Resveratrol-ZnO nanohybrid enhanced anti-cancerous effect in ovarian cancer cells through ROS

Article abstract of DOI:10.1039/c6ra16664d

The use of nanotechnology in medicine and more specifically in drug delivery is expected to spread rapidly. Currently many substances are under investigation for drug delivery and more specifically for cancer therapy. Nano-conjugation of the drug is likely to provide protection against degradation, increasing bioavailability, and improvement in intracellular penetration, enhanced efficacy and control delivery of the drug. In this study, ZnO nanoparticles (NPs) conjugated with a well-known anti-proliferative and chemopreventive trans-resveratrol (RSV) has been designed, characterized and found to be a potential drug for ovarian cancer treatment. Nano-conjugate (RSV-ZnO) has been characterized by FTIR, Raman scattering and Transmission electron microscopy (TEM). Picosecond-resolved fluorescence studies of RSV-ZnO nano-conjugate reveal efficient electron migration from ZnO NPs to RSV, eventually enhancing the ROS activity compared to free RSV. Various in vivo and in vitro studies including MTT assay and apoptosis studies on ovarian cancer (PA1) cell lines reveals the RSV-ZnO nano-conjugate to be more effective in cancer cell death in comparison to free RSV. DCFH assay (in vitro) and DCFDA method (in vivo in PA1 cell lines) demonstrate the huge enhancement of antioxidant property (through ROS) in case of nano-conjugate. JC-1 staining method unravels the increase in depolarization of the mitochondrial membrane in the PA1 cell upon nano-conjugate, consistent with mitochondrial dysfunction. Finally we have performed a Western blot study by expression of some proteins like actin, Bax, Bcl-2 and Caspase-9 to confirm apoptosis.

Full text of DOI:10.1039/c6ra16664d

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DOI: 10.1039/C6RA16664D
Resveratrol-ZnO Nanohybrid Enhanced Anti-cancerous Effect in
Ovarian Cancer Cells through ROS
Mahamuda Khatuna, Susobhan Choudhuryb, Bo Liuc, Peter Lemmensc,d, Samir Kumar Palb,*
and Santasree Mazumdera,*
aDepartment of Biochemistry, Ballygunge Science College,
University of Calcutta, Kolkata 700 019, India
bDepartment of Chemical, Biological & Macromolecular Sciences,
S. N. Bose National Centre for Basic Sciences,
Block JD, Sector III, Salt Lake,
Kolkata 700106, India
&
cInstitute for Condensed Matter Physics and dLaboratory for Emerging Nanometrology,
TU-Braunschweig Mendelssohnstrasse 3,
38106 Braunschweig, Germany
*To whom correspondence should be addressed:
E-mail:skpal@bose.res.in and smbioc@gmail.com
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DOI: 10.1039/C6RA16664D
Abstract
The use of nanotechnology in medicine and more specifically in drug delivery is expected to
spread rapidly. Currently many substances are under investigation for drug delivery and more
specifically for cancer therapy. Nano-conjugation of the drug is likely to provide protection
against degradation, increasing bioavailability, improvement in intracellular penetration,
enhanced efficacy and control delivery of the drug. In this study, ZnO nanoparticles (NPs)
conjugated with a well-known anti-proliferative and chemopreventive trans-Resveratrol
(RSV) has been designed, characterized and found to be a potential drug for ovarian cancer
treatment. Nano-conjugate (RSV-ZnO) has been characterized by FTIR, Raman scattering
and Transmission electron microscopy (TEM). Picosecond-resolved fluorescence studies of
RSV-ZnO nano-conjugate reveal efficient electron migration from ZnO NPs to RSV,
eventually enhancing the ROS activity compared to free RSV. Various in vivo and in vitro
studies including MTT assay and apoptosis studies on ovarian cancer (PA1) cell lines reveals
the RSV-ZnO nano-conjugate to be more effective in cancer cell death in comparison to free
RSV. DCFH assay (in vitro) and DCFDA method (in vivo in PA1 cell lines) demonstrate the
huge enhancement of antioxidant property (through ROS) in case of nano-conjugate. JC1
staining method unravels the increase in depolarization of the mitochondrial membrane in the
PA1 cell upon nano-conjugate, consistent with mitochondrial disfunction. Finally we have
performed Western blot study by expression of some proteins like actin, Bax, Bcl2 and
Caspase9 to confirm apoptosis.
