Catharanthus roseus Aqueous Extract on Jurkat and HT29 Cancer Cell Lines Nanotechnology Congress amp Expo August 1113 2015 Frankfurt Germany Nor Hazwani Ahmad PhD ID: 806417
Download The PPT/PDF document "Cytotoxicity of Silver Nanoparticles Syn..." is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
Cytotoxicity of Silver Nanoparticles Synthesized by Catharanthus roseus Aqueous Extract on Jurkat and HT29 Cancer Cell Lines
Nanotechnology Congress & Expo August 11-13, 2015 Frankfurt, Germany
Nor
Hazwani
Ahmad, PhD
Slide2Research backgroundEffects of Catharanthus roseus Aqueous Extract on Jurkat
Cells and Normal Peripheral Blood Mononuclear CellsBiosynthesis and Characterization of Silver Nanoparticles Using Catharanthus roseus Plant Extracts and Analysis of Cytotoxic Activities2008-20122013-nowSiti Zulaikha GhozaliPostgraduate stu dent(Master of Science (Medical Research))Ira Maya SophiaUndergraduate
stu
(Bachelor of Science (Biology))
Nur
Jalilahtul
Mahfuzah
Postgraduate
stu
dent(Master of Science (Medical Research))
Supervisor: Prof. Ishak MatCo-supervisor: Assoc. Prof. Mustaffa Fadzil Farid Wajidi
Slide3The 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
Slide4The 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.= Downregulated genes= Upregulated genes
= S-phase arrest-induced genes
Gene expression profiling of
C.
roseus
-treated
Jurkat
cells
Slide5Introduction
Slide6Definition: 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 synthesisPhysical (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]
Silver nanoparticles (
AgNPs)
Slide7Problem statement 1: Limitation of conventional anticancer therapy
Problem statement 2: Hazardous chemicals used for AgNPs synthesis
To evaluate the anticancer activity of
C.
roseus
-AgNPs
on
Jurkat
and HT29 cells
Main objective
To examine the effects of
C.
roseus
-AgNPs
on the proliferation of
Jurkat
and HT29 cells
To evaluate early detection of apoptosis in
Jurkat
and HT29 cells treated with
C.
roseus
-AgNPs
To analyze the cell cycle of
Jurkat
and HT29 cells treated with
C.
roseus
-AgNPs
Specific objectives
Slide8MethodologyMTS
Concentrations: 1.96 to 1000 µg/ml (double dilution manner)Incubation times: 24, 48 and 72 hELISA microplate reader Annexin-FITC/PICell cycleConcentrations: 5, 10, 15 µg/ml
Incubation times: 6, 24, 48 and 72 h
FACS
Calibur
flow cytometer (Cell Quest Pro software)
Concentration: 10 µg/ml
Incubation times: 24, 48 and 72 h
FACS
Calibur
flow cytometer (Cell Quest Pro &
ModFit
softwares
)
Preparation of
C.
roseus
-
AgNPs
10% of
C.
roseus
aqueous extract in 5
m
M
of AgNO
3
Statistical analysis
One-way ANOVA, post-hoc
Tukey’s
test
Significance:
p
< 0.05
Preparation of cell lines
Jurkat
cells (4 × 10
5
cells/ml)
HT29
cells
(1
× 10
5
cells/ml)
Characterization
C.
roseus
-AgNPs
Surface
plasmon
(
uv-
vis
spectroscopy)
500 nm
Transmission electron microscopy (TEM)
Shape: Spherical & uniform
Size: 20 to
50 nm
Average of diameter: 30 nm
X-ray diffraction (XRD)
Spectrum 2θ
values: 38.12°, 44.31, 64.45 & 77.41
Plane: 111, 200, 220
& 311
Structure: Crystals in nature
Slide9Results and DiscussionProliferative effects of C. roseus-AgNPs on Jurkat cells
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. Median IC50 value: 5.87 µg/ml
Slide10Proliferative effects of C. roseus aqueous extract on Jurkat cells
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. Median IC50 value: 361.72 µg/ml
Slide11Proliferative effects of C. roseus-AgNPs on HT29 cells
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. Median IC50 value: 13.19 µg/ml
Slide12Proliferative effects of C. roseus aqueous extract on HT29 cells
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. Median IC50 value: 419.26 µg/ml
Slide13C.
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
anti-tubulin properties inhibit formation of mitotic spindles by damaging microtubules cell cycle arrest
[23]
Slide14Induction 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, α-tocopherol, reduced glutathione and antioxidation enzymes) scavenge the ROS [25]
Slide15Expression of externalized
phosphatidylserine on Jurkat cells treated with C. roseus-AgNPsFigure 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.
Slide16Expression 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.
Slide17Expression of externalized
phosphatidylserine on HT29 cells treated with C. roseus-AgNPsFigure 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. **
Slide18Expression of externalized phosphatidylserine on HT29 cells treated with C. roseus aqueous extractFigure 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.
