Polygodial analog induces apoptosis in LNCaP prostate cancer cells

Article abstract of DOI:10.1016/j.ejphar.2018.03.029

Prostate cancer (PCa) is the second leading cause of death in American men. The chemotherapeutic treatment strategies are generally not effective and can lead to side effects. Hence, there is an urgent need to identify novel chemotherapeutic agents. The aim of this study was to synthesize and evaluate the therapeutic effects of a synthetic analog of polygodial (PG), a pungent constituent abundantly present in mountain pepper, water pepper and dorrigo pepper, on LNCaP PCa cell line and its anti-cancer mechanisms in a preclinical study. We evaluated the anti-cancer potential of the PG analog namely DRP-27 using various assays such as cell viability by MTT assay, anchorage independent growth by soft agar assay, reactive oxygen species generation by 2′,7′-dichlorofluorescein probe-based fluorescence assay, and apoptosis by Annexin-V and TUNEL assays respectively. Western blot analysis was performed to identify the molecular mechanism of DRP-27-induced cell death. Our results showed that DRP-27 significantly inhibited LNCaP cell proliferation in a dose-dependent manner at 48 h treatment in vitro. In addition, DRP-27 potently inhibited anchorage-independent growth of these cells. Flow cytometry, Annexin-V and TUNEL assays confirmed that DRP-27 induces apoptosis in LNCaP cells. DRP-27 also induced the activation of intracellular reactive oxygen species. Western blot analysis revealed that DRP-27 downregulated the expression of survivin, while activating Bax and DNA damage marker pH₂AX in LNCaP cells. In conclusion, our study suggests that DRP-27 might be an effective anti-cancer agent for PCa.

Full text of DOI:10.1016/j.ejphar.2018.03.029

Author’s Accepted Manuscript
Polygodial analog induces apoptosis in LNCaP
prostate cancer cells
Subramanyam Dasari, Angela Lincy Prem Antony
Samy, Parnal Narvekar, Venkata Satish Dontaraju,
Ramesh Dasari, Alexander Kornienko, Gnanasekar
Munirathinam
www.elsevier.com/locate/ejphar
PII:
S0014-2999(18)30181-X
https://doi.org/10.1016/j.ejphar.2018.03.029
EJP71733
DOI:
Reference:
To appear in: European Journal of Pharmacology
Received date: 24 January 2018
Revised date: 13 March 2018
Accepted date: 15 March 2018
Cite this article as: Subramanyam Dasari, Angela Lincy Prem Antony Samy,
Parnal Narvekar, Venkata Satish Dontaraju, Ramesh Dasari, Alexander
Kornienko and Gnanasekar Munirathinam, Polygodial analog induces apoptosis
in LNCaP prostate cancer cells, European Journal of Pharmacology,
https://doi.org/10.1016/j.ejphar.2018.03.029
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Polygodial analog induces apoptosis in LNCaP prostate cancer cells
a1
a1
a
Subramanyam Dasari , Angela Lincy Prem Antony Samy , Parnal Narvekar , Venkata
b
c
c
a1*
Satish Dontaraju , Ramesh Dasari , Alexander Kornienko , Gnanasekar Munirathinam
a
Department of Biomedical Sciences, College of Medicine, University of Illinois, Rockford, IL
b
Internal Medicine, Rockford Memorial Hospital, Rockford, IL
c
Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX 78666
*
Corresponding author. Dr. Gnanasekar Munirathinam, Department of Biomedical Sciences,
University of Illinois College of Medicine, 1601 Parkview Avenue, Rockford, IL, 61107. Tel.:
15-395-5773. mgnanas@uic.edu
8
Abstract
Prostate cancer (PCa) is the second leading cause of death in American men. The
chemotherapeutic treatment strategies are generally not effective and can lead to side effects.
Hence, there is an urgent need to identify novel chemotherapeutic agents. The aim of this study
was to synthesize and evaluate the therapeutic effects of a synthetic analog of polygodial (PG), a
pungent constituent abundantly present in mountain pepper, water pepper and dorrigo pepper, on
LNCaP PCa cell line and its anti-cancer mechanisms in a preclinical study. We evaluated the
anti-cancer potential of the PG analog namely DRP-27 using various assays such as cell viability
by MTT assay, anchorage independent growth by soft agar assay, reactive oxygen species
generation by 2′,7′-dichlorofluorescein probe-based fluorescence assay, and apoptosis by
Annexin-V and TUNEL assays respectively. Western blot analysis was performed to identify the
molecular mechanism of DRP-27-induced cell death. Our results showed that DRP-27
significantly inhibited LNCaP cell proliferation in a dose-dependent manner at 48 h treatment in
vitro. In addition, DRP-27 potently inhibited anchorage-independent growth of these cells. Flow
cytometry, Annexin-V and TUNEL assays confirmed that DRP-27 induces apoptosis in LNCaP
cells. DRP-27 also induced the activation of intracellular reactive oxygen species. Western blot
1
Contributed equally to the study
1