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1. Introduction:
DOI: 10.1039/C6RA16664D
Ovarian cancer is the most common cause of cancer mortality in women and one of the
leading reason of death from gynaecological malignancies1-2. In case of maximum patients,
the early stage of malignancy is rarely detected and symptoms are evident after spread out
over the surface of the peritoneal cavity. While patients with early-stage of cancer have
survival rate of 90%, advanced-stage patients have a survival rate of 15% to 30% only3.
Conventional approaches like chemotherapy4 and cytoreductive surgery have limited success
of controlling ovarian cancer due to chemotherapy resistance and high recurrence rate5.
Resveratrol (3,5,4'-trihydroxy-trans-stilbene), the natural phytoalexin present in grapes,
berries, peanuts and red wine, is well known for its antioxidant,6antiaging7, antivira
cardiovascular and neuro protective effectsl8. Several recent studies demonstrated
antiproliferative, anticancer and chemopreventive efficacy of Resveratrol (RSV) against of a
wide variety of tumors including breast9, prostate10, lymphoid11, lung12, pancreas13, colon14
cervical15 and ovarian16 cancer. RSV is believed to directly modulate various molecular
signal transduction pathways that are known to inhibit cancer cell proliferation and induce
cancer cell death17.In some lineages of cancer cell culture, RSV has been shown to induce
apoptosis, revealing RSV is responsible for cancer cell death through antioxidant and anti-
angiogenic properties.
Regardless of the promising results reported in the contemporary literature, the massive use
of RSV has met limited success due to its instability, poor solubility in water, inefficient
systemic delivery, and low bioavailability18-20. To overcome these physicochemical and
pharmacokinetic limitations, the conjugation of RSV with nanoparticles (NPs) is a powerful
strategy21. Zinc oxide (ZnO) NPs is receiving much attention for their good effectiveness in
cancer treatment22 and also for potential targeted drug delivery. It has also been reported that
ZnO NPs selectively induce apoptosis in human cancer cells through reactive oxygen species
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23
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DOI: 10.1039/C6RA16664D
(ROS) . ZnO NPs are being used in the food industry as additives and in packaging due to
their antimicrobial properties24-25. They are also being explored for their potential use as
anticancer drugs and biomedical imaging applications26-27. In mammalian cells, the toxic
effects of ZnO NPs such as membrane injury, inflammatory response, DNA damage and
apoptosis have been demonstrated28–30. In some studies, the toxicity of ZnO NPs has been
ascribed to the release of Zn2+ ions31-32. However, studies have also shown that the toxicity of
ZnO NPs is due to their particulate nature which may give rise to reactive oxygen species29,33
.
ROS generation is linked to DNA damage and cellular apoptosis34-35.By using the advantage
of particulate nature of ZnO Nps we design our research work by forming conjugation of
ZnO and Resveratrol. The treatments available are limited and most of them have toxic/
adverse effect. When the cancer is metastasized, the treatment options are limited, and
chemotherapy remains the only choice of treatment. So it raises a large scope of research on
natural and nontoxic anticancer agents. To convey higher therapeutic payload to the cells,
compared to the RSV molecules, the nanoconjugate of ZnO-RSV is synthesized.
To continue our research focused on the use of natural phenol compounds on ovarian
cancer, in this study we have investigated the ZnO-RSV nano-conjugate as a potential drug
for ovarian cancer treatment on PA1 cell lines. To explore and modulate the effectiveness of
NPs, RSV-ZnO nano-conjugates have been prepared by conjugation of ZnO Nps of ~5 nm
particle size and RSV drug molecules. Small size of the ZnO-RSV is very effective in
interacting with cells. The nano-conjugates have been characterized by different optical
techniques. The sizes of the NPs and crystallinity were confirmed using high-resolution
transmission electron microscopy (HRTEM). FTIR and Raman scattering studies were used
to understand the conjugation at the molecular level. Steady-state fluorescence and
picosecond resolved fluorescence studies unravel the efficient electron transfer from ZnO to
RSV, which probably increase the ROS activity in the nano-conjugate. From our studies it is
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clearly visible that upon conjugation with ZnO NPs the ROS activity of RSDVOI:i1n0c.10re39a/sCe6RsA16664D
drastically compared to free RSV both in vitro and in vivo. Effective cancer cell death in PA1
cell lines by nano-conjugate compared to free RSV was observed through MTT and flow
cytometry (FACS) assays. Western blot assay was performed by expressing Bax Bcl-2,
Caspase9 proteins in PA1 cells in order to confirm the apoptotic pathway of cell death.
Depolarization of the mitochondrial membrane in the PA1 cell was also observed using JC1
staining method. Overall, our present study is an attempt to explore the enhanced anti-cancer
activity of the promising nano-conjugate through ROS generation.