Slide19At 24 h, total percentages of early and late apoptotic cells for each treatment correlate with MTS assay
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.
roseu
s-AgNPs
induced apoptosis earlier than 6 h
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 & 7.82
µg/
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
Slide20Effects of
C. roseus-AgNPs and C. roseus aqueous extract on the cell cycle of Jurkat cellsFigure 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.
Slide21Effects 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.
Slide22The 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 arrestVinca 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 G
2
/M phases
[30, 3
1
]
AgNO
3 arrested G0/G
1
phase (48, 72 h) in HT29 cells
Requires further investigation since it did not inhibit the proliferative activity
Slide23Jurkat and HT29 cells have undergone AgNPs-induced stressFurther 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 evaluatedIn vivo studies are necessary to address the formulation of biogenic AgNPs as an alternative to conventional anticancer drugsExperimental evidence indicating C. roseus-AgNPs have been shown to induce higher cytotoxic effects compared to C. roseus aqueous extractSmall-sized AgNPs have increased its effectiveness to penetrate cells cell deathExpand 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
Conclusion
Slide24ANTHONY, J. J., SITHIKA, M. A. A., JOSEPH, T. A., SURIYAKALAA, U., SANKARGANESH, A., SIVA, D., KALAISELVI, S. & ACHIRAMAN, S. 2013. In vivo antitumor activity of biosynthesized silver nanoparticles using Ficus religiosa as a nanofactory in DAL induced mice model. Colloids and Surfaces B: Biointerfaces, 108, 185-190.
AHAMED, M., ALSALHI, M. S. & SIDDIQUI, M. K. J. 2010. Silver nanoparticle applications and human health. Clinica Chimica Acta, 411, 1841-1848.SINGH, R. & JR., J. W. L. 2009. Nanoparticles-based targeted drug delivery. Experimental and Molecular Pathology, 86, 215-223.SATAPATHY, S. R., MOHAPATRA, P., PREET, R., DAS, D., SARKAR, B., CHOUDHURI, T., WYATT, M. D. & KUNDU, C. N. 2013. Silver-based nanoparticles induce apoptosis in human colon cancer cells mediated through p53. Nanomedicine, 8, 1307-1322.BHATTACHRYYA, S., KUDGUS, R. A., BHATTACHRYYA, R. & MUKHERJEE, P. 2011. Inorganic nanoparticles in cancer therapy. Pharmaceutical Research, 28, 237-259.PRABHU, S. & POULOSE, E. K. 2012. Silver nanoparticles: mechaanism of antimicrobial action, synthesis, medical applicatios, and toxicity effects. International Nano Letters, 2.JEYARAJ, M., SATHISKUMAR, G., SIVANANDHAN, G., MUBARAKALI, D., RAJESH, M., ARUN, R., KAPILDEV, G., MANICKAVASAGAM, M., THAJUDDIN, N., PREMKUMAR, K. & GANAPATHI, A. 2013. Biogenic silver nanoparticles for cancer treatment: An experimental report. Colloids and Surfaces B: Biointerfaces, 106, 86-92.SARANYAADEVI, K., SUBHA, V., RAVINDRAN, R. S. E. & RENGANATHAN, S. 2014. Green synthesis and characterization of silver nanoparticle using leaf extract of Capparis zeylanica. Asian Journal of Pharmaceutical and Clinical Research, 7.GURUNATHAN, S., RAMAN, J., MALEK, S. N. A., JOHN, P. A. & VIKINESWARY, S. 2013b. Green synthesis of silver nanoparticles using
Ganoderma neo-japonicum
Imazeki: a potential cytotoxic agent against breast cancer cells.
International Journal of Nanomedicine,
8
,
4399-4413.
RANI, P. V. A., HANDE, M. P. & VALIYAVEETTIL, S. 2009. Anti proliferative activity of silver nanoparticles.
BMC Cell Biology,
10.
SRIRAM, M. I., KANTH, S. B. M., KALISHWARALAL, K. & GURUNATHAN, S. 2010. Antitumor activity of silver nanoparticles in Dalton's lymphoma ascites tumor model.
International Journal of Medicine,
5, 753-762.VASANTH, K., ILANGO, K., KUMAR, R. M., AGRAWAL, A. & DUBEY, G. P. 2014. Anticancer activity of Moringa oleifera mediated silver nanoparticles on human cervical carcinoma cells by apoptosis induction. Colloids and Surfaces B: Biointerfaces, 117,
354-359.
YEHIA, R. S. & AL-SHEIKH, H. 2014. Biosynthesis and characterization of silver nanoparticles produced by Pleurotus ostreatus and their anticandidal and anticancer activities.
World J Microbiol Biotechnol,
30
,
2797-2803.
MANIVASAGAN, P., VENKATESAN, J., SENTHILKUMAR, K., SIVAKUMAR, K. & KIM, S.-K. 2013. Biosynthesis, antimicrobial and cytotoxic effect of silver nanoparticle using a novel
Nocardiopsis sp.
MBRC-1.
BioMed Research International
.
SANKAR, R., KARTHIK, A., PRABU, A., KARTHIK, S., SHIVASHANGARI, K. S. & RAVIKUMAR, V. 2013.