analysis revealed that DRP-27 downregulated the expression of survivin, while activating Bax
and DNA damage marker pH AX in LNCaP cells. In conclusion, our study suggests that DRP-27
2
might be an effective anti-cancer agent for PCa.
Keywords: Prostate cancer, Polygodial, Reactive oxygen species, Apoptosis
1
. Introduction
Prostate cancer (PCa) is the fourth most frequent neoplastic disease in general and the second
most common cancer among men in western countries (Yoo et al., 2016). Approximately 1.1
million prostate cancer cases were diagnosed worldwide in 2012, accounting for 15% of all
cancers in men (Ferlay et al., 2015) with the occurrence of 1 in 5 new diagnoses (Siegel et al.,
2
015). There are limited treatment options available for PCa. Patients with PCa is usually treated
with surgery, chemotherapy (docetaxel and cabazitaxel) or secondary hormonal therapeutic
agents (abiraterone or enzalutamide). Chemotherapy and radiation therapy are largely ineffective
and metastatic disease invariably recurs in patients (Diaz and Patterson, 2004). However, the
effects of these treatments are less satisfactory, and there is an urgent need for the development
of effective novel anti-prostate cancer agents (Yoo et al., 2016).
Some of the traditional systems of medicine (Ayurveda and Unani) have used natural
plant products for therapeutic purposes because they have many pharmacological effects with
minimal side effects (Choudhury et al., 2010). Numerous studies have reported that certain
compounds present in spices, such as black pepper, ginger, chilli, turmeric, possess anti-cancer
properties (Brown et al., 2010; Choudhury et al., 2010; Sharma et al., 2005). One such natural
compound is polygodial (PG), a pungent component of water pepper, dorrigo pepper and
mountain pepper. It was originally isolated from the plant water pepper (Polygonum hydropiper)
(
Kubo et al., 2001). It is the most widely occurring sesquiterpene dialdehyde, having been found
in flowering plants, ferns, liverworts, fungi, and marine molluscs from all around the world
Huq et al., 2014). PG is known to possess several pharmacological benefits such as antibacterial
(
and antifungal, anti-allergic and anti-inflammatory properties. Its antifungal and antibacterial
properties come from its ability to function as a non-ionic surfactant (Kubo et al., 2005;
Montenegro et al., 2014). It has also been shown to inhibit mitochondrial ATPase, which can
cause cell death of Saccharomyces cerevisiae (Lunde and Kubo, 2000). Previously, we reported
2

that a derivative of PG (9-epipolygodial, DRP-10) exhibits inhibitory activity against non-small
cell lung cancer, melanoma, and glioma (Dasari et al., 2015b). We also reported that a Wittig
derivative of PG (DRP-3) possesses promising activity against drug-resistant cancer cells. It was
found to be effective against apoptosis-resistant cancer cells due to its cytostatic rather than
cytotoxic effects (Dasari et al., 2015a). Based on the preliminary pharmacological evidence of
anticancer activities of PG and its derivatives, we aimed to explore their anti-cancer effects on
PCa cells. In this study, we designed a series of experiments to determine the effect of PG and its
synthetic derivative DRP-27 on cell proliferation and anchorage-independent growth of androgen
dependent PCa cell line (LNCaP). We also intended to identify the mechanism(s) by which
DRP-27 exerts its anti-cancer effects in PCa cells.
2
2
. Materials and Methods
.1. Chemical synthesis
Compounds DRP-3 and DRP-10 were prepared as previously described (Dasari et al., 2015b,
2
1
015a). Compound DRP-27 (diethyl ((E)-2-((1R,4aS,8aS)-1-formyl-5,5,8a-trimethyl-
,4,4a,5,6,7,8,8a-octahydronaphthalen-2yl)vinyl)phosphonate) was prepared by the following
procedure: To a solution of tetraethyl methylenediphosphonate (36.9 mg, 0.128 mM) in THF (2
ml) was added n-BuLi (1.6 M) (80 μl, 0.128 mM) at -78 °C and the mixture was stirred for 20
min. A solution of polygodial (10 mg, 0.0427 mM) in THF (1 ml) was then added dropwise at -
7
8 °C. The resultant mixture was allowed to warm up to 0 °C and stirred for 2 h. After
completion, the reaction was quenched with saturated NH Cl and then extracted with EtOAc.
4
The organic layer was washed with water and dried over anhydrous Na SO and then
2
4
concentrated to give crude residue. The crude product was purified by preparative TLC using
6
0% EtOAc/hexanes solvent system to obtain DRP-27 in 95% yield (14.9 mg).
2
.2. Characterization data:
1
H NMR (400 MHz, CDCl ) δ 9.57 (d, J = 5.0 Hz, 1H), 9.47 (d, J = 4.8 Hz, 0.5H), 7.23 – 7.00
3
(m, 1.5H), 6.51 – 6.41 (m, 1.5H), 5.57 (t, J = 17.4 Hz, 1H), 5.26 (t, J = 17.1 Hz, 0.5H), 4.12 –
3
0
1
.94 (m, 6H), 2.88 – 2.71 (m, 1.5H), 2.49 (dt, J = 20.2, 5.0 Hz, 1H), 2.31 (dt, J = 19.8, 4.8 Hz,
.5H), 2.27 – 2.13 (m, 1.0H), 1.89 – 1.80 (m, 0.5H), 1.78 – 1.64 (m, 3H), 1.63 – 1.55 (m, 1.5H),
1
3
.54 – 1.45 (m, 3H), 1.35 – 1.25 (m, 9H), 1.23 – 1.14 (m, 3H), 1.02 – 0.87 (m, 13.5H); C NMR
3

