Cytotoxicity of Silver Nanoparticles Synthesized by
Catharanthus roseus Aqueous Extract on Jurkat and
HT29 Cancer Cell Lines
Nor Hazwani Ahmad, PhD
Nanotechnology Congress & Expo
August 11-13, 2015
Frankfurt, Germany
Research background
2008-2012
2013-now
Effects of Catharanthus roseus
Aqueous Extract on Jurkat Cells and
Normal Peripheral Blood
Mononuclear Cells
Biosynthesis and Characterization of
Silver Nanoparticles Using Catharanthus
roseus Plant Extracts and Analysis of
Cytotoxic Activities
Supervisor: Prof. Ishak Mat
Co-supervisor: Assoc. Prof. Mustaffa Fadzil Farid
Wajidi
Siti Zulaikha Ghozali
Postgraduate stu dent
(Master of Science (Medical Research))
Ira Maya Sophia
Undergraduate stu
(Bachelor of Science (Biology))
Nur Jalilahtul Mahfuzah
Postgraduate stu dent
(Master of Science (Medical Research))
Gene expression profiling of C. roseus-treated Jurkat cells
The mechanism of apoptosis regulated by genes in C.roseus-treated
Jurkat cells. Adapted from “The role of the Bcl-2 protein family in
cancer” by L. Coultas and A. Strasser, 2003. Seminars in Cancer Biology,
13, p. 116.
= Pro-apoptosis genes
= Inhibition
= Activation
= Upregulated genes
= Downregulated genes
Gene expression profiling of C. roseus-treated Jurkat cells
The differentially expressed genes associated with the progression of cell cycle induced by C.roseus extract in Jurkat cells. Adapted from “The
molecular biology of head and neck cancer” by C. R. Leemans, B. J. M. Braakhuis and R. H. Brakenhoff, 2011, Nature Reviews in Cancer, 11, p. 12.
= S-phase arrest-induced genes
= Upregulated genes
= Downregulated genes
Introduction
Detection,
target &
treatment of
CA [1]
Nanoscale Tx is
advantageous
[2, 3]
Nano
oncology
Overcome
limitation of
conventional
CA therapy [4]
NP-derived
drugs in
different phase
of clinical
development [5]
Silver nanoparticles (AgNPs)
•
•
•
Definition: nanoscale metals sized within 1 to 100 nm [6]
Unique properties: good in conductivity, stability, catalytic & antibacterial [7]
Biomedical application: anti-inflammatory, antioxidant, antimicrobial, medical devices [8,9
•
However, potential application as anticancer agents are still new and remain to be
investigated
]
• Biocompatible to healthy cells and has inhibitory effects on various human CA cell lines:
glioblastoma cells [10], Dalton’s lymphoma ascites [11], cervical carcinoma [12], breast
carcinoma MCF-7 cells [13], HeLa cells [14], lung cancer A549 cells [15].
