoptosis [36,38], and
our data showed an increase in cleaved PARP levels in PC-3 cells treated
with RE indicating an effect of RE to induce apoptosis (Fig. 5). Similar to
our ndings, treatment of 22RV1 and LNCaP prostate cancer cell with
RE resulted in a signicant increase in apoptosis [51]. In addition,
treatment of PC-3 cells with the RE polyphenol carnosic acid [63] and
PC-3 and DU145 cells with the RE polyphenol rosmarinic acid [64]
induced apoptosis as indicated by the increased levels of cleaved PARP.
Furthermore, RE treated PC-3 cells showed a signicant inhibition of
cell migration (56.14 ±3.48 % of control, p <0.0001) that was com-
parable to the response seen with docetaxel (70.92 ±2.35 % of control,
p <0.001) (Fig. 7). No other studies examining the anti-migratory or
anti-metastatic effects of RE in prostate cancer cells currently exist. We
previously found a signicant inhibition of MDA-MB-231 breast cancer
cell migration by RE treatment [65]. In a study by P´
erez-S´
anchez et al.,
Fig. 7. Inhibition of PC-3 prostate cancer cell migration by rosemary extract. PC-3 cells were exposed to 1
μ
g/mL of mitomycin-C for one hour, followed by a wound
induction and treatment without or with 50
μ
g/mL rosemary extract (RE) or 10 nM docetaxel (DTX) for 40 hs. Representative images are shown immediately after
wound induction (0 h) and after 40 h of treatment (A). Wound closure was calculated as indicated in the methods and expressed as a percent of control untreated cells
(B). The data are the mean ±SEM of 3 independent experiments. ***p <0.001, ****p <0.0001.
A. Jaglanian et al.
Biomedicine & Pharmacotherapy 131 (2020) 110717
8
treatment with RE inhibited the migration of HGUE-C-1, HT-29, and
SW480 human colon cells [66]. In prostate cancer metastasis, cells
migrate away from the primary tumor to other tissues. Once in other
tissues, their invasiveness depends on their clonogenic survival. Treat-
ment with RE signicantly reduced both, the cell migration, as assessed
by the wound-healing assay, and the clonogenic survival. Our data
indicate that RE has the potential to reduce prostate cancer cell prolif-
eration and tumor growth as well as reduce their migration and invasion
capabilities.
The expression and activation of the serine/threonine kinase Akt is
often elevated in human prostate cancer. Approximately 40 % of early
cases and 70–100 % of advanced cases of prostate cancer have aberra-
tions in the PI3K/Akt signaling [26–29]. This is often due to mutations
on PI3K [4], Akt [67] as well as the loss of the tumor suppressor gene
PTEN [68], all of which lead to overactivation of Akt resulting in
enhanced proliferation and survival. Our study showed a signicant
inhibition of Akt phosphorylation/activation with RE treatment (Fig. 3).
A search of the literature revealed that no other studied have examined
the effects of RE treatment on Akt in prostate cancer cells. It should be
noted that a signicant inhibition of Akt phosphorylation/activation
was seen in PC-3 prostate cancer cells treated with the RE polyphenol
carnosic acid [63] and carnosol [69]. In previous studies by our lab, we
found a signicant inhibition of Akt phosphorylation/ activation by RE
treatment of A549 lung cancer [53] and MDA-MB-231 breast cancer
cells [65].
It has been reported that the levels of total and phosphorylated
mTOR are twice as great in prostate cancer tissue when compared to
normal prostate epithelium [34]. mTOR is a downstream target of Akt
and its activation leads to increased protein synthesis and cell prolifer-
ation [70]. Our study is the rst to show a signicant inhibition of mTOR
in prostate cancer cells with RE treatment (Fig. 4). Similar to our study, a
signicant inhibition of mTOR phosphorylation/activation was seen in
PC-3 prostate cancer cells treated with the RE polyphenol carnosol [69].