(
100 MHz, CDCl ) δ 205.4, 201.7, 150.6, 150.5, 150.0, 149.9, 141.2, 141.2, 141.0, 140.9, 130.9,
3
1
4
2
3
30.7, 129.3, 129.0, 113.13, 113.1, 111.25, 111.23, 62.61, 62.60, 62.15, 62.14, 61.5 (m) 60.4,
8.6, 44.6, 42.1, 41.8, 40.2, 37.7, 37.4, 36.6, 33.2, 33.1, 32.7, 25.37, 25.36, 24.7, 24.7, 22.2, 21.9,
+
1.2, 21.0, 18.4, 18.0, 16.36 (m), 15.5, 14.2; HRMS m/z (ESI) calcd for C H O P (M+H )
20
34
4
69.2195, found 369.2193.
2
.3. Prostate cancer cell lines
LNCaP, PC-3, DU145 and RWPE-1 cells were obtained from ATCC. PCa cell lines (LNCaP,
PC-3 and DU145) were cultured in RPMI-1640 (Lonza) media supplemented with 10% (v/v)
fetal bovine serum (FBS), 1% (v/v) antimycotic and 0.6% (v/v) antibiotic (Sigma Aldrich) at
3
7⁰C and 5% CO . RWPE-1 cells were cultured in KSFM media (Gibco) supplemented with
2
bovine pituitary extract (25g/ml) and EGF (5ng/ml) at 37⁰C in 5% CO environment.
2
Polygodial (PG) was obtained from Sigma. All drugs were dissolved in dimethyl sulfoxide
(DMSO), purchased from Fisher Biotech, to make 10 mM stock. Further dilutions were made in
plain RPMI-1640 media.
2
.4. Cell viability assay by MTT
To study the effects of PG and its derivatives on cell viability, PCa cells (LNCaP, PC-3 and DU-
45) were treated with PG and its derivatives. Cell viability was determined by measuring 3-
4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) colorimetric dye
1
(
reduction (van Meerloo et al., 2011). PCa cells were cultured in 96-well plates at a cell density of
3
1
x10 /well in complete RPMI-1650 (10% FBS, 1% antimycotic and 0.6% antibiotic) and
o
allowed to incubate at 37 C in 5% CO environment. The cells were treated with various
2
concentrations (0, 2.5, 5, 10 and 20 µM) of PG and its derivatives for 48 h. 10 μl of MTT reagent
(5 mg/ml) was added to each well and the plates were incubated for 3 h at 37 °C and the crystals
were dissolved by using a solubilization solution. The absorbance was measured at 570 nm using
Bio-Rad plate reader. The percentage of cell viability was determined relative to the control.
Each treatment dosage was performed in replicates of six and was repeated three times to obtain
consistent results. To evaluate the anti-cancer mechanism of DRP-27, LNCaP cells were treated
in combination of DRP-27 and caspase inhibitor (CI) (Z-VAD-FMK), antioxidant N-acetyl
cysteine (NAC) or autophagy inhibitor 3-methyladenine (3-MA). Similarly, the cytotoxicity of
4

DRP-27 was evaluated on normal prostate cells (RWPE-1). The RWPE-1 cells were seeded,
treated with various concentrations of DRP-27 and evaluated for cell viability as described
above.
2
.5. Anchorage-independent colony formation assay
Colony formation or soft agar assay was performed to evaluate the effects of PG derivative
(DRP-27) on anchorage-independent growth of PCa cells as described previously (Franken et al.,
3
2
006). LNCaP cells (15-20×10 ) were mixed with 0.7% agarose (ISC Bioexpress) solution in
complete RPMI with 20% FBS and poured on top of solidified 1% base agar (Thermofisher) in
-well plates. Various concentration of PG and its derivatives were also mixed with soft agarose
6
and allowed to incubate for 4-5 weeks. Cells were fed with complete RPMI containing 20% FBS
every 4 days for up to 4 weeks. After the incubation period the colonies were stained with
0
.005% crystal violet and examined with a microscope.
2
.6. Measurement of reactive oxygen species
The relative levels of cellular peroxides induced by DRP-27 were determined using reactive
oxygen species fluorescent probe, 2′,7′-dichlorofluorescin diacetate (DCFDA). LNCaP cells
4
(
2×10 cells/500 μl) were cultured in 8-chamber tissue culture slide and treated with various
o
concentrations of DRP-27 (0, 5, 10 and 20 μM), incubated at 37 C, 5% CO for 48 h. Cells were
2
then washed with PBS and incubated with 10 μM DCFDA dye suspended in serum-free medium
for 30 min at 37 °C. The green fluorescence was detected using a microscope (Olympus). The
intensity of fluorescence was quantified using ImageJ software (v.1.4.3.67).
2
.7. Assessment of apoptosis in LNCaP cells by flow cytometry.
Apoptosis in LNCaP cells after treatment with DRP-27 was determined using flow cytometry as
described previously with some minor modifications (Thirugnanam et al., 2008). In brief,
5
LNCaP cells (6x10 ) were treated with DRP-27 for 48 h, detached and attached cells were
collected and washed twice with ice-cold PBS. The collected cells were then re-suspended in 500
5

μl of 1X binding buffer, 5 μl Annexin V-FITC and 5 μl of propidium iodide were added and
incubated for 15 min at room temperature in the dark. The number of apoptotic cells were
quantified by flow cytometer (Becton Dickinson, USA) and data analyzed by CellQuest
software.
2
.8. Detection of Apoptosis by Annexin V-FITC Staining
Apoptotic cell death was detected by Annexin V-FITC kit (BioVision Inc). For this experiment,
4
2
×10 cells/500 μl of complete RPMI-1640 were cultured in 8-chamber tissue culture slide and
treated with various concentrations of DRP-27 (0, 5, 10 and 20 μM). After 48 h, cells were
stained with 5 μl of Annexin-V-FITC and 5 μl of PI, dissolved in 500 μl of binding buffer per
well, and incubated in the dark at room temperature for 10 min. The cells were washed and
analyzed for the apoptotic cell morphology under a fluorescence microscope.
2
.9. Detection of Apoptosis by TUNEL Assay
The TUNEL assay was performed to visualize the apoptotic cell death using an in-situ cell death
detection kit (AAT Bioquest, Inc. CA, USA). The protocol was followed according to the
manufacturer’s guidelines. The LNCaP cells were grown in a 96-well plate, treated with various
o
concentrations of DRP-27 and incubated for 48 h at 37 C and 5% CO . After treatment, cells
2
were washed and fixed (4% paraformaldehyde) for 30 min at room temperature. Samples were
incubated with 50 µl of TUNEL reagent (TdT-mediated digoxigenin-dUTP nick-end labeling)
o
provided in the kit for 1 h at 37 C in a dark chamber. Samples were washed with PBS and
mixed with the Hoechst stain (30 nM) for 5 min. TUNEL fluorescence was visualized with an
Olympus fluorescence microscope (Culture Microscopes, Olympus, Center Valley, PA, USA).
2
.10. Assessment of active caspase-3
The active caspase-3 level was measured using quantitative caspase-3 ELISA kit (R&D Systems,
Inc. USA). Cells were treated with various concentrations of DRP-27 and vehicle alone (control
group) for 48 h. After incubation, cell extracts were prepared according to manufacturer
instructions. Briefly, cells were mixed with the lysis buffer and cell lysates were transferred into
the wells of a microplate, pre-coated with a monoclonal antibody specific for caspase-3.
Following this, substrate solution (streptavidin-HRP) was added to the wells. The enzyme
6