2. Materials and Methods:
2.1. Materials:
The PA1 cell line was purchased from National Centre for Cell Science (Pune, India).
Resveratrol was from Sigma Aldrich (Saint Louis, MO) and pure Zinc acetate dehydrate
(Zn[CH3COO]2H2O,99%-100%) was from Merck Chemicals (Darmstadt, Germany). Fetal
bovine serum (FBS), Antibiotic (penicillin-streptomycin), EMEM (Eagle’s Minimum
Essential Medium) medium and Dulbecco phosphate buffered saline (PBS) were purchased
from Invitrogen Co, (Carlsbad, CA). MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-
diphenyltetrazoliumbromide), 2,7- dichlorofluorescin diacetate (DCFH-DA), Annexin
V/FITC apoptosis detection kit were from BD Bioscience. JC1fluorescent probe 5,50,6,60-
tetrachloro-1,10,3,30-tetraethylbenzimidazolcarbocyanine iodide, Hoechst and PI stain were
from Sigma Aldrich (St Louis, MO). Anti-caspase, anti-bax, anti-bcl-2, and anti-β-actin
antibodies were obtained from Sigma-Aldrich (St Louis, MO). Secondary antibodies were
bought from Santa Cruz Biotechnology, Inc, (Santa Cruz, CA). All other chemicals used
were of the highest purity available from commercial sources.
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DOI: 10.1039/C6RA16664D
2.2. Synthesis of ZnO NPs and RSV-ZnO nano-conjugate:
ZnO Nps were prepared by the co-precipitation technique using highly pure Zinc acetate
dehydrate (Zn[CH3COO]2H2O, 99% pure) as a precursor with sodium hydroxide solution
following the procedures reported earlie36. With this ZnO solution required amount of RSV
was added into 20 ml of ZnO Nps solution and kept it in a shaker for overnight to formation
of nano-conjugate.
2.3. Characterization Method:
TEM grids were prepared by applying a diluted drop of the ZnO samples to carbon-coated
copper grids. The particle sizes were determined from micrographs recorded at a
magnification of 100000x using an FEI (Technais-Twin, operating at 200kV) instrument. For
optical experiments, the steady state absorption and emission were determined using a
Shimadzu UV-2450 spectrophotometer and a Jobin Yvon Fluorolog, respectively.
Picosecond-resolved spectroscopic studies were done using a commercial time correlated
single photon counting (TCSPC) setup with MCP-PMT from Edinburgh Instruments
(IRF=80 ps) using a 375 nm excitation laser source. Details of the time resolved fluorescence
setup have been depicted in our previous reports37-39. For FTIR study, a JASCO FTIR-6300
instrument of 0.5 cm-1 resolution was used. Raman scattering experiments were carried out
by a Micro Raman setup (Horiba LabRAM) with the excitation wavelength of 532 nm at
room temperature.
2.4. Preparation of dichlorofluorescein and ROS measurements:
DCFH was prepared from DCFH-DA by mixing 0.5 ml of 1.0 mM DCFH-DA in methanol
with 2.0 ml of 0.01 N NaOH. This de-esterification of DCFH-DA proceeded at room
temperature for 30 min, and the mixture was then neutralized with 10 ml of 25 mM
NaH2PO4, at pH 7.4. This solution was kept on ice in the dark until use. All the
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measurements were performed in a total volume of 2 ml water that contained 10 DμOlI:o1f0.1D03C9/FC6HRA16664D
solution, RSV, ZnO and RSV-ZnO.
2.5. Bacterial strain and culture condition:
The viable count assay was performed with E. Coli XL1-blue cells. The cells were cultured at
0
37 C in a liquid Luria-Bertani (LB) medium. When the optical density reached 0.6, the
inoculums was serially diluted ten thousand times with a Luria-Bertani (LB) medium and
plated in different plates containing RSV, ZnO NPs and RSV-ZnO. In order to study the
0
effect of drugs, the plates were then incubated at 37 C for 24 hours. Finally the colonies
were counted after overnight incubation.
2.6. Cell Culture:
The Human ovarian Carcinoma cell lines (PA1) were cultured in EMEM medium
supplemented with 10 % fetal bovine serum (FBS) and 1% antibiotic at 37 0C in a humidified
atmosphere of 95 % air and 5 % CO2.