•
Method of synthesis
• Physical (thermal and laser ablation, sputtering, milling) [16]
• Chemical (sodium borohydride, potassium bitartrate, methoxypolyethelene glycol,
hydrazine). Hazardous Substance Fact Sheet: sodium borohydride cause irritation and
burn, shortness of breath etc [17]
• Biological (Plants, microorganism) [18]
Problem statement 1: Limitation of conventional anticancer therapy
Problem statement 2: Hazardous chemicals used for AgNPs synthesis
Main objective
To evaluate the anticancer activity of C. roseus-AgNPs on Jurkat and HT29 cells
Specific objectives
1) To examine the effects of C. roseus-AgNPs on the proliferation of Jurkat and HT29 cells
2) To evaluate early detection of apoptosis in Jurkat and HT29 cells treated with C. roseus-AgNPs
3) To analyze the cell cycle of Jurkat and HT29 cells treated with C. roseus-AgNPs
Methodology
Preparation Characterization
of C. roseus -AgNPs
Preparation of cell lines
C. roseus-AgNPs
Surface
plasmon
nm
10% of C. roseus
aqueous
extract in 5 mM of AgNO500
3
(uv-vis spectroscopy)
Transmission electron
microscopy (TEM)
MTS
X-ray diffraction (XRD)
Concentrations: 1.96 to
1000 µg/ml (double
dilution manner)
Incubation times: 24, 48
and 72 h
ELISA microplate reader
Jurkat cells (4 × 105 cells/ml)
HT29 cells (1 × 105 cells/ml)
Shape: Spherical & uniform
Size: 20 to 50 nm
Annexin-FITC/PI
Cell cycle
Average of diameter: 30 nm
Spectrum 2θ values: 38.12°, 44.31, 64.45 &
Concentration: 10 µg/ml
Concentrations:
77.41 5, 10, 15
µg/ml111, 200, 220 & 311
Plane:
Incubation times: 24, 48
Structure: Crystals in nature and 72 h
Incubation times: 6, 24,
48 and 72 h
FACS Calibur flow
cytometer (Cell Quest Pro
software)
Statistical analysis
One-way ANOVA, post-hoc Tukey’s test
Significance: p < 0.05
FACS Calibur flow
cytometer (Cell Quest Pro
& ModFit softwares)
Results and Discussion
Proliferative effects of C. roseus-AgNPs on Jurkat cells
Median IC50 value: 5.87 µg/ml
Figure 1: Proliferative effects were evaluated by MTS assay. Jurkat cells were treated with C. roseus-AgNPs at double dilution manner. Untreated
cells were used as negative control while camptothecin was used as positive control. Each value was expressed as mean ± SD of three
replicates. * indicates significant differences (p < o.o5) with respect to untreated group.
Proliferative effects of C. roseus aqueous extract on Jurkat cells
Median IC50 value: 361.72 µg/ml
Figure 2: Proliferative effects were evaluated by MTS assay. Jurkat cells were treated with C. roseus aqueous extract at double dilution manner.
Untreated cells were used as negative control while camptothecin was used as positive control. Each value was expressed as mean ± SD of
three replicates. * indicates significant differences (p < o.o5) with respect to untreated group.
Proliferative effects of C. roseus-AgNPs on HT29 cells
Median IC50 value: 13.19 µg/ml
Figure 3: Proliferative effects were evaluated by MTS assay. HT29 cells were treated with C. roseus-AgNPs at double dilution manner. Untreated
cells were used as negative control while camptothecin was used as positive control. Each value was expressed as mean ± SD of three
replicates. * indicates significant differences (p < o.o5) with respect to untreated group.
Proliferative effects of C. roseus aqueous extract on HT29 cells
Median IC50 value: 419.26 µg/ml
Figure 4: Proliferative effects were evaluated by MTS assay. HT29 cells were treated with C. roseus aqueous at double dilution manner.
Untreated cells were used as negative control while camptothecin was used as positive control. Each value was expressed as mean ± SD of
three replicates. * indicates significant differences (p < o.o5) with respect to untreated group.
•
C. roseus-AgNPs on Jurkat
cells
C. roseus-AgNPs on HT29
cells
C. roseus aqueous extract
on Jurkat cells
C. roseus aqueous extract
on HT29 cells
Med: 5.87 µg/ml
Med: 13.19 µg/ml
Med: 361.72 µg/ml
Med: 419.26 µg/ml
C. roseus-AgNPs produced higher cytotoxicity effects than C. roseus aqueous extract
• Different phytochemicals responsible in the AgNP synthesis have contributed to the cytotoxic
effects on cells (fatty acids, esters & alkaloids) [19]
• Unique features of AgNPs (small size, high surface area to volume ratio, surface
functionalization)  biokinetics of AgNPs & increase cytotoxicity [20]
• Higher number of AgNPs were observed in mouse fibroblasts compared to silver
microparticles  induce ROS  DNA damage  apoptosis [21]
•
Jurkat cells were more sensitive than HT29 cells, either in response to C. roseus-AgNPs or C.