reaction yielded a blue product that turned yellow when a stop solution was added. The optical
density of each well was determined within 30 min, using a microplate reader (Synergy 2 Multi-
Mode Reader, BioTeK, Winooski, Vermont, USA) set to 450 nm with a wavelength correction at
5
40 nm or 570 nm. The active caspase-3 concentrations were calculated from a standard curve
constructed using known concentrations of active caspase-3.
.11. Nuclear morphology detection by Hoechst 33258
2
Hoechst 33258 was employed to label both intact and apoptotic nuclei (Durand and Olive, 1982;
5
Jiang et al., 2014). Cells were seeded in 96-well plates at a density of 1×10 cells/well, followed
by DRP-27 treatment (10 µM). Following treatment, the LNCaP cells were washed in ice-cold
PBS buffer (pH 7.4), fixed with 4% p-formaldehyde and incubated with 1 μg/ml Hoechst 33258
for 3-5 min at room temperature. Condensed and fragmented nuclei were evaluated using
fluorescent probe Hoechst 33258. Visualization was conducted at an excitation and emission
wavelengths of 480 and 520 nm, respectively, by Olympus IMT-2 fluorescence microscopy
(Tokyo, Japan).
2
.12. Detection of phosphoH AX and cleaved caspase-3 by confocal immunofluorescence
2
LNCaP cells were cultured in 8-chamber plates, treated with DRP-27 (10 µM) and incubated at
o
3
5
7 C and 5% CO for 48 h. Cells were fixed (4% formaldehyde) for 15 min and blocked with
2
% goat normal serum (Invitrogen) with 0.3% Triton X- 100 (Sigma–Aldrich) in PBS. Cells
were washed (PBS) and incubated with primary antibodies (1:200) (pH2AX and cleaved
caspase-3) for 1 h. After three successive washings, cells were treated with either 0.1 µg/ml of
anti-mouse IgG or secondary anti-rabbit IgG conjugated with FITC for 1 h. Cells were counter
stained with DAPI (30 nm) for 10-20 min, washed with PBS and a coverslip with Fluorogel
(Electron Microscopy Sciences, Hatfield, PA, USA) was prepared for visual inspection with an
Olympus FluoView confocal microscope. The quantification of the image was done using
ImageJ software.
2
.13. Protein expression profiling using Western blot analysis
DRP-27 treated cells were harvested using cell scrappers and washed with serum free medium.
Total cellular protein was isolated using lysis buffer (BD Pharmingen) (Liu et al., 2014). Protein
concentration was determined using a protein assay kit (Pierce chemicals, USA). Equal amounts
7

of protein with Laemmli sample buffer (Bio-Rad) were loaded in each lane, separated using 12%
SDS-PAGE and transferred to nitro cellulose membranes by semi-dry transfer method. The
membranes were blocked with 5% skim milk in Tris Buffered Saline (TBS) and incubated with
primary antibodies (1:1000). After washing with TBS-Tween-20, the membranes were incubated
with corresponding peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies
followed by detection with enhanced chemiluminescence staining and the bands were observed
on an X-ray film. β-Actin/GAPDH was used to normalize protein loading. The expression level
of protein was quantified by ImageJ software (v1.4.3.67) analysis (Kaur and Bachhawat, 2009).
2
.14. Statistical analysis
Statistical analysis was performed using Student’s t-test. The differences were considered
statistically significant when P<0.05.
3
3
.0 Results
.1. Synthesis of polygodial derivatives
The preparation of compounds used in the current study is shown in Figure 1. DRP-10 was
obtained from PG by acid-catalyzed epimerization at C9 position, which is alpha to the C11-
aldehyde moiety (Fig. 1). This reaction leads to a thermodynamic mixture, from which the
desired compound DRP-10 can be isolated in 40% yield. To obtain DRP-3, PG was reacted with
a stabilized Wittig reagent highly selectively at the C12-aldehyde group (Fig. 1). It appears that
the C11-aldehyde is sterically shielded by the adjacent quaternary center. DRP-3 was produced
in a high 94% yield. Finally, PG was reacted with tetraethyl methylene bisphosphonate to
produce DRP-27 in an excellent 95% yield (Fig. 1). Although the reaction proceeded highly
selectively at the C12-aldehyde, the position C9, alpha to the C11-aldehyde moiety, underwent
epimerization under the highly basic reaction conditions. Unfortunately, our attempts to separate
the epimers were futile. Thus, compound DRP-27 represents a mixture of two epimers at C9.
3
.2. Anti-cancer effects of PG and its derivatives
PG and its derivatives (DRP-3, DRP-10 and DRP-27) inhibit the growth of LNCaP cells. Of the
compounds tested, DRP-27 showed robust inhibition of LNCaP growth with the IC value of 5
5
0
µM (Fig. 2A). We observed that anti-proliferative effects of DRP-27 were significantly
8