2.8. Cytotoxicity assays
i)
Cell viability assay:
The cell viability assay was assessed using the MTT assay following the method of
Mossmann40.The cells were incubated for 24 hrs with different concentration of RSV, ZnO
NPs and RSV-ZnO and
3
hours with MTT[3-(4,5-dimethylthiozoyl-2-yl)-2,5-
diphenyltetrazolium bromide] dye(5mg/ml in PBS). The medium from each well was
discarded and the resulting formazon crystals were solubilised by adding 150 μL of dimethyl
sulphoxide and quantified by measuring absorbance at 530 nm in an Elisa Reader.
Cytotoxicity has been stated as the concentration of corresponding substance as inhibitory
cell growth factor by 50 % (IC50), and all tests and analyses were performed in triplicate.
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The IC50 value of ZnO NPs, RSV and RSV-ZnO nano-conjugate was calculated from MTT
DOI: 10.1039/C6RA16664D
assay.
ii)
Determination of Cell cycle by flow cytometry:
Cell viability under CC50 value of RSV, ZnO NPs, and RSV-ZnO were analyzed on flow
cytometer (FACS Calibur, Beckton Dickinson, SanJose). Cell cycle phase distribution of
nuclear DNA was determined by PI fluorescence (excited with 488 nm argon laser light and
detected the fluorescence at 623 nm) and analysed through CellQuest software (Becton-
Dickinson, san Jose).Ten thousand total events were acquired and histogram display of DNA
content (x-axis, PI fluorescence) vs counts (y-axis) has been displayed.
2.9. Oxidative stress parameters
i)
Measurement of intracellular reactive oxygen species(ROS):
The level of intracellular ROS generation was estimated by using 2,7-dichlorofluorescein
diacetate (DCFDA) dye40. Cells were seeded in a 6-well black bottom plate (104 cells /well).
After 24 hours, the cells were exposed to ZnO NPs, RSV, and RSV-ZnO nano-conjugate for
up to 6 hours. Following exposure, the cells were washed twice with PBS and incubated with
DCFDA dye (20 μM) for 30 minutes at 37 0C. The reaction mixture was then use to measure
the fluorescence intensity by FACS. Intracellular ROS were measured by flow cytometric
analysis as we have previously reported41.
ii)
Measurement of mitochondrial membrane depolarization:
While the transition pore was partially functional, the functional dysfunction of the
mitochondria was detected by measurement of membrane depolarization. To determine
whether there was membrane dysfunction, JC-1 staining was performed by flow cytometry
analysis. Mitochondrial membrane depolarization was detected with the fluorescent probe
5,5/,6,6/-tetrachloro-1,1/,3,3/-tetraethylbenzimidazolcarbocyanine
iodide
(JC-1).
Mitochondrial energization was determined by retention of JC-1dye42. Cells exposed to ZnO
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NPs, RSV and RSV-ZnO with their IC50 value were harvested by trypsinizationDaOnI:d10w.10a3Vs9ieh/wCe6AdRrtAic1le6O66nl4inDe
with PBS twice. The cells were incubated with 10 μM JC-1 for 15 minutes at 37 0C, washed
with PBS and re-suspended in PBS at a concentration of 106cells/ml. The fluorescence
intensity was measured at an emission wavelength of 590 nm upon excitation wavelength at
485 nm using FACS.
2.10. Apoptosis
i)
Annexin V-FITC/PI staining
Annexin V-FITC/PI staining was carried out by AnnexinV-FITC apoptosis detection kit as
per the manufacturer’s protocol. Briefly, the cells were harvested by trypsinization, washed
with PBS and re-suspended in binding buffer at a concentration of 106 cells/ml. Annexin V-
FITC (5 μ) and propidium iodide (10 μL) was added to 500 μL of cell suspension and
incubated for 10 minutes in the dark at room temperature. The cells were immediately
analyzed by FACS. Fluorescence emitted by Annexin V bound FITC and DNA-bound
propidium iodide in each event was detected. Results were analyzed by FACSDiva 6.1.2
software.
ii)
Hoechst staining:
Apoptotic nuclear morphology was also assessed using Hoechst 33342. Cells were fixed with
4% para-formaldehyde at room temperature for 30 minutes, and were washed with PBS and
then stained with 10μg/ml of Hoechst33342 solution at 37 0C for 10 minutes. The cells were
washed and the morphology was observed by fluorescence microscope.