roseus aqueous extract
• Anticancer vinca alkaloids present in C. roseus are commercial anticancer chemotherapeutic
drugs to combat acute lymphoblastic leukemia [20, 22]
• Other possible anticancer alkaloids include vindesine, vinepedine and vinsrosidine  antitubulin properties  inhibit formation of mitotic spindles by damaging microtubules  cell
cycle arrest [23]
C. roseus-AgNPs on Jurkat
cells
Induction: 1.96 µg/ml (72 h)
•
C. roseus-AgNPs on HT29
cells
Induction: 3.91 & 7.82
µg/ml (48 h), 1.96 & 3.91
µg/ml (72 h)
C. roseus aqueous extract on
Jurkat cells
C. roseus aqueous extract
on HT29 cells
No induction of cell
proliferation
No induction of cell
proliferation
Induction of cell proliferation at low concentrations of C. roseus-AgNPs on Jurkat and
HT29 cells
• Differential effects may occur where high dose increases cytotoxicity while low dose
induces cell proliferation. Hormesis = potentially toxic agents cause stimulation in lower
doses [24]
• Crude plant extract used in AgNPs synthesis contains numerous active compounds that
interacting to one another [24]
• Apart from anticancer compounds, non-enzymatic antioxidant molecules (ascorbic acid, αtocopherol, reduced glutathione and antioxidation enzymes)  scavenge the ROS [25]
Expression of externalized phosphatidylserine on Jurkat cells treated with C. roseus-AgNPs
Figure 5: Histograms of the detection of phosphatidylserine by annexin V-FITC/PI staining. Quantitative percentages of viable cells (Each
value was expressed as mean ± SD of three replicates. * indicates significant differences (p < o.o5) with respect to untreated group.
Expression of externalized phosphatidylserine on Jurkat cells treated with C. roseus
aqueous extract
Figure 6: Histograms represent quantitative percentages of viable cells (annexin-/PI-), early apoptotic cells (annexin+/PI-), late apoptotic cells
(annexin+/PI+) and necrotic cells (annexin-/PI+). Each value was expressed as mean ± SD of three replicates. * indicates significant differences (p
< o.o5) with respect to untreated group.
Expression of externalized phosphatidylserine on HT29 cells treated with C. roseus-AgNPs
*
*
Figure 7: Histograms represent quantitative percentages of viable cells (annexin-/PI-), early apoptotic cells (annexin+/PI-), late apoptotic cells
(annexin+/PI+) and necrotic cells (annexin-/PI+). Each value was expressed as mean ± SD of three replicates. * indicates significant differences (p
< o.o5) with respect to untreated group.
Expression of externalized phosphatidylserine on HT29 cells treated with C. roseus
aqueous extract
Figure 8: Histograms represent quantitative percentages of viable cells (annexin-/PI-), early apoptotic cells (annexin+/PI-), late apoptotic cells
(annexin+/PI+) and necrotic cells (annexin-/PI+). Each value was expressed as mean ± SD of three replicates. * indicates significant differences (p
< o.o5) with respect to untreated group.
•
At 6 h, C. roseus aqueous extract produced higher percentages of early apoptotic cells
while C. roseus-AgNPs produced higher percentages of late apoptotic cells
• Indicates that C. roseus aqueous extract started to induce apoptosis after 6 h and C.
roseus-AgNPs induced apoptosis earlier than 6 h
C. roseus-AgNPs on Jurkat
cells
•
C. roseus aqueous extract
on Jurkat cells
C. roseus aqueous extract
on HT29 cells
At 24 h, total percentages of early and late apoptotic cells for each treatment correlate
with MTS assay
C. roseus-AgNPs on Jurkat
cells
•
C. roseus-AgNPs on HT29
cells
C. roseus aqueous extract
on Jurkat cells
C. roseus-AgNPs on HT29
cells
C. roseus aqueous extract
on HT29 cells
At 48 and 72 h, total percentages of early and late apoptotic cells of HT29 treated by C.