abrogated in presence of anti-oxidant N-acetyl cysteine (NAC) and caspase inhibitor (Z-VAD-
FMK) suggesting that the reactive oxygen species and caspase activation are the underlying
mechanisms of DRP-27 effects on LNCaP cells (Fig. 2B). The DRP-27 treatment also induced
marked morphological changes, including cytoplasmic shrinkage and cellular flattening
following the 48 h treatment (Fig. 2C). We also evaluated the cytotoxicity of DRP-27 on PC-3,
DU145 and normal prostate cells (RWPE-1), in which DRP-27 does not exhibit significant
cytotoxicity even at higher concentration (28% of cell viability inhibition at 50 µM) (Fig. 2D).
Of all these cells tested, LNCaP displayed higher sensitivity to DRP-27 compared to DU145 or
PC-3. Therefore, further experiments were performed using LNCaP cells. Results showed that
DRP-27 significantly inhibited the anchorage-independent growth (in vitro tumorigenesis) of
LNCaP cells. Untreated cells grew in large colonies, whereas no colonies were observed in the
DRP-27-treated cells (Fig. 2E). These results suggest that DRP-27 has the potential to inhibit
prostate tumor growth.
3
.3. DRP-27 induces reactive oxygen species generation and apoptotic cell death in LNCaP
cells
When LNCaP cells were treated with DRP-27, the induction of reactive oxygen species could be
observed using immunofluorescent probe (DCFDA). Fig. 3A illustrates a significant induction of
reactive oxygen species production in DRP-27 treated LNCaP cells compared with the untreated
cells. The well-known antioxidant N-acetyl cysteine (NAC), abrogated the effect of DRP-27 by
decreasing the reactive oxygen species production (Fig. 3A) suggesting that oxidative stress as
the potential mechanism of cytotoxicity of DRP-27 in LNCaP cells. A quantitative analysis of
reactive oxygen species production was performed by measuring the DCFDA fluorescence
intensity with ImageJ software (Fig. 3B). Significant differences (*P<0.01) were observed
between control and DRP-27 treatment groups and (#P< 0.01) between DRP-27 alone and NAC
+
DRP-27 treatments. DRP-27 induced internucleosomal DNA fragmentation, which is
associated with chromatin condensation was observed by Hoechst 33258 staining using inverted
fluorescence microscopy. As shown in Fig. 3D, DRP-27 led to condensed and fragmented nuclei
compared with the control, indicating possible apoptosis activation by DRP-27 in LNCaP cells.
To investigate whether apoptosis was triggered by DRP-27 treatment, LNCaP cells were
analyzed by Annexin V FITC/PI flow cytometry. Significant number of apoptotic cells were
9

detected in LNCaP cells treated with DRP-27 (10 and 20 µM) when compared with untreated
cells (Fig. 3E).
3
.4. DRP-27 induces apoptotic cell death via caspase-3 activation
To further investigate the effects of DRP-27 on apoptosis induction, LNCaP cells were treated
with various concentrations of (0, 10 and 20 μM) DRP-27 for 48 h. As shown in Fig. 4A, we
found that DRP-27 induced apoptosis in LNCaP cells as these cells were positive for Annexin V-
FITC (green color) and propidium iodide (red color). DRP-27-induced apoptosis was further
confirmed by terminal nucleotidyl transferase–mediated nick end labeling (TUNEL) assay.
Indeed, we found that DRP-27 induced a dose dependent increase in the apoptosis of LNCaP
cells compared to control cells (Fig. 4A). Quantitative analysis of color intensity produced by
TUNEL stain (red) and Hoechst 33342 stain (blue) was determined by ImageJ software (Fig.
4
B). Caspase-3 is the key executioner of apoptotic process in cancer cells. Therefore, we
quantified the levels of cleaved caspase-3 following DRP-27 treatment in LNCaP cells using
quantitative ELISA method. Figure 4D, shows that at 20µM concentration, DRP-27 induces
significant (P<0.01) increase in active caspase-3 when compared to control cells. In addition to
ELISA, Western blot results (Fig. 4E) showed that procaspase-3 levels were decreased indicating
potential caspase-3 activation. Caspase-3 cleavage in DRP-27 treated LNCaP cells was analyzed
using confocal analysis. Results of confocal immunohistochemistry (Fig. 4F and 4G) showed
that DRP-27 promotes caspase-3 cleavage suggesting that DRP-27 induces apoptosis via
caspase-3 activation.
3
.5. DRP-27 induces DNA damage response by pH AX
2
The DNA damage response was measured by the levels of phosphorylated histone H AX
2
(
pH AX). Immunofluorescence analysis of pH AX labeled with FITC (green color) indicated the
2
2
increased levels of pH AX in DRP-27-treated LNCaP cells compared to untreated LNCaP cells
2
(
Fig. 5A). Increased pH AX (FITC-green color) expression as quantified by Image-J software
2
(Fig. 5B) analysis suggested that DRP-27 induced DNA damage response in LNCaP cells. The
1
0

DNA damage response can be measured by double-stranded breaks (DSBs), which rapidly result
in the phosphorylation of histone H AX (Sharma et al., 2012).
2
3
.6. Protein expression profiling using Western blot analysis
DRP-27-induced apoptosis was confirmed by the cleavage of PARP-1, a substrate for caspase-3
using Western blot. As shown in Fig. 6, the cleavage of PARP-1 in LNCaP cells was detectable
at 10μM DRP-27 by the appearance of 89 KDa cleaved product. As evidence that DRP-27
induces DNA damage, we also examined the activation of pH2AX. Western blot analysis of cell
lysates showed robust induction of pH2AX in LNCaP cells treated with DRP-27. DRP-27 also
down regulated the expression of Survivin, an inhibitor of apoptosis (IAP) family with
concurrent activation of pro-apoptotic protein, Bax. The survivin protein functions to inhibit
caspase activation, thereby leading to negative regulation of apoptosis or programmed cell death
(Mita et al., 2008).
4
.0 Discussion
Previous studies suggest that certain compounds extracted from spices such as ginger, black
pepper, turmeric and chilies possess anti-cancer properties (Brown et al., 2010; Choudhury et
al., 2010; Sharma et al., 2005). One such compound is polygodial (PG), the active compound of
pepper seeds (Dorrigo pepper, mountain pepper and water pepper). Chemically, PG is a
sesquiterpene dialdehyde and is known to possess anti-bacterial, anti-fungal, anti-hyperalgesic
and anti-inflammatory activities (Kubo et al., 2001, 2005). In this study, we evaluated the anti-
cancer activity of a new PG derivative, DRP-27 against androgen sensitive human prostate
cancer cells (LNCaP). Cell viability analysis of PG and its derivatives indicated that PG
derivatives showed more pronounced anti-proliferative activity than the parent PG, with DRP-27
being the most potent. Combinational treatment shows that antioxidant N-acetyl cysteine (NAC),
caspase inhibitor (CI), Z-VAD-FMK significantly abrogated, and autophagy inhibitor 3-MA
partially abrogated the effects of DRP-27, suggesting that reactive oxygen species and caspase
activation are the potential underlying anti-cancer mechanisms of DRP-27 in LNCaP cells. We
also evaluated the cytotoxicity of the DRP-27 on normal prostate cells. Of note, there is no
significant cytotoxic effect of DRP-27 on benign prostate cells (RWPE-1 cells) suggesting its
specific effect on LNCaP cells. Loss of contact inhibition and anchorage-independent growth are
1
1