Origanum vulgare
mediated biosynthesis of silver nanoparticles for its antibacterial and anticancer activity.
Colloids and Surfaces B: Biointerfaces,
108
,
80-84.
References
Slide25MITTAL, A. K., CHISTI, Y., BANARJEE, U. C. 2013. Synthesis of metallic nanoparticles using plant extracts. Biotechnology Advances. 31: 346-356.DURAI, P., CHINNASAMY, A., GAJENDRAN, B., RAMAR, M., PAPPU, S., KASIVELU, G. & THIRUNAVUKKARASU, A. 2014. Synthesis and characterization of silver nanoparticles using crystal compound of
sodium para-hydroxybenzoate tetrahydrate isolated from Vitex negundo. L leaves and its apoptotic effect on human colon cancer cell lines. European Journal of Medicinal Chemistry, 84.SULAIMAN, G. M., MOHAMMED, W. H., MARZOOG, T. R., AL-AMIERY, A. A. A., KADHUM, A. K. H. & MOHAMAD, A. B. 2013. Green synthesis, antimicrobial and cytotoxic effects of silver nanoparticles using Eucalyptus chapmaniana leaves extract. Asian Pacific Journal of Tropical Biomedicine, 3, 58-63.SOTTOMAYOR, M., CARDOSO, I. L., PEREIRA, L. & BARCELÓ, A. R. 2004. Peroxidase and the biosynthesis of terpenoid indole alkaloids in the medicinal plant Catharanthus roseus (L.) G. Don. Phytochemistry Reviews, 3, 159-171.ASHARANI, P., LOW KAH MUN, G., HANDE, M. P. & VALIYAVEETTIL, S. 2008a. Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS nano, 3, 279-290.WEI, L., TANG, J., ZHANG, Z., CHEN, Y., ZHOU, G. & XI, T. 2010. Investigation of the cytotoxicity mechanism of silver nanoparticles in vitro. Biomedical Materials, 5, 044103.DANIELLE, M., PARESH, C. R. & YU, H. 2014. Molecular Toxicity Mechanism of Nanosilver. Journal of Food and Drug Analysis, 22, 116-127.CHUN-HUA WANG, GUO-CAI WANG, YIN WANG, XIAO-QI ZHANG, XIAO-JUN HUANG, DONG-MEI ZHANG, MIN-FENG CHEN & WEN-CAI YE 2012. Cytotoxic dimeric indole alkaloids from Catharantus roseus. Fitoterapia, 83, 765-769.ASMAH, R., ZETI NADIA, M. Z., ABDAH, M. A. & FADZELLY, A. B. M. 2005. Effects of
Catharanthus roseus
,
Kalanchoe laciniata
and
Piper longum
Extracts on the Proliferation of Hormone-dependent Breast Cancer (MCF-7) and Colon Cancer (Cac02) Cell Lines.
Malaysian Journal of Medicine and Health Sciences,
1
,
105-110.
NEJAT, N., VALDIANI, A., CAHILL, D., TAN, Y.-H., MAZIAH, M. & ABIRI, R. 2015. Ornamental Exterior versus Therapeutic Interior of Madagascar Periwinkle (Catharanthus roseus): The Two Faces of a Versatile Herb. The Scientific World Journal, 2015
, 982412.ASHARANI, P. V., LOW KAH MUN, G., HANDE, M. P. & VALIYAVEETTIL, S. 2008b. Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS nano, 3, 279-290.EOM, H.-J. & CHOI, J. 2010. p38 MAPK Activation, DNA Damage, Cell Cycle Arrest and Apoptosis As Mechanisms of Toxicity of Silver Nanoparticles in Jurkat T Cells. Environmental Science & Technology, 44
,
8337-8342.
SHEN, Y., LIU, Q., SUN, H., LI, X., WANG, N. & GUO, H. 2013. Protective effect of augmenter of liver regeneration on vincristine-induced cell death in Jurkat T leukemia cells.
International Immunopharmacology,
17
,
162-167.
SHAO, R. G., CAO, C. X., NIEVES-NEIRA, W., DIMANCHE-BOITREL, M. T., SOLARY, E. & POMMIER, Y. 2001. Activation of the Fas pathway independently of Fas ligand during apoptosis induced by camptothecin in p53 mutant human colon carcinoma cells.
Oncogene,
20
,
1852-1859.
DONG, Y. B., YANG, H. L. & MCMASTERS, K. M. 2003. E2F-1 overexpression sensitizes colorectal cancer cells to camptothecin.
Cancer Gene Ther,
10
,
168-178.
Slide26Acknowledgement
Scienfund Grant, AMDI Research FundProf. Ishak Mat, Dr. Syed Atif Ali for the cell lines provided Co-researchers:Nor Jalilahtul Mahfuzah NoordinIra Maya Sophia NordinShahrul Bariyah Sahul Hamid
Slide27Terima KasihMentransformasikan Pengajian Tinggi Untuk Kelestarian Hari Esok