2.11. Western Blot:
Human ovarian cancer PA1 cells were cultured in six-well plates and exposed to ZnO NPs,
RSV and RSV-ZnO nano-conjugate at the concentration of CC50 value for 24 hours. The
harvested cell pellets were lysed in lysis buffer (50 mM Tris HCL) in the presence of a
protease inhibitor. The total protein concentration was measured by the Bradford method45
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bovine serum albumin as the standard. A 40 μg/lane protein were separated byD1O0I: 1%0.10S39D/CS6R-A16664D
polyacrylamide gel electrophoresis and transferred onto a nitrocellulose blotting membrane
(Bio-rad) for overnight. The membrane was blocked with 5% non fat milk for 1.5 hours at
room temp and probed with anti-mouse primary antibody (1:1000) against beta-actin, BCl2,
Bax, Caspase-9 for 1 hour at room temperature followed by overnight incubation at 40C. The
membrane was then incubated for 1.5 hours at room temperature with secondary antibodies
conjugated to horseradish peroxidise at 1:5000 dilution. The protein bands were visualised on
X-ray film using an enhanced chemiluminescence system.
2.12. Statistical analysis
All quantitative data are expressed as mean ± SD unless otherwise stated. One-way analysis
of variance followed by Tukey’s multiple comparison test was executed for comparison of
different parameters between the groups using a computer program GraphPad Prism (version
5.00 for Windows), GraphPad Software (CA, USA). p < 0.05 was considered significant43.
3. Result and Discussion:
3.1. Structural Characterization of RSV-ZnO nano-conjugate: Figure 1a depicts a
characteristic HRTEM image of the ZnO NPs. From the lattice fringe of a single ZnO NPs,
inter planar distance is found to be approximately 0.31 nm, which corresponds to the spacing
between two (100) planes. The HRTEM image clearly reflects the crystalline nature of ZnO
NPs44. The average particle size was determined from our experimental HRTEM data and
found to be approximately 4.89+0.5 nm as revealing from inset of Figure 1a. The absorption
spectra of ZnO NPs, RSV and RSV-ZnO nano-conjugate are shown in Figure 1b. The major
absorption peak of RSV appears at 327 nm with a shoulder at 311 nm. No distinct peak of
RSV is observed in absorption spectra of nano-conjugate due to the very low concentration of
RSV and large scattering of the ZnO NPs. The drug used in this study, RSV, contains three
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hydroxyl groups, which possesses binding ability to the surface of ZnO45..The FoVuierwieArrticle Online
DOI: 10.1039/C6RA16664D
transform infra-red (FTIR) technique is used to investigate the binding mode of the hydroxyl
group of RSV on the ZnO surface. For the pure drug the stretching frequency of hydroxyl
group is located at 3498 cm-1 as reported earlier. The peak observed at 3430 cm-1 is due to O-
H stretching vibration of ZnO46. The perturbation of peak at 3498 cm-1 of RSV upon attached
to ZnO as shown in figure 2a, is revealing that RSV is attached to ZnO NPs through O-H
functional group. To further investigate the binding between the drug and delivery vehicle,
Raman spectra were collected from RSV, ZnO NPs and RSV–ZnO nano-conjugate as shown
in Figure. 2b. The Raman spectrum of RSV does not show any peak in the wavenumber
range of 300–600 cm-1. However, four vibration peaks at 328, 378, 438, and 577 cm-1 are
observed in the Raman spectrum of ZnO NPs as reported earlier47-48. The strong peak at 438
cm-1 is corresponds to the nonpolar optical phonon, E2 mode of the ZnO NPs at high
frequency, which is associated with oxygen deficiency. After binding to RSV, the peak at 438
cm-1 of ZnO is significantly perturbed. This is indicative of the passivation of ZnO surface
states upon RSV assembly. The presence of other characteristic bands indicates good
retention of ZnO crystal structure and shape during the sensitization of ZnO NPs with RSV.
3.2. Steady-state and Picosecond-Resolved Fluorescence Study: As shown in Figure. 3a
the room temperature steady-state emission of ZnO NPs is very broad around 530 nm upon
excitation above the band-edge (ex=330 nm) due to presence of defect states near the surface
of ZnO. The broad emission is composed of two emission band 500 nm (P1) and 550 nm

(P2)49. While the first one corresponds to singly charged vacancy center V , the other one is
arising due to doubly charged vacancy center V. The emission intensity of ZnO NPs in the
RSV–ZnO nano-conjugate decreases considerably than that of free ZnO NPs which can be
attributed to the efficient electron transfer processes from ZnO NPs to RSV. To investigate
the mechanism we have extended our study with picosecond resolved fluorescence
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spectroscopy. The fluorescence decay profile of ZnO NPs in the presence andDOabI: s10e.1n0c39e/Co6RfA16664D
RSV was obtained upon excitation of a 375 nm laser and monitored at 500 nm (P1) and 550
nm (P2) (Figure 3b and c, respectively). The excited state lifetime of the ZnO NPs quenches
in the RSV–ZnO nanohybrid compared to that of free ZnO NPs. The details of the
spectroscopic parameters and the fitting parameters of the fluorescence decays are tabulated
in Table 1. The faster component in RSV-ZnO nano-conjugates is compared with the earlier
study of ZnO-benzoquinone (BQ), efficiently accepts excited electrons from the surface of
semiconductor quantum dots50. Similar time scales reveal the excited state electron transfer
from ZnO NPs to RSV.