characteristics of tumor cells (Wang, 2004). Therefore, agents that target these malignant
properties of cancer cells could be potential treatment for cancer. Anti-clonogenic potential of
DRP-27 was studied using soft agar assay. Recent studies have reported that piperine, a major
component of pepper, inhibits colony formation in human colon cancer cells (HT-29) and
osteosarcoma cells (HOS and U OS), in a dose depended manner (Yaffe et al., 2015; Zhang et
2
al., 2015). Similarly, in the present study, DRP-27 treatment reduced the size and number of
colonies in LNCaP cells in a dose dependent manner compared with the control cells. Thus,
DRP-27 inhibits the anchorage independent property of androgen dependent PCa cells (LNCaP
cells), thereby indicating its potential anti-tumorigenic activity. It is well established that reactive
oxygen species are known mediators of intracellular cascade signaling for most of the cellular
death mechanisms. The excessive generation of reactive oxygen species induces damages to the
biomolecules and induces oxidative stress, loss of cell functionality and apoptosis (Slater et al.,
1
995). Since NAC abrogated the effects of DRP-27, we postulate that DRP-27 may increase the
levels of reactive oxygen species, which could be involved in apoptosis. In our study, the levels
of reactive oxygen species were significantly increased in a dose dependent manner in DRP-27
treated cells compared to control cells. Our results indicated that reactive oxygen species
generation was effectively diminished by pretreatment with reactive oxygen species scavengers
(NAC) when cells were treated with DRP-27. Therefore, the intracellular reactive oxygen species
appears to be critical for its anti-cancer mechanisms in DRP-27-induced cell death in LNCaP
cells, because oxidative stress is implicated in many biological processes such as cell cycle
arrest, DNA damage and apoptosis (Rasul et al., 2013).
The chemical nature of polygodial is sesquiterpene, a plant derived compound used for
various health conditions such as thyroiditis, asthma, allergies, arthritis, and autoimmune
diseases (Barrosa et al., 2016). Sesquiterpene lactones (SLs) compounds can induce apoptosis of
cancer cell by decreasing the levels of intracellular reduced glutathione (GSH). Treatment of SL
can affect intracellular redox signaling pathways that are directly involved in the apoptosis
mechanism (Gach et al., 2015). It is well established that SL-induced apoptosis is associated
with increased generation of reactive oxygen species, decreased mitochondrial transmembrane
potential, glutathione (GSH) depletion, cytochrome c release, and activation of caspases (caspase
3
, 9 and 7) (Khan et al., 2012; Wen et al., 2002). Mitochondria is an important component of the
apoptosis execution machinery, which involves pro-apoptotic events including decreased
1
2

mitochondrial membrane potential (Δψm), release of cytochrome C and activation of caspase
cascade (Elmore, 2007). It is becoming increasingly apparent that the mitochondria play a
fundamental role in the processes leading to cell death (Wang and Youle, 2009). Previously,
Lunde and Kubo, (2000) reported that plasma membrane disruption is the mechanism of
polygodial-induced cell death. Specifically, polygodial inhibits the mitochondrial ATPase in a
dose-dependent manner at fungicidal concentration (Lunde and Kubo, 2000). Therefore, we
speculate that DRP-27 could target mitochondria in prostate cancer cells to execute apoptosis,
which needs further evaluation.
Apoptosis is a programmed cell death characterized by specific morphological and
energy-dependent biochemical mechanism (Elmore, 2007). Accumulated evidences suggest that
most of the chemotherapeutic agents halt tumor cell growth and proliferation through induction
of apoptosis (Rasul et al., 2013). In this study, we examined whether DRP-27 inhibited growth of
PCa (LNCaP) cells through the induction of apoptosis. DRP-27-induced apoptosis was
determined by flow cytometry, Annexin-V binding, and TUNEL assays. The results of these
assays suggest that 10 µM concentration of DRP-27 was sufficient to induce potent apoptosis in
LNCaP cells. We also found that pre-treatment with NAC completely blocked the apoptotic
effect of DRP-27 indicating that induction of apoptosis is reactive oxygen species-dependent.
Further, we confirmed the activation of caspase-3 and PARP-1 cleavage in DRP-27 treated
LNCaP cells. Activation of Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in cellular
response to DNA damage (Cepeda et al., 2006). In the present study, DRP-27 promoted the
cleavage of PARP-1, indicating that DRP-27 induces DNA damage response in LNCaP cells.
Phosphorylation of the subtype of histone H A, specifically H AX (γH AX), is another important
2
2
2
cellular response to DNA damage involving either cell cycle arrest or apoptosis (Podhorecka et
al., 2010). We observed that DRP-27 elevated the levels of γH AX in LNCaP, which is
2
confirmed by immunofluorescence of γH AX and western blot analysis. The caspases are a class
2
of proteins associated with apoptotic process via activation of the death receptors and
mitochondrial pathways to accomplish the programmed cell death (Cohen, 1997). Caspases are
present in the form of inactive zymogens, which are activated during process of apoptosis.
Among these, caspase-3 is an important and frequently activated protease, catalyzing the specific
cleavage of many key cellular proteins including PARP-1 (Adams, 2003; Porter and Jänicke,
1
999). The results showed that procaspase-3 was cleaved to yield two fragments (17 and 20
1
3