Fig. 8. Inhibition of 22RV1 prostate cancer cell proliferation and survival by RE. 22RV1 cells were treated with 5, 10, 25, 50, 75, 100, or 150
μ
g/mL of rosemary
extract (RE) (A,B) for 72 h followed by xing and staining with 0.5 % crystal violet. The stain was solubilized, and absorbance was read at 570 nm. 22RV1 cells were
treated with 2.5, 5, 10, 15, or 20
μ
g/mL of rosemary extract (RE) (C,D) for 7 days followed by xing and staining with 0.05 % methylene blue. Colonies of more than
50 cells were counted. Data are expressed as percent of control, untreated cells. Data are the mean ±SEM of 5 independent experiments. *p <0.05,
**p <0.01, ****p <0.0001.
Fig. 9. Effect of RE on PNT1A normal prostate epithelial cell proliferation.
PNT1A cells were treated with 5, 10, 25, 50, 75, 100, or 150
μ
g/mL of rosemary
extract (RE) for 72 h, followed by xing and staining with 0.5 % crystal violet.
The stain was solubilized, and absorbance was read at 570 nm. Data are
expressed as percent of control, untreated cells. Data are the mean ±SEM of 3
independent experiments.
A. Jaglanian et al.
Biomedicine & Pharmacotherapy 131 (2020) 110717
9
The mechanisms involved in the RE-induced inhibition of Akt and mTOR
(Fig. 10) is not known. It is possible that components in RE act as allo-
steric inhibitors of Akt and/or mTOR or they act on a step upstream of
Akt. Another possibility is that components in RE increase the activity of
Akt and/or mTOR specic phosphatases [71,72] resulting in their
reduced phosphorylation/activation. It is also possible that the inhibi-
tion of mTOR is due to Akt inhibition. Future studies should examine
these possibilities and elucidate the mechanisms involved in these
inhibitory effects of RE.
PC-3 cells, contrary to 22RV1, contain PTEN mutations leading to
enhanced Akt activation [73]. It is possible that the increased activation
of the PI3K/Akt/mTOR cascade in PC-3 cells may explain their higher
sensitivity to RE treatment compared to 22RV1 cells. The notion that RE
targets prostate cancer cells characterized by increased Akt-mTOR
signaling should be explored in future studies to further dene RE’s
potential as a therapeutic agent.
Apart from a reduction in phosphorylated/activated Akt and mTOR
levels, our study shows a reduction in the total levels of these proteins
with RE treatment (Figs. 3 and 4), which may be due to the inhibition of
gene transcription, inhibition of protein synthesis, upregulation of
Fig. 10. Rosemary extract inhibits proliferation and survival of PC-3 androgen-insensitive and 22RV1 androgen-sensitive prostate cancer cells. RE had no effect on
the proliferation of PNT1A healthy prostate epithelial cells (A). Rosemary extract inhibits Akt and mTOR signaling and enhances PARP cleavage in PC-3 prostate
cancer cells (B).
A. Jaglanian et al.
Biomedicine & Pharmacotherapy 131 (2020) 110717
10
protein degradation or an effect on protein stability. Another study
showed that resveratrol, a polyphenol found in grapes, inhibited total
and phosphorylated levels of Akt in both androgen-dependent and in-
dependent prostate cancer cell lines [74]. Previous studies by our group
performed in A549 lung [53] and MDA-MB-231 breast [65] cancer cells
also observed a signicant reduction of both total Akt and mTOR levels
by RE treatment.
Activation of the PI3K/Akt/mTOR pathway in prostate cancer is
associated with disease progression, resistance to androgen deprivation
therapy, and poor prognosis [4,64] and in recent years many efforts
have been focused on the development of inhibitors that target this
pathway [25,75]. ATP-competitive and allosteric inhibitors of Akt have
been developed and used against prostate cancer [25,75,76]. In a phase
II clinical study the combination of the Akt inhibitor ipatasertib, with
the anti-androgen medication abiraterone acetate, in patients with
metastatic castration-resistant prostate cancer improved
progression-free survival [77]. In another study, it was found that the
dual PI3K/Akt and mTOR inhibitor, NVP-BEZ235 (40 mg/kg), when
used as a combined therapy with paclitaxel (4 mg/kg), resulted in a
greater inhibition of tumor growth in castrated mice xenografted with
C4−2AT6 prostate cancer cells when compared to monotherapy treat-
ments [78]. Treatment with NVP-BEZ235 was shown to overcome
docetaxel resistance in human castration resistant prostate cancer [78].