3.3. In vitro ROS activity: After investigation of the interfacial dynamics and molecular
proximity, we have extended our studies to the anti-microbial activity of RSV-ZnO
composite. RSV exhibits anticancer effect on various human cancer cells through ROS.
However, due to low solubility of RSV in water the anticancer efficacy decreases. In this
juncture we have used RSV-ZnO nano-conjugates as a potential antibacterial agent for the
inhibition of growth of Escherichia coli (E. coli). The upper panel of Figure 4a shows picture
of E. coli cultures treated with DMSO, ZnO NPs, RSV and RSV-ZnO nano-conjugate. The
inhibition in growth of the bacterial culture for RSV-ZnO nano-conjugates is clearly visible.
The culture treated with RSV-ZnO nano-conjugate contains a smaller number of colonies
with respect to the control samples and cultures containing ZnO Nps and RSV. The decrease
of colony forming unit (CFU) is 65% in case of RSV-ZnO nano-conjugate as shown in
Figure 4a. In order to explain the detailed mechanistic view of both antioxidant and
antimicrobial action of RSV-ZnO nano-conjugate, the ROS generation was investigated
directly by dichlorofluorescin–dichlorofluorescein (DCFH–DCF) conversion in aqueous
medium. DCFH is a widely known marker that is used in ROS detection assays51-52. The ROS
generated in the medium, oxidized non-fluorescent DCFH into fluorescent DCF. The
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fluorescence emission intensity of DCF was monitored with respect to time. As RSV itself
DOI: 10.1039/C6RA16664D
generates some ROS, there is some enhancement in fluorescence intensity of DCFH as shown
in the figure 4b. However, maximum enhancement of fluorescence intensity in case of RSV–
ZnO nano-conjugate compared to free RSV as shown in Figure 4b. A control experiment was
performed where ZnO NPs and DCFH only show negligible ROS generation.
3.4. In vitro cytotoxicity: The strategy of investigating cytotoxicity in different cell lines
using MTT is well documented in the literature53-54. We have performed MTT assay on the
ovarian cancer cell line (PA1) to investigate the cytotoxicity of RSV-ZnO nano-conjugates
with respect to its precursors, RSV and ZnO NPs. IC50 values for RSV, ZnO are 150 M for
both in PA1 cell line. However, for RSV-ZnO nano-conjugate the IC50 value is 4 M and
100 M for individual concentration of RSV and ZnO in nano conjugate, respectively.
Clearly, the assay revealed a much high cytotoxic potential of the nano-conjugate compare to
that of RSV only on PA1 cell line as shown in Figure 5. In Figure 6 we have also represented
the morphological change of cells after treatment by phase contrast microscopy. Additionally
our findings also estimate that conjugate creates potent toxicity in PA1 cells more efficiently
than that of RSV. In order to compare the potent toxicity effect of RSV-ZnO compared to
RSV on PA1 cells, we performed the flow cytometric cell cycle phase distribution analysis.
As shown in Figure 7, our results showed significant higher cell death of PA1 cell line with
the treatment of RSV-ZnO at even lower dose in compare to that of RSV. Conjugate generate
41.74% cell death compare to 30.98% with RSV treatment and ZnO NPs separately generate
13.34% of cell death at same concentration.
3.5. Induction of apoptosis: To confirm the findings obtained from MTT assay we
performed phase contrast microscopic imaging of PA1 cells on treatment with RSV, ZnO
NPs and RSV-ZnO nano-conjugate as shown in Figure 6. Our observation showed that nano-
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conjugate produced similar percentage (50%) of cell death at a lower concentration (4 μM
DOI: 10.1039/C6RA16664D
RSV individual concentration in the nano-conjugate) compare to RSV and highlighting that
the efficacy of conjugate to induce death in PA1 cells was superior to that of RSV.