KDa). We further confirmed caspase-3 activation using ELISA, showing that 5 µM
concentration of DRP-27 is sufficient to activate caspase-3 compared to control. These findings
are consistent with previous studies, showing that sesquiterpene lactones (e.g., 9β-
acetoxycostunolide) induce the apoptosis by the activation of caspase-3 (Ma et al., 2009).
Conclusion
In summary, the analog of PG (DRP-27) may be an effective anti-cancer agent for PCa. Based
on our in vitro studies with LNCaP cells, DRP-27 specifically induces reactive oxygen species-
mediated apoptotic cell death (Fig. 7). As follow up to these in vitro results, further studies are
needed to evaluate the therapeutic potential of DRP-27 against PCa in experimental in vivo
models.
Acknowledgement
Authors would like to thank the Department of Biomedical Science, University of Illinois,
Rockford for supporting this study. This study is also partly supported by funding received from
NIH, United States (R21 CA184646-01A1 and R03 CA212890-01A1) and Brovember Inc.
Rockford.
Competing Interest Statement
Authors declare that they do not have conflict of interest.
Author contributions
SD, ALPAS, PN, and RD performed the experiments. SD, ALPAS, SVD, AK, and GM analyzed
and interpreted the data. SD, ALPAS, SVD, AK and GM wrote the paper.
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Fig. 1. Chemical synthesis of PG analogs: DR-P27, DR-P3 and DR-P10.
Fig. 2. Cell viability assay of PG and its derivatives on prostate cancer cells: (A) Derivatives
of PG, especially DRP-27, inhibit the proliferation of prostate cancer cells (LNCaP) more
effectively than PG as determined by MTT assay. Data shown as mean ± SD. *P<0.05
compared to control group. (B) Anti-proliferative activity of DRP-27 against LNCaP cells
were abrogated by NAC and CI. *P<0.001 compared to control. #P<0.01 compared to DRP-
2
7 treatment group. (C) Morphological changes in LNCaP cells after treatment with DRP-27.
Cells were treated for 48 h with various concentrations of DRP-27. Morphological changes
were observed using microscopy (×20 magnification). (D) Cytotoxicity evaluation of DRP-
2
7 on normal prostate cells (RWPE-1) and androgen-independent prostate cancer cells (PC-3
and DU145). Results showed that DRP-27 has minimal toxic effects on RWPE-1 compared
to PC-3 and DU145 PCa cells. *P<0.05 compared to RWPE-1 cells. DRP-27 treatment
reduces anchorage-independent growth of LNCaP cells: (E) Soft agar assay results show that
DRP-27 significantly inhibited the colony formation ability of LNCaP cells. Colonies were
detected using crystal violet staining. (F) Quantitative analysis of clonogenesis in LNCaP
cells against different concentrations of DRP-27. Data are shown as mean ± S.D.
Fig. 3. DRP-27 promotes oxidative stress in LNCaP cells: (A) The reactive oxygen species
generation was detected by adding 2’,7’ –dichlorofluorescin diacetate (DCFDA) fluorescent
probe and observed by flourescent microscope. The fluorescent green color indicates
generation of reactive oxygen species in DRP-27-treated cells compared to untreated cells.
Effect of DRP-27 was abrogated in the presence of anti-oxidant N-acetyl cysteine (NAC).
(B) Quantitative analysis of reactive oxygen species generation in LNCaP cells. Data are
1
7

shown as mean ± S.D. *P<0.001 compared to untreated group. #P<0.01 compared to cells
treated with 10 µM DRP-27. DRP-27 induces DNA fragmentation in LNCaP cells: Apoptotic
morphological evaluation of LNCaP cells by Hoechst 33258 staining (inverted fluorescence
microscopy, ×20). LNCaP cells were treated with 10 µM of DRP-27 for 48 h. Arrows show
condensed and fragmented nuclei. (C) Control untreated cells do not exhibit condensed and
fragmented in nuclei. (D) Treatment with 10 μM DRP-27 leads to condensed and fragmented
nuclei in LNCaP cells. (E) DRP-27 induces apoptosis in LNCaP cells. The cells were
cultured and treated with various concentrations of DRP-27 for a period of 48 h. Following
incubation, the cells were stained with Annexin FITC (FL1-H) and propidium iodide (FL2-
H) and analyzed using flow cytometry. Results indicate that DRP-27 treatment induces
potent apoptosis in LNCaP cells.
Fig. 4. DRP-27 activates apoptosis in LNCaP cells. (A) LNCaP cells were treated with
various concentrations of DRP-27 (10 and 20µM) for 48 h. Cells were stained with Annexin
V-FITC (Green) to detect apoptotic cells and PI (Red) to stain the nuclei. Apoptotic activity
of DRP-27 was abrogated in the presence of NAC (2.5mM). DRP-27 induces apoptosis in
LNCaP cells. (B) LNCaP cells treated with DRP-27 (5 and 10 µM) for 48 h were stained
with TUNEL red to detect apoptotic cells and Hoechst 33342 to stain the nuclei (Blue). (C)
Quantitative analysis of TUNEL color intensity produced in LNCaP cells following DRP-27
treatment. Data are shown as mean ± S.D. *P<0.05 compared to control group. DRP-27
promotes activation of caspase-3 in LNCaP cells. (D) Quantitative analysis of relative levels
of caspase-3 by ELISA. Data showed that DRP-27 promotes caspase-3 activation in a dose
dependent manner. *P<0.05 compared to control group. (E) Western blot analysis showed
that procaspase-3 expression was decreased in DRP-27 treated cells indicating caspase-3
activation. DRP-27 promotes caspase-3 cleavage in LNCaP cells. (F) The cells were cultured
and treated with different concentrations of DRP- 27 and probed with cleaved caspase-3
antibody. The confocal microscope pictures indicate that there is an increase in cleaved
caspase-3 in DRP-27 treated cells when compared to control. (G) ImageJ analysis showed
that cleaved caspase-3 is increased in DRP-27 treated LNCaP cells. *P<0.05 compared to
control cells.
Fig. 5. DRP-27 treatment increases the levels of pH AX. LNCaP cells were treated with
2
DRP-27 and subjected to immunofluorescence microscopy probed with anti-pH AX (green
2
1
8