roseus-AgNPs were higher compared to aqueous extract
• MTS assay - Induction of cell proliferation was observed at low concentrations, 3.91 &
7.82µg/ml (48 h) and 1.96 and 3.91 µg/ml (72 h)
• These concentrations are within the ranges of concentrations used for annexin/PI
staining
Effects of C. roseus-AgNPs and C. roseus aqueous extract on the cell cycle of Jurkat cells
Figure 9: Histograms of cell cycle of Jurkat cells that indicate percentages of cells in G0/G1, S, and G2/M phases. Each value was
expressed as mean ± SD of three replicates. * indicates significant differences (p < o.o5) with respect to untreated group.
Effects of C. roseus-AgNPs and C. roseus aqueous extract on the cell cycle of HT29 cells
Figure 10: Histograms of cell cycle of HT29 cells that indicate percentages of cells in G0/G1, S, and G2/M phases. Each value was
expressed as mean ± SD of three replicates. * indicates significant differences (p < o.o5) with respect to untreated group.
•
C. roseus-AgNPs on Jurkat
cells
C. roseus-AgNPs on HT29
cells
C. roseus aqueous extract on
Jurkat cells
C. roseus aqueous extract
on HT29 cells
Arrest: S-phase (all
incubations)
Arrest: G0/G1 phase (24,
48h), S phase(72h)
Arrest: S-phase (all
incubations)
Arrest: G0/G1 phase (24h), S
phase(48, 72h)
The DNA damage may arrest or suspend the cells in either G1, S or G2 before undergoing
apoptosis, in case the damage cannot be fixed [26]
• Oxidative stress activates p38 MAPK & inflammation of the transcription factors [27]
• Affect mitochondrial dependent jun-N terminal kinase pathway disruption of mitochondrial
respiratory chain  increase ROS & interferes ATP synthesis  damage cellular DNA [26]
•
Phytochemicals in C. roseus plant responsible for the cell cycle arrest
• Vinca alkaloids have anti-tubulin properties  disrupt and interfere microtubules  M-phase
arrest [28]
• Vincristine  G2/M phase arrest [29]
• Arrest was due to other active compounds in C. roseus
•
•
Camptothecin arrested S and G2/M phases [30, 31]
AgNO3 arrested G0/G1 phase (48, 72 h) in HT29 cells
• Requires further investigation since it did not inhibit the proliferative activity
Conclusion
Proliferative
activity
•
Induction of
apoptosis
Cell cycle
arrest
Jurkat and HT29 cells have undergone AgNPs-induced stress
• Further analysis on the detailed mechanism of cytotoxicity and cellular uptake for better
understanding on the cellular interaction
• Major drawback associated with new drug development include lack of specificity and
uncertainty with its cytotoxicity on normal cells  should be further evaluated
• In vivo studies are necessary to address the formulation of biogenic AgNPs as an alternative to
conventional anticancer drugs
•
Experimental evidence indicating C. roseus-AgNPs have been shown to induce higher
cytotoxic effects compared to C. roseus aqueous extract
• Small-sized AgNPs have increased its effectiveness to penetrate cells  cell death
•
Expand the knowledge on the comparison cytotoxic effects between C. roseus-AgNPs and C.
roseus aqueous extract on Jurkat and HT29 cells
• Foundation to develop better strategy of cancer therapeutic agents
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Acknowledgement
•
•
•
Scienfund Grant, AMDI Research Fund
Prof. Ishak Mat, Dr. Syed Atif Ali for the cell lines provided
Co-researchers:
Nor Jalilahtul Mahfuzah
Noordin
Ira Maya Sophia Nordin
Shahrul Bariyah Sahul
Hamid
Terima Kasih
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