To further assess whether the cell death was due to apoptosis or not we examined the
percentage of Annexin-V positive cells by flow cytometry. Flow cytometry analysis was
conducted using AnnexinV-FITC/PI staining to investigate the mode of cell death induced by
RSV, ZnO NPs and RSV-ZnO. PA1 cells were treated with IC50 value of RSV, ZnO and
RSV-ZnO for 24 hrs. The cells treated with RSV, ZnO and RSV-ZnO for 24 hours showed
50.22 %, 19.81 % and 62.74 % apoptotic cells, respectively (Figure 8). Apoptosis was further
confirmed by Hoechst 33342 staining. PA1 cells were treated by RSV and RSV-ZnO nano-
conjugate for 24 hours and examined for nuclear morphology along with a control. As
revealed from Figure 9 treated cells showed marked DNA condensed nuclei and apoptotic
bodies whereas control cells had homogenously stained nuclei.
3.6. Induce apoptosis via ROS generation targeting mitochondria mediated pathway: It
is well established and also supported by many scientific reports that high level of ROS
formation usually inhibit the proliferation of many types of cells55 and RSV has been
identified as a potent antioxidant that can promote ROS generation56. So in the present
experimental set up we first evaluated whether treatment with conjugate could up regulate the
formation of ROS higher than the RSV only in PA1 cells were treated with IC50 value of
RSV and nano-conjugate with different time interval (0, 10 minutes, 30 minutes, 1 hr, 2 hrs, 4
hrs and 6 hrs). The generated ROS was measured using the fluorescence dye DCFH-DA and
the results were analyzed using flow cytometry. Treatment with conjugate produce much
higher ROS with lower concentration in compare to that of RSV in PA1 cells as shown in
Figure 10. Apparently both the ZnO and RSV promoted the formation of ROS in time
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dependent manner but conjugate was found to efficiently increase ROS generation at
DOI: 10.1039/C6RA16664D
significant lower dose than RSV.
After estimation of ROS generation in PA1cells by conjugate treatment, we analyzed the
mitochondrial membrane depolarization in treated cells and found a change of mitochondrial
membrane potential in PA1 cells. Figure 11 reveals the cells treated with RSV, ZnO NPs and
RSV-ZnO for 6 hrs showed a marked increase (p< 0.05 compared to control in all cases) in
percentage of depolarized cell number. There were 35% depolarized cells at IC50 value of
RSV-ZnO as compared to the control cells. The value of depolarised cells in case of RSV-
ZnO is significantly differ from RSV (p< 0.0024) and ZnO NPs (p<0.002) treated systems.
The Taken together our findings suggest that conjugation induced apoptosis is also mediated
through ROS generation and have much effect on PA1 cell line.
3.7. Analysis of key apoptotic protein: Apoptosis can be initiated by intracellular and
extracellular signals that determine whether a cell is to survive or undergo apoptosis. In
previous findings it has been shown that Bcl2 family members are the mediators of cell
survival and apoptosis. The expression level of key apoptotic protein was examined in treated
cells by western blotting. Figure 12 reveals a significant increase in Bax protein expression
level and decrease level of Bcl2 protein expression infers the apoptosis pathway of ovarian
cancer cell death57-58. For further confirmation, we have also performed the expression level
of Caspase 9 protein in PA1 cells upon treatment with RSV ZnO NPs and RSV-ZnO nano-
conjugate. A significant increase in Caspase-9 levels was revealed from our study. Bax and
Bcl2 are pro and anti apoptotic proteins, respectively and caspase-9 is also an apoptotic
protein59. Our results strongly suggest that lose dose conjugate inhibits ovarian cancer cell
growth via apoptosis mechanism as shown in Scheme 1.
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Conclusion: In summary, we have investigated the enhanced efficacy of RSV-DOZIn: 1O0.10n3a9/nCo6R-A16664D
conjugate over free RSV in PA1 cells (ovarian cancer cell). Also we have explored different
mechanism like cytotoxicity, oxidative stress parameters and apoptosis in same cells upon
treatment with RSV- ZnO nano-conjugate. The RSV–ZnO nano-conjugate was characterized
by FTIR spectroscopy, Raman scattering and TEM images. Picosecond-resolved fluorescence
studies on RES–ZnO nano-conjugate also reveal efficient electron migration from ZnO to
RSV, eventually enhancing the ROS activity in the RSV–ZnO nanostructures. PA1 cells
exposed with IC50 value of drugs which showed decrease in cell viability and the mode of
induced cell death was found to be apoptosis and the percentage of apoptosis is higher than
free RSV. RSV-ZnO nano-conjugate also induced high oxidative stress by ROS generation
than free RSV. Mitochondrial membrane depolarization value is also high in case of RSV-
ZnO nano-conjugate than free RSV. Our results evaluate that apoptosis is occurred through
high ROS generation and mitochondrial membrane depolarization. Our findings reveal that
the RSV-ZnO nano-conjugate shows much more efficacy in anticancer effect on PA1 cells
compare to free RSV.