fluorescence) and counter stained with DAPI (blue fluorescence). (A) The level of pH AX
2
was increased in DRP-27 treated LNCaP cells compared to untreated controls. (B) Levels of
pH AX were quantitatively measured by using ImageJ software. Data represented as mean ±
2
S.D. *P<0.05 compared to control group.
Fig. 6. DRP-27 targets apoptosis signaling pathways in LNCaP cells. Cells were treated with
various concentration of DRP-27 and harvested at 48 h and equal amount of proteins were
subjected to SDS-PAGE (12%) and analyzed by Western blot. Results show that DRP-27
upregulates pH AX, and Bax proteins while downregulating the expression of survivin.
2
Fig. 7. Schematic representation of anti-cancer mechanism of DRP-27 in prostate cancer.
DRP-27 induces cell death through reactive oxygen species generation leading to DNA
damage and apoptosis.
1
9

O
P
O
P
EtO
OEt
CHO
O
P
OEt OEt
OEt
OEt
n-BuLi, THF
78 °C to 0 °C
5%
H
-
9
DR-P27
CHO
CHO
11
CHO
CHO
O
PTSA, 4A MS
PhMe, rt, 40%
9
1
2
H
H
DR-P10
PG
CHO
OMe
Ph P=CHCO Me
3
2
PhMe, rt, 94%
H
DR-P3
Fig. 1.
(A)
PG
1
00
DRP-3
DRP-10
DRP-27
80
60
40
20
0
*
*
*
*
*
*
*
*
Control
2.5µM
5µM
10µM
20µM
50µM
Treatment concentrations of PG and its derivatives
Fig. 2. (A)
2
0

(B)
100
#
#
80
60
40
20
0
*
Control DRP-27 10
µM
NAC
2.5mM
DRP-27
10µM +
NAC
CI 25µM
DRP-27 3MA 2.5mM DRP-27µM
10µM + CI
25µM
+ 3MA
2.5mM
2.5mM
Control
DRP-27 5µM
DRP-27 10µM
DRP-27 20µM
DRP-27 50µM
(C)
Fig. 2. (B) and (C)
(
D)
RWPE-1
PC-3
1
00
8
6
4
2
0
0
0
0
0
DU145
*
*
*
*
*
Control
5
10
DRP-27 (μM)
20
50
Fig. 2. (D)
2
1

Control
DRP-27 5µM
DRP-27 10µM
DRP-27 20µM
DRP-27 50µM
(E)
4
(F)
3
2
1
0
Control
5 µM
10 µM
20 µM
50 µM
Treatment concentrations of DRP-27
Fig. 2. (E) and (F).
DRP-27 10µM +
NAC 2.5mM
Control
DRP-27 5µM
DRP-27 10µM
DRP-27 20µM
DRP-27 50µM
NAC 2.5mM
(A)
50
40
30
20
10
0
(B)
*
*
*
*
#
Control
DRP-27
DRP-27
10µM
DRP-27
20µM
DRP-27 NAC 2.5mMNAC 2.5mM
50µM + DRP-27
0µM
5µM
1
Fig. 3. (A) and (B).
2
2

Control
DRP-27 10µM
Fig. 3. (C) and (D)
Control
DRP-27 10μM
DRP-27 20μM
(E)
4.06%
0.39%
4.51%
4.45%
0.69%
5.53%
95.07%
0.48%
53.33%
37.71%
44.89%
48.89%
Annexin V FITC
Fig. 3 E.
2
3

Annexin-V FITC
PI
Merged
A
Control
DRP-27 10µM
DRP-27 20µM
NAC 2.5 mM
+
DRP-27 10µM
Fig. 4 A.
TUNEL Red
(B)
(C)
Control
1
2
8
4
0
TUNEL Red
*
DRP-27 5µM
Control
DRP-27 5µM
DRP-27 10µM
Fig. 4. (B) and (C)
DRP-27 10µM
2
4

6
5
4
3
2
1
0
*
(D)
(E)
DRP-27 (µM)
Control
5
10
20
*
1
0.67
0.31
0.50
procaspase-3
3
5 KDa
Actin
4
5 KDa
Control
DRP-27 5µM DRP-27 10µM DRP-27 20µM DRP-27 50 µM
Fig. 4. (D) and (E).
Control
DRP-27 5µM
DRP-27 10µM
DRP-27 20µM
(F)
(G)
2
1
0
.5
2
LNCaP DRP -27
*
*
.5
1
.5
0
Control
5μM
10μM
20μM
Concentrations of DRP 27
Fig. 4 (F) and (G).
2
5

(
A)
(B)
60
40
20
0
pH2AX
Merged
FITC-pH AX
DAPI
Merged
2
Control
*
Control
DRP-27 10µM
DRP-27 20µM
DRP-27
10µM
DRP-27
0µM
2
Fig. 5. (A) and (B)
DRP-27 (µM)
Control
5
10
20
1
1.36
1.85
2.10
1
5 KDa
pH AX
2
1
0.51
0.15
0.01
PARP-1
116 KDa
1
9.61
9.79
1.02
11.54
Bax
2
0 KDa
1
0.82
0.43
Survivin
Actin
1
6 KDa
4
5 KDa
Fig. 6.
2
6

DRP-27
pH
2
AX
Bax
Mitochondria
Survivin
Cleaved
Caspase-3
Cleaved
PARP-1
Apoptosis
Fig. 7.
2
7