The inhibition of Akt and mTOR by RE treatment is strong and future
studies should explore if RE and its components have effects similar to
the above mentioned Akt and mTOR inhibitors.
A few in vivo animal studies exist examining the effects of RE in
prostate cancer. Treatment of athymic nude mice transplanted with
22RV1 human prostate cancer cells with RE (100 mg/kg/day) resulted
in a 46 % reduction in tumor size compared to the control untreated
mice [51]. Treatment resulted in a signicant reduction in androgen
receptor and prostate specic antigen (PSA) levels [51]. Similarly, mice
xenografted with 22RV1 human prostate cancer cells treated with the RE
polyphenol CA (100 mg/kg/day), showed a 53 % reduction in tumor
growth when compared to the control untreated mice [79]. Addition-
ally, CA decreased androgen receptor levels [79]. Although the studies
examining the effects of rosemary extract and rosemary extract poly-
phenols in vivo are limited, the data/evidence suggests they may be
effective in inhibiting prostate cancer tumor growth and warrants
further investigation.
We recognize that the concentration of bioactive, medicinal in-
gredients in plant extracts, and in this case rosemary extract, may be
inuenced by environmental factors (such as soil quality, water, and sun
exposure) and by the extraction method used. We used high-
performance liquid chromatography (HPLC) and measured the levels
of carnosic acid and rosmarinic acid. Our rosemary extract was found to
contain 2.12 ±0.22 % carnosic acid [80] and 13.39 ±0.23 % rosmarinic
acid [81]. Based on the molecular weight of carnosic acid
(332.42 g/mol) and rosmarinic acid (360.31 g/mol) the cells treated
with 50
μ
g/mL RE would have been exposed to 3
μ
M carnosic acid and
20
μ
M rosmarinic acid. Apart from the aforementioned two, RE contains
many other polyphenols and constituents. The exact bioactive constit-
uents responsible for the anti-cancer properties of RE are not known. We
plan to explore the effects of carnosic acid and rosmarinic acid in future
studies.
5. Conclusions
In the present study we found a signicant inhibition of proliferation
and survival of the PC-3 androgen-insensitive and the 22RV1 androgen-
sensitive prostate cancer cells by RE while the PNT1A normal prostate
epithelial cells were not affected (Fig. 10). Furthermore, treatment with
RE induced apoptosis and reduced migration of PC-3 prostate cancer
cells.
Importantly, RE signicantly reduced the phosphorylation/activa-
tion levels of Akt and mTOR (Fig. 10). Overactivation of the PI3K/Akt/
mTOR pathway in prostate cancer is associated with increased prolif-
eration and survival, resistance to treatment, and overall poor prognosis.
Our data provide strong evidence that treatment with RE targets this
pathway, but it is not known whether this is true in vivo. Future studies
using animal models xenografted with prostate cancer cells should be
performed to investigate this possibility as well as further examine the
anti-cancer properties of RE. Future studies should also examine the
exact polyphenolic constituent(s) of rosemary extract that contribute to
its anti-cancer effects.
Author contributions
E.T conceived and designed the experiments and contributed to data
interpretation and manuscript writing. A.J performed the majority of
the experiments, analyzed the data, prepared the gures and contrib-
uted to manuscript writing. D.T performed the experiments using 22RV1
and PNT1A cells. All authors have read and agreed to the published
version of the manuscript.
Funding
This work was funded by a research grant to E.T from the Prostate
Cancer Fight Foundation, Ontario, Canada.
Declaration of Competing Interest
The authors report no declarations of interest.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.biopha.2020.110717.
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