Acknowledgement: M.K. thanks Maulana Azad National fellowships (MANF), UGC, India
and S.C. thanks CSIR, India for their research fellowships. PL thanks the Braunschweig
International Graduate School of Metrology B-IGSM and the DFG Research Training Group
GrK 1952/1 "Metrology for Complex Nanosystems” for support. We thank UGC-CAS for
central instrumental facility of University of Calcutta. We also thank DST (India) for
financial grants (SB/S1/PC-011/ 2013) and DAE (India) for (2013/37P/ 73/BRNS).
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DOI: 10.1039/C6RA16664D
Table 1: Fluorescence lifetimes (i ) of different systems in two detection wavelengths 500
nm and 550 nm. (ex=375 nm)
System
1 ns (%)
2 ns (%)
3 ns (%)
avg ns
ZnO NPs
1.15 (70)
2.45 (25)
38.67 (5)
3.35
(em@500 nm)
ZnO NPs
(em@550 nm)
RSV-ZnO
(em@500 nm)
RSV-ZnO
0.96 (51)
0.19 (58)
0.21 (59)
3.94 (21)
1.63 (31)
1.86 (29)
44.81 (28)
9.13 (12)
10.99 (12)
13.86
1.17
1.98
(em@550 nm)
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DOI: 10.1039/C6RA16664D
Figure 1: (a) High-resolution TEM (HRTEM) image of ZnO NPs with. Inset shows the size
distribution of the ZnO NPs (b) UV-Vis absorption of ZnO NPs, RSV and RSV–ZnO.
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Figure 2: (a) FTIR and (b) Raman spectra of RSV, ZnO NPs and RSV–ZnO nano-
composites.
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Figure 3: (a) Fluorescence spectra of ZnO NPs and RSV–ZnO nano-composites are shown.
The broad emission band is composed of two components, P1 (500 nm) and P2 (555 nm).
The picosecond-resolved fluorescence transients of ZnO NPs (excitation at 375 nm) in the
absence and in the presence of RSV collected at (c) 500 nm and (d) 555 nm are shown.
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Figure 4: (a) Antibacterial activity of RSV–ZnO, RSV and ZnO. The upper panel shows
images of E. coli plates in presence of the corresponding samples. (b) The DCFH oxidation
with time in the presence of RSV–ZnO, RSV and ZnO and DCFH only.
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Figure 5: Cells were treated with different concentration of RSV, ZnO NPs and RSV-ZnO
nano-conjugate for 24hrs.viability was quantified by MTT assay.
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Figure 6: Cell morphology change after treatment by phase contrast microscopy
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Figure 7: Sub G0 value of samples which describe the % of cell death by apoptosis in cell
cycle study.
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DOI: 10.1039/C6RA16664D
Figure 8: RSV, ZnO, RSV-ZnO induced apoptosis in PA1 cells:Flow cytometric ananlysis of
AnnexinV-FITC/PI stained cells Representative dot plots of four independent experiments
are presented.
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DOI: 10.1039/C6RA16664D
Figure 9: Fluorescence image of cells stained with Hoechst33342. RSV, ZnO and RSV-ZnO
treated cells show apoptotic cells with condensed or fragmented nuclei (indicated by arrow).
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Figure 10: RSV, ZnO, RSV-ZnO treated cells were assessed by flow cytometry for ROS
generation by measuring DCF-fluorescence intensity and the data was represented as line
diagram.
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Figure 11: Flow cytometric analysis of JC1 stained cells to study mitochondrial membrane
potential changes. RSV-ZnO treated cells show high mitochondrial membrane depolarization
in compare to Control, RSV, and ZnO respectively. *Values differ significantly from control
$
#
(p<0.005),
Value of conjugate differ significantly from RSV (p<0.002),
Value of
conjugate differ significantly from ZnO (p<0.002)
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DOI: 10.1039/C6RA16664D
45kD
26kD
23kD
35kD
Figure 12: Western blot analysis of proteins involved in apoptosis: Bax, Bcl-2, and Caspase-9
proteins in PA1 cells treated with RSV, RSV-ZnO and ZnO with their IC50 concentration for
24 hrs. β-actin was used as control.
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